Localization of hatching enzyme in embryos and larvae of the sea-squirt Ciona intestinalis

A polyclonal antibody raised against the hatching enzyme of Ciona intestina lis (D'Aniello et al., 1997) was used on larvae of different ages in whole mount immunofluorescence experiments in order to localize the cells secreti ng the enzyme. After staining with FITC-conjugated second antibody, the lar vae were observed by confocal microscopy. Larvae just before hatching (9-10 hours after fertilization) showed the presence of the enzyme in the periph eral cells of the adhesive papillae. The newly hatched larvae showed fluore scence also in the epidermal cells of the tip of the tail. Higher magnifica tion confocal images of the papillae revealed bright fluorescence both in p eripheral 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 i n the hyaline cap for some hours until the beginning of metamorphosis, wher eas the fluorescence of the tip of the tail disappears. Following application of the antibody to Phallusia mamillata, the periphera l 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 ab ove the papillae and the anterior part of the cephalenteron. We never obser ved fluorescence in the cells of the epidermis of the tail. Retinoic acid (RA) treatment has been used to confirm the localization on t he 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 p ositively reacted to immunofluorescence stain with anti-hatching enzyme ant ibody. On the contrary Phallusia mamillata larvate failed to hatch and did not show anti-hatching enzyme reaction in the tail.