Extended Data Fig. 6: Developmental trajectories of visual cortex nonIT Glut cell types.

(a) Transcriptomic trajectory tree for nonIT clusters starting from the common IMN nonIT antecedent. Nodes are clusters subdivided by synchronized age bins, and edges represent antecedent-descendant relationship between adjacent nodes, with thicker end at the antecedent node and thinner end at the descendant node. Nodes are grouped by subclass, and adult clusters are labeled. Nodes from L6b/CT ENT subclass are not included. (b-d) UMAPs for nonIT cells colored by subclass (b), cluster (c) and synchronized age bin (d). (e) Clusters are grouped together based on similar trajectories. Within each cluster group, all cells along their trajectories, including all antecedent nodes, are shown and are colored by cluster membership. (f) Spatial distribution of nonIT subclasses and clusters within each subclass in P0 and P56 MERFISH data, based on the ABC-WMB Atlas¹⁴. (g) Marker genes illustrating cell type diversification along trajectories. (h) Cluster composition of all nonIT cells at each age. There is a distinct population of L6b like cells with shared expression of subplate markers Cplx3, Lpar1, Nr4a2, but not Ccn2, Nxph4 and Pappa2. This population is more abundant than L6b at E17-P3 (Fig. 2b), with expression of Cyp26b1 and Cobll1, and mapped to adult L6b/CT ENT subclass. Based on Allen Developing Brain Atlas⁹⁴, Cyp26b1 is expressed specifically in the entorhinal cortex at E18.5, and our MERFISH data confirms the localization of L6b/CT ENT neurons in entorhinal cortex at P0 and P56 (Fig. 2e). For the L5 ET subclass, clusters 371–373 (Chrna6) represent the most distinct subset^10,12,18, emerging at P3 with specific expression of TFs Pou6f2 and Otx1. Expression of marker gene Chrna6 begins relatively late, around P9, and peaks in adulthood. Clusters 372 and 373 diverge from 371 after P21, with 373 specifically expressing Hk2. Based on our trajectory analysis, Chrna6⁺ clusters 371–373 share a common origin with clusters 365 and 366, with shared expression of Kctd8. We have identified multiple TFs potentially involved in regulation of different L5 ET clusters, including Foxo1, Bmp5, Lhx2, Zfp804b and Erg. There is no apparent spatial segregation of different L5 ET clusters in visual cortex at P56, while cluster 369 shows enrichment ventrally at P0. The L5 NP subclass contains two clusters, 466 and 468, which are diverged around P3, with Sv2c and Nxph2 enriched in each cluster respectively. Nxph2⁺ cluster 466 appears to be slightly deeper than cluster 468 at P56, while only cluster 468 is present at P0. Unlike most other subclasses of cortical glutamatergic neurons, L5 NP cells do not have long-range projections, and their functions remain elusive^10,95. The L6 CT subclass has three major clusters, 440, 439 and 437, diverging at E17. Interestingly, Nxph2⁺ L6 CT cluster 440 is very distinct from the other L6 CT clusters but more related to L5 NP subclass based on trajectory analysis, with shared expression of TF gene Pou3f2 with L5 ET and L5 NP. The separation between L6 CT clusters 437 and 439 (the dominant L6 CT cluster) is quite subtle transcriptomically, marked by enrichment of Pantr1 and Htr4, respectively, but very distinct spatially: cluster 437 is clearly deeper than 439 at both P56 and P0, and is co-localized with L6b cells. Pantr1, a noncoding RNA gene adjacent to TF gene Pou3f3, is absent in the deep L6 CT cluster 437 and L6b but present in all other more superficial nonIT clusters. In L6b subclass, two major clusters 427 and 428 diverge around E17, with TFs Foxp2, Nr4a2 and Id4 enriched in 427 and Tox enriched in 428. There is no apparent difference in spatial distribution of these two clusters, but 427 is more closely related to L6 CT subclass transcriptomically.