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

TiersABCD

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