Jeremy S. Dasen - US grants

Locomotor behavior in vertebrates requires the establishment of selective connections between motor neurons in the spinal cord and muscle targets in the periphery. A regulatory network of Hox transcription factors has been directly linked with two critical steps in motor differentiation: the establishment of columnar identities which directs motor axons toward a specific target field; and the diversification of neurons within a column into motor pools, each pool targeting a single muscle. The molecular mechanisms by which Hox proteins contribute to motor neuron columnar and pool identities are not known. We have found that the forkhead homeodomain transcription factor FoxP1 is selectively expressed by two Hox-sensitive motor neuron columnar subtypes, the lateral motor column (LMC) and preganglionic column (PGC). The aim of this proposal is to further elucidate how the activities FoxP1 and Hox are coordinately regulated in motor neurons and to elucidate the downstream pathway that are critical in the Hox-dependent programs of motor neuron identity. The first aim of this proposal will explore the regulation of FoxP1 expression by Hox proteins and the mechanisms by which FoxP1 becomes selectively expressed in a subset of motor neuron subtypes. In this aim we will determine the influences of FoxP1 protein levels on the establishment of motor neuron columnar identities through overexpression of FoxP1 in vivo. In the second aim the impact of loss Foxp1 on motor neuron identity and connectivity with muscle targets will be examined. We will use anatomical and histological assays to examine the role of Foxp1 in establishing the initial patterns of motor axon projections in the limb and in defining the selection of synaptic targets. In the third aim biochemical interactions of FoxP1 and Hox proteins in the control of motor neuron-specific gene expression will be explored. The hypothesis that FoxP1 interacts directly with most or all of genes expressed in LMC motor neurons and in specific pools will be examined using chromatin immunoprecipitation assays. We will then examine the consequences of interactions between FoxP1 and Hox proteins using in vitro and in vivo assays. Together, these studies should help to provide a better understanding of how motor neuron diversity is generated and provide some of the basic insights into the mechanisms that determine the synaptic specificity of neurons in other regions of the nervous system.

DESCRIPTION (provided by applicant): The neural circuits that govern behaviors vital to mammals, such as locomotion and respiration, rely on the ability of motor neurons (MNs) within the spinal cord to establish selective connections with dedicated sets of peripheral and central synaptic targets. Signaling pathways acting along the dorsoventral axis of the neural tube have been shown to determine the early identity of MNs and distinguish this class from other neuronal types within the spinal cord. The subsequent diversification of MNs depends on the actions of approximately 20 Hox transcription factors, which appear to be required at distinct phases of MN differentiation. While Hox genes are essential for MN fate specification, the targets of their activities are not known, nor is it understood how they achieve MN-specificity, given their relatively broad roles in patterning along the rostrocaudal axis. Moreover the factors that determine the expression patterns of Hox proteins in MNs are poorly defined. In aim1 we will characterize the direct targets of Hox proteins, assess how they are regulated in motor columns, and determine if and how they intersect with MN-specific gene programs. In aim2 we will dissect the mechanisms of Hox protein specificity in controlling facets of MN identity, focusing on the Hoxc9 protein, a central determinant of MN columnar organization. In aim3 we will test the hypothesis that the organization of Hox-dependent MN subtype relies on graded activities of Polycomb proteins that ensure proper postmitotic Hox expression patterns. These studies will provide basic insights into the mechanisms through which Hox proteins influence MN differentiation, and should allow for the design of strategies to generate MN subtypes from undifferentiated cells.

