Spinal Locomotor Circuits Develop Using Hierarchical Rules Based on Motorneuron Position and Identity

SummaryThe coordination of multi-muscle movements originates in the circuitry that regulates the firing patterns of spinal motorneurons. Sensory neurons rely on the musculotopic organization of motorneurons to establish orderly connections, prompting us to examine whether the intraspinal circuitry that coordinates motor activity likewise uses cell position as an internal wiring reference. We generated a motorneuron-specific GCaMP6f mouse line and employed two-photon imaging to monitor the activity of lumbar motorneurons. We show that the central pattern generator neural network coordinately drives rhythmic columnar-specific motorneuron bursts at distinct phases of the locomotor cycle. Using multiple genetic strategies to perturb the subtype identity and orderly position of motorneurons, we found that neurons retained their rhythmic activity—but cell position was decoupled from the normal phasing pattern underlying flexion and extension. These findings suggest a hierarchical basis of motor circuit formation that relies on increasingly stringent matching of neuronal identity and position

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism

Rostrocaudal Diversification of Spinal Neurons Confers Segment-Specific Spinal Network Architectures

The spinal cord represents the final stage of generating motor behaviors, where descending commands or sensory inputs must be transformed into behaviorally-relevant pattern of motor neuron activity. Networks along the rostrocaudal axis of the spinal cord regulate diverse motor behaviors such as respiration, forelimb, trunk, and hindlimb movements, mediated by stringent innervations of motor neurons to muscle fibers. However, how the network properties of the central nervous system enable these diverse motor outputs remains elusive. To address this question, we set out to investigate whether a cardinal class of spinal cord neurons are diversified in diffeernt spinal segment. This dissertation describes a series of original work that aims to elucidate the diversification of spinal neurons in different segments of the spinal cord. The first chapter is an introduction into the developmental processes of spinal cord development. The second chapter proceeds from this review to explore whether spinal neurons are further diversified in different spinal segments to underlie distinct network operation and motor outputs. In particular, we studied V2a interneurons as a model to address this question. Using viral tracing and RNA-sequencing, we uncovered how V2a interneurons exhibit distinct anatomical connectivity schemes and distinct genetic signatures in forelimb regulating- and hidnlimb regulating-segments of the spinal cord. It is my hope that our studies establish a framework of how diversification of spinal neurons along the rostrocaudal axis underlies distinct intrinsic network properties in different spinal segments that ultimately contribute to diverse motor outputs that the spinal cord regulates

Enteroendocrine cell lineages that differentially control feeding and gut motility.

Peer reviewed: TrueAcknowledgements: We thank Nancy Thornberry and Paul Richards for manuscript comments, Lin Gan (Rochester) for Atoh1-Cre mice, Chris Wright (Vanderbilt) for Ptf1a-Cre mice, Patricia Jensen (NIH) and Nicholas Plummer (NIH) for genotyping advice, members of the Liberles laboratory for experimental advice and assistance, the Boston Children’s Hospital Mouse Gene Manipulation Core (NIH P50 HD105351) for help with generating mice, the Beth Israel Deaconess Medical Center Energy Balance Core (OD028635, P30 DK034854) for help with CLAMS assays, the Harvard Medical School Nikon Imaging Center and Neurobiology Imaging Facility (NINDS P30 NS072030) for help with microscopy, the Biopolymers Facility for help with sequencing, the Immunology Flow Cytometry Core Facility for help with cell sorting, and the Harvard Medical School O2 High Performance Computer Cluster for bioinformatics support. This work was supported by the NIH (DP1AT009497 to SDL, R01DK103703 to SDL, and T32 HL007901 to JAK), and the Food Allergy Science Initiative (to SDL). MH is a fellow of Japan Society for the Promotion for Science. SDL is an investigator of the Howard Hughes Medical Institute.Funder: Food Allergy Initiative; FundRef: http://dx.doi.org/10.13039/100001419Funder: Japan Society for the Promotion of Science; FundRef: http://dx.doi.org/10.13039/501100001691Enteroendocrine cells are specialized sensory cells of the gut-brain axis that are sparsely distributed along the intestinal epithelium. The functions of enteroendocrine cells have classically been inferred by the gut hormones they release. However, individual enteroendocrine cells typically produce multiple, sometimes apparently opposing, gut hormones in combination, and some gut hormones are also produced elsewhere in the body. Here, we developed approaches involving intersectional genetics to enable selective access to enteroendocrine cells in vivo in mice. We targeted FlpO expression to the endogenous Villin1 locus (in Vil1-p2a-FlpO knock-in mice) to restrict reporter expression to intestinal epithelium. Combined use of Cre and Flp alleles effectively targeted major transcriptome-defined enteroendocrine cell lineages that produce serotonin, glucagon-like peptide 1, cholecystokinin, somatostatin, or glucose-dependent insulinotropic polypeptide. Chemogenetic activation of different enteroendocrine cell types variably impacted feeding behavior and gut motility. Defining the physiological roles of different enteroendocrine cell types provides an essential framework for understanding sensory biology of the intestine

