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Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally-distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes, and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily-conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss ofprdm16ormecomcauses fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

The little skate Leucoraja erinacea, a cartilaginous fish, displays pelvic fin driven walking-like behavior using genetic programs and neuronal subtypes similar to those of land vertebrates. However, mechanistic studies on little skate motor circuit development have been limited, due to a lack of high-quality reference genome. Here, we generated an assembly of the little skate genome, with precise gene annotation and structures, which allowed post-genome analysis of spinal motor neurons (MNs) essential for locomotion. Through interspecies comparison of mouse, skate and chicken MN transcriptomes, shared and divergent gene expression profiles were identified. Comparison of accessible chromatin regions between mouse and skate MNs predicted shared transcription factor (TF) motifs with divergent ones, which could be used for achieving differential regulation of MN-expressed genes. A greater number of TF motif predictions were observed in MN-expressed genes in mouse than in little skate. These findings suggest conserved and divergent molecular mechanisms controlling MN development of vertebrates during evolution, which might contribute to intricate gene regulatory networks in the emergence of a more sophisticated motor system in tetrapods.

Spinal motoneurons are a remarkably diverse class of neurons responsible for facilitating a broad range of motor behaviors and autonomic functions. Studies of motoneuron differentiation have provided fundamental insights into the developmental mechanisms of neuronal diversification, and have illuminated principles of neural fate specification that operate throughout the central nervous system. Because of their relative anatomical simplicity and accessibility, motoneurons have provided a tractable model system to address multiple facets of neural development, including early patterning, neuronal migration, axon guidance, and synaptic specificity. Beyond their roles in providing direct communication between central circuits and muscle, recent studies have revealed that motoneuron subtype-specific programs also play important roles in determining the central connectivity and function of motor circuits. Cross-species comparative analyses have provided novel insights into how evolutionary changes in subtype specification programs may have contributed to adaptive changes in locomotor behaviors. This chapter focusses on the gene regulatory networks governing spinal motoneuron specification, and how studies of spinal motoneurons have informed our understanding of the basic mechanisms of neuronal specification and spinal circuit assembly.

The little skate Leucoraja erinacea, a cartilaginous fish, displays pelvic fin driven walking-like behaviors using genetic programs and neuronal subtypes similar to those of land vertebrates. However, mechanistic studies on little skate motor circuit development have been limited, due to a lack of high-quality reference genome. Here, we generated an assembly of the little skate genome, containing precise gene annotation and structures, which allowed post-genome analysis of spinal motor neurons (MNs) essential for locomotion. Through interspecies comparison of mouse, skate and chicken MN transcriptomes, shared and divergent MN expression profiles were identified. Conserved MN genes were enriched for early-stage nervous system development. Comparison of accessible chromatin regions between mouse and skate MNs revealed conservation of the potential regulators with divergent transcription factor (TF) networks through which expression of MN genes is differentially regulated. TF networks in little skate MNs are much simpler than those in mouse MNs, suggesting a more fine-grained control of gene expression operates in mouse MNs. These findings suggest conserved and divergent mechanisms controlling MN development system of vertebrates during evolution and the contribution of intricate gene regulatory networks in the emergence of sophisticated motor system in tetrapods.

The loss of the tail is one of the main anatomical evolutionary changes to have occurred along the lineage leading to humans and to the “anthropomorphous apes”1,2. This morphological reprogramming in the ancestral hominoids has been long considered to have accommodated a characteristic style of locomotion and contributed to the evolution of bipedalism in humans3–5. Yet, the precise genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Primate genome sequencing projects have made possible the identification of causal links between genotypic and phenotypic changes6–8, and enable the search for hominoid-specific genetic elements controlling tail development9. Here, we present evidence that tail-loss evolution was mediated by the insertion of an individual Alu element into the genome of the hominoid ancestor. We demonstrate that this Alu element – inserted into an intron of the TBXT gene (also called T or Brachyury10–12) – pairs with a neighboring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated a mouse model that mimics the expression of human TBXT products by expressing both full-length and exon-skipped isoforms of the mouse TBXT ortholog. We found that mice with this genotype exhibit the complete absence of a tail or a shortened tail, supporting the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype, albeit with incomplete penetrance. We further noted that mice homozygous for the exon-skipped isoforms exhibited embryonic spinal cord malformations, resembling a neural tube defect condition, which affects ∼1/1000 human neonates13. We propose that selection for the loss of the tail along the hominoid lineage was associated with an adaptive cost of potential neural tube defects and that this ancient evolutionary trade-off may thus continue to affect human health today.

