Arid3c identifies an uncharacterized subpopulation of V2 interneurons during embryonic spinal cord development

Abstract

Motor activity is organized by neuronal networks composed of motor neurons and a wide variety of pre-motor interneuron populations located in the brainstem and spinal cord. Differential expression and single-cell RNA sequencing studies recently unveiled that these populations subdivide into multiple subsets. However, some interneuron subsets have not been described yet, and the mechanisms contributing to this neuronal diversification have only been partly deciphered. In this study, we aimed to identify additional markers to further describe the diversity of spinal V2 interneuron populations. Here, we compared the transcriptome of V2 interneurons with that of the other cells of the embryonic spinal cord and extracted a list of genes enriched in V2 interneurons, including Arid3c. Arid3c identifies an uncharacterized subset of V2 that partially overlaps with V2c interneurons. These two populations are characterized by the production of Onecut factors and Sox2, suggesting that they could represent a single functional V2 unit. Furthermore, we show that the overexpression or inactivation of Arid3c does not alter V2 production, but its absence results in minor defects in locomotor execution, suggesting a possible function in subtle aspects of spinal locomotor circuit formation.

Keywords: Arid3c; V2 interneurons; V2c interneurons; embryonic spinal cord; motor activity; motor circuits.

Figure 1

Identification of genes enriched in V2 interneurons (INs) during spinal cord development. (A) Schematic representation of the experimental design for RNA-seq experiment on V2 INs. Spinal cords of Vsx1|tdTomato embryos were collected at embryonic day (E)14.5 from females injected twice with tamoxifen (100 mg/kg) at E9.5 and were dissected and dissociated. FACS sorting of tdTomato⁺ cells enabled the separation of V2 INs from the other cells of the spinal cord. (B) Heat map showing differentially expressed genes across two comparisons (tdT⁺ versus tdT⁻) from each spinal cord sample (n = 6). Each row represents the transformed z-score of one differentially expressed gene across all samples (blue, low expression; red, high expression). (C) Fold-change comparison between V2 INs (tdTomato⁺ cells) and all the other cells of the spinal cord for known V2 IN markers and for a new candidate gene, Arid3c.

Figure 2

Arid3c is expressed specifically in V2 INs, while Arid3a and Arid3b are not. (A) In situ hybridization for Arid3c on transverse cryosections of the spinal cord of E12.5 or E14.5 Vsx1|tdTomato embryos, wherein V2 INs produce tdTomato. Cells expressing Arid3c are located in a ventrolateral position close to the motor columns. Overlay with the tdTomato fluorescence demonstrates specific expression in V2 INs, in the ventral-most population that affixes on the medial boundaries of the motor columns. (B) Arid3c expression is detected by in situ hybridization from E10.5 to E16.5 in a similar location, although it is progressively more restricted at later stages. (C) Arid3a expression is detected by in situ hybridization in the ventral part of the spinal cord from E10.5 to E12.5. Arid3a is broadly expressed in the spinal cord and in the dorsal root ganglia at E10.5 and E11.5 but its expression decreases from E11.5 and becomes undetectable at E14.5. (D) Arid3b expression is detected by in situ hybridization in the ventral part of the spinal cord from E10.5 to E14.5. It is expressed in MNs (black arrowheads), in the dorsal root ganglia (red arrowheads), and in the notochord (green arrowheads) at E10.5 and E11.5. At E12.5, its expression becomes weaker and more diffuse in the spinal cord and the dorsal root ganglia but is detected in the ventral sympathetic chain (blue arrowheads), while it becomes undetectable at E14.5. Scale bars = 400 μm.

