I’ve used WormMine to compile a list of genes with a hypo-osmotic shock sensitivity phenotype. This is relatively short - a couple of CYP genes and unc-29, unc-38, unc-50. There are no
genes involved in cuticle integrity, eg. dpy-18. I believe the cuticle is relatively permeable to water so can I infer that the lack of an apparent association of such genes with
hypo-osmotic sensitivity indicates that defects in genes encoding collagens, PDI, etc, whilst resulting in cuticle-related dpy, sqt phenotypes, etc, don’t affect water flux across the
cuticle significantly?
Hmm, it’s true that I haven’t come across any cuticle genes that cause hypo-osmotic stress sensitivity when inactivated. However, acs-20 has been shown to be required for a wild-type surface barrier. acs-20 mutants are sensitive to hypo-osmotic stress, permeable to a normally cuticle-excluded dye (Hoechst 33342), and exhibit cuticle structural defects by TEM. In the acs-20 paper the authors suggested that the barrier phenotypes could be due to either altered ECM secretion or aberrations in the cuticle barrier itself.
I don’t know if the lack of cuticle genes in the WormMine list reflects that inactivation of cuticle genes does not generally cause hypo-osmotic stress or that many of these genes have not been tested. I will say that the hypo-osmotic stress assay is one of the easiest ones I have ever tried. Put worms in water, watch every five minutes for exploding. Sensitive mutants will die in 5-15 minutes. You could always throw a couple of classic cuticle mutants you have in the lab into water and see what happens.
Thanks - I’ll try the Hoechst 33342 permeability test. Do you know of any CGC-available cuticle-defective strains that would act as a good
+ve control for Hoechst 33342 staining (I could ask for the acs-20 strain but obtaining tm strains is a little more ‘time-consuming’ compared
to a simple CGC request)?
Yeah-bus-8 is a great control and available from the CGC. I used the e2885 allele in a recent PLoS ONE paper and got comparable Hoechst permeability to the acs-20 mutant. Nicely, I got very similar results to those described by Meli et al (2010), so the phenotype seems quite consistent. bus-8 mutants are generally useful to have around, as their permeability makes them useful for drug sensitivity assays.
Thanks for your help on this Jordan. I’ve tried 33342 on N2, bus-8(e2885) and my mutant with mixed results. Initially the results were as expected with bus-8
nuclei staining as expected. However, I’ve not been able to repeat this. I’m wondering whether washes are critical - too many/too large volume - may result in dye efflux.
Have you got an 'idiots guide; to your method?