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First published online March 22, 2004
Journal of Experimental Biology 207, 1523-1532 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00899

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In vitro delayed senescence of extirpated buds from zooids of the colonial tunicate Botryllus schlosseri

Claudette Rabinowitz and Baruch Rinkevich^*

Israel Oceanographic and Limnological Research, National Institute of Oceanography, Tel Shikmona, PO Box 8030, Haifa, Israel

View larger version (44K): [in a new window]   Fig. 1. General morphology of (A) an extirpated Botryllus bud in vitro at blastogenic stage `B' (x100) and (B) extirpated Botryllus zooid at blastogenic stage `D' (x40). as, atrial siphon; sb, secondary bud. Bars, 100 µm.

View larger version (137K): [in a new window]   Fig. 2. Morphological consequences for in vitro maintenance of extirpated buds and migrating cells. (A) Aggregations of hemocytes released from an isolated buds, day 7 of culture (x200). (B) Test cells attached to the plate (arrows) after migrating from an isolated bud, day 3 of culture (x200). (C) A hollow sphere development from an extirpated stage `B' bud (x200). (D) Epithelial monolayer originating from isolated and cultured buds, in 14 day-old culture (x100). (E) Phase contrast photomicrographs of 1-month old culture, with confluent epithelial monolayer grown from a bud isolated at stage `D', showing cuboidal cell morphology with a polygonal shape (x400). (F) A deteriorating monolayer, day 21 (x200). b, bud body; n, nucleus; pc, pigment cell; s, sphere; vc, vacuolated cell. Bars, 50 µm.

View larger version (119K): [in a new window]   Fig. 3. Histology and cytochemistry of extirpated buds and degenerating zooids isolated from Botryllus schlosseri colonies. (A–C) An epithelial monolayer that developed from an isolated zooid in mid-takeover, 14-day culture period (A, x200; B, x400). At 3 weeks incubation (C, x200), the monolayer has started to deteriorate. TUNEL assay did not reveal positive nuclei. (D) An epithelial monolayer originated from stage `C' bud (x200) at the deteriorating stage, 4 weeks in culture. TUNEL assay performed on this monolayer did not reveal positive nuclei. (E,F) Apoptotic cells in histological section carried out on the extirpated stage `D' and TUNEL-stained zooid (E, x200); also stained for general morphology by Azan (F, x200). (G,H) TUNEL-positive cells (arrowhead) in histological sections of 5-week old cultured buds, counterstained by Methyl Green (G, x1000; H, whole bud, x200). cb, cell boundary; dv, disintegrated visceral organs; e, epithelium; me, monolayer edge; s, stomach; v, vacuole; vo, visceral organ; n, nucleus. Scale bars, 50 µm.

View larger version (40K): [in a new window]   Fig. 4. Distribution of PKH-26 fluorescent dye in bud tissues. 2-week-old cultured buds developed two spheres clearly observed under light microscopy (A) but not under fluorescence microscopy (B). Bars, 100 µm (x100). b, bud; s, sphere.

View larger version (19K): [in a new window]   Fig. 5. 3H-thymidine incorporation in cultures of stage `C' extirpated buds assayed 1–14 days after isolation. Values are means ± S.D. The numbers of wells are indicated. On day 7, sphere growth was observed in 2 of 4 wells [7(I)]. Asterisk indicates significant difference (P<0.05, t test).