A Role for the L-Selectin Adhesion System in Mediating Cytotrophoblast Emigration from the Placenta

Developmental Biology 298 (2006) 107–117 www.elsevier.com/locate/ydbio

A role for the L-selectin adhesion system in mediating cytotrophoblast emigration from the placenta ⁎ Akraporn Prakobphol a, Olga Genbacev a, Matthew Gormley a, Mirhan Kapidzic a, Susan J. Fisher a,b,

a Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA b Departments of Anatomy and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA Received for publication 24 February 2006; revised 13 June 2006; accepted 13 June 2006 Available online 15 June 2006

Abstract

Cytotrophoblast (CTB) aggregates that bridge the gap between the placenta and the uterus are suspended as cell columns in the intervillous space, where they experience significant amounts of shear stress generated by maternal blood flow. The proper formation of these structures is crucial to pregnancy outcome as they play a vital role in anchoring the embryo/fetus to the decidua. At the same time, they provide a route by which CTBs enter the uterine wall. The mechanism by which the integrity of the columns is maintained while allowing cell movement is unknown. Here, we present evidence that the interactions of L-selectin with its carbohydrate ligands, a specialized adhesion system that is activated by shear stress, play an important role. CTBs in cell columns, particularly near the distal ends, stained brightly for L-selectin and with the TRA-1-81 antibody, which recognizes carbohydrate epitopes that support binding of L-selectin chimeras in vitro. Function-perturbing antibodies that inhibited either receptor or ligand activity also inhibited formation of cell columns in vitro. Together, these results suggest an autocrine role for the CTB L-selectin adhesion system in forming and maintaining cell columns during the early stages of placental development, when the architecture of the basal plate region is established. This type of adhesion may also facilitate CTB exit from cell columns, a prerequisite for uterine invasion. © 2006 Elsevier Inc. All rights reserved.

Keywords: Cytotrophoblast; L-selectin; TRA-1-81; Carbohydrate ligands; Adhesion/tethering

Introduction Subsequently, integrin function is also required for pattern- ing of the maternal–fetal interface. In humans, this phenomenon During development of the human placenta, the organ's is evident during formation of anchoring chorionic villi, which specialized cells, termed trophoblasts, orchestrate a complex se- physically attach the placenta to the uterus. Specifically, ries of stage-specific adhesive interactions with multiple hetero- mononuclear trophoblast (i.e., cytotrophoblast [CTB]) progeni- typic and homotypic components. We and many other groups tors, which lose their basement membrane attachments, form have focused on heterotypic adhesive processes, which play aggregates, termed cell columns, that bridge the gap between important roles in specialized trophoblast functions from the placenta and the uterus. At sites where columns reach the implantation onward. In this regard, cell–extracellular matrix uterine wall, CTBs detach from one another and invade the receptors are critical. For example, studies of murine blastocysts decidua. Eventually, CTBs occupy the entire endometrium and suggest that integrin-mediated signals generated by interactions the inner third of the myometrium as well as the uterine with fibronectin (Armant, 2005), laminin and other basement vasculature. The latter process, which establishes the utero– membrane components (Miner et al., 2004) regulate murine placental circulation, places these tumor-like placental cells in trophoblast differentiation at the blastocyst stage and, thus, direct contact with maternal blood. As CTBs move from the implantation. placenta into columns with subsequent migration into the uterine wall, they execute dramatic switches in their integrin repertoire that enable both the invasion process and their ⁎ Corresponding author. Department of Cell and Tissue Biology, S-534, University of California, San Francisco, CA 94143-0512, USA. Fax: +1 415 502 eventual adhesion to maternal cells in both the interstitial and 7338. intravascular compartments (reviewed in Red-Horse et al., E-mail address: [email protected] (S.J. Fisher). 2004).

