DEV02804 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Specific anti-β-tubulin immunostaining of neurons, stenotele nematocytes and interstitial cells detected after tyramide labeling on whole-mount hydra. (A,B) Tyramide labelling for 17 minutes is necessary and sufficient for achieving an excellent staining of the neurons (A). By comparison, longer labelling times, here 22 minutes (B), provide an ubiquitous non-specific staining. Notice the difference in the exposure times; views taken on Axioplan2 microscope (Zeiss). (C-F) Confocal views of the different morphological types of neurons (C-E) and nematocytes (F) stained with anti-β-tubulin after a 17 minute tyramide labelling. (G,H) At equivalent tyramide labelling times (17 minutes), anti-α-tubulin (red) and anti-β-tubulin (green) antibodies cross-react with different cell types in tentacles (G), heads (H) and body columns (not shown) of whole hydra. α-tubulin is strongly expressed in ectodermal myoepithelial cells. Scale bars: 10 ?m. (I-L) Anti-β-tubulin staining showing the dramatic reduction in the cell density, namely neuronal cell bodies, nematocytes and interstitial cells in Sf-1 Hydra maintained for 2 days at 26°C. Axioplan2 microscopic views of the apical region (I,J); the peduncle and basal disk (K); and the body column (L).

• Supplemental Figure 2 -

  Fig. S2. Disorganization of the apical nervous system (ANS) in cnox-2(-) hydra. Anatomy of the ANS after anti-β-tubulin staining in control (A,B), Kazal1(-) (C-E) and cnox-2(-) (F-I) hydra exposed five times to dsRNAs. Notice the lower complexity and reduced extension of the neuronal network in F-I compared with A-E: the density of sensory neurons (arrows) is lower, their systematic parallel orientation is lost and their connections to the ganglia neurons that form lines (arrowheads in E) is no longer visible. Views are oblique (A,B,H,I) or sagittal (C-G). Confocal views with maximal projections (C,E,G) Axioplan2 pictures (A,B,D,F,H,I). Scale bars: 50 ?m.

• Supplemental Figure 3 -

  Fig. S3. The early late de novo neurogenesis observed during head regeneration is restricted to the regenerating tip. (A-E) Confocal views of the nervous system in head-regenerating tips after anti-β-tubulin immunostaining. The head-regenerating tips are at the top. Neuronal processes are absent from the tips until 32 hours after mid-gastric section. (C,D) From 24 hours onwards, pairs of dividing interstitial cells are observed, providing neuronal precursors that differentiate (D). (E) The neuronal network surrounding the mouth opening becomes detectable at the time tentacle buds emerge, here 48 hours post-bisection. Scale bars: 100 ?m.

• Movie 1 -

  Movie 1. Anatomical organisation of the apical nervous system in Hydra vulgaris, as detected with anti-β-tubulin staining.