Multiple parallel cell lineages in the developing mammalian cerebral cortex

Abstract

Cortical neurogenesis follows a simple lineage: apical radial glia cells (RGCs) generate basal progenitors, and these produce neurons. How this occurs in species with expanded germinal zones and a folded cortex, such as human, remains unclear. We used single-cell RNA sequencing from individual cortical germinal zones in ferret and barcoded lineage tracking to determine the molecular diversity of progenitor cells and their lineages. We identified multiple RGC classes that initiate parallel lineages, converging onto a common class of newborn neuron. Parallel RGC classes and transcriptomic trajectories were repeated across germinal zones and conserved in ferret and human, but not in mouse. Neurons followed parallel differentiation trajectories in the gyrus and sulcus, with different expressions of human cortical malformation genes. Progenitor cell lineage multiplicity is conserved in the folded mammalian cerebral cortex.

Fig. 1.. Similar cellular composition of GZs across cortical areas and developmental stages.

(A) Germinal layers and known major progenitor cell types and lineages in the developing ferret cortex. (B) Examples of live parasagittal tissue slice microdissections for scRNA-seq analyses and Nissl stains of the SG. Dashed lines indicate borders of GZs. Ctx, cortex; Th, thalamus; Str, striatum. Scale bars, 100 μm. (C) t-Distributed stochastic neighbor embedding (t-SNE) plot of the full dataset; clusters identified as in (D). Clusters in gray were only found in some litters and excluded. (D and E) Uniform Manifold Approximation and Projection (UMAP) and unbiased clustering of scRNA-seq data from cells in the EN lineage (D) and expression level of marker genes for the main cell classes (E). QR code, https://in.umh-csic.es/en/cortevo/, for access to expression of all transcripts. (F) UMAPs of cells from the indicated conditions and frequency of main cell types across conditions. (G) Violin plots for genes differentially expressed between conditions in the three main cell types. Adjusted P values range from 2.2 × 10⁻³⁰⁸ to 0.02 (see table S2).

Fig. 2.. Multiplicity of RGC types across GZs and cortical areas.

(A) Frequency distribution of RGC clusters across conditions, and pair-wise cluster enrichment. Means ± SEM for clusters above ±1 log₂FC is indicated (red indicates adjusted P < 0.05). (B) UMAP of P1 cells from clusters 16 (RGα) and 8 (RGβ) after unbiased subclustering, UMAPs of indicated features, pair-wise subcluster enrichment, and schema of RGC subclass distribution in UMAPs. (C) Gene expression across cell classes at P1 (subclusters indicated). (D) Subclasses of ferret RGCs in G₁ identified at P1, duplicated in VZ and OSVZ. (E to J) UMAPs, violin plots and ISH stains of CRYAB and HOPX expression in RGC subclusters and germinal layers at P1. (K) Mean CRYAB RNA levels, abundance (RNA), and density (protein; P3) of CRYAB⁺ cells (means ± SEM; **P < 0.01, t test). a.u., arbitrary units. (L to N′) tRGs in VZ of P1 (L and M) and P3 (N and N′) ferrets identified with anti-CRYAB immunostains and morphology confirmed by GFP (N). bv, blood vessels. Arrows indicate the tip of the basal process, arrowheads indicate the soma, dotted lines indicate the apical VZ surface. (N′) shows orthogonal view. (O) GFP-labeled RGCs in clarified P2 ferret brains, with tRGs shown in detail. Open arrowheads indicate basal endfoot arbors of aRGCs. (P) Violin plots for marker genes in RGC classes between conditions. P values range from 2.2 × 10⁻³⁰⁸ to 0.01 (see table S4). ILKAP, GDPD2, and DNMT1 are top markers for RGβ and RGα. HOPX distribution defines RGα₂. (Q) RNAscope coexpression analysis at P1 for RGC marker genes between conditions. Green box in indicates area shown at high magnification in a single-plane confocal image. Cells expressing CRYAB or AQP4 are indicated with colored circles. Scale bars, 100 μm (G and J), 60 μm (Q), 30 μm (L and O), 10 μm (M and N).

Fig. 3.. Parallel cell trajectories converge into a single class of newborn neurons.

(A) Three-dimensional (3D) UMAP and cell trajectories of the clustered scRNA-seq ferret dataset. Clustering is the same as in Fig. 1D and in (C). Numbers and greek letters identify key clusters. (B) 3D UMAPs showing the clusters involved in each of the three trajectories identified, each starting from a set of mitotic RGC clusters, and schematic drawings of summarized cell lineages. Circular arrows indicate mitotic cell clusters. (C) 2D UMAP of cell clusters indicating the three trajectories identified in the 3D UMAP [color-coded as in (B)], and pseudo-time and trajectory plots of the complete ferret dataset and individual conditions, indicating the RGCs of origin (pink and red circles) and trajectories present in each case (indicated with colored lines in OSVZ). The total number of cells in each condition is indicated.

Fig. 4.. Lineage relationships in the developing ferret cortex.