A great deal is known about the pathways that lie both Materials and methods upstream and downstream of integrin actions. For example, physiological regulators such as oxygen tension affect Materials trophoblast integrin switching as well as differentiation (Genbacev et al., 1997; Maltepe et al., 2000). It is likely that Mouse monoclonal antibodies to the extracellular domain of L-selectin (DREG-56; unconjugated and fluorescein isothiocyanate [FITC]-conjugated this observation explains, at least in part, defects in the integrin forms) were from Caltag, Burlingame, CA. A goat polyclonal antibody to the C- switching program that are associated with the pregnancy terminus of L-selectin was from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. complication preeclampsia (new onset of maternal high blood Mouse monoclonal antibodies to E- and P-selectin were from R&D Systems, pressure, proteinuria,edema;reviewedinFisher, 2004). Inc., Minneapolis, MN. A rabbit polyclonal antibody to the nicotinic α3 receptor Interactions of integrin with extracellular matrix ligands also was from Research and Diagnostic Antibodies, Benicia, CA. A mouse monoclonal antibody to human integrin α1 (VLA-1) was from Endogen, initiate intracellular signaling cascades. For example, strength- Woburn, MA. Rat monoclonal antibodies that recognize L-selectin ligands ening of blastocyst adhesion to fibronectin rapidly elevates (HECA-452 and MECA-79) were from BD Biosciences, San Jose, CA. The cytoplasmic free Ca2+ (Wang et al., 2002). Additionally, CTB HECA-452 antibody recognizes sLex [Siaα2→3Galβ1→4(Fucα1→3)GlcNAc] x invasion of the uterine wall triggers phosphorylation of the and related structures, including 6-sulfo sLe [Siaα2→3Galβ1→4(Fucα1→3) → ′ x α → → β → α → focal adhesion kinase (Ilic et al., 2001), an important mediator (SO3 6)GlcNAc], 6 -sulfo sLe [Sia 2 3 (SO3 6)Gal 1 4(Fuc 1 3) GlcNAc] and 6′,6-disulfo sLex [Siaα2→3 (SO →6)Galβ1→4(Fucα1→3) of integrin signals. 3 (SO3→6)GlcNAc] (Mitsuoka et al., 1997). The MECA-79 antibody recognizes Usually, the role of homotypic adhesion mechanisms that a high-affinity L-selectin ligand carbohydrate epitope containing SO3→ mediate CTB–CTB interactions in placental development 6GlcNAc (Yeh et al., 2001). Anti-sLex was prepared by culturing the hybridoma receives much less attention. This is despite a great deal of cell line CSLEX-1, obtained from the American Type Culture Collection, circumstantial as well as experimental evidence to suggest that Manassas, VA, as described (Genbacev et al., 2003). This antibody recognizes sLex and other related structures, excluding 6-sulfo sLex and 6′,6-disulfo sLex these interactions also play an important role in placental (Mitsuoka et al., 1997). TRA-1-81, a mouse monoclonal antibody that recognizes development. In particular, CTB aggregation in cell columns sulfate-containing carbohydrate epitopes presented by keratan sulfate (Badcock requires the cells to form strong adhesive interactions with one et al., 1999), was purchased from Chemicon International, Inc., Temecula, CA. A another. Although trophoblasts express VCAM, an immunoglo- mouse monoclonal antibody to human cytokeratin 7 was from DakoCytomation, bulin (Ig) superfamily member that can mediate homotypic Glostrup, Denmark. Horseradish-peroxidase (HRP)-conjugated anti-human IgG, 2+ HRP-conjugated, FITC-conjugated and rhodamine-conjugated anti-mouse IgM, interactions, Ca -dependent adhesion molecules (cadherins) FITC-conjugated anti-rat IgM, FITC-conjugated anti-mouse IgG and FITC- often play dominant roles. In this regard, CTBs in columns conjugated anti-rabbit IgG were from Jackson ImmunoResearch Laboratories, express primarily E-cadherin (Fisher et al., 1989), which is West Grove, PA. Recombinant human L-selectin/Fc chimeras were obtained downregulated as their expression of VE-cadherin is upregu- from R&D Systems Inc. Enhanced chemiluminescence (ECL) detection reagents lated in the decidual compartment (Zhou et al., 1997). and Hyperfilm ECL were obtained from Amersham Biosciences Corp., Piscataway, NJ. Nitrocellulose membranes (0.45 μm) and nonfat dry milk Furthermore, E-cadherin function appears to play a critical were obtained from Bio-Rad, Hercules, CA. Culture plates (6 and 24 wells) were role from the earliest stages of development onward. Tropho- from Corning Inc., Corning, NY. Millicell-CM culture dish inserts (0.4 μm, blast morphogenesis is defective in embryos that carry a 12 mm) and Microcon centrifugal filter devices (3000 molecular weight cut-off) mutation in α-E-catenin, an important part of the downstream were from Millipore Corp., Bedford, MA. Optimal cutting temperature com- signaling cascade initiated by homotypic interactions involving pound (OCT) was from Miles Scientific, Naperville, IL. Vectashield mounting medium with DAPI was from Vector Laboratories, Inc., Burlingame, CA. E-cadherin (Torres et al., 1997). – The unusual cell cell interactions that take place in the Tissue sources columns of anchoring villi prompted us to search for adhesion mechanisms that also permit cell movement because CTBs use Informed consent was obtained from all tissue donors using a protocol that these structures as conduits to reach the uterine wall. Our was approved by the UCSF Committee on Human Research. Placentas from previous work showed that early-gestation CTBs in this region elective terminations of pregnancy (5 to 20 weeks) or from normal births were collected immediately and washed repeatedly in phosphate-buffered saline express L-selectin, which plays an important role in mediating (PBS) containing antibiotics before suspending them in culture medium on ice. interactions with receptive uterine luminal epithelial cells that Placentas with documented or suspected aneuploidies, inflammation/infections express its highly specialized sulfated carbohydrate ligands or other pathologies were excluded. (Genbacev et al., 2003; Rosen, 2004). Our data suggest that CTBs, like leukocytes, which also express L-selectin, are Cytotrophoblast isolation capable of shear-stress-dependent tethering-type adhesion. We hypothesize that these specialized adhesive interactions are Cells were isolated from pools of placentas as previously described (Kliman et al., 1986; Fisher et al., 1989). Briefly, placentas were subjected to a series of among the mechanisms that initiate implantation, subsequently enzymatic digestions that detached CTBs from the underlying stromal cores of triggering integrin-mediated stable adhesion. Presumably, these the chorionic villi. Once detached, the cells were purified over a Percoll gradient. heterotypic adhesive interactions are recapitulated as pregnancy progresses, with numerous cell columns eventually forming Organ culture of chorionic villus explants connections with the uterine wall. We tested the theory that CTBs also express L-selectin carbohydrate ligands that enable Anchoring villi were cultured as previously described (Genbacev et al., 1993). Briefly, small fragments of placental tissue from the maternal–fetal them to use this adhesion system to promote homotypic interface, which contained remnants of severed CTB columns, were dissected interactions within cell columns that facilitate movement of from first-trimester placentas under stereomicroscopic visualization. Then, the CTBs from the placental to the uterine compartment. villi were transferred to Matrigel-coated 12-mm inserts that were placed in 24- A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117 109

