Project Summary/Abstract Retinal vascular disruption, either during development or in adulthood, remains a major cause of blindness, yet the underlying molecular mechanisms guiding their formation and survival remain largely unknown. Retinal ganglion cells (RGCs), being the first to differentiate, express diffusible molecules that trigger the entry of astrocytes and endothelial cells into the retina, leading to formation of the vasculature. Thus, understanding the transcription factor network expressed in RGCs and endothelial cells is critical for formation and maintenance of retinal vasculature. Our group has shown that the transcription factor, TBX3, plays a critical role in this process. We previously found that TBX3 is required for eye formation in frog, yet its role in mammalian eye formation remained unknown. A confounding issue was that the first reported genetic disruption of Tbx3 was actually a hypomorph with incomplete penetrance. Thus, to address the role of TBX3 in eye formation, we have turned to analyze a validated conditional knockout of Tbx3 from retinal tissues. Using this mouse, we have compelling preliminary data showing Tbx3 is required for intrinsically photosensitive retinal ganglion cell (ipRGC) formation and is also expressed by, and essential for, normal retinal endothelial cell formation. In Aim 1, we will determine which RGCs require TBX3 and we will perform an unbiased search for TBX3 transcriptional targets. In Aim 2, we characterize how the loss of Tbx3 affects Wnt signaling in endothelial cells and if TBX3 is required by endothelial cells for retinal vasculature formation. At the completion of this exploratory R21, we will have laid the foundation to determine the cell types and transcriptional targets most affected by Tbx3 loss, so that future experiments can further examine key factors essential for RGC and vascular formation in the retina.