Monte Westerfield, University of Oregon, Eugene, USASponsored by the Office of the Director National Institutes of Health,

the National Human Genome Research Institute, the National Institute of Child Health & Development,

the National Institute on Deafness & Other Communication Disorders, the National Eye Institute,

the Usher 1F Collaborative, and the Megan and Vision for a Cure Foundations

Zebrafish in studies of human health and disease

VisionForACure.com

• Experimental advantages of zebrafish

• Zebrafish as models of human disease

• Gene editing to generate precise models of human genotypes

Zebrafish in studies of human health and disease

18 hour zebrafish!28 day human!

Vertebrate embryos

Study development directly in transparent embryos

(Karlstrom & Kane)

cell-specific promoter GFPTol2 Tol2Tol2kit

Inject into zebrafish

Reporter constructs & highly efficient transgenesis

Screen for rescue Assays: OKR, ERG, tap test, apoptosis, etc.

Models for translational studies

Study physiology directly

(Wilkinson laboratory)

Study blood flow directly in live animals

(Santoro laboratory)

Watch development directly in live animal

(Link laboratory)

Complex behaviors - prey capture

(Engert laboratory)

(Keller & Ahrens, 2013)

Study activity of every neuron in the brain simultaneously

High-throughput optical screening

(Pardo-Martin et al., 2010)

Wild-type!

Mutant!no tail (ntl)!

(Kimmel laboratory)

Body axis development

Wild-type!

Mutant!

.!

Cerebellum!

acerebellar (ace)!

Brain development

(Nüsslein-Volhard laboratory)

Normal! � Cyclops � mutant!

Eye development

(Kimmel laboratory)

Thousands of mutants- a - i :

_ - " " _ _ e

;ffir'trii..-':.

,.. ' f ' . ]t ' :, '

1ps6-{s..$

-:jt**l!'

"\{-{*S*r

lF __=! -r;i3--

ffi

t.* . "* * \ . . Lry"u.lSr. tqy * ".1,-": . ,. "i-".*o* t

rmffir*&e Krurua;#.l:****;*egmo*

Knock out of all protein coding genes

Reverse genetic technology

CRISPRs - Clustered Regularly Interspaced Short Palindromic Repeats

MORPHOLINOS - antisense oligonucleotides

Morpholinos vs gene editing

(Kok et al., 2015)

For 80% of genes, morpholino phenotypes are not observed in mutants

Morpholino CRISPR/TALENAdvantages Disadvantages

Study directly in injected animals

Study in stable lines

Easy to use Requires breeding & genotyping

Blocks maternal message

Requires maternal zygotic mutants to block maternal products

Disadvantages Advantages

Diluted over time – limited to early developmental stages

Genetically stable – can study at all developmental stages including adults

Induces p53 dependent apoptosis

Low toxicity

Off target effects Highly specific

Morpholinos vs gene editing

(Kok et al., 2015)

For 80% of genes, morpholino phenotypes are not observed in mutants

Morpholino CRISPR/TALENAdvantages Disadvantages

Study directly in injected animals

Study in stable lines

Easy to use Requires breeding & genotyping

Blocks maternal message

Requires maternal zygotic mutants to block maternal products

Disadvantages Advantages

Diluted over time – limited to early developmental stages

Genetically stable – can study at all developmental stages including adults

Induces p53 dependent apoptosis

Low toxicity

Off target effects Highly specific

Gene editing is becoming a primary genetic technology in zebrafish research

TALENs

CRISPRs

Morpholinos (x 0.2)

