Blastocysts Don't Go It Alone. Extrinsic Signals Fine-Tune the Intrinsic

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Developmental Biology 280 (2005) 260–280 www.elsevier.com/locate/ydbio Review Blastocysts don’t go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells

D. Randall ArmantT

Departments of Obstetrics and Gynecology and Anatomy and Cell Biology, C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, 275 E. Hancock Avenue, Detroit, MI 48201-1415, USA

Received for publication 21 September 2004, revised 16 January 2005, accepted 8 February 2005

Abstract

The preimplantation embryo floats freely within the oviduct and is capable of developing into a blastocyst independently of the maternal reproductive tract. While establishment of the trophoblast lineage is dependent on expression of developmental regulatory genes, further differentiation leading to blastocyst implantation in the uterus requires external cues emanating from the microenvironment. Recent studies suggest that trophoblast differentiation requires intracellular signaling initiated by uterine-derived growth factors and integrin-binding components of the extracellular matrix. The progression of trophoblast development from the early blastocyst stage through the onset of implantation appears to be largely independent of new gene expression. Instead, extrinsic signals direct the sequential trafficking of cell surface receptors to orchestrate the developmental program that initiates blastocyst implantation. The dependence on external cues could coordinate embryonic activities with the developing uterine endometrium. Biochemical events that regulate trophoblast adhesion to fibronectin are presented to illustrate a developmental strategy employed by the peri-implantation blastocyst. D 2005 Elsevier Inc. All rights reserved.

Keywords: Blastocyst implantation; Trophoblast; Integrins; Extracellular matrix; Cell adhesion; Cell invasion; Epigenetic regulation; Paracrine signaling; Intracellular calcium; Protein trafficking

Introduction and background serve a variety of functions, including invasive remodeling of the maternal vasculature and molecular transport at the The trophoblast lineage is responsible for sustaining a maternal–fetal interface (Adamson et al., 2002; Pijnenborg mammalian conceptus throughout gestation. Beginning with et al., 1981). Thus, the trophoblast is ultimately responsible their progenitors derived from the outer layer of the for ensuring adequate flow of maternal blood to the preimplantation embryo, the trophectoderm, trophoblast placenta, as well as shuttling nutrients, gases, and waste cells perform a multitude of functions essential for products between maternal and fetal blood. blastocyst formation, implantation, and placentation. At While the complexity of trophoblast differentiation has the late blastocyst stage, the trophoblast attaches to the been well described, the biochemical mechanisms that guide endometrium and invades through the uterine epithelium to trophoblast development are still the subject of intense infiltrate the interstitium (Carson et al., 2000). Blastocyst investigation. For example, it is clear that trophoblast stem implantation is the initial step of placentation and the cells are capable of converting to diverse phenotypes trophoblast comprises a major component of the placenta. depending on where in the conceptus they are located. But Trophoblast cells differentiate into several subtypes that how does cell position determine cell behavior? What is the molecular basis of the phenotypic plasticity of the trophoblast? Differentiation of the many trophoblast subtypes, as T Fax: +1 313 577 8554. described below, is highly complex, making these questions E-mail address: [email protected]. difficult to answer. The major influence of extrinsic signals

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.02.009 D.R. Armant / Developmental Biology 280 (2005) 260–280 261 in directing trophoblast differentiation could account for toward an invasive phenotype. Through this directed their sensitivity to positional information. The molecular process, trophoblast behavior can be intricately coordinated (Cross, 2000; Kunath et al., 2004; Red-Horse et al., 2004) with changing conditions in the endometrium. Similar and cellular (Denker, 1993; Sutherland, 2003) aspects of interactive strategies could guide other aspects of tropho- trophoblast differentiation based on studies in experimental blast development and conversion to trophoblast subtypes species have been detailed in previous reviews. Here, we during placentation. will examine trophoblast differentiation during blastocyst implantation in mice with respect to mechanisms that The maternal–embryonic dialogue regulate their adhesion to the extracellular matrix (ECM) as they commence invasion of the endometrium. The Blastocyst implantation is dependent on the intrinsic biochemical process that establishes trophoblast adhesion embryonic program, operating in conjunction with extrinsic provides a paradigm for understanding how the maternal signals emanating from the female reproductive tract and embryonic developmental programs operate in har- (Fig. 1A). Uterine-derived signals maintain the pace of mony. Trophoblast cells incorporate specific molecular cues blastocyst development and mobilize embryonic receptors obtained from their surroundings to advance stepwise used for subsequent signaling (Armant et al., 2000).

