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Modeling the evolutionary loss of erythroid genes by Antarctic icefishes: analysis of the hemogen gene using transgenic and mutant zebrafish
by Michael J. Peters
B.S. in Biology, University of New Hampshire
A dissertation submitted to
The Faculty of the College of Science of Northeastern University
in partial fulfillment of the requirements for the degree of Doctor of Philosophy
June 4, 2018
Dissertation directed by
H. William Detrich, III Professor of Marine Molecular Biology and Biochemistry
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Dedication
For my Oma Oswald, who started this journey with me.
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Acknowledgments
I would like to thank my advisor, Dr. H William Detrich III, for encouraging me to be
innovative and to pursue cutting-edge research. I thank the members of my committee,
Drs. Erin Cram, Rebeca Rosengaus, Steven Vollmer, and Leonard Zon for their many
helpful suggestions. I enjoyed working alongside Sandra Parker and Carmen
Elenberger and appreciate their support. I also enjoyed working with many students
including Caroline Benavides, Carolyn Dubnik, Carmen Elenberger, Laura Goetz, Urjeet
Khanwalkar, Ben Moran, Alessia Santilli, Eileen Sheehan, Margaret Streeter, Kathleen
Shusdock, and Sierra Smith. I especially thank Jonah Levin, who joined the lab as a
high school student and has since continued working with me. I thank Corey Allard and
Drs. Donald Yergeau and Jeffrey Grim for collecting samples that I used in my studies. I
owe thanks to the staff of the Marine Science Center for their support, including Roberto
Valdez, Sonya Simpson, Heather Sears, David Dawson, and Ryan Hill. I thank Drs.
Joseph Ayers and Justin Ries for use of their facilities and Drs. Isaac Westfield and
Ryan Myers for help with scanning electron microscopy. I thank Drs. Camille Berthelot,
Melody Clark, James Monaghan, and Leonard Zon for valuable discussions and
providing important datasets and materials. I thank Dr. Jill de Jong and her lab for
inspiring my interest in zebrafish research. I was pushed to my best by my fellow
graduate students at Northeastern University. I am especially grateful to my Mom and
Dad for their unending support and for reading every word I write. I am grateful to my
siblings Sarah, Katie, Ryan, and Zachary for inspiring me with their passions and
positive energy.
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Abstract of Dissertation
The Antarctic icefishes (Channichthyidae) are the only vertebrate taxon whose
species do not produce red blood cells, thereby providing a natural mutant model to
study the regulators of blood development and disease. To identify novel regulators of
erythropoiesis, I compared RNA-Seq transcriptomes from red- and white-blooded
notothenioids. I find that both icefishes and their sister taxon, the dragonfishes
(Bathydraconidae), model beta-spectrin mutated spherocytic anemia. Icefishes appear
to have evolved morph-biased changes in expression of hematopoietic regulatory
genes, including down-regulation of the histone acetyltransferase p300 and
overexpression of histone deacetylase 1b. In icefishes, I characterize a frameshift
mutation that truncates the P300-binding domain of Hemogen, an important erythroid
transcription factor. Tol2 and CRISPR/Cas9-generated transgenic zebrafish lines reveal
that hemogen is expressed in hematopoietic, renal, neural, and reproductive tissues. I
find that two conserved non-coding elements differentially contribute to hemogen
expression in primitive and definitive waves of hematopoiesis. CRISPR-generated
mutant zebrafish lines, which replicate the C-terminal mutation in icefish hemogen,
show severe anemia and growth defects. Furthermore, I show that the function of
zebrafish Hemogen is dependent on acidic residues within the TAD. Thus, Antarctic
icefishes evolved an intricate system for repression of erythropoiesis that is caused in
part by the loss of Hemogen function.
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Table of Contents
Dedication 2
Acknowledgments 3
Abstract of Dissertation 4
Table of Contents 5
List of Figures 6
List of Tables 9
List of Symbols 10
List of Genes 14
Chapter 1: Morph-biased gene expression and sequence divergence typifies disease-like
traits of Antarctic icefishes 25
Chapter 2: Divergent Hemogen genes of teleosts and mammals share conserved roles in
erythropoiesis: Analysis using transgenic and mutant zebrafish 87
Chapter 3: Erythroid gene discovery using the erythrocyte-null Antarctic icefishes 157
Conclusion 196
References 200
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List of Figures
Introduction
Figure 1 Erythropoiesis in the zebrafish 19
Figure 2 Comparison of red- and white-blooded notothenioids 24
Chapter 1
Figure 1 Peripheral blood smears from Antarctic notothenioid fishes 32
Figure 2 Expression profile heatmap of differentially expressed genes 36
Figure 3 Gene ontology enrichment for differentially expressed genes 40
Figure 4 Association networks for DE genes in the icefish head kidney 44
Figure 5 Three tissue-specific clusters of hematopoietic genes are differentially
expressed in the head kidneys of Ps. georgianus and P. charcoti 48
Figure 6 Differential expression of hematopoietic regulators 52
Figure 7 Deleterious substitutions in erythroid genes from icefishes 55
Figure 8 The dragonfish P. charcoti is a natural mutant model for beta-spectrin
mutated spherocytic anemia 57
Figure 9 Functional mutations occur in the interaction domains of Hemogen, Gata1,
and P300 64
Figure 10 Whole protein acetylation in the head kidneys of red- and white-blooded
notothenioids 68
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Chapter 2
Figure 1 Zebrafish si:dkey-25o16.2 and human hemogen are orthologous and
encode related proteins that differ in size 93
Figure 2 hemogen expression in zebrafish embryos 99
Figure 3 Alternative promoters drive hemogen expression in hematopoietic and
nonhematopoietic tissues in zebrafish 103
Figure 4 Conserved elements in the zebrafish hemogen promoter are predicted
targets for transcription factors 108
Figure 5 Gata1 binds distal and proximal promoter elements to regulate hemogen
expression in zebrafish 111
Figure 6 Promoter elements have distinct roles in driving hematopoietic, renal, and
testicular expression of hemogen in transgenic Tg(hemgn:mCherry)
zebrafish 115
Figure 7 Morpholino targeting of hemogen inhibits erythropoiesis in embryonic
zebrafish 118
Figure 8 CRISPR/Cas9 mutagenesis of the third exon of zebrafish hemogen
impairs primitive and definitive erythropoiesis 124
Chapter 3
Figure 1 The erythroid gene hemogen is mutated in Antarctic icefishes 161
Figure 2 hemogen is expressed in hematopoietic, renal, and neural tissues in red-
blooded notothenioids 164
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Figures 3 A truncated isoform of hemogen is highly expressed in icefishes and is
translated 169
Figure 4 Overexpression of icefish hemogen in zebrafish blocks primitive
erythropoiesis 172
Figure 5 A novel MABP-containing protein (mabpcp) is an RBC-specific gene that
was lost in icefishes 174
Figure 6 Modeling a truncated cd33-related Siglec (cd33rSig) from icefishes in
mutant zebrafish 180
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List of Tables
Chapter 1
Table 1 Hematopoietic genes are differentially expressed in the icefish head
kidney 84
Table 2 GO enrichment of genes under different selective pressures in two red-
and two white-blooded notothenioids 84
Table 3 GO enrichment for genes with deleterious substitutions found in two white-
blooded icefishes but not in two red-blooded notothenioids 85
Table 4 Table of primers 85
Table 5 Icefishes have predicted deleterious substitutions in targets of human
diseases 86
Chapter 2
Table S1 Sequences of primer and oligonucleotides used in experiments 156
Chapter 3
Table S1 Primer Sequences 194
Table S2 Oligos for CRISPR gRNA template 195
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List of Symbols
AGM Aorta gonad mesonephros
ALL Acute lymphocytic leukemia
AML Acute myeloid leukemia
ATP Adenosine triphosphate
B-ALL B-cell acute lymphoblastic leukemia
BWS Beckwith-Wiedemann syndrome
bZIP Basic leucine zipper domain
CBF-AML Core binding factor acute myeloid leukemia
CC Coiled coil domain
CHT Caudal hematopoietic tissue
Ce Corpus cerebelli
CL-XPosure Clear-blue X-ray film
CLL Chronic lymphocytic leukemia
CML Chronic myeloid leukemia
CMP Common myeloid progenitor
COFS Cerebro oculo facio skeletal syndrome
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
CT domain C-terminal cystine knot-like domain
CT-ZF C-terminal zinc finger
CV Caudal vein
Cyto Cytoplasmic domain
C2H2 Cys2-His2 zinc finger
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DA Dorsal aorta
DBA Diamond-Blackfan anemia
DED1 Death effector domain
DLBCL Diffuse large B-cell lymphoma
DS-AMKL Acute megakaryoblastic leukemia in Down syndrome
ECL Enhanced chemiluminescence
EGFP Enhanced green fluorescent protein
G Glomerulus
HCP Hereditary Coproporphyria
HDR Homology directed repair
HK Head kidney
HNSCC Head and neck squamous cell carcinoma
HRP Horseradish peroxidase
HS Hereditary spherocytic anemia
ICM Intermediate cell mass
Ig Immunoglobulin
IgG Immunoglobulin G
ITIM Immunoreceptor tyrosine-based inhibition motif
LDS Lithium dodecyl sulfate
MAE Myoclonic astatic epilepsy
MABP MVB12-associated beta prism domain
MHB Midbrain-hindbrain-boundary
MO Medulla oblongata
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MPN Myeloproliferative neoplasms
NH-terminus Amino-terminus
NHEJ Non-homologous end joining
NLS Nuclear localization signal
PBI Peripheral blood island
PD Pronephric ducts
PHD Plant homeodomain
ProE Proerythroblast
PTK Protein tyrosine kinase
PVDF Polyvinylidene fluoride
RING-finger Really interesting new gene finger domain
SCN Severe congenital neutropenia
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Se Sertoli cells
Siglec Sialic acid-binding immunoglobulin-type lectin
SNF Sucrose non-fermentable
ST Seminiferous tubules
TAD Transactivation domain
TALEN Transcription activator-like effector nuclease
T-ALL T-cell acute lymphoblastic leukemia
T-CLL T-cell chronic lymphoblastic leukemia
TBST Tris-buffered saline and Tween-20
TFBS Transcription factor binding site
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TK Trunk kidney
Tr Transmembrane domain
UDP Uridine diphosphate
WISH Whole-mount in situ hybridization
Zn Zinc
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List of Genes
Add1 Adducin-1
Add2 Adducin-2
AKT2 RAC-beta serine/threonine protein kinase 2
Ank1 Ankyrin-1
Anxa2 Annexin A2
Asxl2 Additional sex combs like 2, transcriptional regulator
Band3/Slc4a1 Band 3 anion transport protein
Bcl11a B-cell lymphoma/leukemia 11a
Bcl2l1/BclxL BCL2-like 1 gene/B-cell lymphoma-extra large 1 gene
Blvrb Biliverdin reductase B
Brca1 Breast cancer 1
Card11 Caspase recruitment domain family member 11
Casp8 Caspase 8
Cdkn1c Cyclin dependent kinase inhibitor 1c
Cd33rSig CD33 related siglec
Chd2 Chromodomain helicase DNA binding protein 2
Cox15 Cytochrome C oxidase assembly homolog
Cpox Coproporphyrinogen oxidase
Edag Erythroid differentiation associated gene
Eml1 Echinoderm microtubule associated protein like 1
Epor Erythropoietin receptor
Ercc1 Excision repair 1
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Ero1lα Endoplasmic reticulum oxidoreductase 1 alpha
Fam161al Family with sequence similarity 161, member A-like
Fech Ferrochelatase
Fes Feline sarcoma oncogene
Fgl1 Fibrinogen-like protein 1
Flt1 Fms related tyrosine kinase 1
Foxo1 Forkhead box protein O1
Foxp1 Forkhead box protein P1
Gata1 GATA-binding factor 1
Gapdh Glyceraldehyde 3-phosphate dehydrogenase
Gfi1 Growth factor independent 1 transcriptional repressor
Gfi1b Growth factor independent 1 transcriptional repressor B
G6PD Glucose-6-phosphate dehydrogenase
Hbba Hemoglobin, beta adult major chain
Hbbe1 Hemoglobin beta embryonic 1.1
Hdac1b Histone deacetylase 1b
Hemgn Hemogen
Hpx Hemopexin
Ikzf1 Ikaros family zinc finger 1
Ikzf2 Ikaros family zinc finger 2
Il7r Interleukin-7 receptor
Klf1 Krüppel-like factor 1
Ldb1 LIM domain-binding protein 1
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Lmo2 LIM domain only 2
Mabpcp1/Dkey:30j10.5 MABP-containing protein 1
Mate1 Multidrug and toxin extrusion protein
Nfe2 Nuclear factor, erythroid derived 2
Nfe2l1 Nuclear factor, erythroid derived 2 like 1
Ntrk1 Neurotrophic receptor tyrosine kinase 1
Numa1 Nuclear mitotic apparatus protein 1
Pphln1 Periphilin 1
Pu.1/Spi1 Spi-1 proto-oncogene
P300 Histone acetyltransferase p300
Rela NFĸB p65 subunit/RELA proto-oncogene
Runx1 Runt related transcription factor 1
Sgk1 Serum and glucocorticoid-regulated kinase 1
Slc25a39 Solute carrier family 25 member 39
Sptb Spectrin beta, erythrocytic
Stat5b Signal transducer and activator of transcription 5B
Tal1 T-cell acute lymphocytic leukemia protein 1
Tf Transferrin
Tfrc Transferrin receptor C
Tgfβ Transforming growth factor beta 1
Trim16l Tripartite motif containing 16 like
Ugt1a1 UDP glucuronosyltransferase 1 family, polypeptide A1
Zfp64 Zinc finger protein 64
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Introduction
1. Ontogeny of hematopoiesis in vertebrates
Hematopoiesis, the production of all blood lineages from pluripotent
hematopoietic stem cells, is a complex developmental process essential to vertebrate
life. Comparison of hematopoiesis in different animal models (e.g. humans, chicken,
mice, zebrafish) has revealed fundamental features and key molecular regulators of
blood development and disease (Detrich, 1999; Paw and Zon, 2000; Zon, 1995).
Several hematopoietic processes and genes originated early in evolution and are
conserved in invertebrates, including both chordates (Pascual-Anaya et al., 2013) and
non-chordates (Evans et al., 2003). In all vertebrates, blood production occurs in two
distinct waves, termed primitive and definitive hematopoiesis (Maximow, 1909).
Primitive hematopoiesis occurs in the yolk sac blood islands in embryos of all
vertebrates except fishes (Zon, 1995). In this first wave, committed progenitors
differentiate into nucleated erythrocytes that supply the early embryo with oxygen as
they complete maturation in circulation (Palis, 2014). Definitive hematopoiesis is defined
as the production of blood lineages from pluripotent hematopoietic stem cells (HSCs),
which originate in the aorta gonad mesonephros (AGM). In humans and other
mammals, HSCs seed and develop in the fetal liver for a short time before colonizing
the bone marrow and thymus (Palis, 2014).
Even though many aspects of blood development are highly conserved across
vertebrate taxa, the evolution of diverse forms of animals was coincident with alterations
to the ontogeny of hematopoiesis. In all vertebrates, primitive hematopoiesis initiates in
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lateral plate mesoderm (LPM) from cells called “hemangioblasts,” which are capable of
forming both blood and vasculature (Sabin, 2002). In sharks and in some teleosts,
primitive hematopoiesis is extraembryonic and commences directly on the yolk sac
(Zon, 1995). However, in most teleost fishes, the first wave of hematopoiesis is
intraembryonic (Oellacher, 1872). The zebrafish has provided an excellent model to
study teleost blood development (Fig. 1A). Primitive erythroid progenitors are produced
in the intermediate cell mass (ICM) (Detrich et al., 1995). Subsequently, a committed
population of definitive erythroid-myeloid progenitors (EMPs) is found in the peripheral
blood island (PBI), intermixed with primitive erythrocytes (Bertrand et al., 2007a; Detrich
et al., 1995). Concurrently, primitive myelopoiesis initiates from anterior lateral plate
mesoderm (ALM) (Bennett et al., 2001). Primitive erythrocytes migrate from the ICM
and are pulled into circulation by the heart through the ducts of Cuvier (Detrich et al.,
1995). At 30 hpf, the first definitive HSCs are produced from hemogenic endothelial
cells from the ventral wall of the dorsal aorta in a region called the aorta gonad
mesonephros (AGM) (Thompson et al., 1998). The HSCs migrate via circulation to
colonize a temporary site of definitive hematopoiesis in the caudal hematopoietic tissue
(CHT) and to seed the thymus, the major site of adult T-cell production (Murayama et
al., 2006). From the CHT, HSCs “crawl” along the pronephric ducts to colonize the
pronephric head kidney, the site analogous to human bone marrow (Bertrand et al.,
2008). Here, lymphoid and myeloid cells are produced within the hematopoietic stem
cell niche between renal tubules. The primary juvenile and adult hematopoietic organs
(kidney, spleen, liver, bone marrow) vary between different species of amphibians,
reptiles, and fishes (Zon, 1995).
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2. Regulators of erythroid differentiation and function
The path to becoming a red blood cell is determined by signaling molecules,
transcription factors, structural proteins, and other factors. Stages of erythroid
differentiation (progenitors in Fig. 1B) are generally characterized by condensation of
the nucleus, a decrease in cell size, and an accumulation of hemoglobin during terminal
differentiation. Each stage is also defined by a unique gene expression profile. As stem
cells mature and lose their pluripotency, factors involved in self-renewal (e.g. myb) are
down-regulated while factors that are critical for heme synthesis and erythroid
differentiation (e.g. gata1) are up-regulated (Hattangadi et al., 2011). Extrinsic factors
within erythroblast islands play an early role in the activation of erythroid differentiation
and these include cytokines and interactions with receptors on neighboring cells or with
the extracellular matrix. The major activator of erythropoiesis is the hormone,
Erythropoietin (Epo), which binds to the erythropoietin receptor (EPOR) on BFU-E and
CFU-E progenitors (D'Andrea et al., 1989; Krantz, 1991; Lin et al., 1996), which signals
up-regulation of erythroid genes and anti-apoptotic factors (Dolznig et al., 2002).
Erythroid transcription factors are also expressed and interact within complexes to
regulate cell differentiation. The Gata1 transcription factor, a master regulator of
erythropoiesis, colocalizes with other nuclear proteins (e.g. Scl/Tal1, Ldb1, Lmo2, Klf1)
at promoters and enhancers to activate erythroid gene transcription via long-range
chromatin interactions (Love et al., 2014; Tijssen et al., 2011). Gata1 also functions as a
transcriptional activator or repressor by recruiting the Hdac1-containing NuRD
(Nucleosome Remodeling Deacetylase) complex (Miccio et al., 2010). During terminal
differentiation, these cofactors up-regulate expression of erythroid genes including anti-
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apoptotic molecules like Bcl-xL, which protects erythroid cells from BAX-induced cell
death (Dolznig et al., 2002; Rhodes et al., 2005).
3. Gene editing and zebrafish mutant models
The zebrafish has provided an excellent model to study the genetic regulators of
hematopoiesis (Kafina and Paw, 2018). Zebrafish spawn 100-200 translucent eggs
whose development may be easily studied following fertilization. The generation of
transgenic zebrafish using the Tol2 system has allowed researchers to track gene
expression and tissue development using fluorescent reporters (Kawakami, 2016).
Forward mutant screens have used chemical mutagens like N-ethyl-N-nitrosourea
(ENU) or retroviral insertion of DNA to generate zebrafish lines with developmental
abnormalities (Detrich, 1999; Frame, 2017.). A series of zebrafish blood mutants were
found to have defective erythropoiesis (e.g. vlad tepesm651, riesling, zinfandel) (Ransom
et al., 1996; Weinstein et al., 1996) and impaired HSC specification (e.g. hi1618 and
hi2335) (Amsterdam et al., 2004). These methods have generally not been site-specific.
The advent of CRISPR technologies (Clustered Regularly Interspaced Short
Palindromic Repeats) provides a highly efficient method for targeted gene disruption
using a small guide RNA (sgRNA) and the Cas9 endonuclease (Ata, 2016; Jinek et al.,
2012). Indeed, this technology permits precise modification of genes of interest to
generate mutant zebrafish lines that model human diseases (Frame, 2017.). Further
improvements of these gene editing technologies have enhanced our ability to
manipulate and study the genome (Carroll, 2017; Sertori et al., 2016).
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4. Defective erythropoiesis in notothenioid fishes
The only vertebrates that do not produce erythrocytes are the family of Antarctic
icefishes (Channichthyidae), a monophyletic clade of the suborder Notothenioidei
(Cocca et al., 1995b; di Prisco et al., 2002; Near et al., 2006b; Zhao et al., 1998b).
Notothenioid fishes adaptively radiated in the Southern Ocean ~34 million years as it
began cooling to the freezing point of seawater (–1.86C ) (Colombo et al., 2015;
Matschiner et al., 2011; Rutschmann et al., 2011) and other fish taxa became locally
extinct (Eastman, 1993). The decline in temperature was likely caused by the
separation of the Antarctic continent and formation of the Antarctic Circumpolar Current
(Kennett, 1977). Today, notothenioids are the most speciose of Antarctic fishes,
comprising ~130 species grouped into 8 families (Eastman and Eakin, 2000; Near et al.,
2003).
Notothenioids share several synapomorphic traits that allowed for their evolution
in a harsh environment. Almost all members of the clade possess antifreeze
glycoproteins (AFGPs) that keep their blood from freezing (Chen et al., 1997; Deng et
al., 2010). Cold adaptation may also explain the convergent evolution of different XY
sex-chromosome systems in several Antarctic notothenioids, which may avoid skewed
sex ratios otherwise induced by temperature-dependent sex determination (Ghigliotti et
al., 2016). All notothenioids lack a gas bladder, the organ through which most teleosts
regulate their buoyancy (DeVries and Eastman, 1978). Nevertheless, the evolution of
decreased bone mineralization and accumulation of lipids to enhance buoyancy
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facilitated the radiation of notothenioid species to occupy diverse niches in the water
column (Albertson et al., 2010). All notothenioids display hematopoietic phenotypes with
reduced hematocrits and hemoglobin concentrations compared to temperate fishes
(Wells et al., 1980). The complete loss of red blood cells by Antarctic icefishes (Fig. 2)
provides a unique opportunity to study the genetic regulators of erythropoiesis.
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Chapter 1: Morph-biased gene expression and sequence divergence typifies disease-
like traits of Antarctic icefishes
Key words: icefish, Antarctic, erythropoiesis, anemia, mutant, gene expression
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Abstract
The family of white-blooded Antarctic icefishes (Channichthyidae) displays
several unusual traits that are reminiscent of human diseases, most notably their severe
anemia. Because most molecular-genetic pathways are shared among vertebrates, the
mutations that cause the icefish traits may be the same targeted pathways in human
diseases. Here I performed a comparative transcriptomics study to identify changes in
gene expression and coding mutations that are morph-biased for red- and white-
blooded notothenioid fishes. I show that both the profoundly anemic icefishes and their
red-blooded sister clade, the dragonfishes, possess predicted deleterious mutations in
genes that are associated with hemolytic anemias in humans (e.g. g6pd, sptb).
However, erythropoiesis in icefishes appears to be stalled early in erythroid
differentiation prior to potential hemolysis. I hypothesize that this may be an adaptation
to abrogate the production of erythrocytes in an environment, the cold, oxygen-rich
Southern Ocean, in which their utility is marginal. Moreover, I propose the icefishes
have shut down terminal erythroid differentiation through decreased expression of
positive regulators of erythroid differentiation and increased expression of pluripotency
markers typically expressed in leukemias. I show that mutations in the transcription
factor hemogen, combined with overexpression of hdac1b and decreased expression of
p300b, are likely to cause an imbalance in regulatory acetylation of Gata1 that
downregulates its activity in icefish hematopoietic tissues. Together, these changes
suggest that Antarctic notothenioids have evolved an intricate repression of
erythropoiesis.
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Introduction
Fish are proven models for studying blood development as they share the same
set of hematopoietic cell lineages as mammals. One exception is the family of Antarctic
icefishes (Channichthyidae), which is the only vertebrate clade that has lost the ability to
produce red blood cells (Cocca et al., 1995a; di Prisco et al., 2002; Near et al., 2006b;
Zhao et al., 1998b). Thus, icefishes provide a natural mutant model for human anemias
(Albertson et al., 2009).
The icefish clade belongs to the Antarctic notothenioid suborder, which radiated
adaptively as the Southern Ocean began cooling ~34 million years ago (Mya) to the
freezing point of seawater (–1.86C ) today; other fish groups became locally extinct
(Eastman, 1993). The notothenioid radiation produced ~136 species belonging to eight
recognized families, among which there are highly divergent phenotypes (Colombo et
al., 2015; Matschiner et al., 2015).
The 16 species of Antarctic icefishes are unique among vertebrates in that they
neither produce the oxygen-carrying pigment hemoglobin nor do they produce typical
mature erythrocytes (Cocca et al., 1995b; di Prisco et al., 2002; Near et al., 2006b;
Zhao et al., 1998b). The high oxygen content in the Southern Ocean facilitated the loss
of erythrocytes in icefishes, but their severe anemia was clearly disaptive (Montgomery
and Clements, 2000), as the icefish co-evolved expanded hearts and highly
vascularized tissues, possibly as a consequence of elevated systemic nitric oxide (NO)
levels (Sidell and O'Brien, 2006). Red-blooded Antarctic notothenioids also have
decreased hematocrits and reduced hemoglobin concentrations compared to temperate
teleost species (Eastman, 1993; Wells et al., 1980). There is evidence for temperature-
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sensitive phenotypic plasticity of erythropoiesis in temperate teleosts; erythropoiesis is
repressed by cold exposure (Kulkeaw et al., 2010; Maekawa et al., 2012). Thus, genetic
fixation of cold-induced anemia in ancestral notothenioids may be an example of West-
Eberhard’s theory of “Increased genetic divergence due to phenotype fixation” (West-
Eberhard, 1989, 2005).
