research article 1381 - journal of cell sciencemyocardial cells, partially rescued the atrial...
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Genetic interaction between pku300 and fbn2bcontrols endocardial cell proliferation and valvedevelopment in zebrafish
Xu Wang1,*, Qingming Yu2,*, Qing Wu1, Ye Bu1, Nan-Nan Chang1, Shouyu Yan1, Xiao-Hai Zhou1, Xiaojun Zhu1
and Jing-Wei Xiong1,`
1Institute of Molecular Medicine, Peking University, Yi He Yuan Lu 5, Hai Dian Qu, Beijing 100871, China2Department of Medicine/Nephrology, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Boston, MA 02129, USA
*These authors contributed equally to this work`Author for correspondence ([email protected])
Accepted 20 December 2012Journal of Cell Science 126, 1381–1391� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.116996
SummaryAbnormal cardiac valve morphogenesis is a common cause of human congenital heart disease. The molecular mechanisms regulatingendocardial cell proliferation and differentiation into cardiac valves remain largely unknown, although great progress has been made on
the endocardial contribution to the atrioventricular cushion and valve formation. We found that scotch tapete382 (scote382) encodes anovel transmembrane protein that is crucial for endocardial cell proliferation and heart valve development. The zebrafish scote382 mutantshowed diminished endocardial cell proliferation, lack of heart valve leaflets and abnormal common cardinal and caudal veins.
Positional cloning revealed a C946T nonsense mutation of a novel gene pku300 in the scote382 locus, which encoded a 540-amino-acidprotein on cell membranes with one putative transmembrane domain and three IgG domains. A known G3935T missense mutation offbn2b was also found ,570 kb away from pku300 in scote382 mutants. The genetic mutant scopku300, derived from scote382, only had the
C946T mutation of pku300 and showed reduced numbers of atrial endocardial cells and an abnormal common cardinal vein. Morpholinoknockdown of fbn2b led to fewer atrial endocardial cells and an abnormal caudal vein. Knockdown of both pku300 and fbn2b
phenocopied these phenotypes in scote382 genetic mutants. pku300 transgenic expression in endocardial and endothelial cells, but not
myocardial cells, partially rescued the atrial endocardial defects in scote382 mutants. Mechanistically, pku300 and fbn2b were requiredfor endocardial cell proliferation, endocardial Notch signaling and the proper formation of endocardial cell adhesion and tight junctions,all of which are crucial for cardiac valve development. We conclude that pku300 and fbn2b represent the few genes capable of regulatingendocardial cell proliferation and signaling in zebrafish cardiac valve development.
Key words: Scotch tape, Cardiac valve development, Endocardial cell proliferation, Zebrafish
IntroductionThe heart tube, at the time of its formation, has a two-layer
structure consisting of an inner endothelium and an outer
myocardium, connected by the extracellular matrix (Fishman
and Chien, 1997). While the myocardium has been the focus of
heart development during the past decades, the origin,
proliferation and differentiation of the endocardium has gained
less attention and so remains largely unknown. Lineage tracing
studies support the suggestion that endocardial and myocardial
progenitors are located near each other ventro-laterally in the
zebrafish blastula (Keegan et al., 2004; Lee et al., 1994; Stainier
et al., 1993). These progenitors migrate to form the cardiac field
(or anterior lateral plate mesoderm) in the early somite zebrafish
embryos, where the anterior Scl+ and Etsrp+ progenitors give rise
to endocardial/endothelial cells and the posterior Nkx2.5+
mesodermal cells to myocardial progenitors (Palencia-Desai
et al., 2011; Schoenebeck et al., 2007). In zebrafish, etsrp,
cloche and lycat are critical for the generation of endocardial and
endothelial progenitors (Palencia-Desai et al., 2011; Stainier
et al., 1995; Xiong et al., 2008). Tie2 (Tek) is required for the
formation of the endocardium, but dispensable for the formation
of vessels in mice (Puri et al., 1999). Hedgehog signaling is
required for the specification and differentiation of endocardial
progenitors in zebrafish (Wong et al., 2012). Scl/tal1 is not
needed for the formation of endocardial progenitors but is
essential for endocardial cell migration (Bussmann et al., 2007).
While VEGF-induced NFATc1 activation promotes endocardial
cell proliferation for sustaining endocardial cell numbers in heart
valve development (Combs and Yutzey, 2009b; Graef et al.,
2001; Johnson et al., 2003; Lee et al., 2006), we have limited
knowledge on how endocardial cell proliferation is regulated
before and during the formation of the atrio-ventricular canal
(AVC) and cardiac valves. The common cardinal vein (CCV) is
on the yolk syncytial layer connecting the posterior cardinal vein
(PCV) and the sinus venosus of the heart. The CCV appears
,24 hours post-fertilization (hpf) and finishes remodeling
,72 hpf (Isogai et al., 2001).
Endocardial differentiation is associated with remarkable
morphological and molecular changes during cardiac valve
development in the zebrafish, chicken, and mouse (Beis et al.,
2005; Gitler et al., 2003; Hu et al., 2000; Markwald et al., 1975;
Mjaatvedt et al., 1998; Person et al., 2005; Timmerman et al.,
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2004). In zebrafish, AVC valve development progresses through
three major and continuous stages: first, AV endocardial cells
extend processes and invaginate into the space between the
endocardium and myocardium around three days post-
fertilization (dpf) (Rutenberg et al., 2006; Scherz et al., 2008);
second, a subset of AVC endocardial cells form endocardial
cushions through the epithelial–mesenchymal transition (EMT)
,6 dpf (Beis et al., 2005; Martin and Bartman, 2009); and third,
endocardial cushions are subsequently remodeled into mature
valve leaflets between 16 and 28 dpf (Martin and Bartman,
2009). RNA in situ analysis in mouse and zebrafish embryonic
hearts reveals that several genes are preferentially expressed in
the endocardial cushions/valves, including vegf, neuregulin,
erbB3, NFATc1, notch1, versican, bmp2/4, tbx2b, foxn4 and
pkd2 (Chi et al., 2008; de la Pompa et al., 1998; Del Monte et al.,
2007; Gitler et al., 2003; Just et al., 2011; Ranger et al., 1998;
Rivera-Feliciano and Tabin, 2006; Walsh and Stainier, 2001).
Analyzing these signaling pathways in mouse and zebrafish
mutants further supports their roles in endocardial cushion and
valve formation (Armstrong and Bischoff, 2004; Chi et al., 2008;
Combs and Yutzey, 2009a; Hinton and Yutzey, 2011;
MacGrogan et al., 2010; Scherz et al., 2008; Smith et al., 2011;
Totong et al., 2011). In the AVC of mouse embryos, myocardial
Bmp2/Tgfb2 and endocardial Notch1 activate Snail1, then
suppress VE-cadherin to promote the EMT and endocardial
cushion formation (for reviews. see Armstrong and Bischoff,
2004; Hinton and Yutzey, 2011; MacGrogan et al., 2010).
