An appropriate molecular environment is essential for stem and progenitor cells to decide whether to remain undifferentiated or to undergo differentiation. We are using the C. elegans germ line as a model for understanding how this decision is influenced by changes in an animal’s environmental conditions.
During larval development in C. elegans, a pool of proliferative germline stem/progenitor cells accumulates, from which gametes are produced. Previously, our lab showed that the DAF-7/TGFβ-related signaling pathway modulates the accumulation of these cells in response to sensory cues, independent of previously defined roles for this pathway in the dauer decision and lifespan regulation. The expression of daf-7 in ASI chemosensory neurons provides a link between environmental changes perceived by the animal and the number of proliferative germ cells. The TGFβ receptor (TGFβR) complex and its downstream transcriptional regulatory effectors act in the distal tip cell (DTC), the germline stem cell niche.
We sought the molecular link between TGFβR signaling in the DTC and the response in the germ line. GLP-1/Notch signaling maintains the proliferative pool of germ cells in response to DSL-family ligands LAG-2 and APX-1 produced by the DTC. However, previously, we found that TGFβR signaling can influence germ cell accumulation in a glp-1-independent manner, suggesting that it acts in parallel to GLP-1/Notch.
More recently, we found that TGFβR signaling can also promote the expression of lag-2 in the DTC. This conclusion is based on results obtained using new transcriptional reporters and fluorescence in situ hybridization analysis. Expression of lag-2 is reduced upon disruption of TGFβ signaling, but is restored in the absence of daf-3 or daf-5, the negatively regulated downstream effectors of DAF-7/TGFβ signaling. By both yeast and bacterial one-hybrid assays, we found evidence for direct interaction between the DNA-binding domain of DAF-3 (a homolog of Smad4, that in DAF-7 pathway acts as a transcriptional repressor) and the lag-2 promoter. By ChIP, we identified potential DAF-3 binding motifs in the same region of the promoter. Currently, we are mutating these sites in vivo by CRISPR/Cas9. Initial results from reporter assays indicate that eliminating these motifs abrogates the response to TGFβR signaling, suggesting that they directly mediate the response of lag-2 to TGFβ.