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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: Acta Histochem. 2005 Sep 21;107(4):243–251. doi: 10.1016/j.acthis.2005.06.009

A Novel Approach to Study Adhesion Mechanisms by Isolation of the Interacting System

Cathy Coyle-Thompson 1, Steven B Oppenheimer 1,*
PMCID: PMC1857332  NIHMSID: NIHMS19783  PMID: 16181663

Summary

For decades most investigations into mechanisms of adhesive interactions have examined whole organisms or single cells. Results using whole organisms are often unclear because it may not be known if a probe used in an experiment is directly affecting the cellular interaction under study or if it is an indirect effect resulting from action on some other structure or pathway. Here we develop a novel approach to isolate the structural components of a cellular interaction by dissecting them out of the organism to study them in a pristine environment away from all confounding factors. We used the adhesion between the archenteron and blastocoel roof of the sea urchin gastrula stage embryo as a model that can be replicated in many other developmental and pathological systems. The isolated components of the cellular interaction and those in the whole organism possessed identical cell surface receptors and adhesive affinities.

Keywords: Isolation of cell adhesion systems, sea urchins, gastrulation, archenteron attachment

Introduction

Although many cell adhesion molecules, ligands and receptors have been isolated and characterized (Cavallaro and Christifori, 2004; Kleen and Schachner, 2004; Gooding, et al., 2004; Martinez-Rico, et al., 2004; Barsegov and Thirumalai, 2005; Salinas and Price, 2005; Jiménez, et al., 2005; Roseman, 2001;Zhao, et al., 2001; Tangemann et al., 1999; Mah, et al., 2005), molecular mechanisms of adhesive interactions that control embryonic development, cancer spread and other cellular recognition events are not fully understood.

One of the major problems in examining mechanisms that control adhesive interactions in vivo is the difficulty in isolating the specific adhesive interaction from the many other interactions that occur in the organism under study. It is often the case that one can not be certain if a specific probe that influences the cellular interaction under study is directly affecting that adhesive contact or is influencing other interactions or molecular pathways in the organism. In addition, a probe that affects the adhesive contact may also affect other vital functions of the organism. Here we describe a novel approach to directly study specific cellular interactions by isolating those interactions from all other factors in organisms that might confound the results. We have developed this approach in the sea urchin embryo model but it can be adapted to many other developmental and pathological systems.

In order to introduce this new approach for studying isolated cellular interactions, we will briefly examine the gastrulating sea urchin embryo where the model interaction that we are studying occurs. Figure 1 shows that gastrulation in sea urchins involves invagination of the vegetal region forming the archenteron (Gustafson, 1963; Oppenheimer and Carroll, 2004). The cellular interaction that is the focus of this report is the adhesion of the secondary mesenchyme cells at the advancing tip of the archenteron to the blasotcoel roof (Figure 1). This interaction is essential for the formation of a functional gut tube.

Figure 1.

Figure 1

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Sea urchin gastrulation (Based on Gustafson, 1963; Oppenheimer and Carroll 2004).

Previous studies from this laboratory suggested that ligands containing D-mannose or D-glucose terminal groups bind to lectin-like receptors for these sugars that are present on the surface of the advancing archenteron and on the blastocoel roof (Latham, et al., 1998; Latham, et al., 1999; Khurrum, et al., 2004). Lens culinaris agglutinin, that binds to D-glucose/D-mannose residues, entered sea urchin gastrula embryos and bound to the archenteron and blastocoel roof, binding that was blocked by α-methyl-mannose (Latham, et al., 1998). This same lectin prevented attachment of the tip of the archenteron to the blastocoel roof resulting in exogastrulation, where the archenteron drops out of the embryo proper, an effect that was also blocked by α-methyl-mannose (Latham, et al., 1999). These results coupled with the finding that α-mannosidase and α-glucosidase also blocked this adhesive interaction (Khurrum, et al., 2004) suggested that glucose /mannose groups and their receptors were involved in the attachment of the archenteron to the blastocoel roof.

