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Study of Developmental Biology using Zebrafish Major advantages of zebrafish as a vertebrate model Where is zebrafish in the world 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 y A few hundreds of eggs per female y y Laying weakly y External fertilization and development y Transparent embryos for easy observation Controllable laying time 3. Small size and easy raising 4. Well-developed genetic and embryonic technologies y y y Mutagenesis Transgenesis Parthenogenesis and androgenesis y y y Lineage tracing Cell transplantation Nuclear transplantation Major Research Directions y Gastrulation/embryonic induction y Segmentation/circadian rhythmicity y Neural patterning and neurobiology: AP patterning, brain development, eye, axonal guidance, neurodegeneration, behavior,and etc. y Organogenesis: heart, liver, pancreas, blood, vasculature, gonads, fin, and etc. y Cell convergency and extension y Germ cells: specification, migration y Disease models: cancers, DiGeorge syndrome, Bardet-Biedl syndrome, and etc. y Toxicity estimation y Evolution Mutagenesis y Cloning of novel genes functioning in a developmental pathway y Demonstration of interactions between genes y Starting materials for microarry, proteomics, drug screening, and etc. y Animal models for human diseases Approaches for creating mutants y Chemical mutagenesis, e.g., N-ethyl-Nnitrosourea (ENU) y Insertional mutagenesis, e.g., DNA microinjection, proviral insertion, and transposon insertion y Physical mutagenesis, e.g., X-ray and r-ray Mutagenesis with N-ethyl-Nnitrosourea (ENU) = O N H3C-CH2-N-C-NH2 = O Alkylating agent Sites of action: O6 guanine O4 thymine N3 adenine PO4 y Large-scale mutagenesis y Identification of important genes DNA Lesions after ENU treatment Mutation E.coli Drosophila Big Blue Mouse mouse germline A.T - T.A 1% A.T - G.C 18% G.C - A.T 74% G.C - C.G 1% A.T - C.G 3% G.C - T.A 1% Other 3% (small deletion) 13% 13% 67% 3% 3% 3% 0% 33% 7% 38% 3% 3% 11% 5% 41% 42% 7% 5% 2% 2% 1% In zebrafish, 1-3 mutations per gene can be obtained per 1000 mutagenized genomes Steps for identifying recessive mutants WT WT Present status of ENU mutagenesis y It is widely used to obtain new mutations y Tissue- or pathway-specific mutagenesis using transgenic materials y Gene-specific mutagenesis y Identify 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 y Single-copy insertion; y High integration efficiency; y Easy cloning of mutant genes y 600 mutants have been generated and 300 mutant genes have been identified (as said on July 2004) Retroviral insertion 正常胚胎 X 基因 完整的 X 基因使胚胎正常发育 突变胚胎 病毒基因组 X 基因被病毒基因组破坏后胚胎异常发育 Enlarged ventricle Long fin reduced circulation, edema around heart, bent body, jaw does not form Small size Apart eyes, looped heart (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. Abnormal liver and swim bladder small head No pigment Edema around eyes Mottled eyes (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. Transgenesis Microinjection of linearized plasmid DNA Promoter/enhancers Test cDNA pA 1-10% transgenic rate; Inducible systems available Infection with recombinant retrovirus 15-50% transgenic rate Examples of transgenic fish GAL4/UAS Inducible Transgenic System y Avoid embryonic lethality y Spatiotemporal control of expression Scheer N, Campos-Ortega JA. Mech. Dev., 80 (1999) 153–158 GAL4 expressing line UAS target gene line New tool: Transposon-based transgenesis/Gene trapping Tol2 transposon was found in medaka y High transgenic efficiency (>50%) y Identify genes expressing in a tissue-specific manner y Provide tissuespecific markers y Create mutants Kawakami K, et al. Dev Cell. 2004 Jul;7(1):133-144 New tool: In vitro cultured sperms for transgenesis yEasy handling yTransgenic rate: 5% Kurita K, et al. Proc Natl Acad Sci USA. 2004 Feb 3;101(5):1263-1267 Uses of transgenic fish y Dissection of cis-regulatory elements y In vivo anatomy/ongenesis of organs y Isolation of tissue-specific cells y 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 Posterio r Dorsal Left Right Ventral Strehlow D and Gilbert W. Nature, 361:451-453, 1993 Fate map of 16cell 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 the ventral 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 left somites; 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 posterior right 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 tb eye dien hb mb 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 y From easily dissectible tissues/organs, e.g., heart, fin, eyes y From sorted cells with a label, e.g., in transgenic fish y mRNA differential display or subtractive cloning y Screen 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: 亚磷酸二酰胺键 High specificity Stable Non-toxic Disadvantages Costly Low successful ratio Block of translation with MOantisense oligo Nasevicius A, Ekker SC. Nat Genet, 26(2):216-220, 2000. Splice morpholinos RNAi in zebrafish Long dsRNA causes nonspecific mRNA degradation GFP mRNA GFP mRNA + dsGFP GFP mRNA + dspouII pouII mRNA + dsGFP pouII mRNA + dspouII Antisense GFP probe Antisense pouII probe pouII mRNA Gene-specific mRNA degradation induced by siRNA y Not activate PKR/RNase L pathways. y Applicable in invertebrates and vertebrates. y High throughput screening. y High cost for synthetic dsRNA y Vector-driven siRNA has not been available yet Cloning and gene knockout in zebrafish Cultured embryonic cells transfection Target construct selection Nuclear transfer Lee K et al. Nat. Biotechnol., Aug;20(8):795-9, 2002 Generation of haploid individuals