Molecular programs of regional specification and neural stem cell fate progression in macaque telencephalon

Abstract

During early telencephalic development, intricate processes of regional patterning and neural stem cell (NSC) fate specification take place. However, our understanding of these processes in primates, including both conserved and species-specific features, remains limited. Here, we profiled 761,529 single-cell transcriptomes from multiple regions of the prenatal macaque telencephalon. We deciphered the molecular programs of the early organizing centers and their cross-talk with NSCs, revealing primate-biased galanin-like peptide (GALP) signaling in the anteroventral telencephalon. Regional transcriptomic variations were observed along the frontotemporal axis during early stages of neocortical NSC progression and in neurons and astrocytes. Additionally, we found that genes associated with neuropsychiatric disorders and brain cancer risk might play critical roles in the early telencephalic organizers and during NSC progression.

Fig. 1.. Cell atlas of macaque telencephalon

(A) Age scheme and representative macaque brains illustrating the regions/areas dissected. Scale bar: 1 cm. (B) From innermost to outmost: UMAP visualizing cell classes; subtype proportions; marker expression; region and age composition; cell classes; subtypes. The region nomenclature is based on the temporal development of the telencephalon (bottom left).

Fig. 2.. Molecular signatures of putative telencephalic organizers

(A-B) Organizer cell subtypes (A) and their markers (B). (C) RNAscope of macaque sagittal brain sections. Scale bar: 500 μm (panoramic) and 200 μm (zoom-in). D: dorsal; LV: lateral ventricle. (D) Predicted TF regulatory network with nodes colored by subtype (left) or signaling (right).

Fig. 3.. Signaling cross-talks between organizers and NSCs

(A) L-R pair modules (M) for selected signaling pathways (i) mediating organizer-NSC cross-talks at E37-43 (ii). (B) Directed L-R mediated interactions (i). RNAscope of macaque sagittal brain sections (ii). Scale bar: 500 μm (panoramic) and 200 μm (zoom-in). (C) Organizer markers enriched in macaque versus mouse (25). (D) RNAscope of macaque and mouse sagittal brain sections. Scale bars as in B. (E) Immunohistochemistry of hCO exposed to FGF8 +/− GALP (left) and highthroughput quantification (± SD, right). Scale bar: 100 μm. One-way ANOVA, Dunnett’s multiple comparison (**: p < 0.01; ns: not significant). DIV: day in vitro.

Fig. 4.. Transcriptomic variation of NSC progression across cortical regions

(A) Cortical NSC subtypes and pseudotime. (B) Region and age proportion of the NSCs (left). Markers for the anterior-enriched PMP22⁺ RG subtype (right). (C) Scheme of NSCs progression. OSVZ: outer SVZ; CP: cortical plate (i). Number of regional DEGs along ventricular NSCs (ii) and oRG (iii) progression. (D) Region-specific gene cascades along the ventricular NSC progression. (E) Region specificity correlation between early (NESCs and vRG_E) or late (vRG_L) NSCs versus oRG. Colors denote the subtypes showing region enrichment. (F) RNAscope of macaque brain sections. Scale bar: 500 μm (panoramic) and 200 μm (zoom-in). Sep: septum; Th: thalamus.

Fig. 5.. Spatiotemporal transcriptomic divergence of cortical neurogenesis

(A) UMAP showing IPCs generating excitatory neurons. (B) Transcriptomic integration of E54-110 and adult macaque PFC excitatory neurons (30). (C) Scheme of neurogenesis (i). Number of regional DEGs along the DL (ii) and UL (iii) neuron trajectories. (D) Region-specific gene cascades along neurogenesis (i and ii). Region/area-specific genes in late excitatory neurons (iii and iv). (E) RNAscope in macaque brain sections. Scale bar: 500 μm (panoramic) and 200 μm (zoom-in). (F) Shared region-specific genes between RG and excitatory neurons.

Fig. 6.. Transcriptomic versatility of gliogenesis across cortical areas

(A-B) UMAP (A) and Monocle2 (B) showing neurogenic and gliogenic trajectories from late RG. (C) Top 10 branch-specific genes. (D) Numbers of DEGs (top) and TFs (bottom) across areas, following one-sided t-test (P<0.001 ***, ns: not significant). (E) Curated area-specific genes in E110 astrocytes. (F) Predicted L-R mediated interactions. (G) Selected L and R expression. n/a/oIPC: neuron/astrocyte/oligodendrocyte IPC.

Fig. 7.. Expression of brain disease-risk genes in early telencephalon

(A) Expression enrichment of disease genesets across cell subtypes (dots); Significance: q value < 0.05. (B) Expression of disease gene markers for a PC subtype expressed in no more than three other subtypes, excluding non-neural cells. Gene-disease association on the left. (C) Expression enrichment cascades of disease genesets along the temporal progression of dorsal and ventral NSCs. (D) Top regionally-enriched (dot colors) disease gene markers.