Unlike embryos of many higher organisms, the sea urchin embryo is completely transparent, allowing direct observation of specific cellular recognition events. It is therefore a suitable model for studying cellular interactions that offer insights into mechanisms of adhesive recognition in higher organisms. Moreover, experiments can be performed in seawater without the requirement of expensive tissue culture systems which is another advantage of using sea urchin embryos for such studies. Billions of embryos are easily obtainable and molecules involved in cellular interactions are easily accessible for molecular probes.

We are engaged in a series of studies designed to investigate the molecular nature of cellular interactions in sea urchins with emphasis on one specific model cellular interaction. The interaction involves attachment of secondary mesenchymal cells at the advancing tip of the archenteron (primitive gut) to cells in the roof of the blastocoel. We have shown previously that the mannose/glucose-binding lectin Lens culinaris agglutinin can enter living sea urchin embryos and cause a phenomenon known as exogastrulation. Exogastrulation is a process by which the developing archenteron, instead of attaching to the roof of the blastocoel, does not attach and instead falls out of the embryo proper.

In the present study, we examine the putative role of sugar-containing ligands and receptors for these ligands in mediating cellular recognition events in sea urchin embryos with emphasis on their role in archenteron development and in the attachment of secondary mesenchymal cells to the blastocoel roof. We have used a variety of approaches including incubations with glycosidases, inhibitors of synthesis of molecules containing carbohydrates, and derivatized beads. We have used derivatized beads in many previous studies. Use of these beads is a novel histochemical approach that does not require standard staining procedures. Here, we also report preliminary data on putative ligands and receptors that may mediate adhesive recognition events that are studied. We provide evidence that these molecules are released into the supernatant when sea urchin embryos are disaggregated into single cells with the use of calcium- and magnesium-free seawater (CMFSW).

Material and methods

Lytechinus pictus sea urchin eggs and sperm were obtained and fertilization was performed as previously described. Embryos were cultured in artificial seawater (ASW) at 15°C. Swimming embryos (24–32 h old) were collected, pelleted and resuspended in CMFSW and washed 3 times in this solution. A pellet of embryos (1 ml) was resuspended in 15 ml CMFSW and pipetted 10–20 times to disaggregate them. Cell suspensions were centrifuged at 120×g for 5 min at room temp and supernatants were decanted from cell pellets. Single cells resulting from the disaggregation procedure were healthy and viable as indicated by ciliary movement of isolated cells when resuspended in ASW. Calcium and magnesium were added to the supernatant to obtain concentrations that are present in ASW (6.4 mg MgCl₂, 2.9 mg MgSO₄, and 3.6 mg CaCl₂, in 12 ml supernatant). Supernatants were either used immediately or frozen at ?20°C. Single cells obtained with the disaggregation procedure were either used immediately in bead experiments as described below or fixed in 1% formaldehyde in ASW and then used at a later time. Fixed or live cells were found to bind in a similar way to derivatized beads.

For embryo incubation experiments, 24–32-h-old swimming embryos were suspended in 10 ml supernatant or 10 ml ASW (control) in 60×15 mm polystyrene Petri dishes and incubated for 24–48 h at 15°C.

For studies using enzymes and/or inhibitors, 24–32-h-old swimming embryos were incubated for 24 h in the presence of 1-5 mg sodium selenate (Sigma, St. Louis, MO, USA) per ml embryo suspension, 0.005-0.085 mg tunicamycin (Sigma) per ml embryo suspension, 0.5-1 mg ?-amylase (Sigma) per ml embryo suspension, 1 mg ?-glucosidase (Sigma) per ml embryo suspension, or 5 mg ?-mannosidase (Sigma) per ml embryo suspension. These concentrations were selected on the basis of preliminary studies generating concentration–effect data to identify concentrations that were effective, whereas embryos were maintained healthy, and swimming. In all cases, the reagents at the concentrations used had specific effects, whereas viability of the embryos was maintained as demonstrated by continued swimming and/or ciliary movement. The reagent concentrations that showed activity were in the mg range except for tunicamycin that was effective at a concentration of 5 ?g presumably because, as has been shown previously, these amounts of reagents are required for the compounds to enter thousands of swimming embryos and to retain activity over a 24-h period. Especially in the case of enzymes, that cleave sugars, enough enzyme molecules have to be present continuously because sugars are likely to be regenerated and then cellular interactions under study may not be blocked.

For the bead experiments, single cells were washed 3 times and mixed (approx. 10⁶ cells/ml) in droplets on glass microscope slides with approx. 1 mg agarose beads that were washed 3 times and were derivatized with L. culinaris agglutinin (Sigma), concanavalin A (Sigma), or mannan (Sigma) in the presence or absence of 0.2 M ?-methyl-mannose or 0.2 M -fucose. Samples in each set of experiments were photographed using an Axiolab photomicroscope (Zeiss, Oberkochen, Germany) with Kodak (Rochester, NY, USA) TMAX400 professional black and white film. All experiments were repeated at least 3 times by 2 or more investigators and results were found to be consistent and reproducible.