Fig. 1. Maternal–embryonic interactions during peri-implantation development. (A) The intrinsic developmental program will direct blastocyst differentiation (yellow shading) to the adhesion competent stage, but extrinsic factors (intersecting yellow arrows) impact the outcome. As development progresses, the nature of the extrinsic factors is altered by embryo location and by physiological changes in both embryonic and uterine tissues. These signals accelerate the rate of trophoblast differentiation and induce cell competence for subsequent interactions with other extrinsic signals by mobilizing new receptors. (B) Growth factors (GFs) and other soluble bioactive molecules secreted by the oviduct and uterus provide paracrine signals to the embryo. These signals could trigger protein trafficking to the surface of trophoblast cells to activate receptors for new extrinsic signals. After hatching from the zona pellucida, juxtacrine signaling can occur at the interacting surfaces of trophoblast and luminal epithelial (LE) cells. Integrin trafficking induced by these interactions imparts competence for blastocyst adhesion to the ECM. As the blastocyst invades through the LE, contact with the basement membrane and decidual ECM activates integrin signaling required for strong trophoblast adhesion and subsequent invasion of the decidua. Later during placentation, extrinsic signaling could account for the differentiation of trophoblast cells along several divergent developmental pathways. 262 D.R. Armant / Developmental Biology 280 (2005) 260–280

Decidualization of the endometrium is, in turn, regulated by blastocyst (Collins and Fleming, 1995; Watson and Bar- the blastocyst (Psychoyos, 1973).Thepresenceofa croft, 2001). The trophectoderm does not contribute any blastocyst is required for uterine expression of heparin- tissues to the fetus, but rather establishes the trophoblast binding EGF-like growth factor (HB-EGF), followed by lineage that produces the placenta and extraembryonic production of betacellulin, epiregulin, neuregulin-1, and membranes. The fetus is derived solely from a cluster of cyclooxygenase-2 as the blastocyst attaches to the luminal non-polar, pluripotent cells situated eccentrically within the epithelium (Dey et al., 2004). This molecular dialogue blastocyst, the inner cell mass (ICM). serves to synchronize embryonic and maternal tissues At the implantation stage in mice, mural trophoblast cells during the peri-implantation period. Like the mature oocyte, (those not contacting the ICM) differentiate into primary the blastocyst is primed to advance in development only trophoblast giant cells (TGCs) that undergo endoreduplica- after it receives an external signal. Rather than a sperm, the tion and initiate invasion of the endometrium (Dickson, blastocyst awaits contact with the receptive endometrium. 1963; Kirby et al., 1967). The primary TGCs anchor the As in the developing zygote, blastocyst differentiation relies embryo in the uterine wall and form the outermost layer of on transcripts synthesized at a prior stage (Armant et al., the parietal yolk sac (Cross et al., 1994; Gardner et al., 2000; Schindler and Sherman, 1981). In this article, 1973). The polar trophoblast cells remain diploid due to evidence will be reviewed illustrating that crucial gene signals, including fibroblast growth factor-4 (FGF-4), they products required for blastocyst implantation are activated receive from the adjacent ICM (Chai et al., 1998; Tanaka et only after the endometrium signals its readiness. al., 1998). As polar trophoblast cells proliferate, those Mammals have evolved to sequentially deliver extrinsic displaced away from the ICM are incorporated into the signals to the preimplantation embryo by taking advantage mural trophoblast and differentiate into primary TGCs of its location within the reproductive tract, temporal (Gardner, 2000). changes in the endometrium, and accessibility of embryonic During mouse placentation, components of the ICM and cells to maternal compartments. Preimplantation embryos polar trophoblast develop into the extraembryonic ectoderm are initially surrounded by an ECM, the zona pellucida, that and ectoplacental cone (EPC), respectively (Adamson et al., limits paracrine signaling; only soluble molecules present in 2002; Gardner et al., 1973). Stem cells residing in the fluids of the oviduct and uterus can penetrate the zona and extraembryonic ectoderm provide a reservoir of trophoblast reach the embryo (Fig. 1B). Once freed of the zona cells for the growing placenta (Tanaka et al., 1998). The pellucida, the blastocyst can interact directly with cells core of the EPC contains diploid trophoblast cells that form and tissues. Growth factors tethered to cell surfaces become the outer spongiotrophoblast and inner labyrinthine layers of accessible for juxtacine signaling (Fig. 1B), widening the the placenta. In the labyrinthine layer, the site of exchange scope of interactions. For example, epithelial cells adjacent between maternal and fetal blood, diploid trophoblast cells to the hatched blastocyst produce the transmembrane form fuse to form syncytiotrophoblasts. At midgestation, a of HB-EGF, which activates receptors on the surface of population of trophoblast glycogen cells appears transiently trophoblast cells (Raab et al., 1996). HB-EGF accelerates within the spongiotrophoblast layer and invade out to the the rate of blastocyst differentiation in vitro, in part by distal regions of the decidua (Adamson et al., 2002; Redline stimulating a5h1 integrin trafficking to the apical surface of and Lu, 1989). Trophoblast cells at the periphery of the trophoblast cells (Wang et al., 2000). Later, the epithelial placenta differentiate into secondary TGCs that closely cells adjacent to the embryo are lost and ECM components resemble the primary TGCs. Secondary TGCs secrete of the underlying basement membrane become accessible to hormones, including placental lactogens and proliferin, the trophoblast (Fig. 1B). We will discuss in this article how and invade the uterine arteries (Adamson et al., 2002; Faria integrin signaling mediated by a5h1 mobilizes other et al., 1990). Using laboratory animal models and tropho- integrins that strengthen trophoblast adhesion to fibronectin. blast stem cells induced to differentiate in vitro, genes have An important second messenger integrating signals that been described that determine each phenotype, ranging from reach the embryo from both growth factors and ECM is undifferentiated trophoblast stem cells to syncytiotropho- Ca2+, which plays a similar role in egg activation by blasts and TGCs (Rossant and Cross, 2001). These initiating protein trafficking. The fine-tuning of trophoblast discoveries reveal the importance of intercellular signaling differentiation by extrinsic signals orchestrates the molec- in placental morphogenesis. ular interactions required for implantation in a precise and metabolically efficient sequence. Blastocyst implantation

