Figure 4

a, Expression heatmaps of known progenitor and neuronal marker genes, as well as selected human RGC-enriched gene sets, from LG⁺ and LG⁻ cells isolated by FACS from the P2 developing ferret cortex (n=2). Enrichment of classic RGC markers and a high degree of similarity between gene sets enriched in human and ferret LG⁺ cells validate the use of LeX and Glast to select RGC from the developing ferret cortex. Notably, however, several genes (black bullets) show distinct expression patterns between the two species (e.g., CXCL12, UNC5B, NTNG2, SEMA5A), suggesting that certain growth factor and other pathways may be expressed in a species-specific manner in RGC. b, Single-cell gene expression profiling of 185 single ferret LG⁺ progenitors was performed using the same gene panel as shown in Figure 3 for human and mouse RGC. As in humans, a substantial fraction of ferret cells in clusters i, iv, and v co-express both RGC markers and Tbr2/Neurog2, consistent with our immunohistochemcial analysis of NEUROG2 expression in the ferret (Fig. 2a) and suggesting this “proneural” RGC transcriptional state is conserved. Similar to human RGC, a subset of these proneural cells also express the downstream factors NEUROD1 and NEUROD4. However, some human ORG-enriched genes (e.g. Rasgrp1) are expressed in fewer ferret RGC, while others (e.g. Plcb4, Sstr2, Gadd45g, Ttyh2) appear more homogenous across all cells. Interestingly, Foxn2, which was detected in nearly all human RGC, appears to mark a distinct subpopulation of ferret apical RGC (clusters i and iv). Overall, while ferret RGC exhibit more diversity of transcriptional states than mouse and generally more similarity to human, they are nonetheless distinct in their relative proportions and composition.