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Fig. S1. Proximal tubule defects cause edema formation. (A) Knockdown of inx10 or the cilia component IFT172 causes severe bloating in uncut RNAi-fed animals 14 days after the last feed. Scale bar: 500 µm. (B) A 10-fold increase in environmental osmolarity for 24 hours phenotypically rescues inx10(RNAi)-induced bloating (bottom right), without noticeable effects on control animals (top right). Randomly selected animals are shown. Scale bar: 1000 µm. (C) Transverse sections through the tail region stained with the protein dye Toluidine Blue. The drastically lowered cell density in bloated inx10(RNAi) animals in a regular environment (center) nearly returned to control levels (left) after 24 hours exposure to high external osmolarity (right). VNC, ventral nerve chord; g, gut. Scale bar: 50 µm.
Fig. S2. EGFR-5 structure and sequence analysis. (A) Domain structure (SMART, http://smart.embl-heidelberg.de/) of SMED-EGFR-5 and its two closest human (HS) and fly (DM) homologs ERBB4 and EGFR, respectively. Note that the N-terminus of the SMED-EGFR-5 sequence is truncated. Fu, Furin-like repeats; TyrKc, tyrosine kinase domain. Dark blue bar, transmembrane domain, which is obscured in ERBB4 owing to overlap with an Fu prediction. Pink rectangles, disordered regions. Scale bar indicates number of amino acids. (B) Blast e-values, obtained by blasting the SMED-EGFR-5 sequence against human (H.s.) or Drosophila melanogaster (D.m.) non-redundant protein sequences. The top hits in both cases were EGFR homologs. Despite the high e-value, SMED-EGFR-5 is unlikely to be an ortholog of vertebrate ERBB4. Preliminary sequence analysis (J.C.R., unpublished) suggests that the five planarian EGFR homologs arose from an independent expansion of this gene family. (C) Amino acid sequence alignment (Clustal-W) between human (Hs) ERBB4, D. melanogaster (Dm) EGFR and SMED-EGFR-5. The N-termini of Hs-ERBB4 and Dm-EGFR were cropped to the length of SMED-EGFR-5. Identical (*), conserved (:) and similar (.) residues are indicated. The tyrosine kinase domain is highlighted in orange, the individual transmembrane domain predictions (SMART) in gray. The regions corresponding to the five Furin-like repeats in the SMED-EGFR-5 sequence are underlined in blue.
Fig. S3. EGFR-5(RNAi) regeneration phenotypes. (A) Protonephridial marker gene expression in regenerated control and EGFR-5(RNAi) animals 14 days after amputation. Each pair was developed under identical conditions. Only regenerated heads and tails are shown. Scale bars: 100 µm. (B) Magnified view of left anterior and left posterior blastema of regenerating trunk fragments of EGFR-5(RNAi) animals at the indicated time points after amputation. Refer to Fig. 4 for control. Dashed lines demarcate the boundary between old and new tissue, as inferred from autofluorescence in the infra-red channel (not shown). α-Tubulin antibody staining (blue) was combined with the flame cell probe (DNAH-β3, red). Images are maximum projections of confocal z-sections. Scale bars: 100 µm. (C) Representative examples of regenerating EGFR-5(RNAi) animals, hybridized with the EGFR-5 antisense probe, at the indicated day of regeneration. EGFR-5 expression was below the detection threshold on day 1, but clearly detectable at day 6. The hazy staining at intermediate time points is likely to be background. The fragments were all treated and developed in an identical manner, except for day 14 animals, which were from a separate experiment. Scale bars: 100 µm.
Fig. S4. Gradual collapse of proximal units in non-regenerating EGFR-5(RNAi) animals. Representative examples of protonephridial units in the tail region from the dataset used to quantify the progressive flame cell loss depicted in Fig. 7C. For each of the indicated days, one control unit and three separate examples of proximal units in EGFR-5(RNAi) animals are shown. The images are confocal maximum projections of α-Tubulin staining. Scale bar: 20 µm.
Fig. S5. Differential RNAi effects in regenerating fragments. (A) Representative examples from a batch of EGFR-5(RNAi) animals 14 days after amputation, showing the four classes of branching phenotype. inx10 in situ hybridization was used to visualize proximal branching. 'Defective branching' was defined by abnormal thickening of branches or projections away from the head margin; blastemas displaying at least one extended branch were scored as 'minimal branching'. Scale bars: 100 µm. (B,C) Color-coded population frequency of the above phenotypic categories in 14-day regenerates exposed to four different RNAi dosage regimes. In order to dilute the RNAi dosage, EGFR-5 dsRNA-expressing bacteria were diluted with bacteria expressing an irrelevant control dsRNA, such that the absolute amount of dsRNA was constant in all cases. '4×′ corresponds to the standard RNAi dosage used throughout this study, and '1×′ is � thereof. The number of animals in the cohort is listed below each bar. Head and tail blastemas were scored separately and the results are shown in B and C, respectively. (D,E) Morphological phenotype assessment of the two RNAi dosage regimes used in Fig. 6C. The population frequencies of the phenotypic categories as defined and color-coded above were scored in the batch of 14-day regenerates used for the flame cell count. The number of animals in each cohort is listed below the bar. Head and tail blastemas were scored separately and the results are shown in D and E, respectively.
Movie 1. Animated 3D projection of protonephridial units stained for inx10, CAVII-1 and α-Tubulin. Shown are inx10 (red) and CAVII-1 (green) expression and anti-α-Tubulin staining (blue). The projection was generated from the same confocal z-stack as shown in Fig. 3C.
Movie 2. Animated 3D projection of protonephridial units stained for inx10, DNAH-β3 and α-Tubulin. Shown are inx10 (red) and DNAH-β3 (green) expression and anti-α-Tubulin staining (blue). The projection was generated from the same confocal z-stack as shown in Fig. 3C.
Movie 3. Animated confocal z-stack of protonephridial units stained for EGFR-5 and α-Tubulin. Shown are α-Tubulin antibody (green) and EGFR-5 expression (red). The movie progresses towards the dorsal surface.