A brief overview of trophoblast differentiation Conversion of trophoblast cells to an adhesive phenotype during peri-implantation development proceeds in a step- Trophectoderm is the first polarized epithelium to appear wise fashion that is coordinately regulated with endometrial during mammalian development. It emerges through the differentiation, as depicted in Fig. 1B. It is crucial that the assembly of tight cellular junctions and inward movement blastocyst becomes competent for implantation during the of water to generate the large cavity characteristic of a brief bwindowQ of uterine receptivity for implantation (Paria D.R. Armant / Developmental Biology 280 (2005) 260–280 263 et al., 1993). The process of implantation may be subdivided to distinguish in vivo (E) from in vitro (GD) development. into phases delineated by critical cellular interactions Cultured blastocysts recapitulate many of the peri-implan- between the trophoblast and uterine endometrium (Schlafke tation stages that occur in utero, but at a considerably slower and Enders, 1975). Initially, tight apposition occurs between rate (Table 1). the embryo and uterine epithelium as the blastocyst lodges The central event of peri-implantation embryonic devel- in a crypt of the uterine folds and localized edema of the opment is the conversion of the apical domain of the endometrium molds an implantation chamber around the trophoblast plasma membrane to an adhesive surface. embryo. During this period, the blastocyst hatches from the Initially, trophoblast cells are not reactive with maternal zona pellucida. The apical surface of the mural trophoblast cells or ECM. As the trophoblast differentiates, the apical then attaches to the opposing surface of the luminal surface first becomes attachment competent, acquiring epithelium. Blastocyst attachment initiates decidualization receptors that mediate interactions with the uterine luminal of fibroblast cells in the surrounding endometrial stroma. epithelium. The attachment reaction appears to be governed The attachment reaction is followed by apoptosis of those by a broad variety of molecules that inhibit or promote the luminal epithelial cells contacting the blastocyst, providing attachment of trophoblast to uterine epithelial cells (Dey et access to the underlying basement membrane. In the final al., 2004; Kimber, 2000; Kimber and Spanswick, 2000). In stage, trophoblast cells undergo changes in cell polarity and the next step, the blastocyst becomes adhesion competent by adhesiveness, allowing them to complete their differentia- activating receptors for ECM on the apical surface of tion to the invasive phenotype. Invading trophoblast cells primary TGCs. Trophoblast adhesion to the uterine base- breach the uterine basement membrane to infiltrate the ment membrane and interstitial ECM has been particularly decidua. amenable to study in vitro. Blastocysts become adhesion While trophoblast differentiation at implantation varies competent in serum-free medium on GD7 (Table 1) and among species, the general processes of apposition, attach- quickly undergo trophoblast dispersion and migration to ment, acquisition of apical adhesiveness, and invasion apply form an outgrowth on surfaces coated with adhesive to human embryos, as well as mice. The basolateral surfaces proteins (Armant et al., 2000). of undifferentiated trophectoderm cells adhere to a basement Using the in vitro experimental approach, a great deal of membrane that surrounds the blastocyst cavity, but their biochemical information has accumulated, indicating the apical surfaces are non-adhesive, typical of epithelial cells. nature and molecular basis of embryo-endometrial inter- After the blastocyst hatches from the zona pellucida, the actions during implantation, as outlined in Fig. 1. Adhesive apical surface of the mural trophoblast differentiates mechanisms can be analyzed by perturbing trophoblast (Armant et al., 1986a; Sherman and Atienza-Samols, outgrowth with specific reagents to yield information about 1978), altering its ability to interact with other cells or the processes that directly mediate trophoblast adhesion ECM. Efforts in the past two decades have focused on (Wang and Armant, 2002) and the developmental program understanding the ontogeny of adhesive mechanisms on the that converts trophoblast cells from an epithelial phenotype apical surface of trophoblast cells during this brief period of to invasive cells (Armant et al., 2000). Combined with data development. These changes are critical for the transition on the in utero localization of key proteins, experimental from an epithelial trophectoderm to the invasive TGC studies of blastocysts in culture have revealed novel phenotype responsible for blastocyst implantation. mechanisms that orchestrate adhesive interactions of primary TGCs with molecular components of the uterine endometrium during implantation. Experimental approaches for investigating implantation

