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First published online 20 July 2005
doi: 10.1242/dev.01941

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Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture

Department of Cell and Developmental Biology and Neuroscience Program, University of Illinois at Urbana-Champaign, B107 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA

View larger version (90K): [in a new window]   Fig. 1. Domain structure and expression patterns of Smed-netR in the intact and regenerating nervous system visualized by whole-mount in-situ hybridization. (A) Domain structure of Smed-netR compared to other members of the DCC family: frazzled (Drosophila), neogenin (mouse) and unc-40 (C. elegans). Smed-netR contains five predicted fibronectin type III repeats (NCBI CDD) (Marchler-Bauer and Bryant, 2004). (B,C) In intact planarians, Smed-netR is expressed in the cephalic ganglia, in the VNCs, in the nerve ganglia of the pharynx (asterisk) and in a few cells around the brain region. (D-I) Smed-netR expression during anterior regeneration. Arrowheads in E point to the brain primordia. (J-L) Smed-netR expression during posterior regeneration. Arrowheads in K point to the regenerated VNCs. Asterisks indicate the new pharynx. (B) Dark field image; (C-L) Nomarski differential interference contrast microscopy. All images are ventral views. (B-C) Anterior to the left. (D-L) Anterior to the top. Scale bars: 400 µm in B; 100 µm in C; 500 µm in D-I; 400 µm in J-L. cg, cephalic ganglia.

View larger version (130K): [in a new window]   Fig. 2. Defects in anterior CNS regeneration after Smed-netR RNAi. (A-F) Eleven-day regenerated cephalic ganglia in control (A-C) and dsRNA-injected (D-F) animals. In A and D, arrowheads point to the brain's anterior commissure and the red lines mark the positions of the VNCs. Note the lateral expansion of the cephalic ganglia in D. (B,E) Nuclear labeling of the same planes as A and D, respectively, showing the peripheral localization of the neuronal cell bodies. (C,F) Higher magnification of merged images of A and B, and D and E, respectively. Arrowhead in C points to a lateral branch projecting from the cephalic ganglia; the branches are reduced or absent in Smed-netR dsRNA-injected animals (F). (G-L) Eighteen-day regenerated VNCs in control (G-I) and dsRNA-injected (J-L) animals. By contrast to the parallel nerve cords observed in G and H, the Smed-netR dsRNA-injected animals regenerate a disorganized neural meshwork (J,K). (I,L) Higher magnification of merged images of G and H, and J and K, respectively. (A,B) Confocal projections through 10.4 µm. (D,E) Confocal projections through 12 µm. (C,F) Merge of two single confocal planes. (G-L) Single confocal planes. Anterior to the left. Scale bar: in L, 100 µm for A-B, D-E, G-H, J-K; 50 µm for C,F,I,L. cg, cephalic ganglia; g, gut; fc, flame cells.

View larger version (139K): [in a new window]   Fig. 3. Effects of Smed-netR RNAi upon uninjured regions of the animal and posterior regeneration. (A-B) Submuscular nerve plexus in the uninjured post-pharyngeal region of control (A) and dsRNA-injected (B) regenerating animals. The arrowheads point to some nerve fibers of the plexus. Asterisks mark the position of the VNCs deeper within the animal. (C-D) VNCs in the post-pharyngeal region of control (C) and dsRNA-injected (D) animals undergoing head regeneration. Ectopic nerve fibers appear between the VNCs of dsRNA-injected animals (arrowheads in D). (E-F) Newly formed VNCs in the regenerated tail 15 days after amputation; control (E) and dsRNA-injected (F) animals. Arrowheads in E and F point to the posterior end of the VNCs. (A) Confocal projection through 2 µm. (B) Confocal projection through 2.5 µm. (C,D) Single confocal planes. (E) Confocal projection through 2 µm. (F) Confocal projection through 2.5 µm. (A-C,E-F) Anterior to the left. (D) Anterior to the top. Scale bar: in F, 50 µm for A-D; 100 µm for E,F.

View larger version (67K): [in a new window]   Fig. 4. Defects in visual axon targeting in regenerating planarians and in phototactic behavior after Smed-netR RNAi. (A) Confocal projections showing the visual axons (VC-1 staining, magenta) relative to the cephalic ganglia (anti-phospho-tyrosine staining, green). In control animals, the visual axons target the brain visual center (arrowheads, n=45/50; 5/50 show minor defects in the visual pattern different from those described below). In Smed-netR dsRNA-injected animals the visual axons do not project posteriorly to the brain visual center (white arrowheads). A black arrowhead in the upper right panel labels an ectopic anterior projection along the midline. All the samples are 2-week regenerants. Anterior to the left. (B) Phototaxis assays. Twenty-five days after Smed-netR RNAi, the negative response to light is significantly slower compared with control and Smed-semcap1 RNAi animals (bottom right panel; *P<0.005; ns, non significant). After Smed-semcap1 RNAi, the anterior commissure is thinner compared with controls and Smed-netR dsRNA-treated animals (white arrows). White arrowheads point to the posterior end of the visual axons, which project more posteriorly after Smed-semcap1 RNAi. Asterisks indicate the position of the eye-cups. Anterior to the top. Scale bar for A: 100 µm. oc, optic chiasm.