Significant mutations in coding sequences, including the deletion of globin genes,
have probably made permanent the anemia of icefishes (Cocca et al., 1995b; Near et
al., 2006b). However, one cannot rule out mutations of erythroid gene regulatory
elements as causes of icefish anemia; indeed, such changes might have initiated red
cell loss. The relaxed selection pressure accompanying regulatory mutations may
explain the high mutation rates observed in morph-biased genes when they become
expressed below a functional level (Helantera and Uller, 2014; Leichty et al., 2012).
Thus, reduction of hemoglobin levels, as seen in more derived notothenioid clades [i.e.,
the family Bathydraconidae (dragonfishes)] due to deletion of globin gene regulatory
elements, may have led ultimately to the evolutionary loss of the globin locus in
Antarctic icefishes (Lau et al., 2012; Near et al., 2006b; Zhao et al., 1998b). However,
the loss of β-globin may be just one of many erythroid specific gene mutations that
occurred in icefishes.
In this study, I compare multi-tissue, RNA-Seq transcriptomes (Berthelot et al.,
2018. Manuscript in preparation) to interrogate morph-biased gene expression and
coding sequence divergence between the derived sister lineages of dragonfishes
(Bathydraconidae) and icefishes (Channichthyidae). The goal was to discover the
genetic determinants of icefish traits, specifically changes to hematopoietic genes that
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may contribute to their anemia. The analysis revealed tissue-specific, differential
expression for genetic pathways that regulate development of blood, brain, muscle,
gonad and kidney. In hematopoietic tissues, decreased expression was observed for
both well-known and previously uncharacterized erythroid genes – several of these
genes contained predicted deleterious substitutions or frameshift mutations. I found that
the dragonfish, Parachaenichthys charcoti, was a natural mutant model for hereditary
spherocytic anemia. The icefish, Chaenocephalus aceratus, has been shown to be
blocked in erythroid differentiation (Yergeau et al., 2005; Yergeau et al., 2006.
Manuscript in preparation). I suggest that the block to differentiation may be caused by
increased expression of pluripotency factors and decreased expression of erythroid
differentiators. Specifically, the block in erythroid differentiation may be caused by
increased expression of hdac1b and by deleterious mutations in the interaction domains
of Hemogen, P300b, and Gata1, which would together promote deacetylation and
deactivation of Gata1. Together, these changes show that notothenioid evolution led
ultimately to an intricate repression of the erythropoietic pathway.
Results
Erythrocyte morphology in notothenioid fishes
All Antarctic notothenioid fishes are anemic, with reduced hematocrits and low
hemoglobin concentrations, compared to temperate fishes (Wells et al., 1980). This is
most apparent in dragonfishes (Bathydraconidae), the sister lineage to icefishes, which
display a more severe anemia (Kunzmann et al., 1991) than other red-blooded
notothenioids. Yet, very little is known about the process of erythroid differentiation in
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notothenioids. Two icefishes, C. aceratus and Dacodraco hunteri, were proposed to
have very rare, senile erythrocytes (Barber et al., 1981). Subsequently, it was
discovered that C. aceratus produces erythroid progenitors but shows a clear block to
terminal erythroid differentiation (Yergeau et al., 2005; Yergeau et al., 2006. Manuscript
in preparation). Nonetheless, the blood cell phenotypes of most notothenioid species
have not been characterized.
I examined the morphology and frequency of hematopoietic cell types in
peripheral blood smears from six species from three families of Antarctic notothenioid
fishes. The red blood cells of two nototheniids, Notothenia coriiceps and Gobionotothen
gibberifrons, were oval shaped, measuring 11.7±0.30 µm (n = 12) and 12.1±0.33 µm (n
= 12) on the longest axis, and were morphologically similar to erythrocytes seen in
temperate teleosts (Fig. 1A,B). Strikingly, erythrocytes of the dragonfish, P. charcoti,
were spherical in shape and significantly smaller (mean diameter 8.7±0.19 µm; n = 14;
Student’s t test, P = 6.1E-06) than those of N. coriiceps. They also had a reduced
surface area of 48.7±3.1 µm2 compared to the oval erythrocytes from N. coriiceps at
77.6±4.1 µm2 (Student’s t test, P = 0.003, n = 6) (Fig. 1C). Interestingly, the spherocytic
erythrocytes of P. charcoti were morphologically similar to the erythrocyte-like cells that
have been described in the icefish Channichthys rhinoceratus (Hureau, 1966; Spillman
and Hureau, 1967). Spherocytic morphology of erythrocytes is typically caused by
defects in erythroid membrane cytoskeletal proteins.
Among the icefishes that I analyzed, blood cell morphologies and frequencies
varied between species (Fig. 1D-F). Mature erythrocytes were not apparent in the
peripheral blood of Pseudochaenichthys georgianus (Ps. georgianus) or C. aceratus,
31
but there were a number of circulating erythroblasts in the blood of C. aceratus (5.7% of
peripheral blood cells, n = 72, Fig. 1E). By comparison, the blood of N. coriiceps did not
contain circulating erythroblasts and reticulocytes were present at a low frequency
(1.8% n = 111, Fig. 1A). The peripheral blood of Champsocephalus gunnari and
Chionodraco rastrospinosus contained abundant myeloid cell-types (56.3% N = 98, and
19.7% n = 65 respectively, Fig. 1). Some myeloid cell-types in C. rastrospinosus had
clear cytoplasm and condensed nuclei in contrast to the polymorphonuclear leukocytes
of C. aceratus (Fig. 1D,E) and were similar to the putative erythrocytes of the icefish, C.
rhinoceratus (Hureau, 1966; Spillman and Hureau, 1967). Thus, blood cell
morphologies are highly variable among notothenioid fishes even between species
within the icefish clade.
32
33
Figure 1. Peripheral blood smears from Antarctic notothenioid fishes. Light
micrographs of Giemsa stained blood cells from N. coriiceps (A), G. gibberifrons (B), P.
charcoti (C), C. rastrospinosus (D), C. aceratus (E), and C. gunnari (F). Note the oval
erythrocytes in two nototheniids (A,B) and spherocytic erythrocytes of the dragonfish
(C,D). Abbreviations: E, eosinophil; Er, erythrocyte; L, lymphocyte; Mon, monocyte; N,
neutrophil; O, orthochromatophilic normoblast; ProE: proerythroblast; T, thrombocyte.
Scale bars = 10 µm (A-F).
34
Comparative transcriptomics reveals tissue-specific, differentially expressed
genes between an icefish and dragonfish
To identify genes that are differentially expressed in icefish tissues, I performed an in
silico comparison of RNA-Seq expression profiles between the transcriptomes of the
icefish, Ps. georgianus, and red-blooded dragonfish, P. charcoti (Berthelot et al., 2018.
Manuscript in preparation). More than 18,700 orthologous genes were identified in the
multi-tissue transcriptomes of these species using OMA stand-alone and a pipeline that
has been described previously (Altenhoff et al., 2013; Altenhoff et al., 2011; Sharma et
al., 2014). For each tissue, the TPM (transcripts per million) normalized expression
values were strongly correlated between tissue replicates within each species (0.78 > R
> 0.98). Expression was correlated for most interspecies comparisons (0.51 > R > 0.92)
except at sites of hematopoiesis and in the liver (Fig. S1-2). Differential expression (DE)
analysis was conducted on the orthologs using edgeR v3.10.2 to normalize and detect
significant differences between read counts in each tissue from Ps. georgianus and P.
charcoti with 2-4 biological replicates per sample. From the list of 18,781 orthologs,
differentially expressed transcripts (significance criterion P ≤ 1E-05, FDR ≤ 0.001) were
identified in brain (2,005), head kidney (2,317), liver (1,960), ovary (784), spleen
(3,108), trunk kidney (2,688), pectoral muscle (2,129), white muscle (1,447), and heart
ventricle (2,005). Significant differential expression of 295 genes (39 up-regulated, 256
down-regulated) was observed in every tissue comparison. Tissue-wide down-
regulation of a gene may hint at a significant genomic alteration or it may occur due to
an incorrect orthology call. Therefore, the orthologies for specific genes of interest were
35
confirmed by genomic synteny and/or by mapping to established zebrafish and
stickleback orthologs. Subsequently, I performed hierarchical clustering of TPM-
normalized expression values for DE genes across tissues from both species (Fig. 2).
This facilitated the isolation of specific clusters of DE genes (cut at 56% height of the
dendogram) with the highest expression in each tissue. Tissue-specific genes were
differentially expressed in brain (432), head kidney (255), liver (102), ovary (138),
spleen (323), trunk kidney (402), pectoral muscle (139), white muscle (56), and heart
ventricle (171). In the head kidney, 152 DE genes were determined to be blood-specific
genes.
36
37
Figure 2. Multi-tissue expression profile heatmap of genes that are differentially
expressed in head kidney from Ps. georgianus and P. charcoti. Differentially
expressed genes were identified in the head kidney RNA-Seq transcriptomes from Ps.
georgianus and P. charcoti (Fisher’s exact test, P < 1E-05, FDR < 0.001). For genes
that are differentially expressed in the icefish head kidney, the relative transcript
abundances in each tissue from both species are shown as the log2 transformed values
of transcripts per million (TPM). Hierarchical clustering identified groups of genes with
similar expression profiles. Tissue-specific clusters of genes with the highest expression
in each tissue were isolated by cutting the dendogram at a height of 56% (dashed line).
Abbreviations: H. Kidney, head kidney; T. Kidney, trunk kidney; Pectoral, pectoral
muscle; W. muscle, white muscle.
38
Gene ontology enrichment of DE genes highlights tissue-specific molecular
processes that underlie icefish phenotypes
The differential expression of molecular processes in each tissue between P.
charcoti and Ps. georgianus may represent lineage-specific adaptations to temperature,
oxidative stress, and/or functional loss of red blood cells. To evaluate these possibilities,
gene ontology (GO) enrichment was used to assess the tissue-specific biological
functions of each DE gene cluster and to identify specific genetic pathways that may be
unique to icefish development (Fig. 3).
At sites of hematopoiesis, in the head kidney (n = 234, Fig. 3A) and spleen (n =
296, Fig. 3B), many of the genetic pathways that control cell survival, proliferation and
differentiation were enriched. More genes involved in the immune system were
differentially expressed in the spleen (n = 68; sum of up- and downregulated genes)
compared to head kidney (n = 27), particularly for genes involved in lymphopoiesis. In
both hematopoietic tissues, the widespread decrease in expression for erythroid genes
highlights the loss of mature erythrocytes in icefishes (Fig. S3).
With the highest number of tissue-specific DE genes (n = 398), the icefish brain
primarily exhibited down-regulation of regulators of nervous system development and
function (Fig. 3C). Decreased expression was observed for several factors involved in
glutamate receptor signaling (e.g. GRM8, GRIK5, GRIA3, GRIA1), which is consistent
with the contraction of this gene family observed in notothenioids (Shin et al., 2014).
Reduced glutamate signaling might inhibit neuronal cell function but may represent an
39
adaptation to prevent excessive generation of reactive oxygen species (ROS)
(Reynolds and Hastings, 1995; Willard and Koochekpour, 2013).
In the trunk kidney (n = 372), gene expression changes were observed for
several metabolic processes (Fig. 3D). For example, the differential expression for
many lipid metabolites (n = 37, e.g. Fabp1) is consistent with the elevated
polyunsaturated fatty acid (PUFA) levels in icefish mitochondrial membranes (O'Brien
and Mueller, 2010).
Tissue-specific DE genes in pectoral red muscle (n = 128, Fig. 3E) and in trunk
white muscle (n = 50, Fig. 3F) were involved in striated muscle development and
function. In most cases, the same processes were strictly up-regulated in pectoral
muscle of the icefish but down-regulated in white muscle compared to the dragonfish.
This may highlight the increased hypertrophy and loss of hyperplasia in icefish pectoral
muscle (Archer and Johnston, 1987). Icefishes generally use their pectoral muscles for
labriform swimming (Archer and Johnston, 1987), whereas Parachaenichthys species
use sub-carangiform swimming and have a heavily muscled trunk (Kuhn et al., 2010).
More genes encoding mitochondrial proteins were differentially expressed in icefish
pectoral muscle, reflecting its higher concentration of mitochondria (Archer and
Johnston, 1991; Lin et al., 1974).
40
41
Figure 3. Gene ontology (GO) enrichment for tissue-specific, differentially
expressed genes between Ps. georgianus and P. charcoti. Enriched GO groups
were identified using STRING (Fishers exact test, P < 0.05, FDR < 0.01). Graphs show
numbers of up-regulated (red) and down-regulated (blue) genes in Ps. georgianus head
kidney (A), spleen (B), brain (C), trunk kidney (D), pectoral muscle (E), and white
muscle (F) for different biological processes.
42
Interaction network for differentially expressed, tissue-specific hematopoietic
regulators in the icefish head kidney
To identify the pathways that control blood development in icefishes, I created a
gene association network for tissue-specific, differentially expressed genes in the icefish
head kidney, the major site of adult definitive hematopoiesis in teleosts (Fig. 4). The
network was created with STRING (Jensen et al., 2009) using annotations of the human
orthologs (see Methods). For genes of interest, orthology was verified by comparative
synteny of the sequenced genomes for N. coriiceps and H. sapiens. In the association
network, K-means clustering (n = 11, Fig. 4) revealed sets of genes that were grouped
consistently with ten GO biological functions (Fig. 4). The network highlights the loss of
expression for groups of genes involved in the erythroid skeletal membrane, heme
synthesis, erythroid transcriptional regulation, chromatin regulation, apoptosis, lipid
metabolism, and in the regulation of adenylate cyclase activity. It also shows a cluster of
signaling molecules, including many with increased expression in the icefish head
kidney (Fig. 4).
Central nodes linking these clusters included tspo (25), hdac1 (18), akt (16), rela
(15), foxo1 (13), gata1 (13), tk2 (13), tfrc (12), fech (10), and ntrk1 (10), all of which
were down-regulated with the exceptions of hdac1 and ntrk1 (Fig. 4). The cluster
centering on gata1 highlights the interactions between down-regulated Gata1
transcriptional targets and Gata1 co-factors that cooperate to drive erythropoiesis
(Ferreira et al., 2005). Previous studies have emphasized the correlation between node
centrality and essential function (Batada et al., 2006; He and Zhang, 2006; Jeong et al.,
43
2001; Raman et al., 2014; Song and Singh, 2013). Thus, differential expression of these
central nodes should highlight the major genetic changes that contribute to the
44
hematopoietic defecefishes.
45
Figure 4. Association networks between tissue-specific DE genes in the icefish
head kidney showing both up-regulated and down-regulated genes. Gene
association networks were created for tissue-specific DE genes in the head kidney with
STRING (Jensen et al., 2009) using annotations from the human orthologs. Colors
represent K-means clusters of gene nodes (n = 11). Genes were generally clustered by
their biological functions.
46
Decreased expression of genes involved in erythroid differentiation in icefishes
The most obvious phenotype that differentiates the icefishes from other
notothenioids is their lack of red blood cells (RBC, erythrocytes). To distinguish the loss
of erythroid-specific genes from early-acting regulators of the hematopoietic stem cell
niche, I examined two clusters of down-regulated genes (C1, C2) in the icefish that had
strong tissue-specific expression in P. charcoti head kidney or peripheral blood (Fig
5A,B). While many erythroid genes function in erythroid progenitors of the head kidney
(Orkin and Zon, 1997), the high concentration of RBC in the peripheral blood of P.
charcoti allowed me to detect both early and late erythroid markers.
Down-regulated genes that were specific to the head kidney included the
hematopoietic stem cell (HSC) markers myb and runx1, both of which are required for
definitive, but not primitive, hematopoiesis (Sood et al., 2010; Soza-Ried et al., 2010).
Decreased expression was also observed for several other genes that play critical roles
in HSC maintenance and differentiation, including relA/p65 (Stein and Baldwin, 2013)
and caspase 3 (Janzen et al., 2006).
Of the down-regulated genes in the icefish head kidney, 149 were blood-specific
markers (Fig. 5A,B, Table 1). The decreased expression of many RBC-specific genes
reflects the loss of erythrocytes in icefishes and included genes encoding globins, heme
biosynthetic enzymes and erythrocyte membrane proteins (n = 27; e.g. hb1, blvrb,
band3, band4.1, alas2, fech, add2, ank1, sptb). The list also included erythroid factors
that are more highly expressed in immature erythroblasts (Kingsley et al., 2013). These
genes regulate erythroid lineage commitment and/or terminal differentiation in the head
47
kidney (n = 51; e.g. gata1, hemogen, klf1, scl/tal1, gfi1b, ldb1, epor, tfrc, tgm2).
Additionally, I identified 31 novel blood-specific genes that have not been previously
associated with erythropoiesis (data not shown).
48
49
Figure 5. Three tissue-specific clusters of hematopoietic genes are differentially
expressed (DE) in the head kidneys of Ps. georgianus and P. charcoti. (A) Heat
map of relative gene expression for tissue-specific DE genes in the icefish head kidney.
Expression was normalized to transcripts per million (TPM). Three clusters show tissue-
specific genes in (C1) dragonfish blood, (C2) dragonfish head kidney, and (C3) icefish
head kidney. (B) Expression profiles for genes in each tissue-specific cluster.
Abbreviations: HK, head kidney; TK, trunk kidney; Liv, liver; Pec, pectoral; WM, white
muscle.
50
Increased expression of pluripotency markers highlights mechanisms of
erythroid inhibition in icefishes
The decreased expression for many erythroid genes in icefish head kidney is in
part caused by the loss of mature erythrocytes by this group. Thus, the genes with
increased expression may portray the cell lineages and developmental processes that
predominate in the icefish head kidney. My results show that icefish kidney expressed
at elevated levels a number of hematopoietic regulatory genes (Fig. 4-5), including cbfb,
ntrk1/trk1, gas6, and dock1. Several of the overexpressed genes in the icefish head
kidney (Table 1) are proto-oncogenes (e.g. flt1/vegfr1, bcr, ntrk1/trka) or leukemia
markers (e.g. hdac1b, dock1, gas6) that are frequently associated with leukemogenesis
(Bradbury et al., 2005; Collins et al., 1987; Dirks et al., 1999; Lee et al., 2017; Wang et
al., 2003).
Signaling pathways that drive hematopoietic proliferation (Van Etten, 2007)
showed altered expression in icefish kidney (Fig. 4). Specifically, the interaction
network of hematopoietic DE genes was enriched for the PI3K-Akt-mTOR signaling
pathway that promotes cell survival and proliferation (Ghosh and Kapur, 2017). This
included up-regulated oncogenes like sgk1 (Orlacchio et al., 2017) and down-regulated
genes, such as the tumor suppressor foxo1 and others (akt2, casp3, catalase) (Fig. 4).
Aberrant cell signaling promotes carcinogenesis (Martin, 2003) and mutations in
regulators of PI3K-Akt-mTOR signaling frequently activate this pathway in leukemias
(Fransecky et al., 2015; Park et al., 2010). In agreement with previous findings, I found
increased expression of TGF-beta signaling molecules (Xu et al., 2015), which may be
51
due to extensive duplications of genes in this pathway in Antarctic notothenioids (Chen
et al., 2008). TGF-β signaling has been shown to activate AKT signaling in many normal
and leukemic cell types (Drabsch and ten Dijke, 2012; Naka et al., 2010). Thus,
erythroid differentiation may stall in icefish due to increased expression of signaling
molecules and pluripotency genes that may mark a proliferative cell-type.
52
53
Figure 6. Differential expression of hematopoietic regulators is represented by
red- and white-blooded notothenioids. Relative expression of hdac1b, p300b, gata1,
and spi1b determined by qPCR in head kidneys from two red-blooded (N. coriiceps, P.
charcoti) and two white-blooded (C. aceratus, Ps. georgianus) notothenioids. Target
gene expression was normalized to beta-actin and error bars represent standard
deviation (n.s., not significant; Student’s t test, P > 0.05).
54
Confirmation of differential expression of hematopoietic regulatory genes across
notothenioid clades
Genes with morph-biased expression may show high variation even between
individuals within a species (Helantera and Uller, 2014). To assess whether differential
expression of hematopoietic genes was a consistent feature of the red- and white-
blooded notothenioids, I employed qRT-PCR to examine kidney expression of
hematopoietic regulatory genes across four representative notothenioid species: the
icefishes Ps. georgianus and C. aceratus, the nototheniid N. coriiceps, and the
dragonfish P. charcoti. The head kidneys of both icefishes showed significant down-
regulation of gata1a and p300b (Student’s t test, P < 0.05; Fig. 6). By contrast,
expression of hdac1b was found to be significantly up-regulated in the head kidneys of
both icefishes (Student’s t test, P < 0.05). Expression of the myeloid marker, pu.1/spi1b,
did not differ significantly between the four species (Fig. 6), consistent with the
comparable numbers of myeloid cells in the head kidneys of red- and white-blooded
notothenioids. In the RNA-Seq transcriptome, I showed that p300b was down-regulated
in all tissues of P. georgianus compared to P. charcoti. In contrast, I found that hdac1b
was specifically up-regulated in the icefish head kidney and spleen but not in non-
hematopoietic tissues (significance criterion P ≤ 1E-05, FDR ≤ 0.001). These findings
suggest that erythropoietic regulatory proteins (e.g., Gata1) may be differentially
acetylated, and hence differentially active, in icefish head kidney.
55
56
Figure 7. Predicted deleterious substitutions and frameshifts in blood genes from
icefishes. Provean was used to predict deleterious substitutions (Provean score < -3)
(Choi and Chan, 2015) that were shared by three white-blooded icefishes but which did
not occur in red-blooded notothenioids. (A) The CD33-related Siglec contained a F753*
frameshift mutation that truncated the transmembrane and cytoplasmic regions in the
icefishes, N.ionah, C. aceratus and Ps. georgianus. Numbers indicate length in amino
acids. (B) Predicted deleterious substitutions in icefish Glucose-6-phosphate
dehydrogenase (G6pd). Lines represent the NADP binding sites. White boxes indicate
the dimer interface. Capital letters represent beta-turns and lowercase letters are alpha
helices. Icefish mutation highlighted in yellow is mutated in human G6PD-deficiency. (C)
Predicted deleterious substitutions in the Transferrin receptor (Tfrc). (D) Predicted
deleterious substitutions in Hemopexin (Hpx). Residue highlighted in yellow is involved
in binding heme. Abbreviations: Cyto, cytoplasmic; C2-set, immunoglobulin c2-set
(constant) domain; Ig, immunoglobulin-like domain; ITIM, immunoreceptor tyrosine-
based inhibitory motif; v-set, immunoglobulin v-set (variable) domain; PA, protease-
associated domain; R, Hpx repeat; S, signal peptide; Tr, transmembrane.
57
58
Figure 8. Erythroid beta-spectrin is mutated in the dragonfish P. charcoti.
Scanning electron micrographs of erythrocytes from (A) N. coriiceps, a nototheniid, and
from (B-C) P. charcoti, a dragonfish. (D) Light micrographs of Giemsa stained triton-
insoluble erythrocyte membrane skeletons from N. coriiceps (Nc) and P. charcoti (Pc).
Flash frozen blood samples were treated with 1% Triton-X, spread on glass coverslips,
fixed with 4% PFA. (E) Predicted deleterious mutations in dragonfish (bold italic) and
icefish (Roman case) Erythroid beta-spectrins. Residues highlighted by yellow boxes
are also mutated in hereditary spherocytic anemia in humans. Scale bars = 10 µm (A,B)
5 µm (C,D).
59
Icefish-specific deleterious substitutions and frameshift mutations occur in
common targets of disease
Deleterious point mutations or frameshifts in erythroid-specific functional domains
are likely to contribute to the profound anemia of icefishes. I identified frameshift
mutations in 16 genes (Table 5) from three icefish species (Neopagetopsis ionah, C.
aceratus, Ps. georgianus) after alignment to the orthologs from red-blooded
notothenioids (P. charcoti, N. coriiceps, H. antarcticus). One blood-specific gene, a
Cd33-related Siglec (Sialic-acid-binding immunoglobulin-like lectin) contained a C-
terminal frameshift that removed its cytoplasmic immunoreceptor tyrosine-based
inhibition motif (Fig. 7A). Loss of CD33 causes slight erythropoietic defects in mutant
mice (Brinkman-Van der Linden et al., 2003).
I identified all nonsynonymous substitutions in 7,049 orthologs that differed
between red- and white-blooded notothenioid lineages and then used Provean to
predict whether these were potentially deleterious mutations. Genes with potentially
deleterious substitutions (Provean-score < -3.0) were significantly enriched for
hematopoietic factors (n = 11; P < 5.380e-2), including regulators of heme metabolism
(n = 6; P < 2.890e-5) and myeloerythroid differentiation (n = 9; P < 1.940e-2) (Table 3,
Table 5, Fig. 7). The mutations were found in important functional domains that have
been associated with human diseases (Table 5). Mutated residues in G6PD (glucose-6-
phosphate dehydrogenase, Fig. 7) and Erythroid beta-spectrin (Fig. 8) are also mutated
in hemolytic anemias in humans (Barisic et al., 2005; Landrum et al., 2016).
