RESUMO
Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses.
Assuntos
Proteínas 14-3-3/metabolismo , Axônios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/metabolismo , Neurônios/citologia , Traumatismos da Medula Espinal/patologia , Proteínas 14-3-3/genética , Aminoácidos , Análise de Variância , Animais , Axônios/efeitos dos fármacos , Proteínas de Bactérias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carbazóis/farmacologia , Células Cultivadas , Quimiotaxia , Galinhas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroporação , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Neurônios/classificação , Neurônios/metabolismo , Piperazinas/farmacologia , Gravidez , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pirazóis/farmacologia , Pirróis/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Simplexvirus/genética , Fatores de Tempo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteína Gli2 com Dedos de Zinco , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The migration of glial chains along the nerve entails directional and coordinated movement. Despite its importance in the formation of the nervous system, this process remains poorly understood, because of the difficulty of manipulating identified cells. Using confocal time-lapse and cell ablation in the whole animal, we provide direct evidence for a discrete number of Drosophila peripheral glial cells acting as pioneers and guiding the rest of the migratory chain. These cells are in direct contact with several follower cells through a very long and stable cytoplasmic extension. The presence of pioneer cells and homotypic interactions at the tip of the chain allows coordinated movement and the formation of a continuous sheath around the nerve. These in vivo data open novel perspectives for understanding the cellular bases of vertebrate glial migration in physiological and pathological conditions.
Assuntos
Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Comunicação Celular/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Fluorescência Verde/biossíntese , Temperatura Alta , Terapia a Laser/métodos , Microscopia Confocal/métodos , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52 degrees mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.