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First published online 21 March 2007
doi: 10.1242/dev.02835

Development 134, 1679-1689 (2007)
Published by The Company of Biologists 2007
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Clathrin-mediated endocytic signals are required for the regeneration of, as well as homeostasis in, the planarian CNS

Takeshi Inoue1,2, Tetsutaro Hayashi1,*, Katsuaki Takechi1,{dagger} and Kiyokazu Agata1,3,{ddagger}

1 Group for Evolutionary Regeneration Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
2 KAN Research Institute, Inc., Kobe MI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
3 Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.


Figure 1
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  		Fig. 1. Expression patterns of DjCHC mRNA and protein. (A-C) DjCHC mRNA, analyzed by whole-mount in situ hybridization. (A) Dorsal view. a, anterior; p, posterior; Ph, pharynx. (B) DjCHC expression at 7 days after X-ray irradiation. (C) DjCHC sense probe. (D) DjCHC protein, analyzed by whole-mount immunostaining. VNCs, ventral nerve cords. (E) Higher magnification of a single section of the confocal image of the head region shown in D. (F) Schematic illustration of the head region of planarian. The planarian CNS, consisting of an inverted U-shaped spongy region (red) and nine lateral branches (green). (G) Upper panels show whole-mount in situ hybridization and lower panels show whole-mount immunostaining of planarians at 1, 2, 3, 5 or 7 days after amputation. Scale bars: 100 µm.

 

Figure 2
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  		Fig. 2. DjCHC-RNAi planarian could not regenerate the CNS. (A) Equal protein loadings of homogenates of 7-day-regenerated head region of either control or DjCHC-RNAi-treated planarians were subjected to SDS-PAGE using 7% gels, and western blots were probed with antibodies against the indicated protein. C, control; R, DjCHC-RNAi treated. (B) Dorsal view of the injected organisms (bright-field images). The left (control) and right (DjCHC-RNAi) panels show the 7-day regenerates of trunk pieces. The control planarian regenerated the tail and the head, as indicated by the dashed lines. The DjCHC- RNAi-treated planarian did not regenerate (arrowheads). a, anterior; p, posterior. (C) Defects of DjCHC-RNAi-treated planarians 7 days after amputation were detected in the CNS architecture, as revealed by immunostaining with anti-DjSYT and anti-DjGß. The dashed line indicates the border between the newly formed region and old stump region. Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. No effects on the tail, pharynx, intestinal tract or muscles of DjCHC-RNAi-treated planarian. (A) Control and DjCHC-RNAi-treated planarian head piece at 7 days after amputation showing tail regeneration. Upper panels show DjAbd-Ba expression (blue) visualized using whole-mount in situ hybridization. Lower panels show VNCs of regenerated head pieces visualized with anti-DjSYT. White arrows, VNCs; black arrows, commissure neurons. VNCs of DjCHC-RNAi-treated planarians were perturbed. The dashed line indicates the border of the newly formed region and old stump region. (B) Pharynx regeneration of the head piece of the control and DjCHC-RNAi-treated planarian 7 days after amputation. The upper panels show pharynx-specific-gene DjMHC-A expression (blue) by whole-mount in situ hybridization. The lower panels show nerves of the pharynx visualized using anti-DjSYT. The dashed line indicates the pharynx. (C) Regeneration of the intestinal tract of a tail piece of a DjCHC-RNAi-treated planarian 7 days after amputation. Schematic drawings in A-C show the original expression patterns. (D) Body-wall musculature of a control and DjCHC-RNAi-treated planarian 7 days after amputation visualized with anti-DjMHC-B. Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. DjCHC-RNAi treatment caused CNS atrophy. (A) DjCHC-RNAi treatment caused atrophy of the head and brain from 7 to 14 days after the injection of intact animals. (B) Numbers of head-atrophied planarians in control, X-ray-irradiated, DjCHC-RNAi-treated and DjCHC-RNAi plus X-ray-irradiated planarians. n=30. (C) BrdU-labeling of the trunk piece of control and DjCHC-RNAi-treated planarians. Samples were fixed 4 or 48 hours after BrdU treatment. a, anterior; p, posterior; ph, pharynx. (D) Frequency of mitosis in uncut planarians detected using anti-H3P. Confocal single projection of the fluorescent image of H3P-positive cells (magenta) and the CNS of the identical animal visualized using anti-DjSYT (green) are shown. Control, DjCHC-RNAi-treated and X-ray-irradiated planarians were fixed 14 days after RNAi treatment or irradiation. Animals are oriented with anterior towards the top. (E) The number of labeled mitoses determined using anti-H3P in planarians. Each column represents the average number (±s.e.m.) of anti-H3P-positive cells from 12 planarians. Similar data were obtained in experiments using BrdU. ***P<0.01. NS, not significant. Scale bars: 100 µm.