However, the cellular and molecular events underlying this
important process remain largely unknown, particularly how
notch signaling is regulated in the endocardium. In this work, we
investigated this important process by positional cloning of an
ethylnitrosourea (ENU)-induced genetic mutant, scotch tapete382
(scote382), and revealed simultaneous mutations of pku300 and
fbn2b in scote382, and their genetic interactions when endocardial
cells proliferate and differentiate into cardiac valves.
ResultsEndocardial and valve morphogenesis are abnormal in the
scote382 mutant heart
To gain insights into the molecular basis of cardiac valve
development, we analyzed the zebrafish genetic mutant scote382,
which was isolated from a large-scale ENU-induced mutagenesis
of the zebrafish genome for cardiovascular mutants (Chen et al.,
1996). Homozygous scote382 mutants were indistinguishable from
their siblings before 24 hpf, but had pericardial edema (Fig. 1B),
dilated heart chambers (Fig. 1D), and abnormal caudal veins in the
trunk (supplementary material Fig. S3B) at 48 hpf. Histological
sections revealed that scote382 mutants had abnormal AVC at
48 hpf (Fig. 1F), and failed to form cardiac valve leaflets at 6 dpf
(Fig. 1H), compared with wild-type siblings (Fig. 1E,G). By
taking high-speed movies of scote382 mutant hearts, we found that
major blood stream followed the direction from the inflow tract to
atrium, ventricle and outflow tract, but a fraction of blood cells
were pushed back from ventricle to atrium, suggesting the
presence of valve defects in the AVC (supplementary material
Movie 1). A systematic examination revealed that ,3%
homozygous mutant embryos with milder pericardial edema and
some circulation at 48 hpf survived to adulthood. Therefore, we
maintained both heterozygous and homozygous adult scote382
mutant zebrafish for subsequent experiments.
Fig. 1. Endocardial and cardiac valve defects
were found in scote382 mutant embryos.
(A,B) Brightfield views of live wild-type (A) and
scote382 mutant (B) embryos at 48 hpf. Note
pericardial edema and heart abnormality (black
arrowhead), as well as abnormal vessels in the
caudal trunk (red arrowhead). (C,D) Confocal
images of Tg(myl7:eGFP) transgenic wild-type
(C) and scote382 mutant (D) hearts at 48 hpf. Note
lack of constricted AVC and dilated heart in scote382
mutant (arrowhead). (E,F) Hematoxylin-eosin
staining of wild-type (E) and scote382 mutant
(F) hearts at 48 hpf on transverse JB4 sections.
Note lack of AVC constriction (arrowhead) in
scote382 mutant heart. (G,H) Valve leaflets
(arrowhead) were labeled and detected by the
Tg(kdrl:eGFP) transgene in wild-type (G), but not
in scote382 mutant (H) embryos at 6 dpf on
vibrotome sections across the heart.
(I–N) Endocardial markers fli1 (I,J) and tie1
(K,L) were expressed in wild-type (I,K) and scote382
mutant (J,L) embryos; and Tg(kdrl:eGFP) labeled
the endocardium in wild type (M) and scote382
mutant (N) embryos at 48 hpf. Note that
endocardial genes (fli1, tie1 and flk1) were reduced
in the atrium of scote382 mutants (J,L,N). Arrows
point to the AVC. a, atrium; v, ventricle.
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To identify cellular and molecular defects in scote382 mutants,we analyzed and compared the expression patterns of endocardial
and myocardial genes between scote382 mutants and controlsiblings. We found that endocardial fli1, tie1 and kdrl werealmost undetectable in the atria of scote382 mutants (Fig. 1J,L,N)while they were normally expressed in both the atria and ventricle
of control siblings at 48 hpf (Fig. 1I,K,M). The myocardial genesmyl7, nkx2.5 and tbx5 were normally expressed in scote382 mutants(supplementary material Fig. S1). To evaluate vascular
development in scote382 mutants, we crossed the Tg(kdrl:EGFP)transgene into wild-type and scote382 mutant zebrafish, and foundthat the overall vasculature formed in both the scote382 mutants and
the Tg(kdrl:eGFP) siblings (supplementary material Movies 6, 7).However, the organization of endothelial cells in the caudal veinwas disrupted in the scote382 mutants at 48 hpf (supplementarymaterial Fig. S3B). Together, our data suggested that primary
endocardial and endothelial cell defects led to abnormal cardiacvalve and caudal vein development in scote382 mutants.
A novel gene pku300 is responsible for the scote382
mutant locus
For genetic analysis, we collected ,25% homozygous mutants
with both abnormal heart and caudal vein from heterozygousscote382 mutant zebrafish crosses. We mapped the scote382 locuson linkage group 22 by bulk segregation analysis withmicrosatellite markers. Fine mapping determined the genetic
interval between Z10673 and Z33723 by genotyping 1632informative meioses. Further genetic and physical walkingidentified closer flanking genetic markers 9 (1/1632) and 6 (2/
1632), as well as marker 7 (0/1632) (Fig. 2A). Therefore, thescote382 locus was defined between markers 9 and 6, whichare covered by the contigs CR751224.20, CR394531.15,
CU570881.11, CU582903.5, CU207311.10, CU929300.5 andCU076074.3 in Zv9 on the Zebrafish Genome Browser Gateway.We identified the four genes calreticulin (calr), LAG1 longevity
assurance homolog 4 (trh1), 182 (ZDB-GENE-070705-222 orENSDART00000147116) and calcium homeostasis endoplasmic
reticulum protein (cherp) from this genetic interval. A recentreport showed that fbn2b is mutated in the scote382 locus
(Mellman et al., 2012), however fbn2b was located outside ofthis genetic interval on our genetic mapping panel (Fig. 2A).
To search for mutations in scote382 mutant embryos, we
isolated full-length coding regions of the four genes by RT-PCR.The 182 gene is predicted as ENSDART00000147116 in theEnsembl. Isolating 182 cDNA from zebrafish embryos at 24 hpf
revealed that the correct 182 mRNA transcript contained 1976nucleotides encoding a peptide with 540 amino acids(supplementary material Fig. S7). A C946T mutation in exon 3led to a premature stop code (TAG), generating a truncated
protein with only 121 amino acids (Fig. 2B). This nonsensemutation was also confirmed by sequencing genomic DNA ofscote382 mutants (Fig. 2C). These findings suggested that the 182
gene was responsible for the scote382 mutant locus. We namedthis 182 cDNA pku300. pku300 is located within CU570881.11(contig 3) and fbn2b is within FP360035.6 (contig 15), where the
two genes are ,570 kb apart in Zv9 (Fig. 2A). UsingInterProScan, we found that the Pku300 protein had onetransmembrane domain, three extracellular immunoglobulin
(IgG) domains, and a short 19-amino-acid cytoplasmic tail(Fig. 2D), suggesting that Pku300 may be a receptor or co-receptor on the cell surface. By blast searching in the human
genome with the zebrafish Pku300 protein as a query, we
identified several homologs of IgG-containing proteins such as
CD44, the hyaluronic acid receptor. However, the sequence
Fig. 2. Positional cloning of pku300 from the scote382 locus in zebrafish.
