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Nature NeuroscienceDevelopmental BiologyPeer-reviewed journal article

Single-Cell Atlas of Early Human Brain Development Highlights Heterogeneity of Human Neuroepithelial Cells and Early Radial Glia

Ugomma C. EzeiD, Aparna BhaduriiD, Maximilian HaeussleriD, Tomasz Jan NowakowskiiD, Arnold R. KriegsteiniD

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Abstract

The human cortex is comprised of diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain, and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 2 (10 Dec 2020) · CC BY-NC-ND 4.0

Single-cell RNA sequencing of 289,000 first-trimester human brain cells identifies nine telencephalic progenitor subpopulations, a transient mesenchymal-like population, human-enriched C1orf61+ and ID4+ progenitors, and low early organoid fidelity.

Single-cell atlases of human cortical development have focused heavily on second-trimester peak neurogenesis, leaving the cellular heterogeneity and signaling dynamics of first-trimester neuroepithelial cells and early radial glia poorly resolved. Profiling human brain regions across Carnegie Stages 12 to 22 alongside embryonic mouse forebrain and cortical organoids reveals early human-enriched progenitor states and benchmarks in vitro model fidelity.

Key results

  1. ShownWhole-brain first-trimester scRNA-seq revealed region-specific transcription factor signatures emerging at Carnegie Stage 13 and resolved 63 telencephalic/cortical cell clusters including a transient mesenchymal-like population.

    Across 289,000 cells from 12 first-trimester embryos (CS12-CS22, GW6-10), regional markers (HOX, PAX7, GBX2, NKX2-1, FOXG1) segregated areas from CS13; clustering 59,000 telencephalon/cortex cells (58,145 in UMAP) identified 63 clusters across neuroepithelial cells, radial glia, IPCs (CS18), neurons (<CS16 MEF2C/LHX5-AS1; >CS16 NHLH1/NEUROD6/BCL11B), and a LUM+/ALX1+ mesenchymal population fading after CS16. · Whole Brain Analysis; Single-cell Sequencing of the Telencephalon; Progenitor Heterogeneity; Figure legends

  2. ShownSubclustering cortical progenitors identified nine SOX2+/VIM+ subpopulations spanning a gradual transition from neuroepithelial cells to radial glia, with RNA-velocity drivers enriched for neurodevelopmental disorder genes.

    Subclustering 30,562 telencephalic progenitors resolved nine clusters present across first-trimester stages, capturing a shift from ZO-1 (TJP1)+/FGF10+ neuroepithelial cells to NES+/HES5+ radial glia (including NTRK3+ Cluster 3 and early DLK1+ Cluster 8), while scVelo lineage stream driver genes were enriched for autism, cortical malformation, and schizophrenia risk genes. · RNA-Velocity Analysis of Lineage; Progenitor Heterogeneity; RNA-velocity; Figure legends

  3. ShownCanonical developmental signaling pathways exhibited stage-specific activation peaks in first-trimester human cortical progenitors.

    Single-cell RNA profiling and immunostaining across CS12-CS22 showed mTOR activity (phosphorylated S6 ribosomal protein) peaking along the cortical plate in the earliest samples and declining by CS22, whereas active Wnt signaling (phosphorylated beta-catenin) and Notch1 cleavage (NICD in the ventricular zone) peaked at CS14-CS16 before diminishing by CS22. · Canonical Developmental Signaling Pathway…; Figure legends

  4. ShownTwo early human cortical progenitor clusters (C1orf61+ Cluster 4 and ID4+ Cluster 7) lacked counterparts in embryonic day 9-10 mouse forebrain, while early human cortical organoids showed low progenitor fidelity.

    Comparing 9 human progenitor clusters to 16,053 E9-E10 (TS14-TS16) mouse forebrain cells matched 7 of 9 clusters, while C1orf61+ Cluster 4 and ID4+ Cluster 7 (sustained from CS13 in human vs peaking at E11.5 in mouse) were absent in TS14-TS16 mouse but detected in E64 macaque and ape organoids; in 108,593 week 3-5 human organoid cells, non-neuronal cells had 0.28 correlation to cortex (vs ~0.5 for excitatory cells). · Abstract; Progenitor Heterogeneity; Conservation of Progenitor Populations Across…; Figure legends

Methods, data and reproducibility

Performs 10x Chromium v2 scRNA-seq across dissected brain structures from first-trimester human embryos (CS12-CS22, GW6-10) and E9-E10 mouse forebrains using CellRanger v2, Seurat v2 batch regression, Jaccard-Louvain clustering, WGCNA, and velocyto.py/scVelo RNA velocity. Validates markers via immunostaining and RNAScope/BaseScope, and correlates clusters against mouse and week 3-5 human organoid data.

  • First-trimester human brain scRNA-seq across dissected regions (telencephalon, diencephalon, midbrain, hindbrain, cerebellum, ganglionic eminences, thalamus, hypothalamus, cortex; CS12-CS22) · human · n = 289,000 cells (59,000 telencephalon/cortex) from 12 individuals · https://assets.nemoarchive.org/dat-0rsydy7
  • Embryonic mouse (CD-1 IGS) forebrain scRNA-seq at embryonic days E9 and E10 (Theiler Stages 14 and 16) · mouse · n = 16,053 cells
  • Previously generated human pluripotent stem cell-derived cortical organoid scRNA-seq dataset (weeks 3 and 5, 4 stem cell lines, 3 protocols) · human · n = 108,593 cells
  • Primary human (CS13-CS22 and GW14), mouse (TS14, TS16), macaque (E64), and chimpanzee/human organoid brain sections for immunostaining and in situ hybridization · multiple species · n = 5 first-trimester human individuals + 1 GW14 sample (1 sample imaged per panel) · https://cells-test.gi.ucsc.edu/?ds=early-brain
Methods & tools
CellRanger v2, Seurat v2, RANN, igraph, veloctyo.py v0.17, scVelo, WGCNA, FIJI Image J, RNAScope Multiplex Fluorescence Assay v2, BaseScope v2 Assay
Reagents & environment
GRCh38-0.1.2 reference genome; CellRanger v2; Seurat v2; veloctyo.py v0.17; scVelo; RANN; igraph; FIJI Image J [S14, S15, S18].
Reproducibility gaps
Custom scripts for Jaccard distance calculation and downstream analysis parameters are not deposited in a public code repository and are available only upon request from the corresponding author [S14, S20].
Limitations (3)
  • For three developmental timepoints only one human individual was sequenced per stage, potentially confounding biological age with capture batch during batch regression [S14]. · stated by the authors
  • Due to the scarcity of first-trimester primary human tissue, each immunostaining panel was performed on a single sample per stage without blinding or statistical sample-size pre-determination [S18, S19, S22]. · stated by the authors
  • Because the dataset captures static developmental snapshots without lineage tracing, both direct neurogenesis before CS16 and the continuous transition gradient from neuroepithelial cells to radial glia remain working hypotheses [S4, S9]. · stated by the authors

Read this if you study early human brain and cortical development, compare rodent and primate neurogenesis, or benchmark cerebral organoid protocols against primary fetal tissue.

Citations
427
FWCI
28.72
Field percentile
top 1%

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