Worm Breeder's Gazette 14(2): 42 (February 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Waksman Institute, Rutgers University, Piscataway, NJ-08855
TGF-b signalling pathways are important in the regulation of cell growth and differentiation, and several aspects of development in various organisms, yet the components of these pathways are relatively unknown. In trying to elucidate the signalling pathway of the dpp/BMP family of TGF-b-like molecules, we are studying three homologous C. elegans genes, sma-2, sma-3 and sma-4 (Savage et al., PNAS 1996), that are believed to be in the pathway, acting downstream of the type II serine-threonine kinase receptor daf-4. These genes give some mutant phenotypes similar to those of daf-4 (small body size, male-tail abnormalities), and mosaic analysis indicates that they are required cell-autonomously and in the same cell as daf-4. Sma-2, Sma-3, and Sma-4, along with the Drosophila Mad gene product (Sekelsky et al., 1995), are members of the dwarfin family: a novel set of proteins characterised by two highly conserved regions with a proline-rich region in between. The functions of these regions are not yet known, but we have shown that the smas act in the daf-4 pathway to define body size. Members of the Dwarfin Family In trying to determine whether these genes represent a pathway conserved in vertebrates, we used degenerate PCR with primers designed against regions of homology conserved between the invertebrate members (Das et al.,WM95, pg.176). We have successfully isolated three murine and three human homologues and one Xenopus homologue. Our analysis of the dwarfin family predicted the existence of multiple members in other organisms, including Drosophila, and, in looking intensively to find these, we have thus far isolated demented, a sma-4-like gene. Since daf-4 mutations show the daf-c and egl phenotypes in addition to the sma and mab phenotypes of the sma genes, we wondered whether there might be any dwarfins acting to transduce the other signals from DAF-4. Recently, two other groups have reported that other genes, known to act in the dauer pathway, are also dwarfins. Furthermore, a search of the shotgun sequences from the C. elegans genome sequencing project has revealed the presence of at least two other members; one maps close to daf-14 and the other to XR (cosmid # F52G3). The latter, tentatively named dwa-1 (for dwarfin-1) does not correspond to any known dauer or small locus. We are in the process of identifying mutations in it. It will be interesting to see if this gene also acts downstream of daf-4, causing either the sma and mab phenotypes, or the daf and egl phenotypes. Functional Characterisation of the Dwarfins We are exploring several methods to outline the possible functions of the dwarfins. We are carrying out genetic screens to determine other phenotypes and other members of the pathway (Savage et al., this gazette). Given the great degree of similarity between these proteins, it is tempting to speculate that they may actually interact with each other in a linear order or function as a complex. To differentiate between the two models, we are using the Brent yeast two-hybrid interaction trap system to look for interactions within the SMAs and between the SMAs and DAF-4. In addition to the experiments on sma-2, -3, and -4 outlined above, we are characterising the vertebrate dwarfins in several ways. We are collaborating on doing in-situ hybridisations against the mouse embryo with the murine dwarfins to look for overlaps in patterns of expression with other components of TGF-b-like pathways, for eg. the ligands BMP-2 and -4 and the receptor BRK-1. We are also collaborating on performing biochemical assays using mammalian cell lines. Since vertebrates have several TGF-b-like pathways, we are working with the vertebrate dwarfins to explore the important issue of whether the dwarfins act specifically in the dpp/BMP pathway or whether they are a more ubiquitous mechanism for transducing TGF-b-like signals.