This project investigated a role for a developmentally regulated sulfatase in modulating FGF dependent gene expression in Xenopus. Using Xenopus to study cell signaling in development affords certain advantages including accessibilty from the earliest stages of development and the ease with which one can carry out embryological manipulations, surrogate genetics and functional assays.
We have fully characterised the expression pattern of XtSulf1 during the development of X.tropicalis (Freeman et al., 2008). and found dynamic expression of XtSulf1 in the hindbrain and floorplate of the neural tube, the pronephros, the posterior mesoderm and somites, some aspects of the neural crest and the retina. This dynamic expression pattern shows that XtSulf1 is highly regulated during development and expressed in regions of the embryo known to be involved in inductive interactions, cell proliferation or cell migration. Together with our findings on the biological activity of XtSulf1 suggests that it could play a role in multiple processes during embryonic development.
We have successfully generated an antibody that can recognise XtSulf1 protein in ELISAs and in Western blots of embryos injected with XtSulf1 mRNA. We are currently working to sufficiently purify the anti-sera to make it useful in detecting endogenous XtSulf1 protein in immunoprecipitations, Western blotting and whole mount immunohistochemistry. In the meantime, we generated a GFP-tagged XtSulf1 in order to determine the subcellular localisation of the XtSulf1 protein. The figure above on the right shows that XtSulf1-GFP localises to the cell membrane as expected for this extracelluar sulfatas
During embryonic development, a single cell (the fertilised egg) gives rise to many different kinds of cells. Cells become different from each other by turning on or off distinct genes or sets of genes and they do this in response to signals, including FGFs. A potential modulator of FGF signals, an enzyme called sulfatase, is tightly controlled during embryonic development and I plan to analyse how this enzyme effects FGF signalling and muscle gene activation. I have chosen to undertake my studies using the related frog species, Xenopus laevis and Xenopus tropicalis because of the many advantages that amphibian embryos hold for the study of embryonic development, such as their large size and that they develop rapidly and outside the mother making the embryos accessible throughout development. Many of the experiments that I propose to do are standard procedures for frog embryologists and are not possible to do in bird embryos and would require long and expensive procedures in mouse embryos. Much of what we know about the earliest signals important during embryonic development has been learned by work in amphibian embryos and this project can be carried out efficiently using this animal model. Moreover, since many of the same genes control development of of all vertebrates, what we learn about frog development is directly applicable to what is happening in humans.
In this project we used the amphibian Xenopus tropicalis to demonstrate that Xtsulf1 expression is highly regulated during development and plays an important role in modulating cell signaling in vivo. We have shown that XtSulf1 is required to restrict FGF signals in embryos. We also have shown for the first time that Sulf1 is an endogenous inhibitor of BMP signaling.
|Acronym||Sulf1regulates FGF signaling |
|Effective start/end date||1/10/04 → 30/06/08|