RESUMO
We investigated whether deep reinforcement learning (deep RL) is able to synthesize sophisticated and safe movement skills for a low-cost, miniature humanoid robot that can be composed into complex behavioral strategies. We used deep RL to train a humanoid robot to play a simplified one-versus-one soccer game. The resulting agent exhibits robust and dynamic movement skills, such as rapid fall recovery, walking, turning, and kicking, and it transitions between them in a smooth and efficient manner. It also learned to anticipate ball movements and block opponent shots. The agent's tactical behavior adapts to specific game contexts in a way that would be impractical to manually design. Our agent was trained in simulation and transferred to real robots zero-shot. A combination of sufficiently high-frequency control, targeted dynamics randomization, and perturbations during training enabled good-quality transfer. In experiments, the agent walked 181% faster, turned 302% faster, took 63% less time to get up, and kicked a ball 34% faster than a scripted baseline.
Assuntos
Robótica , Futebol , Robótica/métodos , Aprendizagem , Caminhada , Simulação por ComputadorRESUMO
Nuclear fusion using magnetic confinement, in particular in the tokamak configuration, is a promising path towards sustainable energy. A core challenge is to shape and maintain a high-temperature plasma within the tokamak vessel. This requires high-dimensional, high-frequency, closed-loop control using magnetic actuator coils, further complicated by the diverse requirements across a wide range of plasma configurations. In this work, we introduce a previously undescribed architecture for tokamak magnetic controller design that autonomously learns to command the full set of control coils. This architecture meets control objectives specified at a high level, at the same time satisfying physical and operational constraints. This approach has unprecedented flexibility and generality in problem specification and yields a notable reduction in design effort to produce new plasma configurations. We successfully produce and control a diverse set of plasma configurations on the Tokamak à Configuration Variable1,2, including elongated, conventional shapes, as well as advanced configurations, such as negative triangularity and 'snowflake' configurations. Our approach achieves accurate tracking of the location, current and shape for these configurations. We also demonstrate sustained 'droplets' on TCV, in which two separate plasmas are maintained simultaneously within the vessel. This represents a notable advance for tokamak feedback control, showing the potential of reinforcement learning to accelerate research in the fusion domain, and is one of the most challenging real-world systems to which reinforcement learning has been applied.
RESUMO
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.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43G298S ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases.
Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Neurais/citologia , Neurônios/citologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Mutação , Neurogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Direct or indirect impairment of breathing in humans by diseases or environmental factors can either cause long-term disability and pain, or can ultimately result in death. Automatic respiratory centers in the brainstem control the highly structured process of breathing and signal to a specialized group of motor neurons in the cervical spinal cord that constitute the phrenic nerves. In mammals, the thoracic diaphragm separates the thorax from the abdomen and adopts the function of the primary respiratory musculature. Faithful innervation by the phrenic nerves is a prerequisite for correct functionality of this highly specialized musculature and thus, ultimately, the viability of the entire organism.To analyze the effects of diseases and genetic defects responsible for deleterious or lethal respiratory phenotypes, accurate imaging of respiratory innervation during embryonic development, e.g., in genetically modified mouse models enables the characterization of specific marker genes and pathways that underlie appropriate wiring of the diaphragm. Among the different available immunostaining techniques, wholemount staining methods provide the advantage of clear and faithful three-dimensional information about the location of the antigens of interest. In comparison to routine histological techniques, however, the researcher has to deal with technical challenges, such as antibody penetration, the stability and availability of the antigen, and clearing of the relevant tissue, and the need to be equipped with state-of-the-art microscope equipment.In this methodological chapter, we explain and share our expertise concerning wholemount processing of mouse embryos and thoracic diaphragms for the analysis of mammalian respiratory innervation.
