Rearing of embryos

Ripe adult P. ochraceus collected from Saanich Inlet, British Columbia were kept in tanks of running seawater at the aquatic facility of the University of Victoria. The animals were kept under constant low light conditions to inhibit spawning and were fed with mussels. When cultures were desired the gonads were removed surgically from the adults and the animals were immediately euthanized by placing them in a –20°C freezer. The ovaries were washed with filtered seawater and placed in seawater containing 1-methyladenine for 75–90 minutes to mature the eggs. Once the germinal vesicles had broken down (indicating egg maturity) the mature eggs were washed by pipetting them into 300 ml of freshly aerated, filtered seawater and allowing them to settle. The process was repeated twice. Finally sufficient eggs to cover 1/2 the bottom of a 400 ml plastic beaker were suspended in 300 ml of seawater.

Testis were placed in plastic Petri dishes and allowed to contract to release ‘dry’ sperm. Sufficient ‘dry’ sperm was suspended in freshly aerated filtered seawater to create a slightly turbid suspension. The sperm were checked for activity using a Leitz phase contrast microscope and each beaker of eggs was fertilized with 5–10 drops of the active sperm suspension. The contents of the beakers were mixed using a meat baster and placed in a 12°C incubator. Once the eggs had settled (1–2 hours) most of the seawater was poured off and replaced with fresh aerated, filtered seawater and the eggs were allowed to develop at 12°C. The embryos were examined daily with a Zeiss Jena dissecting microscope until the mid-gastrula stage (Figure 1) was reached.

Fig. 1.

A semithin section through the gastrulae of the starfish Pisaster ochraceus stained with anti-hyaline layer antibodies and studied by fluorescent light microscopy. Staining is seen in the hyaline layer, lining of the archenteron and blastocoel. Arrowheads show the hyaline layer-positive granules located in the cytoplasm of the embryonic cells; ar, archenteron; arl, lining of archenteron; b, blastocoel; ec, ectoderm embryonic cells; hl, hyaline layer.

Purification of hyaline layer

The ECM of the hyaline layer was purified in accordance with a routine protocol (see Reimer & Crawford, Reference Reimer and Crawford1995). Cultures grown as above were harvested at the gastrula stage by gentle centrifugation (speed 3 on a clinical centrifuge for 3 minutes). The packed embryos were stored frozen at –20°C. The previously stored gastrulae were thawed quickly and solubilized in an equal volume of extraction buffer (TBS with 1% Brij 56 (polyoxethylene 10 cetyl ether (Sigma)) containing the protease inhibitors 1 mM iodoacetamide, 1 mM PMSF (phenylmethlysulphonyl flouride) (Sigma), 1 µg/ml pepstatin A (Sigma), homogenized at 4°C with 10 plunges of a Dounce tissue homogenizer followed by sonication on a Fisher probe sonic dismembrenator for 15 seconds at 45% power. The homogenate was then extracted for 30 minutes at 4°C, centrifuged at 100,000 × g for 1 hour. It was then dialysed against 20 mM Tris–HCL, 0.5 M NaCl, pH 7.4 containing the same protease inhibitor listed above overnight at 4°C. To prepare an antigen preparation that was enriched in ECM the dialysed extract was applied to a 10 ml Concanavalin A–Sepharose 4B column that had been equilibrated with 10 column volumes of TBS buffer and washed with 3 volumes of elution buffer (20 mM Tris–HCL, 0.5 M NaCl, pH 7.4 containing 0.5 M α-methyl mannoside). The column was washed with 10 column volumes of washing buffer (20 mM Tris–HCL, 0.5 M NaCl, pH 7.4) and eluted with 5 ml of the elution buffer. The antigen preparation was then concentrated to 4 mg/ml of protein mixed with an equal volume of Freund's incomplete adjuvant.

Preparation of antiserum

The hyaline layer antiserum was generated in accordance with a routine protocol (see Reimer & Crawford, Reference Reimer and Crawford1995). Antigens were isolated from gastrula larvae and injected intraperitonealy into 4 week old BALB/c mice. One month following the first injection the mice were boosted by second injection. Five days later, antiserum from a tail bleed was diluted in 1/100 in PBS-blotto. For control this antiserum part was used as a primary antibody to stain a 1 µm thick section of a P. ochraceus gastrula that had been fixed by freeze substitution and embedded in LR White (Polysciences, Warrington, Pennsylvania). The slide was then rinsed three times in PBS-blotto and stained with FITC labelled goat anti-mouse IgG diluted 1:256 in PBS-blotto for a further 1 hour at room temperature and observed on a fluorescence microscope. Controls consisted of sections in which non-immunized mouse serum was substituted for that from the immunized mouse.