The initial stages of implantation are largely unknown in Table 1 humans due to ethical limitations on experimentation. Most Pre- and peri-implantation mouse embryonic developmental rates in vivo and in vitro of our understanding of blastocyst development and early trophoblast differentiation derives from work with animal Embryonic stage Time Time in vivo in vitro models, including non-human primates, livestock, and Fertilization E0 rodents (Carson et al., 2000). Owing to its small diameter 2-Cell embryo E1.0 (approx. 100 Am) and location within a relatively large Compacted 8-cell embryo E2.5 uterine lumen, the implanting mouse blastocyst is a rather Early blastocyst E3.5 GD4 intractable subject for in utero biochemical approaches. To Expanded blastocyst and hatching E3.8 GD5 explore the embryonic side of implantation, the in vitro Attachment reaction/attachment competent E4.0 GD6 At basement membrane/adhesion competent E5.2 GD7 culture of blastocysts has provided a useful experimental Invasion of stroma/trophoblast outgrowth E6.2 GD7–10 tool. Newly formed blastocysts are flushed from the uterus For in vitro culture, blastocysts are removed from the uterus on embryonic on embryonic day 3.5 (E3.5), initiating in vitro culture on day (E) 3.5 and then cultured on a surface treated with fibronectin in serum- gestation day 4 (GD4, where a copulatory plug is found on free medium, beginning on gestation day (GD) 4. Note that E3.5 is noon of GD1). The two conventions for marking time are used here GD4. 264 D.R. Armant / Developmental Biology 280 (2005) 260–280

