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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.
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