DEV006635 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Comparison between the innexin genes found in S. mediterranea and D. japonica. (A) Phylogenetic tree for innexins in both planarian species. A rooted tree Homo sapiens pannexin 2 isoform 1 (PANX2), Accession number: AF398510 was constructed using ClustalW (http://www.ebi.ac.uk/clustalw/) and njplot software (http://pbil.univ-lyon1.fr/software/njplot.html). Innexin genes were color-coded according to their expression pattern (blue: mesenchymal, red: nervous, green: digestive and brown: excretory tissues). Scale bar represents the estimated number of changes per site. Note the correspondence between clustering and gene expression of different innexin in both species. (B) Schematic summary comparing innexins of two planarian species. DNA homolog sequences are highlighted (gray background), and expression patterns observed in S. mediterranea (color coded as in A) are compared against those observed in D. japonica (Nogi and Levin, 2005). Note that homolog sequences also shown similar expression patterns in both species. Asterisk denotes that the smedinx-11 (labelled smedxin-11 here) expression pattern is more widely distributed than DjInx-11. Accession number or clone names (Sánchez Alvarado et al., 2002): AY067506; AY067503; AY067167; AY067877; NB.39.10e; NB.24.11b; NB.29.3g; AY967612; AY967558; AY068260; DQ851133 and AY066375 for smedinx-1 to smedinx-12 respectively. D. japonica accession numbers are as previously described (Nogi and Levin, 2005). smedinx-11 and DjInx-11 share considerable similarity at the sequence level; DjInx-11 expression is also irradiation sensitive (data not shown) and matches the spatial distribution observed for dividing neoblasts in both S. mediterranea and D. japonica species (Guo et al., 2006; Newmark and Sánchez Alvarado, 2000; Orii et al., 2005; Reddien et al., 2005b; Salvetti et al., 2000; Salvetti et al., 2005). However, their spatial expression patterns differ between the two planarian species. This interesting particularity has also been observed in other piwi-like genes expressed in irradiation-sensitive cells (Reddien et al., 2005b; Rossi et al., 2006). smedinx-11 is more broadly distributed than DjInx-11 (Nogi and Levin, 2005) and is additionally expressed in postmitotic areas, as has been previously reported for vasa-related genes in germline cells and somatic stem cells in D. japonica this type of expression is particularly difficult to discern in a whole-mount ISH (Shibata et al., 1999). Taken together, these studies suggest that some neoblasts markers might be restricted to a specific subset of stem cells e.g. proliferative − X1 subpopulation (Hayashi et al., 2006; Reddien et al., 2005b) while the broader irradiation-sensitive expression might involve proliferative as well as committed progeny of neoblasts (X1 and X2 cells). This is additionally supported by the fact that committed progeny of neoblasts has been also observed in postmitotic areas (Newmark and Sánchez Alvarado, 2000) and that smedwi-1 transcript is mainly expressed in X1 cells − with a similar spatial distribution to that observed for most neoblast markers in both species, but SMEDWI-1 (protein)-positive cells are detected in both X1 and X2 subpopulations (see Fig. S3) with spatial distribution extended to postmitotic areas (Guo et al., 2006). The significance of the differences in smedinx-11 expression in D. japonica and S. mediterranea is an interesting area that could be addressed after individual roles of DjInx-11 are functionally probed in D. japonica.

