Columnar-Intrinsic Cues Shape Premotor Input Specificity in Locomotor Circuits

Abstract

Control of movement relies on the ability of circuits within the spinal cord to establish connections with specific subtypes of motor neuron (MN). Although the pattern of output from locomotor networks can be influenced by MN position and identity, whether MNs exert an instructive role in shaping synaptic specificity within the spinal cord is unclear. We show that Hox transcription-factor-dependent programs in MNs are essential in establishing the central pattern of connectivity within the ventral spinal cord. Transformation of axially projecting MNs to a limb-level lateral motor column (LMC) fate, through mutation of the Hoxc9 gene, causes the central afferents of limb proprioceptive sensory neurons to target MNs connected to functionally inappropriate muscles. MN columnar identity also determines the pattern and distribution of inputs from multiple classes of premotor interneurons, indicating that MNs broadly influence circuit connectivity. These findings indicate that MN-intrinsic programs contribute to the initial architecture of locomotor circuits.

Keywords: Hox gene; locomotion; motor neuron; neural development; sensory neuron; spinal circuit; transcription factor.

Figure 1. Hox-Dependent Programs Establish the Central Pattern of Sensory Projections

(A) Projections of SNs along the rostrocaudal axis of the spinal cord in control animals at P1. SNs were traced by DiI injection into DRG C7 and T3, and central projections were analyzed at indicated segments. Projections include a medial branch directed towards median motor column (MMC) neurons, and a lateral branch to non-MMC populations (LMC and HMC neurons). Bottom panels show choline acetyltransferase (ChAT) expression in MNs between segments C5–T5. (B) Projection of SNs to MNs in thoracic segments after DiI injections into DRG C7 in control and Hoxc9−/− mice at P0. Approximate MN position is outlined in red, based on ChAT. (C) Quantification of DiI pixel intensity along the mediolateral axis in control and Hoxc9−/− mice. Lines show mean pixel intensity ±SEM from n=14 controls (E18.5, n=7; P0, n=7); and n=6 Hoxc9−/− mice (E18.5, n=4; P0, n=2), ***p=0.0007, T2; ****p<0.0001, T3; ****p<0.0001, T4; ***p=0.0003, T5. Inset shows region where distance (d) and pixel intensity (i) were measured. (D) Quantification of total DiI pixel intensity in medial and lateral regions of the spinal cord. In Hoxc9 mutants there is an increase in lateral projections. (E) Comparison of C7 (DiI) and T3 (DiA) SN projections. Both C7 and T3 SNs project to MNs in Hoxc9−/− mice. (F) Summary of SN projections from T3 and C7 in control and Hoxc9−/− mice. See also Figure S1

Figure 2. MN Columnar Identity Shapes Sensory-Motor Connectivity

(A) DiI tracing from DRG C7 analyzed at thoracic segments T2–T5 in indicated mouse mutants at P0. In Hoxc9^CM; Foxp1^CM double mutants and Foxp1^CM mice, SNs do not project to thoracic MNs. (B) Quantification of DiI traced DRG C7 sensory afferents in ventrolateral quadrant of segments T2–T5. Number of animals analyzed: Control, n=6 (P0, n=6); Hoxc9^CM, n=7 (P0, n=7); Foxp1^CM, n=9 (E18.5, n=3; P0, n=6); Hoxc9^CM; Foxp1^CM, n=4 (P0, n=4). (C) Quantification of total DiI pixel intensity in medial and lateral regions of the spinal cord. Pixel intensities in lateral position of Hoxc9^CM; Foxp1^CM and Foxp1^CM mice are similar to controls. ***p=0.0004, *p=0.0219, T2; ***p=0.0001, *p=0.0274, T3; ***p=0.0002, *p=0.0192, T4; **p=0.0025, ns=0.0747, T5. (D) Analysis of triceps sensory terminals in segment T3 at P5. In Hoxc9^CM; Foxp1^CM mice no CTB⁺ terminals are observed in the ventral spinal cord. n=3 each genotype. See also Figure S2.

Figure 3. Limb pSNs Establish Synapses with Ectopic LMC Neurons in Hoxc9 Mutants

(A) Localization of triceps pSN terminals in control and Hoxc9−/− mice at thoracic levels. Cervical pSN terminals were traced by injection of CTB into triceps muscles at P3 and examined at P5 at segment T3. (B) Triceps pSN terminals establish synapses on thoracic MNs in Hoxc9−/− mice. CTB⁺;vGluT1⁺ are observed on MNs, marked by ChAT. (C) Quantification of CTB⁺ terminals on MNs at segment T3. Total synapses were counted in 30 µm sections. Controls, n=5, 0±0 (mean±SEM); Hoxc9−/− mice, n=5, 225.9± 54.44; **p=0.0032. (D) Quantification of onset latencies (control, 3.23±0.37 msec; Hoxc9−/−, 2.99±0.15 msec), peak time (control, 6.13±0.18 msec; Hoxc9−/−, 6.62±0.22 msec), and peak amplitude (control, 0.05±0.01 mV; Hoxc9−/−, 0.40±0.05 mV; **p=0.0015. Bars on graphs show mean ±SEM. (E) CTB tracing of triceps sensory terminals in thoracic segments of control and Hoxc9^CM mice. (F) Images of CTB traced triceps pSNs in thoracic segments. In Hoxc9^CM mice, CTB⁺;vGluT1⁺ were observed on MNs. (G) Quantification of CTB⁺;vGluT1⁺ terminals on thoracic MNs in control and Hoxc9^CM mice. Controls, n=6, 0±0 (mean ±SEM); Hoxc9^CM, n=8, 45.48±11.09. Bars, mean ±SEM; **p= 0.0043. (H) Traces of T3 ventral root signals upon C7 dorsal root stimulation in control (n=10) and Hoxc9^CM mice (n=4) at P6. Lines show mean ± SEM. Quantification of onset latencies (control, 3.12 ±0.17; Hoxc9^CM, 3.13 ±0.153), peak time (control, 6.52±0.13; Hoxc9^CM, 6.90±0.16), and peak amplitudes (control, 0.1±0.01; Hoxc9^CM, 0.31 ±0.04; ****p<0.0001). Bars show mean ±SEM. See also Figure S3.

Figure 4. MN Columnar Identity Determines Premotor IN Input Pattern

(A) Schematic of viral tracing strategy. AAV-G and RABVΔG-RFP viruses were co-injected into rostral intercostal muscles at P5 and examined at P13. (B) Contour plots representing labeled premotor INs position. The regions with the greatest labeling density are encircled with yellow line. Red dotted line indicates midline of the spinal cord. Distances (µm) from the central canal are shown on X–Y axes. Cervical regions (C1–C8) and thoracic regions (T1–T13) are shown separately. Labeled cell position is shown beneath contour plots. Total number of labeled neurons: Control, n=4 mice, 1385 cells (cervical), 1895 (thoracic); Hoxc9^CM, n=4 mice, 1823 (cervical), 1412 (thoracic); Hoxc9−/−, n=3 mice, 2053 (cervical), 2325 (thoracic). (C) Quantification of ipsilateral vs. contralateral premotor populations at cervical and thoracic levels. Numbers represent averages from n=4 controls, n=4 Hoxc9^CM mice, and n=3 Hoxc9−/− mice. **p<0.001, ***p=0.0001, ****p<0.0001. Bar graphs show mean±SEM. (D) Summary of changes in premotor labeling in Hoxc9 mutant mice. See also Figure S4.