Figure 3

Arid3c is produced in V2c INs and identifies an uncharacterized V2 population. (A) Immunolabeling for Arid3c on transverse cryosections of E12.5 or E14.5 Vsx1|tdTomato embryos. All the Arid3c+ cells are V2 INs, characterized by the production of tdTomato (arrowheads). (B) Immunolabeling for Sox1 and Arid3c on transverse cryosections of E12.5, E14.5, E16.5, or E18.5 embryos or fetuses. Arid3c is detected in some V2c INs, large cells ventrolaterally located and producing high Sox1 levels. Moreover, Arid3c is produced in Sox1⁻ cells located in the vicinity of V2c INs. Arrowheads indicate Arid3c⁺ cells that lack Sox1. Note that Sox1 is also produced in the progenitor cells of the ventricular zone (arrows). (C) Quantification of the proportion of cells that contain only Arid3c, only Sox1, or both Sox1 and Arid3c at E12.5 or E14.5 at brachial, thoracic, or lumbar levels of the spinal cord. n ≥ 5. Scale bars = 50 μm.

Figure 4

Arid3c⁺ cells display molecular characteristics similar to V2c INs. (A) Re-analysis of E9.5–13.5 spinal cord scRNAseq dataset (Delile et al., 2019). Heatmap showing the 100 first genes with the highest min.logFC. detected in Arid3c⁺ clusters as compared to V2a IN (Chx10), V2b IN (Gata3), and the other cells of the spinal cord (others). (B) Immunolabeling for Arid3c, Sox1, and OC1, OC2, OC3, or Sox2 on transverse cryosections at E14.5. OC1 is present in a majority of Arid3c⁺ cells or V2c INs (white and yellow arrows), but not all of them (pink and purple arrows), while OC2 and OC3 are produced in all of them (white, yellow, or cyan arrows). Sox2 is detected with Sox1 in V2c INs and in Arid3c⁺ cells. Note that Sox1 and Sox2 are also produced in the progenitor cells of the ventricular zone. (C) Immunolabeling for Arid3c and calbindin or calretinin on transverse cryosections at E14.5. Arid3c does not colocalize with calbindin or calretinin. Scale bars = 50 μm.

Figure 5

Overexpression of Arid3c does not alter V2a or V2b IN production. (A,B) Immunolabeling for V2a (Chx10) or V2b (Gata3) INs on transverse cryosections of chicken embryonic spinal cord electroporated with the pCIG-Arid3c-IRES-EGFP expression vector at HH15-16 and collected at HH26. GFP in the insets demonstrates electroporation all along the dorsoventral axis. (C) Quantification of the number of V2a or V2b INs on the electroporated side (pCIG-Arid3c-IRES-EGFP, +) of the spinal cord as compared to the control side (−). The overexpression of Arid3c does not significantly impact V2a or V2b IN production. n = 3. Scale bars = 50 μm.

Figure 6

Loss of Arid3c does not alter the number of V2a, V2b, or V2c INs. (A) Immunolabeling of transverse cryosections of the spinal cord of Arid3c^+/+ control or Arid3c^−/− mutant embryos at E12.5 or E14.5. The loss of Arid3c does not alter the number of V2c (ventrolaterally located Sox1⁺ cells), V2b (Gata3), or V2a (Chx10) INs. Note that Sox1 is also produced in the progenitor cells of the ventricular zone. (B) Quantification of the number of V2c, V2b, or V2a INs in Arid3c^+/+ control or Arid3c^−/− mutant embryos at E12.5 or E14.5 at brachial, thoracic, or lumbar levels of the spinal cord. The loss of Arid3c has no impact on the proper production of V2a, V2b, or V2c INs. n ≥ 3. Scale bars = 50 μm.

Figure 7

Loss of Arid3c has no impact on the general locomotor behavior. Behavioral analysis of general or specific motor functions of Arid3c^+/+ control or Arid3c^−/− mutant mice assessed by (A) physiocage, (B) open-field, (C) balance beam, (D) rotarod, (E) grip strength, or (F–H) catwalk tests. (A) The loss of Arid3c results in an increase in the general activity of the mice. (B–G) Mice lacking Arid3c show no alteration in general locomotion but (H) display some minor defects of the hind paw steps. * = adj. p < 0.05; *** = adj. p < 0.001.