Assuntos
Diafragma/inervação , Coloração e Rotulagem/métodos , Tórax/inervação , Animais , Fasciculação Axônica , Orientação de Axônios , Moléculas de Adesão Celular/metabolismo , Diafragma/química , Embrião de Mamíferos , Corantes Fluorescentes/química , Camundongos , Neurônios Motores/metabolismo , Desenvolvimento Muscular , Imagem Óptica , Nervo Frênico/crescimento & desenvolvimento , Tórax/químicaRESUMO
How are precise connectivity to peripheral targets and corresponding sensory-motor networks established during developmental innervation of the vertebrate extremities? The formation of functional sensory-motor circuits requires highly appropriate temporal and spatial regulation of axon growth which is achieved through the combination of different molecular mechanisms such as communication between heterotypic fiber systems, axon-environment, or axon-glia interactions that ensure proper fasciculation and accurate pathfinding to distal targets. Family members of the class 3 semaphorins and their cognate receptors, the neuropilins, were shown to govern various events during wiring of central and peripheral circuits, with mice lacking Sema3-Npn signaling showing deficits in timing of growth, selective fasciculation, guidance fidelity, and coupling of sensory axon growth to motor axons at developmental time points. Given the accuracy with which these processes have to interact in a stepwise manner, deficiency of the smallest cog in the wheel may impact severely on the faithful establishment and functionality of peripheral circuitries, ultimately leading to behavioral impairments or even cause the death of the animal. Reliable quantitative analyses of sensory-motor fasciculation, extension, and guidance of axons to their cognate target muscles and the skin during development, but also assessment of physiological and behavioral consequences at adult age, are therefore a necessity to extend our understanding of the molecular mechanisms of peripheral circuit formation. In this chapter we provide a detailed methodology to characterize class 3 semaphorin-mediated effects on peripheral sensory and motor axon pathfinding and connectivity during embryonic development.
Assuntos
Axônios/fisiologia , Desenvolvimento Embrionário , Semaforinas/fisiologia , Animais , Orientação de Axônios , Feminino , Camundongos , GravidezRESUMO
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.
Assuntos
Orientação de Axônios/fisiologia , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios GABAérgicos/fisiologia , Neuropilina-1/fisiologia , Animais , Células CHO , Moléculas de Adesão Celular/metabolismo , Técnicas de Cocultura , Cricetulus , Humanos , Fatores de Crescimento Neural/metabolismo , Neurogênese/fisiologia , Células de Purkinje/fisiologia , Semaforina-3A/fisiologia , Sinapses/fisiologiaRESUMO
Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the viability of the entire organism. Systemic impairment of Sema3A-Npn-1 (Npn-1 is also known as NRP1) signaling causes excessive branching of phrenic nerves in the diaphragm and into the central tendon region, where the majority of misguided axons innervate ectopic musculature. To elucidate whether these ectopic muscles are a result of misguidance of myoblast precursors due to the loss of Sema3A-Npn-1 signaling, we conditionally ablated Npn-1 in somatic motor neurons, which led to a similar phenotype of phrenic nerve defasciculation and, intriguingly, also formation of innervated ectopic muscles. We therefore hypothesize that ectopic myocyte fusion is caused by additional factors released by misprojecting growth cones. Slit2 and its Robo receptors are expressed by phrenic motor axons and migrating myoblasts, respectively, during innervation of the diaphragm. In vitro analyses revealed a chemoattractant effect of Slit2 on primary diaphragm myoblasts. Thus, we postulate that factors released by motor neuron growth cones have an influence on the migration properties of myoblasts during establishment of the diaphragm.