60
61
The dragonfish P. charcoti is a natural mutant model for spherocytic anemia
The red blood cells of the dragonfish, P. charcoti, were morphologically different
from erythrocytes seen in other red-blooded notothenioids. I employed scanning
electron microscopy to compare erythrocytes from P. charcoti and N. coriiceps. A
nuclear bulge was apparent in erythrocytes from N. coriiceps but not from P. charcoti
(Fig. 8A-C). This indicated a spherocytic morphology for the dragonfish RBCs, which
may result from loss of incorporation of cytoskeletal proteins. Loss of membrane
proteins was evidenced by the size difference between triton-insoluble erythroid
membrane skeletons from P. charcoti (5.1±0.18 µm, n = 10) and N. coriiceps (12.4±0.72
µm n = 9) (Student’s t test, P = 1.8E-05; Fig. 8D). In the dragonfish, these features may
result from seven deleterious substitutions found in erythroid β-spectrin including an
R1037S mutation in the 7th spectrin repeat, which corresponds to an R1035W
substitution (rs143827332) that has been associated with hereditary spherocytic anemia
(HS) in humans (Landrum et al., 2016). Accumulation of deleterious substitutions in
erythroid β-spectrin from dragonfishes and icefishes may initially have been caused by
membrane instability at cold temperatures (Lomako et al., 2015) or may have been
caused by relaxed selection on erythrocyte markers as a result of anemia.
62
Regulators of heme metabolism are under different selection in red- and white-
blooded notothenioids
Morph-biased gene expression is associated with increased rates of mutation,
due to relaxed selection upon genes that are expressed by few individuals of the
population or due to loss of function as a result of neutral selection (Helantera and Uller,
2014; Leichty et al., 2012). To determine the selective pressures on icefish coding
sequences, I compared the rate of non-synonymous to synonymous substitutions (ω,
dN/dS) between orthologous genes from two red-blooded species (P. charcoti and
Harpagifer antarcticus) and two white-blooded species (N. ionah and Ps. georgianus).
To search for genes with variable dN/dS ratios between the notothenioid lineages, I
employed a likelihood ratio test (P < 0.05) to compare a 2-ratio and 1-ratio (null) model
for each set of gene alignments. Most hematopoietic regulators (i.e. gata1, spi1b/pu.1)
were under equally strong purifying selection in red and white-blooded notothenioids
and must be functional in some cell lineages in icefishes (data not shown). However,
several erythroid genes had significantly higher dN/dS ratios in icefishes including three
genes (e.g. tfrc, hpx, slc25a39) involved in heme metabolism (Likelihood ratio test, P <
0.05; Table 2). The increased nonsynonomous substitution rate of the icefish transferrin
receptor illustrates continued genetic drift in a gene that is highly polymorphic in
notothenioids (Trinchella et al., 2008). Adaptive changes to hematopoietic pathways
may serve to combat the negative side-effects of anemia in icefishes. It has been
suggested that stable serotransferrin expression may scavenge free ferric iron (Fe 3+)
in icefish tissues (Kuhn et al., 2016). Likewise, the strong up-regulation of hemopexin
63
(hpx) in the icefish, the significant positive selection acting on its sequence, and putative
functional mutations that remove (H36P, H37G, H79R, H333Q, H364A) or introduce
(Q132H, Q175H, Y246H, D362H, D395H, N434H) histidine residues that may bind
heme indicate that Hemopexin function may be adapted to enhance heme scavenging
in the plasma in response to the loss of hemoglobin formation.
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65
Figure 9. Functional mutations occur in the interaction domains of Gata1,
Hemogen, and P300. (A) Deleterious mutations were discovered in the interaction
domains (brackets) of Gata1, P300, and Hemogen from white-blooded icefishes (N.
ionah, Ps. georgianus) but not in red-blooded notothenioids (P. charcoti, H. antarcticus).
Deleterious mutations were predicted with Provean (Choi and Chan, 2015). (A) Icefish
Gata1 contains a deleterious N319S substitution in the C-terminal zinc finger (C-ZF),
which binds Hemogen and P300 (Zheng et al., 2014). (B) Icefish P300 contains three
deleterious substitutions in the Gata1 binding region (Blobel et al., 1998) which overlaps
with the acetyl transferase domain (spans Br, P, KAT, Z, and CH) (Bordoli et al., 2001).
(C) Icefish Hemogen contains a P174fs frameshift mutation that truncates the C-
terminal domain that is responsible for binding of P300 (Zheng et al., 2014). (D) Model
for molecular repression of Gata1 function and erythroid gene transcription caused by
mutations (marked with X) and by dysregulation of gene expression (arrowheads).
66
Icefish-specific deleterious mutations in the interaction domains of Gata1,
Hemogen, and P300b
The top candidate pathways for the block of erythroid differentiation in icefishes
involve Gata1, which is considered the master regulator of erythropoiesis in vertebrates
(Ferreira et al., 2005; Suzuki et al., 2011). Icefish Gata1 and several of its co-factors
(P300, Hemogen, Runx1, Spi1, Gfi1b, Klf1) contained deleterious mutations that may
affect Gata1 activity. In both N. ionah and Ps. georgianus, Gata1 contained a single
deleterious N319S substitution in the C-terminal Zinc finger (CF) (Fig. 9A), a domain
that is required for DNA-binding and for promoting erythroid differentiation (Omichinski
et al., 1993). The Gata1 C-ZF domain is bound by the erythroid transcription factor,
Hemogen (Zheng et al., 2014), and by the histone acetyl-transferases CBP (Creb-
binding protein) and P300 (Boyes et al., 1998). The erythroid transcription factor,
Hemogen, can recruit P300 to promote acetylation of Gata1 in an immediately adjacent
basic domain, leading to enhanced erythroid gene transcription (Zheng et al., 2014).
Previously, I characterized a C-terminal deletion in icefish Hemogen (See
Chapter 4), a defect that introduces a frameshift and premature stop codon in some
icefish species (Fig. 9A) This frameshift removes a C-terminal transactivation domain
motif that may be required for binding of P300. Icefish P300b also contained seven
deleterious substitutions, including three in the TAZ2/CH3 domain (Transcription
Adaptor putative Zinc Finger/cysteine-histidine), the domain that binds Gata1 (Blobel et
al., 1998). Thus, all of the interaction domains of Gata1, Hemogen, and P300b contain
predicted deleterious mutations. In contrast, Hdac1b was highly conserved between
67
red- and white-blooded species and did not contain any predicted deleterious mutations.
The mutations in Gata1, Hemogen, and P300 may contribute to the differential
expression of all identifiable transcriptional targets of Gata1 (n = 161) and Hemogen (n
= 367) in the icefish, Ps. georgianus.
The up-regulation of hdac1b and down-regulation of p300b may contribute to a
homeostatic imbalance in erythroid-specific acetylation in icefishes. Thus, I employed
Western blotting to assess whole-protein acetylation status in the head kidneys of red-
and white-blooded notothenioid fishes (Fig. 10). Acetylation of most proteins was
comparable in C. aceratus and N. coriiceps. Normal acetylation of most proteins may be
compensated by p300 paralogs (e.g. p300a, cbp, cbp-like) in notothenioids. However,
changes in acetylation for specific targets of Hdac1b and p300b could not be ruled out.
In mice, mutations in the KIX domain of P300 cause severe anemia and erythroid cell
defects (Kasper et al., 2002) whereas Hdac1 is inactivated during terminal
differentiation by P300 (Yang et al., 2012). Thus, the differential expression of Gata1
targets in icefishes may result from (1) decreased expression of Hemogen, P300b, and
Gata1, (2) by deleterious mutations in the interaction domains of all three proteins and
(3) by overexpression of Hdac1b (Fig. 9B).
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69
Figure 10. Protein acetylation in the head kidneys of red- and white-blooded
notothenioids. (A) Protein acetylation was detected in purified protein from head
kidneys of N. coriiceps and C. aceratus. Separated proteins were probed with anti-
acetylated lysine antibody (Santa Cruz Biotechnology, AKL5C1). Signals were
normalized to Ponceau stained bands and calculated as a fold change relative to N.
coriiceps. Arrows mark proteins with increased acetylation in the icefish (> 1.5 fold
change).
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Discussion
Molecular repression of erythropoiesis in Antarctic icefishes
Hemolysis of mature erythrocytes may cause the reduced hematocrits observed
in red-blooded Antarctic notothenioids and may have instigated the evolutionary loss of
red blood cells in icefishes. The dragonfish, P. charcoti, possesses spherocytic
erythrocytes, a feature that may be caused by deleterious mutations that occur in the
functional domains of erythroid β-spectrin including specific residues that are mutated in
hereditary spherocytic anemia (HS) in humans. In both dragonfishes and icefishes, the
accumulation of deleterious substitutions in β-spectrin may have been caused by cold-
induced membrane instability (Lomako et al., 2015) or by relaxed selection due to the
loss of red blood cell function.
Icefishes may have adapted a molecular repression of erythroid differentiation to
avoid the consequences of hemolytic anemias. I identified changes in expression of
hematopoietic regulators in icefishes including overexpression of pluripotency genes
and decreased expression for genes that promote erythroid differentiation. Icefish
hematopoiesis may be disrupted by an acetylation imbalance caused by decreased
expression of the p300 acetyltransferase in all tissues and hematopoietic-specific
overexpression of hdac1b. Histone acetyltransferases (HATs) and histone deacetylases
(HDACs) control gene expression through acetylation and deacetylation of histones and
transcription factors (De Ruijter et al., 2003; Eberharter and Becker, 2002; Vo and
Goodman, 2001). Loss of P300 is embryonic lethal and mutations in the KIX domain of
P300 cause severe anemia and erythroid cell defects in mice (Kasper et al., 2002).
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Hdac1 activity also plays a critical role in early erythroid proliferation (Heideman et al.,
2014) but is inactivated by P300 during terminal differentiation (Yang et al., 2012).
Furthermore, Antarctic icefishes contain predicted deleterious mutations in the
interaction domains of Hemogen, P300, and Gata1. Truncation of the C-terminal
transactivation domain in icefish Hemogen may prevent it from recruiting the P300
acetyltransferase to Gata1 (Zheng et al., 2014). Hdac1 facilitates Gata1-mediated
transcriptional repression by the NuRD complex (Hong et al., 2005; Snow and Orkin,
2009). During terminal differentiation, P300 acetylates and inactivates Hdac1 and
converts this complex to an activator (Yang et al., 2012). Thus, in icefishes, Gata1 may
function solely as a transcriptional repressor due to Hdac1b overexpression. Loss of
Gata1 expression and function in icefishes may prevent formation of active chromatin
hubs (ACH), which are thought to play a global role in erythroid gene transcription
(Schoenfelder et al., 2010). Specifically, the loss of chromatin looping by the LCR (locus
control region) (Krivega and Dean, 2016) may have contributed to the deletion of globin
promoter elements in dragonfishes (Lau et al., 2012) and the complete loss of globin
genes in icefishes (Cocca et al., 1995a).
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Methods
Transcriptome assembly and orthology assignment
Transcript sequences from multi-tissue transcriptomes were previously
generated for two red-blooded (H. antarcticus, P. charcoti) and two white-blooded (Ps.
georgianus, N. ionah) notothenioid species (Berthelot et al., 2018. Manuscript in
preparation). Whole-tissue transcriptomes were assembled with Trinity using default
parameters (Haas et al., 2013). For comparisons between transcriptomes, orthologous
relationships were determined as previously described (Sharma et al., 2014). Briefly,
CD-HIT was used to eliminate gene duplicates (95% similarity) and TransDecoder was
used to identify putative open reading frames (Fu et al., 2012; Haas et al., 2013). The
longest ORF was chosen for each Trinity subcomponent to produce unique proteins by
use of usegalaxy.org (Blankenberg et al., 2010; Giardine et al., 2005; Goecks et al.,
2010). OMA stand-alone v.0.99t (Altenhoff et al., 2013; Altenhoff et al., 2011) identified
7,297 orthologs that were shared in the transcriptomes of all four species (Ps.
geogianus, P. charcoti, N. ionah, H. antarcticus) and 18,781 orthologous groups that
were shared between Ps. georgianus and P. charcoti. Confirmation of orthologous pairs
was done with Blast v2.2.30+ (Altschul et al., 1990; Altschul et al., 1997). A list of
52,959 shared genes was identified in Ps. georgianus and P. charcoti assemblies by
reciprocal blast hit (E value < 10-80) criteria. From this list, 19,665 genes from Ps.
georgianus mapped (E value < 10-40) to the published genome (60.9%) of Notothenia
coriiceps (Shin et al., 2014). Transcriptomes were also mapped (E value < 1010) to the
Swiss Prot database for human proteins Release 2015_9 (UniProt, 2015). Reciprocal
Blast confirmed 15,606 of the 18,781 orthologous genes from the OMA analysis.
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Mapping (E value < 1040) to known zebrafish and stickleback orthologs in ENSEMBL
v74 (Herrero et al., 2016) confirmed 9,735 genes. Gene association networks and gene
ontology enrichment were analyzed by STRING v10 (Jensen et al., 2009) based on the
Swiss-Prot annotations (The UniProt, 2017). Association networks were edited using
Inkscape (www.inkscape.org).
Expression Analysis
Expression of pairwise-orthologs shared by Ps. georgianus and P. charcoti was
directly compared between tissues from each species using the Trinity pipeline (Haas et
al., 2013). Briefly, reads from each tissue were aligned to the respective assembly using
bowtie v1.1.1, and abundance estimation was carried out with RSEM v1.2.21
(Langmead et al., 2009; Li and Dewey, 2011). Differential expression (DE) analysis was
performed on Trinity components (gene level) with edgeR v3.10.2 to normalize and
detect significant differences between Ps. georgianus and P. charcoti read counts in
each tissue with 2-4 biological replicates per sample (Robinson et al., 2010). Differential
expression was considered statistically significant with an exact test P-value of 1E-05
and an FDR < 0.05. Expression profiles of DE genes were normalized across samples
to transcripts per million (TPM) (Haas et al., 2013). Hierarchical clustering was
performed with Gene-E to group similarly expressed genes and generate expression
profile heat maps (http://www.broadinstitute.org). Clusters were cut at a dendogram
height of 56%.
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Quantitative RT-PCR
Whole RNA was purified from flash frozen tissues in TriZol using the Ribopure kit
(Ambion). RNA was reverse transcribed with a polyT(23) primer using Protoscript II RT-
PCR kit (Invitrogen). Target genes were amplified from cDNA in triplicate by quantitative
PCR (Table 4). Standard curves were generated in QuantStudio v3 (Thermo Fischer
Sci) to confirm the efficiency of all primers. One or two biological replicates were used
per notothenioid species. Beta-actin expression was used to normalize expression of
target genes for comparison by the ΔΔCt method. Statistical comparisons were carried
out between red- and white-blooded lineages using a Student’s t test (P < 0.05).
Determination and comparison of dN/dS ratios
Sequence analysis was conducted on the set of 7,297 orthologs that were
shared by two red-blooded (P. charcoti, H. antarcticus) and two white-blooded (Ps.
georgianus, N. ionah) notothenioids. First, ratios of nonsynonymous to synonymous
substitution rates (dN/dS) were determined to identify genes that are under different
selection in each ecotype. Coding and peptide sequences were aligned with T-Coffee
v11.00.8 and back-translated with ParaAT (Notredame et al., 2000; Zhang et al.,
2012b). Substitution rates were determined in PAML v4.8 with codeml (Yang, 2007).
This method does not consider gaps in alignments when estimating substitution rates.
Extremely high dN/dS ratios (>10) may be due to high sequence similarity or short
sequence length and were discarded (Mugal et al., 2014). To test for genes with
variable dN/dS ratios between notothenioid lineages, a likelihood ratio test (P < 0.05)
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was carried out to compare a 2-ratio and 1-ratio (null) model for each set of alignments.
Prediction of deleterious missense mutations
Sequence alignments were scanned for mutations that may have altered the
function of the encoded protein. PAML was used to identify all nonsynonymous
substitutions that supported the division between the red- and white-blooded lineage
branches. The program Provean was used to predict whether these missense mutations
have a neutral or deleterious effect on protein function (Choi and Chan, 2015). The
program was run on all sequence alignments using a custom shell script. Provean
works under the assumption that substitutions in evolutionarily conserved protein
domains are likely to have deleterious effects. A Provean score < -3 was was used as a
threshold to predict deleterious mutations because it had a higher specificity than the
default score (<2.5) and was shown to accurately predict ~84% of deleterious
mutations. Protein domain diagrams were created with Geneious version R10
(http://www.geneious.com) (Kearse et al., 2012).
Identification of frameshift mutations
Frameshift mutations were determined by a Blastx search of the icefish coding
sequences (Ps. georgianus, N. ionah) to the translated protein databases for two red-
blooded species (P. charcoti, N. coriiceps) (Shin et al., 2014). As a requisite, the same
mutation(s) in both icefishes could not occur in either red-blooded species. Mutations
were also checked manually by aligning the genes to the reference genome for N.
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coriiceps (Shin et al., 2014). Mutated genes were isolated and sequenced from genomic
DNA purified from N. coriiceps and another icefish, C. aceratus.
Cloning of genes and cDNAs from Antarctic fish tissues
Genomic DNA (gDNA) was isolated from flash frozen tissues using the HotShot
protocol (Truett et al., 2000). Target genes were amplified from gDNA and cDNA by
PCR with 1 µM primers (Table 4) – the amplification program was 35 cycles of 98°C for
10 s, 57°C for 10 s, and 72°C for 30 s. PCR products were cloned into the pGEM-T
Easy vector (Promega, A1360), plasmids were transformed into 5-α competent cells
(New England Biolabs, C2987H), recombinant plasmids were identified by blue/white
screening and purified with the Wizard Plus SV Miniprep Kit (Promega A1330), and
inserts were sequenced by GeneWiz.
Imaging
Peripheral blood smears were prepared from N. coriiceps and P. charcoti on
glass slides and fixed in 4% paraformaldehyde (PFA) (Yergeau et al., 2005). Cells were
stained with Giemsa according to the manufacturer’s instructions (Sigma Aldrich).
Triton-insoluble erythrocyte membrane skeletons were prepared from flash frozen
peripheral blood samples from N. coriiceps and P. charcoti. Briefly, cells were
resuspended in 1% Triton-X, spread on glass coverslips and fixed with 4% PFA. Blood
smears and triton-shell spreads were imaged with a Nikon Eclipse E800 microscope
using a Photometrics Scientific CoolSNAP EZ camera. Morphological measurements of
cells were made using NIKON NIS-Elements AR 4.20 software. For scanning electron
microscopy, peripheral blood smears were sputter-coated for 5 s with gold and imaged
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directly by Scanning Electron Microscopy at the Marine Science Center of Northeastern
University.
Western blotting of anti-acetylated lysine
Total protein was prepared for sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) from flash frozen notothenioid tissues by homogenization
in lithium dodecyl sulfate (LDS) Bolt buffer (Life Technologies, B007) and NuPAGE
reducing agent (Life Technologies, NP0009) using a pestle and microcentrifuge tube
(USA Scientific, 1415-5390). Samples were boiled for 3 min and centrifuged at top
speed in an Eppendorf 5417R centrifuge for 2 min. Aliquots (15 µg) were
electrophoresed on a 4-12% SDS polyacrylamide gel, and the separated proteins were
transferred to a polyvinylidene difluoride (PVDF) membrane by use of the iBlot system
(Life Technologies, IB21001). Membranes were blocked in maleic acid blocking buffer
(2% Roche blocking reagent, 2% BSA, 0.2% heat treated goat serum, 0.1% Tween-20)
for 1 h at room temperature and then incubated overnight at 4°C with 1:1000 mouse
anti-acetylated lysine (Santa Cruz Biotechnology, AKL5C1). Membranes were washed
in TBST (0.1 M Tris, 0.1 M NaCl, 0.1% Tween-20) and incubated for 2 h with
horseradish peroxidase HRP-conjugated goat anti-mouse IgG (H&L) (Aviva,
OARA04973). Bound antibodies were detected with the Amersham ECL Western
Blotting Analysis System (GE Healthcare, RPN2106) on CL-X Posure film (Thermo
Scientific,34091).
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79
Figures S1. Box plot of RNA-Seq expression profiles in notothenioid tissues.
Values are normalized to transcripts per million (TPM) and log2 transformed.
Abbreviations: HK, head kidney; T. kidney, trunk kidney; Pectoral, pectoral muscle; W.
muscle, white muscle; Gonads, ovary. Ventricle, heart ventricle.
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81
Figure S2. Heatmap of Pearson’s correlation coefficients after comparison of
RNA-Seq gene expression in P. charcoti and Ps. georgianus tissues. Heat map
color represents the Pearson’s correlation coefficient for total gene expression from
each tissue comparison. (A) Gene expression correlation coefficients cluster by tissue
type between P. georgianus and P. charcoti when all genes are assessed. (B) Gene
expression correlation coefficients do not cluster between P. georgianus and P. charcoti
for differentially expressed genes in head kidney.
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83
Figure S3. Down-regulation of genes in the icefish head kidney for gene ontology
(GO) groups related to erythropoiesis. GO enrichment was determined using
STRING (Fishers exact test, P < 0.05, FDR < 0.01).