 

Figure 5
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  		Fig. 5. Molecular analysis of DjCHC-RNAi-treated planarians during regeneration. (A-D) Expression patterns of Djnlg (A), DjvlgA (B), ndk (C) and brain-specific homeobox genes (D) were analyzed by whole-mount in situ hybridization 12, 24 or 48 hours after amputation, as indicated. DjCHC-RNAi-treated planarians showed no differences in the expression patterns of Djnlg, DjvlgA, ndk, DjotxA, DjotxB or Djotp compared to those of control animals. (E) Schematic illustration of the discrete expression pattern of the brain-specific homeobox genes. (F) Defects of DjCHC-RNAi-treated planarians 72 hours after amputation were detected in the brain architecture using anti-DjSYT and anti-DjGß. Arrowheads, inverted U-shaped structure; arrows, lateral branches in regenerated blastema. The dashed line indicates the border of the newly formed region and old stump region. (G) Whole-mount immunostaining with VC-1 anti-planarian visual-neuron monoclonal antibody 7 days after regeneration. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Primary cultured neurons (R4HAC) obtained from control and DjCHC-RNAi-treated planarians by FACS. (A) FACS profiles of R4HAC from control body (left), control regenerated head (middle) and regenerated head of the DjCHC-RNAi-treated (right) planarians at 4 days of regeneration. Black ellipses indicate the R4HAC-specific peak of the head region. (B) Percentage (±s.e.m.) of R4HAC among the total cells. (C) Immunostaining with anti-DjCHC (green) and nuclear staining with Hoechst 33342 (blue) of R4HAC of control (upper panels) and DjCHC-RNAi (bottom panels) planarians. The graphs show the percentage (±s.e.m.) of DjCHC protein-expressing cells. (D) Phase-contrast views of R4HAC from control and DjCHC-RNAi-treated planarians. Arrowheads, debris remaining after neurite regression. (E,F) The percentage (±s.e.m.) of neurite-extending cells and the average neurite length per cell of control (white) and DjCHC-RNAi-treated (black) R4HAC. The percentage (±s.e.m.) of neurite-extending cells and average neurite length per cell of DjCHC-RNAi R4HAC compared to those of the control are indicated by the red lines. Data from 200-300 cells from the in vitro analysis were averaged. (G) Immunostaining of R4HAC using anti-DjCHC and DjSYT (green). For counterstaining, planarian 14-3-3{epsilon} was stained with a specific antibody (red). Nuclei were stained with Hoechst 33342 (blue). ***P<0.01. NS, not significant; DIV, days in vitro. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. TUNEL assay of control and DjCHC-RNAi-treated R4HAC. (A-D) TUNEL-positive signals (green) at 2 DIV were visualized in DjCHC-RNAi-treated R4HAC. Antibody staining showed the expression of planarian 14-3-3{epsilon} or DjCHC as counterstaining (red), and Hoechst 33342 staining showed nuclei (blue). White arrows indicate neurite-extending TUNEL-negative cells. White arrowheads indicate neurite-less TUNEL-positive cells. Yellow arrows indicate the DjCHC-positive cells in DjCHC-RNAi-treated R4HAC. Scale bar: 10 µm. (E) Percentage (±s.e.m.) of TUNEL-positive cells out of total cells during culturing. (F,G) The percentage (±s.e.m.) of TUNEL-positive cells in the DjCHC-protein-expressing and DjCHC-protein-negative cell fractions at 3 DIV. ***P<0.01; NS: not significant.

 

Figure 8
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  		Fig. 8. Clathrin-associated endocytic genes are required for proper brain regeneration. (A) Whole-mount immunostaining with anti-PC2 (Agata et al., 1998Go) of planarians at 7 days of regeneration treated with dsRNA of Djunc13A (AB281585), DjsbpA (AB281586) and Djsyt. (B) The phylogenetic tree was constructed by the neighbor-joining method using the full sequences. The tree includes the amino acid sequences of the mu-1 (µ1) and mu-2 (µ2) genes derived from Dugesia japonica (AB243058, AB243059), Homo sapiens (Q9BXS5, Q9Y6Q5, Q96CW1), Mus musculus (P35585, Q9WVP1, P84091), Drosophila melanogaster (EAL28715, AAF56001) and Caenorhabditis elegans (NP491572, AAA72418, P35603). (C,D) The expression pattern of Djmu-1 (Djµ1; C) and Djmu-2 (Djµ2; D). (E) Whole-mount immunostaining with anti-DjSYT of planarians at 7 days of regeneration that had been treated with RNAi for DjCHC, Djmu-1, Djmu-2, Djmu-1 plus Djmu-2, or DjCHC plus Djmu-2. The numbers in each panel represent the downregulated expression level of each gene analyzed by real-time PCR. The dashed line indicates the border between the newly formed region and the old stump region. Scale bars: 100 µm. (F) The numbers on the y axis indicate the percentage of head-atrophied planarians on each day after dsRNA treatment (x axis). n=30.

 

Figure 9
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  		Fig. 9. Schematic illustration of CNS maintenance and regeneration. In normal planarian regeneration (upper panel), blastema formation is followed by patterning and cell differentiation of neural cells, and then the neural cells are organized into neural circuits, which are maintained by homeostasis. In DjCHC-silenced planarians (lower panel), the patterning and cell differentiation of neural cells occur normally, but neurites regress and neural cells die via apoptosis, resulting in atrophy of the CNS.

 




© The Company of Biologists Ltd 2007