(A) The scote382 locus was mapped on linkage group 22 by bulk segregation
analysis with microsatellite markers. Note that the scote382 locus, which
contains calr, trh1, pku300 and cherp genes, was determined between markers
6 and 9 by genotyping 1632 informative meiosis using SLP and SNP methods.
A G3935T missense mutation of fbn2b is reported to be responsible for the
scote382 locus (Mellman et al., 2012), but fbn2b is located outside of the
genetic interval in our mapping cross DNA panels. fbn2b is about 570 kb
away from pku300. (B) pku300 consists of 6 exons and encodes a 540-amino-
acid protein. Note that a C946T mutation in exon 3 of pku300 led to a
premature stop code (TAG) in the scote382 allele (arrowhead). (C) Sequencing
traces of wild-type and homozygous scote382 mutant embryos. Note a single
base pair change from C to T in the scote382 allele (arrowhead). (D) Pku300
protein was predicted to contain three extracellular IgG domains, a
transmembrane domain and a short intracellular tail. (E–G) pku300 was
broadly expressed, including in the heart (arrowhead) during early
embryogenesis. Staged wild-type 24 (E), 48 (F) and 48 hpf (G) hearts were
subjected to in situ hybridization with anti-sense pku300 riboprobe.
(H) pku300 was expressed in day-2 Tg(myl7:eGFP) embryonic hearts by RT-
PCR. (I) pku300 was reduced in cloche (clo) mutant embryos that have no
endocardial cells. RNA was prepared from siblings (sib) and clo mutant
embryos (clo) from heterozygous clochem39 crosses. (J) Pku300-mCherry
fusion proteins were transiently expressed in the heart of a Tg(kdrl:EGFP)
transgenic embryo. Note the red Pku300-mCherry fusion protein localization
on cell membranes (arrowhead), which was not overlapped with cytoplasmic
localization of EGFP. Pku300-mCherry was driven by the zebrafish kdrl
promoter. a, atrium; v, ventricle. Scale bar: 30 mm.
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homology is very low between zebrafish Pku300 and human
CD44, reflecting the sequence diversity in this family of IgG-
domain-containing proteins across species. We found that pku300
mRNA was widely expressed, including in the heart, during
zebrafish embryogenesis by in situ hybridization (Fig. 2E–G).
Also, RT-PCR showed that pku300 was expressed in the heart
(Fig. 2H), and was reduced in clochem39 mutant embryos that
lack endothelial/endocardial lineages (Fig. 2I), showing that
pku300 is expressed in the endocardial and endothelial cells.
Endocardial expression of the fusion gene pku300-mCherry
revealed a cell membrane localization (Fig. 2J), suggesting that
Pku300 is a cell membrane protein.
To investigate whether both pku300 and fbn2b are responsible
for the phenotypes of scote382 mutants, we carefully evaluated
and classified the mutants into two groups by analyzing 2341
offspring from heterozygous scote382 mutant crosses (Fig. 3;
supplementary material Table S2). Group I mutants represented
6.7% of the total offspring and had defects only in the heart,
lacking most of the Tg(kdrl:EGFP) atrial endocardium (Fig. 3C;
supplementary material Fig. S4C). Group II mutants represented
25.5% of the total offspring and had defects in the heart, lacking
most of the Tg(kdrl:EGFP) atrial endocardium, and abnormalities
in the CCV (Fig. 3B; supplementary material Fig. S4B) and the
caudal vein (supplementary material Fig. S3B; supplementary
material Fig. S5B). Wild-type siblings represented 67.8% of the
total offspring (Fig. 3A; supplementary material Fig. S3A;
supplementary material Fig. S4A; supplementary material Fig.
S5A). These data suggested that more than one gene contribute to
the phenotypes of scote382 mutants. Indeed, sequencing analyses
revealed that group I mutants were all heterozygous for both
genes; group II mutants were all homozygous for both genes; and
wild-type siblings were 1) heterozygous for both genes, 2)
heterozygous for pku300, 3) heterozygous for fbn2b, or 4) wild-
type (supplementary material Table S2). Furthermore,
simultaneous knockdown of fbn2b and pku300 with low-dose
morpholinos led to higher percentages of morphants resembling
group II mutants (supplementary material Fig. S2D), while each
applied separately was less potent (supplementary material Fig.
S2B,C). These data strongly suggest a genetic interaction
between pku300 and fbn2b in regulating the development of
the atrial endocardium.
To further investigate the roles of pku300 and fbn2b in scote382
mutants, we applied loss-of-function and gain-of-function
analyses of the two genes in zebrafish embryos. First, we
attempted to isolate pku300+/2 and fbn2b+/2 zebrafish lines from
the scote382 mutant line by segregating the pku300 and fbn2b
mutant loci. We detected either pku300+/2 or fbn2b+/2 mutant
embryos from wild-type offspring of heterozygous scote382
crosses, suggesting that the two loci can be segregated
(supplementary material Table S2). By sequencing 64 adult
offspring of heterozygous scote382 mutant crosses, we only
isolated a pair of pku300+/2 adult zebrafish, named scopku300.
The pku300+/2 mutants lost most of the Tg(kdrl:EGFP) atrial
endocardium and common cardinal vein (Fig. 3E) but had mild
defects in the caudal vein (supplementary material Fig. S3E),
compared with their controls (Fig. 3D; supplementary material
Fig. S3D). We designed a morpholino targeting the ATG of
pku300 (MO1), and a morpholino targeting the first exon splicing
donor site of pku300 (MO2). Microinjections of either MO1 or
MO2 into embryos phenocopied the scote382 mutant phenotypes
in the heart (Fig. 3G). MO1 appeared to be more potent in
knocking down pku300 function. Therefore we used MO1 to
carry out the rest of the experiments, unless otherwise specified.
pku300MO knockdown led to very few Tg(kdrl:EGFP)
endocardial cells (Fig. 3G), little tie1 or fli1 expression (data
not shown) in the atrial endocardium, an abnormal CCV
(Fig. 3G), and mild defects in the caudal vein (supplementary
material Fig. S3G). Consistent with the previous report (Mellman
et al., 2012), we found that fbn2bMO morphants lost most of the
Tg(kdrl:EGFP) atrial endocardium but had no defect in the
Fig. 3. Mutations of both pku300 and fbn2b contributed to
scote382 mutant phenotypes. The endocardium in the atrium,
ventricle and CCV were labeled by the Tg(kdrl:EGFP) transgene.
