mNG-tagged mls-2 knock-in alleles in C. elegans.

The Caenorhabditis elegans HMX/NKX MLS-2 transcription factor was previously shown to play sequential roles in AWC general identity and the stochastic AWCON/AWCOFF subtype choice during embryogenesis. Here we analyze the expression pattern of endogenous mls-2 during AWC development using mNeonGreen (mNG) knock-in strains. Similar to transgenic GFP::MLS-2, functional mNG::MLS-2 knock-in displayed nuclear localization in AWC precursor cells but was not observed in AWC during the later embryonic stage. These results suggest that the expression of mls-2 is below the detectable level and/or the stability of MLS-2 protein is very low in AWC cells. Copyright:

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).

Figure 1.

(A) Structure of the mls-2 locus, mls-2p::GFP::mls-2 transgene (Jiang et al., 2005), mNG::SEC::mls-2 knock-in, and mNG::mls-2 knock-in. mNG, mNeonGreen. SEC, self-excising cassette containing transcriptional terminators, a dominant roller phenotype marker sqt-1(e1350), Cre driven by a heat shock promoter, and a hygromycin resistance gene. The SEC cassette is flanked by LoxP sites.

(B-H) Representative images of GFP::MLS-2 expression from a mls-2p::GFP::mls-2 extrachromosomal transgene in a first-stage larva (B), mNG expression from mNG::SEC::mls-2 knock-in in a first-stage larva (C), and mNG::MLS-2 expression from mNG::mls-2 knock-in animals in different developmental stages (E-H). Integrated transgenes of hlh-16::H1-wCherry and odr-1p::TagRFP (or odr-1p::DsRed) were used as early and late AWC markers, respectively. Insets in panels B, C, E, F, and G are magnified by 2-fold. Scale bar, 10 um. Anterior to the left and ventral to the bottom in lateral or ventrolateral views of the head region in B, C, and F-H; ventral view in E. (D) Expression of the AWCON marker str-2p::TagRFP from an integrated transgene in wild type and mNG::mls-2 knock-in animals. n, total number of animals scored.

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 AWCON 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.

Methods

mNG::SEC::mls-2 and mNG::mls-2 knock-in were generated using the Cas9-triggered homologous recombination protocol as previously described (Dickinson et al., 2013; Dickinson et al., 2015).

Reagents

Strain	Genotype	Source
IX1119	oyIs44 [odr-1p::DsRed; lin-15(+)] V; vyEx535 [mls-2p::GFP::mls-2::mls-2 3’UTR (Jiang et al., 2005); ofm-1p::DsRed]	This study
IX4507	mls-2(vy247 [mNG::SEC::mls-2 knock-in]) X	This study
IX4506	mls-2(vy248 [mNG::mls-2 knock-in]) X	This study
RW10588	unc-119(ed3); zuIs178 [his-72(1kb 5′ UTR)::his-72::SRPVAT::GFP::his-72 (1KB 3′ UTR) + 5.7 kb XbaI-HindIII unc-119(+)]; stIs10544 [hlh-16::H1-wCherry::let-858 3′ UTR]	Murray et al., 2012
IX5609	stIs10544 [hlh-16::H1-wCherry::let-858 3′ UTR] (Murray et al., 2012); mls-2(vy248 [mNG::mls-2 knock-in]) X	This study
IX4894	vyIs56 [odr-1p::TagRFP] III (Cochella et al., 2014); mls-2(vy248 [mNG::mls-2 knock-in]) X	This study
IX3212	vyIs68 [str-2p::TagRFP; srsx-3p::GFP] III	Cochella et al., 2014