View larger version (91K): [in a new window]   Fig. 5. Defects in visual axon projections and nervous system architecture in intact planarians after long-term Smed-netR RNAi treatment. (A-B) Merged confocal projections (VC-1 staining in green) and Nomarski (DIC) images (in gray), showing the visual axons of control (A) and dsRNA-injected (B) planarians. Arrowheads point to the posteriorly projecting visual axons. (C-J) Cephalic ganglia visualized by anti-phospho-tyrosine and Hoechst staining: (C-F) control animals; (G-J) Smed-netR dsRNA-injected animals. Single confocal sections were taken at the plane in which the ventral region of the brain overlaps with the dorsal portion of the VNCs (asterisks in C). (E,F) Higher magnification of C and D, respectively. The bracket in F indicates the juxtaposition of ventrolateral brain cells and the dorsolateral VNC cells. (G-J) The ventrolateral region of the brain (arrowheads in G and I) appears shifted laterally with respect to the VNCs (asterisks in G and I). (I,J) Higher magnifications of G and H, respectively. The bracket in J indicates the separation between ventrolateral brain cells (at the top of bracket) and the VNC cells (at the bottom of the bracket), yielding two discontinuous rows of nuclei. (K,M) Anti-phospho-tyrosine staining to visualize the submuscular nerve plexus from controls (K) and Smed-netR dsRNA-injected planarians (M). In K and M, arrowheads point to the plexus and the asterisks mark the position of the VNCs out of the focal plane. (L,N) Anti-tubulin staining to visualize the posterior VNCs of controls (L) and Smed-netR dsRNA-injected planarians (N). In N, arrowheads point to ectopic processes. All the samples shown were analyzed 4 weeks after RNAi treatment; C-J, L and N show single confocal planes; K and M show confocal projections through 2.5 µm. (A-B,K-N) Anterior to the top. (C-J) Anterior to the left. Scale bar: in B, 100 µm for A,B; in N, 100 µm for C,D,G,H,L,N and 50 µm for E,F,I,J,K,M.

View larger version (109K): [in a new window]   Fig. 6. Expression patterns of Smed-netrin1 and netrin2 in intact and regenerating CNS visualized by whole-mount in-situ hybridization. (A-D) Expression of Smed-netrin1 (A,B) and netrin2 (C,D) in intact animals. Expression of Smed-netrin2 during anterior regeneration: (E) 2 days; (F) 3 days; (G) 10 days. Arrowheads in F point to positive cells within the blastema. (A-D) Anterior to the left. (E-G) Anterior to the top. Scale bars: 500 µm in A,C; 100 µm in B,D; 100 µm in E-G.

View larger version (113K): [in a new window]   Fig. 7. Effects of Smed-netrin1 and netrin2 RNAi in the regeneration and maintenance of the planarian CNS. (A-B) Normal cephalic ganglia (A) and VNCs (B) regenerate after Smed-netrin1 RNAi. Arrowhead points to the commissure connecting the cephalic ganglia. (C-D) After Smed-netrin2 RNAi the anterior commissure is thickened (arrowhead in C) and the ganglia are wider than normal. The VNCs regenerate in a disorganized meshwork of projections (D). (E) Posterior uninjured region of an anterior regenerating animal showing ectopic processes (arrowhead) between the VNCs after Smed-netrin2 RNAi. (F) Newly regenerated tail region showing abnormal regeneration of the VNCs after Smed-netrin2 RNAi. All regenerants were fixed 2 weeks after amputation. (G-H) Disorganized neural pattern with ectopic axonal processes in the cephalic (G) and post-pharyngeal (H) regions of intact planarians 2 weeks after Smed-netrin2 RNAi. B,D,E,G and H are single confocal planes; A shows confocal projections through 5.6 µm; C,F show confocal projections through 4.8 µm. (A-D,F-H) Anterior to the left. (E) Anterior to the top. Scale bar: 100 µm. cg, cephalic ganglia.

View larger version (108K): [in a new window]   Fig. 8. Defects in visual axon targeting in regenerating and intact planarians and in phototactic behavior after RNAi for Smed-netrins. (A-F) Confocal projections showing the visual axons (VC-1 staining, bright green) relative to the cephalic ganglia (anti-phospho tyrosine staining, pale green) on 14-day regenerants. After Smed-netrin2 (B,C) and Smed-netrin1 + netrin2 (D,E) RNAi no posterior projections (B,E) or shorter (C,D) than in controls are observed. Anterior to the upper left corner. (G) Phototaxis assay. After Smed-netrin2, Smed-netrin1 + netrin2 and Smed-netR RNAi the negative response to light is significantly slower compared with controls and after Smed-netrin1 RNAi. Eighteen days of regeneration. *P<0.05; **P<0.005; ns, non significant. (H). The ratio between the length of the posterior axonal projections of the photosensitive cells and the cephalic ganglia is reduced in intact planarians 4.5 weeks after Smed-netrin1, Smed-netrin2 and Smed-netrin1 + 2 RNAi treatment. *P<0.05; **P<0.005.

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