Locomotion is a fundamental and vital animal behavior which relies on the activities of diverse neuronal classes residing within the brainstem and spinal cord. Spinal motor neurons play a central role in coordinating locomotor behaviors, and are engaged by networks of local interneurons that facilitate the rhythm and pattern of limb muscle activation. The basic genetic programs that govern connectivity between spinal interneurons and motor neurons are poorly understood due in large part to the complexity of the hundreds of muscle groups targeted by spinal locomotor circuits. In this exploratory proposal, we will assess the composition and connectivity of locomotor circuits in the little skate Leucoraja erinacea, a primitive cartilaginous fish that displays walking behaviors highly similar to those of tetrapods. Remarkably, we have found that the molecular profiles of fin-innervating motor neurons in Leucoraja are nearly identical to those of tetrapods. Leucoraja generates bipedal locomotion using 8 anatomically well-defined pelvic fin muscles, although the neural circuits that generate this behavior are uncharacterized. In this proposal we will determine the subtype identities and connectivity of spinal motor neurons, interneurons, and muscle in Leocoraja. We will also explore the utility of Leucoraja for assessing motor circuit connectivity programs using viral trans-synaptic tracing assays and electrophysiology. In Aim 1 we will assess the circuit composition and connectivity of motor neurons and interneurons in Leucoraja, and explore the role signaling through Hox transcription factors in the regional allocation of motor neuron columnar and pool subtype identities. In Aim2 we will use viral tracing assays to determine the pattern of connectivity between spinal interneurons and motor neurons, and explore the workings of the intraspinal central pattern generators (CPGs) that coordinate the activation of fin muscles during walking and swimming. By exploiting the relatively simple neuromusculature architecture of this primitive vertebrate, and building off our in depth knowledge of neural specification programs in the mammalian spinal cord, these studies could provide insights into the basic mechanisms through which motor circuits are assembled.

The neural circuits governing behaviors vital to mammals, such as locomotion and respiration, rely on the ability of motor neurons (MNs) to establish selective connections with target cells both centrally and peripherally. Motor neurons innervating specific muscle targets are specified by the large family of chromosomally arrayed Hox transcription factors. A key aspect of Hox gene function is to segregate motor neurons into topographically organized columnar and pool subtypes. While it has been suggested the somatotopic organization of MNs evolved to facilitate the activation of an increasingly more complex limb musculature, it is largely unknown how MNs cluster into columns, and what role MN position plays in shaping the specificity of connections within motor networks. In this proposal we will investigate the function of Pbx genes, essential co-factors of Hox proteins, in the formation of MN topographic maps and in the development of motor circuits. The major goals of this proposal are to: 1) to assess the role of Pbx genes in the organization and connectivity of spinal motor neurons, 2) to determine the mechanisms through which motor neurons are topographically organized, and 3) to define the role of MN position and identity in spinal circuit assembly. In Aim1 we will define the function of Pbx genes in MN differentiation using genetic manipulations and histological assays. In Aim2 we will identity the targets of Pbx proteins in MNs, and assess their function and mechanisms of regulation. In Aim3 we will assess how motor neuron position and identity influences the specificity of connections with presynaptic interneuron populations. By building off our in depth knowledge of motor neuron specification programs in mammals, these studies should provide basic insights into the mechanisms through which motor circuits are assembled.

DESCRIPTION (provided by applicant): The current proposal describes an advanced predoctoral training program focused on molecular, cellular, and translational neuroscience. The training program's goals are focused on (a) achieving a high-quality education in the fundamental principles and techniques that will prepare trainees for the intensely collaborative and interdisciplinary nature of modern neuroscience research; (b) generating in-depth and state-of-the-art laboratory research opportunities in the focus area, and (c) training in the necessary professional skills often overlooked during graduate education, including critical reading, grant writing, oral presentation, leadership, management, and networking. We will achieve these goals through a combination of advanced coursework, workshops, small group discussions, weekly seminars, trainee presentations, and structured (as well as more informal) meetings with program faculty. A well-thought out mentoring program will aid trainees' progress in the program and prepare the participants for their future careers in science. Neuroscience predoctoral trainees at NYU become an integral part of their research labs as well as the expansive neuroscience community at NYU, especially because the proposed training program brings together 30 faculty trainers from across NYU's major campuses. Although historically two related neuroscience graduate programs co-existed at NYU, faculty from both programs have taken several key steps to integrate their graduate training over the past 5-10 years. With substantial support from the University, we reached a new phase of program integration. The proposed training program will be instrumental in furthering the efforts to unify the extensive NYU neuroscience community, particularly those in the areas of molecular, cellular, and translational neuroscience, whose ranks have recently increased substantially thanks to aggressive faculty recruitment by the NYU Center for Neural Science and the new NYU Neuroscience Institute. This thriving community, and especially the selected training faculty, supports a substantial graduate student population. We seek funding for 4 predoctoral students in their 3rd year or higher within this cohort; each will be appointed for 1 to 2 years, just priorto when we anticipate they will transition to independent funding. This size will provide a critical mass of trainees so as to firmly establish this training program within the context of the larger combined neuroscience graduate programs at NYU. Through our newly integrated graduate program, we provide trainees with a vast and rich intellectual environment and the resources and experience to confidently pursue their own scientific interests, which we hope will lead to future breakthroughs in basic neuroscience and the underlying mechanisms of neurological diseases.