The endocardium was projected by red pseudocolor with Imaris
software. (A–C) 2341 offspring from heterozygous scote382
mutant crosses produced 25.5% mutants that had defects in the
atrial endocardium and CCV (B), 6.7% mutants that had only
reduced atrial endocardium (C), and 67.8% siblings that were
phenotypically normal (A), suggesting more than one gene
mutated in the scote382 locus. Sequencing confirmed that the
mutants (group II) with defects in the heart (endocardium and
CCV) and tail (caudal vein) (supplementary material Fig. S3)
were fbn2b2/2 and pku3002/2 (B), and the mutants (group I)
with defects only in the heart (endocardium) were fbn2b+/2 and
pku300+/2 (C). (D–F) 297 offspring from heterozygous scopku300
mutant crosses produced 25.5% mutants with defects in the atrial
endocardium and CCV (E) and 74.6% normal siblings (D), which
was supported by morpholino knockdown of pku300 with 9.6 ng
pku300MO (F). (G–I) A fbn2bMO (1.6 ng) morphant showed
reduced atrial endocardium but had normal CCV (I), compared
with the control (H) and a pku300 morphant (G). a, atrium; v,
ventricle. Scale bars: 50 mm.
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common cardinal vein (Fig. 3I) and defect in the caudal vein
(supplementary material Fig. S3I), compared with their controls
(Fig. 3H; supplementary material Fig. S3H). These data suggest
critical roles of pku300 in the development of the atrial
endocardium, the CCV, and the caudal vein, as well as fbn2b
in the development of the atrial endocardium and caudal vein.
We then asked whether overexpression of pku300 can rescue the
mutant phenotypes of scopku300 or scote382. Microinjection of pku300
mRNA rescued the Tg(kdrl:EGFP) atrial endocardium and common
cardinal vein of a pku3002/2 mutant (Fig. 4C), compared with an
uninjected mutant (Fig. 3E). Nine pku3002/2 mutants out of 182
offspring from heterozygous scopku300 crosses had little or no rescue
(Fig. 4B), showing that overexpression of pku300 mRNA had
rescued most of the pku3002/2 mutants. The genotypes of pku300
mutants and siblings were confirmed (data not shown). On the other
hand, we failed to rescue mutant phenotypes of scote382 byoverexpression of pku300 mRNA (data not shown). To explore
pku300 transgenic rescue, we generated Tg(kdrl:pku300) transgeniczebrafish where pku300 was overexpressed in the endocardium andendothelial cells. We found that the Tg(kdrl:pku300) transgenerescued endocardial Tg(kdrl:EGFP) expression in the atrium of 24
out of 89 (27%) Tg(kdrl:pku300);scote382/te382 mutant embryos(Fig. 4F,G), while there was no rescue in the scote382/te382 mutant(Fig. 4E). We also generated Tg(myl7:pku300) transgenic zebrafish
that overexpressed pku300 in the myocardium, but none of33 Tg(myl7:pku300);scote382/te382 mutant embryos showedTg(kdrl:EGFP) expression in the atrium. They did not show
additional defects (data not shown). The genotypes of scote382/te382
mutants and siblings as well as transgenic Tg(kdrl:pku300) andTg(myl7:pku300) were confirmed (data not shown). These resultsdemonstrate that pku300 is responsible for scote382 and plays a role in
the development of the endocardium, common cardinal vein andcaudal vein.
Proliferation of endocardial cells gradually declines duringheart looping and chamber formation in scote382 mutants
Previous studies reported that endocardial cells are specified in the
anterior lateral plate mesoderm in early somite embryos, thenmigrate and fuse in the midline ,18 hpf, and turn leftward at,20 hpf (Schoenebeck et al., 2007; Bussmann et al., 2007; Wonget al., 2012). We did not see any abnormality, including formation
and patterning of the endocardium within the heart tube in scote382
mutants at 24 hpf. Consistently, Tg (kdrl: EGFP) endocardial cellsmigrated properly to the heart tube in the midline and then turned
toward the left side in scote382 mutants (supplementary materialMovie 3), as in wild-type embryos (supplementary material Movie2). Live imaging suggested that increased Tg(kdrl:EGFP)
endocardial cells came from cell proliferation and migration alongthe looped heart tube. Those Tg(kdrl:EGFP) endocardial cellscoming from outside the heart tube mostly contributed to the sinus
venosus (supplementary material Movies 2, 4). We did not detectendocardial cell apoptosis in scote382 mutant and wild-type embryosby TUNEL assays (data not shown). These findings suggest that thereduced numbers of atrial endocardial cells were not due to defects in
endocardial cell migration or apoptosis in scote382 mutants. Thereforewe hypothesized that the reduced numbers of atrial endocardium of48 hpf mutant embryos result from defects of endocardial cell
proliferation. By using Tg(fli1a:nuEGFP)y7 transgenic labeling(Roman et al., 2002), we revealed that endocardial cells failed todivide in scote382 mutants from 29 to 36 hpf (supplementary material
Movie 5), compared with an average of 12 dividing endocardial cells(13.3% proliferation rate) in the heart tube of wild-type embryos(supplementary material Movie 4). A set of representative images,from one of the three experiments, showed that there was one
dividing cell between 29 hours 30 minutes (Fig. 5A) and 29 hours40 minutes (Fig. 5B), and three dividing cells between 29 hours40 minutes and 29 hours 50 minutes (Fig. 5C) in wild-type
embryos. In contrast, we did not see any dividing cells in scote382
mutants at the same time points (Fig. 5D–F). The ratio ofproliferating to total endocardial cells was much smaller in scote382
mutants than in wild-type siblings (Fig. 5G). Importantly, we foundthat the numbers of endocardial cells in the atrium and the wholeheart were reduced, while they were slightly increased in the
ventricle, in scote382 mutants compared with wild-type siblings at48 hpf (Fig. 5H–J). In contrast, the numbers of myocardial cells inthe ventricle and atrium were slightly increased in scote382 mutants
Fig. 4. Both pku300 mRNA and transgenic expression of pku300 in the
endocardium rescued the respective mutant phenotypes of scopku300 or
scote382. (A–C) The endocardium in the atrium, ventricle and CCV were labeled
by Tg(kdrl:eGFP). The endocardium was projected as red pseudocolor with
Imaris software. Compared with a wild-type sibling (A), pku300 mRNA rescued
the atrial endocardium and CCV of a scopku300 mutant embryo (C). Nine
homozygous scopku300 mutants out of 182 offspring from heterozygous scopku300
crosses showed little or no rescue in the atrial endocardium and CCV (B). Wild-
type sibling (A) and homozygous scopku300 mutants (B,C) were confirmed by
sequencing. Red arrowheads point to the endocardium. (D–G) The endocardium
was labeled by Tg(kdrl:eGFP) in the atrium and ventricle of wild-type embryos
(D). Note little endocardium in the atrium of scote382 mutant Tg(kdrl:eGFP)
embryos (E), which were partially rescued by overexpressing pku300 using
Tg(kdrl:pku300) transgene (F). (G) Higher magnification of the atrium of panel F
shows the rescued atrial endocardial cells (red arrowheads). The atrium and
ventricle are demarcated with white arrowheads (D–F). a, atrium; v, ventricle;
WT, wild type. Scale bars: 50 mm.