Table 1 nonfat dry milk (T-blotto) to prevent nonspecific binding prior to incubation with Summary of cytotrophoblast L-, P- and E-selectin expression a monoclonal antibody to the TRA-1-81 epitope (5 μg/ml) or the MECA-79 antibody (5 μg/ml). Immunoreactive bands were detected with HRP-conjugated Location of Weeks Number Immunoreactivity a rabbit anti-mouse IgM or HRP-conjugated goat anti-rat IgM, respectively, and CTBs of of L-selectin P-selectin E-selectin visualized by using chemiluminescence (Prakobphol et al., 1998, 2005). As a gestation samples negative control, an identical blot was incubated with the secondary antibody Anchoring villi 6 12 +++ –– alone. 7 8 +++ –– 8 10 +++ –– Identification of CTB ligands for L-selectin 9 5 +++ –– –– 10 5 ++ Nitrocellulose blots of CTB lysates were prepared as described above. –– 12 4 ++ Ligands were identified using published methods (Bertozzi et al., 1997; –– Uterine wall 15 3 ++ Prakobphol et al., 2005). Briefly, nonspecific binding was blocked by incubating 16 4 +/− –– membranes in 5% T-blotto for 1 h at room temperature. A complex of human L- − –– 18 3 +/ selectin/Fc chimeras (4 μg/ml) and HRP-conjugated anti-human IgG (1:500, ––– 20 5 vol/vol) was formed in 5% T-blotto by gentle mixing on a rotator (Cole-Parmer 36 2 ––– Roto-Torque Variable Speed Rotator, Vernon Hills, IL) at room temperature for ––– 38 5 20 min. To identify receptors, the nitrocellulose membranes were overlaid with ––– 40 5 the L-selectin complex and incubated on a rotator for 1 h. Then, the membranes A dash indicates no antibody reactivity. were washed three times with T-PBS. Glycoproteins that supported L-selectin a Immunoreactivity was graded on a comparative scale: the strongest staining binding were visualized by using chemiluminescence. As specificity controls, is indicated by +++ and the weakest by +/−. blots of the same samples were incubated as described above in 10 mM EDTA or in HRP-conjugated anti-human IgG alone. well plates, after which medium (Ham's F-12/Dulbecco's modified Eagle's medium [1:1, vol/vol]) supplemented with an antibiotic/antimycotic mixture (100 U/ml penicillin, 100 μg/ml streptomycin, 1.25 μg/ml amphotericin B) and 10% fetal calf serum was added. The cultures were incubated at 37°C under standard conditions (5% CO2/95% air) for 72 to 96 h. The morphology of the villi and their CTB outgrowths was monitored at regular intervals using an inverted-phase microscope. In some cases, a function-perturbing antibody was added to the medium after outgrowths began to emerge from the explants (∼36 h). For time-lapse video microscopy, the villi were cultured directly on the surfaces of Matrigel-coated 6-well plates.

