Jeremy S. Dasen

Motor neuron columnar fate imposed by sequential phases of Hox-c activity

The organization of neurons into columns is a prominent feature of central nervous system structure and function. In many regions of the central nervous system the grouping of neurons into columns links cell-body position to axonal trajectory, thus contributing to the establishment of topographic neural maps. This link is prominent in the developing spinal cord, where columnar sets of motor neurons innervate distinct targets in the periphery. We show here that sequential phases of Hox-c protein expression and activity control the columnar differentiation of spinal motor neurons. Hox expression in neural progenitors is established by graded fibroblast growth factor signalling and translated into a distinct motor neuron Hox pattern. Motor neuron columnar fate then emerges through cell autonomous repressor and activator functions of Hox proteins. Hox proteins also direct the expression of genes that establish motor topographic projections, thus implicating Hox proteins as critical determinants of spinal motor neuron identity and organization.

Hox Repertoires for Motor Neuron Diversity and Connectivity Gated by a Single Accessory Factor, FoxP1

The precision with which motor neurons innervate target muscles depends on a regulatory network of Hox transcription factors that translates neuronal identity into patterns of connectivity. We show that a single transcription factor, FoxP1, coordinates motor neuron subtype identity and connectivity through its activity as a Hox accessory factor. FoxP1 is expressed in Hox-sensitive motor columns and acts as a dose-dependent determinant of columnar fate. Inactivation of Foxp1 abolishes the output of the motor neuron Hox network, reverting the spinal motor system to an ancestral state. The loss of FoxP1 also changes the pattern of motor neuron connectivity, and in the limb motor axons appear to select their trajectories and muscle targets at random. Our findings show that FoxP1 is a crucial determinant of motor neuron diversification and connectivity, and clarify how this Hox regulatory network controls the formation of a topographic neural map.

Hox networks and the origins of motor neuron diversity.

Motor behaviors are the primary means by which animals interact with their environment, forming the final output of most central nervous system (CNS) activity. The neural circuits that govern basic locomotor functions appear to be genetically hard wired and are comprised of discrete groups of neurons residing within the spinal cord. These local microcircuits coordinate simple reflexive behaviors in response to sensory stimuli and underlie the generation of rhythmic patterns of neural activity necessary for walking. In recent years there have been significant advances in understanding the genetic and molecular programs that determine the specificity of neural connections within the spinal cord that are critical for the emergence of coordinate motor behaviors. The assembly of circuits within the spinal cord requires the generation of diverse cell types to accommodate the intricate sets of interconnections between motor neurons, sensory neurons, interneurons, and muscle. The first and most critical aspect of this process is that motor neurons select their appropriate muscle targets in the periphery with fidelity and precision. All of the subsequent steps in motor neuron connectivity, such as their descending inputs from higher brain centers, their circuits with sensory neurons and interneurons are constrained by the early connections formed between motor neurons and their muscle targets. The actions of a single family of transcription factors, encoded by the chromosomally clustered Hox genes, appear to have a central role in defining the specificity of motor neuron-muscle connectivity. The emerging logic of Hox protein function in motor neuron specification may provide more general insights into the programs that determine synaptic specificity in other CNS regions.

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.

Transcriptional mechanisms controlling motor neuron diversity and connectivity

The control of movement relies on the precision with which motor circuits are assembled during development. Spinal motor neurons (MNs) provide the trigger to signal the appropriate sequence of muscle contractions and initiate movement. This task is accommodated by the diversification of MNs into discrete subpopulations, each of which acquires precise axonal trajectories and central connectivity patterns. An upstream Hox factor-based regulatory network in MNs defines their competence to deploy downstream programs including the expression of Nkx and ETS transcription factors. These interactive transcriptional programs coordinate MN differentiation and connectivity, defining a sophisticated roadmap of motor circuit assembly in the spinal cord. Similar principles using modular interaction of transcriptional programs to control neuronal diversification and circuit connectivity are likely to act in other CNS circuits.

Functional Diversity of ESC-Derived Motor Neuron Subtypes Revealed through Intraspinal Transplantation

Cultured ESCs can form different classes of neurons, but whether these neurons can acquire specialized subtype features typical of neurons in vivo remains unclear. We show here that mouse ESCs can be directed to form highly specific motor neuron subtypes in the absence of added factors, through a differentiation program that relies on endogenous Wnts, FGFs, and Hh-mimicking the normal program of motor neuron subtype differentiation. Molecular markers that characterize motor neuron subtypes anticipate the functional properties of these neurons in vivo: ESC-derived motor neurons grafted isochronically into chick spinal cord settle in appropriate columnar domains and select axonal trajectories with a fidelity that matches that of their in vivo generated counterparts. ESC-derived motor neurons can therefore be programmed in a predictive manner to acquire molecular and functional properties that characterize one of the many dozens of specialized motor neuron subtypes that exist in vivo.

Sustained Hox5 Gene Activity is Required for Respiratory Motor Neuron Development

Respiration in mammals relies on the rhythmic firing of neurons in the phrenic motor column (PMC), a motor neuron group that provides the sole source of diaphragm innervation. Despite their essential role in breathing, the specific determinants of PMC identity and patterns of connectivity are largely unknown. We show that two Hox genes, Hoxa5 and Hoxc5, control diverse aspects of PMC development including their clustering, intramuscular branching, and survival. In mice lacking Hox5 genes in motor neurons, axons extend to the diaphragm, but fail to arborize, leading to respiratory failure. Genetic rescue of cell death fails to restore columnar organization and branching patterns, indicating these defects are independent of neuronal loss. Unexpectedly, late Hox5 removal preserves columnar organization but depletes PMC number and branches, demonstrating a continuous requirement for Hox function in motor neurons. These findings indicate that Hox5 genes orchestrate PMC development through deployment of temporally distinct wiring programs.

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 viral strategy for targeting and manipulating interneurons across vertebrate species

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.

Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis

Hox genes encode transcription factors governing complex developmental processes in several organs. A subset of Hox genes are expressed in the developing lung. Except for Hoxa5, the lack of overt lung phenotype in single mutants suggests that Hox genes may not play a predominant role in lung ontogeny or that functional redundancy may mask anomalies. In the Hox5 paralog group, both Hoxa5 and Hoxb5 genes are expressed in the lung mesenchyme whereas Hoxa5 is also expressed in the tracheal mesenchyme. Herein, we generated Hoxa5;Hoxb5 compound mutant mice to evaluate the relative contribution of each gene to lung development. Hoxa5;Hoxb5 mutants carrying the four mutated alleles displayed an aggravated lung phenotype, resulting in the death of the mutant pups at birth. Characterization of the phenotype highlighted the role of Hoxb5 in lung formation, the latter being involved in branching morphogenesis, goblet cell specification, and postnatal air space structure, revealing partial functional redundancy with Hoxa5. However, the Hoxb5 lung phenotypes were less severe than those seen in Hoxa5 mutants, likely because of Hoxa5 compensation. New specific roles for Hoxa5 were also unveiled, demonstrating the extensive contribution of Hoxa5 to the developing respiratory system. The exclusive expression of Hoxa5 in the trachea and the phrenic motor column likely underlies the Hoxa5-specific trachea and diaphragm phenotypes. Altogether, our observations establish that the Hoxa5 and Hoxb5 paralog genes shared some functions during lung morphogenesis, Hoxa5 playing a predominant role.