The problem with using whole embryos, however, to probe a specific cellular interaction is that many unknowns may be affected by the probes. It would be of widespread interest if we could isolate the cellular interaction under study from the possible confounding factors? That concept formed the basis of the experiments reported here in which we microdissect the archenteron and the blastocoel roof out of the embryo, using insect pins, isolating these pieces for study in a pristine environment away from all of the other embryonic components and interactions. This study provides a new approach to investigate cellular interactions that may be of widespread usefulness in many experimental systems.

Material and Methods

Gamete collection and fertilization

Eggs and sperm of Lytechinus pictus and Strongylocentrotus purpuratus sea urchins were collected by intracoelomic injection of 0.55M KCl (Sigma, St. Louis, MO). Eggs were washed twice in pH 8.0 artificial seawater (ASW) and fertilized with freshly diluted sperm (0.1ml concentrated sperm/1.0ml ASW). Fertilized embryos were incubated at 15°C (Latham, et al., 1998; Latham, et al., 1999; Khurrum, et al., 2004). Samples of the embryos were collected, examined at the appropriate developmental stages and dissected or fixed and dissected.

Fixation of embryos

Embryos at 48–56 hours of incubation were collected in 50 ml and 15 ml FalconTM (B. D. Biosciences, San Jose, CA) centrifuge tubes and fixed with a final concentration of 3.7% formaldehyde (Ted Pella, Redding, CA) in ASW pH 8.0. Fixed embryos were stored at room temperature.

Slide preparation

Frosted on one side, precleaned Clay Adams (Becton Dikinson, Raleigh, NC) microscope slides (3"x 1") were coated with 0.5ml of Sigma coteTM (Sigma, St. Louis, MO) and allowed to dry in a fume hood for 24 hours. Slides were stored in a lint free slide box. Just prior to use, the coated slides were wiped with a KimWipeTM (Kimberly Clark, Dallas, TX).

Washing fixed embryos

A 200 microliter drop of ASW pH 8.0 was placed on the slide coated with Sigma coteTM. 100 microliters of fixed embryos were pipetted into the drop. The surrounding solution was removed and another drop of distilled water or ASW was added to wash the fixative solution off the embryos. Excess solution was removed and an additional drop of distilled water or ASW was added.

Dissection of whole fixed or live embryos

Whole fixed embryos were either dissected in fixative solution, or washed and dissected in distilled water or ASW. Live embryos were dissected in ASW. Embryos were placed in a 200-microliter drop of fixative solution in ASW (fixed embryos), distilled water (fixed embryos), or ASW (fixed or live embryos) on the slide coated with Sigma coteTM and photographed. Fine Elephant brand (Austria, BioQuip, Gardena, CA) size 00 or 000 insect pins were used to puncture the side of the blastocoel and catch the tissue of the embryo. A second pin was used to tease away the roof of the blastocoel from the tip of the archenteron. Careful dissection was performed to prevent the tearing of the archenteron by viewing the specimens during dissection with a Leica FZLIII stereo fluorescence dissecting microscope (Wetzlar, Germany). Dissected pieces were photographed. The dissected pieces were placed together to determine if the pieces would adhere to each other. Pieces that appeared to adhere were dragged through the solution to separate them. Pieces that adhered to each other did not come apart when dragged or bobbled in the solution by wiggling the slide. Pieces that did not adhere came apart.

Binding of lectin and inhibition of binding

A concentration of FITC-Lens culinaris agglutinin (Sigma, St. Louis, MO) of 0.045μg/μl in ASW was determined to be optimal so that no excess would confound the inhibition results. FITC-Lens culinaris agglutinin at a concentration of 0.045μg/μl in ASW was incubated for one hour on a slide coated with Sigma coteTM. A mixture of 0.045μg/μl FITC-Lens culinaris and 0.2M of the inhibitory monosaccharide of Lens culinari,s α-methyl-mannose (Sigma, St. Louis, MO) in ASW was also incubated for one hour on another slide coated with Sigma coteTM. Washed-fixed 52–54 hour embryos were dissected in the solutions. Binding of the lectin was observed after 10 minutes, 20 minutes and 1 and 2 hours and videotaped and photographed.

