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Notch/Delta signaling regulates numerous cell-cell interactions that occur during development, homeostasis, and in disease states. In many cases, the Notch/Delta pathway mediates lateral inhibition between cells to specify alternative fates. Here, we provide new tools for use in C. elegans to investigate feedback between the Notch receptor LIN-12 and the ligand LAG-2 (Delta) in vivo . We report new, endogenously tagged strains to visualize LAG-2 protein and lag-2 transcription, which we combined with endogenously tagged LIN-12 to visualize Notch and Delta dynamics over the course of a stochastic Notch-mediated cell fate decision. To validate these tools in a functional context, we demonstrated that our endogenous lag-2 transcriptional reporter was expressed in ectopic anchor and primary vulval precursor cells after auxin-mediated depletion of LIN-12. This toolkit provides new reagents for the C. elegans research community to further investigate Notch/Delta signaling mechanisms and functions for this pathway in vivo ., (Copyright: © 2022 by the authors.)

Notch signaling mediates cell-cell interactions during development and homeostasis. Methods for visualizing and manipulating Notch activity in vivo are essential to elucidate how the Notch pathway functions. Here, we provide new tools for use in C. elegans to visualize and perturb Notch signaling in vivo using endogenously tagged alleles of the Notch receptor lin-12 . Tagging the endogenous LIN-12 intracellular domain with the fluorescent protein mNeonGreen (mNG) allowed for visualization of both its membrane-localized state and translocation of the Notch intracellular domain into the nucleus upon ligand activation. LIN-12::mNG localized to the nucleus in cells where and when Notch signaling is known to be active and provided a real-time readout of Notch activity in vivo that complements existing biosensors and transcriptional reporters. We also report an allele of endogenous lin-12 that we tagged with both mNG and an auxin-inducible degron, to facilitate conditional LIN-12 protein degradation. This toolkit provides novel reagents for the C. elegans research community to investigate mechanisms of Notch signaling and its functions in vivo ., (Copyright: © 2022 by the authors.)

The helix-loop-helix transcription factor hlh-2 (E/Daughterless) has been shown to play an important role in regulating cell fate patterning, cell cycle, and basement membrane invasion in the context of the development of the C. elegans somatic gonad. Here, using CRISPR/Cas9 genome engineering, we generated a new hlh-2 allele ( hlh-2(Δ-1303-702) ) in the endogenous, GFP-tagged hlh-2 locus. This allele represents a deletion of a 601 bp region in the hlh-2 promoter that contains a putative binding site of the histone deacetylase hda-1 (HDAC) according to publicly available ChIP-sequencing data. Strikingly, we find that HLH-2 expression is virtually absent in the dorsal uterine cells of hlh-2(Δ-1303-702) animals compared to wild type controls. Levels of HLH-2 in the anchor cell and ventral uterine cells are only modestly reduced in the mutant; however, this does not seem to be functionally significant based on the lack of relevant phenotypes and expression levels of a downstream gene, NHR-67 (TLX/Tailless/NR2E1), in these cells. Taken together, these results support growing evidence that HDACs can potentially positively regulate transcription and provide a new reagent for studying hlh-2 regulation., (Copyright: © 2021 by the authors.)