Description

The HMX/NKX MLS-2 transcription factor plays a role in the development of the postembryonic mesoderm, CEPsh glia, tube cells of the excretory system, general AWC identity, and AWC asymmetry in C. elegans (Jiang et al., 2005; Yoshimura et al., 2008; Kim et al., 2010; Abdus-Saboor et al., 2012; Hsieh et al., 2021). The expression pattern of MLS-2 protein has been previously examined by immunohistochemical staining with anti-MLS-2 antibodies, transgenes of GFP-tagged MLS-2 (GFP::MLS-2), and mls-2::GFP fosmid reporter lines (Jiang et al., 2005; Yoshimura et al., 2008; Kim et al., 2010; Abdus-Saboor et al., 2012; Walton et al., 2015; Reilly et al., 2020) (Figure 1A). It was shown that GFP::MLS-2 was expressed in the embryonic AWC lineages from automated lineage analysis and was detected transiently in AWC neurons in first-stage larvae (Kim et al., 2010; Abdus-Saboor et al., 2012; Walton et al., 2015). However, we did not detect expression of GFP::MLS-2 transgenes in AWC neurons in late embryos or the first larval stage (Figure 1B).

To determine the expression pattern of endogenous mls-2 locus in AWC, we generated mNG::SEC::mls-2 knock-in and mNG::mls-2 knock-in animals by tagging the 5’ end of endogenous mls-2 coding region with mNG::SEC or mNG using Cas9-triggered homologous recombination (Dickinson et al., 2013; Dickinson et al., 2015; Dickinson and Goldstein, 2016) (Figure 1A). The mNG::SEC::mls-2 knock-in allele is a transcriptional reporter of mls-2, since the self-excising cassette (SEC) contains transcriptional terminators. mNG::SEC::mls-2 knock-in showed diffuse mNG expression in numerous cells in the head and the M mesoblast of first-stage larvae (Figure 1C). The mNG::mls-2 knock-in allele, generated by the SEC excision of mNG::SEC::mls-2 knock-in, is a translational reporter of MLS-2 protein. mNG::mls-2 knock-in animals displayed wild-type AWC asymmetry as determined by the expression of the AWC^ON marker str-2p::TagRFP (Figure 1D), suggesting that mNG::MLS-2 fusion protein is functional in AWC development. Like GFP::MLS-2 expressed from transgenes, mNG::MLS-2 knock-in was localized in the nucleus of AWC precursor cells in early embryos (Figure 1E) but was not observed in AWC cells in late embryos (Figure 1F) or early-stage larvae (Figure 1G). Our results are consistent with single-cell RNA-seq data showing that mls-2 was briefly expressed at a very low level in AWC during early embryogenesis but not detected in AWC in second-stage larvae (Cao et al., 2017; Packer et al., 2019). It was also shown that MLS-2::GFP expressed from an integrated mls-2::GFP fosmid reporter line was not detected in AWC in late larval stage or young adult-stage using NeuroPAL (Reilly et al., 2020).

Similar to MLS-2 antibody staining and GFP::MLS-2 transgenes (Jiang et al., 2005), mNG::MLS-2 knock-in was localized to the nucleus of a subset of head cells and the M mesoblast in first-stage larvae and adults (Figure 1G and 1H). mNG::SEC::mls-2 knock-in and mNG::mls-2 knock-in strains should help to determine the endogenous expression pattern of mls-2 in different cells during development.