Photography of the embryos

A Zeiss (Oberkochen, Germany) Axiolab compound photomicroscope was used to photograph the whole embryos and the dissected pieces. Kodak TMax 400 or 3200 professional film was used for the black and white photographs and Kodak Max color film ASA 400 or 800 was used for the color photographs. A Nikon fluorescence microscope (Tokyo, Japan) with an Olympus camera (Tokyo, Japan) was used for the photographs in figure 3. A Zeiss fluorescence microscope was used for the photographs in figure 4.

Figure 3.

Figure 3

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Fixed whole 52-hour Lytechinus pictus embryos and pieces labeled with FITC-Lens culinaris agglutinin. 3A, B, C are fixed whole embryos. 3D, E, F are dissected blastocoel roofs and G, H, I are dissected archenterons from these fixed embryos. All bound FITC-Lens culinaris agglutinin after 30 minutes incubation in 0.045μg/μl FITC-Lens culinaris agglutinin in ASW. The embryos bound to the lectin were washed three times in ASW and photographed. The lectin did not appear to wash off. Scale: Diameter of sea urchin embryos @ 100μm.

Figure 4.

Figure 4

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Pieces of fixed Lytechinus pictus embryo archenterons bound to FITC Lens culinaris agglutinin and not to FITC Lens culinaris/α-methyl-mannose. 4A is a fluorescence image of an archenteron with FITC-Lens culinaris agglutinin without α-methyl-mannose. 4B is a bright field image of the same archenteron. 4C is a fluorescence image of an archenteron incubated with FITC-Lens culinaris agglutinin with α-methyl-mannose. 4D is a bright field image of the archenteron in 4C. Little or no fluorescence was observed on the embryos incubated with the FITC-Lens culinaris agglutinin with α-methyl-mannose (4C). Scale: Length of archenteron @ 80 μm.

Results and Discussion

In order to determine if the isolated pieces of the cellular interaction possess similar characteristics to those found in vivo, we asked two questions:

Do the pieces adhere as they do in vivo?

Do fixed and live pieces possess similar cell surfaces to the same structures in vivo?

To answer the first question we used fixed and live embryos. We are reiterating some methods detail here to help clarify the procedural context for each result. We dissected 31 fixed Lytechinus pictus and Strongylocentrotus purpuratus sea urchin gastrula stage embryos, isolating the roof of the blastocoel and the archenteron pieces. We did this with whole fixed embryos that were treated three ways. The first was with embryos fixed with 3.7% formaldehyde and dissected in fixative solution; second the embryos were fixed with 3.7% formaldehyde and washed in distilled water and dissected in distilled water. The third set of 24 embryos were fixed with 3.7% formaldehyde, washed in artificial sea water (ASW), and dissected in ASW. Finally, 39 whole live embryos were dissected in ASW. In all cases embryos were 48–52 hours old at the stage when the tip of the archenteron is about to adhere to the blastocoel roof. In each of the cases, the dissected tip of the archenteron firmly adhered to the dissected blastocoel roof. The adhesion did not break when the adhering pieces were dragged through the solution with the dissecting pins or jostled on the slide by tapping with forceps. The results were the same in five repeated experiments (Figure 2).

Figure 2. Whole fixed embryo dissection.

Figure 2

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Figures 2A, B show intact 52-hour sea urchin embryos. 2C, D, E show the microdissected archenteron (2E) and blastocoel roof (2D) pieces from the embryos in A, B. 2F, G show that these two microdissected "pieces’ from C, D and E adhered to each other when they were isolated from intact fixed embryos. The structural integrity of the fixed pieces was better than those obtained from live embryos (not shown), but in both cases, the pieces adhered to each other. Scale: Diameter of sea urchin embryos @ 100μm.

