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Summary
A polyclonal antibody raised against the hatching enzyme of Ciona intestinalis (D'Aniello et al., 1997) was used on larvae of different ages in whole mount immunofluorescence experiments in order to localize the cells secreting the enzyme. After staining with FITC-conjugated second antibody, the larvae were observed by confocal microscopy. Larvae just before hatching (9–10 hours after fertilization) showed the presence of the enzyme in the peripheral cells of the adhesive papillae. The newly hatched larvae showed fluorescence also in the epidermal cells of the tip of the tail. Higher magnification confocal images of the papillae revealed bright fluorescence both in peripheral cells of the papillae and in the cavity between the tunic and the apex of the papillae (hyaline cap).
The swimming larvae maintain the fluorescence in the peripheral cells and in the hyaline cap for some hours until the beginning of metamorphosis, whereas the fluorescence of the tip of the tail disappears.
Following application of the antibody to Phallusia mamillata, the peripheral cells of the papillae of the newly hatched larvae were fluorescent and a bright fluorescence was also present between the two layers of the tunic above the papillae and the anterior part of the cephalenteron. We never observed fluorescence in the cells of the epidermis of the tail.
Retinoic acid (RA) treatment has been used to confirm the localization on the papillae of the cells secreting the hatching enzyme. The larvae of Ciona intestinalis were able to hatch because the cells of the tip of the tail positively reacted to immunofluorescence stain with anti-hatching enzyme antibody. On the contrary Phallusia tnamillata larvae failed to hatch and did not show anti-hatching enzyme reaction in the tail.