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First published online 14 February 2007
doi: 10.1242/dev.02804

Development 134, 1191-1201 (2007)
Published by The Company of Biologists 2007
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Head regeneration in wild-type hydra requires de novo neurogenesis

Marijana Miljkovic-Licina*, Simona Chera, Luiza Ghila and Brigitte Galliot{dagger}

Department of Zoology and Animal Biology, University of Geneva, Sciences III, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.


Figure 1
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  		Fig. 1. cnox-2 expression in the interstitial cell, mechanoreceptor and neuronal cell lineages. (A) cnox-2 expression in adult polyps of Hv. (B) Apical cnox-2+ pairs of dividing i-cells (arrow) and differentiated neurons. (C) Gastric cnox-2+ i-cells, either single or twins, and clusters of synchronously dividing nematoblasts. (D) cnox-2+ apical multipolar neurons (arrows) detected with anti-ß-tubulin staining (see Fig. S1 in the supplementary material). (E) cnox-2+ gastric cells that correspond to i-cells and nematoblast clusters but not neurons (arrowheads). D and E are confocal views. (F) Distribution of the i-cells and nematoblast clusters among the gastric cnox-2+ cells during starvation counted on ten hydra. (G-J) cnox-2 expression in the sf-1 mutant maintained at permissive (18°C, G,H) or restrictive (26°C, I,J) temperatures for 2 days. (K-N) Absence of cnox-2 expression during oogenesis (K,L, arrows) and spermatognesis (M,N, arrowheads). Scale bars: 400 µm in A,G,I,K,M; 100 µm in H,J,L,N; 20 µm in B,C; 10 µm in D,E. ic, i-cell; nb, nematoblast; ne, neuron; oo, oocyte; te, testes.

 

Figure 2
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  		Fig. 2. cnox-2+ cells are cycling cells in intact hydra. (A-F) cnox-2+ (dark blue) and BrdU-labeled (green) cells detected in the body column (A-C) and the apex (D-F) after 2 (A,D), 24 (B,E) or 48 hours (C,F) continuous BrdU incubation. Arrows indicate i-cells and nematoblasts, arrowheads neurons. (G,H) Percentage of gastric (G) and apical (H) BrdU+/cnox-2+ cells (n=10). Scale bar: 40 µm. ic, single i-cell; 2ic, pairs of i-cells; nb, nematoblast; ne, neuron.

 

Figure 3
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  		Fig. 3. Apical neurogenesis is altered in RNAi cnox-2 knocked-down hydra. (A) RT-PCR assay showing cnox-2 expression levels in whole polyps (upper panel) or upper halves collected immediately after bisection (lower panel), exposed 5x and 9x to control (lanes 1,3,5,7) or cnox-2 (lanes 2,4,5,8) dsRNAs. (B-E) cnox-2 expression pattern in hydra exposed to Kazal1 (B) and cnox-2 (C-E) dsRNAs. Note the reduced size of hydra in C. (F,G) Number of apical (F) and gastric (G) cnox-2+ cells per hydra exposed once or 5x to control (lanes 1,4), Kazal1 (lanes 2,5), cnox-2 (lanes 3,6) dsRNAs. (H-N) Disorganization of the ANS upon cnox-2 silencing detected by anti-ß-tubulin staining (see Fig. S2 and Movie S1 in the supplementary material). (H,I,K,M) In control and Kazal1(-) hydra, the ANS formed of parallel sensory neurons (arrows) and meridien multipolar neurons (arrowheads) extends from the tentacle zone up to the mouth opening. (J,L) Reduced and disorganized ANS after 5x and 7x cnox-2 dsRNAs exposures. (M,N) Apical neurons are no longer detected after 9x cnox-2 dsRNA exposures. H,J,K,L: maximum projection confocal views; I,M,N: axioplan views. Scale bars: 400 µm in B,C; 200 µm in D,E; 50 µm in H-N. mo, mouth opening; tent, tentacules; tz, tentacle zone.

 

Figure 4
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  		Fig. 4. cnox-2 acts as an upstream regulator of the nematocyte and neuronal differentiation pathways. (A) Colocalization of the cnox-2 (green) and hyZic (red) transcripts in nematoblast clusters (a-c) and dividing i-cells (e-g). After 9x cnox-2 dsRNA exposures, expression of cnox-2 and hyZic is either abolished (i-k, hydra1) or residual (m-o, hydra2). In cnox-2(-) hydra1, Kazal1 expression in gland cells (purple, l,p) is similar to control (d,h). Scale bars: 30 µm in a-d,i-o; 5 µm in e-h,p. (B) Expression of genes expressed in the nematocyte (left) and/or neuronal (right) cell lineages of hydra exposed 5x to control (lanes 1,3,5,7) or cnox-2 (lanes 2,4,6,8) dsRNAs. Number of cycles: 18 except hyZic (24), msh (16), hyCOUP-TF (16). (C) Scheme depicting the putative regulators of interstitial stem cells, neuronal progenitors, interstitial cells, neurons and nematoblasts. ic, interstitial cell; ISC, interstitial stem cell; nb, nematoblast; ne, neuron; NPG, neuronal progenitor.