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compared with wild-type siblings at 48 hpf (Fig. 5K–M). By using
BrdU labeling or immunostaining with anti-pH3 we also found that
endocardial cells gradually failed to enter mitotic phases in scote382
mutant hearts from 23 to 33 hpf (supplementary material Fig.
S6E,F). Representative images showed that there were more dividing
endocardial cells in wild-type (supplementary material Fig. S6A,C)
than in scote382 mutant (supplementary material Fig. S6B,D)
embryos at 25 hpf and 30 hpf. Interestingly, we did not find
defects in vascular endothelial cell proliferation in the trunk of
scote382 mutants (supplementary material Movie 7), compared with
wild-type embryos (supplementary material Movie 6) from 23 to
33 hpf. Together, these results suggest that pku300 and fbn2b are
required for endocardial cell proliferation during heart looping and
chamber formation.
Endocardial Notch signaling is defective in the AVC of
scote382 mutant
The presence of cardiac valve defects in scote382 mutants urged us
to address the underlying molecular mechanisms. A previous
report showed that Notch1b and bmp4 are restricted in the
respective endocardium or myocardium of the AVC during heart
valve development in zebrafish (Walsh and Stainier, 2001).
Endocardial notch 1b and its ligand dll4 were weakly expressed
in the ventricle at 48 hpf and gradually restricted within the AVC
from 48 to 72 hpf (Fig. 6A–C,M,N; data not shown), however
they were ectopically expressed in the ventricle of scote382 mutant
hearts (Fig. 6D–F,O,P). In contrast, myocardial bmp4 were
normally expressed in the inflow tract, AVC and outflow tract
in both wild-type (Fig. 6G–I) and scote382 (Fig. 6J–L) embryos
from 48 to 72 hpf. To assess whether the endocardial–
mesenchymal transition (EMT) is affected in scote382 mutants,
we examined the expression of secreted phosphoprotein 1 (spp1),
a previously known factor that is upregulated in the AVC
endocardial cushion in zebrafish (Just et al., 2011; Peal et al.,
2009). We found that spp1 was not expressed in the AVC (red
arrowhead) while was reduced in the outflow tract (black
arrowhead) of a scote382 mutant (Fig. 6R), compared with those
in a wild-type embryo (Fig. 6Q), suggesting that EMT was also
Fig. 5. Atrial Tg(fli1:nuEGFP) endocardial numbers were reduced while ventricular cardiomyocyte numbers were increased in scote382 mutant embryos.
(A–F) Endocardial nuclei were labeled by Tg(fli1:nuEGFP) in wild-type (wt) (A–C) and scote382 mutant (D–F) embryos at 29 hours 30 minutes, 29 hours
40 minutes and 29 hours 50 minutes post fertilization. A representative set of time-lapse images showed one divided cell from 29 hours 30 minutes to 29 hours
40 minutes (1 in B), and three divided cells from 29 hours 40 minutes to 29 hours 50 minutes (2, 3 and 4 in C) in wild-type Tg(fli1:nuEGFP) embryos. Note that
there were no divided cells from 29 hours 30 minutes to 29 hours 50 minutes in scote382 mutant embryos (D–F). (G) The endocardial proliferation rate
(proliferating endocardium from 29 to 36 hpf to the total endocardium at 29 hpf) in scote382 was very low, compared with their siblings. n53; mean 6 s.e.m.;
Student’s t-test. (H–J) The endocardium, labeled by Tg(fli1:nuEGFP), was divided into ventricular, AVC and atrial parts. Note fewer endocardial cells in the
mutant AVC and atrium (I–J). (K–M) Myocardial nuclei were labeled by Tg(myl7:nuDsRed). Note more cardiomyocytes in the mutant ventricle (L–M). (J,M)
n58–12; mean 6 s.e.m.; Student’s t-test. Scale bars: 30 mm (A–F); 50 mm (H–L).
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affected in scote382 mutants. Together, these data support a notion
that the compromised expression of AVC endocardial and valve
genes led to abnormal development of the endocardial cushion
and cardiac valve in scote382 mutants.
We then asked whether endocardial notch signaling was
accordingly changed in the AVC of scote382 mutants. The Notch
reporter zebrafish line Tg(Tp1:mCherry) is generated in which
mCherry is placed downstream to 12 repeated RBP-Jk sites
(Parsons et al., 2009). To determine Notch signaling in scote382
mutant hearts, we crossed the Notch reporter transgene into
scote382 mutant zebrafish to make scote382/te382; Tg (kdrl:eGFP);
Tg (Tp1:mCherry) double transgenic mutant zebrafish. In wild
type Tg (kdrl: eGFP) transgenic embryos, we found endocardial
Notch signaling was initially activated in the endocardium of the
AVC and ventricle at 48 hpf (Fig. 7A), and gradually
concentrated in the AVC at 60 hpf and 72 hpf (Fig. 7C,E).
However, Notch signaling was not activated in the endocardium
of the AVC of scote382 mutant hearts (Fig. 7B,D,F) while was
ectopically activated in the ventricles of scote382 mutant hearts at
60 and 72 hpf (Fig. 7D,F), which is consistent with dll4 and
notch1b ectopic expression in mutant ventricles (Fig. 6D–F,O,P).
In particular, Tg(Tp1:mCherry) positive cells did not overlap
with the endocardial cells (Fig. 7D,F), suggesting that the Notch
signaling might be ectopically activated in the myocardium in
scote382 mutants. These data support the idea that pku300 and
fbn2b are required for the endocardial Notch signaling during
cardiac cushion and valve development in zebrafish.
