Table 1.
C. elegans strains used in this study.
Figure 1.
POPTOP is expressed in the L4 somatic gonad.
GFP and POPTOP expression are shown in (A–D) egl-17::GFP; POPTOP expressing animals or (E–H) fkh-6::GFP; POPTOP expressing animals. POPTOP co-localizes with fkh-6::GFP in the L4 spermatheca and in cells surrounding the vulva. Asterisks indicate the position of the vulva. Scale bar is 25 µm.
Figure 2.
CACN-1 is a negative regulator of POPTOP reporter expression.
POPTOP animals were grown on E. coli HT115(DE3) carrying the (A) control RNAi vector or (B) cacn-1 RNAi vector. Asterisks indicate the L4 developing vulva. 3-dimensional representations of POPTOP expression with peak height corresponding to maximum intensity of fluorescent reporter in (C) control RNAi or (D) cacn-1 RNAi treated animals. (E) Individual points represent the number of POPTOP expressing nuclei for a single animal for a given RNAi treatment. (F) Individual points represent mean pixel intensity (a.u.) per pixel for a single animal for a given RNAi treatment. Error bars indicate mean ± standard error of the mean. *** indicates statistical significance of P<0.001. ** indicates statistical significance of P<0.005. Scale bar is 25 µm.
Figure 3.
POPTOP expression requires POP-1 and SYS-1.
(A) POPTOP or pop-1(q624); POPTOP animals were grown on E. coli HT115(DE3) carrying control RNAi or cacn-1 RNAi vector. (B) POPTOP animals were grown on E. coli HT115(DE3) carrying control RNAi, sys-1 RNAi, wrm-1 RNAi, bar-1 RNAi, or hmp-2 RNAi vector. (C) POPTOP animals were grown on E. coli HT115(DE3) carrying cacn-1+sys-1 RNAi, cacn-1 RNAi or sys-1 RNAi. Individual points represent mean pixel intensity (a.u.) per pixel for a single animal for a given RNAi treatment. Error bars indicate mean ± standard error of the mean. *** indicates statistical significance of P<0.0001, * indicates statistical significance of P<0.05. n.s. is not significant.
Figure 4.
Loss of CACN-1 leads to nuclear accumulation of GFP::POP-1.
sys-1p::GFP::POP-1 animals were grown on E. coli HT115(DE3) carrying (A) control RNAi or (B) cacn-1 RNAi vector. Asterisks indicate L4 developing vulva. 3-dimensional representations of GFP expression with peak height corresponding to maximum intensity of fluorescent reporter in the uterus and vulva of (C) control RNAi or (D) cacn-1 RNAi treated animals (E) Western blots of extracts from control RNAi or cacn-1 RNAi-treated L4 animals probed with anti-POP-1 (94I) and anti-Actin antibodies. Scale bar is 25 µm.
Figure 5.
LIT-1 and CACN-1 both negatively regulate POPTOP reporter activity.
POPTOP, lit-1(or131); POPTOP, or lit-1(ne1991); POPTOP animals were grown on E. coli HT115(DE3) carrying control RNAi or cacn-1 RNAi. Individual points represent mean pixel intensity (a.u.) per pixel for a single animal for a given RNAi treatment. Error bars indicate mean ± standard error of the mean. *** indicates statistical significance of P<0.0001, ** indicates statistical significance of P<0.005.
Figure 6.
cacn-1 RNAi reduces LIT-1::GFP expression.
LIT-1::GFP animals were grown on E. coli HT115(DE3) carrying (A, B) control RNAi or (C, D) cacn-1 RNAi vector. Asterisks indicate L4 developing vulva. 3-dimensional representations of GFP expression with peak height corresponding to maximum intensity of fluorescent reporter in the uterus and vulva of (E) control RNAi or (F) cacn-1 RNAi treated animals. (G) Western blots of extracts from control RNAi or cacn-1 RNAi-treated LIT-1::GFP animals were probed with anti-GFP (LIT-1::GFP) and anti-Actin antibodies. Scale bar is 25 µm.
Figure 7.
CACN-1 is localized to seam cells.
(A) CACN-1 expression is shown in seam cells of cacn-1::GFP; ajm-1::mCherry animals. (B) cacn-1::GFP expression in seam cells and differentiated hypodermal cells. Scale bar is 20 µm.
Figure 8.
CACN-1 is required for normal seam cell proliferation.
Representative fluorescence images of seam cells in scm-1p::GFP animals grown on E. coli HT115(DE3) carrying the (A) control RNAi, (B) pop-1 ORF RNAi or (C) cacn-1 RNAi vector. (D) scm-1p::GFP animals were grown on E. coli HT115(DE3) carrying empty vector, pop-1 ORF RNAi, cacn-1 RNAi or cacn-1+pop-1 double RNAi vector. Individual points represent the number of seam cells for a single animal for a given RNAi treatment. Error bars indicate mean ± standard error of the mean. *** indicates statistical significance of P<0.001, and ** indicates statistical significance of P = 0.0013 compared to control RNAi. Scale bar is 20 µm.
Figure 9.
cacn-1 RNAi alters the asymmetrical localization of GFP::POP-1 in seam cells.
sys-1p::GFP::POP-1 animals were grown on E. coli HT115(DE3) carrying (A) control RNAi or (B) cacn-1 RNAi vector. White lines indicate V3.pp(a/p), V4.pa(a/p) and V4.pp(a/p) seam cell daughters. In cacn-1 animals the asterisks indicate equal POP-1 in both daughter cells of V4.pa(a/p) and V4.pp(a/p) divisions. Scale bar is 20 µm. (C) Individual points represent the posterior/anterior ratio of GFP expression in each seam cell pair for a given RNAi treatment. * indicates statistical significance of P = 0.011.