Assuntos
Diafragma/inervação , Diafragma/metabolismo , Desenvolvimento Muscular , Neuropilina-1/metabolismo , Semaforina-3A/metabolismo , Transdução de Sinais , Animais , Fasciculação Axônica , Diafragma/embriologia , Embrião de Mamíferos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Neurônios Motores/metabolismo , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nervo Frênico/metabolismo , Receptores Imunológicos/metabolismo , Células-Tronco/metabolismo , Tendões/metabolismo , Proteínas RoundaboutRESUMO
During development of the CNS, stem and progenitor cell proliferation, cell fate designation, and patterning decisions are tightly regulated by interdependent networks of key transcriptional regulators. In a genetic approach we analyzed divergent functionality of the PAI and RED sub-domains of the Pax6 Paired domain (PD) during progenitor zone formation, motor and interneuron development, and peripheral connectivity at distinct levels within the neural tube: within the hindbrain, mutation of the PAI sub-domain severely affected patterning of the p3 and pMN domains and establishment of the corresponding motor neurons. Exit point designation of hypoglossal axons was disturbed in embryos harboring either mutations in the PD sub-domains or containing a functional Pax6 Null allele. At brachial spinal levels, we propose a selective involvement of the PAI sub-domain during patterning of ventral p2 and pMN domains, critically disturbing generation of specific motor neuron subtypes and increasing V2 interneuron numbers. Our findings present a novel aspect of how Pax6 not only utilizes its modular structure to perform distinct functions via its paired and homeodomain. Individual sub-domains can exert distinct functions, generating a new level of complexity for transcriptional regulation by one single transcription factor not only in dorso-ventral, but also rostro-caudal neural tube patterning.
Assuntos
Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Tubo Neural/embriologia , Fatores de Transcrição Box Pareados/genética , Sistema Nervoso Periférico/embriologia , Proteínas Repressoras/genética , Alelos , Animais , Axônios/metabolismo , Axônios/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas do Olho/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Interneurônios/metabolismo , Camundongos , Neurônios Motores/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/fisiologia , Fenótipo , Estrutura Terciária de Proteína , Proteínas Repressoras/fisiologia , Rombencéfalo/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
The correct wiring of neuronal circuits is of crucial importance for precise neuromuscular functionality. Therefore, guidance cues provide tight spatiotemporal control of axon growth and guidance. Mice lacking the guidance cue Semaphorin 3F (Sema3F) display very specific axon wiring deficits of motor neurons in the medial aspect of the lateral motor column (LMCm). While these deficits have been investigated extensively during embryonic development, it remained unclear how Sema3F mutant mice cope with these errors postnatally. We therefore investigated whether these animals provide a suitable model for the exploration of adaptive plasticity in a system of miswired neuronal circuitry. We show that the embryonically developed wiring deficits in Sema3F mutants persist until adulthood. As a consequence, these mutants display impairments in motor coordination that improve during normal postnatal development, but never reach wildtype levels. These improvements in motor coordination were boosted to wildtype levels by housing the animals in an enriched environment starting at birth. In contrast, a delayed start of enriched environment housing, at 4 weeks after birth, did not similarly affect motor performance of Sema3F mutants. These results, which are corroborated by neuroanatomical analyses, suggest a critical period for adaptive plasticity in neuromuscular circuitry. Interestingly, the formation of perineuronal nets, which are known to close the critical period for plastic changes in other systems, was not altered between the different housing groups. However, we found significant changes in the number of excitatory synapses on limb innervating motor neurons. Thus, we propose that during the early postnatal phase, when perineuronal nets have not yet been formed around spinal motor neurons, housing in enriched environment conditions induces adaptive plasticity in the motor system by the formation of additional synaptic contacts, in order to compensate for coordination deficits.
Assuntos
Axônios/patologia , Proteínas de Membrana/fisiologia , Neurônios Motores/patologia , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Comportamento Animal , Toxina da Cólera/química , Eletromiografia , Marcha , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Destreza Motora , Mutação , Proteínas do Tecido Nervoso/genética , Medula Espinal/patologia , Sinapses/fisiologia , Fatores de TempoRESUMO
Engrailed-1 (En1) is expressed in the ventral ectoderm of the developing limb where it plays an instructive role in the dorsal-ventral patterning of the forelimb. Besides its well-described role as a transcription factor in regulating gene expression through its DNA-binding domain, En1 may also be secreted to form an extracellular gradient, and directly impact on the formation of the retinotectal map. We show here that absence of En1 causes mispatterning of the forelimb and thus defects in the dorsal-ventral pathfinding choice of motor axons in vivo. In addition, En1 but not En2 also has a direct and specific repulsive effect on motor axons of the lateral aspect of the lateral motor column (LMC) but not on medial LMC projections. Moreover, an ectopic dorsal source of En1 pushes lateral LMC axons to the ventral limb in vivo. Thus, En1 controls the establishment of limb innervation through two distinct molecular mechanisms.
