CHAPTER 10, EARLY DEVELOPMENT AND AXIS FORMATION IN AMPHIBIANS PART-2 ZOO3603C Progressive Determination of the Amphibian Axes The specification of axes Vegetal proteins  VegT destroyed--- entire embryo develops as an epidermis  Vg1 lacked --- no endoderm nor dorsal mesoderm The axes are specified by events triggered at fertilization and realized during gastrulation. Concept of regulative development  Blastomere has a potency greater than its normal embryonic fate.  Fate is determined (induced) by interactions between neighboring cells.  How is it induced?  What factor cause the induction? Spemann’s demonstration of nuclear equivalence in newt cleavage After 14 days, each side had a normal embryo. Ligature at 8-cell stage Entering a single nucleus to the other side at 16-cell stage Nuclei at 16-cell stage are genetically identical and still totipotent in the newt. Asymmetry in the amphibian egg Separation at left-right axis Separation at ventral-dorsal axis Gray crescent is essential for normal development. Containing epidermal, blood, mesenchymal and gut cells. Determination of ectoderm during newt gastrulation Normal development Secondary neural plate Regulative development Autonomous development What does cause the determination? Hans Spemann and Hilde Mangold: Primary Embryonic Induction Organization of a secondary axis by dorsal blastopore lip tissue Dorsal blastopore is the self-differentiating tissue. • Organizer • Primary embryonic induction • How does the organizer form? • The dorsal signal: β-catenin • The vegetal TGF-β-like signal • The mesodermal signal Mechanisms of Axis Determination in Amphibians Questions on the mechanisms  How did the organizer get its properties?  What caused the dorsal blastopore lip to differ from any other region of the embryo?  What factors were being secreted from the organizer to cause the formation of the neural tube and to create the anterior-posterior, dorsal-ventral and left-right axes? Mesodermal induction by vegetal endoderm Animal cap cells generate mesodermal tissue. Factors from vegetal cells Mesodermal induction by vegetal endoderm On dorsal side, a signal is released by Nieuwkoop center.Signals for the axial determination  The dorsal signal: β-catenin  The vegetal TGF-β-like signal  The mesodermal signal, Nodal-relating proteinsThe vegetal cells are responsible for causing the initiation of gastrulation Rescue by transplantation of dorsal vegetal blastomeres which can induce another axis in the embryo. Vegetal cells induce the organizer which induces axis. The regional specificity of mesoderm induction D1 most induced dorsal mesoderm. Nieuwkoop Center is in D1 cell What makes dorsal/ventral mesoderm? The role of Wnt pathway in ventral-dorsal axis specification 2-Cell β-catenin Blastula β-catenin localize in nuclei on dorsal, but not on ventral. β-catenin dorsal localization persists through gastrula stage. How this localization happen? Dorsal Ventral Model of the mechanism of localization of β-catenin in the dorsal portion Dsh, Dishevelled protein GBP, GSK3-binding protein GSK3, glycogen synthase kinase 3 Cortical rotation causes the sift of the Dsh complex. on microtubules cortical rotation Model of the mechanism of localization of β-catenin in the dorsal portion 1. Dsh inhibits GSK3 2. GSK3 degrade β-catenin 3. β-catenin initiate the organizer Dsh localization cause the ventral-dorsal axis Model of the mechanism of localization of β-catenin in the dorsal portion Inactivation of GSK3 on both blastomeres of 2 cell. Formation of 2nd axis. GSK3 is one of the key to determine the ventral-dorsal axis through β-catenin. Induction of the organizer in the dorsal mesoderm • Tcf3, ubiquitous transcription factor • β-catenin/Tcf3 complex can activate the transcription. • Goosecoid protein, transcription factor which can activate genes in organizer. Difference of β-catenin expression cause the ventral-dorsal axis. Mesoderm induction and organizer formation by β-catenin and TGF-β proteins Xnr, nodal-related gene induced by β-catenin, Veg1 and Vg1. Goosecoid induction • Induction of neural ectoderm and dorsal mesoderm: BMP inhibitors • Noggin • Chordin • Follistatin Functions of the Organizer Four major ability of the organizer  Self-differentiate dorsal mesoderm.  Dorsalize the surrounding mesoderm into paraxial mesoderm.  Dorsalize the ectoderm, including the formation of neural tube.  Initiate the movements of gastrulation. Ability of goosecoid mRNA to induce a new axis Gastrula Control Injected goosecoid mRNA Single blastopore Double blastopore Control +goosecoid Two dorsal axes Two heads Goosecoid can induce the axis. Neural structures induced in presumptive ectoderm Ectoderm Dorsal lip Filter membrane Incubation of ectoderm with dorsal lip without the direct contacts induced neural structures. Secreted factors from dorsal lip induced the neural differentiation. What is the secreted factors from dorsal lip? Rescue of dorsal structures by Noggin protein Exposure to UV causes no the cortical rotation.  No organizer Control + noggin mRNA Noggin mRNA injection rescue the dorsal structure in dosage-related fashion Localization of noggin mRNA in the organizer tissue In situ Hybridization Gastrulation Involution Convergent extension Blastopore lip Prechordal plate & pharyngeal endoderm Extend beneath the ectoderm Localization of chordin mRNA In situ Hybridization Just prior to gastrulation Beginning gastrulation, Dorsal blastopore lip Later gastrulation, organizer tissues Chordin expression is activated by β-catenin Chordin, one of the organizer protein Model for the action of the organizer Immuno-histo chemistry of Smad1 which is downstream of the cascade BMP4. Organizer molecules block the action of BMP4. Organizer molecules induce ventral-dorsal axis through BMP4 signaling pass way. Control of neural specification by the levels of BMPs Whole mount in situ hybridization of Sox2, which is a specific mRNA in neural tube Control Hyper BMPs by morpholino. Control Hypo BMPs Lack of Sox2 expression in neural tube Lack of ventral-dorsal axis in neural tube Epidermis is instructed by BMP signaling, and the organizer works by blocking the BMP signal from reaching the ectoderm above it. • The determination of regional differences • The