Hox genes: choreographers in neural development, architects of circuit organization

Abstract

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.

Figure 1. Hox Expression Patterns in the Hindbrain and Spinal Cord

(A) In vertebrates 39 Hox genes are distributed across 4 clusters. Each Hox gene is expressed in discrete rostrocaudal domains within the hindbrain and spinal cord. Color coding of Hox genes represents expression domains along the rostrocaudal axis. (B) In the hindbrain, Hox genes from paralog groups 1–5 are expressed and anterior expression limits correspond to rhombomere boundaries. Higher color intensity denotes higher expression. Hoxa1 expression is transient. Hindbrain motor nuclei develop within specific rhombomeres and are shown within their rhombomeres of origin. IV=trochlear, V=trigeminal, VI=abducens, VII=facial, IX=glossopharyngeal, X=vagus, XI=accessory, X=hypoglossal. (C) In the spinal cord, expression of Hox4-Hox11 genes aligns with MN columnar and pool subtypes. PMC=phrenic motor column, LMC=lateral motor column, HMC=hypaxial motor column, PGC=preganglionic motor column, MMC=medial motor column. Although technically a pool, we define phrenic MNs as a column due to their unique trajectory and because they do not reside within a larger columnar group. Peripheral targets of motor columns are indicated. LMC MNs further diversify in ~50 motor pools targeting limb muscles at brachial and lumbar levels.

Figure 2. Mutations in Hox Genes Cause Defects in MN Development, Migration and Axon Guidance

(A) Mutations in Hox1-Hox3 genes result in misspecification, disorganization and abnormal projections of hindbrain MNs. The schematics are composites, showing both early segmentation defects and subsequent MN defects. Facial MNs (VII) caudally migrate to r6 between embryonic day (e)11-e14. The facial (VII) nucleus is absent in Hoxb1 mutants and reduced in Hoxa1, Hoxb2 and Hoxa2 mutants. Ectopic trigeminal nuclei are generated in Hoxa1, Hoxb1 and Hoxb2 mutant mice but are subsequently cleared by apoptosis. Trigeminal (V) axons are misrouted in Hoxa2 mutants and the abducens (VI) nucleus is absent in Hoxa1 and Hox3 mutants. In Hoxa1 mutants, “rx” denotes a hybrid region with no clear rhombomeric identity, in Hoxb1 mutants r4 is transformed to r2/r3-like and in Hox3 mutants r6 acquires an r4 identity. (B) Mutations in Hox5-Hox10 genes result in transformation or reduction of distinct motor columns. Hox5 genes control PMC development, Hoxc9 determines thoracic MN identities while Hox6, Hoxc8 (at brachial levels) and Hox10 (at lumbar levels) genes define aspects of LMC identity. Hoxc6 and Hoxc8 are also required for the specification of pools defined by expression of Pea3 and Scip. In Foxp1−/− mice all Hox-dependent programs are disrupted with the exception of PMC specification. MMC neurons are not depicted as their development is thought to be Hox-independent.

Figure 3. Regulation of Hox Gene Expression in the CNS

(A) Gradients of FGF and RA establish initial patterns of Hox gene expression in the early embryo. Regions of RA signaling are inferred from expression of the Raldh2 gene in somitic mesoderm. Color coding of Hox genes denotes paralog groups regulated by indicated morphogens. RA induces primarily Hox1-Hox5 genes, FGF Hox6-Hox9, and Gdf11/FGF8 Hox10-Hox11 genes. (B) Genes within a Hox cluster are sequentially activated along the rostrocaudal axis in a manner that is spatially and temporally linked to their chromosomal position. Colinear activation is linked to removal of repressive chromatin marks (H3K27me3). Figure on right shows Hox expression is nested in more caudal regions of the embryo. (C) Anterior limits of Hox gene expression are established and maintained through the actions of Polycomb repressive complexes (PRCs). In stem cells, Hox genes are repressed by PRCs. At the progenitor phase RA and FGF act to clear PRC2 associated methylation marks from Hox genes. PRC1 activities are required to maintain Hox gene repression in postmitotic cells. PRC1 function may be distinct in MN progenitors (pMNs, indicated in gray) as depletion of PRC1-Bmi1 only affects Hox expression in post-mitotic MNs (Golden and Dasen, 2012). Cross-repressive effects of Hox genes are also indicated. (D) Cross-repressive interactions of Hox genes define rostrocaudal boundaries and contribute to MN diversification. At thoracic levels Hoxc9 excludes the expression of multiple brachially-expressed Hox genes. At brachial levels, repressive interactions between multiple Hox genes determine motor pool fates. Motor pool identity appears to be specified prior to clustering, and individual MNs within a pool are indicated in yellow, green, and red. (E) Feed-forward and auto-regulatory interactions amongst Hox1 and Hox2 paralogs ensure restricted expression of Hoxb1 in r4. Retinoic acid receptor (RAR) mediates the activation of Hoxa1 and Hoxb1. Hoxb1 is maintained through autoregulation.