Assuntos
Membro Anterior/inervação , Proteínas de Homeodomínio/metabolismo , Animais , Axônios/metabolismo , Embrião de Galinha , Galinhas , Ectoderma/metabolismo , Embrião de Mamíferos/metabolismo , Membro Anterior/metabolismo , Membro Anterior/patologia , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Camundongos , Neurônios Motores/química , Neurônios Motores/metabolismo , Mutação , Receptor EphA4/metabolismoRESUMO
The correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1(cond) and Npn1(Sema-) to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre(+);Npn1(cond-/-) mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1(Sema-) mutants appeared anatomically normal. Furthermore, Olig2-Cre(+);Npn1(cond) mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1(Sema-) mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre(+);Npn1(cond-/-) mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1(Sema-) mutants to compensate for their developmental deficits.
Assuntos
Neurônios Motores/metabolismo , Neuropilina-1/metabolismo , Semaforina-3A/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/fisiologia , Axônios/ultraestrutura , Peso Corporal/genética , Peso Corporal/fisiologia , Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , Osso e Ossos/embriologia , Osso e Ossos/inervação , Osso e Ossos/metabolismo , Membro Anterior/embriologia , Membro Anterior/crescimento & desenvolvimento , Membro Anterior/inervação , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Atividade Motora/genética , Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/inervação , Fibras Nervosas/metabolismo , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Neuropilina-1/genética , Semaforina-3A/genética , Transdução de Sinais/genética , Fatores de TempoRESUMO
Antigen-specific tolerance induction is a desired therapy for uveitis patients. Our relapsing-remitting rat model of experimental autoimmune uveitis (EAU) induced with IRBP peptide R14 enables us to test the effect of oral tolerance on the prevention of relapsing uveitis. We investigated several peptides overlapping the sequence of R14 for prevention and different doses of R14 for therapy to determine the tolerogenic epitope and the most effective therapeutic regimen for uveitis. Lewis rats were immunized with R14-CFA to induce EAU. Oral tolerance was induced prior to immunization (prevention) or after onset of EAU to prevent relapses (therapy). Therapeutic feeding was performed with high and/or low doses of oral antigen for clonal deletion of effector and induction of regulatory T cells. Uveitis was determined clinically and histologically; mesenteric lymph node (mLN) cells of tolerized rats were tested for surface markers, cytokines and Foxp3 expression. Preventive feeding of R14 and its major epitope R16, but none of the overlapping peptides significantly suppressed EAU and also prevented relapses, irrespective of their pathogenicity. Therapeutic feeding with R14 dramatically reduced relapses, while only the consecutive feeding of high and low-dose R14 had an ameliorating effect on the first course of disease. IL-10-producing T cells from mLN decreased after oral tolerization, and with R14-stimulation in vitro the TCRαß+/Foxp3+ population increased in the low-dose fed group. No mLN population could be clearly assigned to successful oral tolerance induction during active autoimmune uveitis.