Immunolocalization

Placental tissues or cultured explants were fixed in 4% paraformaldehyde for 90 min, washed three times in PBS, infiltrated with 5 to 15% sucrose followed by OCT compound and frozen in liquid nitrogen. Sections (5 μm) were prepared using a cryomicrotome (Leica Microsystems, Bannockburn, IL). Isolated CTBs were spun onto Shandon cytoslides using a Cytospin apparatus (Thermo Electric Corp., Pittsburgh, PA) and fixed with 4% paraformaldehyde for 15 min. Placental tissues, villus explants and isolated CTBs were processed using immunolocalization protocols that we published previously (Ilic et al., 2001). Nonspecific antibody reactivity was blocked by incubation in 3% bovine serum albumin in PBS for 1 h. Most of the primary antibodies were added at a concentration of 5 μg/μl. The exceptions were anti-cytokeratin, anti-integrin α1 and HECA-452, which were used at a concentration of 2 μg/μl, and anti- nicotinic α3 receptor, which was used at a dilution of 1:10 (v/v) per the manufacturer's instructions. After the specimens were incubated at 4°C overnight, they were washed three times in PBS and incubated in the appropriate species-specific FITC- or rhodamine-conjugated secondary antibody. In some cases, individual samples were stained with two primary antibodies raised in different species. Additionally, a subset of the samples was Fig. 1. First-trimester human cytotrophoblasts variably express MECA-79- incubated with FITC-conjugated DREG-56 (diluted 1:10, vol/vol). After reactive selectin ligands. Tissue sections of anchoring villi (A and C) or isolated staining, antibody reactivity was evaluated by using a Leica DM 500B cytotrophoblasts (CTBs) (E) were stained with the MECA-79 monoclonal fluorescence microscope (Leica Microsystems). antibody, which recognizes specialized carbohydrate structures that are high- affinity selectin ligands. Nuclei were visualized by staining with DAPI (B, D and Electrophoretic separation and immunoblotting F). In all locations, various CTB subpopulations reacted, usually weakly, with the MECA-79 antibody. (A) In some areas, CTB progenitors that were attached CTB columns were obtained by dissection of first-trimester placentas. to the trophoblast basement membrane exhibited plasma membrane staining. Columns dissected from 2 to 3 placentas were pooled, washed twice in PBS and The overlying syncytiotrophoblast (STB) layer was negative. (C) Many CTBs in macerated in loading buffer. The lysates were separated by SDS-PAGE (10% cell columns (CC) failed to react with MECA-79. The area outlined is enlarged separating gel and 4% stacking gel) under reducing conditions and transferred to in the inset (upper right corner). As shown in the inset, those that did react often nitrocellulose membranes (Prakobphol et al., 1998, 2005). The membrane had a punctate staining pattern. (E) Likewise, only a small subset of isolated replicas were incubated in PBS with 0.1% Tween-20 (T-PBS) containing 5% CTBs stained with MECA-79, also in a punctate pattern. 110 A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117

Time-lapse video microscopy (Genbacev et al., 2003). Furthermore, no staining for either E- or P-selectin was observed. These results are summarized in After the anchoring villus explants attached and CTBs began to migrate Table 1. Therefore, with regard to this class of receptors, CTBs away from column remnants, typically by 36–40 h, the cultures were transferred to an environmentally controlled chamber. Then, the chamber was affixed to the express predominantly L-selectin. stage of a Leica DM IRE2 phase-contrast microscope (Leica Microsystems), Next, we stained tissue sections of placental samples (5 to 20 equipped with a time-lapse video imaging system that was controlled by weeks of gestation) with a monoclonal antibody (MECA-79) Improvision Openlab software (Improvision, Inc., Lexington, MA). Images that specifically reacts with high-affinity L-selectin carbohy- were collected every 12 min for 24 to 30 h into a layered file format. Then, layers drate ligands. The results showed that villous and column CTBs were compiled into QuickTime movies. To study the role of the L-selectin adhesion system on CTB migration, the explants were pre-incubated with also express these epitopes (Figs. 1A and C, respectively). In function-perturbing antibodies 1 h before video images were collected. Prior to contrast to that of L-selectin, the staining pattern was variable: these experiments, sodium azide was removed from the antibody solutions. only a subset of the CTBs exhibited weak immunoreactivity, Briefly, they were diluted in PBS and the antibodies were isolated using a which was lost during the mid second trimester. Since sLex and Microcron centrifugal filter device. Control explants prepared from the same its structurally related carbohydrate epitopes can serve as L- placenta were incubated in the absence of antibody. As an additional control, explants from the same placenta were incubated with anti-nicotinic α3 receptor selectin ligands (Feizi, 2001; Prakobphol et al., 1998; Renkonen, at a dilution of 1:10 (v/v). 1998; Rosen, 2004), we also stained tissue sections of the aforementioned basal plate biopsies with two additional monoclonal antibodies: anti-sLex and HECA-452. HECA-452 Results recognizes sLex and a subset of the sulfated L-selectin carbohydrate ligands. The results showed very weak anti-sLex First-trimester villous and column CTBs express both immunoreactivity, whereas the HECA-452 antibody had a L-selectin and its carbohydrate ligands staining pattern that was very similar to that of MECA-79, except for syncytial reactivity (data not shown). First, we evaluated trophoblast expression of L-selectin family members in situ by immunoanalysis of tissue sections Villous and column CTBs express the TRA-1-81 carbohydrate prepared from placentas and basal plate biopsies obtained antigen, which binds L-selectin chimeras in vitro between 5 and 20 weeks of gestation and at term. In accord with our previous findings, CTBs in all locations expressed In a search for additional glycoproteins that presented L- L-selectin. Typically, the strongest staining was observed in selectin ligands, we assessed CTB expression of carbohydrate the distal regions of cell columns. Immunoreactivity was signi- epitopes that are markers of human embryonic stem cell (hESC) ficantly downregulated duringthemidsecondtrimester pluripotency (Brivanlou et al., 2003; Hoffman and Carpenter,

