Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

Abstract

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. Coordinated motor behaviors—as complex as playing a musical instrument or as simple as walking—rely on the ability of motor neurons within the spinal cord to navigate towards and establish specific connections with muscles in the limbs. The establishment of connections between motor neurons and limb muscles is mediated through the actions of genes encoding Hox proteins, a large family of transcription factors conserved amongst all metazoans. However, the specific requirements for Hox genes in motor neuron specification and patterns of muscle connectivity are poorly understood. We have found that members of the Hox6 gene paralog group (Hoxa6, Hoxc6, and Hoxb6) contribute to diverse aspects of motor neuron subtype differentiation. Hox6 gene activity is required during two critical phases of motor neuron development: first as motor axons select a trajectory toward the forelimb and second as they choose specific muscles to innervate. At the molecular level, these two functions are encoded by distinct peptide domains within Hox proteins. This work indicates that Hox proteins execute their critical functions in motor neurons through intrinsic modules that confer distinct specificities and that these activities are central in the genetic network required for motor neuron differentiation.