Endocardial cell adhesion and tight junctions areectopically expressed during endocardial cushion
development in scote382 mutants
During the first three days of development, endocardial
invagination is formed as a temporary structure in the AVC to
secure directional blood flow in the heart, and EMT might take
place at larval stages after 6 dpf (Martin and Bartman, 2009;
Fig. 6. Cardiac valve genes were abnormally expressed in the AVC of scote382 mutant hearts. (A–R) Wild-type (A–C,G–I, M–N,Q) and scote382
(D–F,J–L,O–P,R) embryos were analyzed by in situ hybridization with cardiac valve markers notch1b (A–F), bmp4 (G–L), dll4 (M–P) and spp1 (Q–R) at 48 hpf
(A,D,G,J), 60 hpf (B,E,H,K,M,O), and 72 hpf (C,F,I,L,N,P–R). Note that endocardial notch1b and dll4 were ectopically expressed in the ventricle of scote382
mutants but myocardial bmp4 was not affected in scote382 mutants from 48 to 72 hpf. spp1, an endocardial–mesenchymal transition marker gene, was not
expressed in the AVC of scote382 mutants (red arrowhead in R). The atrium and ventricle are demarcated with red arrowheads. Black arrowheads (Q,R) point to the
outflow tract. a, atrium; v, ventricle; wt, wild type. Dotted lines outline the heart.
Fig. 7. Notch signaling was not activated in the AVC of scote382 mutant
hearts. (A–F) Tg(Tp1bglob:hmgb1-mCherry)jh11, called Tg(Tp1:mCherry),
transgenic zebrafish (Parsons et al., 2009) were bred with scote382/te382;
Tg(kdrl:eGFP) transgenic zebrafish to generate scote382/+; Tg(Tp1:mCherry)/+;
Tg(kdrl:eGFP)/+ zebrafish. Images were taken with agarose-embedded
embryos using Zeiss 700 upright confocal microscope. Endocardial cells
labeled by Tg(kdrl:eGFP) were used to project the heart shapes. Note that
Tg(Tp1:mCherry) were gradually restricted in the AVC of wild-type hearts at
48 hpf (A), 60 hpf (C) to 72 hpf (E), but were ectopically expressed in the
ventricle of scote382 mutant hearts at 48 hpf (B), 60 hpf (D) and 72 hpf (F). a,
atrium; v, ventricle. Red arrowheads point to the AVC.
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Scherz et al., 2008). However, it is unclear how the endocardial
invagination is related to the cardiac valve during heart
development, and what are the underlying cellular and molecular
mechanisms engaging in cardiac valve development during the
first three days. EMT is normally involved in cell adhesion, cell
junctions and cell polarity (Combs and Yutzey, 2009a). From the
previous studies (Beis et al., 2005; Just et al., 2011; Peal et al.,
2009) and our data (Fig. 6Q), endocardial cushion and the EMT
might take place during the first three days of development
in zebrafish. We found endocardial cell adhesion gene cdh5
(VE-cadherin) was expressed in the whole heart at 48 hpf,
downregulated and restricted in the AVC of wild type hearts
from 60 to 72 hpf (Fig. 8A,C,E), while it remained ectopically
expressed in the endocardium of scote382 mutant ventricles from 48
to 72 hpf (Fig. 8B,D,F). To address morphological changes of cell
junctions, we assessed ZO1 expression during the first three days
of development by immunostaining. We found that ZO1 was
normally expressed around the AV endocardial cells at 48 hpf
(n513) and was gradually reduced at 60 hpf (n512) and 72 hpf
(n515); ZO1 was not expressed in the basal part of the
endocardium of wild type embryos at 60 hpf and was not
expressed in both basal and epical parts of the endocardium of
wild type embryos at 72 hpf (Fig. 8G,I,K). However, in scote382
mutant embryos at 48 hpf (n518), ZO1 was slightly upregulated;
at 60 hpf (n57) and 72 hpf (n511), ZO1 was abnormally
expressed around endocardial cells in the AV canal, and was
particularly upregulated in the basal side of endocardial cells in the
AVC (Fig. 8J,L). These data suggest that abnormal endocardial
cell adhesion and tight junction likely interfered endocardial EMT
or morphological changes that are required for heart valve
formation in scote382 mutant hearts.
DiscussionOur major findings in this work were (1) positional cloning of
scote382 as a novel gene, pku300, that encodes a putative
transmembrane protein, and mutations of both pku300 and
fbn2b contribute to the defects in the atrial endocardium and
caudal vein in scote382 mutants; (2) deciphering pku300 as a novel
player in endocardial cell proliferation; and 3) demonstrating the
critical roles of pku300 and fbn2b in regulating endocardial
Notch signaling, endocardial cell adhesion and tight junctions
during valve morphogenesis. Our data support the hypothesis that
endocardial cell proliferation is critical for correct lining of the
endocardium during heart tube expansion and chamber
formation. Also we demonstrated that dramatic morphological
changes and possible endocardial EMT in the AVC take place in
the first three days of heart development in zebrafish. Both
pku300 and fbn2b play essential roles in this important process in
the AV endocardial cells, partly by regulating the temporo-spatial
patterning of endocardial Notch signaling.
By positional cloning, we clearly demonstrated that scote382
encoded a novel putative transmembrane protein by revealing a
Fig. 8. Endocardial cell adhesion and tight junctions were ectopically expressed in the AVC of scote382 mutant hearts. (A–F) Endocardial cell adhesion gene cdh5/
ve-cadherin was analyzed by in situ hybridization in wild-type embryos at 48 hpf (A), 60 hpf (B) and 72 hpf (C); and scote382 mutant embryos at 48 hpf
(D), 60 hpf (E) and 72 hpf (F). Note gradual restriction of cdh5 in the AVC in wild-type embryos and ectopic expression of cdh5 in the ventricle of scote382 mutant
embryos. Red arrowheads point to the AVC. (G–L) Wild-type and scote382 mutant Tg(kdrl:EGFP) transgenic embryos on vibratome sections were subjected to
immunostaining with anti-ZO1 antibody. Note that ZO1 was expressed in the AVC of wild-type hearts at 48 hpf (G) and downregulated at 60 (I) and 72 hpf
(K), but remained in the AVC of scote382 mutant hearts at 48 (H), 60 (J) and 72 hpf (L). a, atrium; v, ventricle; arrowhead points to the AVC. Dotted lines outline the heart.
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nonsense mutation in scote382 mutants, phenocopying scote382
mutant phenotypes by antisense pku300 morpholino knockdown,
rescuing scote382 mutant phenotypes by overexpressing the wild-type pku300 gene in the endothelium and endocardium, anddetermining pku300 mRNA expression in the endocardium. Asreported recently (Mellman et al., 2012), we also found the
G3935T missense mutation of fbn2b in our scote382 mutant.Mutational analyses of fbn2b and pku300 allowed us to furtherclassify two groups of mutants in scote382, where group I mutants
were heterozygous for both genes and group II mutants werehomozygous for both genes (supplementary material Table S2;Fig. 3A–C). But only ,13% of embryos that were heterozygous
for both genes showed group I mutant phenotypes. Together withthe work by Mellman et al., our data support the conclusion thatsimultaneous mutations of both fbn2b and pku300, which were,570 kb apart in Chromosome 22, contributed to the defects in
scote382 mutants. This very rare event, presumably induced byENU, provides an important lesson that the possibility of morethan one causative gene mutation should be explored if the
mutant phenotypes from a particular locus are not identical.Furthermore, the genetic interaction between fbn2b and pku300
will guide future studies on how the two proteins interact in
regulating endocardial cell proliferation.
