Transcript sonic hedgehog

Amphibians & Fish
Early Development and Axis
Formation
Chapter 7 - Part 2
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FISH
DEVELOPMENT
ZEBRAFISH
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Early Zebrafish Development
• Used in extreme mutagenesis studies
• Each mutagenized male bred to wild
type female
• Dominant mutant genes expressed F1
• Recessive mutant genes not expressed F1
– Are expressed in F3 (25% chance)
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Early Zebrafish Development
7.37 Zebrafish
development occurs
very rapidly
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Early Zebrafish
Development
7.38 Screening protocol for
identifying mutations of
zebrafish development
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Early Zebrafish Development
• Using GFP (green fluorescent protein)
– Reporter genes are easily fused into
zebrafish
– Express GFP with regulatory sequences
• Permeable to small molecules in water
– Can test drugs/effects on development
– Malformations with retinoic acid or ethanol
• These identified genes operate during
human development
– Mariner gene – myosin VIIA
• Causes deafness in zebrafish and humans
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Early Zebrafish Development
7.39 A reporter gene at work in living zebrafish embryos,
Sonic hedgehog gene
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Cleavage In Fish Eggs
• Telolecithal
– Most of the egg cell is in yolk
• Cleavage only on blastodisc
– Thin yolk-free region at animal pole
– Incomplete cell division
– Called Meroblastic (“part divided”)
• This type of meroblastic cleavage is
called Discoidal
– Disc part on top
– Only part to become the embryo
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Cleavage In Fish Eggs
7.40 Discoidal meroblastic cleavage in a zebrafish egg
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Cleavage In Fish Eggs
• Rapid divisions taking about15 minutes
– Meridional cleavage
– Equatorial cleavage
• After first 12 divisions: synchronously
forming mound of cells on top of animal
pole
– Called blastoderm
– Sitting on one large yolk cell
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Cleavage In Fish Eggs
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Maternal mRNA’s establish
1. Embryonic Polarity
2. Cell Division
3. Cell Cleavage pattern
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Without actin microfilaments, it does not form
Microtubular components are critical to formation
Mid blastula transition
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At  10th division
Zygotes genes start transcription
Cell division slows
Cell movement becomes evident
3 distinct populations can be seen
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Cleavage In Fish Eggs
1. Yolk syncytial layer
(YSL)
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Produces a ring of
nuclei at edge of vegetal
part of blastoderm and
yolk cell
All within yolk cell
Later blastoderm
expands to surround
yolk cell
Some nuclei move to
under blastoderm and
form an internal YSL
Other nuclei move
vegetally to form
external YSL
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Cleavage In Fish Eggs
2. Enveloping layer
(EVL)
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Second cell population
Superficial cells of
blastoderm
Form epithelial sheet
1 cell layer thick
Extraembryonic
protective covering
Sloughs off later
3. Deep cells
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Between EVL and YSL
Gives rise to the embryo
proper
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Cleavage In Fish Eggs
7.41 Fish blastula
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Cleavage In Fish Eggs
• A lot of cell mixing during cleavage
– Difficult to trace cell fates
– Early blastomeres can give rise to an
unpredictable variety of tissue descendants
– Cell fate can be determined shortly before
gastrulation begins
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Gastrulation and Formation of the
Germ Layers
• First cells move by epiboly of the
blastoderm cells over the yolk
– Move ventrally and envelop the yolk
– EVL is tightly joined to the YSL and drags
it along with it
• Spring back to the top of the yolk
• YSL continues its expansion around the yolk
– E-cadherin is critical to movement and
adhesion of the EVL to deep cells
– Expansion of YSL depends in part on
microtubules network
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Gastrulation and Formation of the
Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish
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Gastrulation and Formation of the
Germ Layers
• After blastoderm cells expand over half
of the yolk cell
– Thickening occurs throughout the epiboly
margin
– This thickening is called Germ ring
• Epiblast – superficial layer
• Hypoblast – inner layer
– Contains precursors for endoderm and mesoderm
– Forms in a synchronous “wave” of internalization
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Gastrulation and Formation of the
Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish
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Gastrulation and Formation of the
Germ Layers
• Involution begins at future dorsal portion of embryo
• Thus cells of the blastoderm undergo epiboly around
the yolk
– Internalizing it
– Blastoderm margin cells form the hypoblast
• Hypoblast cells reverse their direction
– Proceeding toward the animal pole
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Gastrulation and Formation of the
Germ Layers
Figure 7.43 Cell migration
of endodermal and
mesodermal precursors
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Gastrulation and Formation of the
Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish
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Gastrulation and Formation of the
Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish
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Gastrulation and Formation of the
Germ Layers
• Cells of Epiblast and Hypoblast
– Intercalate on dorsal side
– Form localized thickening with
non involuting cells
– Embryonic shield
– This is “functional blastopore lip”
of amphibians
• Blastoderm margin converges
anterior and dorsally toward
embryonic shield
• Movement forms
chordamesoderm
– Precursor of notochord
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Gastrulation and Formation of the
Germ Layers
7.44 Convergence and extension in the zebrafish gastrula
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Gastrulation and Formation of the
Germ Layers
• Ventral side
– Hypoblast ring moves toward the vegetal
pole and migrating underneath epiblast
– Eventually the ring closes at vegetal pole
• Internalizing the yolk
– Endoderm arises from most marginal
blastomeres of late blastula-stage embryo
• Involute and occupy deep layers of hypoblast
• Directly over yolk syncytial
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Axis Formation In Zebrafish
• DV axis formation:
Embryonic shield and
Nieuwkoop center
– Embryonic shield
• Critical to DV axis in fish
• Can convert lateral and ventral
mesoderm into dorsal
mesoderm
• Can convert ectoderm into
neural tissue
• Similar experiment to Hans
Spemann/Heidi Mangold
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Axis Formation In Zebrafish
• The Fish Nieuwkoop center
– Amphibians
• Endoderm cells beneath dorsal lip accumulate caterin (maternal)
• Critical to induce cells above it to become dorsal
blastopore lip
– Zebrafish embryonic shield
• Endodermal cells beneath embryonic shield
(i.e. Nieuwkoop center)
– accumulate -caterin
• Accumulate in dorsal YSL
• -caterin accumulates on ventral side of egg
• Causes dorsalization and second embryonic axis
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Axis Formation In Zebrafish
• Left-right axis formation
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Both differ anatomically and embryonically
Fish heart is on left
Different left and right regions of brain
Left side of body
• Info by notch and nodal signals
• Pitx 2 factors
– Right side of body
• Info by FGF (fibroblast growth factors)
– Accomplish asymmetry
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Axis Formation In Zebrafish
– Different vertebrates accomplish asymmetry
by
• Currents produced by cilia also contribute to left
and right in all vertebrates
• Dynein genes for cilia
– Ventral portion of node – mouse, chicken, frog, and
zebrafish
– Zebrafish
• Nodal structures housing cilia (for left and right
asymmetry control) is a transient fluid filled organ
• Called Kupffer’s vesicle
• Arise from dorsal cells near embryonic shield after
gastrulation starts
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Axis Formation In Zebrafish
• Experiments to prove this:
– Blocking ciliary formation/function
• Prevents dynein motor molecule synthesis
• Prevents left and right axis formation
– Why leftward flow?
• Possible clockwise rotation of cilia
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• Show short clips
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