Blastocyst adhesion to ECM Trophoblast integrins

The culture of blastocysts in vitro and formation of While many classes of proteins participate in tropho- trophoblast outgrowths was first developed in the 1960s as blast–epithelial interactions during the attachment reaction an in vitro model of implantation (Gwatkin, 1966; Gwatkin (Dey et al., 2004; Kimber, 2000), trophoblast adhesion to and Meckley, 1966). Trophoblast outgrowth is easily ECM appears to be mediated predominantly by integrins observed during blastocyst culture as a spreading monolayer (Sutherland, 2003; Wang and Armant, 2002). Integrins are a of cells around the base of the embryo concomitant with family of heterodimeric (a/h) transmembrane glycoproteins disappearance of the spherical blastocyst morphology. This that bind extracellularly to the ECM and intracellularly to experimental approach was used by several laboratories to adapter proteins that link them to cytoskeletal and signaling examine morphological and biochemical aspects of peri- proteins (Giancotti and Ruoslahti, 1999; Hynes, 2002). A implantation blastocyst development (Enders et al., 1981; role for integrins in trophoblast adhesion was initially Sherman and Atienza-Samols, 1978; Spindle and Pedersen, implicated by the inhibitory activity of an antibody 1973; Surani, 1979). Although there was much interest in (GP140) recognizing a complex of integrins (Richa et al., understanding the molecular basis of trophoblast adhesion 1985) and a small peptide (GRGDSP) representing a in vitro, the original media used for blastocyst culture was sequence within the tenth type III homology repeat of supplemented with serum, which contains adhesive ECM fibronectin (FIII-10) recognized by integrins (Armant et al., components, including fibronectin and vitronectin. For this 1986c). Conversely, trophoblast outgrowth on fibronectin is approach to be viable, it was necessary to develop more specifically inhibited by a peptide corresponding to the defined culture conditions. Toward that end, successful region of the h1 integrin subunit that recognizes the RGD trophoblast outgrowth was reported in serum-free medium sequence (Shiokawa et al., 1999). The RGD sequence when culture plates were coated with collagen (Jenkinson appears in other ECM proteins and mediates their binding to and Wilson, 1973; Wordinger and McGrath, 1979), estab- integrins during cell adhesion cooperatively with other lishing that a major function of serum was the provision of a sequence motifs (Plow et al., 2000; Ruoslahti, 1996). The biologically active substrate for cell adhesion. Later, peptide GRGDSP specifically inhibits trophoblast out- fibronectin, laminin, vitronectin, entactin/nidogen, and other growth on fibronectin, vitronectin, type II collagen, and ECM components were used as surface coatings to generate entactin/nidogen (Armant et al., 1986c; Carson et al., 1988; trophoblast outgrowths in defined, serum free-medium for Yelian et al., 1993). However, outgrowth on laminin 1 is not the purpose of understanding the biochemical basis of blocked by this peptide (Armant et al., 1986c). The E8 trophoblast adhesion to specific ECM proteins (Armant et domain of laminin 1, which is devoid of an RGD sequence, al., 1986a,c; Yelian et al., 1993). is responsible for that outgrowth activity (Armant, 1991). Trophoblast cells adhere to complex ECM and basement Evidence suggests that mouse trophoblast cells adhere to membranes (Cammarata et al., 1987; Wordinger et al., laminin 1 through the a7h1 integrin (Klaffky et al., 2001). 1991), and are capable of differentiating on and migrating The diverse array of adhesive ECM ligands and integrin through ECM purified from mouse endometrium (Armant receptors provides functional redundancy to ensure that and Kameda, 1994). Ultrastructural evidence has established trophoblast cells are able to adhere to and invade the that mouse trophoblast cells adhere to the uterine basement interstitial components of the endometrium. A challenge for membrane around E5.2 in vivo (Blankenship and Given, investigators is to now decipher individual roles of the ECM 1992), after the juxtaembryonic uterine epithelium is lost to components in directing the movements and organization of apoptosis (Schlafke et al., 1985; Parr et al., 1987; Welsh and trophoblast cells during placentation. Studies of knockout Enders, 1991). During blastocyst implantation, the newly mice lacking specific laminin subunits suggest that laminin formed decidua secretes a rich ECM composed of collagens, isoforms have different functions in directing trophoblast proteoglycans, fibronectin, laminin, and entactin/nidogen morphogenesis (Miner et al., 2004). Transgenic models like (Kisalus et al., 1987; Leivo et al., 1980; Rider et al., 1992; these could be useful for revealing the physiological impact Wartiovaara et al., 1979; Wewer et al., 1986; Wu et al., of interactions between specific integrin–ligand partners. 1983), which are available to support trophoblast adhesion Based on experiments using proteolytic fragments of during the invasive phase. Therefore, the interactions fibronectin and peptide inhibitors, the specific target of observed between trophoblast cells and ECM components adhering primary TGCs has been defined as the RGD site in in vitro most likely correspond to adhesive interactions that a 120-kDa fragment of fibronectin (FN-120) containing the occur in utero as the primary TGCs initially adhere to the FIII-10 repeat (Yelian et al., 1995). Adhesion is unsupported basement membrane and commence their invasion of the by another fragment containing the IIICS segment, which underlying decidua. A challenge to investigators is to serves as an alternate (non-RGD) cell recognition site. understand how trophoblast cells efficiently adhere to the These findings are consistent with the observed expression complex mixture of ECM proteins encountered in vivo. in mouse preimplantation embryos of RGD-recognizing Studies of adhesion to fibronectin, described below, provide integrins (a5h1, aVh3, and aIIbh3), but not a4h1ora4h7 some insight into how that could be accomplished. that recognize the IIICS repeat of fibronectin (Schultz et al., D.R. Armant / Developmental Biology 280 (2005) 260–280 265

