The Role of Lineage in Development of Chicken Retina and High Acuity Area

PROJECT SUMMARY / ABSTRACT The primary goal of this proposal is to understand how lineage contributes to cell diversity in different regions of the chick retina including the high acuity area (HAA). While previous studies have traced the output of individual retinal progenitor cells (RPCs) from early in development, the patterns of cell divisions that emerge from these progenitors and the resulting lineage “trees” that describe the complete series of divisions that a single progenitor and its progeny undergo remain unknown. By reconstructing these large, multi- generational lineage trees, we will be able to identify intra-clonal relationships and generate models of how cell diversity is produced across the retina. Our lab has developed a new lineage tracing tool called SCRIBBLE in order to re-create these lineage trees. Preliminary testing of SCRIBBLE in the mouse retina indicates that it is working as intended and suggests that it can accurately re-create the larger, lineage trees. Aim 1 will focus on adapting SCRIBBLE for use with the spatial transcriptomics platform Stereo-seq. Single cell technologies, including spatial transcriptomics like Stereo-seq, only capture a limited number of mRNA molecules from each cell. Accurate lineage reconstruction requires the recovery of enough SCRIBBLE barcodes to reconstruct the entire lineage tree. First, we will perform benchmarking experiments and determine how efficient SCRIBBLE barcode transcripts are captured on Stereo-seq. Next, we will benchmark the efficiency of SCRIBBLE itself as a lineage tracing tool. Together, we will use these results to determine whether SCRIBBLE is sufficient to re-create high resolution clonal lineage trees or whether modifications need to be made in order to use SCRIBBLE for spatial-transcriptomic analysis of the chick retina. In Aim2, we will use SCRIBBLE to perform lineage tracing of early RPCs in the chick retina. We will begin lineage tracing at embryonic day 2 and collect samples at embryonic day 18 when retinal cell identity can be confidently determined transcriptomically. Using Stereo-seq, we will analyze these specimens making sure to include clones in regional domains including the HAA and the surrounding dorsal, ventral, nasal, and temporal peripheral regions. We will analyze lineage tree reconstructions, looking for evidence of higher order organization which might exist in the form of nested, multi-generational patterns of cell divisions and/or spatial biases. We will also compare lineages across different retinal regions to determine whether regional factors might influence lineage decisions and therefore cell diversity across the retina. Together, this proposal seeks to gain foundational insights into the origins of cellular diversity in the retina. The observations made in these studies will help generate future hypotheses regarding the underlying mechanisms that create cell diversity, including how the cellular composition supporting high acuity vision is produced. Such insights might help improve clinical models of vision loss including age-related macular degeneration and contribute to improved therapeutics.