Pani, Ariel M et al. (2022) MicroPubl Biol

Gibney, Theresa V, Pani, Ariel M, Goldstein, Bob, Medwig-Kinney, Taylor N, Matus, David Q

[MicroPubl Biol, 2022]

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.

Sawa, Hitoshi et al. (2019) International Worm Meeting "Reciprocal control of Wnt and Frizzled asymmetry during cell polarization."

Goldstein, Bob, Sawa, Hitoshi, Onami, Mayumi, Pani, Ariel, Matsuo, Misato

[International Worm Meeting, 2019]

Wnts are secreted signaling proteins that regulate cell polarity in many contexts. Wnt signaling between neighboring cells can instructively regulate cell polarity, but how a long-range Wnt signal can regulate polarity at a distance is unclear. To address how cells interpret Wnt gradient information to generate cell polarity, we turned to C. elegans seam cells, which polarize and asymmetrically divide in response to three Wnts: egl-20, cwn-1, and cwn-2. These Wnts are partially redundant in most seam cells despite different expression patterns, and mis-expression experiments suggested that these Wnts may be permissive signals for seam cell polarization (Yamamoto, et. al., 2011). Here, we report that Wnts also have instructive functions in seam cell polarization and that these roles may depend on an unexpected positive feedback reinforcement of asymmetric Wnt and Frizzled localizations to the posterior ends of polarizing cells. We first found that Wnts can instructively regulate seam cell polarity in the absence of the Frizzled homolog LIN-17: Wnt mis-expression in a lin-17 mutant enhances seam cell polarity reversals. This result suggests a permissive function for LIN-17 might obscure an underlying instructive role for Wnt. Based on the finding that EGL-20/Wnt localizes to the posterior ends of many seam cells (Pani and Goldstein, 2018), we hypothesized that asymmetric Wnt enrichment could explain how a long-range Wnt gradient might instruct cell polarity. To investigate the cell biological basis for instructive Wnt signaling, we quantified localization of endogenously tagged Wnts in seam cells prior to their first asymmetric division. We found that EGL-20 and CWN-1 are enriched at the posterior ends of most seam cells, including cells far from the posterior Wnt sources. We discovered that EGL-20/Wnt and MOM-5/Frizzled are reciprocally required for posterior ligand and receptor enrichment in seam cells. We propose that positive feedback can reinforce subcellular asymmetries in Wnt and Frizzled localizations, which allows cells to robustly interpret Wnt gradient information to instruct cell polarity and fates following asymmetric divisions.

Medwig-Kinney, Taylor N et al. (2022) MicroPubl Biol

Matus, David Q, Gibney, Theresa V, Pani, Ariel M, Sirota, Sydney S, Medwig-Kinney, Taylor N

[MicroPubl Biol, 2022]

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 .