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We unexpectedly found that PRC2 is dispensable to preserve the morphogen-induced positional fates of spinal motor neurons (MNs), while PRC1 is essential for the specification of segmentally-restricted subtypes. Mutation of the core PRC1 componentRing1in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities inRing1mutants is due to the suppression of Hox networks by derepressed caudalHoxgenes. These results indicate that PRC1 can function independently ofde novoPRC2-dependent histone methylation to maintain chromatin topology and transcriptional memory at the time of neuronal differentiation.

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs (Hoxa13, Hoxb13, Hoxc13, Hoxd13). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate.

DNA methylation is an epigenetic mark that plays pivotal roles in gene regulation, but its functions in neural fate decisions are poorly understood. In this issue of Cell Stem Cell, Ziller et al. (2018) show that the de novo methyltransferase Dnmt3a ensures efficient generation of motor neurons from stem cells.

There was an error published in Development (2017) 144, [dev152686][1] ([doi:10.1242/dev.152686][2]). In Table 1B,C, the headings were incorrectly changed to match the heading of Table 1A. Corrected: Table 1A. Ratio of genotypes of offspring from Hoxa5 flox/+; Dermo1 +/Cre× Hoxa5 flox/flox

Execution of motor behaviors relies on the ability of circuits within the nervous system to engage functionally relevant subtypes of spinal motor neurons. While much attention has been given to the role of networks of spinal interneurons on setting the rhythm and pattern of output from locomotor circuits, recent studies suggest that motor neurons themselves can exert an instructive role in shaping the wiring and functional properties of locomotor networks. Alteration in the distribution of motor neuron subtypes also appears to have contributed to evolutionary transitions in the locomotor strategies used by land vertebrates. This review describes emerging evidence that motor neuron-derived cues can have a profound influence on the organization, wiring, and evolutionary diversification of locomotor circuits.

Topographic maps are a basic organizational feature of nervous systems, and their construction involves both spatial and temporal cues. A recent study reports a novel mechanism of topographic map formation which relies on the timing of axon initiation.

Nervous systems control locomotion using rhythmically active networks that orchestrate motor neuron firing patterns. Whether animals use common or distinct genetic programs to encode motor rhythmicity remains unclear. Cross-species comparisons have revealed remarkably conserved neural patterning systems but have also unveiled divergent circuit architectures that can generate similar locomotor behaviors.

Sensory-motor reflex circuits are the basic units from which animal nervous systems are constructed, yet little is known regarding how connections within these simple networks are established. In papers in Cell Reports and in this issue of Neuron, Zheng et al. (2015a, 2015b) demonstrate that coordinate activities of Hox genes in sensory neurons and interneurons govern connectivity within touch-reflex circuits in C. elegans.

Evolutionary modifications in nervous systems enabled organisms to adapt to their specific environments and underlie the remarkable diversity of behaviors expressed by animals. Resolving the pathways that shaped and modified neural circuits during evolution remains a significant challenge. Comparative studies have revealed a surprising conservation in the intrinsic signaling systems involved in early patterning of bilaterian nervous systems but also raise the question of how neural circuit compositions and architectures evolved within specific animal lineages. In this review, we discuss the mechanisms that contributed to the emergence and diversity of animal nervous systems, focusing on the circuits governing vertebrate locomotion.

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.

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.

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.

SummaryCultured 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.

SummaryThe 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.

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.

Control of movement is a fundamental and complex task of the vertebrate nervous system, which relies on communication between circuits distributed throughout the brain and spinal cord. Many of the networks essential for the execution of basic locomotor behaviors are composed of discrete neuronal populations residing within the spinal cord. The organization and connectivity of these circuits is established through programs that generate functionally diverse neuronal subtypes, each contributing to a specific facet of motor output. Significant progress has been made in deciphering how neuronal subtypes are specified and in delineating the guidance and synaptic specificity determinants at the core of motor circuit assembly. Recent studies have shed light on the basic principles linking locomotor circuit connectivity with function, and they are beginning to reveal how more sophisticated motor behaviors are encoded. In this review, we discuss the impact of developmental programs in specifying motor behaviors governed by spinal circuits.

