Post‐crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord

Abstract

The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post-crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post-crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post-crossing dI1 axons in mice, using the Atoh1 enhancer-based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post-crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post-crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.

Structure of long-range direct and indirect spinocerebellar pathways as well as local spinal circuits mediating proprioception

“…We find some interesting features of locally-projecting Atoh1-lineage neurons and compare and contrast our findings with those of a previous study using an Atoh1-enhancer driving CRE recombinase electroporated into mouse embryos (Kaneyama and Shirasaki, 2018). First, we find that thoracolumbar Atoh1-lineage neurons project mostly locally, consistent with the findings by Kaneyama and Shiraskai, who found that crossing dI1 axons are intersegmental with only a few traveling far from the soma.…”

Diverse spinal commissural neuron populations revealed by fate mapping and molecular profiling using a novel Robo3Cre mouse

“…Goat polyclonal antibodies raised against Lhx2 (1:200, Santa Cruz #sc‐19,344), Robo3 (1:200, R&D Systems #AF3076), transient axonal glycoprotein (TAG)‐1 (1:200, R&D Systems #AF4439), tropomyosin receptor kinase (Trk)‐A (1:200, R&D Systems #AF1056), TrkB (1:50, R&D Systems #AF1494), and TrkC (1:40, R&D Systems #AF1404) were used. The anti‐Lhx2 antibody () was raised against a C‐terminus peptide of Lhx2 of human origin and has been used to specifically label Lhx2‐positive dI1 neurons in the developing spinal cord (Kaneyama & Shirasaki, ). The anti‐Robo3 antibody () was raised against the extracellular domain of recombinant human Robo3, and specificity was validated in Robo3 conditional knockout mutants (Figure ).…”

Electrophysiological properties of neurons in the intermediate thoracolumbar spinal cord mediating proprioception

“…Atoh1 -lineage neurons have classically been subdivided into a contralaterally-projecting Medial (dI1c) and ipsilaterally-projecting Lateral (dI1i) population (Bermingham et al, 2001; Wilson et al, 2008). However, recent anatomical studies challenge this view with at least a subset of Atoh1 -lineage Lateral cells projecting potentially both ipsi- and contralaterally (Kaneyama and Shirasaki, 2018; Pop et al, 2022). Without unique molecular markers to distinguish subsets of the Atoh1 -lineage population, we set out to evaluate whether the Medial and Lateral cells had distinct electrophysiological signatures.…”

“…Atoh1 Medial cells, also known as dI1c, are thought to be contralaterally-projecting while Atoh1 Lateral cells, also known as dI1i, are ipsilaterally-projecting. However, recent evidence suggests that subsets of the Atoh1 Lateral are also contralaterally-projecting (Kaneyama and Shirasaki, 2018; Pop et al, 2022). Therefore, using whole cell patch clamp and molecular genetic tools in mice, we set out to determine if Atoh1 -lineage Medial and Lateral neurons had distinguishing electrophysiological signatures.…”