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First published online 14 February 2006
doi: 10.1242/jcs.02807

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Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human SPINK1 pancreatic phenotype

Simona Chera^*, Renaud de Rosa^*,, Marijana Miljkovic-Licina, Kevin Dobretz, Luiza Ghila, Kostas Kaloulis and Brigitte Galliot^¶

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

View larger version (32K): [in a new window]   Fig. 1. Structure and sequence of the hydra Kazal1 protein. (A) Schematic view of the Kazal1 cDNAs and the deduced Kazal1 protein structure. The gray box highlights the divergent non-coding 5' sequences of H. magnipapillata. (B) Alignment of Kazal1 to selected KD-containing sequences. The signal peptide is underlined and the cleavage site indicated by an inverted triangle.

View larger version (90K): [in a new window]   Fig. 2. Kazal1 is expressed in gland cells of intact H. vulgaris. (A-G) Kazal1-expressing cells were detected after whole-mount in situ hybridization with Fast-Red either in fluorescence (A-C,F,G, red) or in bright-fields conditions (A,C). To highlight the regions and the cells that do not express Kazal1, hydra were subsequently immunodetected with anti--tubulin (B,E, green). Arrows indicate the lower (A) and the upper (B) limits of the Kazal1 expression domain along the body column. In C, a closer view of the body column region shows that Kazal1-expressing cells are restricted to the endodermal layer. end, endoderm; ect, ectoderm; mes, mesoglea. (D-G) Confocal views of the endodermal layer. Kazal1-expressing cells contain secretory vacuoles (arrows) characteristic of gland cells. The star indicates the nucleus of a gland cell that does not express Kazal1. Bars, 150 µm (A,B), 40 µm (C), 16 µm (D-G).

View larger version (27K): [in a new window]   Fig. 3. The dsRNAi feeding strategy in hydra. (A) Scheme describing the production of dsRNAs in bacteria and the feeding of hydra with the bacteria-agarose mix. (B) Evolution of the size of the H. vulgaris populations exposed three times a week to unc-22 dsRNAs over 5 weeks; s.d. values correspond to four populations (50 hydra at t0) followed in two distinct experiments. (C) Variations in hydra body length over the first 2 weeks of unc-22 dsRNA treatment. The values correspond to the percentages of the animal lengths at each time point over their size at t0; s.d. values correspond to ten hydra. (D) Appearance of tentacle rudiments in head-regenerating halves exposed either 1x, 3x, 8x or 19x to unc-22 dsRNAs (15 hydra per condition).

View larger version (91K): [in a new window]   Fig. 4. Silencing of the Kazal1 gene in intact adult H. vulgaris. (A-E,H,I) Kazal1 expression was detected after whole-mount in situ hybridization with (A,B) NBT-BCIP and (C-E,H,I) Fast-Red. A decrease in the level of Kazal1 expression was observed after 2 to 5 exposures to Kazal1 dsRNA (A,C,D), but not in hydra treated with (B) unc22 dsRNA or (E) agarose. (F,G) Tsp1 expression was not altered in hydra exposed 5x to Kazal1 dsRNA (upper) when compared to untreated animals (lower). Confocal views showing the residual or undetectable level of Kazal1 expression in gland cells of hydra exposed 5x to Kazal1 dsRNA (I) when compared to control hydra treated with agarose (H). DAPI staining (blue), -tubulin staining (green). Bars: 150 µm (C-E,F), 4 µm (H,I).

View larger version (44K): [in a new window]   Fig. 5. Survival and budding rates of Kazal1(–) H. magnipapillata. (A) Sizes of the hydra populations exposed to Kazal1 dsRNAi. After each feeding hydra were counted and the percentage of Kazal1-silenced hydra over the control population exposed to the empty vector dsRNA was calculated. Each curve corresponds to a different experiment; 100, 65 and 60 hydra were initially taken for RNAi-a, RNAi-b and RNAi-c, respectively. (B) Budding rates observed in Kazal1(–) and control hydra populations in (a) RNAi-a, (b) RNAi-b and (c) RNAi-c experiments. The budding rate corresponds to the ratio between the number of new hydra produced by budding over the total number of hydra. Dark bars, control hydra; light bars, Kazal1(–) hydra. (C) Duplication of the peduncle and basal region observed in two different Kazal1(–) hydra after three feedings (RNAi-b).