Spinal motor neurons acquire specialized "pool" identities that determine their ability to form selective connections with target muscles in the limb, but the molecular basis of this striking example of neuronal specificity has remained unclear. We show here that a Hox transcriptional regulatory network specifies motor neuron pool identity and connectivity. Two interdependent sets of Hox regulatory interactions operate within motor neurons, one assigning rostrocaudal motor pool position and a second directing motor pool diversity at a single segmental level. This Hox regulatory network directs the downstream transcriptional identity of motor neuron pools and defines the pattern of target-muscle connectivity.

Pituitary gland development is controlled by signals that guide expression of specific combinations of transcription factors that dictate serial determination and differentiation events. One class of factors is paired-like homeodomain factors. Two that have been investigated are the repressor Hex1/Rpx and activator prophet of Pit-1 (Prop-1), which exert selective roles during pituitary development. The opposing actions of these factors provide one aspect of pituitary organogenesis.

Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed ∼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.

Summary Motor output varies along the rostro-caudal axis of the tetrapod spinal cord. At limb levels, ∼60 motor pools control the alternation of flexor and extensor muscles about each joint, whereas at thoracic levels as few as 10 motor pools supply muscle groups that support posture, inspiration, and expiration. Whether such differences in motor neuron identity and muscle number are associated with segmental distinctions in interneuron diversity has not been resolved. We show that select combinations of nineteen transcription factors that specify lumbar V1 inhibitory interneurons generate subpopulations enriched at limb and thoracic levels. Specification of limb and thoracic V1 interneurons involves the Hox gene Hoxc9 independently of motor neurons. Thus, early Hox patterning of the spinal cord determines the identity of V1 interneurons and motor neurons. These studies reveal a developmental program of V1 interneuron diversity, providing insight into the organization of inhibitory interneurons associated with differential motor output.

Nat. Neurosci. 19, 1743–1749 (2016); published online 31 October 2016; corrected after print 29 November 2016 In the version of this article initially published, authors Joshua S. Grimley, Anne-Rachel Krostag and Ajamete Kaykas were missing. These authors have been inserted into the author list after Jianhua Chu; they are at the Allen Institute for Brain Science, Seattle, Washington, USA, and performed experiments related to hESCs.

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 (rAAV) 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 GABA-ergic function in virtually any vertebrate species.

The establishment of spinal motor neuron subclass diversity is achieved through developmental programs that are aligned with the organization of muscle targets in the limb. The evolutionary emergence of digits represents a specialized adaptation of limb morphology, yet it remains unclear how the specification of digit-innervating motor neuron subtypes parallels the elaboration of digits. We show that digit-innervating motor neurons can be defined by selective gene markers and distinguished from other LMC neurons by the expression of a variant Hox gene repertoire and by the failure to express a key enzyme involved in retinoic acid synthesis. This divergent developmental program is sufficient to induce the specification of digit-innervating motor neurons, emphasizing the specialized status of digit control in the evolution of skilled motor behaviors. Our findings suggest that the emergence of digits in the limb is matched by distinct mechanisms for specifying motor neurons that innervate digit muscles.

Polycomb repressive complexes (PRCs) establish and maintain gene repression through chromatin modifications, but their specific roles in cell fate determination events are poorly understood. Here we show an essential role for the PRC1 component Bmi1 in motor neuron (MN) subtype differentiation through dose-dependent effects on Hox gene expression. While Bmi1 is dispensable for generating MNs as a class, it has an essential role in specifying and determining the position of Hox-dependent MN columnar and pool subtypes. These actions are mediated through limiting anterior Hox expression boundaries, functions deployed in post-mitotic MNs, temporally downstream from morphogen gradients. Within the HoxC gene cluster, we found a progressive depletion of PRC-associated marks from rostral to caudal levels of the spinal cord, corresponding to major demarcations of MN subtypes. Selective ablation of Bmi1 elicits a derepression of more posterior Hox genes, leading to a switch in MN fates. Unexpectedly, Hox patterns and MN fates appear to be sensitive to absolute PRC1 activity levels; while reducing Bmi1 switches forelimb lateral motor column (LMC) MNs to a thoracic preganglionic (PGC) identity, elevating Bmi1 expression at thoracic levels converts PGC to LMC MNs. These results suggest that graded PRC1 activities are essential in determining MN topographic organization.

Respiration in mammals relies on the rhythmic firing of neurons within 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.

