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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