The neural circuits that govern locomotor behaviors rely on the establishment of orderly sets of connections between motor neurons (MNs) and their peripheral and central synaptic targets. A critical and early step in the emergence of locomotor circuitry is the selection of specific muscle targets by a diverse array of MN subtypes. Three organizational features of MNs emerge during embryonic development that contributes to the specificity of their connections with target cells. First, MNs that project axons to common peripheral targets are organized into columns longitudinally arrayed along the rostrocaudal axis of the spinal cord 
. For example, MNs that project into the limb are contained within the lateral motor columns (LMCs), which are generated specifically at brachial and lumbar levels of the spinal cord. LMC neurons subsequently segregate into medial and lateral divisions, a program that dictates whether motor axons project into dorsal or ventral compartments of the limb mesenchyme 
. Finally, cells within each division further segregate into MN pools, each pool a cluster of stereotypically positioned MNs that innervates one of the ~100 muscles in the limbs 
. MNs must therefore acquire a sufficient level of subtype diversity to ensure the appropriate muscle connectivity required for the emergence of coordinate locomotor behavior.
Within the developing spinal cord, Hox proteins exert central roles in the specification of MN columnar and pool subtypes 
. Nearly half of the 39 Hox
genes are expressed by MNs, with subsets of related paralogs functioning at distinct levels of the MN differentiation pathway 
. Three paralog groups, Hox6
, and Hox10
genes have been implicated in the early columnar organization of MNs and contribute to the specificity of their initial projections into the periphery 
. The actions of a much larger group of ~20 Hox
genes contribute to the specification of MN pools, in part, through the induction of intermediate transcription factors 
. During these programs of MN diversification, Hox proteins mediate both the selective activation of downstream targets and the exclusion of other determinants through mutual cross repression, two distinct activities that appear to be intrinsic to Hox proteins 
. Despite significant progress towards defining roles for Hox proteins in MNs, the mechanisms by which they control diverse features of MN subtype identity are largely unknown.
Studies in Drosophila
indicate two key mechanisms through which Hox proteins regulate target genes 
. The first involves the selection of DNA target sites. Hox proteins typically display low affinity for DNA in vitro, with high fidelity binding requiring cooperative interactions with the TALE-domain containing homeodomain factors extradenticle and homothorax (Pbx and Meis proteins in vertebrates) 
. While TALE-domain protein interactions increase the affinity and selectivity of Hox proteins for DNA, they have only a subtle influence on the specificity of site selection in vitro, particularly amongst Hox proteins expressed in more caudal regions of the embryo 
. Recent evidence, however, suggests that in vivo specificity can be achieved by sequences N-terminal to the homeodomain, which mediate contacts with the minor groove at target sites 
. Once bound to a target gene, the activities of Hox/Pbx complexes can be further modulated through the actions of ancillary transcription factors that typically bind in proximity to Hox targets 
. In this mode of action, a Hox protein may not depend as much on DNA site selection for specificity, but rather on how it interacts with factors it engages at a target sequence.
Some insights into the mechanisms by which Hox proteins regulate target genes in MNs have emerged through analysis of mice mutant for a single thoracically expressed Hox
. Hoxc9 is required for the appearance of thoracic-level MN columnar subtypes including preganglionic column (PGC) and hypaxial motor column (HMC) neurons 
. A critical aspect of Hoxc9 function is to establish the boundary between thoracic and forelimb-level MN populations through cross repression, as in the absence of Hoxc9
all brachial Hox
genes are derepressed at thoracic levels, and MNs acquire an LMC fate. This broad repressive activity appears to be mediated by direct interactions of Hoxc9 with multiple sites in the HoxA
loci. Genome-wide analysis of Hoxc9 binding revealed a consensus binding motif which matches a high affinity Hox/Pbx site (TGATTTAT) identified by several groups through in vitro site selection 
. This sequence engages a wide range of Hox paralogs, raising the issue of how the in vivo specificities of Hox proteins in MNs are achieved if they are not dependent on the recognition of specific genomic target sites.
The problem of Hox specificity in MNs is particularly relevant at limb levels of the spinal cord, where individual neurons express multiple Hox proteins at the time of their differentiation 
. In this context Hox proteins appear to contribute to both gene programs common to all LMC neurons as well as more restricted actions necessary for diversification of LMC neurons into MN pools. At limb levels of the spinal cord the actions of Hox6
genes have been implicated in the initiation of the LMC program at brachial and lumbar levels respectively, through activation of the gene encoding the transcription factor FoxP1 
. FoxP1 is subsequently required for the expression of the gene encoding the retinoic acid synthesizing enzyme Raldh2
. This MN-derived source of retinoids is necessary for the Lim homeodomain protein-mediated segregation of the LMC into medial and lateral divisions 
. Thus the deployment of the LMC program at forelimb and hindlimb levels is mediated by two distinct sets of Hox paralogs that activate a common set of downstream pathways required for MN columnar and divisional specification.
While Hox proteins seem to be critical for LMC specification, it is less clear how they contribute to MN pool diversity. At brachial levels the LMC is broadly divided into rostral and caudal domains by expression of Hox5
) and Hoxc8
, respectively; and the actions of these Hox
genes are necessary for delineating the rostrocaudal position of MN pools 
. Within a given segment a repression-based network of Hox4–Hox8
proteins are thought to promote the intrasegmental diversity of MNs, by defining specific molecular codes for each pool subtype. For example, misexpression studies in chick have provided evidence that Hoxc6 is selectively required for the intrasegmental differentiation of pools within the caudal (Hoxc8+) half of the LMC 
. Thus the same Hox6
paralog group that determines the early columnar identity of forelimb-innervating MNs contains members that promote motor pool fates.
In this study we sought to address several unresolved issues concerning the function and specificity of Hox6 genes during MN columnar and pool specification programs. First, what are the specific contributions of the three murine Hox6 genes to MN fate specification? Second, to what extent are the diverse activities of a Hox protein unique, or are they shared amongst gene paralogs within a cluster? Third, are there motifs intrinsic to Hox proteins that subfunctionalize in vivo specificities? To address these questions we analyzed mice in which all Hox6 genes are mutated, as well as employed an in vivo approach to dissect functional domains required for Hox specificity in MNs. We find that although LMC specification is retained in mice lacking Hox6 genes, Hoxc6 has a specific role in promoting MN pool identity and appropriate patterns of limb connectivity. The preservation of LMC fate in Hox6 mutants appears to be mediated by a diverse group of Hox5–Hox8 genes expressed at brachial levels. Dissection of a single Hox protein reveals in vivo specificity relies on motifs that ensure deployment of programs common to all LMC neurons, as well as distinct modules that contribute to MN pool identity.