In the developing spinal cord, regional and combinatorial activities of Hox transcription factors are critical in controlling motor neuron fates along the rostrocaudal axis, exemplified by the precise pattern of limb innervation by more than fifty Hox-dependent motor pools. The mechanisms by which motor neuron diversity is constrained to limb levels are, however, not well understood. We show that a single Hox gene, Hoxc9, has an essential role in organizing the motor system through global repressive activities. Hoxc9 is required for the generation of thoracic motor columns, and in its absence, neurons acquire the fates of limb-innervating populations. Unexpectedly, multiple Hox genes are derepressed in Hoxc9 mutants, leading to motor pool disorganization and alterations in the connections by thoracic and forelimb-level subtypes. Genome-wide analysis of Hoxc9 binding suggests that this mode of repression is mediated by direct interactions with Hox regulatory elements, independent of chromatin marks typically associated with repressed Hox genes., National Institutes of Health (U.S.) (P01NS055923)

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.

The emergence of coordinated locomotor behaviors in vertebrates relies on the establishment of selective connections between discrete populations of neurons present in the spinal cord and peripheral nervous system. The assembly of the circuits necessary for movement presumably requires the generation of many unique cell types to accommodate the intricate connections between motor neurons, sensory neurons, interneurons, and muscle. The specification of diverse neuronal subtypes is mediated largely through networks of transcription factors that operate within progenitor and postmitotic cells. Selective patterns of transcription factor expression appear to define the cell-type-specific cellular programs that govern the axonal guidance decisions and synaptic specificities of neurons, and may lay the foundation through which innate motor behaviors are genetically predetermined. Recent studies on the developmental programs that specify two highly diverse neuronal classes-spinal motor neurons and proprioceptive sensory neurons-have provided important insights into the molecular strategies used in the earliest phases of locomotor circuit assembly. This chapter reviews progress toward elucidating the early transcriptional networks that define neuronal identity in the locomotor system, focusing on the pathways controlling the specific connections of motor neurons and sensory neurons in the formation of simple reflex circuits.

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.

The emergence of coordinated locomotor behaviors in vertebrates relies on the establishment of selective connections between discrete populations of neurons present in the spinal cord and peripheral nervous system. The assembly of the circuits necessary for movement presumably requires the generation of many unique cell types to accommodate the intricate connections between motor neurons, sensory neurons, interneurons, and muscle. The specification of diverse neuronal subtypes is mediated largely through networks of transcription factors that operate within progenitor and postmitotic cells. Selective patterns of transcription factor expression appear to define the cell-type-specific cellular programs that govern the axonal guidance decisions and synaptic specificities of neurons, and may lay the foundation through which innate motor behaviors are genetically predetermined. Recent studies on the developmental programs that specify two highly diverse neuronal classes-spinal motor neurons and proprioceptive sensory neurons-have provided important insights into the molecular strategies used in the earliest phases of locomotor circuit assembly. This chapter reviews progress toward elucidating the early transcriptional networks that define neuronal identity in the locomotor system, focusing on the pathways controlling the specific connections of motor neurons and sensory neurons in the formation of simple reflex circuits.

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.

Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteiniz-ing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1 (ref. 1)f which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report we have identified four CPHD families with homozy-gosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.

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.

▪ Abstract During the development of the pituitary gland, distinct hormone-producing cell types arise from a common population of ectodermal progenitors, providing an instructive model system for elucidating the molecular mechanisms of patterning and cell type specification in mammalian organogenesis. Recent studies have established that the development of the pituitary occurs through multiple sequential steps, allowing the coordinate control of the commitment, early patterning, proliferation, and positional determination of pituitary cell lineages in response to extrinsic and intrinsic signals. The early phases of pituitary development appear to be mediated through the activities of multiple signaling gradients emanating from key organizing centers that give rise to temporally and spatially distinct patterns of transcription factor expression. The induction of these transcriptional mediators in turn acts to positionally organize specific pituitary cell lineages within an apparently uniform field of ectodermal progenitors. Ultimately, pituitary cell types have proven to be both specified and maintained through the combinatorial interactions of a series of cell-type-restricted transcription factors that dictate the cell autonomous programs of differentiation in response to the transient signaling events.