Spleens were harvested from animals that showed positive staining and hybridomas were produced according to published methods (Reimer, Reference Reimer1994). Clones were checked for antibody activity by staining sections as described above using the tissue culture medium from each isolated clone as the primary antibody. Clones that exhibited a strong staining reaction with the hyaline layer were then expanded into 10 cm plastic Petri dishes. Once the cultures had reached logarithmic growth, the tissue culture medium was harvested, the cells were removed by centrifugation (800 × g for 10 minutes) and the media were stored at –20°C. For staining of sections the media were used directly.

Freeze substitution

Immunogold studies were performed on material fixed using the freeze substitution technique (Campbell et al., Reference Campbell, Crawford and Reimer1991; Crawford & Burke, Reference Crawford, Burke, Ettensohn, Wessel and Wray2004). Embryos for immunogold studies were reared and concentrated as described above. For fixation, the embryos were incubated in seawater containing 15% 2, 3 butanediol (Sigma) for 15–20 minutes in a conical centrifuge tube at 4°C. During this time the embryos settled to the bottom of the tube and most of the seawater was removed. Roughly 1 µl of the concentrated embryos was pipetted on to a freeze fracture grid held in very fine tipped cross-closing forceps. As much of the remaining seawater as possible was removed from the grid with the tip of a piece of filter paper. The grid was then plunged into liquid propane in liquid nitrogen. Following this 3–4 grids were rapidly transferred to absolute ethanol saturated with Alcian blue (Marivac/Caneco, Halifax, Nova Scotia) at –85°C and maintained at –85°C for 5 days to undergo substitution. The grids were then slowly warmed to room temperature and embedded in LR White (Polysciences, Warrington, Pennsylvania). Embryos were cut from the polymerized blocks, oriented and mounted on aluminium stubs with epoxy glue, sectioned and mounted on slot grids as described below.

Transmission electron microscopy

Gastrula stage larvae were concentrated by gentle centrifugation (250 × g for 1 minute) and the surplus water was removed by pipette. The cell suspension was fixed in 2.5% glutaraldehyde buffered with 0.2 M cacodylate buffer (pH 7.4). The osmomolarity of solution was adjusted to 1250 milliosmoles by adding NaCl. The materials were then post-fixed for 2 hours with 1% OsO₄ buffered with seawater. After dehydration in alcohol and acetone, the fixed cell suspension was embedded in Epon in large flat moulds. The Epon was polymerized for 48 hours at 60°C and the embryos were located using a dissecting microscope, cut out of the plate with a coping saw, oriented and mounted on the metal stubs. The embryos were then sectioned on a Sorvall Porter-Blum ultramicrotome MT-1 using a diamond knife. The sections were collected on metal slot grids covered by Formvar (Polysciences, Warrington, Pennsylvania) film, stained with uranyl acetate and lead citrate and photographed on a Hitachi H-7000 TEM.

Immunological staining

Material for immunofluorescence studies was sectioned at 1 µm and mounted on glass slides. The sections were incubated for 10–15 minutes in phosphate buffered saline containing 0.2% Carnation instant milk powder (PBS-blotto). They were then treated for 1 hour at room temperature in 1–2 drops of anti-hyaline layer hybridoma supernatant (full strength tissue culture medium in which high concentrations of hybridoma cells had been grown) and rinsed in two 10–15 minute changes of PBS-blotto. Following this the sections were incubated in FITC goat anti-mouse serum diluted 1:256 in PBS-blotto for 1 hour, rinsed in PBS and mounted in Gelvatol (Mosanto). Controls consisted of a normal (non-immunized) mouse serum in place of the anti-hyaline layer tissue culture medium.

The grids were incubated on droplets laid out on sheets of Parafilm (Fisher Scientific, Ottawa) in a moist chamber. The grids were then incubated for 10 minutes on two changes of PBS-blotto. This was followed by 1 hour incubation in the primary antibody which consisted of anti-hyaline layer hybridoma supernatant. The grids were then washed in PBS-blotto and incubated for 1 hour in the secondary goat anti-mouse IgG (Sigma) antibody conjugated to colloidal gold (size 20–25 nm) prepared according to the methods of Slot & Geuze (Reference Slot and Geuze1985). Following this the grids were washed in PBS followed by a rinse in distilled water. They were further stained with uranyl acetate and lead citrate and photographed as above. Controls were made by substituting normal mouse serum instead of primary antibody solution.