the early development of zebrafish
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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 - PowerPoint PPT PresentationTRANSCRIPT
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The Early Development of Zebrafish
Gilbert - Chapter 11
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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.
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• Whereas echinoderm and amphibian eggs used holoblastic cleavage, fish, birds and reptiles utilize meroblastic cleavage
• Danio rerio (Zebrafish)
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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
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Zebrafish Cleavage
• Eggs are telolecithal– Mostly yolk– Meroblastic, discoidal cleavage occurs– REVIEW
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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
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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
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Zebrafish Cleavage (Gilbert, fig. 11.4)
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• 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
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Zebrafish Gastrulation
• Gastrulation begins by epiboly of the blastoderm over 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
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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
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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?
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• Hypoblast will become Mesoderm + endoderm
• The first cells to become hypoblast will become notochord (chordamesoderm)
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• Time lapse Video from Vade Mecum
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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
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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!
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• 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
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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
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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.
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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
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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
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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