Reproducibility with zebrafish models of human health and...

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 of Deafness & Other Disorders

the National Eye Institute, and the Megan and Vision for a Cure Foundations

Reproducibility with zebrafish modelsof human health and disease

VisionForACure.com

• Why zebrafish?

• Current use in biomedical research

• Improving reproducibility

Reproducibility with zebrafish modelsof human health and disease

zebrafish fly

human

zebrafish

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 cellular interactions

Study blood flow directly in live animals

(Santoro laboratory)

(Keller & Ahrens, 2013)

Study activity of every neuron in the brain simultaneously

High-throughput optical screening

(Pardo-Martin et al., 2010)

• Why zebrafish?• in vivo analyses at high resolution

• rapid, inexpensive

• Current use in biomedical research

• Improving reproducibility

Reproducibility with zebrafish modelsof human health and disease

Growth of zebrafish research

H. sapiens/130

M. musculus/20

D. melanogaster

D. rerioC. elegans

Acute radiation syndromeAge-related macular degenerationAlcoholismAlcoholic liver diseaseAlzheimers diseaseAnophthalmiaAnxiety disordersAtherosclerosisAutismAutosomal dominant hearing diseaseAxenfeld-Rieger syndromeBardet-Biedl syndromeBarth syndromeBicuspid aortic valve diseaseBiliary atresiaCancer

Colorectal cancerHead and neck squamous cell carcinomaGliomaInvasive carcinomaLEOPARD syndromeLeukemiaLiver cancerLymphoblastic leukemiaMelanomaMyologenous leuekmiaNeuroblastomaPancreatic cancerRhabdomyosarcoma

Centronuclear myopathiesCerebral cavernous malformationsCholeraChronic granulomatous diseaseChronic kidney diseaseCirculatory diseasesColitisCongenital cataractCongenital glycoslyation diseasesCore binding factor leukemia

Corneal dystrophiesCranial facial diseasesCrohns diseaseCutis laxa Cystic fibrosisDelayed pubertyDiabetes Diamond Blackfan anemiaDuchenne muscular dystrophyEnd stage renal diseaseEmphysemaEpilepsy Extrinsic asthmaEye diseases

Facioscapulohumeral muscular dystrophyFanconi anemiaFatty liver diseaseFetal alcohol syndromeFocal segmental glomerulosclerosisFraser syndromeGanglioside sialidase deficiency diseaseGAPO progeroid syndromeGlaucomaGlioblastomaHeart disease

Atrial fibrillationCardiac arrhythmiaCardiac conduction diseasesCardiomegalyCongestive heart failureHypertrophic cardiomyopathySudden cardiac arrest

Hepatic steatosisHereditary retinal degenerationsHirschsprung diseaseHIVAIDSHolt Oram syndromeHyperammonemia

Hypertension Inflammatory bowel diseaseIntracranial aneurysm Joubert syndromeJuvenile hydrocephalusKidney diseasesLong QT syndromeLysosomal disease mucolipidosis IIMicrophthamiaMidfacial cleft diseaseMultiple sclerosisMucolipidosis type IVMuscular dystrophyMyotonic dystrophyNephronophthisisNonsyndromic cleft lip and palateNoonan syndromeObesityOral cleft diseasesOsteoporosisParkinson diseasePolycystic kidney diseasePseudoxanthoma elasticumRefractory anemiaSchizophreniaScoliosisSkeletal diseasesSleep disordersSpinal cord regenerationSpinal muscular atrophyStroke Treacher Collins syndromeTuberculosisUsher syndromeUveal colobomaVascular thrombosisvon Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Acute radiation syndromeAge-related macular degenerationAlcoholismAlcoholic liver diseaseAlzheimers diseaseAnophthalmiaAnxiety disordersAtherosclerosisAutismAutosomal dominant hearing diseaseAxenfeld-Rieger syndromeBardet-Biedl syndromeBarth syndromeBicuspid aortic valve diseaseBiliary atresiaCancer

Colorectal cancerHead and neck squamous cell carcinomaGliomaInvasive carcinomaLEOPARD syndromeLeukemiaLiver cancerLymphoblastic leukemiaMelanomaMyologenous leuekmiaNeuroblastomaPancreatic cancerRhabdomyosarcoma

Centronuclear myopathiesCerebral cavernous malformationsCholeraChronic granulomatous diseaseChronic kidney diseaseCirculatory diseasesColitisCongenital cataractCongenital glycoslyation diseasesCore binding factor leukemia