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Table 1. Hematopoietic genes are differentially expressed in the icefish head kidney
GO:0030097 hemopoiesis 20 1.00E+00 1.76E-02
Up in Icefish GeneID logFC Pvalue Human homolog
GAS6 4.30 6.55E-14 growth arrest-specific protein 6 NTRK1 3.53 3.63E-06 high affinity nerve growth factor receptor
BAX 2.89 9.88E-08 apoptosis regulator BAX
DOCK1 2.66 1.80E-07 dedicator of cytokinesis protein 1
CBFB 2.02 5.88E-05 core-binding factor subunit beta
Down in Icefish
MYB -2.03 4.24E-05 transcriptional activator Myb
CBFA2T3 -2.50 1.31E-07 protein CBFA2T3
CASP3 -2.55 9.78E-10 caspase-3
GATA1 -2.60 2.53E-07 GATA-1 CD28 -3.33 3.45E-05 T-cell-specific surface glycoprotein CD28
FECH -3.37 6.30E-09 ferrochelatase
ALAS2 -4.53 6.72E-14 5-aminolevulinate synthase
KLF2 -4.59 1.05E-20 Krueppel-like factor 2
KLF1 -4.70 4.05E-16 Krueppel-like factor 1 TFRC -4.88 1.93E-15 transferrin receptor protein 1
GFI1B -2.02 0.000101 zinc finger protein Gfi-1b
Table 2. GO enrichment of genes under different selective pressures in two red- and
two white-blooded notothenioids
GO Enrichment for genes under Different Selection
GO:0030097 hemopoiesis 11 5.66E-02
GO:0034101 Erythrocyte homeostasis 7 6.49E-05
GO:0042168 Heme metabolic process 4 4.10E-04
GO:0033572 Transferrin transport 3 8.50E-03
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Table 3. GO enrichment for genes with deleterious substitutions found in two white-
blooded icefishes but not in two red-blooded notothenioids
GO Enrichment for Deleterious Mutations
GO.0030099 myeloid cell differentiation 31 0.000286
GO.0030097 hemopoiesis 56 0.00165
GO.0006915 apoptotic process 94 0.0074
GO.0055114 oxidation-reduction process 85 0.0231
GO.0007010 cytoskeleton organization 72 0.0284
5220 Chronic myeloid leukemia 11 0.0473
4380 Osteoclast differentiation 19 0.008
Table 4. Table of Primers
Gene Oligo Sequence (5’ – 3’) Gene Method
Sptb_F Sptb_R Sptb_R_exon19
CCAGGCCTTCATGGCTGAG CGCACCTGGTTCTCCGTC GATGCTTCTTGAGCAAGATG
sptb (Notothenioid)
PCR gDNA
Hdac1b_F Hdac1b_R
GAGGAGGCCTTCTACACCAC CGACTCGTCGTCAATACCGT
hdac1b (Notothenioid)
qPCR
Spi1_F Spi1_R
GGATCCAAACCTTGGGGCAC GTGGATACACAGGCCGAGG
spi1b (Notothenioid)
qPCR
Gata1a_F Gata1a_R
CCACAGCCGAGCGCCTCC GCCCCGTCCAGCAGCTGC
gata1a (Notothenioid)
qPCR
SGK1_F SGK1_R
CTGAAGCCTGAGAACATCC CCATAGAGCATCTCGTAGAG
sgk1 (Notothenioid)
qPCR
PML-L_F PML-L_R
TGACCTGGAGGCCACTGG CCTGCAGGTCAGACCCG
pml-like (Notothenioid)
qPCR
P300b_F P300b_R
CCCGAGAAACGGAAGCTGAT TTTTTCAGCGGCAGGCAAAC
p300b (Notothenioid)
qPCR
ZFP64_F ZFP64_R
GCCTTACACTGTGAGGAGG AACTCCTCATTGTGGGAGG
zfp64 (Notothenioid)
qPCR
ERO1α_F ERO1α_R
GCAGGTGCTTCTGTCAG GTTTGGAGAAGAGCTGGTTG
ero1α (Notothenioid)
qPCR
Fam161a_F Fam161a_R
TTTAAGGCGAGACCCATG CACCATCTCAATGGAAACC
fam161a (Notothenioid)
qPCR
CD33rSig_F CD33rSig_R
CTGCTCATTAGAGATTGATGA GAAGGTTATTGTGGAGGTC
cd33rSig (Notothenioid)
qPCR
Bact_F Bact_R
CAGATCATGTTCGAGACCTTCAAC TCACCRGARTCCATGACGATA
beta-actin (Notothenioid)
qPCR
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Table 5. Icefishes have predicted deleterious substitutions in targets of human diseases Gene GO Mutation Provean Domain Associated Diseases Reference
ADD1 MH A246T -3.692 Aldolase II, NH-head domain HS Robledo et al. 2008,Anong et al. 2009
ANXA2 MH Y113F -3.105 Annexin domain AML, B-ALL Olwill et al. 2005
ASXL2 M G1082R -3.637 Proximate to PHD CBF-AML Jean-Baptiste et al. 2014
BCL11A H E14G -3.353 AML, CML Yin et al. 2016
CARD11 H E258G -6.062 GBP_C "guanylate-binding protein” DLBCL Lenz et al. 2008
CASP8 MH C22R -3.965 DED1 domain HNSCC Ando et al. 2013
CDKN1C MH G202E -4.246 BWS Yatsuki et al. 2013
CHD2 H N502I -5.68 SNF2 domain CLL, MAE Rodriguez et al. 2015, Trivisano et al. 2015
EML1 H Y36D -3.204 T-ALL De Keersmaecker et al. 2005
ERCC1 H R414Y -3.021 COFS, ALL Jaspers et al. 2007, Wang et al. 2006
FOXP1 MH V77F -3.126 B-ALL Put et al. 2011
G6PD MH P495N -4.957 G6PD deficiency, hemolytic anemia Beutler et al. ,
GATA1 MHN N319S -4.756 CT-Zn finger Thrombocytopenia, DS-AMKL, DBA Nichols et al. 2000, Crispino 2005
GFI1 MH C217Y -6.278 3rd C2H2 Zn finger AML, CLL, bleeding disorder, SCN Moroy et al. 2015
GFI1B MH E136A -3.387 1st C2H2 Zn finger Macrothrombocytopenia Kitamura et al. 2016
IKZF1 M P145A -4.715 Proximate to Zn finger 1 B-ALL, ALL Kastner et al 2013
NFE2 MH A380V -3.633 Coiled-coil, bZIP DNA binding MPN Jutzi et al. 2013,Shyu et al. 2006
RUNX1 MN L342H -5.353 TAD domain AML, B-ALL, CML Gaidzik et al. 2011
SPI1 MH V56P -3.414 Acidic TAD domain AML Mueller et al. 2002, Lamandin et al. 2002
STAT5B HN P198A -3.563 All-alpha domain Lymphomas Kucuk et al. 2015
TF MH E382A -4.57 C-lobe, disulfide bond Atransferrinemia, Alzheimer's Lee et al. 2001, Giambattistelli et al. 2012
TFRC MH E140V -3.759 ZN-peptidase transferrin receptor Iron deficiency anemia Roetto et al. 2001, Jabara et al. 2016
BLVRB P G145R -3.626 BVR/FR (Flavin reductase) domain Thrombopoiesis WU et al. 2016, O'Brien et al. 2015
COX15 P L235F -3.623 Transmembrane region Leigh syndrome, cardiomyopathy Antonicka et al. 2003, Bugiani et al. 2005
CPOX P P364H -8.838 Coproporphyrinogen III oxidase HCP, harderoporphyria Martasek et al. 1994, Schmitt et al. 2005
HPX P G52E -7.765 Hpx repeat 1 Diabetic macular edema Mehta et al. 2015, Hernandez et al. 2013
NFE2L1 P S301F -4.455 - Cancer, neurodegenerative disease Han et al. 2012, Taniguchi et al. 2017
SLC25A39 P L230F -3.055 1st solcar repeat Anemia, epilepsy Nilsson et al. 2009, Slabbaert et al. 2016
UGT1A1 P R332G -4.98 UDP-glucoronosyltransferase 1-1 Crigler-Najjar (CN), Gilbert (GILBS) Servedio 2005
IKAROS2 G108D -6.144 Zn finger 1 ALL Zhang et al. 2007,Chen et al. 2013
FES R585C -5.823 Catalytic domain of PTK, ATP binding AML Cheng et al. 2001,Sangrar et al. 2005
FLT1 C186R -4.337 CT domain AML, CML Choi et al. 2005, Fragoso et al. 2006
Gene logFC GO Mutation Domain Associated Diseases Reference
PPHLN1 0.57 L265fs Intrahepatic cholangiocarcinoma Sia et al. 2014
PAPPS2 na A43fs Brachyolmia, Prostate cancer Miyake et al. 2012, Ibeawuchi et al. 2015
MATE1 -1.18 F467fs Environmental toxin clearance, CML Chen et al. 2009
ZFP64 0.46 L615fs Amyotrophic lateral sclerosis Schymick et al. 2007
ERO1la -5.06 T37fs Adenocarcinoma Endoh et al. 2004
FAM161Al 3.07 H291fs C-terminus Retinitis pigmentosa 28 Karlstetter et al. 2014, Van Schil et al. 2015
CD33l -1.46 F753fs ITIM domain Alzheimer's, AML (expression) Stefania De Propris et al. 2011
NUMA1 -0.09 E895fs Osteosarcoma, AML Kovac et al. 2015, Strehl et al. 2012
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Chapter 2: Divergent Hemogen genes of teleosts and mammals share conserved
roles in erythropoiesis: Analysis using transgenic and mutant zebrafish
Michael J. Peters1, Sandra K. Parker1, Jeffrey Grim1,2, Corey A. H. Allard1,3, Jonah
Levin1,4, H. William Detrich, III1*
1Department of Marine and Environmental Sciences, Northeastern University, Nahant,
MA 01908, USA
2Present address: Department of Biology, The University of Tampa, Tampa, FL 33606,
USA
3Present address: Department of Biochemistry and Cell Biology, Geisel School of
Medicine at Dartmouth College, Hanover, NH 03755, USA
4Present address: Department of Biochemistry, McGill University, Montreal, Quebec
H3G1Y6, CA
Published:
Peters MJ, Parker SK, Grim J, Allard CAH, Levin J, Detrich HW III. 2018. Divergent
Hemogen genes of teleosts and mammals share conserved roles in erythropoiesis:
Analysis using transgenic and mutant zebrafish. Biology Open bio.035576 doi:
10.1242/bio.035576
88
Summary Statement
Transgenic and mutant zebrafish lines were created to characterize the
expression and functions of Hemogen, a transcription factor involved in the formation of
red blood cells and other processes.
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ABSTRACT
Hemogen is a vertebrate transcription factor that performs important functions in
erythropoiesis and testicular development and may contribute to neoplasia. Here we
identify zebrafish Hemogen and show that it is considerably smaller (~22 kDa) than its
human ortholog (~55 kDa), a striking difference that is explained by an underlying
modular structure. We demonstrate that Hemogens are largely composed of 21-25
amino acid repeats, some of which may function as transactivation domains (TADs).
Hemogen expression in embryonic and adult zebrafish is detected in hematopoietic,
renal, neural, and gonadal tissues. Using Tol2- and CRISPR/Cas9-generated
transgenic zebrafish, we show that Hemogen expression is controlled by two Gata1-
dependent regulatory sequences that act alone and together to control spatial and
temporal expression during development. Partial depletion of Hemogen in embryos by
morpholino knock-down reduces the number of erythrocytes in circulation.
CRISPR/Cas9-generated zebrafish lines containing either a frameshift mutation or an
in-frame deletion in a putative, C-terminal TAD display anemia and embryonic tail
defects. This work expands our understanding of Hemogen and provides mutant
zebrafish lines for future study of the mechanism of this important transcription factor.
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INTRODUCTION
Hemogen (Hemgn) is a vertebrate transcription factor that is expressed in
mammalian hematopoietic progenitors (Lu et al., 2001; Yang et al., 2001) and has been
implicated in erythroid differentiation and survival (Li et al., 2004). Originally identified in
mice and subsequently described in humans as EDAG (Erythrocyte Differentiation
Associated Gene), Hemogen has also been implicated in testis development in
mammals and chickens (Nakata et al., 2013; Yang et al., 2003), and in osteogenesis in
rats (Kruger et al., 2002; Kruger et al., 2005). Here we analyze the developmental roles
of teleost Hemogen using the zebrafish model system and its powerful suite of reverse-
genetic technologies.
Teleost Hemogen was discovered using a subtractive hybridization screen
designed to isolate novel erythropoietic genes from fish belonging to the largely
Antarctic suborder Notothenioidei (Detrich and Yergeau, 2004; Yergeau et al., 2005).
Sixteen species belonging to the icefish family (Channichthyidae) are unique among
vertebrates because they are white-blooded ‒ they fail to execute the erythroid genetic
program or produce hemoglobin (Cocca et al., 1995a; Near et al., 2006a; Zhao et al.,
1998a). Forty-five candidate erythropoietic cDNAs were recovered using
representational difference analysis (Hubank and Schatz, 1999) applied to kidney
marrow transcriptomes of two notothenioid species, one red-blooded and the other
white-blooded (Detrich and Yergeau, 2004; Yergeau et al., 2005). One of the unknown
genes, clone Rda130, was similar to mammalian Hemogen and was expressed only by
the red-blooded notothenioid.
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Although Hemogen is clearly involved in hematopoiesis, its mechanism remains
incompletely understood. In human cell lines, Hemogen activates erythroid gene
transcription in part by recruiting the histone acetyltransferase P300 to acetylate Gata1
(Zheng et al., 2014). Like Gata1, Hemogen protects erythroid cells from apoptosis by
upregulating anti-apoptotic factors (e.g., Nf-κB, Bcl-xL) that are critical for terminal
differentiation (Li et al., 2004; Rhodes et al., 2005; Zhang et al., 2012a).
The regulation of Hemogen expression is of interest because it is overexpressed
frequently in patients with a variety of cancers and leukemias (An et al., 2005; Forbes et
al., 2017; Li et al., 2004). This putative oncogene, which is located in a human
chromosomal region (9q22) of leukemia-associated breakpoints, has been linked to
proliferation and survival of leukemic cells and to induction of tumor formation in mice
(Chen et al., 2016; Lu et al., 2002). Thus, somatic mutations in Hemogen or its
regulators may contribute to neoplasia.
The zebrafish is a well-established model organism for studying hematopoiesis in
vertebrates because it produces the same blood lineages as mammals (de Jong and
Zon, 2005; Paffett-Lugassy and Zon, 2005). In zebrafish, erythropoiesis occurs in
sequential waves at unique anatomical locations in embryos and adults that correspond
to analogous sites in mammals (Galloway and Zon, 2003). Many of the molecular
players that orchestrate the erythroid program appear to be conserved between
zebrafish and mammals, but relatively few have been functionally characterized in
zebrafish. Nevertheless, mutant zebrafish models accurately phenocopy human blood
diseases caused by mutations in major erythroid factors, such as Gata1 (Lyons et al.,
2002) and Erythroid beta-spectrin (Liao et al., 2000).
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The purpose of this study is to characterize the regulation of Hemogen
expression and the function of the Hemogen protein in zebrafish. We identify the
zebrafish Hemogen ortholog, which despite being only 40% as large as the human
protein, contains similarly arranged functional motifs. Hemogen is expressed in blood,
testis, ovaries, kidney, and the central nervous system in zebrafish. Two tissue-specific,
alternative Hemogen promoters are associated with conserved noncoding elements
(CNEs) and have distinct regulatory functions in primitive and definitive hematopoiesis
and other processes. By analysis of morphant and mutant zebrafish, we show that
Hemogen is required for normal erythropoiesis and that this role depends in part on a
cluster of acidic residues within a putative, C-terminal transactivation domain (TAD).
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Figure 1. Zebrafish Si:dkey-25o16.2 and human Hemogen are orthologous and
encode related proteins that differ in size. (A) Structure of the zebrafish Hemogen-
like gene, Si:dkey-25o16.2. Two conserved noncoding elements (C1 and C2; black
boxes) were identified in a 2-kb segment proximal to the start codon (see Results, Figs.
4-6). Coding exons, white boxes; noncoding exons, gray boxes. Numbers indicate
length in bp. (B) Synteny of loci for zebrafish Si:dkey-25o16.2 on chromosome 1 and
Hemogen on human chromosome 9 (region q22). Transcriptional orientations indicated
by arrows. (C) Alternative splicing of zebrafish Hemogen-like transcripts showing
sequenced regions. Introns are shown as chevrons. Transcripts 1 and 2 differ by
retention of 12 bp of intron (red). (D) Modular structures of zebrafish and human
Hemogen proteins each encoded by four exons (numbered boxes). Locations of
truncating mutations found in some human cancers (Forbes et al., 2017) are indicated
by asterisks. Predicted regions and motifs: green, coiled coil; blue, nuclear localization
signal; red, four residues introduced by alternative splicing; yellow, tandem peptide
repeats; brown, acidic repeat with transactivation domain (TAD) motif; gray, no
prediction. (E) Three-dimensional ab initio models of Hemogens. The ribbon diagram of
the zebrafish protein, color-coded as in panel D, is superimposed on the gray, space-
filling model for the human protein (See Materials and Methods).
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RESULTS
Teleosts contain a single Hemogen-like gene that is syntenic with human Hemogen
Chromosomal synteny is an important criterion when assigning gene
relationships across divergent taxa. Despite the whole-genome duplication (WGD) that
coincided with the separation of teleosts from more basal ray-finned fishes and
tetrapods (Postlethwait et al., 2000), the sequenced genomes of nearly all fishes retain
a single Hemogen-like gene. We cloned zebrafish Hemogen-like cDNAs and found that
they corresponded to the predicted gene Si:dkey-25o16.2 on chromosome 1 of the
zebrafish genome (Howe et al., 2013). When we compared the synteny of the putative
teleost and mammalian orthologs, represented in Figure 1B by zebrafish Si:dkey-
25o16.2 (chromosome Dr1) and human Hemogen (chromosome Hs9), we found that
the flanking genes and their transcriptional orientations were conserved, which strongly
supported Si:dkey-25o16.2 as the zebrafish Hemogen ortholog.
Structure of the zebrafish Hemogen gene
The basic structure of the Hemogen gene of teleosts and mammals was also
found to be highly conserved – four coding exons were separated by three introns (Fig.
1A), and two introns were found in the 5’-UTR. Two transcription start sites were
predicted to occur within 2-kb upstream of the Hemogen start codon in zebrafish (Fig.
1A) ‒ these appear to correspond to the hematopoietic- and testis-specific Hemogen
promoters (noncoding exons 1H and 1T, respectively) described for mammals (Yang et
al., 2003). Alignment of Hemogen genes from 10 teleost species (Yates et al., 2016)
revealed two conserved non-coding elements, CNE1 and CNE2, that overlapped with
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zebrafish exons 1T and 1H, respectively (Fig. 1A). We hypothesized that these
elements function individually or together to regulate transcription of Hemogen.
Transcription of the zebrafish Hemogen gene yields multiple mRNA isoforms
We confirmed transcription from both promoters in zebrafish by isolating and
sequencing four splicing variants (Fig. 1C). Three isoforms were transcribed from the
proximal promoter (exon 1H, Fig. 1A,C), each containing the same 5’-untranslated
region (5’-UTR). Alternative splicing of the second coding exon produced transcripts 1
and 2, which differ by four additional codons in the latter (Fig. 1C, red); the shorter
version has not been described in mammals. Transcript 3 retained the entire third intron
(156 bp), which introduced a premature translation-termination codon. A fourth isoform
was transcribed from the distal promoter (1T) located ~1.65 kb upstream of the
translation start codon (Fig. 1A,C). Splicing of exons 1T and 1H to form the 5’-UTR of
transcript 4 made use of canonical donor (AT-GT) and acceptor (AG-TT) splice sites.
Teleost and mammalian Hemogen proteins differ markedly in size but share structural
motifs
Teleost Hemogen-like genes encoded shorter proteins (194-289 amino acids)
than the annotated Hemogen genes of mammals (417-827 amino acids), and the
overall amino acid sequence similarity between teleost and mammalian orthologs was
modest (18%-38%). Despite this heterogeneity in length and sequence, Hemogens of
teleost fish and mammals shared predicted structural motifs, as shown in Figure 1D,E
for zebrafish (198 aa, 22 kDa) and human (484 aa, 55 kDa) orthologs, respectively.
Their N-termini (zebrafish residues 1-74, human 1-78) were substantially conserved
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(51% sequence similarity; Fig. S1) and contained two predicted coiled-coil (CC) forming
alpha-helices, the second of which was a putative nuclear localization signal (NLS)
(Yang et al., 2001) (Fig. 1D; Fig. S1). By contrast, their C-termini (zebrafish residues 75-
198, human 79-484) were weakly conserved in sequence (13% similarity), but both
were rich in Pro and Glu residues (Figs. S1-S2), consistent with intrinsic disorder of
these regions (Dyson and Wright, 2005). Furthermore, the C-termini shared modular
structures – each was built of several 21-25 amino acid motifs, three in zebrafish and
nine in humans, with distinct but related consensus sequences
(PEXXXIAEXXXXXQEVXPQXXLVP and YSXEXYQEXAEPEDXSPETYQEIPX,
respectively) (Fig. 1D,E, Figs. S1-S2). Thus, the size heterogeneity between zebrafish
and human Hemogens was largely attributable to the number of repetitive segments
contained within each.
Within the C-termini of teleost Hemogens, we identified a conserved acidic region
(zebrafish residues 119-169, 35-49% similarity across 10 species) that was similar to an
acidic region of the mouse protein (Yang et al., 2001). Given the transactivation
functions of Hemogen in humans (Zheng et al., 2014), we investigated whether the
zebrafish and human proteins possessed TAD motifs based on the consensus
sequences ϕϕxxϕ or ϕxxϕϕ, where ϕ is a bulky hydrophobic residue (Dyson and Wright,
2016). The acidic C-termini of both Hemogens contained one TAD motif. Four additional
TAD motifs were distributed in other regions of the human protein (Fig. S1).
To assess the three-dimensional conformations of zebrafish and human
Hemogens, although in a static context, we generated ab initio tertiary structural models
with I-Tasser (Yang et al., 2015) using the best of ten predicted templates (Fig. 1E, see
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Materials and Methods). The structures for zebrafish and human Hemogens had
template modeling scores (TM-scores) of 0.45 and 0.55, respectively, where a TM-
score > 0.3 indicates significantly different (P < 0.001) from random structures (Xu and
Zhang, 2010). When the two models were superimposed, amino acid sequences shared
by human and zebrafish Hemogens showed 98% coincidence and a TM-score of 0.71.
The N-termini of the zebrafish and human Hemogens presented exposed CC domains
that may serve as binding sites for Gata1 (Zheng et al., 2014). The “disordered” C-
termini of Hemogens from zebrafish and humans were comprised of two distinct
elements: proline-rich repeats (yellow) and an acidic, C-terminal repeat containing the
TAD motif (maroon) (Fig, 1E, Fig. S1). The former may coalesce as rigid linkers to
extend the TAD motif to binding partners. These features are common to transcription
factors, as epitomized by the structure of p53 (Wells et al., 2008).
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Figure 2. Hemogen expression in zebrafish embryos. (A-H) Wild-type embryos,
WISH. (A) Epiboly at 9 hpf. Hemogen expression was not detected. (B) 10-somite
stage. Hemogen transcripts along the lateral plate mesoderm (LPM). (C) 20 hpf.
Hemogen staining in the intermediate cells mass (ICM) and posterior blood island (PBI).
The inset shows a sense probe control. (D) 33 hpf. Hemogen-positive primitive
erythrocytes of the peripheral blood (PB) exited the Ducts of Cuvier (DC) onto the yolk.
Staining at the midbrain-hindbrain boundary (MHB) was observed. (E) 144 hpf.
Hemogen expression in the caudal hematopoietic tissue (CHT) and pronephric kidney
(PK), and in erythrocytes in the heart (H). The asterisk indicates the plane of the cross
section in panel F. (F) 144 hpf. Cross section of embryo in panel E showing heavily
stained pronephric ducts. (G) 48 hpf. Lateral aspect of tail. Hemogen transcripts in the
CHT and pronephric tubule duct (PD). (H) Kidney touch print from adult fish. Hemogen
expression was observed in proerythroblasts (ProE) and normoblasts (N) but not in
erythrocytes (E). (I) 48 hpf. View of circulating EGFP+ erythrocytes in the dorsal aorta
(DA) of Tg(Lcr:EGFP)cz3325Tg zebrafish after staining for Hemogen protein by indirect
immunofluorescence. Hemogen (red signal) accumulated in nuclei (Nu) of erythrocytes
whereas the cytoplasm (C) was marked by EGFP. Other abbreviations: CV, caudal
vein; DA, dorsal aorta; G, gut; M, myotomes; NC, notochord; PK, pronephric kidney; SB,
swim bladder; SC, spinal cord. Scale bars: (A-F) 250 µm; (G) 1 mm; (H) 100 µm; (I) 50
µm.
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Hemogen expression tracks the ontogenetic progression of hematopoiesis in zebrafish
The spatial and temporal patterns of Hemogen expression were evaluated in
zebrafish between 2 and 144 hours post fertilization (hpf) by whole-mount in situ
hybridization (WISH) (Fig. 2A-H). Hemogen transcripts were not apparent prior to
somitogenesis (Fig. 2A) but first appeared at the 10-somite stage in punctate,
intersomitic foci in the lateral plate mesoderm (LPM; Fig. 2B). By 20 hpf, Hemogen was
expressed throughout the intermediate cell mass (ICM) and posterior blood island (PBI)
(Fig. 2C), the sites of primitive hematopoiesis (Bertrand et al., 2007b; Davidson and
Zon, 2004). Primitive erythrocytes expressed Hemogen as they entered circulation at 33
hpf (Fig. 2D).
Definitive hematopoiesis in zebrafish embryos commences in the aorta gonad
mesonephros (AGM) region at 30 hpf with the emergence of hematopoietic stem
progenitor cells (HSPCs) that subsequently seed the caudal hematopoietic tissue (CHT)
and the thymus (Murayama et al., 2006). By 144 hpf, HSPCs migrate from the CHT to
establish a niche associated with the pronephric glomeruli (Bertrand et al., 2008).
Although we did not detect Hemogen mRNA in the AGM (Fig. 2D), we observed strong
expression in cells of the CHT at 48 and 144 hpf (Fig. 2E,G) and in the region of the
pronephric glomeruli at 144 hpf (Fig. 2E,F). In the adult zebrafish kidney, Hemogen was
strongly expressed in progenitor cells in the interstitial hematopoietic stem cell niche
between pronephric tubules (Fig. 2H). Hemogen expression was robust in progenitors
but absent in mature erythrocytes (Fig. 2H), whereas an anti-sense riboprobe for βe1-
globin hybridized exclusively to mature erythrocytes but not to progenitor cells (data not
shown).
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Hemogen has been shown to function as a nuclear transcription factor in
mammals (Zheng et al., 2014). To determine whether or not Hemogen is likely to play
the same role in zebrafish, we examined Tg(Lcr:EGFP)cz3325Tg embryos at 48 hpf by
indirect immunofluorescence microscopy using an antibody specific for Hemogen.
Tg(Lcr:EGFP)cz3325Tg zebrafish have been used to track both primitive and definitive
erythrocytes (Ganis et al., 2012). Figure 2I shows that Hemogen accumulated in the
nuclei (red signal) of GFP-labeled circulating erythrocytes in the dorsal aorta; thus, its
role in transcription is likely to be conserved in zebrafish.
Alternative promoters regulate Hemogen expression in zebrafish hematopoietic and
reproductive tissues
In zebrafish, we also detected Hemogen expression in the hindbrain and in the
pronephric tubules of embryonic zebrafish between 30 and 48 hpf (Fig. 3A,B) and in
adult zebrafish reproductive tissues (Fig. 3G-H). The alternative Hemogen promoters
found in zebrafish probably correspond to the hematopoietic and testis-specific
Hemogen promoters of mammals (Yang et al., 2003). To quantify relative levels of
transcription from each promoter in zebrafish (Fig. 3I), we performed qRT-PCR on total
RNA from adult peripheral blood, testis, and ovaries (Fig. 3J) using primer pairs specific
for exons 1H and 1T. Because all of exon 1H was included in transcripts initiated from
exon 1T, one must infer transcription from the proximal promoter by difference.
Transcription from the proximal promoter was greatest in peripheral blood; the presence
of transcripts from this promoter in testis and ovarian tissue may be due to
contaminating blood RNA. The distal promoter was highly active in both peripheral
blood and in testes but not in ovaries.
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Figure 3. Alternative promoters drive Hemogen expression in hematopoietic and
nonhematopoietic tissues in zebrafish. WISH of wild-type embryos (A-B) and adult
tissues (C-H). (A) 48 hpf. Hemogen expression in the pronephric kidney glomeruli (PG),
pronephric tubule duct (PD), caudal hematopoietic tissue (CHT), and brain (Br). (B) 48
hpf. Section showing strong Hemogen expression in the hindbrain (HB) but at low levels
in the midbrain (MB). (C) Dorsal and (D) ventral views of the adult zebrafish brain after
staining for Hemogen transcripts. (E) Schematic drawing of the dorsal view. Hemogen
was highly expressed at the midbrain-hindbrain boundary within the eminentia
granularis (EG), in the crista cerebellaris (CC), and in the hypothalamus (Hy). The
asterisk indicates the plane of the cross section in panel F. (F) Section of the hindbrain
showing Hemogen expression in the periventricular gray zone (PGZ). (G) Hemogen
was expressed by Sertoli cells (Se) between the seminiferous tubules (ST) of the testes.
