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Fig. 6. smedinx-11(RNAi) gradually reverses the endogenous AP mitotic
gradient. (A) Confocal projections of whole-mount immunostained
animals using an antibody against the phosphorylated form of Histone-3 (H3P)
(green dots are positive signal). Control (left); smedinx-11(RNAi)
worms at different times after dsRNA exposure (7, 11 and 14 days). H3P signal
is disappearing in a gradient, anterior to posterior, in a time-dependent
manner. Each time-point consisted of n
7 worms. (B)
Schematic of worm regions quantified in C. (C) H3P signal
quantification from different anatomical areas - pre-pharyngeal (PP),
pharyngeal (P) and post-pharyngeal (T) - from control and
smedinx-11(RNAi) animals. The H3P signal was quantified
(Reddien et al., 2005b) at
different time points after dsRNA exposure. H3P signal as detected in
whole-mount immunostaining is disappearing gradually and inhomogenously
throughout the worm. In controls (7/7 worms at each time-point), H3P signal
from the PP area always contained the highest numbers compared with other
regions. By contrast, in smedinx-11(RNAi) worms, the H3P signal from
the PP region always contained the smallest number (7/7 worms at each
time-point). Mitotic activity completely disappeared by >14 days
post-smedinx-11(RNAi). Values are average, and error bars represent
s.d.; groups were compared using the Student's t-test
(***P<0.001, **P<0.02,
*P<0.08, and ND for no difference). RNAi in planarians
(either by feeding or microinjections) results in uniform distribution
throughout the body once the dsRNA molecules are incorporated into the worm
(Reddien et al., 2005a;
Reddien et al., 2005b). Thus,
specific gene expression disappears spatially uniformly after RNAi, in a
time-dependent manner. Interestingly, the inhibition by smedinx-11 of
both neoblast marker expression (S. mediterranea piwi genes,
smedcyclinB and smedbruli) and of mitotic activity was
characterized by a progressive AP disappearance of the signal patterned along
the AP axis (i.e. first depletion of pre-pharyngeal areas followed by
most-posterior ones) as the phenotype progressed. This response was
consistently observed in neoblasts but not in markers of differentiated
tissues - in animals subjected to RNAi for a given gene followed by ISH
(Fig. 2A and
Fig. 4B,C). In addition,
similar results in AP neoblast depletion were obtained as a consequence of
microinjections targeting pre- or post-pharyngeal areas. Thus, the activity
gradients we describe are specific to smedinx-11 and not a general
feature observed in any other RNAi-induced phenotype or irradiation treatment
described in the literature. Scale bar: 0.1 mm in A.
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