In C. elegans DBL-1 is a member of the TGF-<font face=symbol>b</font> superfamily closely related to Drosophila Dpp and vertebrate BMP-2/BMP-4 (Suzuki et al., 1999, Development 126, 241-250). The DBL-1 signaling pathway regulates body size and patterning of male-specific copulatory structures. In regulating body size DBL-1 acts as a dosage-dependent regulator (Suzuki et al., 1999). Loss of
dbl-1 activity results in smaller animals, while overexpression of
dbl-1 results in longer animals. From microarray analysis 242 genes regulated by
dbl-1 were identified. From these, 159 genes are repressed but only 83 genes are activated (Liang et al., 2007, Developmental Biology, In Press). We are interested in determining the biological function of some of these putative target genes. We are focusing on collagens and genes involved in fat metabolism. Collagen genes are involved in body size regulation. Cuticle collagens are synthesized by the hypodermis and secreted on the surface of the worm body as an exoskeleton. In the C. elegans genome, about 175 genes encode collagen-like polypeptides. Mutation of these genes causes defects in body morphogenesis, e.g. dumpy, roller, blister, and embryonic lethality (Myllyharju and Kivirikko, 2004, Trends Genet. 20, 33-43). From the microarray analyses three collagen genes were identified as
dbl-1-regulated target gene,
col-141,
rol-6 and
col-41. It is believed that
dbl-1 represses
col-141 and activates
rol-6 and
col-41 (Liang et. al., 2007). We plan to confirm the gene regulation of these genes by initially performing RT-PCR and then do RNAi to test their function. We are also focusing on genes involved in fat metabolism and transport, specifically
vit-5,
vit-6,
fat-6,
fat-7, and
ins-7 (Liang et. al., 2007). We are interested in whether the
dbl-1 pathway regulates body size in part by regulation of fat storage and metabolism. In addition these genes are believed to play a key role in C. elegans aging.
vit-5,
vit-6, and
ins-7 are upregulated by the
dbl-1 pathway, and
fat-6 and
fat-7 are downregulated by it, and this regulation has been confirmed by RT-PCR for some of these genes. To examine these genes we will use fat staining, GFP-reporters, and RNAi feeding. During the fat staining procedure, worms ingest fluorescent particles, which are temporarily stored in the intestinal cells. The cells can then be seen under a fluorescent microscope, and analyzed for number and degree of fat content. GFP reporters will be used on
fat-6,
fat-7 and
ins-7 to locate the physical expression of the gene.