PROJECT SUMMARY / ABSTRACT The retina is a cellularly complex tissue comprised of over 100 different cell types which work together to enable proprer vision. Bipolar cells are a diverse class of interneurons that connect rod and cone photoreceptors to retinal ganglion cells, the projection neurons of the retina, which then in turn send their output to the brain. There are currently 15 known subtypes of bipolar cells that differ in their connectivity, physiological responses to light, morphology, abundance, and gene expression profiles. A recent developmental study from our lab performed birthdating analysis to determine when different bipolar cell subtypes were born and found that bipolar cell subtype genesis was arranged into concentrated domains that changed over developmental time. When these domains were compared over time, they formed a wave-like temporospatial pattern of bipolar subtype genesis that spread over the entire extent of the retina. This result suggests a hierarchical model of bipolar subtype genesis in which early retinal progenitor cells generate a series of sub-lineages off-set in developmental time, that undergo a temporally ordered production of bipolar subtypes. To investigate this potential model, I have invented a novel molecular tool called SCRIBE (Sequential Combinatorial Recorder for Iterative Barcode Evolution) that functions as an “evolvable” lineage recorder. By iteratively adding barcode fragments to a genomic target site, SCRIBE aims to generate an evolving, heritable sequence that can be used to distinguish between sister sub-lineages across multiple levels of a clonal lineage tree. The goal of this proposal is to investagate how bipolar cell subtype specification is achieved in development. During the mentored portion of this proposal (K99), I will further develop SCRIBE for use in the in vivo retina and use it to investagate the temporal ordering of bipolar subtype production and the lineage relationships between postnatal RPCs that generate bipolar cells. In the independent portion of the proposal (R00), I will determine whether bipolar subtype specification is intrinsic or extrinsic, and investigate potential molecular regulators of bipolar subtypes specification. The completion of these aims will provide me with training in the fields of retinal development and cutting-edge molecular tool development, complementing my previous training experiences. I will perform the mentored portion of this proposal under the supervision of Dr. Constance Cepko who has been a leader in the fields of retinal development, lineage tracing, and in vivo molecular tool development. Furthermore, the broader scientific environment surrounding the Cepko lab at Harvard Medical School will provide outstanding training opportunities. Together, these experiences will provide me with a solid foundation that will support my long-term career goal of leading my own independent academic laboratory focused on studying the molecular bases of ce...