Enteroendocrine cell lineages that differentially control feeding and gut motility.

Funder: Food Allergy InitiativeEnteroendocrine cells are specialized sensory cells of the gut-brain axis that are sparsely distributed along the intestinal epithelium. The functions of enteroendocrine cells have classically been inferred by the gut hormones they release. However, individual enteroendocrine cells typically produce multiple, sometimes apparently opposing, gut hormones in combination, and some gut hormones are also produced elsewhere in the body. Here, we developed approaches involving intersectional genetics to enable selective access to enteroendocrine cells in vivo in mice. We targeted FlpO expression to the endogenous Villin1 locus (in Vil1-p2a-FlpO knock-in mice) to restrict reporter expression to intestinal epithelium. Combined use of Cre and Flp alleles effectively targeted major transcriptome-defined enteroendocrine cell lineages that produce serotonin, glucagon-like peptide 1, cholecystokinin, somatostatin, or glucose-dependent insulinotropic polypeptide. Chemogenetic activation of different enteroendocrine cell types variably impacted feeding behavior and gut motility. Defining the physiological roles of different enteroendocrine cell types provides an essential framework for understanding sensory biology of the intestine

Microscopy ambient ionization top-down mass spectrometry reveals developmental patterning

There is immense cellular and molecular heterogeneity in biological systems. Here, we demonstrate the utility of integrating an inverted light microscope with an ambient ionization source, nanospray electrospray desorption ionization, attached to a high-resolution mass spectrometer to characterize the molecular composition of mouse spinal cords. We detected a broad range of molecules, including peptides and proteins, as well as metabolites such as lipids, sugars, and other small molecules, including S-adenosyl methionine and glutathione, through top-down MS. Top-down analysis revealed variation in the expression of Hb, including the transition from fetal to adult Hb and heterogeneity in Hb subunits consistent with the genetic diversity of the mouse models. Similarly, temporal changes to actin-sequestering proteins β-thymosins during development were observed. These results demonstrate that interfacing microscopy with ambient ionization provides the means to perform targeted in situ ambient top-down mass spectral analysis to study the pattern of proteins, lipids, and sugars in biologically heterogeneous samples

Satb2 Is Required for the Development of a Spinal Exteroceptive Microcircuit that Modulates Limb Position

Motor behaviors such as walking or withdrawing the limb from a painful stimulus rely upon integrative multimodal sensory circuitry to generate appropriate muscle activation patterns. Both the cellular components and the molecular mechanisms that instruct the assembly of the spinal sensorimotor system are poorly understood. Here we characterize the connectivity pattern of a sub-population of lamina V inhibitory sensory relay neurons marked during development by the nuclear matrix and DNA binding factor Satb2 (ISR(Satb2)). ISR(Satb2) neurons receive inputs from multiple streams of sensory information and relay their outputs to motor command layers of the spinal cord. Deletion of the Satb2 transcription factor from ISR(Satb2) neurons perturbs their cellular position, molecular profile, and pre- and post-synaptic connectivity. These alterations are accompanied by abnormal limb hyperflexion responses to mechanical and thermal stimuli and during walking. Thus, Satb2 is a genetic determinant that mediates proper circuit development in a core sensory-to-motor spinal network

Stimulating intestinal GIP release reduces food intake and body weight in mice.

