The Early Development of Zebrafish

1. The Early Development of Zebrafish Gilbert - Chapter 11

2. Goals • Become familiar with the cleavage and gastrulation patterns in fish • Compare patterns of gastrulation between various species • Discuss the influence of the amount of yolk on development • Describe the evolutionary relationships between amount of yolk and location of development of the organism.

3. Whereas echinoderm and amphibian eggs used holoblastic cleavage, fish, birds and reptiles utilize meroblastic cleavage • Danio rerio (Zebrafish)

4. Zebrafish • Typical teleost development (bony fish) • Why this organism? • Rapid development • Easy to obtain large number of embryos • External fertilization • Clear embryos • Can perform genetic screens! • Can mate mutants, develop lineages that contain a mutation

5. Zebrafish Cleavage • Eggs are telolecithal • Mostly yolk • Meroblastic, discoidal cleavage occurs • REVIEW

6. Meroblastic (Incomplete) Cleavage • Occurs in Telolecithal eggs • Dense yolk throughout most of the egg (why?) • Ex. Birds, fish, reptiles, molluscs • Only a portion of the cytoplasm is cleaved • Cleavage furrow does not penetrate through the whole egg

8. Zebrafish Cleavage • The only portion of the egg that cleaves is a thin yolk-free region of cytoplasm • Called Blastodisc • Located in Animal pole • First divisions highly reproducible, synchronous, rapid (every 15 minutes) • Form a mound of cells at the animal pole = Blastoderm • Large yolk cell remains underneath

10. Zebrafish Cleavage (Gilbert, fig. 11.4)

11. After about the 10th cleavage • The YSL (yolk syncitial layer) forms • Large cells in the yolk – no membranes • Important during gastrulation • The EVL (enveloping layer) forms • Outermost layer of blastodisc • Single epithelial sheet • Protective coating for embryo - sloughed off later • Beneath EVL are Deep cells • Deep cells form embryo proper • The midblastula transition (MBT) occurs

14. Zebrafish Gastrulation • Gastrulation begins by epibolyof the blastodermover the yolk • The YSL expands downward, pulling the EVL along with it • Deep cells fill the space between YSL and EVL • One side of the blastoderm becomes thicker = Dorsal side of embryo

16. Zebrafish Gastrulation • At about 50% epiboly • A population of cells begins to migrate to form a second layer of cells • The deep cells that don’t migrate = epiblast • The migratory cells = hypoblast

18. Zebrafish Gastrulation: Forming the Germ Layers • On the dorsal side of the embryo, the hypoblast and epiblast mix together to form a thickening called the embryonic shield • Embryonic shield has the same function as the dorsal lip of the blastopore in amphibians • WHAT DOES THIS MEAN?

20. Hypoblast will become Mesoderm + endoderm • The first cells to become hypoblast will become notochord (chordamesoderm)

24. Time lapse Video from Vade Mecum

25. The Organizer in Fish:How do they initiate gastrulation? • Remember - the embryonic shield is equivalent to the dorsal blastopore lip • Homologous • When transplanted to the ventral side of an embryo, it induces a second axis • Like the dorsal blastopore lip, these cells (first migratory hypoblast cells) form the notochord • EMBRYONIC SHIELD can be thought of as the ORGANIZER in fish

27. ß-catenin - again!? • In zebrafish, the nuclei in the dorsal-most YSL have accumulated ß-catenin • Where in Xenopus is ß-catenin localized to the nuclei? Nieuwkoop center!

29. Nuclear ß-catenin in the dorsal-most YSL serves as a transcription factor that activates expression of 2 genes • Squint, Bozozok - these are similar to the genes activated by ß-catenin in Xenopus • HOMOLOGOUS!! • Induces the organizer - just like the Niewkoop center in Xenopus

30. Zebrafish Gastrulation: Summary • 3 germ layers have been formed • Endoderm: gut tissues • Mesoderm: somites - muscle, bones chordamesoderm - notochord lateral plate mesoderm - limbs • Ectoderm: nervous system skin • Embryonic body plan is established • Axes

31. Lab Activity - Zebrafish Early Development (15 points) • Use the prepared slides & DVD to draw: • Early and Late Cleavage: • Label structures we have just discussed • 1 picture of Gastrulation • Label structures we have just discussed • When finished, put in inbox, work on review sheet - Xenopus Molecular Components of Early Dev.

32. Genetic Screens in Zebrafish • As with Drosophila, genetic screens can be used to find mutations in genes that affect embryonic development • Zebrafish are the first vertebrate organism in which a large scale genetic screen has been performed • Give us more insight into the molecular genetics of vertebrate development

33. Zebrafish Mutagenesis & Screening • Males of the parental generation are fed a mutagen • Random mutations are created in the germ line (passed on through the sperm) • These males are mated to wild-type females to create F1 generation • Heterozygous offspring (if recessive will not show mutation) • F1 fish are mated with wild type to produce F2 males and females • Some of these fish carry mutation • When mated a small percentage of fish will be homozygous recessive for the mutation

36. Other advantages of zebrafish • Genes are susceptible to antisense and RNAi molecules to knock out gene function in particular tissues or at specific times • Small molecules like alcohol & retinoic acid (vitamin A derivative) can permeate • We can determine if molecules are teratogenic • Easy to observe in clear embryo