A critical step in the assembly of the neural circuits that control tetrapod locomotion is the specification of the lateral motor column (LMC), a diverse motor neuron population targeting limb musculature. Hox6 paralog group genes have been implicated as key determinants of LMC fate at forelimb levels of the spinal cord, through their ability to promote expression of the LMC-restricted genes Foxp1 and Raldh2 and to suppress thoracic fates through exclusion of Hoxc9. The specific roles and mechanisms of Hox6 gene function in LMC neurons, however, are not known. We show that Hox6 genes are critical for diverse facets of LMC identity and define motifs required for their in vivo specificities. Although Hox6 genes are necessary for generating the appropriate number of LMC neurons, they are not absolutely required for the induction of forelimb LMC molecular determinants. In the absence of Hox6 activity, LMC identity appears to be preserved through a diverse array of Hox5–Hox8 paralogs, which are sufficient to reprogram thoracic motor neurons to an LMC fate. In contrast to the apparently permissive Hox inputs to early LMC gene programs, individual Hox genes, such as Hoxc6, have specific roles in promoting motor neuron pool diversity within the LMC. Dissection of motifs required for Hox in vivo specificities reveals that either cross-repressive interactions or cooperativity with Pbx cofactors are sufficient to induce LMC identity, with the N-terminus capable of promoting columnar, but not pool, identity when transferred to a heterologous homeodomain. These results indicate that Hox proteins orchestrate diverse aspects of cell fate specification through both the convergent regulation of gene programs regulated by many paralogs and also more restricted actions encoded through specificity determinants in the N-terminus.

Summary The emergence of limb-driven locomotor behaviors was a key event in the evolution of vertebrates and fostered the transition from aquatic to terrestrial life. We show that the generation of limb-projecting lateral motor column (LMC) neurons in mice relies on a transcriptional autoregulatory module initiated via transient activity of multiple genes within the HoxA and HoxC clusters. Repression of this module at thoracic levels restricts expression of LMC determinants, thus dictating LMC position relative to the limbs. This suppression is mediated by a key regulatory domain that is specifically found in the Hoxc9 proteins of appendage-bearing vertebrates. The profile of Hoxc9 expression inversely correlates with LMC position in land vertebrates, and likely accounts for the absence of LMC neurons in limbless species such as snakes. Thus, modulation of both Hoxc9 protein function and Hoxc9 gene expression likely contributed to evolutionary transitions between undulatory and ambulatory motor circuit connectivity programs.

Summary The clustering of neurons sharing similar functional properties and connectivity is a common organizational feature of vertebrate nervous systems. Within motor networks, spinal motor neurons (MNs) segregate into longitudinally arrayed subtypes, establishing a central somatotopic map of peripheral target innervation. MN organization and connectivity relies on Hox transcription factors expressed along the rostrocaudal axis; however, the developmental mechanisms governing the orderly arrangement of MNs are largely unknown. We show that Pbx genes, which encode Hox cofactors, are essential for the segregation and clustering of neurons within motor columns. In the absence of Pbx1 and Pbx3 function, Hox-dependent programs are lost and the remaining MN subtypes are unclustered and disordered. Identification of Pbx gene targets revealed an unexpected and apparently Hox-independent role in defining molecular features of dorsally-projecting medial motor column (MMC) neurons. These results indicate Pbx genes act in parallel genetic pathways to orchestrate neuronal subtype differentiation, connectivity, and organization.

Throughout development, neuronal identity is controlled by key transcription factors that determine the unique properties of a cell. During embryogenesis, the transcription factor Prox1 regulates VIP-positive cortical interneuron migration, survival, and connectivity. Here, we explore the role of Prox1 as a regulator of genetic programs that guide the final specification of VIP interneuron subtypes in early postnatal life. Synaptic in vitro electrophysiology in male and female mice shows that postnatal Prox1 removal differentially affects the dynamics of excitatory inputs onto VIP bipolar and multipolar subtypes. RNA sequencing reveals that one of the downstream targets of Prox1 is the postsynaptic protein Elfn1, a constitutive regulator of presynaptic release probability. Further genetic, pharmacological and electrophysiological experiments demonstrate that removing Prox1 reduces Elfn1 function in VIP multipolar but not in bipolar cells. Finally, overexpression experiments and analysis of native Elfn1 mRNA expression reveal that Elfn1 levels are differentially controlled at the post-transcriptional stage.Thus, in addition to activity-dependent processes that contribute to the developmental trajectory of VIP cells, genetic programs engaged by Prox1 control the final differentiation of multipolar and bipolar subtypes. Significance StatementThe transcription factor Prox1 generates functional diversification of cortical VIP interneuron subtypes in early postnatal life, thus expanding the inhibitory repertoire of the cortex.