Fig. 2. S. mediterranea innexin expression studies in intact and dissociated worms. (A) Representative images of smedinx-11 whole-mount in situ hybridization in intact animals under different conditions. From left to right: wild type (antisense probe), irradiated (antisense probe on worms 7 days post-irradiation with 6000 rad), sense smedinx-11 probe and antisense probe on worms 7 days after smedinx-11(RNAi). Notice that the smedinx-11 signal was strongly downregulated after RNAi and was practically undetectable after irradiation or after hybridization with sense probe. (B) qRT-PCR analyses showing expression of S. mediterranea innexin genes from controls (un-irradiated) and worms 7 days after -irradiation (6000 rad, which destroys neoblasts). Notice that, after -irradiation, three different responses in gene expression were observed (downregulation, no change and upregulation). Only two out of the 12 innexin genes were downregulated (highlighted with gray background in smedinx-11 and smedinx-2. t-test, control versus irradiated, P=0.002 and 0.04, respectively), suggesting their possible association with neoblasts. For clarity, data for smedinx-6-9 are not shown. Gene expressions are relative to the ubiquitously expressed clone H.55.12e (Reddien et al., 2005b). Notice that a baseline signal is not indicative of a lack of expression but rather lack of change in expression, because the y-axis values are relative to the internal control. (C) smedinx-11 qRT-PCR analyses from wild-type FACS-isolated cells (X1, X2 and Xins; see text for nomenclature). smedinx-11 was expressed most-strongly in the neoblast X2 cell population but was also detected in the dividing neoblasts (X1 cells), whereas no signal (NS) was observed in irradiation-insensitive cells (Xins). qRT-PCR results are from triplicate experiments; values represent average and error bars s.d. Gene expressions are relative to the ubiquitously expressed clone H.55.12e (Reddien et al., 2005b). (D) smedinx-11 in situ hybridization in FACS-isolated cells. Percentage of smedinx-11-positive cells from different FACS-isolated cell populations (X1, 129/210; X2, 216/885; and Xins, 1/298) are shown. Notice that, importantly, smedinx-11 is enriched in X1 and X2 cells. Representative pictures of individual cells (X1, X2 and Xins) using the smedinx-11 probe are also shown above each bar. Notice that smedinx-11 expression can be detected in both X1 and X2 but not in Xins. Scale bars: 0.2 mm in A; 10 µm in D.