Figure 1. A toolkit to simultaneously visualize endogenous Delta and Notch dynamics in vivo:(A) Simplified schematic of Notch-Delta signaling. Binding of the Delta-like ligand LAG-2 (cyan) to the Notch receptor LIN-12 (yellow) results in cleavage of the Notch intracellular domain (NICD) by
x263;-secretase (represented by scissors) and NICD translocation to the nucleus. (B-C) Schematics of the endogenously tagged
lag-2 (B) and
lin-12 (C) alleles used in this study. The
lag-2 transcriptional reporter was generated by including a self-cleaving viral peptide (P2A) and histone H2B (HIS-58) between LAG-2 and mT2 (Ahier & Jarriault, 2014). (D) Representative micrographs showing localization of the tagged LAG-2 and LIN-12 alleles in anchor cell/ventral uterine cell (AC/VU) precursors. mT2- and mNG-tagged LAG-2 form puncta on the cell membrane while the transcriptional reporter (LAG-2::P2A::H2B::mT2) is nuclear-localized. LIN-12::mNG is expressed on the cell membrane and exhibits nuclear localization of the Notch intracellular domain (NICD) upon activation. (E) Cartoon of AC/VU cell fate specification (top) as well as primary (1°) and secondary (2°) fated vulval precursor cells (VPCs) (bottom) in a wild-type animal. (F-G) Micrographs depicting LAG-2::mT2 (F) or LAG-2::P2A::H2B::mT2 (G) in concert with LIN-12::mNG reveal Delta and Notch dynamics during the AC/VU decision pre- and post-specification as well as during AC invasion. Asterisks: AC/VU precursors; arrowheads, specified AC and VU cells with colors matching panel E. (H) Endogenous
lag-2 transcriptional reporter expression marks AC and 1° VPC fates in animals harboring LIN-12::mNG::AID and a ubiquitously expressed TIR1::T2A::mCh::HIS-11 transgene. Depleting LIN-12 by treatment with auxin (1 mM K-NAA) leads to specification of a second AC and ectopic 1° VPCs that express LAG-2::P2A::H2B::mT2. Solid bracket, P6.p-derived primary-fated VPCs; dotted bracket, ectopic primary-fated VPCs. Scale bars: 5 µm.