Project Summary/Abstract The current proposal describes an advanced predoctoral training program focused on molecular, cellular, and translational neuroscience. The training program?s goals are focused on (a) achieving a high-quality education in the fundamental principles and techniques that will prepare trainees for the intensely collaborative and interdisciplinary nature of modern neuroscience research; (b) generating in-depth and state-of-the-art laboratory research opportunities in the focus area, and (c) training in the necessary professional skills often overlooked during graduate education, including critical reading, grant writing, oral presentation, leadership, management, and networking. We will achieve these goals through a combination of advanced coursework, workshops, small group discussions, weekly seminars, trainee presentations, and structured (as well as more informal) meetings with program faculty. A well-thought out mentoring program will aid trainees? progress in the program and prepare the participants for their future careers in science. Neuroscience predoctoral trainees at NYU become an integral part of their research labs as well as the expansive neuroscience community at NYU, especially because the proposed training program brings together 30 faculty trainers from across NYU?s major campuses. Although historically two related neuroscience graduate programs co-existed at NYU, faculty from both programs have taken several key steps to integrate their graduate training over the past 5-10 years. With substantial support from the University, we reached a new phase of program integration. The proposed training program will be instrumental in furthering the efforts to unify the extensive NYU neuroscience community, particularly those in the areas of molecular, cellular, and translational neuroscience, whose ranks have recently increased substantially thanks to aggressive faculty recruitment by the NYU Center for Neural Science and the new NYU Neuroscience Institute. This thriving community, and especially the selected training faculty, supports a substantial graduate student population. We seek funding for 4 predoctoral students in their 3rd year or higher within this cohort; each will be appointed for 1 to 2 years, just prior to when we anticipate they will transition to independent funding. This size will provide a critical mass of trainees so as to firmly establish this training program within the context of the larger combined neuroscience graduate programs at NYU. Through our newly integrated graduate program, we provide trainees with a vast and rich intellectual environment and the resources and experience to confidently pursue their own scientific interests, which we hope will lead to future breakthroughs in basic neuroscience and the underlying mechanisms of neurological diseases.

The neural circuits governing motor functions vital to mammals, including walking and breathing rely on the ability of spinal motor neurons (MNs) to acquire specific subtype identities and establish selective connections with peripheral and central synaptic targets. Signaling pathways acting along the dorsoventral axis of the neural tube have been shown to determine the early identity of MNs and distinguish this class from other neuronal types within the spinal cord. The subsequent diversification of MNs depends on the actions of ~20 Hox transcription factors, which orchestrate genetic programs essential for MN organization, identity, and connectivity. During development, expression of Hox genes is induced through the actions of secreted morphogens which act though removing repressive chromatin marks from Hox clusters. These repressive marks are established and maintained through the actions of the large family of Polycomb group (PcG) proteins. Although removal of Polycomb repressive marks is associated with the activation of specific Hox genes, the functions and mechanisms of action of these complexes are poorly understood. In Aim1 we will examine the effects of removal of Polycomb repressive complex 1 (PRC1) activities from MNs, through selective genetic ablation of Ring1 genes. In Aim2 we will determine the targets of PRC1 actions, focusing on Hox genes, and assess how misregulation of PRC targets affects MN differentiation. In Aim3 we will explore the hypothesis that distinct PRC1 configurations determine the organization and identity of MN subtypes through differentially regulating Hox genes along the rostrocaudal axis. These studies should provide basic insights into the mechanisms by which chromatin modifications influence neural specification. Understanding the mechanisms of PcG protein function could improve the current strategies for generating MN subtypes from undifferentiated cells.