View larger version (65K): [in a new window]   Fig. 6. Cellular disorganization of gland cells in Kazal1(–) intact hydra. (A-F) Confocal images of H. magnipapillata gland cells exposed up to 7x either to (A,D,F) Kazal1 or to (B,C,E) empty vector dsRNAs, subsequently immunodetected with (A,B) anti-RSK, (C,D) anti-–tubulin, (E,F) 6C4 anti-LBPA antibodies (green) and stained with (E,F) Hoechst 33342 dye (blue) and the mitotracker dye MitoFluor Red 589. (F) Anomalies from four distinct gland cells; arrows indicate fused vacuoles, arrowheads cytoplasmic portions that are expelled from the cell. Bars, 10 µm (A-D), 5 µm (E,F). (G) Evolution of the gland-cell index over the course of three independent RNAi experiments (RNAi-a, -b, -c). For each condition, the percentage of gland cells over doublets of dividing interstitial cells was measured in control and Kazal1(–) hydra. This percentage was stable in control hydra with a 90% to 100% value.

View larger version (94K): [in a new window]   Fig. 7. Cellular disorganization of digestive cells in Kazal1(–) intact hydra. (A-G) Endodermal epithelial cells from H. magnipapillata (A,B,G) and H. vulgaris (C-F) exposed up to 7x either to (A,C,D,G) Kazal1 or to (B,E,F) empty vector dsRNAs, subsequently immunodetected with either the (A,B) anti-RSK, or the anti--tubulin (C-G) antibodies (green). Stars indicate phagosomes, arrowheads digestive vacuoles mostly abundant at the apical pole, arrows point to the unusual vacuoles that appear after 4x exposures to Kazal1 dsRNAs. (G) Two different confocal sections of a highly disorganized digestive cell that exhibits a giant vacuole (arrow), nu: nucleus. (H) Ectodermal epithelial cells of H. vulgaris exposed up to 5x either to Kazal1 dsRNAs immunodetected with the anti--tubulin antibody (green). Bars, 8 µm. (I,J) Endodermal epithelial cells from H. magnipapillata exposed 4x either to Kazal1 (J) or to the empty vector (I) dsRNAs, labeled with the late-endosome marker anti-LBPA antibody (green) and a mitotracker MitoFluor Red 589 (red). Arrows indicate mitochondria that are either (I,Ja) cytoplasmic or (Jb-j) intra-vacuolic; arrowheads show the disruption of the cellular basal pole in c, two vacuoles that undergo fusion in h. Bars, 10 µm (I, Ja-c,e,h,j); 2 µm in (Jd,f,g,i). In every panel, the cellular basal pole is at the top and nuclei were counterstained with Hoechst 33342 dye (blue).

View larger version (75K): [in a new window]   Fig. 8. Kazal1 silencing in regenerating hydra. (A-C) Kazal1 expression in wild-type regenerating hydra fixed 4, 6 and 30 hours after mid-gastric section. Notice the strong level of expression in the endodermal cells of the (A,B, arrowheads) foot-regenerating tips and (A,B,C, arrows) head-regenerating tips. (D,E) Kazal1 expression in H. magnipapillata having regenerated 4 hours is similar to the wild-type pattern in control hydra exposed 6x to (D) empty vector but strongly silenced after 6x exposures to (E) Kazal1 dsRNAs. The arrow and arrowhead indicate the residual expression in head- and foot-regenerating tips, respectively. (F) In intact hydra 4x exposures to Kazal1 dsRNAs (right) suffice to completely silence Kazal1 expression when compared with the control hydra exposed to empty vector (left). Bars, 150 µm. (G) Kinetics of tentacle rudiments appearance were similar in regenerating H. magnipapillata exposed 3x to unc-22 () or to Kazal1 () dsRNAs. (H) Appearance of tentacle rudiments recorded 42 hours (left) and 44 hours (right) after mid-gastric section of hydra exposed up to 9x either to empty vector () or to the Kazal1 () dsRNAs.

View larger version (82K): [in a new window]   Fig. 9. The Kazal1(–) cellular phenotype is enhanced after amputation. (A,B) Confocal images of cells isolated from (a-j) head-regenerating tips (HR) and (k-o) foot-regenerating tips (FR) of H. vulgaris, 1 hour after bisection following 3 exposures to (a-e) empty vector or (f-o) Kazal1 dsRNAs. Cells were stained with Hoechst 33342 dye (blue) and immunodetected with the anti-hyCREB (red), anti-RSK (green) antibodies. Bars, 16 µm. (B) Staging of the Kazal1(–) endodermal phenotype. Stage (st.) 0, normal, elongated shape, basal nucleus, homogenous RSK-staining; st.1, perinuclear vacuolization; st. 2, multiple non-confluent cytoplasmic vacuoles; st. 3, confluent vacuoles occupying most of the cytoplasm. Small digestive vacuoles that provide false red staining (star) were occasionally noted. (C) Frequencies of the respective phenotypes in control and Kazal1(–) hydra during head- and foot-regeneration. For each condition at least 300 endodermal epithelial cells were counted.

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