Zebrafish Cleavage in Fish Eggs ? eggs are telolecithal - most of the egg cell occupied by yolk ? cleavage can take place only in the blastodisc - thin region of yolk-free cytoplasm at animal pole of the egg ? cell divisions do not completely divide the egg - cleavage = meroblastic ? only the blastodisc becomes the embryo - discoidal cleavage ? calcium wave initiated at fertilization stimulate contraction of actin cytoskeleton to squeeze non-yolky cytoplasm into animal pole ? calcium ions coordinate mitotic apparatus with actin cytoskeleton ? help propagate cell divisions across cell surface ? needed to deepen the cleavage furrow ? heal the membrane after the separation of the blastomeres ? 1st 12 divisions occur synchronously, forming a mound of cells that sits at the animal pole of a large yolk cell ? constitute the blastoderm ? initially, all the cells maintain some open connection with one another and with the underlying yolk cell - molecules can pass freely from one blastomere to the next ? Mid-Blastula Transition ? first detected at 10th cell division ? zygotic gene transcription begins, cell divisions slow, cell movement becomes evident ? at this time: 3 distinct cell populations can be distinguished ? Yolk Syncytial Layer (YSL) ? formed at 9th or 10th cell cycle - when the cells at the vegetal edge of the blastoderm fuse with the underlying yolk cell ? Internal YSL: ? as the blastoderm expands vegetally to surround yolk cell, some yolk syncytial nuclei move under the blastoderm to form this ? External YSL: ? yolk syncytial nuclei move vegetally, staying ahead of the blastoderm margin ? Enveloping Layer (EVL) ? made up of the most superficial cells from the blastoderm ? form an epithelial sheet a single layer thick ? extraembryonic protective covering that is sloughed off during later development ? Deep Cells ? between the EVL and YSL ? cells that give rise to the embryo proper Gastrulation in Fish Embryos ? 1st cell movement = epiboly of the blastoderm cells over the yolk ? deep cells of the blastoderm move outward to intercalate with the more superficial cells ? later, moves egetally over the surface of the yolk cell and envelops it completely ? the EVL is tightly joined to the YSL and dragged along with it ? Formation of Germ Layers ? Germ Ring - thickening that occurs throughout the margin of the epibolizing blastoderm ? composed of a superficial layer (the epiblast); and an inner layer (the hypoblast) ? once hypoblast has formed, cells of epiblast and hypoblast intercalate on the future dorsal side to form localized thickening - embryonic shield ? Embryonic Shield ? functionally equivalent to the dorsal blastopore lip of amphibians - can organize a secondary embryonic axis ? hypoblast cells converge and extend anteriorly, eventually narrowing along the dorsal midline of the hypoblast ? forms the chordamesoderm (precursor of the notochord) ? cells adjacent to this - paraxial mesoderm cells - are the precursors of the mesodermal somties ? endoderm arises from the most marginal blastomeres of the late blastula-stage embryo Axis Formation in Fish Embryos ? Dorsal-Ventral Axis Formation: The Embryonic Sheidl and Nieuwkoop Center ? Embryonic shield ? critical in establishing the dorsal-ventral axis ? can convert lateral and ventral mesoderm (blood and connective tissue precursors) into dorsal mesoderm (notochord and somites) ? can cause ectoderm to become neural rather than epidermal ? forms the prechordal plate and the notochord ? precursors of these 2 regions are responsible for inducing ectoderm to become neural ectoderm ? BMPs and certain Wnts made in ventral and lateral regions would normally induce the ectoderm to become epidermis ? notochord secrete factors that block this induction (ectoderm becomes neural) ? BMP2 - induces embryonic cells to acquire ventral and lateral fates ? Wnt8 - ventralizes, lateralizes, and posteriorizes the embryonic tissues ? Chordino - secreted by chordamesoderm; binds and inactivates BMP2B ? if mutated, neural tube fails to form ? if bmp2b is mutated, dorsal structures expand at the expense of ventral structures ? Insulin-Like Growth Factors ? production of anterior neural plate ? upregulate Chordino and goosecoid while restricting the expression of bmp2b ? receptors found predominantly in the anterior portion of the embryo ? Zebrafish vs. Amphibians: ? Both... ? use similar molecular tools to undergo cleavage, gastrulation, and axis specification ? use beta-catenin and Nodal-related proteins to form dorsal mesoderm and enable this mesoderm to express the organizer genes ? use BMPs and Wnts to lateralize and vegetalize the embryo, and in both groups, the organizer genes encode proteins such as Chordin, Noggin and Dickkopf that antagonize BMPs and Wnts ? The Fish Nieuwkoop Center ? nuclei in that part of the yolk syncytial layer that lies beneath the cells that will become the embyonic shield similarly accumulate beta-catenin ? presence of beta-catenin distinguishes dorsal YSL from alteral and ventral YSL regions ? inducing beta-catenin in ventral side causes dorsalization and a second embryonic axis ? beta-catenin of embryonic shield activates Squint and Bozozok ? Squint - Nodal-like paracrine factor ? Bozozok - homeodomain protein similar to Amphibian Siamois ? can repress BMP and Wnt genes that would promote ventral functions ? supresses a transcriptional inhibitor allowing the organizer genes to function ? with Squint, they act individually to activate the chordino gene and act synergistically to activate other organizer genes such as goosecoid, noggin and dickkopf ? Embryonic shield equivalent to the amphibian organizer and the dorsal part of the yolk cell, with the dorsal marginal blastomeres, can be thought of as the Nieuwkoop Center ? maternal beta-catenin also initiates/coordinates the cell movements of the mesoderm and endoderm during gastrulation ? Dorsal mesoderm cells derived from embryonic shield generate notochord posteriorly and the prechordal plate mesoderm anteriorly ? anterior cells are highly motile and lead the mesoderm into the embryo ? cells that form notochord are not motile, but they do undergo convergent extension ? both are regulated by Stat3 -transcription factor subunit regulated positively by beta-catenin ? regulates expression of small GTPases such as RhoA ? critical components of planar cell polarity pathway - cells become differentiated along their medial-lateral axes ? polarization required for intercalation, also causes cell divisions to align themselves along the animal-vegetal axis ? Anterior-Posterior Axis Formation ? patterning of neural ectoderm along anterior-posterior axis results from the interplay of an FGF, a Wnt and retinoic acid ? 2 separate processes: ? Wnt signal represses the expression of anterior genes ? Wnt, Retinoic acid, and FGF are required to activate the posterior genes ? regulation coordinated by retinoic acid-4-hydroxylase: an enzyme that degrades retinoic acid ? prevents the accumulation of retinoic acid at the embryo's enterior end, blocking the expression of posterior genes there ? FGFs and Wnts inhibit the expression of retinoic acid-4-hydroxylase ? Left-Right Axis Formation ? cells on the left side of the body are given that information by Notch and Nodal signaling and by the Pitx2 transcription factor ? cells on the right side of the body are exposed to FGF signaling ? currents produced by cilia in the node may be responsible for left-right axis formation ? Kupffer's Vesicle ? nodal structure housing cilia that control left-right asymmetry ? fluid-filled organ ? blocking ciliary function by preventing synthesis of dynein or by ablating the precursors of the ciliated cells prevented normal left-right axis formation ? leftward flow of nodal current
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