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.

Grant Number: 1R21EY037867-01
NIH Institute/Center: NIH
Principal Investigator: CONSTANCE CEPKO