BIOL 4330 1st Edition Lecture 7 Outline of Last Lecture I. Amphibian GastrulationII. Amphibian Axes FormationIII. The OrganizerIV. SummaryOutline of Current Lecture I. Intro to Zebrafish DevelopmentII. Axis SpecificationIII. Nodal SignalingIV. Bozozok ProteinV. Axis FormationCurrent LectureZebrafish 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 andepidermal 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.Common Fish and Xenopus molecular tools for cleavage, gastrulation, and axis specification:- Use beta-catenin and Nodal related proteins to form the dorsal mesoderm and enable this mesoderm to express the organizer genes- Use BMPs and Wnts to lateralize and ventralize the embryo and organizer genes encode proteinssuch as Chordin, Noggin, and Dickkopf that antagonize (block) the BMPs and Wnts- Later in development, a specific Wnt protein is used to posteriorize the ectoderm, forming the trunk neural tubeMaternal mRNA for Nodal-related protein is initially found throughout the egg cytoplasm. These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.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.Nodal Signaling- Critical for dorsal axis formation- Maternal mRNA for the Nodal protein (Nodal-related-1 (Ndr1) is transported along microtubules into the dorsal most blastomeres of the 8-cell embryo- Critical for endoderm formation (Squint and Cyclops, another Nodal-related protein) induce the Bon and GATA5 TFs- These are co-expressed in the marginal domain of the late blastula and regulate the expression of the downstream genes that form the endodermBeta-catenin activates organizer genes in the zebrafish.In the late zebrafish blastula, nuclear localization of beta catenin is seen in the yolk syncital layer beneaththe 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.Actions of the Bozozok Protein- Acting alone bozozok can repress BMP and WNT genes that would promote ventral functions- It suppresses a transcriptional inhibitor (vega1 gene) allowing the organizer genes to function- Bozozok and Squint act individually to activate other organizer genes; chordino as well as goosecoid, noggin, and dickkopf genes; these encode the proteins that block BMPs and Wnts and allow the specification of the dorsal mesoderm and neural ectoderm.Organizer and Nieuwkoop Center Equivalents- Embryonic shield is considered equivalent to the amphibian organizer- The dorsal part of the fish yolk synctial layer together with the dorsal marginal blastomeres (the precursors of the Kupffer cells) can be considered equivalent to the Nieuwkoop center of the fishembryoAnterior and Posterior AxesPatterning of the neural ectoderm along the anterior-posterior axis results in the interplay between FGFs, Wnts and RA (retinoic acid) similar to amphibians2 separate processes in fish:1) A Wnt signal represses the expression of anterior genes2) Wnts, A, and FGFs are required to activate the posterior genesThe 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.Left-right Axis Formation in Fishes:- In all vertebrates left and right sides differ both anatomically and developmentally- In fish, heart is on the left side and different structures exist in the L and R-side of the brain- L-side of the body are given info by Notch and Nodal signaling and by the Pitx2 TF- R-side are exposed to FGF signaling- Currents produced by cilia may be responsible for L-R axis formation- In zebrafish the nodal structure housing the cilia that control L-R asymmetry is a transient fluid-filled organ called Kupfferʼs vesicleNodal 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