Fig. 4. smedinx-11 is required for neoblast maintenance. (A) Differences in expression of neoblast markers, analyzed by qRT-PCR using total RNA extracted at 7 and 14 days after smedinx-11(RNAi) injection. Downregulation of smedwi-1 is observed within the first week after smedinx-11(RNAi), whereas a slight increase in smedwi-2 expression was noted during this time. At 1 week later, the expression for both S. mediterranea piwi genes was severely reduced. qRT-PCR experiments were the result of triplicate experiments; values represent the difference between control and smedinx-11(RNAi); error bars represent s.d. Gene expressions are relative to the ubiquitously expressed clone H.55.12e (Reddien et al., 2005b). (B) Expression changes in neoblast X1 markers at different days after smedinx-11 dsRNA injection. Representative whole-mount ISH results using the smedwi-1 probe are shown. Control (left-most) gives a regular signal distributed throughout the mesenchyme (Reddien et al., 2005b). Notice that changes in smedwi-1 expression are revealed by a gradual disappearance of the signal in a time-dependent manner. Within the first 2 weeks after smedinx-11 dsRNA exposure, the smedwi-1 signal is dramatically reduced and, as the phenotype progresses, no signal is detected (>14 days) in the whole organism. In all cases, anterior end is up. At least n=7 worms were included at each time-point. (C) Spatial expression for different markers detected in un-/differentiated tissues after smedinx-11(RNAi). Differentiated (excretory, smedinx-10; CNS, smedinx-3; and digestive, smedinx-9) and undifferentiated (smedwi-1, smedcyclinB and smedbruli) tissue probes were assayed in control animals (upper row) and 14 days after smedinx-11(RNAi) (bottom row). Although expression for differentiated tissue markers remains similar to their control counterparts (5/5 each), the signal for neoblast markers was strongly reduced in smedinx-11(RNAi) worms (5/5 each). Interestingly, the component of smedbruli expression that is observed in differentiated tissue (i.e. CNS) remained, whereas its neoblast-related expression was reduced. (D) FACS profiles highlighting X1 subpopulations (insets from each profile) from dissociated planarians; control (top), 7 days post-irradiation (6000 rad; middle) and 10 days after smedinx-11(RNAi) (bottom). Notice that the population of dividing neoblasts was sharply reduced after irradiation and smedinx-11(RNAi). (E) Percentage of cells from different FACS-isolated cell populations from control, irradiated and smedinx-11(RNAi), 10 and 14 days after first injection (see Fig. 5 for corresponding FACS profiles showing all subpopulations). Interestingly, the number of X2 cells 10 days after smedinx-11(RNAi) is comparable to the control animals. However, as the phenotype progressed, the X2 cell numbers were reduced to levels comparable to the irradiated group. In all FACS experiments, n=20 animals were dissociated for each condition. Scale bars: 0.2 mm.