Ladher, Rajesh; (1997) Patterning the Xenopus embryo: The role of Xom, a novel homeobox gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The body plan of the Xenopus embryo is established by a series of inductive events. The identity of the signalling molecules that mediate these events are known, however little is known as to the transcriptional response the cell makes to a particular signal. In an attempt to clone transcription factors that were induced as a response to various signals, two approaches were used. The first involved subtraction of cDNA libraries. A cDNA library made from animal caps cultured in media was used to remove commonly occurring sequences from a cDNA library made from animal caps cultured in the presence of FGF. This enabled genes to be identified that were specifically induced by FGF. Unfortunately this procedure was hampered by cross-hybridisation of vector sequences between the two pools of cDNA. The other approach involved the cloning of homeobox-containing genes from the early embryo. By using a degenerate primer recognising sequences in the homeobox, a gene was isolated that is expressed at high levels in the Xenopus gastrula. At the early gastrula stage expression of this gene, Xom, is excluded from the organiser. This expression pattern is very similar to the expression pattern of BMP-4. Activation of Xom transcription is inhibited if embryos are dissociated during blastula stages, but addition of BMP-4 to the medium rescues expression. This rescue occurs even in the absence of protein synthesis. Procedures which disturb BMP signalling block the expression of Xom. Animal caps that have been loaded with Xom and then treated with activin, do not undergo the extensive gastrulation-like movements seen in control animal caps treated with activin. Injection of Xom RNA into the dorsal cells of the four cell embryo results in the loss of anterior structures and the inhibition of notochord formation. Both assays resemble the ventralising effects seen when BMP-4 is injected into the Xenopus embryo. This suggests that Xom is not only an immediate early response of BMP-4, it may also mediate some of the effects of BMP-4.

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