Assuntos
Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Tolerância Imunológica/imunologia , Imunoterapia , Uveíte/imunologia , Uveíte/terapia , Administração Oral , Sequência de Aminoácidos , Animais , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/patologia , Relação Dose-Resposta Imunológica , Citometria de Fluxo , Imunofluorescência , Imunização , Linfonodos/patologia , Mesentério/patologia , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/imunologia , Ratos Endogâmicos Lew , Recidiva , Uveíte/induzido quimicamente , Uveíte/patologiaRESUMO
Motor neurons in the vertebrate spinal cord are stereotypically organized along the rostro-caudal axis in discrete columns that specifically innervate peripheral muscle domains. Originating from the same progenitor domain, the generation of spinal motor neurons is orchestrated by a spatially and temporally tightly regulated set of secreted molecules and transcription factors such as retinoic acid and the Lim homeodomain transcription factors Isl1 and Lhx1. However, the molecular interactions between these factors remained unclear. In this study we examined the role of the microRNA 9 (miR-9) in the specification of spinal motor neurons and identified Onecut1 (OC1) as one of its targets. miR-9 and OC1 are expressed in mutually exclusive patterns in the developing chick spinal cord, with high OC1 levels in early-born motor neurons and high miR-9 levels in late-born motor neurons. miR-9 efficiently represses OC1 expression in vitro and in vivo. Overexpression of miR-9 leads to an increase in late-born neurons, while miR-9 loss-of-function induces additional OC1(+) motor neurons that display a transcriptional profile typical of early-born neurons. These results demonstrate that regulation of OC1 by miR-9 is a crucial step in the specification of spinal motor neurons and support a model in which miR-9 expression in late-born LMCl neurons downregulates Isl1 expression through inhibition of OC1. In conclusion, our study contributes essential factors to the molecular network specifying spinal motor neurons and emphasizes the importance of microRNAs as key players in the generation of neuronal diversity.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MicroRNAs/metabolismo , Neurônios Motores/fisiologia , Fatores de Transcrição Onecut/metabolismo , Medula Espinal/embriologia , Análise de Variância , Animais , Sequência de Bases , Embrião de Galinha , Eletroporação , Fluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Hibridização In Situ , Luciferases , MicroRNAs/genética , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Fatores de Transcrição Onecut/genéticaRESUMO
BACKGROUND: GABAergic interneurons regulate the balance and dynamics of neural circuits, in part, by elaborating their strategically placed axon branches that innervate specific cellular and subcellular targets. However, the molecular mechanisms that regulate target-directed GABAergic axon branching are not well understood. RESULTS: Here we show that the secreted axon guidance molecule, SEMA3A, expressed locally by Purkinje cells, regulates cerebellar basket cell axon branching through its cognate receptor Neuropilin-1 (NRP1). SEMA3A was specifically localized and enriched in the Purkinje cell layer (PCL). In sema3A(-/-) and nrp1(sema-/sema-) mice lacking SEMA3A-binding domains, basket axon branching in PCL was reduced. We demonstrate that SEMA3A-induced axon branching was dependent on local recruitment of soluble guanylyl cyclase (sGC) to the plasma membrane of basket cells, and sGC subcellular trafficking was regulated by the Src kinase FYN. In fyn-deficient mice, basket axon terminal branching was reduced in PCL, but not in the molecular layer. CONCLUSIONS: These results demonstrate a critical role of local SEMA3A signaling in layer-specific axonal branching, which contributes to target innervation.
Assuntos
Cerebelo/citologia , Interneurônios/citologia , Semaforina-3A/metabolismo , Transdução de Sinais , Animais , Axônios , Cerebelo/metabolismo , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Ácido gama-Aminobutírico/metabolismoRESUMO
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.
