The Early Development of Zebrafish Gilbert - Chapter 11
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.
Whereas echinoderm and amphibian eggs used holoblastic cleavage, fish, birds and reptiles utilize meroblastic cleavage • Danio rerio (Zebrafish)
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
Zebrafish Cleavage • Eggs are telolecithal • Mostly yolk • Meroblastic, discoidal cleavage occurs • REVIEW
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
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
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
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
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
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?
Hypoblast will become Mesoderm + endoderm • The first cells to become hypoblast will become notochord (chordamesoderm)
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
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!
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
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
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.
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
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
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