Fig. 2. In the cell column region, TRA-1-81-reactive carbohydrate epitopes and L-selectin have overlapping staining patterns. Tissue sections of first-trimester anchoring villi (AV) were doubly stained with TRA-1-81 and anti-L-selectin (DREG-56), which specifically recognizes L-selectin. (A) A subset of villous (vCTBs) and cell column (CC) CTBs expressed TRA-1-81 antigens, with the brightest staining consistently observed near the distal ends of the columns. (B) DREG-56 immunoreactivity showed that L-selectin was expressed in the same pattern. (C) Merging of the images shown in panels A and B allowed visualization of the areas of overlapping expression of the antigens, which appear in yellow. (D) Nuclei were visualized by DAPI staining. The arrow indicates the direction of invasion; the arrowhead lies at the distal end of the cell column, which was dissected away from the original site of uterine attachment. STB, syncytiotrophoblast. A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117 111

452. In addition, there was a gradient of antibody reactivity: CTBs at the distal ends of columns near sites of uterine attachment stained more brightly than did cells in the proximal portions where the CTBs first emerge from the placenta (Fig. 2A). Next, we asked whether there was overlapping expression of the TRA-1-81 carbohydrate epitopes and L-selectin. The results of these double staining experiments revealed a high degree of co-localization (Figs. 2A–C). The subset of cells that stained strongly with TRA-1-81 also stained strongly with the anti-L- selectin, that is, the highest immunoreactivity was observed in the distal regions of cell columns. Furthermore, expression of both L-selectin and the TRA-1-81 epitope was downregulated Fig. 3. Lysates of first-trimester cytotrophoblasts contain TRA-1-81-reactive midway through the second trimester. In additional experiments glycoproteins that interact with L-selectin chimeras in vitro. The lysates were that addressed the issue of cell surface expression of these separated by SDS-PAGE (10% acrylamide running gel; 4% acrylamide stacking carbohydrate epitopes, we stained freshly isolated CTBs with gel) and transferred to nitrocellulose. Arrows indicate the top and bottom of the the TRA-1-81 antibody. The results showed that the immunor- stacking gel. (Lane 1) High-molecular-weight glycoproteins that remained eactivity, which was patchy, was strong on some cells and within the stacking gel reacted with the TRA-1-81 antibody. (Lane 2) No immunoreactivity was detected when a nitrocellulose replica of the same lysate moderate to weak on others (data not shown), presumably a sample was incubated with the secondary antibody alone. (Lane 3) The TRA-1- reflection of the gradient of expression observed in situ. 81-reactive glycoproteins that were retained in the stacking gel interacted with To obtain molecular weight information about the TRA-1- the L-selectin chimeras, suggesting that they present the specialized carbohy- 81-reactive molecules, protein extracts of cell columns that drate ligands with which this receptor interacts. were dissected from first-trimester placentas were separated by SDS-PAGE and transferred to nitrocellulose membranes, which 2005). In contrast, staining tissue sections of basal plate were probed with the TRA-1-81 antibody. As shown in Fig. 3, biopsies from first-trimester placentas with TRA-1-81, a mono- lane 1, this antibody detected a high-molecular-weight band that clonal antibody that recognizes sulfated epitopes on keratan after prolonged electrophoresis entered the stacking gel. This sulfate (Badcock et al., 1999), revealed a clear pattern. Both result is consistent with the fact that selectin ligands are villous and column CTBs stained with this antibody at a much displayed by mucin-type glycoproteins such as salivary Muc- higher intensity than that observed with MECA-79 or HECA- 5b, which has a molecular weight >1,000,000 (Prakobphol et

Fig. 4. Cytotrophoblast outgrowths of explanted anchoring villi stain with TRA-1-81 and DREG-56, which specifically recognizes L-selectin. Anchoring villi were cultured under standard conditions for 72–80 h then fixed in 4% paraformaldehyde. Histological sections of the explanted villi were stained with (A) TRA-1-81, which detected carbohydrate epitopes, or (B) anti-L-selectin (DREG-56). The cytotrophoblast outgrowths reacted with both antibodies. (C) Merging of the two images reveal, in yellow, areas of overlapping antigen expression in the distal region of the column. (D) Nuclei were visualized by DAPI staining. The arrow indicates the direction of invasion; the arrowhead lies at the distal end of the outgrowth of invasive CTBs (iCTBs) that formed in vitro. 112 A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117 al., 2005). No immunoreactive bands were detected when a L-selectin adhesive interactions facilitate CTB emigration replica that contained the same samples was incubated with the through the cell columns of anchoring villi secondary antibody alone (Fig. 3, lane 2). Finally, nitrocellulose replicas of the same extracts were probed with the MECA-79 When anchoring villi are cultured on extracellular matrix antibody. Either faint or no immunoreactive bands were substrates such as Matrigel, CTBs emigrate from the cell detected (data not shown), a finding that was consistent with column remnants. The analogous in vivo process is the first step the immunolocalization results. in placental attachment, which precedes uterine invasion (Aplin, Next, we asked whether the TRA-1-81-reactive glycopro- 1993; Aplin et al., 1999; Genbacev et al., 1993; Vicovac et al., teins interacted with L-selectin. A different replica of the 1995). To determine if this model would be useful for studying same samples was incubated with a complex formed from the role of L-selectin in CTB homotypic adhesive interactions in human L-selectin/Fc chimeras and HRP-conjugated anti- cell columns, first-trimester anchoring villus explants that had human IgG, an in vitro method for identifying glycoproteins been cultured on Matrigel substrates were fixed, sectioned and with L-selectin ligand activity (Bertozzi et al., 1997; stained with antibodies that recognized either L-selectin Prakobphol et al., 2005). As shown in Fig. 3, lane 3, the (DREG-56), its specialized carbohydrate ligand (MECA-79) TRA-1-81-reactive band also interacted with the L-selectin or other sulfated carbohydrate species (TRA-1-81). The results chimeras. As a control for the specificity of L-selectin showed that villous and column CTBs as well as invasive CTBs receptor activity, which is Ca2+ dependent, EDTA was added within the Matrigel reacted with all three antibodies (Fig. 4). In to the incubation solution in which a replica of the same general, the staining patterns were similar to those observed in samples was processed in parallel. Chimera binding to the situ. MECA-79 immunoreactivity was variable and weak (data high-molecular-weight band was decreased by ∼60% (data not shown). In contrast, a gradient of TRA-1-81 staining was not shown). Together, these results suggested that the TRA-1- observed: the proximal portions of cell columns stained weakly, 81 epitopes carried by CTB high-molecular-weight glycopro- and the distal ends demonstrated more intense immunoreactiv- teins are the cells' principal L-selectin ligands. ity (Fig. 4A). Double staining to detect L-selectin expression

