Worm Breeder's Gazette 16(5): 43 (February 1, 2001)
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.
|1||Samuel Lunenfeld Research Institute, Mount Sinai Hospital,, Toronto, Ontario, Canada|
|2||International Institute of Genetics and Biophysics, Naples, Italy|
|3||Department of Biochemistry and Molecular Biology, University of Calgary,, Calgary, Alberta, Canada|
We have previously described a C. elegans gene with significant similarity to VEGF-like growth factors from mammals (Tarsitano et al., WBG 16(2) page 36 (1999)).The next step was to look for potential VEGF-like receptors. A second motivation was that neuropilin, a cell surface glycoprotein that acts as a receptor for collapsin/semaphorin and is involved in axon guidance, also acts as a receptor for some members of the VEGF family of ligands. Popovici et al. (Genome Research (1999a) 9, 1026-1039) have collected some 28 sequences from the C. elegans genome that show significant similarity to receptor mammalian tyrosine kinase receptors. Among this collection, they point out four genes with similarity to mammalian VEGF receptors. Moreover, they have reported that one of these genes (T17A3.1) is expressed in head and tail neurons, including neurons of the amphid and phasmid (WBG 16(1) page 41 (1999b)). We have focused on a tandem pair of VEGFR-like genes found on cosmid F59F3 (X chromosome). Sequence alignments (see also Popovici et al., 1999a) show anywhere from 22-33% amino acid identity (38-48% similarity) to VEGF Receptors 1 and 2, as well as to PDGF receptors A and B. Although presumably the result of a gene duplication event, the two genes (F59F3.5 and F59F3.1) show only 43% amino acid identity (59% similarity) to each other. Northern blots show that mRNAs have the size predicted from AceDB. To determine expression pattern, we fused 4 or 5 kb of 5'-flanking region of each gene to one of Andy Fire's GFP/lacZ reporter constructs and produced a number of transgenic strains. At least as monitored by GFP, the upstream gene (F59F3.5) is expressed intensely in the gut, beginning in the embryo, and in only a few cells outside of the gut. Expression of the downstream gene (F59F3.1), again as monitored by GFP, begins in the gut as early as the 4E-8E cell stage and expression remains intense in the gut of later stages. However, beginning roughly at the start of morphogenesis phase, high levels of expression are seen in many, possibly most, cells of the embryo; expression declines by hatching but in L1 larvae, expression can easily be detected in neurons and hypodermis along with many other cells. Expression declines rapidly thereafter (possibly complicated by reporter protein perdurance) but with gut expression remaining strong. The Kohara data base also reports strong gut expression. A developmental Northern also shows highest expression in embryos. A probable knockout of the downstream gene (F59F3.1) has been isolated (deletion of exons 2,3 and part of 4, leading to loss of N-terminal immunoglobulin domains). The only obvious phenotype is an ectopic male ray 1 (anterior to the normal ray 1 position) and a short ray 3. The penetrance of these defects is roughly 30% and 10%, respectively. RNAi attempts only allow us to say that there is no obvious embryonic phenotype, despite the rather intense embryonic expression. A deletion that should remove the kinase domain of the upstream gene (F59F3.5) is in the last steps of sib-selection. The future will include making antibodies, both to define expression patterns in more detail and to investigate localization within the expressing cells. Is there any reason to think that these receptors interact with the previously described VEGF-like ligand? What are the receptors doing in the early gut and why is there such a burst of widespread expression during morphogenesis? Finally, similar ray defects have been observed in mutants of semaphorin 1a and plexin 1a (Dalpe, Ginzburg, and Culotti, in preparation) and we plan to make and examine the appropriate double mutants with the latter genes.