1997; Sutherland et al., 1993; Yelian et al., 1995). However, integrins on the embryo surface during peri-implantation a4 is expressed on E8.0 in the chorion, and a4 integrin- development. The assay is useful for comparative analysis mediated adhesion is indispensable for chorio-allantoic of adhesion throughout development if embryos are first union (Downs, 2002; Yang et al., 1995). Many integrins treated with heparinase to eliminate microsphere binding to are expressed as early as the oocyte stage, including the heparan sulfate proteoglycans. As would be expected, subunits a5, a6, aV, h1, and h3(Evans et al., 1995; Fusi et fibronectin-binding activity parallels trophoblast outgrowth al., 1993; Hierck et al., 1993; Sutherland et al., 1993; Tarone by first appearing on GD7 (Schultz et al., 1997). Agonists et al., 1993), demonstrating that the preimplantation embryo capable of accelerating the rate of blastocyst outgrowth accumulates integrins capable of binding to fibronectin and concomitantly shift the onset of fibronectin-binding activity other ECM proteins long before the implantation stage. from GD7 to GD6, substantiating the physiological Although integrin genes are highly expressed during relationship between this activity and the adhesion com- preimplantation development, we will see that the presence petence of blastocysts (Wang et al., 1998, 1999, 2000). of integrin proteins at the outer surface of the embryo is restricted. Integrin trafficking appears to be a rate-limiting Molecular interactions between the trophoblast and step in establishing blastocyst adhesion to the ECM. fibronectin As adherent cells spread and migrate, integrins are first clustered in the plasma membrane and then organized into Fibronectin is abundant in the mouse endometrial base- focal adhesions that anchor the actin cytoskeleton to the ment membrane and stromal ECM on E4.5–E5.5, when the ECM to regulate cell shape and migration (Sastry and trophoblast breaches the luminal epithelium and commences Burridge, 2000). a5h1, aVh3, and aIIbh3 all appear in invasion (Wartiovaara et al., 1979). As peri-implantation focal adhesions formed by trophoblast cells during fibro- development proceeds, fibronectin accumulates in the ECM nectin-mediated outgrowth, and blocking antibodies against subjacent to the basement membrane and becomes remod- the h1orh3 subunits delay outgrowth (Rout et al., 2004; eled in the stroma (Rider et al., 1992; Slater and Murphy, Sutherland et al., 1993; Yelian et al., 1995). Although gene 1999). In vitro studies of mouse E7.0 decidua explants knockout studies suggest that none of these integrins are reveal constitutive levels of fibronectin synthesis and essential for implantation (Hynes, 1996), the three integrins deposition (Babiarz et al., 1996), indicative of its dynamic appear to operate synergistically in trophoblast cells adher- role during blastocyst implantation. Other ECM compo- ing to fibronectin (Rout et al., 2004). Details of the latter nents, including laminins and collagens, also undergo study will be discussed below. remodeling (Farrar and Carson, 1992; Glasser et al., 1987; Wewer et al., 1986) and are deposited in isoform-specific Determining the onset of blastocyst adhesion patterns that could influence trophoblast morphology as placentation commences (Miner et al., 2004). Fibronectin A more refined approach than the blastocyst outgrowth colocalizes with invasive extravillous trophoblast cells of model is required to investigate the adhesive activity of the early human implantation site (Earl et al., 1990; integrins at the apical surface of mural trophoblast cells Feinberg et al., 1991) and it promotes anchorage of before they dissociate and migrate outward. To address this placental villous explants (Aplin et al., 1999). Some in dilemma, a fibronectin-binding assay was devised using vitro experiments suggest that fibronectin promotes troph- fluorescent polystyrene microspheres coated with FN-120 oblast adhesion, which may restrict migration, while other that are incubated for 30 min at 48C with intact blastocysts studies indicate that it facilitates motility (Burrows et al., (Schultz and Armant, 1995). The fibronectin-binding assay 1993; Damsky et al., 1994; Irving et al., 1995; Stephens et is ideal to investigate the onset of adhesion at the apical al., 1995; Yelian et al., 1995). Fibronectin has the ability to surface of trophoblast cells, since it is performed rapidly modulate both integrin-mediated adhesion (detailed below) and detection does not require any cellular activity beyond and protease activity (Zhang et al., 1996) of trophoblast ligand binding. Image analysis is used to quantify the cells. In vivo, it could have diverse functions at different intensity of the fluorescence imparted by the bound locations within the implantation site, depending on integrin microspheres. Binding is restricted to the mural trophoblast expression and proximal levels of matrix-degrading pro- where TGCs first differentiate. The specificity of fibronec- teases. Therefore, fibronectin is a relevant and instructive tin-binding activity is demonstrated by competitive inhib- model ligand for studies of mouse primary TGC adhesion to ition with FN-120 or the GRGDSP peptide, but not other ECM. proteins or fragments of fibronectin lacking FIII-10. A key question addressed in studies of trophoblast Importantly, binding activity is specifically blocked by interactions with fibronectin is how the non-adhesive antibodies against the integrin subunits a5, aV, aIIb, h1, surface of the early blastocyst acquires its adhesiveness and h3, which comprise the RGD-recognizing integrins during peri-implantation development. Since some fibro- a5h1, aVh3, and aIIbh3(Rout et al., 2004; Schultz and nectin-binding integrins are expressed on the apical surface Armant, 1995). The fibronectin-binding assay provides an earlier during development, it is important to understand important tool for assessing the