The fixed whole embryo gastrulas are shown in figures 2A and 2B. The archenteron tip is close to the roof of the blastocoel. The embryos were dissected and pieces of the roof of the blastocoel were separated from the tip of the archenteron as shown in figures 2C, 2D and 2E. The cells lining the tip and the sides of the archenteron are clearly observed. Figures 2F and 2G show the pieces of the tip of the archenteron adhering to the roof of the blastocoel. Dissected pieces of fixed embryo tissue adhered together when dissected in fixative solution, distilled water or ASW. This experiment was repeated 11 times with 48–56 hour Lytechinus pictus and 13 times with 48–56 hour Strongylocentrotus purpuratus gastrula stage embryos. In all experiments, pieces of the tip of the archenteron adhered to pieces of the roof of the blastocoel.

Live embryos were also dissected in ASW. However, the morphology of the tissue was not as structurally well defined (data not shown.). Live embryo pieces of the archenteron tip and blastocoel roof adhered together in ASW. A total of 21 (48–52 hour) live Lytechinus pictus embryos and 18 (48–52 hour) live Strongylocentrotus purpuratus embryos were dissected and the pieces did adhere to each other. The pieces stayed alive as indicated by cilia movement and did not de-adhere when moved through the solution. These results indicate that both fixed and live pieces adhere in at least two different species of sea urchin.

It was more advantageous to use fixed material in these studies, as live pieces move and change and begin to lose their structural integrity. In order to answer the second question and determine if fixed and live pieces possess similar cell surface lectin receptors as those found in vivo, we observed the binding of the lectin from Lens culinaris agglutinin to fixed dissected pieces of sea urchin embryos. We knew from previous studies on live embryos, that the archenteron and blastocoel roof strongly bound to FITC-Lens culinaris agglutinin, the lectin that also caused exogastrulation (Latham, et al., 1998; Latham, et al., 1999). Binding of Lens culinaris agglutinin to these structures was inhibited by α-methyl-mannose, which also blocked the exogastrulation-inducing effect of the lectin (Latham, et al., 1998; Latham, et al., 1999). Previous studies (Latham, et al., 1998; Latham, et al., 1999) therefore suggested that Lens culinaris agglutinin receptors are involved in the adhesion of the tip of the archenteron to the blastocoel roof because this lectin blocked that interaction.

Here we show that the fixed pieces also strongly bound FITC-Lens culinaris agglutinin and this binding was inhibited by α-methyl-mannose (Figures 3, 4). In addition, the fixed pieces with bound FITC-Lens culinaris agglutinin were much less adhesive to each other than when the lectin was blocked from binding with α-methyl-mannose, suggesting that Lens culinaris agglutinin binds to adhesion-mediating ligands, as also suggested in the live whole embryo study (Latham, et al., 1998; Latham, et al., 1999). As seen in figures 3 and 4, Lens culinaris agglutinin bound to the entire archenteron and blastocoel roof as also found in live whole embryos (Latham, et al., 1998). This finding indicates widespread presence of receptors for this lectin that may function in many interactions in addition to the one under study. We speculate that these receptors are only one component in the adhesive interaction between the archenteron tip and blastocoel roof. The other component(s) may be more restricted to the tip of the archenteron and/or a more restricted region of the blastocoel roof. This would be a simple possible explanation for any observed adhesive specificity between these structures.

Whole and pieces of fixed 54-hour Lytechinus pictus embryos dissected in FITC-Lens culinaris in ASW, bound to the lectin as shown in Figure 3. These results were identical to those with live whole embryos (Latham, et al., 1998). A total of 20 Lytechinus pictus embryos were dissected in FITC-Lens culinaris in 4 repeated experiments. The FITC-Lens culinaris at a concentration of 0.045μg/μl was determined to be optimal so that no excess would confound the inhibition results below.

