10.17912/MICROPUB.BIOLOGY.000075
Roy, Vincent
Vincent
Roy
Gagné, Olivier
Olivier
Gagné
Hamiche, Karim
Karim
Hamiche
Labbé, Jean-Claude
Jean-Claude
Labbé
Narbonne, Patrick
Patrick
Narbonne
Expression pattern of endogenous PAR-4A & C after CRISPR/Cas9-mediated genome editing
microPublication Biology
2018
Journal Article
2018
PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al. 2011), and metabolism (Narbonne and Roy 2009). Its expression pattern and subcellular localization has been determined in fixed animals by antibody staining (Watts et al. 2000). Here, we used CRISPR/Cas9-mediated genome editing to fluorescently label the two longest endogenous PAR-4 isoforms, PAR-4A and PAR-4C, with monomeric Neon Green (mNG) (Shaner et al. 2013), using the self-excising cassette (SEC) system (Dickinson et al. 2015) (Fig. 1A). We found ubiquitous mNG::par-4a & c expression and cytoplasmic and cortical enrichment of mNG::PAR-4A & C proteins in the germ line and early embryos (Fig. 1B-H), as previously described (Watts et al. 2000). Interestingly, we find that mNG::PAR-4A & C cortical enrichment is transiently lost in the pachytene area of the germ line (Fig. 1F), although it remains unclear whether this is functionally relevant.
The intermediate strain that still contains the SEC (Dickinson et al. 2015) is predicted to be null for par-4a & c, potentially leaving the shorter par-4b isoform functional. To evaluate the requirement for par-4a & c we examined the embryonic lethality (at 25°C) of the generated SEC-containing and SEC-excised strains. We found that the self-progeny of homozygous SEC-containing (e.g. par-4a & c null) animals is viable (Fig. 1H). This suggests that par-4b alone is sufficient to establish embryonic polarity and sustain the essential function of par-4. Consistent with this, to our knowledge, all existing par-4 alleles that impair embryonic development disrupt par-4b (Morton et al. 1992; Watts et al. 2000).