(A) Lineage barcodes were delivered by electroporation at E34; by E37, EGFP-positive cells were extracted; and lineage barcode and transcriptome libraries were sequenced. (B) UMAP embedding of all sampled cells with expression of known marker genes (HES1/VIM: RGC; EOMES: IPC; VGLUT2 and NEUROD6: EN). (C) Cell class annotation after transferring previous labels using correlation-based label transfer. (D) Arrangement of cell trajectories identified previously on the UMAP of the new library. (E) UpSet plot showing observed combinations of clones in different cell clusters, size of the interaction (top), and absolute number of clones per cell cluster (right). (F) Heatmap showing lineage coupling correlation scores between all cell clusters. z scores larger than ±2 are more than 2 SDs from random, hence high-confidence interactions. (G) Cell cluster network visualization, with edges representing lineage coupling correlation scores. Position of nodes was set manually. Interactions shown are those with z score > 0.5. (H) Position of cells in the UMAP embedding of example clones, as well as coupling subnetwork of clusters involved, for each of the three lineages. Interactions shown are those with a z score > 0.5. (I) UMAP highlighting cells from clones containing only ENs plus RGC-A (clusters 23 + 11) or ENs plus RGC-B (clusters 22 + 9). (J) PCA of ENs from (I) color-coded according to their clone type of origin. (K) Number of DEGs between EN types as in (J) (red line), and frequency distribution of DEGs following random distribution of clone cells (500 repetitions; gray bars).

Fig. 5.. Conserved RGC classes in ferret and human.

(A) Sources of integrated scRNA-seq datasets (16, 18). NCx, neocortex. (B) UMAP of integrated human (H), mouse (M), and ferret (F) datasets and unbiased cell clustering. (C) Distribution of cells from each species within the integrated UMAP and expression of genes identifying major cell classes. (D) Integrated UMAP highlighting human cells identified as vRG and oRG in (16), frequency distribution in the integrated clusters, and subset of ferret and mouse cells belonging to the same integrated cluster. (E) UMAP of ferret dataset highlighting cells from iCl.4 (red) and iCl.12 (purple) and their frequency distributions among ferret clusters (f) and P1 sample types. (F and G) Details from the integrated UMAP highlighting P1 ferret G₁ RGCs collected from VZ and OSVZ or belonging to fRGα and fRGβ and their frequency distributions in the indicated integrated cluster compared to human vRG and oRG. (H) Reactome-based PCA of iCl.4 and iCl.12 in the three species. (I) Violin plots for genes expressed in the indicated integrated cluster in each species. Adjusted P values range from 2.2 × 10⁻³⁰⁸ to 0.002 (see table S6). (J) Integrated UMAPs (detail) of CRYAB and HOPX expression in the indicated species. (K) Individual and merged distribution of RGC classes in the integrated UMAPs (detail) and schematic of their overlap.

Fig. 6.. Conserved and divergent RGC classes in ferret, human, and mouse.

(A) UMAP of integrated human, mouse, and ferret (P1) datasets with unbiased cell clustering (showing only cells in the EN lineage) and expression level of marker genes for major cell types. (B and C) Frequency distribution of integrated RGC clusters within species (B) and pair-wise cluster enrichment between species (C). Clusters above ±2 FC are highlighted (means ± SEM). Red font indicates statistical significance (adjusted P < 0.05; see table S3). (D) Pseudo-time and trajectory plot for the multispecies integrated UMAP, indicating the mitotic RGCs of origin (pink and red circles). (E and F) UMAPs highlighting the progenitor cell clusters involved in each of the three trajectories (colored lines) identified in the integrated dataset (E) and summary of trajectories (F). (G) Pseudo-time and trajectory plots for ferret, human, and mouse cells within the integrated UMAP. All three trajectories existed in ferret and human, but only one in mouse. (H) Schematic drawings of summarized cell lineages found in human and ferret versus mouse.

Fig. 7.. Different newborn neuron classes in the gyrus and sulcus.

(A) UMAP of subclustered IPC-newborn neurons from the ferret dataset. (B to D) UMAPs highlighting mature upper layers and immature lower layers cluster sets (B), pseudo-time (C), and cells from LS versus SG (D) and pair-wise cluster enrichment between conditions (B and D). Clusters above ±2 FC are indicated (means ± SEM); red font indicates statistical significance (adjusted P < 0.05; see table S3). In (C), cluster numbers indicated in blue are enriched in LS, and those in red are enriched in SG; arrows indicate estimated pseudo-time trajectory of cells from each region. (E) Violin plots for DEGs between immature neurons in SG and LS and RNAscope analysis at the corresponding ages. Adjusted P values range from 2 × 10⁻¹³⁹ to 6.5 × 10⁻²⁵ (see table S2). Scale bar, 30 μm.

Fig. 8.. RGC types and lineages in the cerebral cortex.

Schematic summary of key findings and interpretations in this study. (A) Conserved diversity of RGC types in ferret and human cortex, including amplificative (RGα), neurogenic (RGβ), general (RGγ), and truncated (tRG). (B) Three distinct cell lineages operate in parallel during development.