adhesive activity of exactly how their activities are regulated during conversion 266 D.R. Armant / Developmental Biology 280 (2005) 260–280 to an adhesive trophoblast surface. The evidence to be So when are the genes that direct this aspect of presented here suggests that trophoblast differentiation is trophoblast development transcribed? This question can be controlled at three levels: (1) the intrinsic developmental addressed by collecting embryos 24 h earlier, at the morula program of the blastocyst, (2) responses to extrinsic stage, for treatment with a-amanitin. Embryos exposed for 8 developmental triggers, and (3) subsequent autocrine signal- htoa-amanitin late on GD3, but not earlier in the day, ing by the trophoblast to reinforce extrinsic input. exhibit significantly reduced fibronectin-binding activity after culture to GD7 (Schultz et al., 1997). This experiment defines a brief period during blastocyst formation when Regulation of adhesion by the intrinsic developmental genes required several days later for trophoblast adhesion to program fibronectin are transcribed. Based on similar experiments using cycloheximide, it has been determined that the period It is first important to establish the nature of the intrinsic of development when protein synthetic activity is required program that advances development during the blastocyst for trophoblast adhesion occurs late (between 16 and 24 h) stage in the context of trophoblast adhesiveness to on GD4 (Schultz et al., 1997), which is in agreement with an fibronectin. What cellular processes are required to unfold earlier investigation that measured outgrowth rates (Blake et the entire program? Is progression through the peri- al., 1982). Results of studies using a-amanitin and cyclo- implantation period dependent on new gene expression? heximide suggest that genes required for the adhesive and Assessments of outgrowth and fibronectin-binding activity migratory activity of primary TGCs are translated about 24 have been used in conjunction with metabolic inhibitors to h after they are transcribed (Table 2). Whether the tran- examine the role of gene transcription, translation, and scripts are sequestered as ribonucleoprotein complexes for protein trafficking in regulating trophoblast development. later translation is not known. Global gene expression analysis of preimplantation Regulation of gene expression development using microarrays indicates that relatively few mRNAs increase in abundance after the 8-cell stage The RNA polymerase II inhibitor, a-amanitin, was ori- (Hamatani et al., 2004a) with the possible exception of a ginally found to have no effect on blastocyst outgrowth burst of transcript accumulation between the 16-cell and when present during any 24-h period between GD4 and GD7 blastocyst stages (Wang et al., 2004), the period of (Schindler and Sherman, 1981). A less specific inhibitor of development when a-amanitin impacts trophoblast differ- transcription, actinomycin D, is also a poor inhibitor of entiation. The 16-cell morula to 32-cell blastocyst transition trophoblast outgrowth (Glass et al., 1976). Conditions of a- encompasses the period of outer cell commitment to the amanitin treatment have been established that completely trophoblast lineage, since blastomeres at the 16-cell stage inhibit poly(A+) RNA synthesis within 3 h in mouse retain totipotency (Ziomek et al., 1982). Therefore, it is not blastocysts without affecting total RNA synthesis (Khidir surprising that transcription at that time is critical for et al., 1995). Culture of blastocysts with a-amanitin between trophoblast function. Discerned patterns of global gene GD4 and GD7 has no effect on the appearance of expression during the preimplantation period support a fibronectin-binding activity early on GD7 (Schultz et al., bwave of activationQ hypothesis in which transcription of 1997), suggesting that genes regulating trophoblast adhesion gene clusters occurs well before execution of their devel- are expressed prior to the early blastocyst stage, over 72 h opmental function (Hamatani et al., 2004a). Trophoblast before the event occurs. De novo gene expression during the adhesion at implantation appears to be regulated in this blastocyst stage could impact ICM development, TGC manner. endoreduplication, or postimplantation events, but appears to have little effect on the progression of trophoblast cells through adhesion and outgrowth. Table 2 The congruent findings of Schindler and Sherman Effect of metabolic inhibitors on trophoblast differentiation (1981) and Schultz et al. (1997) stand in stark contrast Stage of development Effective Required metabolic to transcriptional inhibition studies at an earlier devel- inhibitors processes opmental stage. Treatment of 16-cell morulae with a- Late cavitation (GD3) a-Amanitin mRNA transcription amanitin within 2 h of the onset of cavitation inhibits Early blastocyst (GD4) Cycloheximide Protein synthesis blastocyst formation (Khidir et al., 1995; Kidder and Mid-blastocyst (GD5–6) Tunicamycin N-glycosylation McLachlin, 1985), demonstrating temporal regulation by Late blastocyst (GD6–7) Brefeldin A Protein trafficking de novo gene expression. The effectiveness of a-amanitin Adhesion competent (GD7) Brefeldin A Protein trafficking at the 16-cell morula stage diminishes concern that the [exposed to fibronectin] drug failed to inhibit transcription of critical genes at the Morulae or blastocysts cultured to GD7 were inhibited in their ability to express strong fibronectin-binding activity or form trophoblast outgrowths blastocyst stage, and strengthens the view that adhesive when treated during the indicated stages of embryonic development with differentiation of the trophoblast relies on genes expressed the metabolic inhibitors shown. Cycloheximide and a-amanitin were both earlier during development. ineffective when used before or after the indicated stages. D.R. Armant / Developmental Biology 280 (2005) 260–280 267

