Fig. 5: Cell cycle analyses highlight NEC tripotency and reveal a distinctive ‘neural G0’ dCPEC signature.

Human ‘neural G0’ transcriptomic analysis⁴⁵ (A–F) with confocal maximal projections in derived cells (G) and 23 pcw tissue (H). A, B UMAP of all cells across timepoints, color coded for subtype and timepoint (A) or ‘neural G0’ phase (G0, G1, G2M/S), with major progeny classes encircled. The tripotent NEC progeny class structure is again evident (A). Across timepoints, dCPECs and neurons have predominant G0 signatures (blue), while NECs and progenitors are predominantly in G1 or G2M/S. C, D 3D ‘neural G0’ PCA plots of subtype clusters across timepoints, color coded for subtype as in A (C) or for predominant cell cycle phase(s) (D); see Supplementary Fig. 7A, B for details. The three branches of NEC progeny are evident from neural G0 analysis alone (C). While dCPECs and neurons are principally G0 cells (orange), neural G0 analysis also distinguishes dCPECs from neurons (D). E Lineage model for tripotent NECs (Fig. 3G) with predominant cell cycle signatures added. F 2D ‘neural G0’ PCA scatterplot of all cells from the earliest timepoint (31 div), color coded for cell type (upper) or cell cycle phase (lower). NEC tripotency and dCPEC-neuron distinction are evident by 31 div. G KI67 status of dCPECs at an intermediate timepoint in vitro (TTR-FOXN4-MAP2-KI67 ICC; 49 div). KI67 (red) labels cells outside of dCPEC islands (middle panel), but not dCPECs (purple) or neurons (green in right panel). FOXN4 + C2 cells (arrow in left panel) also lack KI67 expression. H KI67 status of CPECs at midgestation in vivo (MCIDAS-KI67 IHC; 23 pcw); middle and right panels are immediately adjacent. Nuclear KI67 is present in some stromal cells (arrow in left panel), but not in CPECs, including MCIDAS + C2 cells (arrowheads in right panel). See Fig. 6 for C2 marker studies using FOXN4 and MCIDAS.