Corneal dystrophiesCranial facial diseasesCrohns diseaseCutis laxa Cystic fibrosisDelayed pubertyDiabetes Diamond Blackfan anemiaDuchenne muscular dystrophyEnd stage renal diseaseEmphysemaEpilepsy Extrinsic asthmaEye diseases

Facioscapulohumeral muscular dystrophyFanconi anemiaFatty liver diseaseFetal alcohol syndromeFocal segmental glomerulosclerosisFraser syndromeGanglioside sialidase deficiency diseaseGAPO progeroid syndromeGlaucomaGlioblastomaHeart disease

Atrial fibrillationCardiac arrhythmiaCardiac conduction diseasesCardiomegalyCongestive heart failureHypertrophic cardiomyopathySudden cardiac arrest

Hepatic steatosisHereditary retinal degenerationsHirschsprung diseaseHIVAIDSHolt Oram syndromeHyperammonemia

Hypertension Inflammatory bowel diseaseIntracranial aneurysm Joubert syndromeJuvenile hydrocephalusKidney diseasesLong QT syndromeLysosomal disease mucolipidosis IIMicrophthamiaMidfacial cleft diseaseMultiple sclerosisMucolipidosis type IVMuscular dystrophyMyotonic dystrophyNephronophthisisNonsyndromic cleft lip and palateNoonan syndromeObesityOral cleft diseasesOsteoporosisParkinson diseasePolycystic kidney diseasePseudoxanthoma elasticumRefractory anemiaSchizophreniaScoliosisSkeletal diseasesSleep disordersSpinal cord regenerationSpinal muscular atrophyStroke Treacher Collins syndromeTuberculosisUsher syndromeUveal colobomaVascular thrombosisvon Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Alzheimers Disease

Acute radiation syndromeAge-related macular degenerationAlcoholismAlcoholic liver diseaseAlzheimers diseaseAnophthalmiaAnxiety disordersAtherosclerosisAutismAutosomal dominant hearing diseaseAxenfeld-Rieger syndromeBardet-Biedl syndromeBarth syndromeBicuspid aortic valve diseaseBiliary atresiaCancer

Colorectal cancerHead and neck squamous cell carcinomaGliomaInvasive carcinomaLEOPARD syndromeLeukemiaLiver cancerLymphoblastic leukemiaMelanomaMyologenous leuekmiaNeuroblastomaPancreatic cancerRhabdomyosarcoma

Centronuclear myopathiesCerebral cavernous malformationsCholeraChronic granulomatous diseaseChronic kidney diseaseCirculatory diseasesColitisCongenital cataractCongenital glycoslyation diseasesCore binding factor leukemia

Corneal dystrophiesCranial facial diseasesCrohns diseaseCutis laxa Cystic fibrosisDelayed pubertyDiabetes Diamond Blackfan anemiaDuchenne muscular dystrophyEnd stage renal diseaseEmphysemaEpilepsy Extrinsic asthmaEye diseases

Facioscapulohumeral muscular dystrophyFanconi anemiaFatty liver diseaseFetal alcohol syndromeFocal segmental glomerulosclerosisFraser syndromeGanglioside sialidase deficiency diseaseGAPO progeroid syndromeGlaucomaGlioblastomaHeart disease

Atrial fibrillationCardiac arrhythmiaCardiac conduction diseasesCardiomegalyCongestive heart failureHypertrophic cardiomyopathySudden cardiac arrest

Hepatic steatosisHereditary retinal degenerationsHirschsprung diseaseHIVAIDSHolt Oram syndromeHyperammonemia

Hypertension Inflammatory bowel diseaseIntracranial aneurysm Joubert syndromeJuvenile hydrocephalusKidney diseasesLong QT syndromeLysosomal disease mucolipidosis IIMicrophthamiaMidfacial cleft diseaseMultiple sclerosisMucolipidosis type IVMuscular dystrophyMyotonic dystrophyNephronophthisisNonsyndromic cleft lip and palateNoonan syndromeObesityOral cleft diseasesOsteoporosisParkinson diseasePolycystic kidney diseasePseudoxanthoma elasticumRefractory anemiaSchizophreniaScoliosisSkeletal diseasesSleep disordersSpinal cord regenerationSpinal muscular atrophyStroke Treacher Collins syndromeTuberculosisUsher syndromeUveal colobomaVascular thrombosisvon Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Autism

Acute radiation syndromeAge-related macular degenerationAlcoholismAlcoholic liver diseaseAlzheimers diseaseAnophthalmiaAnxiety disordersAtherosclerosisAutismAutosomal dominant hearing diseaseAxenfeld-Rieger syndromeBardet-Biedl syndromeBarth syndromeBicuspid aortic valve diseaseBiliary atresiaCancer

