Development Biology: Fertilization to Blastula

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The Role of localized Cytoplasmic Determinants in the Xenopus Egg

Localized Cytoplasmic determinants:

Regulatory molecules that are differentially distributed in the cytoplasm of the cell such that in cell division the two daughter cells inherit deifferent regulatory molecules and consequently enter different states of commitment.

Daughter cells inherit cytoplasmic determinants Asymmetrically of symmetrically depending on the orientation of the mitotic spindle.

Determination of cell fates

The Xenopus egg is radially symmetric with the Animal pole (darker side) roughly corresponding to the anterior axis and the Vegetal pole (lighter side) the posterior axis. Each of the poles has a different fate: the animal pole will become ectoderm and the vegetal pole will become endoderm. This is because the two poles have different CYTOPLASMIC DETERMINANTS.

Cortical rotation:

The actin-rich cortex of the egg rotates about 30 degrees so that the vegetal cortex opposite the sperm entry point rotates towards the animal pole

In Xenopus, ferilization sets up the body axes via cortical rotation. The rotation is greatest in the plane defined by the sperm entry point. Cortical rotation triggers the formation of a SIGNALLING CENTER (the nieuwkoop Centre) in the ventral region opposite the sperm entry point. The NIEUWKOOP CENTRE defines the side that it occupies as the future dorsal side.

Signalling centre:

Localized region of the embryo the exerts a special influence on surrounding tissues and can thus determine how they develop

Cortical rotation triggers the active transport of vesicles in the vegetal pole to the side opposite the sperm entry point. These vesicles contain DISHEVELLED, a key protein in the Wnt signalling pathway. Wnt 11 mRNA is also present on the dorsal side following cortical rotation. Vegetal cells that inherit a large number of vesicles containing dishevelled and that are exposed to Wnt 11 will become part of the Nieuwkoop centre

Some mRNAs are localized to the vegetal pole others will move during cortical rotation

Veg T is a transcription factor

Wnt 11 is a ligand for a cell surface receptor

Wnt signalling

When DISHEVELLED is inactive phosphorylates beta-caterin is polyubiquitylated and degraded in proteasomes

When DISHEVELLED is activated unphosphorylated beta-caterin accumulates and migrates to nucleus it displaces groucho and associates with coactivator

Beta-catenin

Increased dishevelled protein and Wnt 11 on the dorsal side results in increased bete-catenin on the dorsal side. Bete-catenin is a transcription factor. By the 16 cells stage it can be detected within the nuclei on the dorsal side.

Injecting beta-catenin into the ventral vegetal cells will specify a new Nieuwkoop centre, generating a twinned embryo with two heads

The importance of cortical rotation

Treatments that reduce the rotation of the cortex such as UV light produce a ventralized embryo made up of blood forming mesoderm.

Treatments that activate the Wnt pathway such as lithium produce dorsalized embryo's as lithium inhibits GSK3.

UV ventralized embryos can be rescued by a localized injection of lithium

Cleavage in Xenopus

After CORTICAL ROTATION the egg subdivides into smaller cells by repeated mitosis in a process called CLEAVAGE. The cell cycle is ~20-30 minutes with no transcription. Maternal mRNAs are translated and DNA is synthesized.

The first cleavage plane bisects the NIEUWKOOP CENTRE to generate daughter cells which each contain molecules from the centre.

If you separate the first two daughter cells, they will each from a normal embryo.

If you force the first cleavage plane to bypass the NIEUWKOOP CENTRE the division will be asymmetric as only the daughter cell that inherits the centre will develop dorsal structures

The NIEUWKOOP CENTRE is required for normal development.

Forming a blastula in Xenopus

On the surface of the embryo, tight junctions create a permeability barrier between the embryo and the environment. Gap junctions between the embryonic cells allow ions to flow freely between cells. By the 16 cell stage, Na+ is pumped from the exterior environment into the spaces between cells. Water follows because of the increase in osmotic pressure. The inwards flow of water creates a cavity inside the embryo called the BLASTOCOEL. The embryo, that now consists of an epithelial sheet several cells thick surrounding the BLASTOCOEL is called a BLASTULA