Neal, Katherine Alison; (1999) Cell signalling and gene regulation in early Xenopus development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The two activin type I receptors ALK-2 and ALK-4 have distinct effects on gene expression in the Xenopus animal cap assay. Constitutively active ALK-4 (ALK-4*) can reproduce the effects of activin treatment including the dose dependent induction of progressively more dorso-anterior markers, whilst constitutively active ALK-2 (ALK-2*) induces only ventral mesodermal markers and counteracts the dorsalising effects of ALK-4. The dorsal inducing properties of ALK-4* can be transferred to ALK-2* by transfer of a small peptide loop (the β4-β5 loop) from the kinase domain of one receptor to the other. In contrast, I show, that the equivalent region of the ALK-2* receptor, when transferred to ALK-4*, cannot effectively counteract the dorsalising effects of ALK-4*, suggesting that other regions of type I receptors are also involved in determining signal specificity. The chimeric receptor created by transfer of the ALK-2* GS domain and its flanking sequences into ALK-4* (C9), and the chimeric receptor created by transfer of the activation loop of the ALK-2* kinase (C11) into ALK-4* cannot induce the full range of ALK-4* responses, although they contain the ALK-4* β4-β5 loop. My results suggest that ALK-4* regulates at least two different signalling events, one of which is specified by C9 and C11. The Xenopus Brachyury (Xbra) gene is induced in response to the mesoderm inducing factors FGF and activin (and by the activin receptors ALK-2* and ALK-4*). I have examined the transcriptional regulation of a pseudoallele of Xbra, Xbra2, in response to FGF. My results indicate that 381 bp 5' of the Xbra2 transcription start site are sufficient to confer responsiveness to FGF (and activin). Examination of the Xbra2 5' flanking sequence revealed a serum response-like element. This suggested that activation of Xbra2 in response to FGF may occur in a similar way to the activation of c-fos, through the activation of a ternary complex bound to an SRE- like element. My results show that this is unlikely. However, I provide preliminary evidence that a serum response-like factor functions as a negative regulator of mesoderm formation in Xenopus embryos.

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