Here, we provide new insights into the basic helix-loop-helix transcription factor atonal in hypodermis development. Historically, atonal had been identified as a proneural gene, contributing to neural fate determination in various animals. For example, in Caenorhabditis elegans ( C. elegans ) , the atonal homolog lin-32 initiates the neurogenesis touch neurons (Chalfie and Sulston 1981; Mitani et al. 1993). We also have previously reported that lin-32 is essential for optimizing avoidance behaviors via AIB interneuron development (Hori et al. 2018). The atonal gene and its homolog also contribute to patterning and cell fate determination in specialized epithelial cells like those in the retina (Robertson et al. 2012), intestine (VanDussen and Samuelson 2010), and the inner ear sensory epithelia (Bermingham et al. 1999). However, its function in the hypodermis remains unclear.

The epithelial system of C. elegans is constituted by the hypodermis and specialized epithelial cells. In this study, to determine the role of atonal in hypodermis development, we examined the morphology of the head hypodermis in lin-32 null mutants. lin-32 null mutants exhibited morphological changes in the head hypodermis, presenting external bulges and an internal cavity (Fig. 1A). The phenotype was rescued by the expression of LIN-32 driven by its own promoter (Fig. 1B,C). DNA microarray analysis revealed that lin-32 is expressed in a restricted cell lineage within a narrow time window during embryogenesis (Murray et al. 2012). In the hypodermis, lin-32 is predicted to be expressed in hyp4 and hyp6, which are derived from the ABarpapa lineage (Murray et al. 2012). To assess lin-32 -expression in the peripheral area in embryos, we used a lin-32 promoter-driven EGFP and have observed fluorescence in hyp6 and additional cells that have not yet been identified. These results provide insight into the function of lin-32 in the differentiation of the hypodermis. However, we could not identify the causal cell lineage that induced morphological changes in the head, because the number and location of the hypodermal nuclei were substantially altered in the lin-32 null mutants. Considering the principal role of hyp6 in the morphology of the anterior hypodermis (Cinar and Chisholm 2004), we speculate that lin-32 also affects hyp6 development or localization. These findings reveal a novel role of atonal , which is well known as a proneural gene, in a broader range of tissues than initially thought.

Nematodes and maintenance

We cultured C. elegans strains using modified standard techniques as described previously (Brenner 1974; Hori et al. 2018). The lin-32 ( tm2044 ) mutants were backcrossed with the wild-type animal N2, five times. The information on the strains used is summarized in Table 1.

Microscopy

C. elegans adults were immobilized with M9 buffer containing sodium azide 50 mM on a 5% agarose pad containing 10 mM sodium azide (Wako, 195-11092) (Fig. 1 A,B,C). Eggs were isolated from gravid adults by treating them with a bleaching solution (Fig. 1D). The bleaching solution contained 0.2 mL 4N NaOH (Wako, 194-18865), 0.3 mL 6% bleach (Kao, o17246), and 0.5 mL M9 buffer. Differential interference contrast (DIC) and fluorescence images were obtained using a BX51 microscope equipped with a DP30BW charge-coupled device (CCD) camera (Olympus Optical).

Plasmid construction

In the own promoter rescue experiment (Fig. 1C), pFX_P lin-32 ::LIN-32 was used (Hori et al. 2018). To analyze lin-32 expression (Fig. 1D), pFX_P lin-32 (4.2K)::EGFP was constructed by cloning the lin-32 promoter (from 4.2 kbp of the genomic sequence upstream of the mature lin-32 sequence), which was amplified using PCR, with the N2 genomic DNA as the template. Cloning primers are as follows; ggttccgcgtggatccCTGAAAATTAGAAACTAAATGAG, GCTCACCATgcggccgcCCATGGTTGGTCTGACTGAAAACGAC. The pFX_EGFP plasmid and the PCR fragment of the lin-32 promoter were cut using two restriction enzymes, BamHI (Takara, 1010A) and NotI (Takara, 1166A), and then ligated using ligase (Takara, 6021).

Transgenic lines and strains

To generate lin-32(tm2044); tmEx3143 transgenic animals (Fig. 1C), pFX_P lin-32 ::LIN-32 (20 ng/μl), and pBluescript KS(+)T1(100 ng/μl) were co-injected, along with pFX_ Pnmr-1:: mCherry (80 ng/μl), into FX15206 animals (Hori et al. 2018). To generate tmEx3090 transgenic animals (Fig. 1D) , pFx_P lin-32 (4.2 kb)::EGFP (150 ng/μl) was injected along with pFX_P nmr-1 ::mCherry (50 ng/μl), which acts as an injection marker, into N2 animals.

Quantification and statistical analysis

Statistical analyseswere performed using the GraphPad Prism 6 software (GraphPad software). Pairwise comparisons of frequencies within two groups wereperformed using Fisher’s exact test (Fig. 1B,C).In both analyses,theDIC images were randomized and their normalcy was ascertained in a blinded manner.Figures 1B and 1C are the experimental results of sampling on different days. There is a variable penetrance of incidence and severity of the morphological defect in the lin-32 mutants(Fig. 1B,C). Therefore, we speculate that the penetrance is responsible for the difference in the two results(Fig. 1B,C).The statistical information and the total number of animals analyzed per experiment are indicated in Figure legend.