(H) Hemogen was expressed in early (I-III) but not late (IV) stage oocytes. Transcripts
accumulated around the germinal vesicle (GV). (I) Schematic of the Hemogen
noncoding exons 1T and 1H (gray) upstream of the first coding exon (white); bent
arrows, transcription initiation sites. Arrowheads mark primer binding sites for qPCR
amplification of transcripts initiated from exons 1T or 1H. (J) Expression of transcripts
from alternative promoters determined by qRT-PCR using RNA from blood, testes, and
ovaries of adult TU zebrafish. Expression in three biological replicates were normalized
to β-actin and calculated relative to ovaries. Error bars represent the standard deviation.
Transcription initiated from 1H must be inferred by difference [1H – 1T] because the 1H
primers also amplified 1T transcripts. Other abbreviations: Ce, corpus cerebelli; MO,
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medulla oblongata; OB, olfactory bulb; OT, optic tectum; SR, superior raphe; Te,
telencephalon; TS, torus semicircularis. Scale bars = 250 µm (A, B, F-H); 1 mm (C,D).
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Hemogen CNEs are predicted targets for transcription factors that regulate
erythropoiesis and spermatogenesis
In teleosts, we identified two evolutionarily conserved non-coding elements,
CNE1 and CNE2, that were tightly associated with exons 1T and 1H, respectively (Fig.
1A, Fig. 4A). These elements may function as core promoters and/or enhancers to
regulate transcription of the different Hemogen isoforms in zebrafish. To identify
potential regulators of Hemogen transcription, we used ConTra v2 (Broos et al., 2011)
to predict transcription factor binding motifs in the aligned Hemogen CNEs from two
mammals and nine teleosts (Yates et al., 2016) (Fig. 4B,C). Each CNE contained
binding motifs for transcription factors involved in erythropoiesis and/or
spermatogenesis.
In zebrafish CNE2, two Gata1 binding sites, located +59 and +127 bp
downstream relative to the transcription start site, aligned with Gata1 sites known to be
active in the mammalian Hemogen promoter (Fig. 4C) (Yang et al., 2006). Each Gata
motif was paired with a predicted E-box - this motif in Hemogen CNE2 is a known target
of the Ldb1-erythroid-complex recruited by Scl (Soler et al., 2010). CNE2 also contained
binding sites for Klf4, a driver of zebrafish primitive erythropoiesis (Gardiner et al.,
2007), for Myb, a regulator of zebrafish definitive hematopoiesis (Soza-Ried et al.,
2010), and for HoxB4, a regulator of Hemogen expression in mammalian hematopoietic
stem cells (Jiang et al., 2010).
The distal CNE1 of teleosts possessed a similar suite of transcription factor
binding motifs in roughly the same arrangement as the proximal CNE but with the
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notable addition of binding sites for Sox9 and the Androgen receptor (Fig. 4B), both of
which play roles in zebrafish spermatogenesis (Hossain et al., 2008; Rodriguez-Mari et
al., 2005). CNE1, like CNE2, contained pairs of E-box and Gata motifs downstream of
the zebrafish transcription start site (+15 and +48 bp, respectively). CNE1 may function
as an enhancer for the Hemogen gene and/or act as the core promoter for exon 1T.
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Figure 4. Conserved elements in the zebrafish Hemogen promoter are predicted
targets for transcription factors. (A) Schematic of the zebrafish Hemogen gene.
CNEs, black; coding exons, white; noncoding exons, gray; transcription initiation sites,
bent arrows. Numbers indicate length in bp. (B, C) Sequence alignments of CNE1 and
CNE2, respectively, from 9 teleost species, mice, and humans. ConTra software (Broos
et al., 2011) predicted transcription factor binding sites for the Androgen receptor (light
green), Brca1 (cyan), Foxl2 (pink), Gata1 (dark blue), Gfi1 (orange), HoxB4 (sky blue),
Hnf1a (dark green), Klf4 (yellow), Myb (dark gray), P300 (red), Sox9 (purple),
Scl/Lmo2/Ldb1 complex (light gray). Splice donor sites are highlighted black. Species
abbreviations: Dr, Danio rerio; Ca, Cynoglossus semilaevis; Gm, Gadus morhua; Ga,
Gasterosteus aculeatus; Ol, Oryzias latipes; Xm, Xiphophorus maculatus; On,
Oreochromis niloticus; Tr, Takifugu rubripes; Tn, Tetraodon nigroviridis; Mm, Mus
musculus; Hs, Homo sapiens.
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Hematopoietic and neural expression of Hemogen in zebrafish is dependent on Gata1
binding to the promoter CNEs
In mammals, transcription of Hemogen from the proximal promoter is tightly
regulated by Gata1 in hematopoietic cells (Yang et al., 2006). To investigate whether
Gata1 regulates Hemogen in zebrafish, we analyzed a Gata1 ChIP-seq dataset that
was generated to assess Gata1 activity in adult zebrafish erythrocytes (Yang et al.,
2016). Figure 5A shows that Gata1 bound to CNE1 and CNE2 at sites overlapping their
Gata motifs (red lines), which indicates strongly that Gata1 is required for transcription
of Hemogen in zebrafish. Corroboration that CNE1 and CNE2 were active chromatin
regions was provided by ATAC-seq and DNase I hypersensitive site analysis (Yang et
al., 2016) (Fig. 5A). Our data reveal that Gata motifs in CNE1, like those in CNE2, are
important regulators of Hemogen expression in zebrafish erythrocytes.
We performed WISH to compare the expression of Hemogen and Embryonic
beta-globin (βe1-globin) in embryos produced by the Gata1-null mutant, vlad tepes
(vltm651) (Lyons et al., 2002). At 33 hpf, Hemogen was expressed normally in circulating
blood cells and in the hindbrain of wild-type siblings (Fig. 5B), and βe1-globin was
abundant in the blood (Fig. 5B, inset). Homozygous vltm651 mutant siblings, by contrast,
failed to express Hemogen in the blood and brain (Fig. 5C). This result mimicked the
loss of βe1-globin in vltm651 mutants, with the exception that βe1-globin expression
persisted in the PBI (Fig. 5C, inset), as has been demonstrated for α1-globin, Scl and
Gata1 (Jin et al., 2009).
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Figure 5. Gata1 binds distal and proximal promoter elements to regulate
Hemogen expression in zebrafish. (A) Gata1 ChIP-sequencing showing enriched
binding of Gata1 at CNE1 and CNE2 (C1 and C2, red lines) in the Hemogen promoter
in adult zebrafish red blood cells (Yang et al., 2016). DNase-sequencing and ATAC-
sequencing showing colocalization of the active chromatin regions (Yang et al., 2016).
(B) Hemogen expression by WISH of wild-type (n = 16/21) and (C) homozygous mutant
(n = 5/21) siblings (33 hpf) from in-crossed Gata1+/- vltm651 mutants. Insets show βe1-
globin expression in mutant (n = 4/10) and wild-type (n = 6/10) siblings. Scale bar = 250
µm (C).
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Tg(Hemgn:mCherry) zebrafish reveal the functions of the two Hemogen promoters
To determine the tissue-specific regulatory profiles of the two Hemogen
promoters, we generated transgenic zebrafish embryos
[Tg(Hemgn:mCherry,myl7:EGFP)] in which the mCherry reporter was controlled by the
putative promoter elements (Fig. 6). The dual promoter, P1 (2,248 bp), spanned the
upstream, non-coding region to the Hemogen start codon and contained both CNEs.
Transgenic fish were outcrossed to wild-type TU zebrafish, and offspring with the
strongest mCherry expression were selected as founders. In the early embryo, the P1
transgene drove expression of mCherry in primitive blood cells of the ICM and the PBI
(20 hpf; Fig. 6B) and in primitive erythrocytes in circulation (Movie 1). Between 2-8 dpf,
mCherry was expressed strongly throughout the pronephric ducts (Fig. 6C) and was
present in the proximal convoluted tubule at 72 hpf (Fig. 6D). In adult transgenic fish,
the head and trunk kidneys were positive for the reporter (Fig. 6H), as were Sertoli cells
surrounding the seminiferous tubules of the testes (Fig. 6I). Therefore, the ~2.2 kb P1
transgene contained all of the regulatory elements necessary to recapitulate Hemogen
expression (Fig. 6B-I). We note that the dual promoter did not confer detectable ovarian
or neural expression, which may require more distal sequences.
We found that the same expression profile was driven by the endogenous
Hemogen promoter in embryonic zebrafish by using CRISPR/Cas9 technology to insert
the mCherry gene (containing a polyadenylation motif) two codons downstream of, and
in frame with, the Hemogen start codon (See Methods, Fig. S3A,C). Homology-directed
integration of the transgene, confirmed by sequencing of the locus, produced mCherry+
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cells in the CHT and in the kidney in 10% (n = 15/150) of embryos at 3 dpf (Fig. S3B)
and at a lower frequency in circulating RBC (n = 3/150, data not shown).
To characterize hematopoietic cell lineages that express Hemogen, the P1
reporter plasmid was injected into embryos of Tg(CD41:EGFP)Ia2Tg or
Tg(Lcr:EGFP)cz3325Tg zebrafish, which have been used to track hematopoietic
progenitors (Lin et al., 2005) and primitive and definitive erythrocytes (Ganis et al.,
2012), respectively. We did not observe mCherry expression in the AGM, in the thymus,
or in CD41+ HSPCs colonizing the thymus or pronephros (Bertrand et al., 2008).
However, the reporter was strongly expressed in a subset of LCR+ erythroid and
CD41+ myeloid-biased progenitors in the CHT (Fig. 6E,F), a tissue that supports
myelopoiesis (Gekas and Graf, 2013; Medvinsky et al., 2011). This lends support to
previous findings that Hemogen is a marker and promoter of myeloerythroid, but not
lymphoid, lineages (Li et al., 2007; Lu et al., 2001). Maturing mCherry+ primitive
progenitors peaked in brightness just prior to leaving the caudal plexus and entering
circulation at 72 hpf (observed by time-lapse imaging; data not shown). However,
mature definitive erythrocytes expressed little mCherry in adult transgenics (Fig. 6G),
which supports prior observations that Hemogen expression is limited to primitive
erythrocytes and immature definitive progenitors (Lu et al., 2001).
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Figure 6. Promoter elements have distinct roles in driving hematopoietic, renal,
and testicular expression of Hemogen in transgenic Tg(Hemgn:mCherry)
zebrafish. (A) Schematic of the zebrafish Hemogen gene. CNEs, black; coding exons,
white; transcription initiation sites, bent arrows. Three Tg(Hemgn:mCherry,myl7:EGFP)
transgenes driven by portions of the Hemogen promoter were transfected into one-cell
TU embryos by Tol2 transposase-mediated insertion. Numbers indicate length of
promoter elements and arrows show gene direction. (B) 20 hpf. P1 transgene
expression in the peripheral blood island (PBI). (C) 72 hpf. P1 transgene expression in
the pronephric ducts (PD). (D) 5 dpf. P1 transgene expression in the proximal
convoluted tubule (PCT). (E,F) 72 hpf. colocalization of mCherry and EGFP in
progenitors in the CHT of Tg(Hemgn-P1:mCherry,Lcr:GFP) or Tg(Hemgn-
P1:mCherry,CD41:EGFP) zebrafish. (G) Transgene expression in mature erythrocytes
from adult zebrafish. (H) Transgene expression in adult head kidney (HK), trunk kidney
(TK), and tail kidney (T) near the EGFP+ heart (H). (I) Transgene expression in adult
Sertoli cells (Se) that surround the seminiferous tubules (ST). (J) Proportion of embryos
expressing transgenes P1, P2, or P3 in ICM, kidney, CHT, and circulating primitive
erythrocytes (RBC). Scale bars = 100 µm (B,D-F,I); 500 µm (C,H); 25 µm (G).
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Hemogen promoters have different functions in primitive and definitive erythropoiesis in
zebrafish
We evaluated the separate and combined contributions of the two Hemogen
promoters, including CNE1 or CNE2, to the observed tissue-expression profiles by
injecting wild-type embryos with one of three Tg(Hemgn:mCherry,myl7:EGFP) reporter
constructs in which mCherry expression was driven: 1) by the dual promoter (P1); 2) by
a 2-kb fragment (P2) containing the distal promoter including CNE1; or 3) by a 188-bp
fragment (P3) containing the proximal promoter including CNE2 (Fig. 6A). Transgenic
embryos were screened for EGFP+ hearts, and mCherry transcription was confirmed by
RT-PCR and sequencing.
mCherry fluorescence was examined in four cell types: 1) erythroid progenitors in
the ICM at 1 dpf; 2) primitive erythrocytes in the peripheral blood at 3 dpf; 3) erythroid
progenitors in the CHT at 3 dpf; and 4) renal cells of the kidney tubules at 3 dpf. Fig. 6J
shows that the dual promoter (P1) supported strong expression of the mCherry reporter
in erythroid cells of the ICM and peripheral blood (RBC), in the CHT, and in renal cells
of the kidney. By contrast, the distal promoter (P2 construct) containing CNE1 failed to
drive reporter expression in these tissues. Finally, the proximal promoter (P3 construct)
containing CNE2 alone produced strong expression of the reporter in the CHT and in
kidney cells but was not active in cells of the ICM and peripheral blood. Together, these
results indicate that the proximal promoter containing CNE2 is necessary and sufficient
to drive expression in definitive hematopoiesis and in the kidney, whereas the full 2.2-kb
sequence including both promoters and CNEs is required in primitive erythropoiesis.
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Figure 7. Morpholino targeting of Hemogen inhibits erythropoiesis in embryonic
zebrafish. Embryos were injected with 2-4 ng antisense MO targeted to the first 25
coding nucleotides of Hemogen. (A-B) O-dianisidine staining of erythrocytes was
decreased in morphants (MO) relative to wild-type embryos (WT) or embryos rescued
with 500 pg synthetic Hemgn mRNA (zHem) at 24 hpf. (ANOVA, Tukey post hoc test, P
< 0.001). Live wild-type (C), Hem1 MO-injected (D), and Hem1mm mismatch MO-
injected (E) Tg(Lcr:EGFP)cz3325Tg embryos at 20 hpf. Morphants showed decreased
EGFP expression in the ICM compared to the wild-type and mismatch MO controls. Live
wild-type (F), Hem1 MO-injected (G), and Hem1mm MO-injected (H) embryos at 72 hpf.
Morphant embryos have fewer EGFP+ cells in circulation compared to the two controls.
The dorsal aortas of embryos (insets above F-H) were magnified 20x to permit
quantitation of EGFP+ erythrocytes. Background red (D,G) and green (E,H)
fluorescence was generated by the fluorescent labels on the MOs. (I) In vivo flow
quantitation of EGFP+ erythrocyte concentrations between 3-6 dpf in Hem1-injected (n
= 9,7,7,7), Hem1mm-injected (n = 13,14,11,11), and uninjected (n = 5,10,10,9)
embryos. Data shown as means ± s.e.m. (* P ≤ 0.05, ** P ≤ 0.001, ANOVA, Tukey-
Kramer post hoc test). Scale bars = 500 µm (A-F) 100 µm (inset).
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Morpholino knock-down of Hemogen protein expression partially disrupts erythropoiesis
in zebrafish
To perturb Hemogen function in zebrafish, we first injected wild-type zebrafish
embryos at the one cell stage with an antisense morpholino oligonucleotide (MO), Hem-
1, targeted to the translation start codon of the Hemogen transcript (Hem1). MO
treatment significantly reduced Hemogen protein levels by 19% at 33 hpf (Student’s t-
test, P < 0.05; Fig. S4A,B) and steady-state levels of βe1-globin mRNA at 3 dpf
(Student’s t-test, P < 0.05; Fig. S4C). At 24 hpf, 61% of morphants were anemic
compared to 35% of uninjected zebrafish (Fig. 7A,B). Red cell levels were restored to
wild-type by co-injection of the MO with 500 pg of synthetic zebrafish Hemogen mRNA
containing silent mutations in the MO target site. Both the uninjected and rescue
treatments differed significantly from the MO treatment (ANOVA, Tukey post hoc test, P
< 0.001; Fig. 7A).
We used Tg(Lcr:EGFP) cz3325Tg zebrafish to visualize the red blood cell population
in Hem1-treated morphants from 0-6 dpf. Control embryos were injected with a 5-bp
mismatch MO (Hem1mm) or were uninjected. At 20 hpf, EGFP+ erythrocytes appeared
to be reduced in the ICM/PBI of 75% of Hem1 morphants (n = 56) but not in mismatch
or uninjected control embryos (n = 14 and 63, respectively) (Fig. 7C-E). At 2 dpf,
morphant embryos had few erythrocytes in circulation compared to controls (Fig. 7F-H,
Movie 2). Using quantitative in vivo flow analysis (Fig. 7I), we found that morphant
embryos at 3-5 dpf had fewer than 50% of the circulating EGFP+ erythrocytes as the
uninjected and Hem1mm-injected controls, whereas the controls did not differ
statistically from each other (ANOVA, Tukey-Kramer post hoc test, P < 0.05).
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A conserved C-terminal domain in Hemogen is required for hematopoiesis and prevents
apoptosis in embryonic tissues
The function of the putative C-terminal transactivation domain of zebrafish
Hemogen was investigated using CRISPR/Cas9 mutagenesis. We generated zebrafish
lines with mutations in the conserved region near the end of the third coding exon of
Hemogen, immediately downstream of the TAD motif (Fig. 8A-D, Fig. S1). Founders
(F0) were out-crossed to wild-type TU zebrafish and mutant alleles were genotyped in
the F1 generation by high resolution melting analysis and by sequencing the locus (Fig.
8E, Fig. S1). One line, Hemgnnuz2, had a 5-bp deletion (Δ5) that produced a frameshift
mutation, thereby introducing a premature stop codon (Fig. 8E, Fig. S1). PolyA-tailed
transcripts of the Δ5 allele were detected at equivalent steady-state levels relative to the
wild-type allele in peripheral blood from individual adult heterozygotes (Fig. 8F).
Western blot analysis revealed, however, that truncated Hemogen protein was almost
undetectable in peripheral blood from single heterozygous adults (data not shown) and
in pooled 33-hpf embryos from a heterozygous in-cross (Fig. 8G). Therefore, if the
truncated Hemgnnuz2 transcripts were translated, then the protein must have been
rapidly degraded. The second line, Hemgnnuz4, contained an in-frame 12-bp deletion
(Δ12), which deleted an acidic cluster (EEED) in the last repeat that is conserved in
teleost species (Fig. S1). Hemogen protein was detected in the blood of homozygous
Δ12 adults by Western Blot (data not shown).
To evaluate the effects of the mutant Hemogen alleles on erythropoiesis during
development, we examined embryos from mutant crosses by microscopy and
genotyped them between 20-48 hpf (Fig. 8A-C) - mutant genotypes were recovered
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near the expected Mendelian ratios (Fig. S5A), but homozygous Δ5 hemgnnuz2 mutants
could not be raised to adulthood. To classify the mutants, we assessed the relative
numbers of blood cells and relative concentrations of hemoglobin beginning at 2 dpf
(Ransom et al., 1996). Embryos from a heterozygous in-cross were scored for
hypochromic blood (paler blood) and decreased numbers of circulating cells on the yolk
sac and in the vasculature. Erythrocyte levels were reduced to about 25-75% of normal
levels in frameshift Hemgnnuz2/+ mutants (n = 8) at 24 hpf compared to wild-type siblings
(n = 7) (Fig. 8C). At 48 hpf, 59% of heterozygous (n = 49) and 50% of homozygous (n =
12) Hemgnnuz2 mutants had reduced numbers of circulating erythrocytes (Fig. 8H, Movie
3) and homozygotes could be distinguished by their more severe anemia. Comparable
numbers of anemic individuals were observed for heterozygotes and homozygotes of
the Δ12 Hemgnnuz4 allele – 64% (n = 25) and 60% (n = 10), respectively (Fig. 8H). In all
cases, the proportion of anemic mutant embryos was significantly different from that for
wild-type (* P ≤ 0.05, ** P ≤ 0.005, Chi square).
Erythrocyte levels in adult mutants were partially suppressed in heterozygotes.
Hemgnnuz2/+ and Hemgnnuz4/+ adults gave average erythrocyte counts of 2.2 ± 1.0 x 106
cells µl-1 and 2.1 ± 0.8 x 106 cells µl-1, respectively, whereas wild-type zebrafish had 4.3
± 1.0 x 106 cells µl-1 (Fig. 8J,K). Homozygous Δ12 Hemgnnuz4 gave average erythrocyte
counts of 2.2 ± 1.2 X 106 cells µl-1 (Fig. 8K). Taken together, the erythroid defects of
embryonic and adult zebrafish carrying the CRISPR-generated mutant alleles support
the conclusion that the conserved C-terminus of Hemogen functions as a TAD, but the
mechanism of action of these mutations remains to be determined.
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Both the Δ5 and Δ12 mutant Hemogen alleles also caused mild to severe
developmental defects in the nototchord and the trunk of heterozygotes and
homozygotes (Fig. 8A-B, Fig. S5B). Embryos had kinked notochords and exhibited
increased cellular refractility consistent with apoptotic cell death. Elevated apoptotic cell
death was apparent in Hemgnnuz2/+ mutants as detected by staining with acridine orange
(Fig. S5C). Apoptosis occurred throughout the embryo, including sites of embryonic
hematopoiesis. Nevertheless, viable heterozygotes for both alleles could be raised to
adulthood; they were slightly smaller than wild-type siblings (Fig. 8I). Impaired growth
was significant in homozygous Δ12 Hemgnnuz4 adult mutants (Student’s t test, P = 0.04,
N = 3, Fig. S5D,E).
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Figure 8. CRISPR/Cas9 mutagenesis of the third exon of zebrafish Hemogen
reduces primitive and definitive erythropoiesis. Embryos were injected with Cas9
mRNA and a guide RNA to establish lines with mutations in exon three of zebrafish
Hemogen. (A) 20-hpf. Representative wild-type and mutant siblings with notochord
defects (arrow) (B) 48 hpf. Mutant Δ12 embryos with an in-frame deletion showing
kinked notochords (arrow). (C) 24 hpf. Wild-type and Δ5/+ mutant embryos stained with
diaminofluorene. Production of erythrocytes was reduced in heterozygotes. (D)
Schematic of CRISPR/Cas9 target in the third exon (red arrowhead) of zebrafish
Hemogen. (E) Sequences of founder mutations aligned at the CRISPR target site: Δ5
(Hemgnnuz2); Δ12 (Hemgnnuz4). The sequence traces show the Δ5 and Δ12 mutant
alleles. PAM, blue and underlined; Δ, deletions (highlighted in red). (F) Relative
expression of wild-type and Δ5 transcripts in blood from single adult, heterozygous
Hemgnnuz2/+ mutants determined by qRT-PCR with allele specific primers. Three
biological replicates were normalized to β-actin. Error bars represent the standard
deviation. (G) Western blot of Hemogen in pooled 33 hpf wild-type embryos or pooled
embryos from a Δ5 Hemgnnuz2/+ heterozygous in-cross. We calculated that the protein
would run 6.5 kDa above its molecular weight at 28.5 kDa because of its high acidic
composition (Guan et al., 2015). Arrows show the calculated sizes of wild-type and
truncated alleles. (H) Proportion of genotyped mutants and wild-type sibling embryos at
2 dpf that were anemic (black) or phenotypically normal (white) (* P ≤ 0.05, ** P ≤ 0.005,
Chi square). (I) Wild-type and mutant zebrafish heterozygous for the Δ5 and Δ12
alleles. (J) Red blood cells from adult Hemgnnuz2/+ mutant zebrafish and wild-type
siblings. (K) Erythrocyte counts in adult heterozygous Hemgnnuz2 (Δ5, n = 12),
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heterozygous Hemgnnuz4 (Δ12, n = 4) mutants, homozygous Hemgnnuz4 (Δ12, n = 2)
mutants, and wild-type (n = 9) siblings (* P ≤ 0.05, ANOVA, Tukey post hoc test). Scale
bars = 500 µm (A-C); 50 mm (I); 20 µm (J)
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DISCUSSION
The zebrafish is a compelling model for understanding the pleiotropic functions of
Hemogen in the context of vertebrate development. Our results show that zebrafish
Hemogen is considerably smaller than its human ortholog, a distinction true for teleost
and mammalian Hemogens in general. Hemogen is expressed in multiple zebrafish
tissues from the early embryo to the adult under the control of at least two promoters.
Both primitive and definitive erythropoiesis are affected by depletion of Hemogen and by
targeted mutation of a putative, C-terminal TAD. The transgenic and mutant zebrafish
lines that we have generated will contribute to a mechanistic understanding of this
important transcription factor.
Hemogen ‒ small or large, it’s built of related modules and has a conserved role in
erythropoiesis
We show that the divergent Hemogens of zebrafish and human are largely, but
not entirely, built of 21-25 residue repeats; the number of repeats largely determines
protein size. The repeat consensus sequences are distinct, but they appear to have
evolved from an 8-10 amino acid core motif (Fig. S2). Although all repeats are acidic
(Figure S2), the terminal repeat of each Hemogen is particularly so (> 38% Asp and Glu
for zebrafish, > 29% for human), and these repeats contain TAD motifs. Together, these
features suggest that Hemogens possess flexible, intrinsically disordered TADs, as is
true of many transcription factors (e.g., p53, HIF-1α, NF-κB, etc). The multivalent
structure of Hemogen provides opportunities for cooperative binding to single or multiple
protein partners, including P300 (Zheng et al., 2014).
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Hemogen interacts with a variety of proteins to stimulate the transcription of
genes involved in terminal erythroid differentiation and other processes. In humans,
Hemogen contributes to transcription of erythroid genes in part by recruiting P300 to
acetylate and activate Gata1 (Zheng et al., 2014). Our results show that nonsense (Δ5)
and deletion (Δ12) alleles of Hemogen vicinal to the zebrafish TAD motif cause
significant reductions of erythrocyte levels in embryos and adults. The Δ12 allele may
be hypomorphic, but we have not determined whether the protein that is expressed has
reduced activity.