Colorectal cancerHead and neck squamous cell carcinomaGliomaInvasive carcinomaLEOPARD syndromeLeukemiaLiver cancerLymphoblastic leukemiaMelanomaMyologenous leuekmiaNeuroblastomaPancreatic cancerRhabdomyosarcoma

Centronuclear myopathiesCerebral cavernous malformationsCholeraChronic granulomatous diseaseChronic kidney diseaseCirculatory diseasesColitisCongenital cataractCongenital glycoslyation diseasesCore binding factor leukemia

Corneal dystrophiesCranial facial diseasesCrohns diseaseCutis laxa Cystic fibrosisDelayed pubertyDiabetes Diamond Blackfan anemiaDuchenne muscular dystrophyEnd stage renal diseaseEmphysemaEpilepsy Extrinsic asthmaEye diseases

Facioscapulohumeral muscular dystrophyFanconi anemiaFatty liver diseaseFetal alcohol syndromeFocal segmental glomerulosclerosisFraser syndromeGanglioside sialidase deficiency diseaseGAPO progeroid syndromeGlaucomaGlioblastomaHeart disease

Atrial fibrillationCardiac arrhythmiaCardiac conduction diseasesCardiomegalyCongestive heart failureHypertrophic cardiomyopathySudden cardiac arrest

Hepatic steatosisHereditary retinal degenerationsHirschsprung diseaseHIVAIDSHolt Oram syndromeHyperammonemia

Hypertension Inflammatory bowel diseaseIntracranial aneurysm Joubert syndromeJuvenile hydrocephalusKidney diseasesLong QT syndromeLysosomal disease mucolipidosis IIMicrophthamiaMidfacial cleft diseaseMultiple sclerosisMucolipidosis type IVMuscular dystrophyMyotonic dystrophyNephronophthisisNonsyndromic cleft lip and palateNoonan syndromeObesityOral cleft diseasesOsteoporosisParkinson diseasePolycystic kidney diseasePseudoxanthoma elasticumRefractory anemiaSchizophreniaScoliosisSkeletal diseasesSleep disordersSpinal cord regenerationSpinal muscular atrophyStroke Treacher Collins syndromeTuberculosisUsher syndromeUveal colobomaVascular thrombosisvon Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Obesity

Acute radiation syndromeAge-related macular degenerationAlcoholismAlcoholic liver diseaseAlzheimers diseaseAnophthalmiaAnxiety disordersAtherosclerosisAutismAutosomal dominant hearing diseaseAxenfeld-Rieger syndromeBardet-Biedl syndromeBarth syndromeBicuspid aortic valve diseaseBiliary atresiaCancer

Colorectal cancerHead and neck squamous cell carcinomaGliomaInvasive carcinomaLEOPARD syndromeLeukemiaLiver cancerLymphoblastic leukemiaMelanomaMyologenous leuekmiaNeuroblastomaPancreatic cancerRhabdomyosarcoma

Centronuclear myopathiesCerebral cavernous malformationsCholeraChronic granulomatous diseaseChronic kidney diseaseCirculatory diseasesColitisCongenital cataractCongenital glycoslyation diseasesCore binding factor leukemia

Corneal dystrophiesCranial facial diseasesCrohns diseaseCutis laxa Cystic fibrosisDelayed pubertyDiabetes Diamond Blackfan anemiaDuchenne muscular dystrophyEnd stage renal diseaseEmphysemaEpilepsy Extrinsic asthmaEye diseases

Facioscapulohumeral muscular dystrophyFanconi anemiaFatty liver diseaseFetal alcohol syndromeFocal segmental glomerulosclerosisFraser syndromeGanglioside sialidase deficiency diseaseGAPO progeroid syndromeGlaucomaGlioblastomaHeart disease

Atrial fibrillationCardiac arrhythmiaCardiac conduction diseasesCardiomegalyCongestive heart failureHypertrophic cardiomyopathySudden cardiac arrest

Hepatic steatosisHereditary retinal degenerationsHirschsprung diseaseHIVAIDSHolt Oram syndromeHyperammonemia

