Biol 4330 1st EditionFinal Exam Study Guide Lectures: 7 - 13Lecture 7 (Mar 24)Zebrafish development occurs very rapidly. A reporter gene (GFP) fused to the regulatory region of the sh gene at work in living zebrafish embryos. During gastrulation ventral mesoderm secretes BMP28 to induce the ventral and lateral mesodermal and epidermal differentiation. The dorsal mesoderm secretes factos (e.g. Chordi) that lock BMP28 and dorsalize the mesoderm and ectoderm (converting the ectoderm into Neural tissue). The notochords (dorsal mesoderm, chordamesoderm) of both fish and amphibians secrete factors (Chordino (analog of Chordin, as well as noggin and follistatin)) BMP antagonists that block ventral and lateral induction, thereby allowing the ectoderm to become neural.Maternal mRNA for Nodal-related protein is initially found throughout the egg cytoplasm. Initially found throughout the cytoplasm, begins to acculmulate at 4-cells and by 8-cells is localized in only 2 dorsal most blastomeres in early gastrulation. Nodal related protein is found almost entirely in those cells specifying the dorsal region of the embryo.Beta-catenin activates organizer genes in the zebrafish. In the late zebrafish blastula, nuclear localization of beta catenin is seen in the yolk syncital layer beneath the future embryonic shield. Beta-catenin activates squint, Bozozok (arrogant youth on motorcycle) genes whose proteins activate organizer-specific genes as the Stat3 genes whose product is necessary for gastrulation movement. Patterning of the neural ectoderm along the anterior-posterior axis results in the interplay between FGFs, Wnts and RA (retinoic acid) similar to amphibians. The Cyp26 gene encodes RA-4-hydroxulase, a cytochrome family enzyme that degrades RA. In late blastula stage, the Cyp26 gene is confined to the anterior region by FgF and Wnts from the margin. After the start of gastrulation, convergent extension takes the margin farther from the anterior. RA accumulates in this region and activates genes associated with the posterior neural ectoderm.Pathway through which a boundary can form between anterior Cyp26, Otx-expressing and posterior hoxb1-meis-expressing neural ectoderm. In the posterior region, Fgfs and/or Wnt suppress anterior genes such as Otx2. The Fgf/Wnt signal in the posterior also suppresses Cyp26 (encodes enzyme that degrades RA [retinoic acid-4-hydroxylas]) and enhances expression of aldh1a2 (encodes enzyme that makes RA), so RA accumulates (due to the absence of RA-4-hydrolase) and activates posterior genes.Nodal cilia in Kupferʼs vesicle create a current causing a release of Ca2+ on L-side. Ca2+ ions stimulate Notch and BMP4 pathways on the L-side and activate the Pitx2 transcription factor in the left-hand mesoderm. FGF expression is seen predominantly on the Rhand side.Lecture 8 (Mar 31)Amniotes allow the egg to be laid on land instead of in water where buoyancy compensates it. Aristotle was the first to document the details of the 3-week development of the domestic chicken Gallas galas. Chick embryos are a very inexpensive surrogate to human embryos. Between the blastoderm and yolk is a space called the subgermal cavity formed when the blastoderm cells absorb water from the albumen and secrete it between themselves and the yolk.Deep-cells are shed, leaving the one-cell thick area pellucid (which forms most of the embryo). area opaca – perpherial ring of cells that have not shed their deep cells. marginal zone sits between the area pellucid and area opaca. All these structures absorb water and start to differentiate the yolk from the embryo itself. Amniotes have evolved a set of extraembryonic tissues: the hypoblast, the yolk-sac, and the amnion. The Neiwokoop center in frogs sends the signals to the blastocore organizer area, and is analogous to the hypoblast in chicks. The major structure characteristic of avian, reptilian, and mammalian gastrulation is the primitive streak. Koller’s sickle will extend and eventually become Henson’s node.Cells converge to form the primitive streak by first forming the primitive groove which serves as an opening for migrating. Primitive streak defines the axes of the embryo and extends from the posterior to the anterior, migrating cells enter through the dorsal side and move to its ventral side. Henson’s node (primitive knot) and cells move through the funnel shaped depression (primitive pit) to form the notochord and prechordal plate; and separate the left portion of the embryo from the right. Henson’s node = dorsal lip, (the organizer in amphibians, the embryonic shield in fishes)Centralization of the primitive streak is activated by the Wnt planar polarity pathway in the epiblast next to the Koller’s sickle at the posterior of the embryo. Wnt in the epiblast is activated by FGFs from the hypoblast. SEM show epiblast cells passing into the blastocoel and extending their apical ends to becomebottle cells. Hox gene activation begins when the mesodermal precursor cells are still in the epiblast. Hoxgenes lay down the segmentation of whether that segment will be limbs, the neck vertebrae, coccyx, andthe that the somites are going to contain legs. They identify what each of those veterbraes will ultimatelycontain.While streak is being formed and cells are moving inward, the ectodermal precursors proliferate and migrate to surround the yolk by epiboly (Herculean task). This takes 4 days to complete. At the end the ectoderm surrounds the yolk, the endoderm ahs replaced the hypoblast and the mesoderm is between these two regions. Conversion of the radially symmetrical blastoderm into a bilaterally structure is determined by gravity. As the ovum passes through the reproductive tract it is rotated for about 20 hoursin the shell gland (10-12 revs/h) – shifts the yolk such that the lighter components (stored maternal components) lie beneath one side of the blastoderm and that end becomes the posterior portion of the embryo. PMZ-posterior marginal zone – where gastrulations and primitive streak formation begins.. analogous to the Nieuwkoop Center. Wnt8c and Wg1 act together to induce expression of Nodal (another secreted TGF-beta protein) in the future embryonic epiblast next to the Koller’s sickle and the PMZ (pattern similar to amphibians). Nodal Activity is needed to intiate the primitive streak and that it isthe secretion of Cerberus – (an antagonist to Nodal) – by the primary hypoblast cells that prevents primitive