Hemogen – targeted mutation of the acidic C-terminus impairs erythropoiesis, but not
completely
Our CRISPR-generated zebrafish mutant lines show that nonsense (Δ5) and
deletion (Δ12) alleles of Hemogen caused a decrease in erythrocyte levels in embryos
and adults. However, these phenotypes were incompletely penetrant ‒ in both
heterozygous and homozygous Hemogen mutants the proportion of anemic embryos
was 50-65%, compared to 20% for wild-types. If Hemogen were essential for
erythropoiesis, one would anticipate an erythroid-null phenotype for homozygous
mutants, as observed for the Gata1 mutant, vlad tepesm651 (Lyons et al., 2002). Rather,
the Hemogen phenotype resembles the variable reduction of red cells in zebrafish
zinfandel (zinte207) mutants that harbor a mutation in a regulatory region at the globin
locus (Ransom et al., 1996), a known target of both Hemogen and Gata1 transcription
factors (Zheng et al., 2014). Loss of Hemogen in zebrafish contributes to decreased
expression of Embryonic beta-globin (Fig. S4), which may explain the hypochromic
state of Hemogen mutants.
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The most plausible explanation for the incomplete penetrance of anemia in
Hemogen mutants is the phenomenon of genetic compensation, which may occur when
genes are knocked out as opposed to knocked down (El-Brolosy and Stainier, 2017;
Rossi et al., 2015). Although the mechanisms are poorly understood, genetic
compensation entails changes in gene expression (e.g., upregulation of paralogous
genes or functionally related genes) that at least partially offset the phenotype caused
by the mutant protein. Compensation through elevated expression of other erythroid co-
activators is an attractive possibility that might maintain erythrocyte production in
Hemogen mutants. The functional loss of Hemogen could be mitigated by Gata1
homodimerization and/or by direct recruitment of CBP/P300, both of which enhance
Gata1 activity (Ferreira et al., 2005; Nishikawa et al., 2003).
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Similar design and regulation of Hemogen and Gata1 genes
Comparison of the expression of Hemogen and of Gata1 throughout zebrafish
development reveals a remarkable degree of overlap in tissue and cellular specificity.
For example, Gata1 mRNA appears in cells of the LPM at the two-somite stage (Detrich
et al., 1995), immediately prior to the onset of Hemogen expression at ten somites.
Furthermore, Hemogen and Gata1 are co-expressed in primitive erythrocytes and
definitive hematopoietic progenitors (Ferreira et al., 2005; Lu et al., 2001), in Sertoli
cells (Nakata et al., 2013; Wakabayashi et al., 2003), and at the midbrain-hindbrain
boundary (Volkmann et al., 2008). Interestingly, both Hemogen and Gata1 genes
possess hematopoietic- and testis-specific promoters (Wakabayashi et al., 2003). The
temporal and spatial co-incidence of Hemogen and Gata1 expression almost certainly
results from their similar regulatory architectures and also through regulatory crosstalk.
Our results and studies conducted by others (Ding et al., 2010; Yang et al., 2006; Zheng
et al., 2014) indicate that reciprocal transcriptional activation of Hemogen and Gata1
may form a positive feedback loop that drives erythropoiesis.
Strikingly, the two CNEs of Hemogen are organized like, and have the same
functions as, the distal and proximal enhancers of the Gata1 gene (McDevitt et al.,
1997; Onodera et al., 1997; Suzuki et al., 2009). The proximal Gata1 promoter functions
exclusively in definitive erythropoiesis (McDevitt et al., 1997), as does CNE2 of
zebrafish Hemogen. In contrast, transcription of Gata1 in primitive erythrocytes requires
both the proximal promoter and a distal enhancer comparable to Hemogen CNE1
(McDevitt et al., 1997). Fig. S6 presents a model for the transition from primitive to
definitive hematopoiesis based on chromatin looping at the Hemogen locus We propose
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that the transition from primitive to definitive erythropoiesis involves a switch from a loop
conformation to a linear conformation, mediated by the Gata1/Ldb1-complex at
erythroid transcription factories (Osborne et al., 2004; Schoenfelder et al., 2010). This
model may also apply to the Gata1 enhancer, which is another known target of the
Ldb1-complex (Love et al., 2014). The zebrafish lines produced in this study may help
clarify the cell-specific Hemogen expression profile driven by different Gata1-containing
complexes and the functions of Hemogen in different cell types.
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MATERIALS AND METHODS
Fish husbandry
Wild-type (SAT, AB, TU) zebrafish (Danio rerio), the transgenic lines
Tg(Lcr:EGFP)cz3325Tg (Ganis et al., 2012) and Tg(CD41:EGFP)Ia2Tg (Traver et al., 2003),
and the mutant vlad tepesm651 (Lyons et al., 2002) were all generously provided by Dr.
Leonard I. Zon (Howard Hughes Medical Institute and Harvard Medical School, Boston).
Animal procedures were carried out in full accordance with established standards set
forth in the Guide for the Care and Use of Laboratory Animals (8th Edition). The animal
care and use protocol for live zebrafish embryos was reviewed and approved by
Northeastern University’s Institutional Animal Care and Use Committee (Protocol No.
15-0207R). The animal care and use program at Northeastern University has been
continuously accredited by AAALAC Int. since July 22, 1987, and maintains the Public
Health Service Policy Assurance number A3155-01.
Cloning and sequence analysis of zebrafish Hemogen cDNAs
Total RNA was isolated from wild-type AB zebrafish embryos and adult tissues
(kidney, blood, brain, ovary, intestine) using TRI reagent (Sigma, T9424) and the
Ribopure Kit (Ambion, AM1924). Total cDNA was produced from mRNA using M-MuLV
reverse transcriptase (NEB, M0253S) and an oligo(dT)23 primer. Hemogen cDNA was
amplified by PCR from total cDNA with 1 µM primers (Table S1) – the amplification
program was 35 cycles of 98°C for 10 s, 57°C for 10 s, and 72°C for 30 sec. PCR
products were cloned into the pGEM-T Easy vector (Promega, A1360), plasmids were
transformed into 5-α competent cells (New England Biolabs, C2987H), recombinant
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plasmids were identified by blue/white screening and purified with the Wizard Plus SV
Miniprep Kit (Promega A1330), and inserts were sequenced by GeneWiz.
Bioinformatic comparison of vertebrate Hemogen genes and Hemogen proteins
We utilized the murine gene nomenclature for comparing orthologs from different
vertebrate species. We used Blast+ (Altschul et al., 1990) to identify Hemogen in the
zebrafish genome (assembly GRCz11) (Howe et al., 2013). Chromosomal synteny
comparisons were performed using the Synteny Database with a sliding window of 200
genes (Catchen et al., 2009) and Ensembl Genomes v74 (Kersey et al., 2016).
Hemogen promoter alignments were obtained from whole genome alignments for 10
teleost species (ENSEMBL v74) (Yates et al., 2016). Transcription factor binding motifs
were predicted using the program ConTra with the default similarity matrix of 0.75
(Broos et al., 2011). Transcription start sites were predicted using NNPP v2.2 with a
score cutoff of 0.98 (Reese, 2001).
Protein domains in zebrafish were identified using annotated human Hemogen
(Yang et al., 2001), or they were predicted using HHpred (Soding et al., 2005) and the
Conserved Domain Database (CDD) (Marchler-Bauer et al., 2015). Peptide repeats
were predicted with RADAR (Heger and Holm, 2000). The 9aaTAD Prediction Tool was
first used to predict transactivation domain (TAD) motifs, starting with low stringency
DFx repeats (Piskacek et al., 2016). These were then culled by ϕϕxxϕ or ϕxxϕϕ criteria,
where ϕ is a bulky hydrophobic motif (Dyson and Wright, 2016). We refer to the latter
five amino acid consensus sequences as “TAD motifs,” in contrast to larger, functionally
defined “transactivation domains” (TADs). Ab initio tertiary structure models were
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created for zebrafish and human Hemogen proteins with I-Tasser (Yang et al., 2015)
based on the X-ray structure for the secretory component of Immunoglobulin A
(PDB:3chnS), which was the best of ten predicted structural templates determined by
LOMETS (Wu and Zhang, 2007). The 3D models were superimposed using TM-align
(Zhang and Skolnick, 2005) and Geneious version R10 (Kearse et al., 2012).
MO knock-down of Hemogen in zebrafish and rescue of the morphant phenotype
The antisense MO Hem1 (5’-TCTCTTTCTCCAACGGGTCTTCCAT-3’), which
targets the first 25 base pairs of the zebrafish Hemogen open reading frame, was
designed according to the manufacturer’s instructions (Gene Tools, LLC). The control
MO (Hem1mm; 5’-TCTgTTTgTCCAtCGGcTCTTCgAT-3’) targeted the same sequence
but contains five mismatched bases to prevent efficient binding to Hemogen mRNA.
MOs were labeled with lissamine or fluorescein so that the quality of injections could be
monitored by fluorescence microscopy. MOs were injected (2-8 ng) into embryos at the
single-cell stage using a PLI-100 Picoinjector (Medical Systems Corporation, 65-0001)
and a micromanipulator (Narishige, MN-151). Injected embryos were sampled from 0-6
dpf for subsequent analyses.
Rescue of the morphant phenotype was tested by co-injection of the Hem1 MO
with 500 pg synthetic zebrafish Hemogen mRNA transcribed from a zebrafish Hemogen
cDNA cloned into pGem-T Easy (Promega). Primers (Table S1) introduced five silent
mutations within the MO target site. The clone was digested with Spe1, and mRNA was
transcribed, capped, and polyadenylated in vitro using the mMessage T7 kit (Ambion,
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AM1340) and the Poly(A) Tailing Kit (Ambion, AM1350). mRNA was purified with the
MEGAclear kit (Ambion).
In-situ hybridization
The spatial and temporal patterns of expression of selected genes were analyzed
by whole-mount in situ hybridization (WISH) of zebrafish embryos following standard
protocols (Jacobs et al., 2011). These methods were adapted to evaluate Hemogen
expression in tissues, peripheral blood smears, and pronephric kidney prints prepared
from euthanized adult fish [200 mg L-1 tricaine methane sulfonate (MS222; Sigma-
Aldrich, 886862)] (Detrich and Yergeau, 2004; Gupta and Mullins, 2010). For sectioning,
embryos and tissues were embedded in a solution containing 0.25 g gelatin, 30 g
albumin, 22 g sucrose, 2.5% glutaraldehyde (v/v) per 100 ml phosphate buffered saline
(PBS). Sections were cut with a vibrating blade microtome (Leica, VT1000S).
Digoxigenin-labeled antisense and sense RNA probes were transcribed from zebrafish
cDNA clones using the DIG RNA Labeling Kit (Roche Diagnostics, 11175025910).
Indirect Immunofluorescence
Zebrafish embryos were fixed in 4% paraformaldehyde (PFA) at 48 hpf. Embryos
were incubated with 1:1000 rabbit anti-Hemogen primary antibody (Aviva,
ARP57794_P050) followed by 1:1000 goat anti-rabbit IgG Alexafluor 488 secondary
antibody (Life Technologies, A11034) as previously described (Westerfield, 2000). The
specificity of the Hemogen antibody was validated both by Clontech and by our
laboratory by Western blotting of zebrafish protein extracts.
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Hemoglobin staining
To detect red blood cells in circulation, embryos were stained with o-dianisidine (Iuchi
and Yamamoto, 1983) or diaminofluorene (McGuckin et al., 2003).
Western blotting
Total embryonic protein was prepared for sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) from dechorionated, 33-hpf embryos (n =80) by
homogenization in lithium dodecyl sulfate (LDS) Bolt buffer (Life Technologies, B007)
and NuPAGE reducing agent (Life Technologies, NP0009) using a pestle and
microcentrifuge tube (USA Scientific, 1415-5390). Samples were boiled for 3 min and
centrifuged at top speed in a centrifuge for 2 min. Aliquots (15 µg) were electrophoresed
on a 4-12% SDS polyacrylamide gel, and the separated proteins were transferred to a
polyvinylidene difluoride (PVDF) membrane with the iBlot system (Life Technologies,
IB21001). Membranes were blocked in maleic acid blocking buffer (2% Roche blocking
reagent, 2% BSA, 0.2% heat treated goat serum, 0.1% Tween-20) for 1 hour at room
temperature and then incubated overnight at 4°C with 1:1000 rabbit anti-Hemogen
(Aviva, ARP57794_P050) or with 1:1000 mouse anti-GAPDH (Aviva, OAE00006)
antibodies. Membranes were washed in tris-buffered saline and Tween 20 (TBST) and
incubated for 2 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG
(H&L) (Aviva, ASP00001) or HRP-conjugated goat anti-mouse IgG (H&L) (Aviva,
OARA04973), respectively. Bound antibodies were detected with the Amersham ECL
Western Blotting Analysis System (GE Healthcare, RPN2106) on CL-X Posure film
(Thermo Scientific,34091).
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Tol2 generation of Tg(Hemgn:mCherry) zebrafish
To identify the regulatory elements that drive Hemogen expression in zebrafish,
three different Tg(Hemgn:mCherry) reporter plasmids were created using Gateway
Cloning Technology (Invitrogen, 11791020) (Hartley et al. 2000). First, the proximal
Hemogen promoter (~2.2 kb) was amplified from wild-type SAT zebrafish using 1 µM
primers (Table S1). The promoter sequence spanned the upstream, non-coding region
before, but not including, the Hemogen translation start codon. The promoter was
cloned between KpnI/SpeI restriction sites in the p5e-MCS vector (Tol2kit, #228) using
the Tol2kit vector system (Kwan et al., 2007) to generate the entry clone, p5e-Hemgn-1.
The resulting plasmid was digested with NaeI/KpnI or NaeI/SpeI to remove each of two
conserved non-coding elements (CNE1 or CNE2) from the promoter. Each new
construct was blunt-ended with Q5 Hot Start High-Fidelity 2x Master Mix (NEB) and
religated with T4 DNA Ligase (NEB) to create p5e-Hemgn-2 and p5e-Hemgn-3. Each of
the three entry clones were cloned in front of the mCherry gene within the
pDestTol2CG2 destination vector (Tol2kit, #395). The pCS2FA-transposase clone
(Tol2kit, #396) was digested with PmeI, and Tol2 transposase mRNA was transcribed,
capped, and polyadenylated in vitro using the mMessage SP6 kit (Ambion, AM1340)
and the Poly(A) Tailing Kit (Ambion, AM1350). mRNA was purified by precipitation using
2.5 M LiCl. Transposase mRNA (37 ng µL-1) and each of the
Tg(Hemgn:mCherry,myl7:EGFP) expression clones (25 ng µL-1) were co-injected into
one-cell wild-type zebrafish embryos. Founders were raised and out-crossed to wild-
type TU zebrafish for two generations.
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CRISPR/Cas9 generation of transgenic and mutant zebrafish
Optimal targets for CRISPR-Cas9 mutagenesis were identified within the first and
third exons of zebrafish Hemogen using the program CHOPCHOP (Labun et al., 2016;
Montague et al., 2014). The templates for multiple small guide RNAs were produced by
a cloning-free method as previously described (Table S1) (Hruscha et al., 2013; Talbot
and Amacher, 2014). Guide RNAs were transcribed with the T7 MaxiScript Kit (Ambion,
AM1312) and purified by LiCl precipitation.
A donor construct for homology directed repair was created containing the
mCherry gene and polyadenylation signal flanked by 199 bp and 253 bp homology arms
that were PCR amplified from the sequence surrounding exon 1 of Hemogen from wild-
type AB zebrafish (Table S1). The homology arms and mCherry gene were PCR
amplified with primers that added AvrII and ClaI restriction sites, ligated, and cloned into
the pGem-T Easy vector (Promega). Tg(Lcr:EGFP)cz3325Tg embryos were co-injected at
the single-cell stage with EcoRI linearized donor plasmid (25 ng µl-1), two exon-1
targeting guide RNAs (150 ng µl-1), and Cas9 mRNA (300 ng µl-1) (Trilink). Embryos
were checked for fluorescence between 1 and 3 dpf. To confirm integration, the locus
was PCR amplified with internal and external primers (Table S1) and cloned into the
pGem-T Easy vector for sequencing.
Wild-type (TU) embryos were co-injected with a guide RNA (150 ng µl-1) targeting
exon 3, Cas9 mRNA (300 ng µl-1), and mCherry mRNA (30 ng µl-1) to identify successful
injections. Embryos were raised and adults were tail-clipped for haplotyping by high-
resolution melting analysis (HRMA) as previously described (Talbot and Amacher,
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2014). PCR amplification was run using 1 µM primers (Table S1) with PowerUp SYBR
MasterMix (Applied Biosystems, A25742) on a QuantStudio 3 Real-time PCR system
(ThermoFisher, A28137). Founder mutants were outcrossed to wild-type (TU) fish. The
offspring were raised and mutations were characterized by HRMA and sequencing of
the locus.
Imaging of zebrafish embryos
Fixed embryos were mounted in 80% glycerol and imaged with a dissecting
microscope (Nikon, SMZ-U) and a CCD digital camera (Diagnostic Instruments,
SPOT32). Live embryos were embedded in 0.1% agarose in embryo medium (EB) with
0.01% tricaine and imaged with an epifluorescence-equipped microscope (Nikon,
Eclipse E800). Movies (0.01 sec interval) and time-lapse images (1 min interval) were
obtained using a Photometrics Scientific CoolSNAP EZ camera and NIKON NIS-
Elements AR 4.20 software. Methods for in vivo flow analyses were adapted to quantify
fluorescently labeled red blood cells in MO-injected Tg(Lcr:EGFP)cz3325Tg zebrafish
(Schwerte et al., 2003; Zeng et al., 2012). Briefly, 100 frame videos were taken set at a
500 μs exposure time with no delay. The field of view (20x) was centered on the dorsal
aorta adjacent to the cloaca. The summed maximum intensity images of all frames were
used to create “casts” of the dorsal aorta and the average volume was calculated
assuming cylindrical vasculature. EGFP+ cells were converted to binary objects (6.66
µm diameter, contrast 180) and counted within the region of interest.
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qRT-PCR
RNA was purified from adult zebrafish tissues or 10-30 pooled embryos at 3 or 4
dpf in TriZol (Sigma-Aldrich, T9424) using the PureLink RNA purification Kit (Ambion).
DNase treated RNA was reverse transcribed with a polyT(23) primer using Protoscript II
RT-PCR kit (New England Biolabs, M0368S). Target genes were amplified in triplicate
from cDNA by qRT-PCR with 1 µM primers (Table S1). Standard curves were
generated to confirm primer efficiencies. Target gene expression was normalized to
beta-actin for comparison by the ΔΔCt method. Three or four biological replicates were
used for each treatment for statistical comparisons.
Statistical analyses
Data were analyzed as means ± s.e.m. or means ± s.d. as noted. Statistical tests
applied to the results are provided with each experiment. Differences with a p-value ≤
0.05 were considered significant.
GenBank accession numbers
Zebrafish Hemgn isoform 1, JZ970258; zebrafish Hemgn isoform 2, JZ970260;
zebrafish Hemgn isoform 3, JZ970259; and zebrafish Hemgn isoform 4, JZ970257.
Zebrafish ZFIN IDs
Transgenic construct Tg(hemgn:mCherry,myl7:EGFP), ZDB-TGCONSTRCT-170726-1;
zebrafish line nuz1Tg, ZDB-ALT-170726-1; zebrafish line hemgnnuz2, ZDB-ALT-170726-
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2; zebrafish line hemgnnuz3, ZDB-ALT-170726-3; zebrafish line hemgnnuz4, ZDB-ALT-
170726-4
Acknowledgements
We thank Dr. Leonard Zon and Christian Lawrence at Children’s Hospital in Boston for
providing zebrafish and plasmids. We thank Dr. John Postlethwait, Dr. Leonard Zon,
and Christopher Wells for helpful discussion. We thank Dr. Johanna Farkas and Carly
Ching for their technical contributions. We thank Dr. Leonard Zon, Dr. Yi Zhou, and
colleagues at Boston Children's Hospital, Stem Cell and Regenerative Biology
Department, Harvard Medical School and Harvard University for providing ATAC-seq,
ChIP-seq, and DNase I-seq datasets.
Funding
This research was supported by a Graduate Research Grant from the College of
Sciences and the Office of the Vice Provost of Graduate Studies at Northeastern
University awarded to MJP and by NSF grants PLR-1247510 and PLR-1444167
awarded to HWD. This is contribution number 380 from the Northeastern University
Marine Science Center.
Authors’ Contributions
MJP designed and carried out experiments, created expression plasmids, did in vivo
flow analysis, generated zebrafish lines, and analyzed and interpreted results. SKP
created expression plasmids and contributed to MO-knockdown and WISH experiments
and analyses. JL constructed expression plasmids, helped create transgenic zebrafish,
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and did immunofluorescence microscopy. JG and CAHA designed and carried out MO
experiments and rescues. MJP and CAHA isolated transcripts. HWD conceived the
study and participated in its design and interpretation. MJP and HWD drafted and
revised the manuscript. All authors reviewed the manuscript.
Author Disclosure Statement
No competing financial interests exist.
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Supplemental Figure 1. Alignment of the amino acid sequences of wild-type and
mutant Hemogens in zebrafish with the orthologous proteins from other
vertebrate species. Transactivation domain (TAD) motifs are boxed and were identified
in human and zebrafish Hemogens by ϕϕxxϕ or ϕxxϕϕ, where ϕ is a bulky hydrophobic
motif. Alleles are shown for Hemgnnuz2 (Δ5) and Hemgnnuz4 (Δ12) mutant zebrafish
lines. Predicted motifs: green, coiled coil; blue, nuclear localization signal; maroon, four
residues introduced by alternative splicing; yellow, tandem peptide repeats; box, TAD
motif; purple, TAD motif conserved in teleosts; bold italic, acidic region; red,
frameshifted residues; red dashes, deletion. Species abbreviations: H. sapiens, Homo
sapiens; M. musculus, Mus musculus; G. aculateus, Gasterosteus aculeatus; G. gallus,
Gallus gallus; C. milii, Callorhinchus milii; D. rerio, Danio rerio
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Supplemental Figure 2. Analysis of peptide repeats (A) Alignment of predicted
tandem peptide repeats from zebrafish and human Hemogens. Conserved residues are
shaded black. Each predicted repeat in human Hemogen can be divided into two more
repeats. Conserved regions of peptide repeats between zebrafish and human
Hemogens are boxed. Repeats are most similar within species but repeats 1 and 3 are
similar between human and zebrafish Hemogens (marked with asterisks). (B) Amino
acid composition of zebrafish Hemogen. The repeat region is enriched for glutamic acid
and proline. The acidic C-terminal repeat is enriched for glutamic acid and aspartic acid.
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Supplemental Figure 3. CRISPR/Cas9-mediated replacement of zebrafish
Hemogen with the mCherry transgene recapitulates endogenous Hemogen
expression in zebrafish. (A) Schematic showing insertion of the mCherry transgene at
the CRISPR target site within exon 1 of zebrafish Hemogen. Integration of the mCherry
transgene was confirmed by sequencing the locus with internal and external primers
(arrows; Table S1). (B) 3 dpf. Representative image of the tail segment. mCherry+
mutant cells were present in the CHT and the pronephric duct (PD) and at a low
frequency in circulation in the dorsal aorta (DA, dashed outline) (n = 15 embryos). (C)
Sequence across the insertion, showing part of the Hemogen promoter (blue), the first 7
codons of the mCherry transgene (red), and a linker sequence (black). Scale Bar = 100
µm (B).
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Supplemental Figure 4. (A) Representative Western blot of Hemogen from pooled
morphants (MO) or wild-type (WT) embryos at 33 hpf. (B) Average Hemogen protein
expression from three experiments. GAPDH served as the internal control. (*, P ≤ 0.05,
Student’s t test). (C) Relative βe1-globin expression in pooled morphant or wild-type
embryos at 3 dpf as determined by qRT-PCR. Four samples of 10 pooled embryos were
amplified per treatment. Signals were normalized to β-actin and shown relative to wild-
type. Error bars represent the standard deviation (*, P ≤ 0.05, Student’s t test).
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Supplemental Figure 5. Hemogen mutant zebrafish have increased cell death
during embryonic development. (A) Genotypic ratios of 2 dpf embryos produced from
heterozygous incrosses of Hemgnnuz3 (Δ5) or Hemgnnuz4 (Δ12) mutants. (B) Proportion
of genotyped mutants and wild-type sibling embryos at 2 dpf that were apoptotic (black)
or phenotypically normal (white) (* P ≤ 0.05, ** P ≤ 0.005, Chi square). (BC) Acridine
orange staining for apoptotic cells is increased in the bodies and in the peripheral blood
island (outlined) in 20 hpf heterozygous Hemgnnuz2 mutant zebrafish (n = 3) compared
to wild-type siblings (n = 3). (D) Comparison of adult wild-type and homozygous
Hemgnnuz4 (Δ12) mutant. (E) Average body length of adult wild-type and Hemgnnuz4
(Δ12) mutants. Error bars represent standard error (*, P ≤ 0.05, Student’s t test). Scale
bars = 100 µm (E), 50 mm (D)
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Supplemental Figure 6. Proposed models for regulation of Hemogen expression
by promoter elements. (A) Linear, two-promoter model. (B) Chromatin looping model.