Fig. 5. A diagrammatic depiction of the cell–cell interactions that occur when CTBs emigrated from a cell column as shown in Movie 3. Stationary cells are depicted in yellow; the rest of the CTBs appear in grey. Individual cells with colored nuclei can be clearly tracked in the movie. For example, the CTB with the red nucleus, which tethered to the cells at the boundary of the column, ended up in the outermost cell cluster. The CTB with the black nucleus migrated the furthest distance and eventually left the column (data not shown). Tethers are depicted as red bars, and the direction of migration is indicated by the red arrows. The elapsed time is shown at the lower right corner of each panel. A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117 113

Fig. 6. L-selectin function is required during the formation of cytotrophoblast outgrowths in vitro. Anchoring chorionic villi were cultured under standard conditions for 36–40 h. Then, the explants were transferred to an environmentally controlled chamber that was placed on the stage of a microscope equipped with a video imaging system. Pictures of the cytotrophoblast outgrowths were taken every 12 min for the next 24–30 h. (A) In control explant cultures, CTBs rapidly emerged from the cell column remnants, as shown by the steady increase over time in the diameter of the outgrowth, outlined by dashed yellow lines. Initially, cells at the periphery of the outgrowths extended long processes (38 h). At later times (44, 56 and 68 h), these areas became finger-like projections of invasive CTBs. (B) When explants were cultured in the presence of anti-L-selectin (DREG-56, 30 μg/ml), which disrupts L-selectin receptor activity, the outgrowths did not enlarge, and formation of the finger-like projections of invasive CTBs was almost entirely inhibited.

(Fig. 4B) and merging anti-L-selectin (DREG-56) and TRA- outgrowths by using a tethering motion in which each cell made 1-81 patterns revealed the regions of overlapping expression transient contacts with its neighbors (Movies 2 and 3). Fig. 5 is a of L-selectin and the TRA-1-81 epitopes (Fig. 4C), schematic diagram illustrating the cell–cell interactions that suggesting that these carbohydrate epitopes likely serve as occur as CTBs move through and emigrate from a cell column ligands for L-selectin. as shown in Movie 3. These data, which suggested that the L-selectin adhesion To dissect the role of L-selectin and its ligands in the latter system functions in the distal column region, prompted us to use process, we examined the effects of several function-perturbing video microscopy to carry out a detailed analysis of the cell–cell antibodies. First, we incubated the explants with anti-L-selectin interactions that lead to CTB emigration from the placenta. (DREG-56). Within 5–6 h after antibody addition, CTB Once explants attached and the CTBs started to migrate away emigration from the cell columns slowed dramatically (compare from cell columns, generally 36–40 h after they were placed in Fig. 6A with B and Movie 4 with Movie 5). Rather than culture, images were captured every 12 min for 24 to 30 h. At invading the Matrigel, the cells piled up at the distal ends of low magnification, CTB outgrowths had a lava-like appearance the columns. as they migrated into the Matrigel (Movie 1). At higher Next, we examined the effects of function-blocking magnification, it was evident that CTBs moved through the antibodies that disrupt the actions of the TRA-1-81 epitopes.

Fig. 7. TRA-1-81-reactive carbohydrate epitopes are required during formation of cytotrophoblast outgrowths in vitro. The experimental conditions were identical to those described in the legend to Fig. 6, except that TRA-1-81 (15 μg/ml) rather than DREG-56 was added to the cultures shown in panel B. The results with TRA-1-81 were similar to those of DREG-56 in inhibiting L-selectin function. Additionally, CTB processes that were initially extended (38 and 44 h) eventually retracted (50 and 66 h). 114 A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117

Fig. 8. TRA-1-81-reactive carbohydrate epitopes are required during formation of cytotrophoblast outgrowths in vitro. The experimental conditions were identical to those described in the legend to Fig. 7, except that the images were captured at higher magnification.

