Fig. 4: Alpha motor neurons sub-cluster into eight transcriptional groups.

a Alpha MNs can be re-clustered into 8 sub-clusters. b UMAPs displaying the alpha MN clusters in each separately barcoded segment of the spinal cord. Clusters 2 and 7 are most prominent in cervical, 3 and 4 are sparse or missing from thoracic. c Dot plot showing the top marker genes for each cluster. No markers were identified for clusters 0 or 1. Genes in bold are shown by ISH. d Quantification of cluster marker expression in alpha MNs retrogradely labeled from various muscles (Axial = lumbar extensors of the spine, TA = tibialis anterior). For each muscle, N = 2 animals were injected with the retrograde tracer FastBlue. Percent of FastBlue+ alpha MNs, based on Stk32a expression, expressing cluster markers from each injected muscle. Cpne4 (axial n = 66 neurons, digit n = 56, phrenic n = 175, soleus n = 91, TA n = 78) and Erbb4 (axial n = 138 neurons, digit n = 77, phrenic n = 177, soleus n = 63, TA n = 119) stand out as markers for digit-innervating and phrenic MNs, respectively. Grm5 was relatively enriched in alpha MNs projecting to the soleus compared with the other muscles (axial n = 138 neurons, digit n = 77, phrenic n = 177, soleus n = 63, TA n = 119), and C1qtnf4/Gulp1 was expressed in double the number of alpha MNs projecting to digit muscle (axial n = 94 neurons, digit n = 67, phrenic n = 142, soleus n = 72, TA n = 100). e ISH examples of images quantified in d. Alpha MNs innervating the digits express the cluster 4 marker Cpne4 (yellow) but lack the cluster 5 marker C1qtnf4 (cyan). f Alpha MNs innervating the diaphragm (phrenic MNs) express the cluster 7 marker Erbb4 (yellow) but lack the cluster 2 marker Grm5 (cyan). *p = 0.0053 by Shapiro-Wilk test comparing by muscle. #p = 0.0031 by Shapiro-Wilk test comparing by the gene. Scale bars, e 50 µm, f 25 µm.