The formation of complex tissue and organs systems in developing organisms depends on the ability of dividing stem and progenitor cells to properly integrate extracellular signals present in the embryonic environment. The combined actions of these signals in turn direct diverse processes such as progenitor maintenance, differentiation, and assignment of specific cell fates. A key step towards understanding the basis of and means for preventing birth defect thus lies in defining how different signaling pathways mechanistically intersect, permitting the activation of one signaling pathway to influence responses to other signals. Moreover, the combinatorial activities have the potential to extend the range of outcomes possible from a limited range of developmental signals. Our studies in the developing spinal cord have identified an unexpected role for Notch receptor signaling in modulating the response of cells to the tissue morphogen Sonic hedgehog (Shh). Both activation and inactivation of the Notch pathway alters the dorsoventral register of neural progenitors, leading to corresponding changes in neuronal and glial cell fates (Kong et al. Dev Cell, 2015). In tracking down the mechanism behind these effects, we discovered that Notch signaling influences the trafficking of the Shh receptor Patched1 (Ptch1) and the key downstream Shh effector Smoothened (Smo) to primary cilia, leading to changes in downstream Shh pathway activities. Importantly, this role for Notch can be seen in multiple cell types including mouse and human neural progenitors, fibroblasts, and skeletal myoblasts, suggesting it may be a general feature of mammalian cells. Collectively, our studies reveal a novel and surprisingly proximal role for Notch in shaping the interpretation of the Shh morphogen gradient and thereby impacting cell fate determination. The means by which Notch influences the trafficking of Shh signaling proteins, however, remains unknown. Our preliminary data suggest that these actions of Notch are most likely transcriptionally mediated, raising the questions of what are the target genes regulated by Notch, and how do they impact the trafficking of Shh pathway components, and possibly other signaling proteins, to primary cilia? Moreover, do defects in this Notch-mediated pathway contribute to congenital defects affecting ciliary transport, collectively termed ciliopathies? Our proposed studies will address these questions, first by identifying the transcriptional targets of Notch and its downstream effector Hes1 that coincide with changes in ciliary trafficking, and second by developing a platform for investigating the function of these newly identified Notch target genes in Shh signaling through CRISPR/Cas9-mediated deletions. With this approach, we will: a) reveal the nature of the functional intersection between the Notch and Shh transduction pathways in neural fate selection and b) identify new regulators of Shh signaling and protein trafficking to primary cilia more generally.
The formation and organization of tissue and organ structures such as the brain and spinal cord depends on the ability of dividing stem cells to appropriately respond to a variety of instructive chemical signals present in developing embryos. In the proposed research, we will investigate how one of the most important signals for stem cell maintenance, Notch, modulates cellular responses to a second vital signal, Sonic hedgehog, to direct the formation of different cell types in the nervous system. A better understanding of how signals such as Notch and Sonic hedgehog collaborate in tissue formation will facilitate efforts to both understand the root causes of developmental defects and harness these mechanisms to repair the diseased or damaged nervous system.
