RESUMO
Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.
Assuntos
Encéfalo , Microglia , Axônios , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Macrófagos/fisiologia , Microglia/patologia , MorfogêneseRESUMO
In response to repulsive cues, axonal growth cones can quickly retract. This requires the prompt activity of contractile actomyosin, which is formed by the non-muscle myosin II (NMII) bound to actin filaments. NMII is a molecular motor that provides the necessary mechanical force at the expense of ATP. Here, we report that this process is energetically coupled to glycolysis and is independent of cellular ATP levels. Induction of axonal retraction requires simultaneous generation of ATP by glycolysis, as shown by chemical inhibition and genetic knock-down of GAPDH. Co-immunoprecipitation and proximal-ligation assay showed that actomyosin associates with ATP-generating glycolytic enzymes and that this association is strongly enhanced during retraction. Using microfluidics, we confirmed that the energetic coupling between glycolysis and actomyosin necessary for axonal retraction is localized to the growth cone and near axonal shaft. These results indicate a tight coupling between on-demand energy production by glycolysis and energy consumption by actomyosin contraction suggesting a function of glycolysis in axonal guidance.
Assuntos
Actomiosina , Axônios , Glicólise , Citoesqueleto de Actina , Actomiosina/metabolismo , Trifosfato de Adenosina , Axônios/fisiologiaRESUMO
BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.
Assuntos
Axônios/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Animais , Axônios/classificação , Axônios/fisiologia , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hipocampo/citologia , Humanos , Laminina/farmacologia , Camundongos , Técnicas Analíticas Microfluídicas , Proteínas do Tecido Nervoso/administração & dosagem , Netrina-1 , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Tálamo/citologiaRESUMO
Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.
Assuntos
Córtex Cerebral/fisiologia , Vias Neurais/fisiologia , Tálamo/fisiologia , Fatores Etários , Animais , Axônios/fisiologia , Padronização Corporal/genética , Calbindina 2 , Córtex Cerebral/citologia , Contactina 2/metabolismo , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular , Complexo Antígeno L1 Leucocitário/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Fatores do Domínio POU/genética , Proteínas Repressoras/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Semaforinas , Proteínas com Domínio T , Tálamo/citologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Wnt3A/genética , Proteínas tau/genéticaRESUMO
How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.