The development of the pituitary gland has provided an instructive model system for exploring the mechanisms by which differentiated cell types arise from a common primordium in response to extrinsic and intrinsic signals. Recent studies have established that organ commitment, early patterning, proliferation and positional determination of cell types in the developing pituitary are mediated through the integral actions of multiple signaling gradients acting on an initially uniform ectodermal cell population. Studies of the cell-autonomous transcriptional mediators of the transient signaling events have also provided insight into the molecular mechanisms by which overlapping patterns of transcription factor expression can positionally specify pituitary cell lineages. There is emerging evidence for a morphogenetic code for the development of the pituitary gland based on the cooperative and opposing actions of multiple signaling gradients, mediated by corresponding expression patterns of temporally and spatially induced transcription factors.

The development of the hormone-secreting cell types in the pituitary gland provides an excellent model system in which to explore the complex transcriptional mechanisms underlying the specification and maintenance of differentiated cell types in mammalian organogenesis. Pituitary development is orchestrated through the combinatorial actions of a repertoire of signaling-gradient-induced transcription factors which, on the basis of their distinct and overlapping expression patterns, and functional interactions, ultimately has led to the generation of functionally distinct cell phenotypes from a common ectodermal primordium.

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes1. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 (ref. 3) is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT4,5, mSin3A/B6,7,8 and histone deacetylases6,7,8 and a co-activator complex that includes the CREB-binding protein (CBP)9 and p/CAF10. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, thehistone acetyltransferase functions of CBP11,12 or p/CAF10 are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

The gene apparently responsible for a heritable form of murine pituitary-dependent dwarfism (Ames dwarf, df) has been positionally cloned, identifying a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (Prop-1). The df phenotype results from an apparent failure of initial determination of the Pit-1 lineage required for production of growth hormone, prolactin or thyroid-stimulating hormone, resulting in dysmorphogenesis and failure to activate Pit-1 gene expression. These results imply that a cascade of tissue-specific regulators is responsible for the determination and differentiation of specific cell lineages in pituitary organogenesis.

The human COX5B gene encodes subunit Vb of cytochrome c oxidase (COX). COX Vb is 1 of the 10 subunits of the mitochondrial COX complex encoded by a nuclear gene. We have defined a region in the human COX5B promoter essential for gene expression and shown by phylogenetic footprinting of 11 primate COX5B promoters that many cis -regulatory elements in this region are evolutionarily conserved. The transcription start site of human COX5B was mapped 58 bp upstream of the initiation Met codon by primer extension using a thermostable reverse transcriptase. A 475-bp region (−456 to +20) of the human COX5B gene was shown to function as a promoter for the chloramphenicol acetyl transferase (CAT) gene in expression vectors when transfected into HeLa cells. The human COX5B gene is located in a CpG island and contains several potential binding sites for the transcription factor Sp1, but no consensus TATA box element. Several sequence elements associated with the transcriptional regulation of respiratory genes were also found in the promoter and 5′ flanking region, including a single NRF-1 site and two 9-bp direct repeats containing binding sites for ets -domain proteins, such as NRF-2/GABP. Many features of the human COX5B promoter are conserved in the COX5B promoters of primates, in particular, the presence of a single binding site for NRF-1 and multiple sites for Sp1 and NRF-2/GABP. Electrophoretic mobility shift assays demonstrate that the conserved NRF-1 site in primate COX5B promoters is specifically recognized by a factor present in HeLa nuclear extracts. Phylogenetic footprinting has identified additional conserved elements that may also function as binding sites for regulatory factors.

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., Author Summary Coordinated motor behaviors—as complex as playing a musical instrument or as simple as walking—rely on the ability of motor neurons within the spinal cord to navigate towards and establish specific connections with muscles in the limbs. The establishment of connections between motor neurons and limb muscles is mediated through the actions of genes encoding Hox proteins, a large family of transcription factors conserved amongst all metazoans. However, the specific requirements for Hox genes in motor neuron specification and patterns of muscle connectivity are poorly understood. We have found that members of the Hox6 gene paralog group (Hoxa6, Hoxc6, and Hoxb6) contribute to diverse aspects of motor neuron subtype differentiation. Hox6 gene activity is required during two critical phases of motor neuron development: first as motor axons select a trajectory toward the forelimb and second as they choose specific muscles to innervate. At the molecular level, these two functions are encoded by distinct peptide domains within Hox proteins. This work indicates that Hox proteins execute their critical functions in motor neurons through intrinsic modules that confer distinct specificities and that these activities are central in the genetic network required for motor neuron differentiation.