In C.elegans studies of hindgut development indicate the EGL-38 Pax transcription factor directly regulates
lin-48 , a gene encoding an Ovo related zinc finger transcription factor. To better understand the function of these two genes, we have made clones that include a heat shock promoter ( hsp 16-2 and hsp 16-41 ) and cDNA for each gene. These clones will allow us to test the effect of ectopic expression for each gene. Since
egl-38 and
lin-48 are initially expressed in embryos, we wanted to test the effect of heat shock on embryos. In preliminary experiments, we observed embryos are very sensitive to the commonly used conditions of 33 degC for 1+ hours. Consequently, we wanted to identify a heat shock (HS) condition that could both induce heat shock expression and minimize lethality. To identify a good heat shock condition, a gfp cDNA was cloned into the
hsp16-2 and hsp 16-41 promoter vectors. After the injection of the animals and the production of transgenic lines, the animals were heat shocked under various conditions (Table 1.1). We found the greatest gfp expression with minimum amount of lethality due to the heat shock was observed when the animals were incubated at 35 deg C for half an hour. The above heat shock conditions were also not too detrimental for the animals if they were heat shocked everyday for 24 hours through out their developing life. HS conditions Clone % lethality after HS % lethality w/out HS gfp expression 33 degC for 1 hour
hsp16-2::gfp 7% 6% +/- 33 degC for 1 hour
hsp16-41::gfp 9% 5% +/- 35 degC for 1 hour
hsp16-2::gfp 39% 7% + 35 degC for 1 hour
hsp16-41::gfp 55% 0% + 35 degC for 1/2 hour
hsp16-2::gfp 9% 1% + 35 degC for 1/2 hour
hsp16-41::gfp 6% 5% + Table 1.1 These conditions were then used on transgenic animals carrying a clone with
hsp16-2::
egl-38 , and animals with
hsp16-41::
lin-48 . Although we found neither transgene significantly affected embryonic variability, both affected adult male spicule development (Table 1.2),
hsp16-2::
egl-38 also affects larvae viability. Genetic results indicate that
lin-48 plays an inhibitory role in the development of the male spicules because
lin-48 mutant males have ectopic spicule-like structures. The similarity in effect for
egl-38 and
lin-48 is most likely due to the fact that
egl-38 is a transcription factor for
lin-48. Therefore, over expressing
egl-38 results in an over expression of
lin-48, and there by yielding the deformed or absent male spicule phenotype. Clone % abnormal males after HS % abnormal males w/out HS
hsp16-2::
lin-48 59% 1.6% Control 3% 0% Table 1.2