The Pku300 protein was predicted to contain threeextracellular IgG domains, a single transmembrane domain, anda short intracellular tail without any conserved kinase motifs
according to InterProScan (http://www.ebi.ac.uk/interpro/scan.html), which is similar to a large class of cell adhesion moleculesand receptors, including CD44, one of the hyaluronan receptors
(Bechara et al., 2007). Blast searching with human CD44 proteinas bait did not identify any potential homologs in the zebrafishgenome. IgG-domain-containing proteins are generally sequence
diversified. Our current hypothesis is that Pku300 functions as ahyaluronic acid (HA) receptor that participates in cardiac jellyformation and then modulates Notch, Fbn2b and other signaling
pathways during heart valve development. However, true Pku300protein homologs in other vertebrates remain to be identified.
Cardiac jelly is the extracellular matrix between endocardiumand myocardium, and its major components are
glycosaminoglycans (GAGs) and proteoglycans. GAGs arelong-chain sulfated sugar polymers of three subtypes:chondroitin sulfate (CS), heparan sulfate (HS) and HA. The
generation and breakdown of cardiac jelly are preciselycontrolled, as they are essential for proper heart development.Mice lacking hyaluronan synthase-2, the enzyme required for the
production of mucopolysaccharide hyaluronan, do not developtrabeculae and have significantly reduced cardiac jelly(Camenisch et al., 2000; Mjaatvedt et al., 1998). This is mainlydue to the failure of the cardiac endothelium to undergo EMT
with inactivation of ERBB2/ERBB3 and RAS signaling(Camenisch et al., 2002). Hyaluronan binds to either two majorreceptors (CD44 and RHAMM) or other receptors at the cell
surface (Toole, 2004). In zebrafish, ENU-induced mutagenesisscreens have led to the identification of many valve mutants, ofwhich two are known to have cardiac jelly defects: scotch tape
and jekyll (Chen et al., 1996; Walsh and Stainier, 2001). Jekyll isa null mutation of UDP-glucose dehydrogenase, which isrequired for the synthesis of HS, CS, and HA (Walsh and
Stainier, 2001). Jekyll mutants indeed show abnormal expressionof several valve markers at 48 hpf, suggesting that cardiac jelly isinvolved in heart valve development at very early embryonic
stages. As reported previously (Chen et al., 1996), we foundcardiac jelly was reduced in scote382 mutant hearts, however, CS,
a component of cardiac jelly, was normally expressed in thesemutants (data not shown). The other two components, HS andHA, need to be examined in scote382 mutant hearts when the
required assay reagents become available. Identifying the roles ofthe pku300 and fbn2b genes here will allow further study of themolecular basis of cardiac jelly defects and their relationship toheart valve development.
Our data suggest that both pku300 and fbn2b are required forendocardial cell proliferation but are dispensable for vascular
endothelial cell proliferation in the trunk. To our knowledge, thisis one of the very few reports on identifying regulators inendocardial cell proliferation. Previous studies have shown thatmyocardial Nfat2/3/4 represses Vegf expression in the AVC that
allows local EMT, and that later endocardial Calcineurin-Nfatc1promotes valve elongation and remodeling in mice (Chang et al.,2004). Early studies suggested that VEGF-Nfatc1 is essential for
endocardial cell proliferation (Lee et al., 2006; Combs andYutzey, 2009b). On the other hand, others reported that VEGF isdispensable for endocardial cell proliferation, cell death and
Nfatc1 nuclear localization in the endocardial cells of elongatingmitral valves (Stankunas et al., 2010). Therefore, the underlyingsignaling pathways that regulate endocardial cell proliferationremain largely unknown. Identifying pku300 and fbn2b as
essential genes for endocardial cell proliferation will likely helpto further delineate related molecular mechanisms in the future.
As in jekyll mutants, cardiac valve genes were abnormallyexpressed in the AVC of scote382 mutants. Previously, Hey1 andHey2 were shown to suppress Bmp2/4 and then Tbx2 expressionin the heart chambers, so Bmp2/4 and Tbx2 are restricted to the
myocardium of the AVC where Hey1 and Hey2 are not expressed(Fischer et al., 2007; Kokubo et al., 2007; Rutenberg et al., 2006).On the other hand, it remains unknown how endocardial notch1 is
regulated in the AVC. Here, we found that endocardial Notchsignaling was not activated and dll4/notch1b were not properlyexpressed in the AVC of scote382 mutant hearts, suggesting that
pku300 and fbn2b modulate Notch signaling during endocardialcell proliferation and valve development. Ectopic Notch activity,particularly in the ventricle of scote382, might promote ventricular
myocyte proliferation, which is consistent with the finding ofmore myocytes in the mutant ventricle (Fig. 5K–M) and Notchactivation of cell cycle entry and proliferation in mammaliancardiac myocytes (Campa et al., 2008). Previous studies suggest
that the TGFb/BMP and Notch signaling pathways are essentialfor endocardial EMT and valve development (Combs andYutzey, 2009a; MacGrogan et al., 2010; Rivera-Feliciano and
Tabin, 2006; Timmerman et al., 2004). In addition,overexpression of a constitutively active Notch intracellulardomain in zebrafish embryos increases endocardial cell
proliferation (Timmerman et al., 2004). The lack of endocardialNotch signaling and the endocardial EMT marker spp1 in theAVC substantiates the roles of fbn2b and pku300 function in
valve development. Therefore, our work provides anotherimportant entry point to determine endocardial signaling incardiac valve morphogenesis.
Materials and MethodsZebrafish strains
scote382 is a cardiac valve mutant isolated from an ENU-induced mutagenesisscreen in Tubingen (Chen et al., 1996). We acquired this mutant fish from MarkFishman’s laboratory at Massachusetts General Hospital, Boston. Both
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heterozygous and homozygous scote382 fish were maintained. Transgenic lineTg(kdrl:EGFP) that EGFP was driven by the zebrafish kdrl promoter (Choi et al.,2007; Cross et al., 2003) was crossed with heterozygous scote382/+ fish. Individualfish of Tg(kdrl:EGFP)/+, scote382/+ and Tg(kdrl:EGFP);scote382/+ were identified.Heterozygous scopku300/+ was derived from offspring of heterozygous scote382/+
crosses. Tg(myl7:EGFP) and Tg(myl7:nuDsRed) lines were from Geoffrey Burnsat Massachusetts General Hospital (Burns et al., 2005). Tg(fli1:nuEGFP) wasprovided by Feng Liu at Institute of Zoology, Chinese Academy of Sciences(Roman et al., 2002). The Notch reporter transgenic line Tg(Tp1:mCherry) wasobtained from Dr Michael Parsons, Johns Hopkins University (Parsons et al.,2009). Tg(kdrl:pku300-mCherry), Tg(kdrl:pku300) and Tg(myl7:pku300)transgenic zebrafish lines were generated by using Tol2-based transgenesis asdescribed (Kawakami et al., 2004). Individual fish of Tg(kdrl:pku300)/+;scote382/te382
and Tg(myl7:pku300);scote382/+ were isolated from crosses between scote382/te382 andtransgenic zebrafish. Zebrafish were raised and handled in accordance with the PekingUniversity and Massachusetts General Hospital institutional guidelines.
