The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs.

Subcellular target recognition in the CNS is the culmination of a multiple-step program including axon guidance, target recognition, and synaptogenesis. In cerebellum, basket cells (BCs) innervate the soma and axon initial segment (AIS) of Purkinje cells (PCs) to form the pinceau synapse, but the underlying mechanisms remain incompletely understood. Here, we demonstrate that neuropilin-1 (NRP1), a Semaphorin receptor expressed in BCs, controls both axonal guidance and subcellular target recognition. We show that loss of Semaphorin 3A function or specific deletion of NRP1 in BCs alters the stereotyped organization of BC axon and impairs pinceau synapse formation. Further, we identified NRP1 as a trans-synaptic binding partner of the cell adhesion molecule neurofascin-186 (NF186) expressed in the PC AIS during pinceau synapse formation. These findings identify a dual function of NRP1 in both axon guidance and subcellular target recognition in the construction of GABAergic circuitry.

Major depressive disorder (MDD) is a complex and debilitating illness whose etiology remains unclear. Small RNA molecules, such as micro RNAs (miRNAs) have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified miRNA expression by small RNA sequencing in the anterior cingulate cortex and habenula of individuals with MDD and psychiatrically-healthy controls. Thirty-two miRNAs showed significantly correlated expression between the two regions (False Discovery Rate < 0.05), of which four, miR-204-5p, miR-320b, miR-323a-3p, and miR-331-3p, displayed upregulated expression in MDD. We assessed the expression of predicted target genes of differentially expressed miRNAs in the brain, and found that the expression of erb-b2 receptor tyrosine kinase 4 (ERBB4), a gene encoding a neuregulin receptor, was downregulated in both regions, and was influenced by miR-323a-3p in vitro. Finally, we assessed the effects of manipulating miRNA expression in the mouse ACC on anxiety-and depressive-like behaviors. Mice in which miR-323-3p was overexpressed or knocked-down displayed increased and decreased emotionality, respectively. Additionally, these mice displayed significantly downregulated and upregulated expression of Erbb4, respectively. Overall, our findings indicate the importance of brain miRNAs in the pathology of MDD, and emphasize the involvement of miR-323a-3p and ERBB4 in this phenotype. Future studies further characterizing miR-323a-3p and neuregulin signaling in depression are warranted.

The neuromuscular junctions are the specialized synapses whereby spinal motor neurons control the contraction of skeletal muscles. The formation of the neuromuscular junctions is controlled by a complex interplay of multiple mechanisms coordinately activated in motor nerve terminals and in their target myotubes. However, the transcriptional regulators that control in motor neurons the genetic programs involved in neuromuscular junction development remain unknown. Here, we provide evidence that the Onecut transcription factor HNF-6 regulates in motor neurons the formation of the neuromuscular junctions. Indeed, adult Hnf6 mutant mice exhibit hindlimb muscle weakness and abnormal locomotion. This results from defects of hindlimb neuromuscular junctions characterized by an abnormal morphology and defective localization of the synaptic vesicle protein synaptophysin at the motor nerve terminals. These defects are consequences of altered and delayed formation of the neuromuscular junctions in newborn mutant animals. Furthermore, we show that the expression level of numerous regulators of neuromuscular junction formation, namely agrin, neuregulin-2 and TGF-ß receptor II, is downregulated in the spinal motor neurons of Hnf6 mutant newborn animals. Finally, altered formation of neuromuscular junction-like structures in a co-culture model of wildtype myotubes with mutant embryonic spinal cord slices is rescued by recombinant agrin and neuregulin, indicating that depletion in these factors contributes to defective neuromuscular junction development in the absence of HNF-6. Thus, HNF-6 controls in spinal motor neurons a genetic program that coordinates the formation of hindlimb neuromuscular junctions.

The control of all our motor outputs requires constant monitoring by proprioceptive sensory neurons (PSNs) that convey continuous muscle sensory inputs to the spinal motor network. Yet the molecular programs that control the establishment of this sensorimotor circuit remain largely unknown. The transcription factor RUNX3 is essential for the early steps of PSNs differentiation, making it difficult to study its role during later aspects of PSNs specification. Here, we conditionally inactivate Runx3 in PSNs after peripheral innervation and identify that RUNX3 is necessary for maintenance of cell identity of only a subgroup of PSNs, without discernable cell death. RUNX3 also controls the sensorimotor connection between PSNs and motor neurons at limb level, with muscle-by-muscle variable sensitivities to the loss of Runx3 that correlate with levels of RUNX3 in PSNs. Finally, we find that muscles and neurotrophin 3 signaling are necessary for maintenance of RUNX3 expression in PSNs. Hence, a transcriptional regulator that is crucial for specifying a generic PSN type identity after neurogenesis is later regulated by target muscle-derived signals to contribute to the specialized aspects of the sensorimotor connection selectivity.