OBJECTIVE: Glucose dependent insulinotropic polypeptide (GIP) is well established as an incretin hormone, boosting glucose-dependent insulin secretion. However, whilst anorectic actions of its sister-incretin glucagon-like peptide-1 (GLP-1) are well established, a physiological role for GIP in appetite regulation is controversial, despite the superior weight loss seen in preclinical models and humans with GLP-1/GIP dual receptor agonists compared with GLP-1R agonism alone. METHODS: We generated a mouse model in which GIP expressing K-cells can be activated through hM3Dq Designer Receptor Activated by Designer Drugs (DREADD, GIP-Dq) to explore physiological actions of intestinally-released GIP. RESULTS: In lean mice, Dq-stimulation of GIP expressing cells increased plasma GIP to levels similar to those found postprandially. The increase in GIP was associated with improved glucose tolerance, as expected, but also triggered an unexpected robust inhibition of food intake. Validating that this represented a response to intestinally-released GIP, the suppression of food intake was prevented by injecting mice peripherally or centrally with antagonistic GIPR-antibodies, and was reproduced in an intersectional model utilising Gip-Cre/Villin-Flp to limit Dq transgene expression to K-cells in the intestinal epithelium. The effects of GIP cell activation were maintained in diet induced obese mice, in which chronic K-cell activation reduced food intake and attenuated body weight gain. CONCLUSIONS: These studies establish a physiological gut-brain GIP-axis regulating food intake in mice, adding to the multi-faceted metabolic effects of GIP which need to be taken into account when developing GIPR-targeted therapies for obesity and diabetes

Stimulating intestinal GIP release reduces food intake and body weight in mice

Objective: Glucose dependent insulinotropic polypeptide (GIP) is well established as an incretin hormone, boosting glucose-dependent insulin secretion. However, whilst anorectic actions of its sister-incretin glucagon-like peptide-1 (GLP-1) are well established, a physiological role for GIP in appetite regulation is controversial, despite the superior weight loss seen in preclinical models and humans with GLP-1/GIP dual receptor agonists compared with GLP-1R agonism alone. Methods: We generated a mouse model in which GIP expressing K-cells can be activated through hM3Dq Designer Receptor Activated by Designer Drugs (DREADD, GIP-Dq) to explore physiological actions of intestinally-released GIP. Results: In lean mice, Dq-stimulation of GIP expressing cells increased plasma GIP to levels similar to those found postprandially. The increase in GIP was associated with improved glucose tolerance, as expected, but also triggered an unexpected robust inhibition of food intake. Validating that this represented a response to intestinally-released GIP, the suppression of food intake was prevented by injecting mice peripherally or centrally with antagonistic GIPR-antibodies, and was reproduced in an intersectional model utilising Gip-Cre/Villin-Flp to limit Dq transgene expression to K-cells in the intestinal epithelium. The effects of GIP cell activation were maintained in diet induced obese mice, in which chronic K-cell activation reduced food intake and attenuated body weight gain. Conclusions: These studies establish a physiological gut-brain GIP-axis regulating food intake in mice, adding to the multi-faceted metabolic effects of GIP which need to be taken into account when developing GIPR-targeted therapies for obesity and diabetes

Satb2 Is Required for the Development of a Spinal Exteroceptive Microcircuit that Modulates Limb Position

Motor behaviors such as walking or withdrawing the limb from a painful stimulus rely upon integrative multimodal sensory circuitry to generate appropriate muscle activation patterns. Both the cellular components and the molecular mechanisms that instruct the assembly of the spinal sensorimotor system are poorly understood. Here we characterize the connectivity pattern of a sub-population of lamina V inhibitory sensory relay neurons marked during development by the nuclear matrix and DNA binding factor Satb2 (ISR(Satb2)). ISR(Satb2) neurons receive inputs from multiple streams of sensory information and relay their outputs to motor command layers of the spinal cord. Deletion of the Satb2 transcription factor from ISR(Satb2) neurons perturbs their cellular position, molecular profile, and pre- and post-synaptic connectivity. These alterations are accompanied by abnormal limb hyperflexion responses to mechanical and thermal stimuli and during walking. Thus, Satb2 is a genetic determinant that mediates proper circuit development in a core sensory-to-motor spinal network