Positional cloning of pku300 gene
About 25% siblings from heterozygous scote382 mapping zebrafish were mutantswith defects in both the heart and caudal vein, which were sorted for generatingour mapping DNA panels. The scote382 locus was mapped between themicrosatellite markers Z10673 and Z33723 on chromosome 22 using bulksegregation analysis. Using sequence length polymorphism (SLP) and singlenucleotide polymorphism (SNP) markers (supplementary material Table S1), wenarrowed down the scotch tape genetic interval into about 0.18 cM betweenmarkers 6 and 9. SLP markers were assayed by agarose gel electrophoresis of PCRproducts. SNP markers were analyzed by sequencing PCR products. Marker 6 wasin the 4th intron of pku300. The scote382 genetic interval was covered andsequenced in the assembled zebrafish genome sequence by the Sanger Institute,UK, where four genes were determined, including calr, trh1, pku300 and cherpgenes. fbn2b was ,570 kb away from pku300.
Morpholinos and capped mRNA Injections
Two antisense morpholinos were synthesized to target the zebrafish pku300 ATGsite (pku300MO1): AAATCATACCTCAGCGAAGCTCACA; the first splicingdonor site (pku300MO2): GTGTTGTAGCACCTGTAATTTTCA; fbn2b start sitetargeted morpholino (fbn2bMO): GCGACTCCTGAAGCGCCGGTAAATG(Mellman et al., 2012); and a control morpholino (CtrMO): GCAGC-GGGCACTGCTGGTGGAAGT (Gene Tools, LLC). The full length of wild typeand mutant pku300 cDNAs were cloned into T7TS vector and linearized, and cappedmRNA was synthesized using Ambion mMESSAGE mMACHINE mRNAtranscription synthesis kits. Morpholinos or mRNA were injected into 1- or 2-cell-stage wild-type or scote382 or scopku300 mutant embryos. Injected embryos wereincubated at 28.5 C for examination of phenotypes or were fixed for in situhybridization and immunostaining as described (Xiong et al., 2008). Photographswere taken using a Leica MZ16 fluorescence microscope.
RNA in situ hybridization and histology
In situ hybridization was done using antisense myl7, tbx5, nkx2.5, ve-cadherin, fli1,tie1, notch 1b, dll4, versican, bmp4, spp1, pku300 and tbx2b RNA probes.Histology was done by JB4 section according to a protocol by the manufacturer(Polysciences, Inc., Warrington, PA, USA).
Reverse transcription PCR
Wild type TL, scote382 and clochem39 mutant embryos were collected. Hearts fromTg(myl7:eGFP) embryos at 48 hpf were isolated under the florescencemicroscope. Total RNA was isolated from embryos and hearts using Trizolreagents (Invitrogen). First-strand cDNA was synthesized using the Superscript IIRT system (Invitrogen), followed by a standard PCR.
Endocardial cell labeling using Tg(fli1:nuEGFP) transgenic embryos
For endocardial cell labeling, one-cell Tg(fli1:nuEGFP) or Tg(kdrl:EGFP)transgenic embryos were microinjected with 0.96 ng of tnnt2aMO to stop heartbeating. For live imaging, embryos were embedded in 1.5% low-melting-temperature agarose with 0.16 mg/ml tricaine (Sigma) in E3 water. When theagarose cooled completely, extra agarose were removed leaving the embryo headfacing E3 water with tricaine. The embryos were imaged with Zeiss 700 confocalmicroscope, following up to 10 hours at 5–6 minutes time intervals using a 206water immersion objective. Z-stack movies were assembled in 3D time lapse usingImaris7.0 (Bitplane).
Cell proliferation assay
BrdU labeling for DNA synthesis and phosphorylation of histone 3 for cell cycleswere carried out to determine endocardial cell proliferation. For BrdU pulsechasing, scote382 mutants and siblings were incubated with 10 mM BrdU for10 hours, and then subjected to immunostaining with anti-BrdU (1:500, Sigma) asdescribed (Laguerre et al., 2005). For detecting phosphorylation of histone 3,
staged scote382 mutants and siblings were fixed and subjected to immunostainingwith anti-pH3 (1:500, Millipore).
Immunostaining of zebrafish heart
For anti-ZO1 antibody staining, all embryos were put into 0.12% tricaine (Sigma,St. Louis, USA) to stop and relax the heart before fixation. Embryos were fixed in4% paraformaldehyde (PFA) at room temperature for 2 hours, embedded in 3%low melting agarose gel and sectioned into 100 mm slices with a vibratome (LeicaVT 1,000s, Bannockburn, USA). Embryos were blocked with a blocking solution[10% normal goat serum (NGS) plus 2% blocking reagent (BR) (RocheDiagnostics, Indianapolis, USA) in maleic acid buffer (MAB) (150 mM maleicacid, 100 mM NaCl, pH 7.5)]. Immunostaining with anti-ZO1 (Invitrogen,Eugene, USA) (1:100) antibody was performed in an incubation solution (2%NGS plus 2% BR in MAB) at 4 C overnight. Goat anti-rabbit IgG(H+L) (VectorLaboratories, Burlingame, USA) (1:2000) was used as secondary antibody. Imageswere taken under the confocal microscope (Zeiss LSM 5 Pascal or Zeiss LSM510).
AcknowledgementsWe acknowledge Dr Jinrong Peng and members of Dr Xiong’slaboratory for comments on this manuscript and Dr Iain Bruce for theEnglish editing.
Author contributionsX.W. and Q.Y. equally designed and carried out the experiments anddata interpretation and manuscript writing; Q.W., Y.B., N.-N.C.,S.Y., X.-H.Z. and X.Z. designed and carried out some experiments;J.-W.X. conceived, designed and wrote up this work.
FundingThis research was funded by the National Key Basic ResearchProgram of China [grant numbers 2010CB529503 and2012CB944501]; the National Natural Science Foundation ofChina [grant numbers 30971662 and 31271549]; a NationalInstitutes of Health (NIH) training grant [grant number T32-DK07540-22]; the March of Dimes Birth Defects Foundation[grant number 1-FY2007-471]; and the Milton Fund from HarvardUniversity. Deposited in PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.116996/-/DC1
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