Assuntos
Fator 6 Nuclear de Hepatócito/fisiologia , Neurônios Motores/fisiologia , Junção Neuromuscular/crescimento & desenvolvimento , Animais , Sequência de Bases , Técnicas de Cocultura , Primers do DNA , Imunofluorescência , Hibridização In Situ , Locomoção , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Reação em Cadeia da PolimeraseAssuntos
Relações Enfermeiro-Paciente , Cateterismo Urinário/enfermagem , Cateterismo Urinário/psicologia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Meningomielocele/enfermagem , Meningomielocele/psicologia , Pais/educação , Educação de Pacientes como Assunto/métodos , Autocuidado/psicologia , Bexiga Urinaria Neurogênica/enfermagem , Bexiga Urinaria Neurogênica/psicologia , Infecções Urinárias/enfermagem , Infecções Urinárias/prevenção & controle , Infecções Urinárias/psicologiaRESUMO
During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas com Homeodomínio LIM/metabolismo , Neurônios Motores/fisiologia , Fatores de Transcrição Onecut/metabolismo , Medula Espinal/citologia , Fatores de Transcrição/metabolismo , Animais , Embrião de Galinha , Imunoprecipitação da Cromatina , Primers do DNA/genética , Eletroporação , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , CamundongosRESUMO
During development, fibroblast growth factors (FGF) are essential for early patterning events along the anterior-posterior axis, conferring positional identity to spinal motor neurons by activation of different Hox codes. In the periphery, signaling through one of four fibroblast growth factor receptors supports the development of the skeleton, as well as induction and maintenance of extremities. In previous studies, FGF receptor 2 (FGFR2) was found to interact with axon bound molecules involved in axon fasciculation and extension, thus rendering this receptor an interesting candidate for the promotion of proper peripheral innervation. However, while the involvement of FGFR2 in limb bud induction has been extensively studied, its role during axon elongation and formation of distinct nervous projections has not been addressed so far. We show here that motor neurons in the spinal cord express FGFR2 and other family members during the establishment of motor connections to the forelimb and axial musculature. Employing a conditional genetic approach to selectively ablate FGFR2 from motor neurons we found that the patterning of motor columns and the expression patterns of other FGF receptors and Sema3A in the motor columns of mutant embryos are not altered. In the absence of FGFR2 signaling, pathfinding of motor axons is intact, and also fasciculation, distal advancement of motor nerves and gross morphology and positioning of axonal projections are not altered. Our findings therefore show that FGFR2 is not required cell-autonomously in motor neurons during the formation of initial motor projections towards limb and axial musculature.
Assuntos
Axônios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Medula Espinal/embriologia , Animais , Fasciculação/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genótipo , Camundongos , Microscopia de Fluorescência/métodos , Modelos Genéticos , Semaforina-3A/metabolismo , Transdução de SinaisRESUMO
Interaction of the axon guidance receptor Neuropilin-1 (Npn-1) with its repulsive ligand Semaphorin 3A (Sema3A) is crucial for guidance decisions, fasciculation, timing of growth and axon-axon interactions of sensory and motor projections in the embryonic limb. At cranial levels, Npn-1 is expressed in motor neurons and sensory ganglia and loss of Sema3A-Npn-1 signaling leads to defasciculation of the superficial projections to the head and neck. The molecular mechanisms that govern the initial fasciculation and growth of the purely motor projections of the hypoglossal and abducens nerves in general, and the role of Npn-1 during these events in particular are, however, not well understood. We show here that selective removal of Npn-1 from somatic motor neurons impairs initial fasciculation and assembly of hypoglossal rootlets and leads to reduced numbers of abducens and hypoglossal fibers. Ablation of Npn-1 specifically from cranial neural crest and placodally derived sensory tissues recapitulates the distal defasciculation of mixed sensory-motor nerves of trigeminal, facial, glossopharyngeal and vagal projections, which was observed in Npn-1(-/-) and Npn-1(Sema-) mutants. Surprisingly, the assembly and fasciculation of the purely motor hypoglossal nerve are also impaired and the number of Schwann cells migrating along the defasciculated axonal projections is reduced. These findings are corroborated by partial genetic elimination of cranial neural crest and embryonic placodes, where loss of Schwann cell precursors leads to aberrant growth patterns of the hypoglossal nerve. Interestingly, rostral turning of hypoglossal axons is not perturbed in any of the investigated genotypes. Thus, initial hypoglossal nerve assembly and fasciculation, but not later guidance decisions depend on Npn-1 expression and axon-Schwann cell interactions.