Faculty Bibliography

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell-cycle inhibitors p16Ink4a and p19Arf (ref. ). However, deletion of Ink4a/Arf only partially rescues Bmi1-null phenotypes, indicating that other important targets of BMI1 exist. Here, using the continuously growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.

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 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.

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.

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.

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

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

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 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