Retinal cell fate decisions are governed by transcriptional networks that guide multipotent retinal progenitor cells (RPCs) into seven major retinal cell classes. Among these, retinal ganglion cells (RGCs) are the retina’s sole output neurons and depend on a cascade of transcription factors for specification and differentiation. The basic helix-loop-helix factor Atoh7 has an established role in conferring competence to RPCs, while downstream factors such as Pou4f2 and Isl1 promote terminal differentiation and subtype specification. Earlier work implicated the SoxC family (Sox4, Sox11, Sox12) in RGC genesis, but how SoxC factors integrate with the Atoh7-driven regulatory hierarchy remained unclear. This study used conditional genetics, immunohistochemistry, transcriptomics, and genome-binding profiling to define the roles of SoxC factors in multiple early retinal lineages and to test their relationship with Atoh7 in RGC formation.
To interrogate SoxC function specifically in the developing retina, the authors employed a retina-restricted Vsx2-Cre driver to generate targeted deletions. They produced a Sox4/Sox11 double conditional knockout (dcKO) to remove two principal SoxC paralogs in the retina, and a Sox4/Sox11/Atoh7 triple knockout (tKO) to evaluate genetic interactions between SoxC factors and the bHLH factor Atoh7. These conditional models enabled assessment of lineage outcomes, progenitor behavior, and molecular programs in the absence of SoxC activity alone or in combination with loss of Atoh7.
Immunohistochemical analyses and marker-based lineage characterization were used to quantify changes in cell populations and progenitor states across genotypes. The study reports widespread lineage disruption in both dcKO and tKO retinas. Marker analysis documented severe reductions in several early inner retinal neuronal classes and complementary changes in other cell types, indicating a shift in the balance of retinal neurogenesis when SoxC genes are lost.
Loss of Sox4 and Sox11 had notable effects on RPC behavior. The dcKO and tKO retinas displayed markedly reduced progenitor proliferation and decreased cell survival. These changes in basic progenitor properties are consistent with the observed downstream deficits in multiple early retinal lineages and suggest that SoxC factors contribute to maintaining RPC populations and/or their capacity to produce specific neuronal fates during early retinal development.
Across SoxC-deficient retinas, genesis of RGCs and of horizontal and amacrine (H&A) cells was severely compromised. In contrast, production of photoreceptor cells (PHCs) increased. These opposing lineage shifts indicate that Sox4 and Sox11 help coordinate the generation of discrete early retinal lineages, promoting inner retinal neuronal fates while restraining photoreceptor production in the contexts examined.
A key observation was that, similar to the Atoh7-null retina, RGC precursors still formed in the SoxC dcKO retina. However, in the Sox4/Sox11/Atoh7 tKO, RGC genesis was nearly completely abolished. This outcome indicates that the SoxC factors act in parallel with Atoh7 as a major upstream regulatory input to initiate RGC fate, rather than being strictly downstream mediators of Atoh7. In other words, both inputs contribute nonredundantly to the initiation and execution of RGC differentiation programs.
Bulk RNA sequencing was performed to define transcriptional programs dependent on SoxC activity. The bulk RNA-seq data revealed SoxC-dependent transcriptional programs and signaling pathways that corroborate the cell-type and marker-level changes demonstrated by immunohistochemistry. The transcriptomic shifts aligned with reductions in inner retinal neuron markers and upregulation of photoreceptor-associated programs, consistent with the lineage distribution changes observed in dcKO and tKO retinas.
To identify direct genomic targets, the authors applied CUT&Tag profiling for Sox11 and mapped genome-wide binding sites. This analysis nominated candidate target genes and regulatory loci through which Sox11—and by extension SoxC factors—may regulate progenitor behavior and lineage choices across multiple retinal cell states and types during development. CUT&Tag results thus provide mechanistic leads linking SoxC occupancy to transcriptional outcomes.
Collectively, the genetic, histological, transcriptomic, and genome-binding data position SoxC transcription factors as central regulators of coordinated early retinal lineage generation. Sox4 and Sox11 influence progenitor proliferation and survival, bias fate allocation toward inner retinal neurons (RGCs, H&A), and restrain photoreceptor production. Their parallel action with Atoh7 establishes SoxC factors as a major upstream regulatory input required for robust RGC genesis. The CUT&Tag and RNA-seq datasets provide resources for identifying downstream effectors and pathways mediating these roles.
This report is a preprint and has not undergone peer review. The abstract and main text summarize principal findings, but detailed experimental parameters, quantitative metrics, statistical analyses, and full datasets are presented in the preprint document itself. Those seeking experimental specifics, replication details, or additional data should consult the deposited preprint and associated supplementary materials.