Whole and pieces of fixed 54-hour Lytechinus pictus embryos dissected in 0.045μg/μl FITC-Lens culinaris which was first incubated for one hour in a 0.2M solution of the Lens culinaris inhibitory monosaccharide, α-methyl-mannose in ASW failed to bind to the lectin as found in whole live embryos (Latham, et al., 1998). Figure 4 shows the dissected archenterons of two embryos. Figures 4A and B show the archenteron incubated with FITC-Lens culinaris and Figure 4C and D show the archenteron incubated with FITC-Lens culinaris and α-methyl-mannose. A total of 16 Lytechinus pictus embryos were dissected in a total of three repeated experiments with identical results.

The results indicate that the fixed pieces behave as do the structures in vivo in terms of binding affinity with each other. In each case for two species of sea urchin, the fixed tip of the archenteron adhered to the fixed roof of the blastocoel; the live pieces also adhered to each other. Furthermore, the Lens culinaris agglutinin receptor binding sites were preserved in the fixed tissue as shown by the fluorescent Lens culinaris staining pattern shown in Figures 3 and 4. This Lens culinaris binding is also inhibited in the fixed tissue by incubation of the lectin with the monosaccharide inhibitor α-methyl mannose, as in the living embryo (Latham, et al., 1998).

Thus this model system of isolated interacting pieces displays, like the living embryo, surface binding sites for Lens culinaris agglutinin, sites that have been implicated in mediating adhesion of the archenteron and blastocoel roof (Latham, et al., 1998; Latham, et al., 1999; Khurrum, et al., 2004).

Decades of studies that have used the sea urchin embryo as a model have provided numerous insights into cellular interactions that control developmental events (Mah, et al., 2005; Gustafson, 1963; Oppenheimer and Carroll, 2004; Latham, et al., 1998; Latham, et al., 1999; Khurrum, et al., 2004; Marsden and Burke, 1998; Harden and McClay, 1990; McClay, et al., 2004; Alliegro and Alliegro, 1991; Akasaka, et al., 1980; Angerer and Angerer, 2003; Asao and Oppenheimer, 1970; De Simone and Spiegel 1986; Hawkins, et al., 1995; Hertzler and McClay, 1999; Huggins and Lennarz, 2001; Ingersoll and Ettensohn, 1994; Katow, 1995; Kinoshita and Saiga, 1979; Latham , et al., 1995; McClay, et al., 2000; Miller and McClay, 1997; Neri, et al., 1975; Oliveri, et al., 2003; Oppenheimer and Meyer, 1982; 1982a Oppenheimer and Odencrantz, 1972; Roberson and Oppenheimer, 1975; Roberson, et al., 1975; Schneider, et al., 1978; Sherwood and McClay, 1999; Solursh, et al., 1986; Spiegel and Burger, 1982; Wessel and McClay, 1987). Still however, the molecular basis of the interaction between the archenteron tip and blastocoel roof is not well understood. This is probably the result of past failure to study this interaction in an isolated system, away from confounding factors. Our past work using live intact embryos (Latham, et al., 1998; Latham, et al., 1999) showed that Lens culinaris agglutinin caused exogastrulation and failure of attachment of the tip of the archenteron to the blastocoel roof. Even this work could not definitively determine if Lens culinaris agglutinin receptors were directly involved in the adhesive interaction or were involved in some step leading to the interaction. The current study directly examines the adhesive interaction and not more complex phenomena such as exogastrulation. This system, thus, allows study of the primary adhesive interaction (s) only.

The use of isolated structural components to study adhesive mechanisms is novel and elegant and could become a method of choice in many experimental systems.

Acknowledgments

Supported by grants from NIH NIGMS SCORE, NIGMS RISE, and the Joseph Drown Foundation. We thank Maria Elena Zavala for use of her fluorescence photomicroscope and Astrid Hernandez, Maria Khurrum, and Azaila Contreras for assistance with embryo preparation.

Footnotes

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