The DBL-1 ligand, a TGF-b homolog in C. elegans, is secreted by neurons and is necessary for body size regulation, mesodermal patterning, innate immunity, reproductive life span, and male tail sensory ray identity. DBL-1 signals via SMA-6 and DAF-4, the type I and type II receptors respectively. The Smads SMA-2, SMA-3, and SMA-4 form a heterotrimeric complex and are shuttled to the nucleus. In the nucleus, this complex is suspected to bind DNA along with SMA-9 (zinc finger transcription factor) to regulate DBL-1 target genes.The mechanisms of DBL-1 target gene regulation are still relatively undefined. For example, which target genes are directly or indirectly regulated by Smads is currently unknown. Here we propose to look at direct/indirect regulation of
col-41, a DBL-1 target gene, which is hypodermis specific in worms. As previously established, the hypodermis is the main tissue responsible for body size regulation via the DBL-1 pathway. Using microarray analysis and tissue specific promoter constructs we have established
col-41 as a DBL-1 target gene. Here we explore whether
col-41 is a direct or indirect target of the DBL-1 pathway. The hypodermal specific
col-41p::2xNLSmcherry construct was used to monitor
col-41 expression. Regulatory region analysis of
col-41 revealed 6 conserved regions containing multiple putative GTCT Smad binding sites. Upon deletions of the conserved regions changes in
col-41 expression were observed. For further analysis of the regulatory region of
col-41, we constructed GST fusions with SMA-2, SMA-3, and SMA-4. We aim use these constructs to determine whether the Smads bind directly to probes composed of fragments of the upstream regulatory region of
col-41. We have presented strong evidence of regulation of
col-41 by the DBL-1 pathway. Elements 1, 2, 3, and 6 in the
col-41 upstream region are required for
col-41 expression. Future investigation will involve testing the upstream regulatory region for binding with Smads and with ELT-1, a known master transcription factor responsible for hypodermal cell-fate that may be involved in Smad recruitment.