Fig. 5: miR-232 regulates excretory canal morphogenesis by repressing NHR-25.

A Expression of miR-232-pr. B mir-232 deletion allele. C Comparison of excretory canal length (visualized by a membrane-localized mNG) between wild-type and mir-232(dev280) animals at the L4 stage. D Quantitative comparison of excretory canal length at postembryonic stages. Data are represented as mean ± SD. Statistics: Mann–Whitney U test, two-tailed. E Micrograph showing the cystic canal phenotype of the mir-232 mutant. F, G Changes of miR-232 reporter expression upon RNAi against ceh-6 and pros-1. Data are represented as mean ± SD. Statistics: Mann–Whitney U test, two-tailed. H Micrographs showing the expression of a protein-fusion reporter of NHR-25 in the four excretory cells with the epithelial identity. The cell lineage diagram on the right shows the corresponding cells’ developmental origin and NHR-25 expression status. I Top: Micrographs illustrating the difference in NHR-25 protein expression in the excretory canal cell between the wild-type and mir-232(dev280) embryos. Bottom: Enlarged views of NHR-25 expression in the canal cell. J Quantifying NHR-25::GFP expression in the excretory canal of wild-type and mir-232(dev280) embryos. Data are presented as mean ± 95% CI, Loess smoothed. Statistics: Wilcoxon rank sum test, two-tailed. K Left: Changes in canal length after ectopic expression of NHR-25 protein in the excretory canal at the L4 stage. Stars indicate animals with an abnormally short canal. Right: quantification of excretory canal length. Data are represented as mean ± SD. Statistics: Mann–Whitney U test, two-tailed. L Top: Quantifying mNG intensity expressed from the nhr-25 3′UTR reporter with or without miR-232 binding sites driven by a ubiquitous promoter (ubq-1). Data presentation and statistics are identical to (J). Bottom: mNG expression in the excretory canal at representative times. M Changes in excretory canal length at the L4 stage after mutating miR-232 binding sites in the endogenous nhr-25 3′UTR. Stars indicate animals with an abnormally short canal. N The proportion of animals with a shortened canal (≤3.5) after mutating miR-232 binding sites in the endogenous nhr-25 3′UTR. Statistics: Chi-square test, two-tailed. O Proposed function model of miR-232. Source data are provided as a Source Data file.