Study of Developmental

Biology using Zebrafish

Major advantages of zebrafish as a vertebrate model

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Natural habitats of zebrafish

still water (currents, 0 m–sec to 0.1 m–sec) at 27°C to 34°C and pH 7.9–8.2; widths of water bodies ranged from 1 to 12 m, and depths ranged from 16 to 57 cm; water was relatively clear (transparent to 35 cm).5-6 months for sexual maturation.

Engeszer RE, et al. Zebrafish, 4:21-40, 2007

1. Rapid development

=28d human embryo

Movie of Zebrafish Embryogenesis

2. High reproductivity

A few hundreds of eggs per femaleLaying weaklyControllable laying timeExternal fertilization and developmentTransparent embryos for easy observation

3. Small size and easy raising

4. Well-developed genetic and embryonic technologies

Mutagenesis

Transgenesis

Parthenogenesis and androgenesis

Lineage tracing

Cell transplantation

Nuclear transplantation

Major Research DirectionsGastrulation/embryonic inductionSegmentation/circadian rhythmicityNeural patterning and neurobiology: AP patterning, brain development, eye, axonal guidance, neurodegeneration, behavior,and etc. Organogenesis: heart, liver, pancreas, blood, vasculature, gonads, fin, and etc.Cell convergency and extensionGerm cells: specification, migrationDisease models: cancers, DiGeorgesyndrome, Bardet-Biedl syndrome, and etc.Toxicity estimationEvolution

MutagenesisCloning of novel genes functioning in a developmental pathwayDemonstration of interactions between genesStarting materials for microarry, proteomics, drug screening, and etc.Animal models for human diseases

Chemical mutagenesis, e.g., N-ethyl-N-nitrosourea (ENU)Insertional mutagenesis, e.g., DNA microinjection, proviral insertion, and transposon insertionPhysical mutagenesis, e.g., X-ray and r-ray

Approaches for creating mutants

Mutagenesis with N-ethyl-N-nitrosourea (ENU)

H3C-CH2-N-C-NH2=

N

=

O

O

Alkylating agentSites of action: O6 guanine

O4 thymineN3 adeninePO4

Large-scale mutagenesis

Identification of important genes

DNA Lesions after ENU treatment

Mutation E.coli Drosophila Big Blue Mousemouse germline

A.T - T.A 1% 13% 33% 41%A.T - G.C 18% 13% 7% 42%G.C - A.T 74% 67% 38% 7%G.C - C.G 1% 3% 3% 5%A.T - C.G 3% 3% 3% 2%G.C - T.A 1% 3% 11% 2%Other 3% 0% 5% 1%(small deletion)

In zebrafish, 1-3 mutations per gene can be obtained per 1000 mutagenized genomes

Steps for identifying recessive mutantsWT

WT

Present status of ENU mutagenesis

It is widely used to obtain new mutationsTissue- or pathway-specific mutagenesis using transgenic materialsGene-specific mutagenesisIdentify mutants with peculiar defects, e.g., sight, circadian rhythm, and behavior

New tool: Targeted mutagenesis

Cecelia Moens, Fred Huthinson Cancer Research Center, WU, Tilligen, Inc.

Weinholds E et al. Science, 297:99-102, 2002

Retroviral insertional mutagenesis

Single-copy insertion；

High integration efficiency；

Easy cloning of mutant genes

600 mutants have been generated and

300 mutant genes have been identified

(as said on July 2004)

正常胚胎 突变胚胎 X 基因 病毒基因组 完整的 X 基因使胚胎正常发育 X 基因被病毒基因组破坏后胚胎异常发育

Retroviral insertion

(A) Ten-week-old wild-type (top) vs. dominant mutant hiD862 with long fins. (B) Wild-type (left) vs. bubble brain at day 2. (Arrowhead) Region of enlarged ventricle in mutants. (C) Wild-type (top) vs. two no knack mutant embryos at day 4. (D) Nineweek-old wild-type (top) vs. a nearly normal sibling, one of ∼10% of the homozygotes that survived. (E) Wild-type (top) vs. hi37 mutant at day 4.

Long fin

reduced circulation, edema around heart, bent body, jaw does not form

Enlarged ventricle

Small size Apart eyes, looped heart

(F)Wild-type (top) vs. hi43 at day 5. (Arrowhead) Liver that is abnormal in the mutant. (G) Closer view of wild-type (top) vs. hi43 liver region. (H) Wild-type (top) vs. hi63 mutant at day 3. (I) Wild-type (left) vs. hi96 mutant embryo at day 4. (Arrowhead) Unusual edema with pooled blood around eye. Edema around body of mutant is also visible. (J) Wild-type (left) vs. bleached blond mutant at day 4. (K) Closer view of eyes of bleached blond at day 4 showing mottled appearance.