Hypertension Inflammatory bowel diseaseIntracranial aneurysm Joubert syndromeJuvenile hydrocephalusKidney diseasesLong QT syndromeLysosomal disease mucolipidosis IIMicrophthamiaMidfacial cleft diseaseMultiple sclerosisMucolipidosis type IVMuscular dystrophyMyotonic dystrophyNephronophthisisNonsyndromic cleft lip and palateNoonan syndromeObesityOral cleft diseasesOsteoporosisParkinson diseasePolycystic kidney diseasePseudoxanthoma elasticumRefractory anemiaSchizophreniaScoliosisSkeletal diseasesSleep disordersSpinal cord regenerationSpinal muscular atrophyStroke Treacher Collins syndromeTuberculosisUsher syndromeUveal colobomaVascular thrombosisvon Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Schizophrenia

Acute radiation syndromeAge-related macular degenerationAlcoholismAlcoholic liver diseaseAlzheimers diseaseAnophthalmiaAnxiety disordersAtherosclerosisAutismAutosomal dominant hearing diseaseAxenfeld-Rieger syndromeBardet-Biedl syndromeBarth syndromeBicuspid aortic valve diseaseBiliary atresiaCancer

Colorectal cancerHead and neck squamous cell carcinomaGliomaInvasive carcinomaLEOPARD syndromeLeukemiaLiver cancerLymphoblastic leukemiaMelanomaMyologenous leuekmiaNeuroblastomaPancreatic cancerRhabdomyosarcoma

Centronuclear myopathiesCerebral cavernous malformationsCholeraChronic granulomatous diseaseChronic kidney diseaseCirculatory diseasesColitisCongenital cataractCongenital glycoslyation diseasesCore binding factor leukemia

Corneal dystrophiesCranial facial diseasesCrohns diseaseCutis laxa Cystic fibrosisDelayed pubertyDiabetes Diamond Blackfan anemiaDuchenne muscular dystrophyEnd stage renal diseaseEmphysemaEpilepsy Extrinsic asthmaEye diseases

Facioscapulohumeral muscular dystrophyFanconi anemiaFatty liver diseaseFetal alcohol syndromeFocal segmental glomerulosclerosisFraser syndromeGanglioside sialidase deficiency diseaseGAPO progeroid syndromeGlaucomaGlioblastomaHeart disease

Atrial fibrillationCardiac arrhythmiaCardiac conduction diseasesCardiomegalyCongestive heart failureHypertrophic cardiomyopathySudden cardiac arrest

Hepatic steatosisHereditary retinal degenerationsHirschsprung diseaseHIVAIDSHolt Oram syndromeHyperammonemia

Hypertension Inflammatory bowel diseaseIntracranial aneurysm Joubert syndromeJuvenile hydrocephalusKidney diseasesLong QT syndromeLysosomal disease mucolipidosis IIMicrophthamiaMidfacial cleft diseaseMultiple sclerosisMucolipidosis type IVMuscular dystrophyMyotonic dystrophyNephronophthisisNonsyndromic cleft lip and palateNoonan syndromeObesityOral cleft diseasesOsteoporosisParkinson diseasePolycystic kidney diseasePseudoxanthoma elasticumRefractory anemiaSchizophreniaScoliosisSkeletal diseasesSleep disordersSpinal cord regenerationSpinal muscular atrophyStroke Treacher Collins syndromeTuberculosisUsher syndromeUveal colobomaVascular thrombosisvon Willebrand disease

Human diseases studied with zebrafish models

(NIH funded grants, 2013)

Sleep disorders

• Why zebrafish?• in vivo analyses at high resolution

• rapid, inexpensive

• Current use in biomedical research• widespread and growing

• Improving reproducibility

Reproducibility with zebrafish modelsof human health and disease

Forward genetics: thousands of mutants

Reverse genetics: morpholinos knockdown gene function

Translationblocker

Spliceblocker

mRNA

Problems for reproducibility:• toxicity• effectiveness diluted over time• genomic polymorphisms• off-target effects

Translationblocker

Spliceblocker

mRNA

Reverse genetics: morpholinos knockdown gene function

Knock out of all protein coding genes

CRISPRs provide precise genome editing

(Ebermann et al.)

Zebrafish models of human patients

(Ebermann et al.)

Potential genetic interaction between PDZD7 & USH2A

(Ebermann et al.)

Zebrafish model validates genetic interaction between PDZD7 & USH2A

New tools increase reproducibility

• complete maps of all SNPs

• rapid, low cost sequencing

• isogenic strains

• Why zebrafish?• in vivo analyses at high resolution

• rapid, inexpensive

• Current use in biomedical research• widespread

• Improving reproducibility• precise models of human patients

• improved tools needed

Reproducibility with zebrafish modelsof 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 of Deafness & Other Disorders

the National Eye Institute, and the Megan and Vision for a Cure Foundations

Reproducibility with zebrafish modelsof human health and disease

VisionForACure.com