Fig. 3. CSR-1 isoforms function independently of one another.

a Schematic representation of two deletion alleles of csr-1a. cmp135 removes 20 bp of coding sequence, including the start codon. cmp143 removes ~500 bp of promoter sequence and 20 bp of coding sequence. b Brood-size assay performed on wild-type and csr-1a mutant animals at 20 and 25 °C. Three replicates were performed for each genotype, with ten animals per replicate. For each biological replicate, brood sizes for all ten animals are shown, with bar graphs representing mean brood size and error bars indicating standard deviation. Two-tail t-tests were performed to determine statistical significance. n.s. denotes not significant and indicates a p value >0.05 and * indicates a p value ≤0.05. See Supplementary Data 7 for more details regarding statistical analysis. c An in vitro sperm activation assay was performed on spermatids from wild-type, csr-1a(cmp135), csr-1a(cmp143), mCherry::CSR-1A, and mCherry::CSR-1A csr-1b(cmp258) mutants. Animals were raised at 25 °C for either one or two generations and at least 80 spermatids were counted for each replicate at each generation. Two biological replicates are shown, with bar graphs representing the mean. d Schematic representation of 2xFLAG::CSR-1B in a csr-1a(cmp135) mutant. The 2xFLAG tag was introduced immediately after the CSR-1B start codon in a csr-1a(cmp135) mutant animal. e Immunofluorescence staining for 2xFLAG::CSR-1B and P granules (PGL-1) in a csr-1a(cmp135) mutant at L4 stage. The experiment has been reproduced with at least five individual germlines imaged. Scale bar, 5 μM. f Western blot for CSR-1 expression levels, in wild-type and csr-1a(cmp135) mutant animals at L4 stage. Actin is shown as a loading control. This blot has been reproduced. g Schematic representation of disrupting csr-1b expression in a 2xHA/mCherry::CSR-1A strain. The csr-1b start codon was mutated from methionine to isoleucine (M1I) using CRISPR, which also introduces a M164I point mutation in the CSR-1A protein. h Live imaging of mCherry::CSR-1A in a wild-type and csr-1b(cmp258) mutant L4 hermaphrodite. At least five individual germlines were imaged for each strain. Scale bar, 25 μM. Source data are provided as a Source Data file.