A molecular atlas of adult C. elegans motor neurons reveals ancient diversity delineated by conserved transcription factor codes

Abstract

Motor neurons (MNs) constitute an ancient cell type targeted by multiple adult-onset diseases. It is therefore important to define the molecular makeup of adult MNs in animal models and extract organizing principles. Here, we generate a comprehensive molecular atlas of adult Caenorhabditis elegans MNs and a searchable database. Single-cell RNA sequencing of 13,200 cells reveals that ventral nerve cord MNs cluster into 29 molecularly distinct subclasses. Extending C. elegans Neuronal Gene Expression Map and Network (CeNGEN) findings, all MN subclasses are delineated by distinct expression codes of either neuropeptide or transcription factor gene families. Strikingly, combinatorial codes of homeodomain transcription factor genes succinctly delineate adult MN diversity in both C. elegans and mice. Further, molecularly defined MN subclasses in C. elegans display distinct patterns of connectivity. Hence, our study couples the connectivity map of the C. elegans motor circuit with a molecular atlas of its constituent MNs and uncovers organizing principles and conserved molecular codes of adult MN diversity.

Keywords: C. elegans; CP: Neuroscience; molecular diversity; motor neurons; mouse; neuropeptides; single-cell RNA sequencing; transcription factors.

Figure 1.. Strategy for scRNA-seq of adult C. elegans MNs

(A) Schematic of C. elegans MNs in RVG, VNC, and PAG. (B) Fluorescence micrographs: acr-2p::GFP and lin-39p::RFP transgene expression. Scale bar, 50 μm. (C) Single-neuron reporter data for each transgene depicted in (B). n = 10. (D) Workflow for scRNA-seq. Ganglionic and VNC MNs were isolated separately from acr-2::gfp and lin-39::rfp transgenic animals. Two biological replicates. (E) UMAP plot: molecular separation of all eight MN classes. (F) Dot plot showing log expression and percentage of cells (VNC and ganglionic MNs) expressing known MN class-specific genes. (G) UMAP plot in (E), but colors depict strain of origin. (H) Dot plot showing log expression and percentage of cells expressing neurotransmitter identity genes.

Figure 2.. scRNA-seq identifies striking MN diversity and a subclass-specific TF code

(A) UMAP and table showing 24 cholinergic MN subclasses (left) and five GABAergic MN subclasses (right) in adult C. elegans VNC and ganglia. (B) Gene Ontology (GO) categories (wormcat.com) over-represented (Fisher’s exact test, p < 0.01) in adult MN subclasses. (C) Chart depicting TF numbers detected in adult MNs. (D) Dot plots showing scaled expression and proportion of cells expressing MN subclass-specific TFs. TF families (left).

Figure 3.. Hox gene expression delineates adult MN subclasses

(A) C. elegans Hox cluster. (B) Expression of endogenous fluorescent reporters for all six Hox genes in every adult MN (VNC and ganglionic). (C) Tables showing strong concordance for Hox gene expression between scRNA-seq and endogenous reporter analysis. Colored boxes denote scRNA-seq expression. +, complete agreement between the two methods; −, disagreement. “Yes” or “no” indicates whether or not all subclasses of a given class can be solely defined by Hox codes. (D) Feature plots showing log Hox expression in VA MNs. (E) UMAP: all VA subclasses (color coded) and Hox expression. (F) UMAP: elt-1/GATA1–3 in VA. (G) Endogenous elt-1::mNG and NeuroPAL expression in RVG and anterior VNC MNs, elt-1/GATA1–3 scRNA-seq data (bottom). Red: MNs not captured by FACS. Expression pattern: average of nine biological replicates. Scale bar, 10 μm.

Figure 4.. Combinatorial expression of extra-synaptic signaling genes delineates adult MN subclasses

(A and B) Dot plots: scaled expression and proportion of cells expressing genes encoding neuropeptides (A) or neuropeptide receptors (B) in adult MNs (ganglionic and VNC). (C) Fluorescence micrographs and endogenous reporter expression for nlp-11(syb4759). TPM, transcripts per million. scRNA-seq transcript detection displayed as color code (light yellow, 1–500 TPM; red, >5,000 TPM). Red, MNs not captured by FACS. Expression pattern: average of nine biological replicates. Scale bar, 10 μm.

Figure 5.. DA and DB MN subclasses display distinct connectivity patterns

(A, C, and E) Schematics: soma location for DA (A), DB (C), and VC (E) MNs and their respective morphologies derived from EM reconstructions (wormatlas.org; wormwiring.org). v/dBWMs, ventral/dorsal body wall muscle. (B, D, F) Neural network diagrams of DA (B), DB (D), and VC (F). Network diagrams from nemanode.org using complete adult EM data.^, Connections depicted with at least one chemical or electrical synapse. Red, cholinergic; blue, GABAergic; yellow, glutamatergic; gray, unknown.

Figure 6.. HD TFs delineate adult mouse MN diversity

(A) Schematic: cholinergic MN populations of the adult mouse spinal cord. Blue, visceral; green, skeletal. (B) Pie chart: number of HD TF genes from each family detected in adult mouse MNs. (C and D) Dot plot: scaled expression and proportion (%) of cells expressing the 76 HD TF genes (C) or the 42 NHR TF genes (D) detected in either visceral or skeletal MNs. Scaled expression: centered, scaled expression for each gene across all MN subclasses. Values and corresponding colors reflect standard deviations below (negative values) or above (positive values) mean expression. Left: HD TF families. Right: in situ hybridization data from Allen Brain Atlas for each HD TF gene. YES, clear MN expression; NA, no data available; NO, no MN expression. (E and F) Clustering dendrogram based on averaged expression of each HD TF gene (E) or each NHR TF gene (F) across all visceral and skeletal MN subclasses.