Publ

icat

ions

0

10

20

30

40

50

Year

2010 2011 2012 2013 2014 2015

• Experimental advantages of zebrafish

• Zebrafish as models of human disease

• Gene editing to generate precise models of human genotypes

Zebrafish in studies of human health and disease

zebrafish fly

human

zebrafish

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Alzheimers Disease

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Autism

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Cancers

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Obesity

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Schizophrenia

Acute radiation syndrome Age-related macular degeneration Alcoholism Alcoholic liver disease Alzheimers disease Anophthalmia Anxiety disorders Atherosclerosis Autism Autosomal dominant hearing disease Axenfeld-Rieger syndrome Bardet-Biedl syndrome Barth syndrome Bicuspid aortic valve disease Biliary atresia Cancer Colorectal cancer Head and neck squamous cell carcinoma Glioma Invasive carcinoma LEOPARD syndrome Leukemia Liver cancer Lymphoblastic leukemia Melanoma Myologenous leuekmia Neuroblastoma Pancreatic cancer Rhabdomyosarcoma Centronuclear myopathies Cerebral cavernous malformations Cholera Chronic granulomatous disease Chronic kidney disease Circulatory diseases Colitis Congenital cataract Congenital glycoslyation diseases Core binding factor leukemia

Corneal dystrophies Cranial facial diseases Crohns disease Cutis laxa Cystic fibrosis Delayed puberty Diabetes Diamond Blackfan anemia Duchenne muscular dystrophy End stage renal disease Emphysema Epilepsy Extrinsic asthma Eye diseases Facioscapulohumeral muscular dystrophy Fanconi anemia Fatty liver disease Fetal alcohol syndrome Focal segmental glomerulosclerosis Fraser syndrome Ganglioside sialidase deficiency disease GAPO progeroid syndrome Glaucoma Glioblastoma Heart disease Atrial fibrillation Cardiac arrhythmia Cardiac conduction diseases Cardiomegaly Congestive heart failure Hypertrophic cardiomyopathy Sudden cardiac arrest Hepatic steatosis Hereditary retinal degenerations Hirschsprung disease HIVAIDS Holt Oram syndrome Hyperammonemia Hypertension

Inflammatory bowel disease Intracranial aneurysm Joubert syndrome Juvenile hydrocephalus Kidney diseases Long QT syndrome Lysosomal disease mucolipidosis II Microphthamia Midfacial cleft disease Multiple sclerosis Mucolipidosis type IV Muscular dystrophy Myotonic dystrophy Nephronophthisis Nonsyndromic cleft lip and palate Noonan syndrome Obesity Oral cleft diseases Osteoporosis Parkinson disease Polycystic kidney disease Pseudoxanthoma elasticum Refractory anemia Schizophrenia Scoliosis Skeletal diseases Sleep disorders Spinal cord regeneration Spinal muscular atrophy Stroke Treacher Collins syndrome Tuberculosis Usher syndrome Uveal coloboma Vascular thrombosis von Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Usher syndrome

Usher syndrome - the leading cause of deafblindness

• Prevalence ≈ 1 per 6,000 births in the US(more common than ALS or Huntington’s Disease)

• Congenital deafness (~4% of deaf have Usher) Sensorineural hearing loss Vestibular dysfunction

• Retinitis pigmentosa Loss of rod photoreceptors Progressive tunnel vision as cones die

R245X mutation truncates the PCDH15 protein causing Usher syndrome type 1F

R245X is common in the Ashkenazi Jewish community- accounts for ~65% of Usher 1 patients

Exon skipping as strategy for therapy

171 bp(57aa - in frame)

12 Kb 80 Kb

site of premature termination

codon in R245X patients

aso1 aso2

E7 E8 E9

E7 E9

Exon 8 deleted from transcript

PCDH15 pre-mRNA

PCDH15 mature mRNA

Exon skipping as strategy for therapy

12 Kb 80 Kb

R245X

E7 E9E8

Zebrafish gene

Precise model ofhuman mutation

Human exon + Flanking intron sequences

CRISPR

E8

12 Kb 80 Kb

E7 E9E8

R245X

aso1 aso2

E8

Zebrafish homologyarms

+

• Experimental advantages of zebrafish

• Zebrafish as models of human disease

• Gene editing to generate precise models of human genotypes - 377 genes targeted since 2011

Zebrafish in studies of human health and disease

Monte Westerfield, University of Oregon, Eugene, USASponsored by the Office of the Director National Institutes of Health,

the National Human Genome Research Institute, the National Institute of Child Health & Development,

the National Institute on Deafness & Other Communication Disorders, the National Eye Institute,

the Usher 1F Collaborative, and the Megan and Vision for a Cure Foundations

Zebrafish in studies of human health and disease

VisionForACure.com