Single cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human specific features

Key results

1. ShownKnowledge-matrix classification of 43,485 neural cells recovered the expected dorsoventral progenitor and neuronal domains, and mouse and human cells of the same type correlated best on transcription factor expression.

   Cells were assigned via a binarized marker knowledge matrix (Antler) after unsupervised clustering failed. On 1:1 orthologous transcription factors, Pearson correlation peaked for matched mouse/human types; progenitors resembled adjacent domains, neurons grouped by excitatory/inhibitory class. Human ventral pseudo-bulk CS12 best matched in vitro motor neuron differentiation Day 5, CS14 Day 9 (Spearman). · Identification of cell types in developing human…; Classification of central nervous cells; Mouse and human correlation analysis; Comparison to bulk in vitro RNA-seq data

2. ShownHuman embryos differ from mouse in several patterning genes: PAX7 is expressed in floor plate, NKX6.2 spans most ventral progenitor domains rather than p1 alone, and PHOX2B+ visceral motor neurons express little TBX20.

   Human PAX7 was detected in floor plate at CS12, CS14 and CS17 but at no mouse timepoint; CDH7, TXLNB, CDHR3, PIFO and RRAD correlate with PAX7 in human floor plate and are not expressed in mouse floor plate. Human NKX6.2 was found in p1, p2, pMN, p3 and floor plate, declining over time in pMN and p3 (RNAscope on CS13/CS15 sections). Human PHOX2B+ neurons (n=170) express TBX2 and TBX3 but little TBX20. · Specific features of gene expression in the…; Correlation analysis for PAX7; RNAScope FISH with immunohistochemistry; Figure legends

3. SuggestedSpinal neurogenesis follows a similar overall course and temporal transcription factor code in human and mouse, but human begins with relatively more pMN and p3 progenitors and keeps dorsal neurogenesis going longer.

   After stages were aligned by developmental events, the ratio of p3 and pMN to pI and pD progenitors was higher in human than mouse at CS12 and CS14 and comparable by CS17; dorsal neurons outnumbered ventral by CS19. In human, Onecut factors marked CS12 neurons, POU2F2 and ZFHX2-4 appeared by CS14, NEUROD2/6 and NFIA/B only by CS19 (NFI in MNs from CS14); late markers were sparse in dI3-dI6. · Dynamics of neurogenesis in the human spinal…; A conserved temporal code for the specification…

4. SuggestedOLIG2/NKX2.2 double-positive progenitors are more common in early human than early mouse neural tube, look like a mix of pMN and p3 identities, and carry gliogenic markers, consistent with early oligodendrocyte precursors.

   In mouse, double positives were under 5% of Sox2+ cells expressing Olig2 or Nkx2.2 until E11.5 (rising to ~15% at E12.5), vs ~10% in human at equivalent stages, where the fraction fell at CS17 and CS19. Human double positives were closer to pMN than p3 at CS12-CS14, with p3 similarity rising later, and kept high FABP7, OLIG1 and OLIG2 and a higher gliogenic module score than pMN or p3. · Co-expression of Olig2 and Nkx2.2 in the…; Cell-cycle and gene module scoring

5. SuggestedRNA velocity and CellRank reconstruct a single convergent progenitor-to-neuron path for seven spinal domains in both species, with broadly similar ordering of gene expression in latent time.

   scVelo and CellRank were run on mouse E9.5-E13.5 and human CS12, CS14 and CS19 (CS17 dropped for low counts per gene) for p3, pMN, p2, p1, dI4, dI3 and dI2. In the pMN-to-MN path of both species Olig2 came first in latent time, then Neurod4, then Elavl3 and Mnx1; latent-time heatmaps for V3, V2, V1, dI4, dI3 and dI2 were broadly alike. Human trajectories were less well resolved. · Transcriptional dynamics during neurogenesis in…; RNA Velocity; CellRank Trajectory Analysis