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

FISH DEVELOPMENT Zebrafish

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)

Early Zebrafish Development 7.37 Zebrafish development occurs very rapidly

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

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

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

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

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

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

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

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

Cleavage In Fish Eggs Enveloping layer (EVL) Deep cells Second cell population Superficial cells of blastoderm Form epithelial sheet 1 cell layer thick Extraembryonic protective covering Sloughs off later Deep cells Between EVL and YSL Gives rise to the embryo proper

Cleavage In Fish Eggs 7.41 Fish blastula

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

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

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

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

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

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

Gastrulation and Formation of the Germ Layers Figure 7.43 Cell migration of endodermal and mesodermal precursors

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

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

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

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

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

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

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

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

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

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

Show short clips