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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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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/β-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., Competing Interests: MK, JW, JL, KL, AJ, KM No competing interests declared, (© 2021, Kurashina et al.)

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.