The C. elegans genome encodes two Notch receptors, GLP-1 and LIN-12 (Greenwald and Kovall, 2013). These receptors function in multiple tissues to regulate development, behavior, and reproduction. GLP-1 controls cell fate decisions in the germline (Kimble and Seidel, 2013) and early embryo (Priess, 2005). LIN-12 controls development of the vulva (Greenwald, 2005; Sternberg, 2005). These receptors also act outside the context of development in post-mitotic neurons to regulate chemosensation (Singh et al., 2011), locomotion (Chao et al., 2005), and life-history decisions (Ouellet et al., 2008). Additional processes regulated by Notch signaling include oocyte growth (Nadarajan et al., 2009) and ovulation (McGovern et al., 2018). Understanding the many functions of Notch signaling therefore requires a global picture of Notch receptor expression.Previous efforts to visualize Notch receptor expression have focused on
lin-12 expression in the soma and
glp-1 expression in the germline and early embryo. LIN-12 has been visualized successfully using LIN-12::GFP fusion proteins introduced via traditional, multi-copy transgenic techniques (e.g. Levitan and Greenwald, 1998; Sarov et al., 2012). Such techniques have been difficult for visualizing
glp-1 expression in the germline, until recently (Cinquin et al., 2015; Gutnik et al., 2018), due to germline silencing of multi-copy transgenes (Kelly et al., 1997; Merritt and Seydoux, 2010). Visualizing GLP-1 expression in the germline has therefore relied on antibody staining, primarily using an antibody raised against the GLP-1 extra-cellular domain (Crittenden et al., 1994). This antibody reveals easily detectable GLP-1 protein at the plasma membrane in the distal germline and early embryos (Crittenden et al., 1994; Evans et al., 1994), but it does not allow visualization of the GLP-1 nuclear intracellular domain (NICD), which moves into the nucleus upon receptor activation. This antibody is therefore not useful for identifying cells that have directly received active GLP-1 signaling. To overcome this limitation, and to broaden the toolkit for visualizing
glp-1 expression, we created five strains expressing tagged
glp-1 alleles, including transgenes and CRISPR tags at the endogenous locus. We report here the expression patterns of the tagged
glp-1 alleles, focusing on the adult gonad.We created five tagged
glp-1 alleles, each expressing GLP-1 protein tagged with one of the following tags: sfGFP (~27 kDa), Halotag (~33 kDa), 4xV5 (~7 kDa), 3xOLLAS (~4.4 kDa), or 6xMyc6xHis (~8 kDa). The 6xMyc6xHis tag was placed at the C-terminus of
glp-1, downstream of the GLP-1 PEST domain (Figure 1A). The remaining tags were placed C-terminal to the GLP-1 Ankyrin repeats but upstream of the PEST domain (Figure 1A). These sites place the tags at or near the C-terminus of the NICD. Strains expressing
glp-1::sfGFP,
glp-1::Halotag, and
glp-1::6xMyc6xHis were created via Mos1-mediated single-copy insertion of the transgene into the genome. Strains expressing
glp-1::4xV5 and
glp-1::3xOLLAS were created by CRISPR-mediated insertion of the tag into the endogenous
glp-1 locus.To assess functionality of the tagged GLP-1 proteins, we tested each for rescue of the
glp-1 loss-of-function phenotype, which includes infertility and maternal-effect embryonic lethality. Rescue by
glp-1::sfGFP,
glp-1::Halotag, and
glp-1::6xMyc6xHis was assessed by crossing each transgene into animals carrying the null allele
glp-1(
q46) (Kodoyianni et al., 1992). Rescue by the CRISPR-generated alleles (
glp-1::4xV5 and
glp-1::3xOLLAS) was assessed by examining animals homozygous for the tagged allele. We observed strong rescue for all tagged
glp-1 alleles: Animals expressing
glp-1::4xV5 or
glp-1::3xOLLAS were all fertile (n > 50 hermaphrodites per allele) and showed no noticeable embryonic lethality; animals expressing
glp-1::6xMyc6xHis,
glp-1::sfGFP or
glp-1::Halotag were all fertile, but showed a low penetrance embryonic lethality (Figure 1B). We conclude that all five tagged
glp-1 alleles encode functional GLP-1 proteins...