In accord with the immunolocalization data, which showed subpopulation that exhibited the highest L-selectin immunor- variable expression of these specialized oligosaccharide eactivity consistently expressed carbohydrate epitopes that structures (Figs. 1 and 4), the results obtained after the addition reacted with the TRA-1-81 antibody, a marker of “stemness” of MECA-79 were inconsistent (data not shown). In contrast, in the human embryonic stem cell system (Badcock et al., dramatic effects were observed in every experiment in which 1999). Although the exact structures of the TRA-1-81 epitopes the TRA-1-81 antibody was added to cultured villus explants. In are not known, they are expressed together with other sialylated accord with the immunolocalization data (Figs. 2 and 4), which carbohydrate epitopes (recognized by TRA-1-60) on a keratan consistently demonstrated strong expression of these oligosac- sulfate proteoglycan. Therefore, the TRA-1-81 epitopes appear charide epitopes at the distal ends of cell columns, CTB to fit the sulfate requirement that applies to high-affinity selectin emigration from these structures was almost entirely inhibited ligands (Rosen, 2004). (compare Fig. 7Awith B, Fig. 8Awith B, Movie 6 with Movie 7 At a functional level, our results provide evidence for the and Movie 8 with Movie 9). As observed in Movie 9, at high concept that CTBs use L-selectin adhesion system to carry out a magnification, it became clear that the emigration of CTBs from novel type of tethering adhesion that facilitates homotypic the distal end of the columns, where most of the cell surface was rather than heterotypic interactions. Given the highly specia- accessible to the antibody, was significantly decreased to the lized nature of cell adhesion within the columns of placental point that their forward progression was halted. In contrast, anchoring villi, we theorize that these receptor–ligand interac- addition of an antibody to another CTB cell surface molecule, tions could play several important roles. For example, the nicotinic receptor α3 subunit, did not interfere with the expression of L-selectin family members is upregulated as cells' emigration from the columns (data not shown). CTBs move through the columns. Thus, it seems possible that Finally, we asked whether disruption of L-selectin-mediated these unique tethering-type adhesive interactions could play a adhesion affected CTB interactions with the extracellular role in extension of cell columns, which takes place in an matrix. Tissue sections prepared from control explants and environment in which shear stress is generated by maternal explants cultured in the presence of the TRA-1-81 antibody blood flow through the intervillous space. Additionally, this were stained with a monoclonal antibody to integrin α1, an adhesion system could also function during the initial stages in adhesion molecule that is upregulated as cytotrophoblasts which cell columns attach to the uterine wall. Although little is move into columns. Under both conditions, strong staining known about the surface antigens that are expressed by human was noted in the expected pattern, suggesting that disruption of uterine epithelial cells at points of cell column attachment, our L-selectin-mediated adhesion did not affect cytotrophoblast group has shown that, during the receptive phase of the cycle, interactions with the extracellular matrix. As an additional these cells express the high-affinity L-selectin ligands that are control, we used an immunoblot approach to analyze the lot of recognized by the MECA-79 antibody (Genbacev et al., 2003). Matrigel that we used for potential TRA-1-81-reactive epitopes Since the abundance of these specialized oligosaccharide units and detected none. is hormonally regulated (Prakobphol et al., 2005), it is possible that their expression is maintained during pregnancy. In this Discussion case, they would be ideally placed to function as ligands for CTBs at the termini of cell columns, which stain brightly for Our results show that the subset of cell column CTBs that L-selectin. exhibited the brightest L-selectin staining also exhibited weak The discovery that the L-selectin system functions during variable reactivity with MECA-79, an antibody that recognizes reproduction is an unexpected finding. E-, L- and P-selectin this receptor's high-affinity ligands. Additionally, the CTB were originally thought to be expressed exclusively by A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117 115 hemangioblast descendents (reviewed in Rosen, 2004). E- specific carbohydrate motifs that serve as ligands for L-selectin selectin is expressed on activated endothelial cells and mediates (Prakobphol et al., 1998, 2005; Thomsson et al., 2002). For adhesion of neutrophils and monocytes. P-selectin is expressed example, MG2 carries the sLex epitope, which we showed on the surfaces of activated platelets and endothelial cells and mediates leukocyte rolling and tethering in a range of shear can also mediate adhesion of neutrophils and monocytes, as stresses that are comparable to those predicted to be generated well as certain lymphocyte subsets. L-selectin is the “homing within the oral cavity (Prakobphol et al., 1995, 1998). MG1 receptor” for lymphocytes that mediates their tissue-specific (Muc-5b, Mr ∼1,000,000) carries MECA-79-reactive epitopes, adherence to high endothelial venules. Each selectin molecule which also support interactions with L-selectin, assayed using contains a lectin-like N-terminal domain followed by an the nitrocellulose overlay method employed here (Prakobphol epidermal growth factor-like motif, a series of consensus et al., 2005). In this regard, it is interesting to note that our repeats similar to those in complement-regulatory proteins, a microarray analysis of gene expression at the maternal–fetal