• Supplemental Figure S2 -

  Fig. S2. Schematic of the indirect laterality-based GJC permeability assay in Xenopus. To test the GJC properties of these innexins, we used an assay in which the behavior of wild-type, dominant-negative and constitutively active gap junction proteins is very well characterized, thus allowing us to compare the behavior of smedinx-11 to known gap junction genes in a context that is more physiological (related to control of patterning) than in vitro cell-pairing assays. In frog embryos, the endogenous difference in gap junctional coupling − connectivity along the dorsal blastomeres and isolation between the ventral blastomeres (A) is crucial to normal laterality and asymmetric positioning of the visceral organs (Levin and Mercola, 1998; Levin and Mercola, 1999). Establishing ectopic gap junctional coupling by injection of connexin mRNA into the ventral blastomeres into the 4-cell stage (B) results in an independent randomization of subsequent organ positioning (heterotaxia) because of the breaking of the necessary isolation on the ventral side (Esser et al., 2006; Fukumoto et al., 2005; Levin et al., 2006). (C) By contrast, introduction of additional junctions by injection of connexin mRNA on the dorsal side does not affect asymmetry because those cells are normally coupled anyway. This same strategy was used to test smedinx-11: when injected on the ventral side, it randomized the left-right axis just as connexin-based gap junctions do (35% incidence of heterotaxia, P<<0.01), while control injections on the dorsal side or injections of a non-functional mutant of this innexin protein had no effect on asymmetry (3% and 4% heterotaxia, respectively). This behavior exactly mimics the behavior of connexin-based gap junctions (but not most proteins) in this assay, and indicates that the ability of this innexin to randomize asymmetry is dependent on its modulation of the native gap junctional communication in the embryo.

• Supplemental Figure S3 -

  Fig. S3. smedinx-11 is expressed in the mesenchyme including postmitotic areas and regenerating blastema. Representative in situ hybridization on longitudinal section (8 μm thickness): bright-field pictures showing smedinx-11 signal (small dots of purple precipitation), left column with respective PI nuclei counterstaining (middle) and merge images (right) in A, B and C, respectively. (A) Anterior areas of the worm. Photoreceptors and pigment cup are labeled as PR and cephalic ganglia as CG. Note that smedinx-11 expression is extensively distributed at different levels throughout the mesenchyme, in tissue anterior to the photoreceptors and nervous tissue (some positive cells are pointed with arrows). Dorsal is up and the most-anterior part is to the left in all panels. (B) In situ hybridization of smedinx-11 showing the pharynx (c-shaped structure). Some positive cells (purple precipitation) are indicated with arrows. Dorsal is up and anterior is left. Scale bar is 20 μm. (C) smedinx-11 expression during regeneration. In situ hybridization on longitudinal section 4 days after amputation. The plane of amputation is shown with a white dotted line. Note the presence of smedinx-11-positive cells in newly formed tissue (right side of the picture). Scale bar in all cases is 20 μm. To improve visualization, purple signal in merge image was pseudocolored to green using Adobe Photoshop image software.

• Supplemental Figure S4 -

  Fig. S4. Immunostaining with anti-SMEDWI-1 antibody in FACS-isolated cells. Different cell populations after flow cytometry experiment were obtained (X1, X2 and Xins) from wild-type animals and immunostaining with anti-SMEDWI-1 (kindly provided by P. Newmark) was performed. Percentages of SMEDWI-1-positive cells are shown for each cell population (X1: 541/556, X2: 407/429 and Xins: 12/423). Note that SMEDWI-1 protein is enriched in X1 and X2 but not in Xins cells. Nuclei counterstaining is shown with Hoechst (blue signal, left most column), anti-SMEDWI-1 antibody (green signal, middle column) and merge image (right-most column). In each case, some positive and negative cells are shown (green signal +/−, respectively). Scale bars are 20 μm.

• Supplemental Figure S5 -

  Fig. S5. smedwi-2(RNAi) does not affect smedinx-11 expression. Gene expression analysis by qRT-PCR using total RNA extracted 8 days after smedwi-2 dsRNA exposure. Expression patterns in control and smedwi-2(RNAi) worms are shown. Note that smedwi-2 expression after RNAi is dramatically reduced compared to the control, while expression for smedinx-11 (labelled smedxin-11 here) remains unchanged. Values represent average of triplicate experiments and error bars indicate s.d. Gene expressions are relative to the ubiquitously expressed clone H.55.12e (Reddien et al., 2005b).