Abnormal liver and swim bladder small head

No pigmentEdema around eyes

Mottled eyes

Transgenesis

Microinjection of linearized plasmid DNA

Infection with recombinant retrovirus

Promoter/enhancers Test cDNA pA

1-10% transgenic rate; Inducible systems available

15-50% transgenic rate

Examples of transgenic fish

GAL4/UAS Inducible

Transgenic System

GAL4 expressing line

UAS target gene line

Avoid embryonic lethalitySpatiotemporal control of expression

Scheer N, Campos-Ortega JA. Mech. Dev., 80 (1999) 153–158

New tool: Transposon-based transgenesis/Gene trapping

Tol2 transposon was found in medaka

High transgenic efficiency (>50%)Identify genes expressing in a tissue-specific mannerProvide tissue-specific markersCreate mutants

Kawakami K, et al. Dev Cell. 2004 Jul;7(1):133-144

New tool: In vitro cultured sperms for transgenesis

Easy handlingTransgenic rate: 5%

Kurita K, et al. Proc Natl Acad Sci USA. 2004 Feb 3;101(5):1263-1267

Uses of transgenic fish

Dissection of cis-regulatory elements

In vivo anatomy/ongenesis of organs

Isolation of tissue-specific cells

Mutagenization of specific pathways

Cell fate mapping of early blastomeres by single-cell injection

Injecting dye into one cell of 8-cell embryo

Observing distribution of labeled cells at 24 hpf

Cell fate mapping of late blastula by single-cell injection

Study of cell fate by labeling and transplantation

Useful tool for addressing cell autonomous or nonautonomous

Fate map of 8-cell stage zebrafish embryo

Anterior Posterior

Dorsal

Ventral

Left

Right

Strehlow D and Gilbert W. Nature, 361:451-453, 1993

Fate map of 16-cell stage embryo

NOTO, notochord; HATCH, hatching gland; OLF, olfactory epithelium; TEL, elencephalon;BLOOD, blood; HEART, heart; MES, mesencephalon; RHOM,rhombencephalon; YOLK, epithelium over the yolk; NEPH,pronephric ducts; LLENS, left lens; RLENS, right lens; DFIN, epithelium over the dorsal fin region; VFIN, epithelium over theventral fin region; LRET, left retina; RRET, right retina; SPI, spine; HEAD, epithelium over the head region; ALSOM, muscle cells of the anterior left somites; ARSOM, muscle cells of the anterior right somites; DTRU, epithelium over the dorsal trunk; VTRU, epithelium over the ventral trunk; MLSOM, muscle cells of the middle leftsomites; MRSOM, muscle cells of the middle right somites; LOTO, left otocyst; LGILL, left primordial gill slits; PLSOM, muscle cells of the posterior left somites; PRSOM, muscle cells of the posteriorright somites; ROTO, right otocyst; RGILL, right primordial gill slits; LATRU, epithelium over the lateral trunk; Number of injections indicates the number of times each designated blastomere was injected. Total number of injections is 112.

Strehlow D, et al. Development,120:1791-8, 1994

Fate map of zebrafish gastrulas

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Study of induction by transplantation

(A) Schematic representation of the operations. The labeled YC, from which the blastoderm had been removed, was transplanted on top of the animal-pole region of unlabeled embryos. (B–C) Induction of gsc expression by the transplanted normal YC. Four figures were obtained from the same specimen. (B) Ectopic gsc expression (arrowheads) was observed near the donor YSL of the YC. (C) After avidin-biotin peroxidase staining, ectopic expression (arrowheads) was observed in the unlabeled host cells.

Mizuno T et al. Mech. Dev., 81 (1999) 51–63

Identification of tissue- or organ-specific genes

From easily dissectible tissues/organs, e.g., heart, fin, eyesFrom sorted cells with a label, e.g., in transgenic fishmRNA differential display or subtractive cloningScreen by whole-mount in situ hybridization

Thisse group, Laboratoire de Biologie Moleculaire du Developpement, INSERM U368, France, has analyzed 10,089 cDNAs and identified 2,605 cDNAs with a restricted expression pattern.

Morpholino knockdown

Advantages:High specificityStableNon-toxic

DisadvantagesCostlyLow successful ratio

亚磷酸二酰胺键

Block of translation with MO-antisense oligo

Nasevicius A, Ekker SC. Nat Genet, 26(2):216-220, 2000.

Splice morpholinos

RNAi in zebrafish

Antisense GFP probe

Antisense pouII probe

GFP mRNA GFP mRNA + dsGFP GFP mRNA + dspouII

pouII mRNA pouII mRNA + dsGFP pouII mRNA + dspouII

Long dsRNA causes nonspecific mRNA degradation

Gene-specific mRNA degradation induced by siRNA

Not activate PKR/RNase L pathways.Applicable in invertebrates and vertebrates.High throughput screening.High cost for synthetic dsRNAVector-driven siRNA has not been available yet

Cloning and gene knockout in zebrafish

Target construct

Cultured embryonic cells

transfection

selection

Nuclear transfer

Lee K et al. Nat. Biotechnol., Aug;20(8):795-9, 2002

Generation of haploid individuals