Characterisation of skn-1b(tm4241) and SKN-1B:GFP.
A) Expression levels of skn-1 isoforms in WT and skn-1b(tm4241) determined by Q-PCR. Combined data from 6 biological replicates shown. Error bars show st. dev. Two-tailed t-test compared to WT control *p<0.05, **p< 0.001, ***p<0.0001, NS not significant. B-D) Brood size of WT and skn-1b mutants at three different temperatures. skn-1b mutants are fully fertile, so can be maintained as homozygotes. Combined data from 3 biological replicates shown, n>30 worms per group. Error bars show st. dev. Two-tailed t-test *p<0.05, **p< 0.001, ***p<0.0001, NS not significant. E) Age-specific fecundity in WT and skn-1b mutants. F) Expression pattern of the Scarlet::SKN-1B reporter in day 1 adults under fed conditions shows SKN-1B in ASI neurons. Our lab also generated an endogenous NeonGreen::SKN-1C reporter but cannot detect expression of SKN-1C in neurons (available on request). No significant differences were observed using a two-tailed t-test on any day between genotypes. G-J) SKN-1B::GFP is expressed differentially during growth and in additional neurons in response to bacterial deprivation. H) SKN-1B::GFP observed in additional neurons in response to fasting. I) Quantification of the number of visible neurons in SKN-1B::GFP expressing worms in response to fasting. A total of 52 fed and 64 fasted worms were examined. J) DiI staining confirms SKN-1B::GFP in the ASI neurons and identified two of these additional neurons (counted in S3I Fig) as being the ADLs. Recently, others have identified SKN-1B in AIY neurons as a regulator of chemosensory processes and behaviour, showing that animals lacking the skn-1a, c and b do not chemotax towards NaCl, butanone or temperature, or move towards thicker bacterial lawns (as a WT worms would) [57]. We tested our skn-1b specific mutant in a NaCl chemotaxis assay and got similar results but have no evidence that SKN-1B is endogenously expressed in the AIYs in fed conditions. It is possible that SKN-1B signals from the ASI—AIY neurons to mediate this response or that some SKN-1B::GFP expressing neurons in fasted conditions are AIYs. As the no-food conditions in NaCl chemotaxis assays is sufficient to induce SKN-1B expression in these cells, this could mediate the effects.
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