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

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.

调控四足动物运动的神经环路组装过程中，一个核心步骤是外侧运动柱（lateral motor column, LMC）的特化——该结构是一类靶向肢体肌肉的多样性运动神经元群体。Hox6旁系同源组基因已被证实为脊髓前肢水平LMC命运的关键决定因子，其可通过促进LMC限制性基因Foxp1与Raldh2的表达，并通过排除Hoxc9来抑制胸段神经元命运，从而发挥该调控作用。然而，Hox6基因在LMC神经元中的具体功能与调控机制仍未明确。本研究证实，Hox6基因对LMC身份特征的多个维度至关重要，并明确了其体内特异性所需的调控基序。尽管Hox6基因对于生成数量恰当的LMC神经元是必需的，但它们并非诱导前肢LMC分子决定因子的绝对必要条件。在缺失Hox6活性的情况下，LMC的身份特征似乎可通过一系列多样的Hox5–Hox8旁系同源基因得以维持；这类基因足以将胸段运动神经元重编程为LMC命运。与早期LMC基因程序可兼容多种Hox输入的表观现象不同，单个Hox基因（如Hoxc6）在促进LMC内部的运动神经元池多样性方面发挥特定作用。对Hox体内特异性所需基序的解析表明，交叉抑制相互作用或与Pbx辅因子的协同作用足以诱导LMC身份特征；当将N端结构域移植至异源同源结构域时，其仅能促进柱状身份而非神经元池身份的形成。上述结果表明，Hox蛋白可通过两种方式调控细胞命运特化的多个维度：一是由众多旁系同源基因协同调控的基因程序的趋同调控，二是通过N端的特异性决定子编码的更为局限的靶向作用。