Transcript Developmental Biology

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
Zebrafish Cleavage (Gilbert, fig. 11.4)
• 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 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
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)
• Time lapse Video from Vade Mecum
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 dorsalmost 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