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Movie 1. Circulating erythrocytes in Tol2-generated transgenic Tg(Hemgn-
1:mCherry,myl7:EGFP) zebrafish at 2 dpf. 4x magnification.
Movie 2. Comparison of circulating EGFP+ erythrocytes in the dorsal aorta of Hemogen
morphant and wild-type Tg(Lcr:EGFP)cz3325Tg zebrafish embryos at 3 dpf. The dorsal
aorta is highlighted, and EGFP+ erythrocytes are marked with a dot. 20x magnification.
Movie 3. Comparison of circulating erythrocytes in, Hemgnnuz2/+ zebrafish embryos (Δ5
frameshift), Hemgnnuz4/+ embryos (Δ12 deletion) and wild-type siblings at 24 hpf. 10x
magnification.
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Table S1. Sequences of primer and guide oligonucleotides used in experiments
Gene Oligo Sequence (5’ – 3’) Method
hemgn F1 hemgn F2 hemgn F3 hemgn R1 hemgn R2
CTTTCTTCTGTGAGTATTGTGC GAGAAAGAGATCCCACCAACTG GACATGATTGTGAACACGCCC TTGTTTCCATAGTAAGGAGGTG TCTGAGTCGCCGCCGAATTCC
RT-PCR of Hemgn
hemgn F1 hemgn F2 hemgn R
GACATGATTGTGAACACGCCC CTGTGAGTATTGTGCCAAGTCC TCTGAGTCGCCGCCGAATTCC
qRT-PCR of Hemgn
hemgn-Kpn1F hemgn-Spe1-R
ATCATGGGTACCCACATCCAGAAATGAGACAT ATCATGACTAGTTTTGTAGTCCTGTCACATGA
PCR Promoter
hemgn F mCherry R
CTGTGAGTATTGTGCCAAGTCC GAACTCCTTGATGATGGCC
RT-PCR transgene
hemgnMM F4 hemgn R1
ACCATGGAGGATCCGCTGGAGAAAGAGA TTGTTTCCATAGTAAGGAGGTG
zHemgn rescue cDNA
βe1-globin F βe1-globin R β-actin F β -actin R
TCGCCAAGGCTGACTACGA CGGCATTGTAGGTTTCCAA CGAGCAGGAGATGGGAACC CAACGGAAACGCTCATTGC
qRT-PCR of Morphants
LarmF LarmR-AvrII-R Rarm-ClaI-F RarmR mCherry-AvrII-F PolyA-ClaI-R
CTGTGAGTATTGTGCCAAGTCC ATCATGCCTAGGGTCTTCCATTTTGTAGTCC ATGTACATCGATCCTTAGCATTAAACATCAATCAC CCATGCCTAGTGTCAGGATC ATCATGCCTAGGATGGTGAGCAAGGGCG ATGTACATCGATCTTGTTTATTGCAGCTTATAATGGTTAC
Constructing Donor Plasmid
hemgn F mCherry R
GCTCGCTTGTTGTTTACTCT GAACTCCTTGATGATGGCC PCR Knock-in
sgRNA AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
CRISPR template
sgRNA Hemgn ex1a
GAAATTAATACGACTCACTATAGGTGGGATCTCTTTCTCCAAGTTTTAGAGCTAGAAATAGC
CRISPR template
sgRNA Hemgn ex1b
GAAATTAATACGACTCACTATAGGAATAAAAGATTCAGATGAGTTTTAGAGCTAGAAATAGC
CRISPR template
sgRNA Hemgn ex3
GAAATTAATACGACTCACTATAGGATCTGGGGCCAGATGAGGGTTTTAGAGCTAGAAATAGC
CRISPR template
hemgn ex3 F hemgn ex3 R
GGTGCCTGAAGAAGCAATAAGTG CATTCATGAACAAGACGTTTCAGC
HRMA
hemgn ex1 F hemgn ex1 R
GCATGAATGTAAGCGGGC GTGATTGATGTTTAATGCTAAGG
HRMA
hemgn WT F hemgn Δ5 F hemgn Δ12 F hemgn Both F hemgn Both R
TGAGGATCTGGGGCCAGATG GGATCTGGGGCCAGGAG AGGATCTGGGGCCAGATATGC GATTGAGGATCTGGGGCCAG GGTGCTGGAGCAAACATTGG
qRT-PCR of
mutant alleles
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Chapter 3: Erythroid gene discovery using the erythrocyte-null Antarctic icefishes
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Abstract
The molecular regulators of erythropoiesis have been carefully studied in
different animal models. However, only the most important erythroid genes or the most
highly expressed markers have been well characterized. The complete loss of functional
red blood cells in Antarctic icefishes provides an opportunity to discover and
characterize new erythroid genes. I previously identified 31 novel erythroid-specific
genes by transcriptomic comparison of hematopoietic tissues from red- and white-
blooded notothenioids. Here, I characterize the loss of the erythroid gene hemogen and
two novel blood genes, mabcp1 and cd33rSig, from icefishes.
My studies reveal a truncating frameshift mutation in hemogen from icefishes,
which may alter its function in hematopoietic cells, kidney, brain, and testis where it is
expressed. This defect may have resulted from overexpression of a short hemogen
isoform (hemgn-s) that is specific to icefishes. I show that overexpression of icefish
hemogen in zebrafish embryos impairs primitive erythropoiesis. Finally, I characterize
the loss of two more erythroid genes, a novel mabp (MVB12-associated β-prism)-
containing protein with the second highest expression in notothenioid red blood cells
and the teleost ortholog of cd33 which is truncated in icefishes.
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Introduction
The “white-blooded” Antarctic icefishes (Channicthyidae), are the only
vertebrates that do not produce hemoglobin nor typical mature erythrocytes (Cocca et
al., 1995b; Near et al., 2006b; Zhao et al., 1998b). Loss of the globin genes in icefishes
(Cocca et al., 1995b) may be one of several erythropoietic defects that contributed to
their anemia. The defective molecular pathways in icefishes may reveal novel regulators
of erythroid development and disease (Albertson et al., 2009).
The study of blood development has helped uncover fundamental aspects of cell
differentiation and function including transcriptional regulation, chromatin regulation,
heme synthesis, the cytoskeleton, and cell survival. The process of hematopoiesis
provided the first model of cell differentiation from pluripotent stem cells (Till and
McCulloch, 1980). Erythrocytes also provided the first model to study the cytoskeleton
and important membrane-associated proteins (e.g. Spectrin, Actin, Band3, Band 4.1,
Band7) (Steck, 1974).
The severe anemia of Antarctic icefishes makes these animals the ideal “mutant
model” for erythroid gene discovery (Detrich and Yergeau, 2004). I identified 31 novel
erythroid genes by transcriptomic comparisons of red- and white-blooded notothenioid
fishes (See Chapter 1). In the current study, I characterize three blood-specific genes
that were mutated in Antarctic icefishes. I evaluate the functions of each gene using the
zebrafish model. This study provides a first analysis of three novel erythroid genes that
were lost by Antarctic icefishes.
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Results
hemogen (hemgn)
Isolation of the mutated hemogen gene from Antarctic icefishes
Teleost hemogen was originally discovered as a candidate erythroid gene that
was strongly down-regulated in Antarctic icefishes (Detrich and Yergeau, 2004;
Yergeau et al., 2005). In all vertebrates, hemogen is found as a single-copy, four exon
gene (Fig.1A) at a locus that is highly syntenic in fish and mammals (Fig. S1).
To characterize hemogen in notothenioids, I isolated and sequenced the
genomic locus from N. coriiceps and C. aceratus. Alignments of the two orthologs
revealed a 90-bp deletion and 1-bp insertion in exon 3 of icefish hemogen that
introduced a frameshift and premature stop codon (Fig. 1B-D). The same frameshift
mutation was found in hemogen in the RNA-Seq transcriptome for N. ionah. However,
the ortholog of the icefish, Ps. georgianus, only contained a 90-bp in-frame deletion,
which removed 30 amino acids (176P_206P). Thus, hemogen from icefishes first
evolved a 90-bp deletion which was followed by a 1-bp insertion in some species.
Strikingly, the hemogen gene from the dragonfish, P. charcoti, contained a 6-bp
deletion at the same site as the icefish mutation which removes two amino acids
(191V_192Pdel). Thus, mutations accumulated in the third exon of hemogen in a region
that may constitute a functional domain with an erythropoietic role. Furthermore, these
data show that loss of residues in this domain began prior to the divergence of
dragonfishes and icefishes.
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Figure 1. The erythroid gene hemogen is mutated in Antarctic icefishes. (A) Gene
structure of zebrafish hemogen on chromosome 1 from assembly GRCz11 (O'Leary et
al., 2016). Coding exons, green boxes; introns, green lines. CRISPR/Cas9 targets are
highlighted red. RNA-Sequencing shows strong expression of hemogen in zebrafish
blood and other tissues. Values are Log2 transformed RPKM (reads per kilobase of
transcript per million mapped reads). (B) Protein domains of Hemogen from Notothenia
coriiceps. In the icefish, C. aceratus, a frameshift mutation occurs at a putative
transactivation domain (TAD). Numbers indicate length in amino acids. Abbreviations:
CC, coiled-coil domain; NLS, nuclear localization signal; R, peptide repeats; 4AA, four
amino acides introduced by alternative splicing. (C) 3D model of Hemogen from N.
coriiceps (color coded as in panel B) designed with I-tasser (Yang et al., 2015) using
human secretory component of immunoglobulin g as a template (PDB:3CHN:S). Red
and yellow regions mark the deleted and frameshifted sequences in the icefish,
respectively. (D) Exon structures of hemogen genes from N. coriiceps and C. aceratus.
The frameshift mutation occurs in exon 3 (red arrow) in icefish hemogen.
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The deleted domain in icefish Hemogen contains a transactivation domain motif
Teleost Hemogens possess conserved functional domains at the N- and C-
termini separated by a linker formed by proline-rich peptide repeats (Peters et al.,
2018). The N-terminus contains predicted coiled-coil forming nuclear localization
signals. In most teleosts, the C-terminus contains a conserved transactivation domain
(TAD) motif with the consensus sequence φφxφ, where φ is a strong hydrophobic
residue. Residues within this conserved region of Hemogen were found to be critical for
normal erythropoiesis in zebrafish (Peters et al., 2018). However, notothenioid
Hemogens lack this specific TAD motif. Instead, notothenioid Hemogens possess a
TAD motif within the peptide repeat that was deleted in icefishes (Fig. 1B).
To predict the tertiary structure of Hemogen from N. coriiceps, ab initio, 3D
models were created with I-Tasser using the X-ray structure for the secretory
component of immunoglobulin g (PDB 3CHN:S) as a template (See Methods, Fig. 1C).
The structures had TM-scores of 0.48+0.15 (TM-score > 0.3 is significant). Mutations in
Hemogens from icefishes remove the proline-rich linker (highlighted red, Fig. 5C)
including the TAD motif. I predict that loss of the proline-linker and C-terminal globular
domain including the putative TAD may disrupt Hemogen activation of erythropoiesis in
C. aceratus.
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Figure 2. hemogen is expressed in hematopoietic, renal, and neural tissues in
embryonic and adult notothenioids. (A) 1 month post fertilization. Whole-mount in
situ hybridization (WISH) of hemogen in embryos of the red-blooded nototheniid, N.
coriiceps. Transcripts were detected with an anti-sense riboprobe synthesized from C.
aceratus hemogen cDNA. (B) 1 week. WISH of hemogen transcripts in the brain of
embryos from the icefish, C. aceratus. (C) Northern blot detection of hemogen
transcripts in tissues from N. coriiceps using an antisense riboprobe for C. aceratus
hemogen. Four alternative transcripts were detected in different tissues. (D) Western
blot detection of Hemogen protein in spleen from adult individuals of N. coriiceps (Ncor)
and C. aceratus (Cace). Specific bands were detected in both species at the molecular
weight for full-length Hemogen (~36 kDa). (E) In situ hybridization of hemogen
transcripts in spleen prints from C. aceratus with a digoxigenin-labeled antisense
riboprobe for C. aceratus hemogen. Cytoplasmic staining was seen in different
hematopoietic cell types in the icefish.
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In situ hybridization of hemogen in notothenioid embryos and in spleen from
icefishes
To determine the tissues that express hemogen in Antarctic notothenioid fishes
and that may be affected by the mutation in icefishes, I employed whole-mount in situ
hybridization to detect hemogen transcripts in embryos from N. coriiceps and C.
aceratus. In N. coriiceps embryos, at the onset of blood circulation (~1 month post
fertilization), hemogen was detected in the pronephric tubules, in the brain, and in
circulating blood cells in the vasculature and on the yolk sac (Fig. 2A). The same
expression profile is found in zebrafish embryos at 48 hpf (Peters et al., 2018). In C.
aceratus embryos, prior to the onset of circulation (~1 week post fertilization), hemogen
expression was detected in the brain but not in blood cells of the intermediate cell mass
(ICM) (Fig. 2B). In adult spleen prints from C. aceratus, hemogen transcripts were
detected in different hematopoietic cell types (Fig. 2E).
Tissue-specific isoforms of hemogen are expressed in red- and white-blooded
notothenioids
To characterize the hemogen transcripts that were expressed in notothenioids, I
performed Northern blotting of hemogen transcripts in tissues from N. coriiceps (see
Methods). Multiple alternative isoforms of hemogen were detected in different tissues
from N. coriiceps. The first transcript (~1.35 kb) was highly expressed in blood and head
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kidney (Fig. 2C) and corresponded to the expected size (1,331 kb) of N. coriiceps
hemogen (XM_010775526.1). A second transcript was found at ~1.8 kb and was
expressed in all tissues. This isoform may correspond to the 1,777-bp hemogen
transcript that retains intron 2. A third transcript was detected at 1.25 kb in testis and
ovary (Fig. 6C), and this may correspond to the testis-specific isoform found in zebrafish
(Peters et al., 2018) and in mammals (Yang et al., 2003). A fourth transcript was
detected at 900 bp in brain from N. coriiceps (Fig. 2C). Our laboratory isolated a fifth
short isoform (hemgn-s) by RT-PCR that occurred in icefishes but not in red-blooded
notothenioids (data not shown). This transcript splices around the deleted region in
icefish hemogen but produces the same frameshift and premature stop codon.
Normal and short Hemogen protein variants are expressed in icefishes
To identify Hemogen protein in notothenioids, I ran Western Blots on different
tissues from N. coriiceps and C. aceratus using an antibody directed against the
conserved N-terminal region of human Hemogen (see Methods). Hemogen was
specifically detected at 36 kDa in both spleen and brain from N. coriiceps and in spleen
from C. aceratus (Fig. 2D). The absence of a size difference for Hemogens from N.
coriiceps and C. aceratus could not be explained. I also detected a short Hemogen
isoform at ~12 kDa (Fig. 3A) that was only expressed by icefishes and likely
corresponded to the short isoform (hemgn-s). I employed quantitative PCR to measure
expression of the normal and short isoforms of hemogen in head kidneys from red- and
white-blooded notothenioids (Fig. 3B). Expression of normal hemogen was significantly
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down-regulated in icefish head kidney compared to that of red-blooded species (~35
fold change, Fig. 3B). By contrast, expression of the truncated isoform was significantly
higher in icefishes (~2,634 fold change, Fig. 3B). Therefore, down-regulation of normal
hemogen in icefishes was associated with up-regulation of the truncated isoform
(hemgn-s).
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Figures 3. The short isoform of hemogen is overexpressed in icefishes and is
translated into a truncated protein. (A) Western blot detection of a short variant of
Hemogen (Hemgn-s, 12 kDa). The Hemgn-s protein was expressed in head kidney
(HK) and spleen of the icefish, C. aceratus (Ca), but not in the red-blooded nototheniid,
N. coriiceps, nor in the peripheral blood (PB) of either species. (B) Relative expression
of full-length hemogen (hemgn) and short hemogen (hemgn-s) isoforms in head kidney
from notothenioid fishes detected by quantitative PCR. Target gene expression was
normalized to beta-actin and error bars represent the standard deviation of one or two
biological replicates. Significant differences were seen between red- and white-blooded
phenotypes (Student’s t-test, P < 0.05,*).
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Overexpression of icefish hemogen disrupts erythropoiesis in zebrafish embryos
To characterize the developmental abnormalities caused by icefish Hemogen,
zebrafish embryos were injected with synthethic, icefish hemogen mRNAs (200 pg) with
the endogenous Kozak sequence. Blood production was assessed by o-dianisidine
staining at 48 hpf and injected embryos were compared with wild-type siblings or
embryos injected with mCherry mRNA (Fig. 4). Red blood cell production was reduced
in ~40% of embryos injected with hemogen mRNA from the icefish (Ca-Hemgn)
compared to 10% of embryos injected with mCherry mRNA and 2% of uninjected
zebrafish (Fig. 4A,B). Thus, icefish Hemogen inhibits primitive erythropoiesis and may
function as a dominant negative allele.
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Figure 4. Overexpression of icefish hemogen in zebrafish blocks primitive
erythropoiesis. (A) 48 hpf. TU embryos were injected with a mix of synthetic,
polyadenylated hemogen mRNA from C. aceratus (Ca-Hemgn) and mCherry mRNA.
Controls were uninjected TU wild-type (WT) siblings or embryos injected with mCherry
mRNA alone. O-dianisidine staining of erythrocytes was reduced in Ca-Hemgn injected
embryos. (B) Graph showing the proportion of Ca-Hemgn injected or control embryos
that had decreased erythrocyte production (P < 0.01,* P < 0.001,**; chi square test of
proportions).
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Figure 5. A novel MABP-containing protein (mabpcp) is a RBC-specific gene in
notothenioid fishes. (A) Gene structure of the zebrafish mabpcp ortholog (si:dkey-
30j10.5) on chromosome 3 from assembly GRCz11 (O'Leary et al., 2016).
CRISPR/Cas9 targets in the gene are highlighted red. RNA-Sequencing shows strong,
specific expression of si:dkey-30j10.5 in zebrafish blood. Values are the log2
transformed RPKM. (B) Protein domains of Mabpcp from Notothenia coriiceps.
Numbers indicate length in amino acids. Two gaps (marked X) were found in the
assembled transcript from both icefishes, C. aceratus and Ps. georgianus.
Abbreviations: non-cyto, non-cytoplasmic domain; S, signal peptide; MABP, MVB12-
Associated β-prism domain. (C) Color-coded, 3D model of MABP-containing protein
from Notothenia coriiceps (LOC104952319) designed using I-tasser (Yang et al., 2015).
The model is superimposed on the MABP domain from human MVB12B (PDB:
3TOW:A). Yellow arrows, beta sheet; red, gaps in icefish sequence; blue, lipid-binding
residues; white, no prediction. (D) 24 hpf. Whole-mount in situ hybridization of dkey-
30j10.5 in wild-type zebrafish. Sense probe control shown as inset. (E) 20 hpf. Wild-type
TU and CRISPR-injected sibling embryos. Mutants were injected with Cas9 and a
gRNA targeting dkey-30j10.5 as in Panel A. (F) CRISPR target sequences in zebrafish
dkey-30j10.5. Protospacer-adjacent motif (PAM, underlined and highlighted blue).
Abbreviations: MB, midbrain; HB, hindbrain; Vent, brain ventricle
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MABP-CONTAINING PROTEIN (MABPCP)
I identified a novel RBC-specific gene in the RNA-Seq transcriptomes of
notothenioid fishes, which had the second highest expression level in P. charcoti
peripheral blood (63,503 TPM) and head kidney (36,283 TPM), second only to beta-
globin (267,053 TPM and 76,609 TPM) and 11 times higher than all other blood-specific
genes. The two-exon gene mostly encodes an MVB12-Associated β-prism domain
(MABP, InterPro IPR023341) that is related to the MABP domain of the DENND4c
protein (DENN domain-containing protein 4C) found in all vertebrates. Thus, we named
the notothenioid protein MABP-containing protein (MABPCP). The icefish ortholog was
fragmented and not strongly expressed (< 0.39 TPM, Fig. 5B) in the transriptomes of
both Ps. georgianus and N. ionah. In the icefish ortholog, two gaps were present in the
assembled contig at residues that correspond to W115 and Y176 (Fig. 5B). The gaps in
the assembly may have been caused by repetitive sequences and/or significant
genomic alterations at the gap loci.
The MABP domain is a lipid-binding structure that localizes proteins to
membranes and which has been implicated in endocytic transport (de Souza and
Aravind, 2010). A variety of MABP-containing proteins are found in eukaryotes and also
in bacteria (de Souza and Aravind, 2010) but no direct ortholog of notothenioid
MABPCP occurs in mammals. In teleosts, including notothenioid fishes, multiple
duplicated paralogs are found as one or two-exon genes. The nototheniid N. coriiceps
contains the RBC-specific protein and two more paralogs, LOC104952319 and
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LOC104956446 – the latter was specifically expressed in the trunk kidneys of P.
charcoti and Ps. georgianus.
dkey:30j10.5 (Acc. XM_001335220.7) was identified as the MABPCP ortholog in
zebrafish, and was also found to have strong, specific expression in peripheral blood
(Fig. 6A). dkey:30j10.5 occurs as a single-copy, two-exon gene located on chromosome
3 at a locus that is a well-known erythroid gene cluster on chromosome 17 in humans
(Fig. S1). Thus, the loss of red blood cells in icefishes was coincident with the loss of a
teleost-specific erythroid gene.
Protein domain and structure of MABPCP from notothenioids
Ab initio, tertiary structure models were created for notothenioid MABPCP (Fig.
5C) using I-Tasser based on the solved X-ray structure for the MABP domain of human
Multivesicular body subunit 12B (MVB12B, PDB 3TOW:A) (Boura and Hurley, 2012).
The model and its template had a template modeling score (TM-score) of 0.34±0.11
(TM-score > 0.3 = P < 0.001). In the structure, the MABP domain from notothenioid
MABPCP contains the same hydrophobic β2-β3 loop seen in MVB12B that has been
predicted to insert itself within lipid membranes (Boura and Hurley, 2012) (Fig. 5C). The
gaps in icefish MABPCP occur at electropositive residues that anchor this domain to
membranes (Boura and Hurley, 2012) (Fig. 5B,C). Thus, changes to this domain in
icefishes may disrupt MABPCP binding to cell membranes.
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Expression profile of mabp-containing protein in zebrafish embryos
The spatiotemporal expression profile of zebrafish mabpcp (dkey:30j10.5) was
evaluated in wild-type TU embryos by whole mount in situ hybridization (WISH) at 20
hpf (Fig. 5D). Sibling embryos were fixed and hybridized with sense or anti-sense
digoxigenin-labeled riboprobes targeting a portion of the dkey:30j10.5 gene (See
Methods). Faint expression was specifically detected in the central nervous system with
an anti-sense probe (Fig. 5D) but not with a sense probe (Fig. 5D inset). The highest
expression occurred in the midbrain and hindbrain, specifically in cells associated with
the brain ventricles (Fig. 5D). The intermediate cell mass (ICM) and peripheral blood
island (PBI) did not show strong expression, which indicates dkey:30j10.5 is not
associated with primitive erythropoiesis in embryos.
CRISPR/Cas9 targeting of mabp-containing protein in zebrafish
To analyze the developmental role of mabpcp, we employed CRISPR/Cas9 gene
editing to target zebrafish dkey-30j10.5 (Fig. 6A,E,F). Wild-type TU zebrafish embryos
were co-injected with a guide RNA (100 ng µl -1), Cas9 mRNA (1000 ng µl -1), and
mCherry mRNA (100 ng µl -1) (See Methods and Table 2). At 20 hpf, 88% of injected
mutants had shortened tails and severe deformities compared to uninjected, wild-type
siblings (n = 25, Fig. 5E) - these deformities are common phenotypes produced from
off-target effects. Furthermore, the deformities (Fig. 5E) were not restricted to the sites
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of dkey-30j10.5 expression in embryonic zebrafish (Fig. 5D), which also suggested they
were non-specific phenotypes.
Several MABP-domain containing proteins have been found in eukaryotes and
bacteria and were shown to bind cell membranes (Allaire et al., 2010; Boura and Hurley,
2012; Denef et al., 2008; Rosado et al., 2007). In the dragonfish, P. charcoti, mabpcp is
the second highest expressed gene in red blood cells. This protein may have a function
in the ESCRT machinery (endosomal sorting complexes required for transport) as do
other MABP-containing proteins (Boura and Hurley, 2012). The ESCRT pathway plays
an important role in mitochondrial removal during erythroid maturation and defects in
this process can cause anemia (Mortensen et al., 2010).
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Figure 6. Modeling a truncated CD33-related Siglec (CD33rSig) from icefishes in
mutant zebrafish. (A) Gene structures of the zebrafish cd33rSig paralogs, dkey-
238d18.10 and LOC101884840, on chromosome 15 from assembly GRCz11 (O'Leary
et al., 2016). CRISPR targets are highlighted red. RNA-Sequencing shows specific
expression of dkey-238d18.10 in blood and LOC101884840 in brain. Values are the
log2 transformed RPKM. (B) Protein domains of the CD33rSig ortholog from N.
coriiceps. The F753* mutation truncates CD33rSig in both icefishes, C. aceratus and
Ps. georgianus. Numbers indicate length in amino acids. (C) 20 hpf. Wild-type TU
embryo (D) 20 hpf. CRISPR-injected embryo targeting dkey-238d18.10. Note the
enlarged peripheral blood island (PBI, outlined) in the mutant. (E) 20 hpf. CRISPR-
injected embryo targeting LOC101884840. (F) 3D model of CD33rSig from N. coriiceps
created with I-tasser (Yang et al., 2015) using human CD33 as a template (PDB:
5IHB:A). Red marks the truncated region in icefish CD33rSig. (G) High resolution
melting curve showing decreased melting of the mutant (red) dkey-238d18.10 alleles
compared to wild-type (blue). Inset shows the difference curves for several mutants. (H)
Sequence TRACE result of dkey-238d18.10 from CRISPR-injected TU zebrafish.
