Hox Genes: Choreographers in Neural Development, Architects of Circuit Organization

The neural circuits governing vital behaviors, such as respiration and locomotion, are comprised of discrete neuronal populations residing within the brainstem and spinal cord. Work over the past decade has provided a fairly comprehensive understanding of the developmental pathways that determine the identity of major neuronal classes within the neural tube. However, the steps through which neurons acquire the subtype diversities necessary for their incorporation into a particular circuit are still poorly defined. Studies on the specification of motor neurons indicate that the large family of Hox transcription factors has a key role in generating the subtypes required for selective muscle innervation. There is also emerging evidence that Hox genes function in multiple neuronal classes to shape synaptic specificity during development, suggesting a broader role in circuit assembly. This Review highlights the functions and mechanisms of Hox gene networks and their multifaceted roles during neuronal specification and connectivity

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

A Latent Propriospinal Network Can Restore Diaphragm Function After High Cervical Spinal Cord Injury

Spinal cord injury (SCI) above cervical level 4 disrupts descending axons from the medulla that innervate phrenic motor neurons, causing permanent paralysis of the diaphragm. Using an ex vivo preparation in neonatal mice, we have identified an excitatory spinal network that can direct phrenic motor bursting in the absence of medullary input. After complete cervical SCI, blockade of fast inhibitory synaptic transmission caused spontaneous, bilaterally coordinated phrenic bursting. Here, spinal cord glutamatergic neurons were both sufficient and necessary for the induction of phrenic bursts. Direct stimulation of phrenic motor neurons was insufficient to evoke burst activity. Transection and pharmacological manipulations showed that this spinal network acts independently of medullary circuits that normally generate inspiration, suggesting a distinct non-respiratory function. We further show that this “latent” network can be harnessed to restore diaphragm function after high cervical SCI in adult mice and rats

Coordinated cadherin functions sculpt respiratory motor circuit connectivity.

Breathing, and the motor circuits that control it, is essential for life. At the core of respiratory circuits are Dbx1-derived interneurons, which generate the rhythm and pattern of breathing, and phrenic motor neurons (MNs), which provide the final motor output that drives diaphragm muscle contractions during inspiration. Despite their critical function, the principles that dictate how respiratory circuits assemble are unknown. Here, we show that coordinated activity of a type I cadherin (N-cadherin) and type II cadherins (Cadherin-6, -9, and -10) is required in both MNs and Dbx1-derived neurons to generate robust respiratory motor output. Both MN- and Dbx1-specific cadherin inactivation in mice during a critical developmental window results in perinatal lethality due to respiratory failure and a striking reduction in phrenic MN bursting activity. This combinatorial cadherin code is required to establish phrenic MN cell body and dendritic topography; surprisingly, however, cell body position appears to be dispensable for the targeting of phrenic MNs by descending respiratory inputs. Our findings demonstrate that type I and II cadherins function cooperatively throughout the respiratory circuit to generate a robust breathing output and reveal novel strategies that drive the assembly of motor circuits

Parallel Pbx-Dependent Pathways Govern the Coalescence and Fate of Motor Columns.

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

Parallel Pbx-Dependent Pathways Govern the Coalescence and Fate of Motor Columns.

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

Pbx-dependent and -independent strategies for generating LMC neurons.

<p>(A) Expression of Hoxc6NΔ91 at thoracic levels activates <i>Foxp1</i> and <i>Raldh2</i>, but fails to induce <i>Pea3</i> or repress <i>Hoxc9</i>. (B) Expression of Hoxc6IM (YPWM->AAAM mutation) at thoracic levels activates <i>Foxp1</i>, <i>Raldh2</i>, <i>Pea3</i> and represses <i>Hoxc9</i>, similar to wildtype Hoxc6. The ability of Hoxc6IM to activate Pea3 could reflect a Pbx-independent program that specifies the Pea3+ pool. (C) Expression of Hoxc6NΔ91+IM fails to repress <i>Hoxc9</i> or induce LMC identity. (D) Interpretation of results. Hoxc6 normally induces LMC-specific genes in a Pbx-dependent manner, and contributes to the exclusion of <i>Hoxc9</i>. When the Pbx interaction domain is mutated, <i>Hoxc9</i> is still repressed. As Hoxc9 normally acts to dampen <i>Foxp1</i> expression, the absence of Hoxc9 allows Hox proteins resident to the thoracic spinal cord to induce <i>Foxp1</i> and activate the LMC program. When both the Pbx interaction motif and <i>Hoxc9</i> repression domain are deleted, <i>Hoxc9</i> is expressed, ensuring <i>Foxp1</i> is not activated, and preventing LMC specification.</p

Effects of native and chimeric Hox proteins on columnar and pool specification.

<p>(A–B) Electroporation of Hoxc6 induces <i>Pea3</i> expression at thoracic levels. (C–F) Expression of Hoxa7 and Hoxc8 at thoracic levels induces <i>Foxp1</i> but not <i>Pea3</i> expression. Similar results were obtained with Hoxa5 (data not shown). (G–H) A chimera of the Hoxc6 N-terminus (including the YPWM motif) and the Hoxc4 homeodomain (HD) induces high levels of <i>Foxp1</i> expression. (I–J) The Hoxc6:c4 chimera fails to induce <i>Pea3</i> expression at thoracic levels and shows an attenuated capacity to repress Hoxc9. (K–L) A chimera of the Hoxc6 N-terminus to the homeodomain of Hoxc8 induces LMC MNs, but fails to induce <i>Pea3</i> expression. (M) Model for Hox interactions in MN subtype specification. During LMC specification multiple Hox paralogs converge on the regulation of pan-LMC genes such as FoxP1 and Raldh2. In motor neuron pools more specific Hox activities are employed.</p