transmembrane domain and a cytoplasmic tail. The lectin interface revealed expressions of several mucin genes (Winn domains mediate a Ca2+-dependent interaction between the and Fisher, submitted for publication), such as Muc-5ac and selectin molecules and their ligands under shear flow. To date, Muc-5b, that encode for high-molecular-weight mucins that are known E- and P-selectin ligands include PSGL-1 (Asa et al., candidates for being the scaffolds that present the L-selectin 1995; Moore et al., 1995). L-selectin ligands include CD34 carbohydrate ligands. This theory is consistent with our finding (Baumheter et al., 1993), GlyCAM-1 (Lasky et al., 1992), that the L-selectin chimera-reactive band was a high molecular MAdCAM-1 (Berg et al., 1993), podocalyxin (Sassetti et al., species that did not migrate beyond the initial portion of the 1998), endoglycan (Sassetti et al., 2000) and a heparin-like stacking gel (Fig. 3, lane 3). Second, we have data (Carson et molecule (Norgard-Sumnicht et al., 1993). Our studies added al., 2006) showing that mucin-1 (Muc-1), a cell-surface human salivary mucins MG1/Muc-5b and MG2/Muc-7 to this glycoprotein expressed on uterine luminal epithelial cells list (Prakobphol et al., 1998, 2005). Owing to the scarcity of (Hild-Petito et al., 1996), carries both sLex and MECA-79 purified forms of most of these molecules, information on the epitopes. Third, on the fetal side, we showed that the carbohydrate-binding preference of the selectins has mainly trophectoderm, the earliest trophoblast lineage, expresses L- come from studies of “surrogate” molecules\both glycolipids selectin (Genbacev et al., 2003). Additional experiments and synthetic carbohydrate mimetics (reviewed in Feizi, 2001). addressing function provided evidence that blastocyst-stage For example, L-selectin, which recognizes sLex (reviewed in embryos use this receptor to mediate initial adhesive interac- Renkonen, 1998), prefers the sulfate-containing synthetic tions with the uterine wall, which we theorize start the saccharide 6′,6-disulfo-lactose (Bertozzi et al., 1995; Bruehl implantation process (Genbacev et al., 2003). During the first et al., 2000), known from metabolic labeling studies to be trimester, CTBs in the villi and cell columns continue to express present on GlyCAM-1 (Hemmerich et al., 1994). L-selectin, for which staining is greatly reduced after the mid The ability of CTBs to migrate, adhere to and finally invade second trimester. These findings suggest that L-selectin may the uterine wall is thought to be due, in part, to their specialized also play an important role during early placentation, a period adhesive properties. Our earlier studies focused on molecules when cell columns of anchoring villi are continuously formed that belong to three of the four major eukaryotic cell adhesion and the process of their uterine attachment is repeated until the systems—integrins, cadherins and the Ig superfamily. The mature architecture of the basal plate has been established. results indicated that CTBs have a remarkable ability to Additionally, our in vitro experiments showed that the TRA- modulate their adhesion molecule repertoire as they move 1-81 epitope, in the context of the CTB cell surface through cell columns and acquire the ability to invade the glycoproteins on which it is presented, can mediate interactions uterine wall and the blood vessels that traverse this region with L-selectin chimeras. Thus, it appears that the TRA-1-81- (Damsky and Fisher, 1998). In essence, these ectodermal reactive oligosaccharide structures together with their protein derivatives assume many of the cell surface properties of scaffold(s) constitute novel L-selectin ligands. Whether the endothelial cells. same motifs presented on the hESC surface can interact with L- Recently, we expanded our studies to include adhesion selectin remains to be determined, as does the functional molecules in the selectin family, the fourth major cell adhesion importance of this observation. Nevertheless, it is interesting to system that governs rolling and tethering of blood cells. There find that hESCs are capable of elaborating complex carbohy- are several reasons why we are interested in the concept that drate motifs with highly specialized functions in a tethering type selectin–ligand interactions play important roles in formation of of cell adhesion. the maternal–fetal interface. First, the results of other work from In summary, our results suggest that the L-selectin adhesion our laboratory show that L-selectin and its carbohydrate ligands system plays an autocrine role in CTB homotypic adhesive mediate important functions in the oral cavity (Prakobphol et interactions that occur within cell columns, particularly near al., 1998, 2005). In these studies, we analyzed in detail the their distal ends where both receptor and ligand expression oligosaccharide structures carried by two highly glycosylated peaks. We theorize that this specialized adhesion system is a components of human saliva—the high- and low-molecular- vital component of the molecular mechanisms that play weight mucins, MG1 (Muc-5b) and MG2 (Muc-7), respectively. important roles in critical functions of cell columns, including The analyses showed that these molecules carry a hetero- attaching the placenta to the uterus and providing a route by geneous mixture of oligosaccharide chains, some of which bear which CTBs reach the uterine wall, which they subsequently 116 A. Prakobphol et al. / Developmental Biology 298 (2006) 107–117 invade. The specialized nature of L-selectin-mediated adhesive Fisher, S.J., 2004. The placenta problem: linking abnormal cytotrophoblast interactions seems ideally suited for simultaneously maintaining differentiation to the maternal symptoms of preeclampsia. Reprod. Biol. 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