Frameshifts occur at the protospacer-adjacent motif (PAM, underlined). Abbreviations:
S, sigal peptide; Ig, immunoglobulin-like domain; C2-set, immunoglobulin c2-set
(constant) domain; v-set, immunoglobulin v-set (variable) domain; Tr, transmembrane;
cyto, cytoplasmic; ITIM?, putative immunoreceptor tyrosine-based inhibitory motif.
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CD33-RELATED SIGLEC (CD33rSig)
One of the blood-specific gene was identified as a member of the multi-gene
family of CD33-related Sialic-acid-binding immunoglobulin-like lectins (CD33rSiglec)
and had strong, specific expression in peripheral blood cells from P. charcoti (42.04
TPM). This CD33rSiglec was found as a single copy gene and is orthologous to
LOC104953882 from the genome of the red-blooded nototheniid, N. coriiceps (Shin et
al., 2014). In the transcriptomes of three icefishes, (N. ionah, Ps. georgianus, and C.
aceratus), the corresponding orthologs contain a C-terminal frameshift mutation created
by a 1-bp deletion in exon 9, which introduced a frameshift and an immediate premature
translation termination codon (Fig. 7B).
The family of Siglecs is made up of diverse cell surface receptors that are
expressed on the membranes of different hematopoietic lineages (Crocker et al., 2007).
In humans, CD33 (Siglec-3) is restricted to myeloid lineages and is over-expressed in
acute myeloid leukemias (De Propris et al., 2011). No obvious cd33 ortholog is found in
teleosts but tandem duplication of an ancestral CD33-like gene produced numerous
CD33rSiglec-extended (CD33e) genes in fishes that are conserved with mammalian
CD33rSiglecs, Siglec-4/MAG (myelin-associated glycoprotein), and Siglec-2/CD22 (Cao
et al., 2009). Recently, a new study discovered the same CD33rSig in another teleost
(rock bream, Oplegnathus fasciatus), and this gene was designated as the functional
ortholog of mammalian CD33 (Jeswin et al., 2018). As for notothenioid fishes, rock
bream CD33 was specifically expressed in leukocytes of the peripheral blood and was
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found to be up-regulated during the immune response (Jeswin et al., 2018). Thus,
Antarctic icefishes appear to have lost the functional ortholog of CD33.
In the zebrafish genome assembly GRCz10 (Howe et al., 2013), a tandem pair of
paralogs, si:dkey-238d18.10 and LOC101884840, are both related to N. coriiceps
LOC104953882 and are located at the CD33rSiglec cluster on chromosome 15 (Fig.
7A,S1). This locus shares synteny with the genomic loci of both MAG and CD33 on
human chromosome 19 (Fig. S1). High, specific expression in blood is seen for
zebrafish si:dkey-238d10.10 but not for LOC101884840 (Fig. 6A). Together these data
indicate that notothenioid LOC104953882 and zebrafish si:dkey-238d18.10 are
candidates for the functional orthologs of the myeloid Siglec, CD33.
Protein structures and domains of CD33rSig from notothenioids
Ab initio, tertiary structure models were created for the notothenioid CD33rSiglec
(LOC104953882) with I-Tasser using the X-ray structure for human CD33 (PDB 5IHB:A)
as a template (Dodd, 2016, to be published). The structures for the zebrafish and
human proteins had template modeling scores (TM-scores) of 0.726, (TM-score > 0.3 =
P < 0.001) (Xu and Zhang, 2010). The frameshift mutation and premature stop codon in
icefish CD33rSiglec occurs in the most C-terminal C2-set immunoglobulin domain,
which removes the transmembrane and cytoplasmic domains from this cell surface
protein (Fig. 6B,F). In red-blooded species, the cytoplasmic domain contains two
tyrosine residues that likely function as immunoreceptor tyrosine-based inhibitory (ITIM)
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or activator (ITAM) motifs (Fig. 6B,F), which carry out the function of most CD33rSiglecs
(Paul et al., 2000). The loss of this inhibitory domain in icefishes may affect immunity,
hematopoietic proliferation and cell survival (Nguyen et al., 2006; Varki and Angata,
2006; Vitale et al., 2001).
CRISPR/Cas9 targeting of CD33rSig in zebrafish
To determine the developmental role of cd33rSiglec in fishes, I employed
CRISPR/Cas9 gene editing to target the zebrafish paralogs, si:dkey-238d18.10 and
LOC101884840 (Fig. 6A). Wild-type TU zebrafish embryos were co-injected with a
guide RNA (100 ng µl -1), Cas9 mRNA (1000 ng µl -1), and mCherry mRNA (100 ng µl -1)
(See methods and Table 2). Mutant and wild-type siblings were imaged and genotyped
by high-resolution melting analysis (HRMA) (Fig. 6G) and by sequencing of the locus
(Fig. 6H). At 20 hpf, enlarged peripheral blood islands (PBI) were seen in 33% of
injected mutant si:dkey-238d18.10 zebrafish (n = 12) (Fig. 6D). Additionally, 33% of
mutants had shortened tails with a reduced PBI, and 25% were deformed due to cell
death (Fig. 6D). While some of these phenotypes may result from off-target effects, the
uncontrolled proliferation of PBI blood cells in si:dkey-238d18.10 mutant zebrafish is an
uncommon trait and may evidence a role for this CD33rSiglec in primitive
hematopoiesis. The phenotype of si:dkey-238d18.10 mutants contrasted sharply with
that of LOC101884840 mutants (Fig. 6D). Shortened tails occurred in 71% of
LOC101884840 mutants and 57% were developmentally delayed (n = 7, Fig. 6E).
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In mammals, truncation of CD33 and loss of the cytoplasmic ITIM has been
shown to prevent internalization of the receptor thereby increasing CD33 expression on
the cell surface (Walter et al., 2008). CD33 is highly expressed in normal and malignant
myeloid lineages and is a common target for immunotherapy – internalization of a drug-
linked CD33 antibody (Gemtuzumab ozogamicin) promotes cell death (Geiger and
Rubnitz, 2015). The functions of CD33 in myeloid cells are not well understood
(Ulyanova et al., 1999). Many Siglecs are involved in erythroblast island formation
(Rhodes et al., 2008) and CD33 knockout mice display a slight erythroid defect
(Brinkman-Van der Linden et al., 2003). The loss of cd33 may affect the survival of
myeloerythroid progenitors in icefishes.
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Methods
Fish husbandry
Wild-type (SAT, AB, TU) and transgenic Tg(lcr:egfp) zebrafish (Danio rerio), were
generously provided by Dr. Leonard I. Zon (Howard Hughes Medical Institute and
Harvard Medical School, Boston). Animal procedures were carried out in full
accordance with established standards set forth in the Guide for the Care and Use of
Laboratory Animals (8th Edition). The animal care and use protocol for live zebrafish
embryos was reviewed and approved by Northeastern University’s Institutional Animal
Care and Use Committee (Protocol No. 15-0207R). The animal care and use program
at Northeastern University has been continuously accredited by AAALAC Int. since July
22, 1987, and maintains the Public Health Service Policy Assurance number A3155-01.
Cloning and sequence analysis of zebrafish and notothenioid cDNAs
Total RNA was isolated from wild-type AB zebrafish embryos or flash frozen
tissues from Antarctic notothenioid species using TRI reagent (Sigma, T9424) and the
Ribopure Kit (Ambion, AM1924). Total cDNA was produced from mRNA using M-MuLV
reverse transcriptase (NEB, M0253S) and an oligo(dT)23 primer. cDNAs were amplified
by PCR from total cDNA with 1 µM primers (Table S1) – the amplification program was
35 cycles of 98°C for 10 s, 60°C for 10 s, and 72°C for 30 s. PCR products were cloned
into the pGEM-T Easy vector (Promega, A1360), plasmids were transformed into 5-α
competent cells (New England Biolabs, C2987H), recombinant plasmids were identified
by blue/white screening and purified with the Wizard Plus SV Miniprep Kit (Promega
A1330), and inserts were sequenced by GeneWiz.
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Comparison of vertebrate genes
We used Blast+ (Altschul et al., 1990) to identify genes from Antarctic
notothenioid fishes in the zebrafish genome (assembly GRCz11) (Howe et al., 2013).
Chromosomal synteny comparisons were performed using the Synteny Database with a
sliding window of 200 genes (Catchen et al., 2009). Protein domains were predicted
using InterProScan (Jones et al., 2014). Ab initio tertiary structure models were created
for proteins from N. coriiceps including Hemogen, LOC104953882, and
LOC104952319. 3D models were created with I-Tasser (Yang et al., 2015) based on
the X-ray structures for the secretory component of immunoglobulin G (PDB:3CHN:S),
CD33 (PDB:5IHB:A), and the MABP domain of MVB12B (PDB:3TOW:A), respectively.
The 3D models were superimposed using TM-align (Zhang and Skolnick, 2005) or
Geneious version R10 (Kearse et al., 2012).
In-situ hybridization
The spatial and temporal patterns of expression of selected genes were analyzed
by whole-mount in situ hybridization (WISH) of zebrafish and notothenioid embryos
following standard protocols (Jacobs et al., 2011). These methods were adapted to
evaluate Hemogen expression in spleen prints prepared from adult notothenioid fishes
after they were euthanized in 200 mg L-1 tricaine methane sulfonate (MS222; Sigma-
Aldrich, 886862) (Detrich and Yergeau, 2004; Gupta and Mullins, 2010). Digoxigenin-
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labeled antisense and sense RNA probes were transcribed from cDNA clones using the
DIG RNA Labeling Kit (Roche Diagnostics, 11175025910).
Northern Blotting
Total mRNA was purified from flash frozen tissues using TRI reagent (Sigma,
T9424) and the Ribopure Kit (Ambion, AM1924). mRNA (5 µg) was electrophoresed in a
denaturing gel. Replicate lanes were cut out and stained with ethidium bromide to
assess RNA quality. Separated mRNAs were transferred overnight to nylon paper by
upward capillary transfer. Blots were hybridized with digoxigenin-labeled antisense or
sense RNA probes following a standard protocol (Alwine et al., 1977).
Western blotting
Total protein was prepared for sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) from flash frozen notothenioid tissues or fresh zebrafish
tissues by homogenization in lithium dodecyl sulfate (LDS) Bolt buffer (Life
Technologies, B007) and NuPAGE reducing agent (Life Technologies, NP0009) using a
pestle and microcentrifuge tube (USA Scientific, 1415-5390). Samples were boiled for 3
min. and centrifuged at top in an Eppendorf 5417R centrifuge speed for 2 min. Aliquots
(15 µg) were electrophoresed on a 4-12% SDS polyacrylamide gel, and the separated
proteins were transferred to a polyvinylidene difluoride (PVDF) membrane with the iBlot
system (Life Technologies, IB21001). Membranes were blocked in maleic acid blocking
buffer (2% Roche blocking reagent, 2% BSA, 0.2% heat treated goat serum, 0.1%
189
Tween-20) for 1 hour at room temperature and then incubated overnight at 4°C with
1:1000 rabbit anti-Hemogen (Aviva, ARP57794_P050) or with 1:1000 mouse anti-
GAPDH (Aviva, OAE00006) antibodies. Membranes were washed in TBST (0.1 M Tris,
0.1 M NaCl, 0.1% Tween-20) and incubated for 2 h with horseradish peroxidase (HRP)-
conjugated goat anti-rabbit IgG (H&L) (Aviva, ASP00001) or HRP-conjugated goat anti-
mouse IgG (H&L) (Aviva, OARA04973), respectively. Bound antibodies were detected
with the Amersham ECL Western Blotting Analysis System (GE Healthcare, RPN2106)
on CL-X Posure film (Thermo Scientific,34091).
Overexpression of icefish hemogen in zebrafish
The natural hemogen kozak sequence in notothenioids was added to hemogen
cDNA clones from N. coriiceps and C. aceratus by PCR using 1 µM primers (Table S1)
– the amplification program was 35 cycles of 98°C for 10 s, 60°C for 10 s, and 72°C for
30 s. PCR products were cloned into the pGEM-T Easy vector (Promega, A1360),
plasmids were transformed into 5-α competent cells (New England Biolabs, C2987H),
recombinant plasmids were identified by blue/white screening and purified with the
Wizard Plus SV Miniprep Kit (Promega A1330), and inserts were sequenced by
GeneWiz. Sense mRNAs were transcribed, capped, and polyadenylated in vitro using
the mMessage SP6 kit (Ambion, AM1340) and the Poly(A) Tailing Kit (Ambion,
AM1350). mRNA was purified by precipitation using 2.5 M LiCl. Hemogen mRNAs (30
ng µL-1) and mCherry mRNA (30 ng µl-1) were co-injected into one-cell, wild-type SAT
zebrafish embryos. Treated and control embryos were stained with o-dianisidine using
190
previously established methods (Yergeau et al., 2005) and micrographed between 20-
48 hpf.
CRISPR/Cas9 generation of mutant zebrafish
Optimal targets for CRISPR-Cas9 mutagenesis were identified in zebrafish
si:dkey-30j10.5, si:dkey-238d18.10/CD33, LOC101884840, and tyrosinase using the
program CHOPCHOP (Labun et al., 2016; Montague et al., 2014). The templates for
multiple small guide RNAs (Table S2) were produced by a cloning-free method as
previously described (Hruscha et al., 2013; Talbot and Amacher, 2014). Guide RNAs
were transcribed with the T7 MaxiScript Kit (Ambion, AM1312) and purified by LiCl
precipitation.
Wild-type (TU) embryos were co-injected with a guide RNA (150 ng µl-1), Cas9
mRNA (300 ng µl-1), and mCherry mRNA (ng µl-1) to identify successful injections.
Embryos were raised and adults were tail-clipped and genotyped by high-resolution
melting analysis (HRMA) as previously described (Talbot and Amacher, 2014). PCR
amplification was run using 1 µM primers (Table S1) with PowerUp SYBR MasterMix
(Applied Biosystems, A25742) on a QuantStudio 3 Real-time PCR system
(ThermoFisher, A28137). PCR amplicons were sequenced by Genewiz.
Imaging
Fixed zebrafish or notothenioid embryos were mounted in 80% glycerol and
imaged with a dissecting microscope (Nikon, SMZ-U) and a CCD digital camera
191
(Diagnostic Instruments, SPOT32). Live zebrafish embryos were embedded in 0.1%
agarose in embryo medium (EB) with 0.01% tricaine and imaged with an
epifluorescence-equipped microscope (Nikon, Eclipse E800) using a Photometrics
Scientific CoolSNAP EZ camera and NIKON NIS-Elements AR 4.20 software.
Quantitative PCR
RNA was purified from flash frozen notothenioid tissues or fresh zebrafish
tissues in TriZol (Sigma-Aldrich, T9424) using the PureLink RNA purification Kit
(Ambion). DNase treated RNA was reverse transcribed with a polyT(23) primer using
Protoscript II RT-PCR kit (New England Biolabs, M0368S). Target genes were amplified
in triplicate from cDNA by qRT-PCR with 1 µM primers (Table S1). Standard curves
were generated to confirm primer efficiencies. Target gene expression was normalized
to beta-actin for comparison by the ΔΔCt method. Three or four biological replicates
were used for each treatment for statistical comparisons.
Statistical analyses
Data are displayed as means±s.e.m. or means±s.d. or as noted. Differences with
a p-value ≤ 0.05 were considered significant for all statistical tests.
192
193
Figure S1. Synteny maps comparing the chromosomal loci of novel RBC-specific
genes in zebrafish and humans. (A) Syntenic Hemogen loci on zebrafish
chromosome 1 and human chromosomal region 9q22.33. (B) Synteny of loci for
zebrafish dkey-30j10.5 on chromosome 3 and the corresponding region on human
chromosome 17. No direct ortholog was identified in humans. (C) Synteny of loci for
zebrafish dkey-238d18.10 and LOC101884840 paralogs on chromosome 15 and
human CD33 on chromosome 19.
194
Table S1. Primer Sequences
Gene Oligo Sequence (5’ – 3’) Gene Method
Ncor130For NcHemgn_R2 NcHemgnR1050
GGAGGAGACATTTCAAC CTAACAGGATGCACACTAACC AGATACCCGTCATTCAGGA
hemgn (Notothenioid)
PCR gDNA
NcHemgnR2.2 NcHemgnR2.1 NcHemgn5utrF Icefish_For5utr
CCTCAGAAGATCCCTGTCAC CACGTAACCGGCGACGGATC ATGCCCTCACACAACTTGAC GTGTCCCCGAGGTTATAATAC
hemgn (Notothenioid)
PCR gDNA
30j10_F 30j10_R 30j10_F3 30j10_R_mp1
CCAGCACTGCGGTTCAG GAGATATGGAAAAAGGTCTGGAGG GACCAGGATCAGTTTTCATTC AGATTCTTCTTGACCTGCTCGT
dkey:30j10.5 (Zebrafish)
RT-PCR
30j10_F 30j10_R
CCACCACTAAAGATGAGGAGGA CCACAGATTGATTTTGTCTCCA
dkey:30j10.5 (Zebrafish)
HRMA
Dkey238F Dkey238R
GTGCACTATTATTTGCACGCTC CCCGATTTAAACCAGAAAGTGT
dkey:238 (Zebrafish)
HRMA
LOC101884840F LOC101884840R
CCACAGCTGCAATTTACAGAAC CTGATACCACACAACTCTGCGT
LOC101884840 (Zebrafish)
HRMA
Hemgn_F_kozak NcHemgn_R2
AATTCATAGCAGGACTCAGAATGGAGGAGACATTTCAAC CTAACAGGATGCACACTAACC
hemgn (Notothenioid)
PCR gDNA
CD33rSig_F CD33rSig_R
CTGCTCATTAGAGATTGATGA GAAGGTTATTGTGGAGGTC
cd33rSig (Notothenioid)
PCR gDNA
Drhemex3Fb CCTCAAGAGGAGTTTTTGATTGAGG hemgn
(Zebrafish) PCR
β-globin_F β-globin_R
TCGCCAAGGCTGACTACGA CGGCATTGTAGGTTTCCAA
beta-globin (Zebrafish)
qPCR
β-actin_F β-actin_R
CGAGCAGGAGATGGGAACC CAACGGAAACGCTCATTGC
beta-actin (Zebrafish)
qPCR
β-act_F β-act_R
CAGATCATGTTCGAGACCTTCAAC TCACCRGARTCCATGACGATA
beta-actin (Notothenioid)
qPCR
Hemgn_short_F NcHemgn_R2
GACTAACCAGTGGGTTTTAAGCC CTAACAGGATGCACACTAACC
hemgn (Notothenioid)
qPCR
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Table S2. Oligos for CRISPR gRNA template
Gene Oligo Sequence (5’ – 3’) Gene Method
30j10_gRNA1 30j10_gRNA2
GAAATTAATACGACTCACTATAGGAAAGATCGCGTCTTCCTCGTTTTAGAGCTAGAAATAGC GAAATTAATACGACTCACTATAGGAGGCAGCTGGGTACGAGCGTTTTAGAGCTAGAAATAGC
dkey:30j10.5 (Zebrafish)
CRISPR
LOC101884840_gRNA GAAATTAATACGACTCACTATAGGAACCTTGGAGGCCGTGAAGTTTTAGAGCTAGAAATAGC
LOC101884840 (Zebrafish)
CRISPR
Dkey238_gRNA GAAATTAATACGACTCACTATAGGTTGGACTCTCTTTCTGACGTTTTAGAGCTAGAAATAGC
dkey:238d18.10 (Zebrafish)
CRISPR
gRNA_common AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
CRISPR template
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Conclusion
The evolution of Antarctic notothenioid fishes appears to have involved a gradual
reduction in the activity of the erythropoietic pathway (Eastman, 1993; Lau et al., 2012;
Wells et al., 1980), which culminated in the complete loss of production of typical
mature red blood cells in the derived monophyletic clade of icefishes (Channicthyidae).
Mutations in key erythroid genes or genetic pathways may have instigated the
evolutionary loss of red blood cells. Genes that are targets in human anemias are prime
candidates, but there may also be unknown genes whose mutation caused or
contributed to icefish anemia. As an example of the former, I found that the Antarctic
dragonfish P. charcoti (dragonfishes are the sister clade to the icefishes), produces
abnormal spherocytic erythrocytes and has mutations in erythroid beta-spectrin that are
identical to several that cause human hereditary spherocytic anemia. Moreover, some
mutations in icefishes, like the deletions of the adult alpha and beta globin genes
(Cocca et al., 1995b; di Prisco et al., 2002; Zhao et al., 1998b), may be a consequence
of relaxed selection due to the loss of globin transcriptional regulation (Lau et al., 2012)
or due to the loss of globin-expressing erythrocytes. Alternatively, one may speculate
that some regulator(s) of globin transcription (e.g. Lcr, Gata1, Hemgn, etc.) became
functionally compromised, and this led to the loss of globin expression and the
subsequent deletion of the locus.
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Chapter 2
Teleost hemogen was first discovered as a marker that was strongly expressed
in the hematopoietic tissues of red-blooded notothenioids but not in the derived lineage
of white-blooded Antarctic icefishes (Detrich and Yergeau, 2004; Yergeau et al., 2005) –
this finding implicated teleost hemogen in erythropoiesis. In my preliminary research, I
identified a frameshift mutation in the icefish hemogen gene, a defect that truncates the
putative transactivation domain (TAD) of the encoded transcription factor. The hemogen
gene is present in the genomes of all vertebrates except the superclass of jawless
fishes (Agnatha), and is found as a highly conserved, single-copy, four exon gene.
Except in a few species, the hemogen gene has been preserved in the same state for
over 450 million years and is likely to be a gene that is crucial for vertebrate
development. In support of this, I found that the expression pattern of hemogen was
conserved between fishes and mammals. In zebrafish, hemogen expression is driven
by Gata1 in differentiating erythrocytes, in Sertoli cells of the testis, in the brain, and in
renal cells of the kidney. Two conserved non-coding elements function individually and
together to regulate hemogen expression in primitive and definitive waves of blood
development in zebrafish.
In icefish Hemogen, deletion of the putative TAD is likely to impair the
recruitment of P300 to the erythroid transcription factor, Gata1 (Zheng et al., 2014). To
determine the effects of the icefish hemogen mutation on erythroid development, I used
the CRISPR/Cas9 gene editing system to generate hemogen mutant zebrafish that
recapitulate the icefish mutation. I showed that the frameshift mutation in zebrafish
hemogen was a dominant-negative allele, which caused partial anemia in embryos and
198
adults. Therefore, intact Hemogen appears to be required for erythropoiesis and may
contribute to the anemia of Antarctic icefishes.
The transgenic zebrafish lines produced in this study provide the first in vivo
animal models to analyze the function of Hemogen during embryonic and adult
development. These zebrafish models may be useful in identifying causes and
treatments of human blood diseases that have been associated with hemogen
overexpression.
Chapter 1
To discern the mutation events that led to the deletion of the globin genes and
the loss of red blood cells in icefishes, I performed comparative transcriptomics of red-
and white-blooded notothenioid species. I show that the mutation in icefish hemogen
was one of several genetic defects in a shared molecular pathway that may contribute
to an intricate repression of erythropoiesis. Notably, icefish erythropoiesis may be
blocked by an acetylation imbalance caused by down-regulation of P300 and
overexpression of Hdac1b, two proteins that are known to regulate the activity of Gata1
(Boyes et al., 1998). Furthermore, both P300 and Gata1 contain predicted deleterious
substitutions in the domains that bind Hemogen, which suggest that this activating
complex was lost by icefishes. These mutations and the truncation of the Hemogen
TAD in icefishes may hint at the loss of a multi-protein complex formed by Hemogen,
Gata1, and other cofactors that bind the globin locus control region (Lcr).
199
Chapter 3
In red-blooded Antarctic notothenioid fishes, hemogen is expressed in the same
tissues at sites of embryonic and adult hematopoiesis, in the brain, and in renal cells of
the kidney. Despite their severe anemia, icefish embryos produce hemgn+ primitive
erythroid cells in lateral plate mesoderm and express the truncated allele at very low
levels compared to red-blooded species. Overexpression of icefish hemogen severely
impairs erythropoiesis in a zebrafish model, which indicates that this truncated hemogen
is a dominant negative allele. Interestingly, icefishes produce another truncated isoform
of hemogen (hemgn-s) through alternative splicing, which is not seen in red-blooded
species. It is likely that this truncated Hemogen isoform also operates as dominant
negative protein to disrupt erythropoiesis in icefishes. This unique mechanism of
evolution is strikingly similar to splicing mutations that cause some human blood
diseases (Conboy, 2017). One might speculate that overexpression of the hemgn-s
isoform may have pre-empted the permanent deletion and truncation of the Hemogen
TAD in icefishes.
The evolutionary loss of red blood cells in Antarctic icefishes facilitated the
discovery of 31 novel erythroid genes. In icefishes, three blood-specific genes (hemgn,
cd33rsig, and mabp-like) contain nonsense mutations that disrupt important functional
domains. The hemgnuz2 and hemgnnuz4 mutant zebrafish lines produced in this study
demonstrate a critical role for the Hemogen C-terminal TAD in erythropoiesis.
Generation of stable mutant zebrafish lines for the cd33rsig and mabp-like alleles from
icefishes may also reveal novel roles for these genes in erythropoiesis.
200
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