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First published online 1 August 2007
doi: 10.1242/dev.006635

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smedinx-11 is a planarian stem cell gap junction gene required for regeneration and homeostasis

Center for Regenerative and Developmental Biology, Forsyth Institute, and Developmental Biology Department, Harvard School of Dental Medicine, 140 The Fenway, Boston, MA 02115, USA.

View larger version (61K): [in this window] [in a new window]   Fig. 1. S. mediterranea innexin gene expression analysis. (A) Representative S. mediterranea innexin expression patterns in wild-type animals were organized into four groups: (1) nervous: central nervous system (CNS) and marginal cells (smedinx-3 and smedinx-4), CNS and pharynx opening (smedinx-5); (2) mesenchymal tissue (smedinx-1, smedinx-2, smedinx-6, smedinx-7, smedinx-8, smedinx-11 and smedinx-12), which is widely distributed and might include more than one cell type (e.g. pharynx, CNS and diverse sub-epithelial cells); (3) excretory system: flame cells (smedinx-10); and (4) digestive: gastro-vascular system (smedinx-9). In all panels, anterior is up and, except for the CNS group, which is ventral, dorsal views are shown. For each probe, n18 animals (results were consistent in each case). (B) Anterior transverse section (8 µm) at the level of the photoreceptors/brain showing specific expression of smedinx-11 in individual cells. Red arrowheads indicate representative expressing cells. CG, cephalic ganglia; PR, photoreceptor. Scale bars: 0.2 mm in A; 20 µm in B.

View larger version (36K): [in this window] [in a new window]   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.

View larger version (60K): [in this window] [in a new window]   Fig. 3. smedinx-11(RNAi) phenotype. (A) Early phenotype: lack of regeneration. Regenerating fragments at 7 days post-amputation; control animal (left) formed AP blastemas (lightly pigmented new tissue indicated with white arrows), whereas smedinx-11(RNAi) fragments failed to create new tissue (30/30) even at 3 weeks post-amputation (red dotted lines represent plane of amputation, animals were amputated 8 days after first injection). (B) Late phenotype. Intact animals; control (left) and smedinx-11(RNAi) representative images show progression of the phenotype. Top scale indicates days after first injection of smedinx-11 dsRNA. Initial signs of the phenotype are visible 2 weeks after first injection and were characterized by a contraction of the pre-pharyngeal region (white arrows at 14 days). At 21 days, the lateral and posterior edges of the worms curled under ventrally (white arrows) (50/50). Unlike irradiated, smedwi-2(RNAi) or smedbruli(RNAi) worms, animals with the smedinx-11(RNAi) phenotype do not develop head regression even 1 month after first exposure to smedinx-11 dsRNA (13/15 worms, yellow arrow at the front of the animal at 31 days). Simultaneous smedinx-11 + smedinx-2 double-knockdown phenotype in both intact and regenerating fragments did not show differences when compared to smedinx-11(RNAi) alone (data not shown). All animals died 35-40 days after first injection. In all cases, anterior end is up. Scale bars: 0.2 mm.

View larger version (51K): [in this window] [in a new window]   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.

View larger version (29K): [in this window] [in a new window]   Fig. 5. smedinx-11(RNAi) gradually depletes the neoblast compartment. FACS profiles using Hoechst blue versus calcein, highlighting X1 (blue rectangle), X2 (red rectangle) and Xins (yellow rectangle) subpopulations from dissociated control planarians (left-most), planarians at 7 days post-irradiation (6000 rad; irradiated), and at 10 and 14 days after smedinx-11(RNAi). Notice that the numbers of dividing X1 neoblasts were sharply reduced after irradiation and at 10 days after smedinx-11(RNAi), but this did not occur for X2 cells (see Fig. 4E for quantification). However, as the phenotype progressed, the X2 cells were reduced to levels comparable to irradiated worms >14 days after dsRNA smedinx-11 exposure (data not shown).

View larger version (51K): [in this window] [in a new window]   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 n7 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.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Summary schematic of smedinx-11 function in intact and regenerating planarians. (A) In intact animals, smedinx-11 is required to maintain proliferative neoblasts. Representative phenotype progression after smedinx-11(RNAi) shows that smedinx-11 is required for the maintenance of neoblasts with the capability to proliferate (red dots). Our data suggest that intact animals possess an anterior-posterior endogenous gradient of mitotic neoblasts that is gradually reversed as the neoblasts lose their proliferative capabilities after smedinx-11(RNAi). For simplicity, smedinx-11 postmitotic expression is not represented. (B) smedinx-11 is specifically required for blastema formation. Animals amputated 1 week after smedinx-11(RNAi) failed to regenerate even though proliferative neoblasts were present, suggesting a loss of normal neoblast behavior even prior to their disappearance. Although mitotic neoblasts disappear by about 2 weeks after smedinx-11(RNAi), the RNAi-treated animals survived longer than 1 month in both intact and trunk fragments, but were unable to mount a regeneration response, demonstrating the distinct requirement for smedinx-11 in blastema formation. (C) Summary of the key features of different cell populations (X1, X2 and Xins) obtained in flow cytometry experiments. Asterisk denotes the fact that approximately 10-12% of X2 cells can be positive for smedwi-1 and smedwi-2 gene expression while none will be positive for smedcyclinB (Reddien et al., 2005b). (D) Schematic model of the dynamics of different cells (X1, X2 and Xins) obtained in flow cytometry experiments. Proliferative neoblasts (X1) are shown in red, X2 cells in green and blue, and differentiated cells (Xins) in gray. The graded bar represents the level of smedinx-11 expression; the percentages of smedinx-11-positive cells are indicated. Neoblast-related cells are included in X1 and X2 subpopulations that express smedinx-11. The proposal that X2 cells represent the progeny of the proliferative neoblasts is based on: (i) irradiation-sensitive smedinx-11 expression is observed in both proliferative and postmitotic areas; (ii) the time-dependent manner in which neoblasts disappear after smedinx-11(RNAi) (i.e. first X1 cells disappear followed by X2); (iii) smedwi-1 gene expression is restricted to proliferative cells, but its protein signal is broadly observed in both proliferative and postmitotic irradiation-sensitive cells, consistent with the switching off of the normal smedwi-1 expression by cells as they exit X1 but retaining the protein in postmitotic stages prior to protein turnover; and (iv) smedinx-11 expression overlaps with that of smedwi-1 at both the gene and protein level. Therefore, it is possible that X2 cells might represent a transitional stage between X1 and Xins cells, which is consistent with results obtained in vitro for gap junction genes using mammalian neuronal progenitor cells.