RESUMO
Precise neuronal wiring is critical for the function of the nervous system and is ultimately determined at the level of individual synapses. Neurons integrate various intrinsic and extrinsic cues to form synapses onto their correct targets in a stereotyped manner. In the past decades, the nervous system of nematode (Caenorhabditis elegans) has provided the genetic platform to reveal the genetic and molecular mechanisms of synapse formation and specificity. In this review, we will summarize the recent discoveries in synapse formation and specificity in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios/metabolismo , Sinapses/fisiologia , Animais , Transdução de SinaisRESUMO
The auxin-inducible degron (AID) provides reversible, spatiotemporal control for the knockdown of target proteins. Here, we present a protocol for AID-mediated protein knockdown in Caenorhabditis elegans. We describe steps for generating the knock-in mutants using two CRISPR-Cas9 genome editing techniques and preparing the auxin-containing nematode growth media (NGM) plates. We also detail AID-mediated spatiotemporal protein knockdown. For complete details on the use and execution of this protocol, please refer to Kurashina et al. (2021).1.
Assuntos
Caenorhabditis elegans , Ácidos Indolacéticos , Animais , Caenorhabditis elegans/genética , Ácidos Indolacéticos/farmacologia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodosRESUMO
Neuronal cell fate determinants establish the identities of neurons by controlling gene expression to regulate neuronal morphology and synaptic connectivity. However, it is not understood if neuronal cell fate determinants have postmitotic functions in synapse pattern formation. Here we identify a novel role for UNC-4 homeobox protein and its corepressor UNC-37/Groucho, in tiled synaptic patterning of the cholinergic motor neurons in Caenorhabditis elegans. We show that unc-4 is not required during neurogenesis but is required in the postmitotic neurons for proper synapse patterning. In contrast, unc-37 is required in both developing and postmitotic neurons. The synaptic tiling defects of unc-4 mutants are suppressed by bar-1/ß-catenin mutation, which positively regulates the expression of ceh-12/HB9. Ectopic ceh-12 expression partly underlies the synaptic tiling defects of unc-4 and unc-37 mutants. Our results reveal a novel postmitotic role of neuronal cell fate determinants in synapse pattern formation through inhibiting the canonical Wnt signaling pathway.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Correpressoras/genética , Proteínas de Homeodomínio/metabolismo , Mitose , Neurônios/fisiologia , Sinapses/fisiologia , Fatores de Transcrição/metabolismoRESUMO
During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.