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Apoptosis – a death-inducing mechanism tightly linked with morphogenesis in Hydractina echinata (Cnidaria, Hydrozoa)

Developmental Biology of Animals, Faculty of Biology, University of Kaiserslautern, Erwin-Schroedinger-Strasse, Building 13, D-67663 Kaiserslautern, Germany

View larger version (75K): [in a new window]   Fig. 1. Pattern of apoptosis during CsCl-induced metamorphosis of planulae of Hydractinia echinata (fluorescence images). Animals were induced as described in 116 mM CsCl, fixed and subjected to TUNEL. (A) non-induced larva, (B) 30 minutes post induction (p.i.), (C) 60 minutes p.i., (D) 2 hours p.i., (E) 2.5 hours p.i. (anterior cap), (F) 3 hours p.i., (G) 6 hours p.i., (H) 12 hours p.i., (I) 19 hours p.i. In A-F the anterior larval end is located on the left side. a, anterior; p, posterior; h, hypostome anlagen; t,, tentacle anlagen. Scale bars, 100 µm.

View larger version (46K): [in a new window]   Fig. 2. Verification of the TUNEL assay (fluorescence images). Animals were induced as described (116 mM CsCl) and tested using (A,D) propidium iodide staining, (B,E) TUNEL reaction, and (C,F) BrdU labelling. (A-C) Larvae 3 hours p.i. The anterior larval end is on the left. (D-F) Small primary polyps 24 hours p.i. h, hypostome; s, stolon; t, tentacle. Scale bars, 100 µm.

View larger version (95K): [in a new window]   Fig. 3. Apoptotic DNA fragmentation in metamorphosing larvae of Hydractinia echinata. (A-C) Nucleosomal DNA (DNA laddering), in a 2% agarose gel, of larvae harvested over (A) the first hour p.i., (B) 6 hours p.i., (C) 24 hours p.i.; residual genomic DNA of high molecular mass is seen at the top end of each lane. (D-E) Nucleosomal aberrations in apoptotic cells (fluorescence of TUNEL-stained larvae). Arrows indicate micronuclei and arrowheads indicate DNA marginalisation of fragmented DNA at the nuclear envelope. Scale bars 20 µm.

View larger version (44K): [in a new window]   Fig. 4. Apoptosis and posterior morphogenesis (head formation). (A) Schematic of transverse dissection. Selected anterior fragments are termed ‘cap’, ‘quarter’, and ‘half’ of the animal. (B) TUNEL analysis of dissected ‘quarter’animals; "p", the new posterior end produced by dissection. (C) TUNEL-stained control animal; p, posterior. Scale bars, 100 µm. (D) Frequency of apoptosis and head development in the new ‘posterior ends’ of dissected larvae. Left, without wound healing; right, with 15 hours wound healing of dissected larvae, n=approx. 25. Yellow, animals positive for metamorphosis of a polyp head, green, larvae with apoptosis clustered in the (new) posterior end of the larva (TUNEL assay). Levels of significance are shown as: *0.1P0.01; **0.01P0.001; ***P0.001, statistical analysis was performed using G-test. Bars indicate 95% confidence limits.

View larger version (49K): [in a new window]   Fig. 5. Apoptosis and anterior morphogenesis (formation of stolons and basal plate). (A) Immunocytochemical (anti-GLWamide) staining of a partially metamorphosed larva with a larval anterior end. Arrows, ectodermal neurons of larval origin (anterior larval end); arrowheads, endodermal neurons of the primary polyp (formerly posterior larval end, now polyp head). (B-F) Pattern of apoptosis during GLWamide-induced metamorphosis of planulae of Hydractinia echinata. Animals were induced as described (33 µM GLWamide) fixed and subjected to TUNEL. (B) 30 minutes post induction (p.i.), (C) 3 hours p.i., (D) 6 hours p.i., (E) 12 hours p.i., (F) 24 hours p.i. Scale bars 100 µm. The anterior larval end is on the left. (G) Frequency of aberrant polyps obtained by induction with GLWamide (red bars, n=120) and Cs+ ions (blue bars, n=200). (H) Frequency of animals with non-apoptotic and apoptotic anterior halves compared to those undergoing aberrant (‘larval end’) or normal (‘stolons’) anterior metamorphosis, respectively. Blue bars, animals induced with CsCl (n=50); red bars, animals induced with GLWamide (n=70). Bars indicate 95% confidence limits.