View Program - 63rd Annual Drosophila Research Conference

Notch signaling is a critical regulator of multiple developmental processes through its ability to control gene expression, and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Notch signaling is activated through binding of the transmembrane ligand, Delta, to the Notch extracellular domain (NECD) of the transmembrane Notch receptor. Ligand engagement and trans-endocytosis into the signal-sending cell drives removal of the labile NECD, and concomitant cleavage of the Notch intracellular domain (NICD). Upon subsequent translocation to the nucleus, the NICD regulates target gene expression through its interaction with Suppressor of Hairless Su(H). Although Notch signaling has been shown to play a role in regulating single minded (Sim) expression in the embryonic mesectoderm, Notch activity and function in the directly-adjacent cells of the ventral mesoderm remain unknown. Considering that Delta endocytosis and NECD trans-endocytosis are required for Notch signal activation, and are restricted to the ventral mesoderm in the early embryo, we investigated the role of Notch signaling in the ventral mesoderm. To test our hypothesis, we have used a combination of Optogenetics, quantitative RNA fluorescent in situ hybridization (FISH), and qPCR. Through in silico analysis, we identified 13 potential Notch target genes based on the following three criteria: 1) expression in the embryonic ventral mesoderm, 2) promoter-resident Su(H) binding site(s), and 3) differential expression during ventral mesoderm formation. We have validated these candidate Notch target genes, which include WntD, Asph, Heartless, Traf4, Tinman, Stumps, Mef2, Mes2, mir-1, Neurotactin, and NetrinA, by comparing the level of gene expression between loss-of-function Notch mutants and wild-type embryos through two orthogonal techniques; FISH and qPCR. Consistent with a role for Notch signaling, expression of these mesoderm-specific genes is reliant upon Notch. These results prompted us to ask whether Notch signaling is sufficient to drive expression of these mesodermal target genes. To address this, we have developed and validated a set of novel Optogenetic tools to ectopically activate Notch signaling in a precise spatio-temporal manner. Consistent with previous findings, we observed that ectopic activation of Notch signaling is sufficient to drive Sim, a Notch target gene normally restricted to the mesectoderm. Interestingly, we have observed a stage-dependent expansion of Sim expression into both the ectoderm and the ventral mesoderm of the early embryo, along with a change in the position of the ventral mesodermal boundary, demonstrating an effect of precocious Notch activity on embryonic patterning.

Investigating the role of Notch signalling in the development of the ventral mesoderm in Drosophila melanogaster