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Vertebrate Embryonic Patterning 1 Amphibian Gastrulation: Classical Observations & Experiments Amphibian Development • Fertilization initiates cortical rotation • Cortical rotation relocalizes determinants • These determinants act to specify the dorsal-most regions of the embryo Establishing Asymmetry Localized mRNAs Early Cleavages in Xenopus gray crescent Tiers A B C D Fate Map of Xenopus Blastula Primary Tissue Layers • Ectoderm – skin, neural • Mesoderm – notochord, somites, blood, kidney • Endoderm – gut, prechordal plate Gastrulation Movements Xenopus Gastrulation Movements Gastrulation (again) Xenopus laevis Classic Experiments • Hans Spemann (1903) • Constriction of cleavage stage embryos & fertilized eggs • Demonstrated asymmetry in fertilized egg Embryo Constriction Experiment Egg Constriction Experiment Constriction of the egg becomes the 1st cleavage plane. Constriction of egg has to bisect the gray crescent in order for twinning to occur. If division occur perpendicular to this, only ½ of the egg develops into an embryo. Fate Map of Gray Crescent • dorsal blastopore lip • point where gastrulation begins • What is it about the gray crescent that makes it so important? Spemann’s Specification Experiments Presumptive neural plate ectoderm in the early gastrula was specified as ectoderm, but undetermined. Later gastrula neuroectoderm was determined to a neural fate. Dorsal Lip Transplantation Perturbations of Axis Formation: Making Two Dorsal Axes Cool Perturbations of Normal Development • Ventralization: – UV irradiation of vegetal pole shortly after fertilization – blocks cortical rotation by crosslinking cytoskeletal proteins – DAI Scale – rating of 0 to 5 of dorsal inhibition • 0 is normal, 5 is completely ventralized • embryo develops as a lump of endoderm & ectoderm More Cool Perturbations • Anterior/Dorsalization: – LiCl treatment • increases the amount of anterior/head tissue at the expense of posterior-dorsal mesoderm & ventral tissues • Posterior/Ventralization – Retinoic Acid treatment • decreases anterior/head structures Implications • Strategies for cloning genes involved in dorsal/ventral specification – Subtractive library screens between dorsalized and ventralized embryos – Functional library screens • Rescue of ventralized embryos • Induction of secondary axis Properties of Dorsal Lip Region • Autonomously specified • Dorsal lip never respecified by tissue at location of transplantation • Donor cells always contribute to notochord (mesoderm) • Secondary neural tube forms from recruited host cells • Donor lip cells organize host cells Mesoderm Induced by Vegetal Cells Nieuwkoop’s Experiment Nieuwkoop’s Center Rescues Dorsal Axis in UV & Duplicates Axis in Wild-type Inducing Centers in the Blastula “Spemann” (General) Fate Map of Organizer and Nieuwkoop’s Center