Figure 4. Binding Specificity and Cofactors Mediate Hox Activity in the CNS

(A) Binding of Hox proteins to Pbx cofactors enhances binding specificity to target sequences. Binding preferences for the indicated Hox paralogs groups are inferred from Drosophila homologs (Slattery et al., 2011). Interaction of Meis proteins with Hox/Pbx dimers can occur in the absence of Meis binding sites. (B) Cofactor interactions determine the specificity of transcriptional outputs in Hox target gene regulation. In Drosophila, Engrailed (En) allows the Hox protein AbdA to repress the distalless (Dll) gene. Foxp1 appears to act as a collaborative factor for many LMC-specific genes. The Foxp1/Hox interactions are speculative. At the Hoxb1 gene, Meis proteins displace corepressors from Hox/Pbx dimers and recruit coactivators. (C) Hox proteins display distinct specificities in determining MN subtype identities.

Figure 5. Hox Effectors Control Aspects of Subtype-Specific Neuronal Identity

(A) LMC neurons are disorganized and project haphazardly in Foxp1−/− mice, due to the loss of adhesion and guidance molecules, such as EphA4. (B) Hoxc6−/− and Hoxc8−/− mice have a reduced and disorganized Pea3+ pool, due to loss of cadherin expression. (C) MN-specific Hox5 deletion results in defects in multiple aspects of PMC development, likely due to downregulation of target effectors such as ALCAM and PTN. (D) Tangentially migrating pontine neurons express Robo2 which prevents premature ventral migration by responding to Slit2/3 repulsive signals secreted from the facial motor nucleus (VII). In the absence of Hox2 genes, both Robo2 and Slit2/3 are downregulated resulting in abnormal pontine neuron migration. (E) Hoxa2 inactivation perturbs anteroventral cochlear neuron (AVCN) axonal pathfinding to the medial nucleus of the trapezoid body (MNTB) in the superior olive, resulting in decreased contralateral and increased ipsilateral targeting of MNTB due to the downregulation of Rig1/Robo3, the main axon guidance receptor required for midline crossing.

Figure 6. Hox Genes Contribute to Neuronal Identity in the CNS of Drosophila

(A) Hox genes are essential for the generation of several types of peptidergic neurons in the ventral nerve cord of the fly embryo. Hox expression is shown relative to thoracic (T1-T3) and abdominal (A1-A8) segments. Peptidergic neurons are identified by expression of Apterous (Ap) and Capability (Capa) as well as other markers in Va and dMP2 populations. The distribution of these neurons is affected in Hox mutant embryos. (B) Graded Antp activities contribute to the innervation of leg muscles in the fly. Elevation or depletion of Antp affects the branching patterns of motor axons at proximal and distal regions of the femur.

Figure 7. A Model for the Emergence of Motor Neuron Diversity in Vertebrates

(A) Ancestral MNs were characterized by the expression of a core set of transcription factors including Hb9, Isl1/2, and Lhx3, which are conserved in MNs of many invertebrates. (B) Agnathan species lacked paired appendages and likely contained MNs required for the innervation of dorsal and ventral muscles at all segments. For simplicity we indicate these populations as “MMC” and “HMC” neurons, although they likely lacked a columnar organization. The specification of ventrally projecting MNs may have required the exclusion of Lhx3, which defines dorsally projecting MMC neurons in tetrapods. (C) Zebrafish pectoral fins are innervated by MN populations that appear to be generated in registry with Hox6 and Hox9 expression domains. MMC- and HMC-like MNs are not organized into columns. (D) Tetrapod MNs display a columnar organization. Foxp1 likely played a key role in establishing both the columnar organization and multiple aspects of MN specification. (E) In mammals Hox5 genes specify PMC MNs in a Foxp1-independent manner.

Figure 8. Hox Genes in the Assembly of Neural Circuits

(A) Multiple Hox genes mediate the assembly of respiratory networks in the hindbrain and spinal cord. The pontine respiratory group (PRG) relies on Hoxa2 expression, while Hoxa1 is involved in the development of the rhythmogenic parafacial respiratory group (pFRG) and Hox5 genes control phrenic MN identity. (B) Hoxa2 is required for topographic projections from the trigeminal principal sensory nucleus (PrV) to the ventral posterior medial (VPM) nucleus of the thalamus. In the absence of Hoxa2, the maxillary (Mx) branch of the trigeminal nerve fails to arborize at the r3-derived domain of the PrV, which is ectopically targeted by the mandibular (Md) branch that normally innervates the r2-derived PrV. Projections from the r3-derived PrV are also mistargeted in the VPM and both the PrV and VPM lose their topographic organization. (C) In the auditory system, r4-derived structures (red), such as the posteroventral cochlear nucleus (PVCN) and the ventral lateral lemniscus (VLL) form circuits that mediate sound perception while r2/r3 and r5-derived structures (green) such as the anteroventral cochlear nucleus (AVCN) and the superior olivary complex (SOC) are primarily involved in circuits encoding sound localization. Mutations in Hoxb1/Hoxb2 affect the sound perception pathway while Hoxa2 is involved in the wiring of the sound-localizing circuits. SOC is shown as one structure for simplicity. (D) In mouse, but not chick, target-derived nerve growth factor (NGF) induces expression of Hoxd1 in nociceptors. In the absence of Hoxd1, both peripheral and central connectivity of mouse nociceptors is altered to resemble chick nociceptor properties.