Extended Data Fig. 16: Gene regulatory networks for GABAergic and Glial cell types.

(a,d) TF DNA-binding motif enrichment for chromatin accessibility peak modules with different cell type and temporal specificities in GABAergic (a) and Glial (d) cell types. Within each panel, the dot plot to the left shows the average motif frequency for each peak module, dot size indicates the frequency, and color corresponds to the log-odds of motif occurrence in each module relative to random chance. The large heatmap at the bottom shows the average accessibility for each peak module (in rows) across each subclass-by-age group (in columns). The heatmap at the top shows the average expression of specific TFs corresponding to the motif across each subclass-by-age group. The values in the heatmaps are normalized per peak module or gene with 1 indicating the maximum value, and 0 indicating no accessibility or expression. (b,e) Gene regulatory networks for GABAergic (b) and Glial (e) cell types. Nodes represent genes, with triangles denoting TFs and circles denoting other genes. Each node is colored according to the subclass in which the gene is most highly expressed. Activation interactions are colored in green, repression in orange, and edge widths reflect interaction strengths. (c,f) Expression of TF regulators on UMAPs for GABAergic (c) and Glial (f) cell types. Here we also provide additional notes regarding specific TF regulators for all cell types. Nr4a2 is identified as a key regulator of the CLA-EPd-CTX Car3 subclass, with its motif enriched in both early and late Car3 subclass-specific peak modules, targeting marker genes including Car3, Oprk1, and Nr2f2 (Fig. 7a,b). Nr2f2, activated after Nr4a2, is predicted to regulate other late Car3 markers such as Synpr and Col11a1, suggesting a feed-forward pathway involved in maturation. The bHLH neurogenic motifs, shared by TFs such as Neurog1/2, Neurod1/2/4/6 and Bhlhe22 (also known as Bhlhe5), may exhibit subtle differences depending on other bHLH dimerization partners. These motifs are depleted in peak modules enriched in L2/3 IT and Car3 subclasses (Fig. 7a). While all these TFs are highly expressed in the IP or IMN populations, they are downregulated to various degrees in later stages. Neurod1 and Bhlhe22 are downregulated in deep-layer neurons, while Neurod6 is reduced in upper layers (Fig. 7e). Prdm8, a member of histone methyltransferase family, is strongly co-expressed with Neurod1 and Bhlhe22. Bhlhe22 is known to recruit Prdm8 to repress target genes, and loss of either leads to similar neuronal mistargeting phenotypes⁹⁹. Our analysis suggests Bhlhe22 represses deep-layer markers in upper-layer neurons, and when it is downregulated in deep layers, Neurod6 may activate the same targets (Fig. 7b). This dynamic likely fine-tunes layer-specific gene expression during postnatal IT neuron development. We found POU-III class TFs, Pou3f1/2/3, as key regulators for upper layer neurons, consistent with their crucial roles in specifying and maintaining the identity of these neuronal populations⁶¹, and predicted Cux2 as a downstream target (Fig. 7a,b). While Pou3f2 (Brn2) is mostly reported as activators, it has been reported to act as a repressor to downregulate Cdh13 and Mitf in melanoma cells^100,101, and our analysis suggests that these TFs may repress other deep layer markers, such as Cobl. Rfx3 is predicted as another regulator of upper layer neurons and is predicted to be a downstream target of Pou3f1. Etv1 is predicted to act as an activator in the L5 IT subclass, with subclass-specific targets such as Myl4 and Arhgap25. Fosl2 is identified as a candidate regulator of the L6 IT subclass; while most IEGs increase expression after eye opening, Fosl2 is activated early in L6 IT neurons soon after their divergence from IMN IT (Fig. 7b, Extended Data Fig. 12). Tcf4 and Sox4 are upregulated in IP stage and gradually decrease after IMN stage. We predicted that Sox4 simultaneously represses the premature expression of certain neuronal markers, particularly L6 CT markers (Fig. 7c,d), to help maintain cells in the IMN state. It was shown previously that Sox4 can act as a transcriptional repressor by interfering with the assembly of transcriptional machinery at promoters¹⁰². We predicted that Tcf4 supports early neurogenesis and maturation while preventing premature activation of late-stage genes that are typically expressed after eye opening (Fig. 7f,g). It was shown previously that loss of Tcf4 leads to elevated baseline levels of cFos¹⁰³ and profound changes in the structure and excitability of adult neurons. We identified Mafb and Sox6 as major MGE regulators and Nfib and Nr2f2 as CGE regulators. Mafb is known to regulate MGE interneuron fate and function¹⁰⁴. Members of the Nuclear Factor I family, Nfib, Nfia, and Nfix, are known to be co-expressed specifically in CGE-derived interneurons¹⁴, and our data show that they are activated in this class by E16.5. In the oligodendrocyte trajectory, we observed significant enrichment of SOX motifs, particularly Sox6, Sox8, and Sox9, in oligodendrocyte-specific but not OPC-specific peak modules. The motif for Sox10, a close homolog of Sox8, was not significantly enriched, possibly due to its low quality in the JASPAR motif database. Sox8 and Sox10 are expressed selectively in the OPC-Oligo class. In contrast, Sox6 and Sox9 are broadly expressed in RG, glioblasts, astrocytes, and OPCs, but are turned off during oligodendrocyte maturation, Sox9 in late OPCs and COPs and Sox6 in NFOLs. Based on these patterns, we propose that Sox8 and Sox10 promote oligodendrocyte maturation, while Sox6 and Sox9 act as stage-specific repressors of maturation. Sox8, Sox9, and Sox10 belong to the SOXE group of TFs, which are known to often function as dimers and regulate diverse developmental processes¹⁰⁵. Sox9 may inhibit oligodendrocyte maturation until appropriate developmental signals, which in turn leads to its downregulation. Similarly, Sox6 has been shown to repress oligodendrocyte maturation in mouse spinal cord¹⁰⁶. Our results highlight the intricate interplay among SOX family TFs in guiding stage-specific transitions during oligodendrocyte development.