DEV01941 Supplementary Material

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• Supplemental Figure 1 - Fig.S1. Efficiency and specificity of RNAi-mediated gene silencing. (A) Smed-netR RNAi: expression of Smed-netR is downregulated in the newly regenerated cephalic region and throughout the animal. Seven days after amputation. Anterior to the left. (B) Smed-netrin2 RNAi: expression of Smed-netrin2 is downregulated in the newly regenerated cephalic region and throughout the animal. The expression of Smed-netrin1 is not affected. Seven days after amputation. Anterior to the left.

• Supplemental Figure 2 - Fig.S2. Alignment between Smed-netR and other members of the DCC family. (A) Genomic map of Smed-netR including 25 exons distributed along a genomic region >30 kbp, as predicted by whole genome shotgun sequences from S. mediterranea. Discontinuous lines represent current gaps in genomic sequence. (B) Identical amino acid residues are boxed in black; conserved residues are boxed in gray. Mouse neogenin, Drosophila frazzled and C. elegans UNC-40. Arrowheads point to the conserved cysteine residues in the four predicted Ig repeats. A putative transmembrane (TM) region is underlined. The intracellular conserved domain P3 is boxed. Alignment performed using ClustalW v1.8 (Thompson et al., 1994). Smed-netR accession number is DQ086493. (C) Maximum likelihood tree of several members of the DCC family of Netrin receptors. Values at the nodes obtained from 1000 quartet-puzzling replicates (Tree-Puzzle5.2). Accession numbers: Drosophila frazzled, U71001; C. elegans UNC-40, U70618; Mouse neogenin, NM_008684; Chicken neogenin, XM_413704; Zebrafish neogenin, AY029280; Rat DCC U68725; Human DCC, X76132; Xenopus DCC, U10986.

• Supplemental Figure 3 - Fig.S3. Expression pattern of Smed-semcap-1 and RNAi phenotype. (A,B) Smed-semcap-1 is expressed throughout the planarian CNS and also in the pharynx. (C,D) The anterior commissure (asterisks) connecting both cephalic ganglia is thinner after Smed-semcap-1 RNAi. (A,B) Anterior to the left. (C,D) Anterior to the top. Accession number for Smed-semcap-1 is DN294110.

• Supplemental Figure 4 - Fig.S4. Alignment between predicted planarian netrin proteins and other members of the Netrin family. (A) Full-length Smed-netrin1 is 2206 bp in length and contains an ORF of 683 aa; it is organized in six exons contained in a genomic region of ~19.6 kbp. The partial cDNA of Smed-netrin2 is 1806 bp in length and contains an ORF of 602 aa; it is organized in four exons contained in a genomic region of >13.6 kbp. The discontinuous line indicates a gap in genomic sequence. (B) Both genes show high degrees of similarity to netrins. Identical amino acid residues are boxed in black; conserved residues are boxed in gray. The conserved N terminal laminin domain VI, three EGF-like domains V, and a C domain are indicated. Alignment performed using ClustalW v1.8 (Thompson et al., 1994). Accession numbers for Smed-netrin1 and netrin 2 are AY945304 and AY945305, respectively. (C) Maximum likelihood tree of several members of the Netrin family. Values at the nodes obtained from 1000 quartet-puzzling replicates (Tree-Puzzle5.2). Accession numbers: Drosophila netrinA, U60316; Drosophila netrinB, U60317; Chicken netrin1, L34549; Mouse netrin1, NM_008744; Amphinetrin, AJ252166; Mouse netrin3, NM_010947; C. elegans unc06, NM_076764; Leech netrin1, AF101029.