 

Figure 5
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  		Fig. 5. Concomittant de novo neurogenesis and cnox-2 expression at the early-late stage of head regeneration. (A) Anti-ß-tubulin immunostaining of the nervous system in head-regenerating halves (see also Fig. S3 in the supplementary material). Neuronal processes are absent from the tips (arrowhead, boundary indicated with dots) up to 32 hpa when neuronal precursors differentiate (arrows). (B-G) cnox-2 expression in the presumptive head region (arrows) detected about 20 hours before TBs emerge during both head regeneration at 24 hpa (B,D), and budding at stage 3 (G, arrows). (B) Enlarged views of the head-(arrows) and foot-(arrowhead) regenerating tips depicted in C. Note the absence of cnox-2+ cells in the 16 hpa head-regenerating tip. (D-G) cnox-2+ cells appear first as pairs of dividing i-cells (D), subsequently as neuronal percursors (E,G) and finally as differentiated neurons (arrows in F) in regenerating tips (D-F) and distal buds (G). (H-J) Transient variations in the cnox-2+ cell distribution in regenerating halves (percentages calculated over DAPI-stained ectodermal cells). (H) Decrease in the gastric cnox-2+ cell number. (I) Increase in cnox-2+ single i-cells at 16 hpa. (J) Early-late increase in cnox-2+ cells in head-regenerating tips. Scale bars: 200 µm in C; 100 µm in A,B,G; 20 µm in D,E,G right panel; 10 µm in F. ic, i-cell; ne, neuron; np, neuronal precursor.

 

Figure 6
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  		Fig. 6. cnox-2+ cells in head-regenerating tips that have just traversed the cell cycle. (A-C) Head-regenerating tips oriented toward the top, showing increasing density of BrdU-labeled (green) and cnox-2+ (red) cells, ultimately surrounding the mouth opening at 44 hpa (B,C). (D-F) Confocal views of BrdU+/cnox-2+ apical cells (green/red), which appear as asymmetrical pairs (D), differentiating processes (E, arrow) or dividing i-cells (F). Hydra (Hv), BrdU-labeled before amputation, were fixed at 20 hpa (A,D,E), 32 hpa (F) and 44 hpa (B,C). Scale bars: 10 µm. mo, mouth opening.

 

Figure 7
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  		Fig. 7. Correlation between the cnox-2+ cell number in head-regenerating sf-1 mutants and the regeneration phenotype. (A) Kinetics of head regeneration as assessed by TB emergence in wild-type hydra (black squares), sf-1 hydra maintained at 18°C (red diamonds) or 28°C (green circles). (B) Number of cnox-2+ apical cells in amputated sf-1 hydra maintained at 28°C and sorted according to their regeneration stage (n=22, 17, 16, 31, 14). (C-K) cnox-2 expression in amputated sf-1 hydra maintained at 28°C (C-H) or 18°C (I-K). (C) ball-shape stage-1; (D,I) elongated stage-2; (E,G,H,J) TB1 stage-3; (F) TB2 stage-4; (K) fully-regenerated stage-5. Endodermal staining is artefactual as samples were overstained. Scale bars: 100 µm.

 

Figure 8
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  		Fig. 8. Head regeneration is altered in cnox-2 RNAi hydra. (A) Delay in head regeneration after 5x (n=12) and 9x (n=15) cnox-2 dsRNA exposures. (B-J) Cellular analyses of head-regenerating hydra amputated after 5x exposures to dsRNAs. (B,C) Decrease in the number of cnox-2+ cells in head-regenerating tips (B) and body column (C) of lower halves at 30 and 48 hpa. (D-H) cnox-2 expression in head-regenerating hydra exposed to Kazal1 (D,F) or cnox-2 (E,G,H) dsRNAs. Right panels: enlarged views of tips (arrows), TBs (arrowheads) and body column (D,E). Note the small size and the delayed regeneration of animals shown in G,H when compared with F. (I,J) Anti-ß-tubulin staining of head-regenerating tips at 39 hpa in hydra exposed to cnox-2 (I) or control (J) dsRNAs. Results depicted in panels A,B-H,I,J correspond to three independent experiments. Scale bars: 200 µm in D-H; 100 µm in I,J.

 

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© The Company of Biologists Ltd 2007