1. Amphibians & Fish Early Development and Axis Formation
Chapter 7 - Part 2

2. FISH DEVELOPMENT
Zebrafish

3. 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)

4. Early Zebrafish Development
7.37 Zebrafish development occurs very rapidly

5. Early Zebrafish Development
7.38 Screening protocol for identifying mutations of zebrafish development

6. 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

7. Early Zebrafish Development
7.39 A reporter gene at work in living zebrafish embryos, Sonic hedgehog gene

8. Cleavage In Fish Eggs Telolecithal Cleavage only on blastodisc
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

9. Cleavage In Fish Eggs
7.40 Discoidal meroblastic cleavage in a zebrafish egg

10. 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

11. Cleavage In Fish Eggs Maternal mRNA’s establish
Embryonic Polarity
Cell Division
Cell Cleavage pattern
Without actin microfilaments, it does not form
Microtubular components are critical to formation
Mid blastula transition
At  10th division
Zygotes genes start transcription
Cell division slows
Cell movement becomes evident
3 distinct populations can be seen

12. Cleavage In Fish Eggs Yolk syncytial layer (YSL)
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

13. Cleavage In Fish Eggs Enveloping layer (EVL) Deep cells
Second cell population
Superficial cells of blastoderm
Form epithelial sheet cell layer thick
Extraembryonic protective covering
Sloughs off later
Deep cells
Between EVL and YSL
Gives rise to the embryo proper

14. Cleavage In Fish Eggs
7.41 Fish blastula

15. 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

16. 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

17. Gastrulation and Formation of the Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish

18. 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

19. Gastrulation and Formation of the Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish

20. 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

21. Gastrulation and Formation of the Germ Layers
Figure Cell migration of endodermal and mesodermal precursors

22. Gastrulation and Formation of the Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish

23. Gastrulation and Formation of the Germ Layers
7.42 Cell movements during gastrulation of the Zebrafish

24. 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

25. Gastrulation and Formation of the Germ Layers
7.44 Convergence and extension in the zebrafish gastrula

26. 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

27. 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

28. 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

29. Axis Formation In Zebrafish
Left-right axis formation
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

30. 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

31. 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

32. Show short clips