Project Summary The neural circuits controlling motor behaviors vital to mammals, including walking, breathing, and balance, rely on the ability of neurons within the spinal cord to establish selective connections during development. Work over the past decade has provided a fairly comprehensive understanding of the genetic pathways that determine the identity of each major neuronal class within the neural tube. The mechanisms through which neurons acquire subtype identities necessary for the incorporation into a particular motor circuit are, however, still poorly defined. Our studies on the specification of spinal motor neurons indicate that the large family of Hox transcription factors play key roles in generating the hundreds of subtypes required for selective innervation of muscle. Hox proteins orchestrate genetic programs that control diverse aspects of motor neuron maturation, including their topographic organization, peripheral target muscle specificity, and presynaptic partners. Emerging studies from our group also indicate that Hox genes function in multiple neuronal classes to shape synaptic specificity during development, suggesting a broader role in circuit assembly. The overall goals of the proposed research are to elucidate the mechanisms of neural diversification and connectivity within the spinal cord, and to determine how Hox-dependent and -independent genetic programs establish the circuit architectures necessary for motor control. Ultimately, we hope to uncover the pathways through which genetically encoded developmental programs contribute to the emergence of specific motor behaviors. Our approach integrates selective genetic manipulations of neuronal subtypes, genome-wide interrogation of regulatory networks, modern circuit-tracing methods, comparative analyses in multiple vertebrate organisms, and rigorous analyses of behavior. Elucidating the basic mechanisms of motor circuit assembly will provide foundational insights relevant to the design of therapeutic strategies to treat degenerative diseases of spinal neurons or repair motor circuits damaged by injury.

This is a proposal to renew an advanced predoctoral training program focused on Integrative Approaches to Explore Cellular Interactions in Neural Circuits. The goal of this program is to endow trainees with diverse research interests and scientific backgrounds with skills necessary for the study of neural circuits using multidisciplinary approaches and rigorous methodologies. This integrative program specifically targets trainees with research interests at the interface between molecular/cellular and systems/behavioral neuroscience by emphasizing scientific approaches that span multiple levels of analysis and employ multiple experimental models. The program's goals will be accomplished through a tailored curriculum that includes advanced coursework in the study of neural circuits, training in rigorous neuroscience research techniques and quantitative analysis, and mentorship in science communication and career planning. In a course that has been specifically designed for this training grant, trainees will learn from contemporary studies of neural circuits that incorporate diverse approaches, including cell and molecular neurobiology, genetics, synaptic physiology, and behavior. In this renewal, we add a new training component for PhD candidates that brings their research project to a biostatician for in-depth feedback on scientific methodologies and rigor in data analysis, in the context of the trainee's own experimental results. This training program enables predoctoral students to prepare for the intensely collaborative and interdisciplinary nature of modern neuroscience research by providing: (a) high-quality scientific education in the fundamental principles of neurobiology, state-of-the-art techniques to study neural circuits, and statistical approaches to rigorous experimental design; (b) mentoring that aids trainees? progress toward their future careers in science, and (c) training in the professional skills that are necessary for success in academic research, including critical reading, grant writing, oral presentation, leadership, management, and networking. Four trainees will be selected by an Executive Committee on the basis of their excellence in research, interest in neural circuits, and potential for future leadership. Each trainee will be appointed for one year and will continue to receive mentoring and career development support in subsequent years. Thirty-one faculty from the NYU School of Medicine and NYU Arts and Science campus will participate in this multidisciplinary training program. Faculty mentors lead strong NIH-supported research programs and use cellular, molecular, and genetic approaches to reveal basic principles of neural circuit assembly and function. The environment at NYU strongly supports the goals of this training program, and education continues to be a core mission of our neuroscience community.