Two genes indispensable for trophoblast differentiation Trophoblast differentiation could be regulated in the are the caudal-related homeodomain transcription factor, absence of new gene induction through the degradation of Cdx2 (Chawengsaksophak et al., 1997), and the mouse RNA or proteins that maintain the non-adhesive pheno- homologue of a T-box transcription factor, eomesodermin type. Targeted protein turnover is accomplished through (mEomes)(Russ et al., 2000), which are both expressed the ubiquitination pathway, which directs specific proteins exclusively in the trophectoderm on E3.5 (Beck et al., to the proteosome for degradation (Jackson et al., 2000). A 1995; Hancock et al., 1999). Blastocysts lacking either of component of the ubiquitination pathway, the E2 (ubiquitin these genes fail to outgrow in vitro. Later, the genes are conjugating) enzyme, is among genes induced at the early expressed in the extraembryonic ectoderm in response to blastocyst stage by 0.1% ethanol (Rout et al., 1997), a FGF-4 signaling and are downregulated as trophoblast treatment that activates intracellular Ca2+ signaling to stem cells differentiate (Cross, 2000; Tanaka et al., 1998). accelerate blastocyst outgrowth in vitro and increase The regulation of Cdx2 and mEomes and the identity of implantation rates in vivo (Stachecki et al., 1994). their target genes are not well understood. Cdx2 is Ubiquitinated proteins accumulate in mouse blastocysts involved in mesodermal pattern formation and activates specifically within the trophoblast (Sutovsky et al., 2001), expression of certain Hox genes (Lohnes, 2003). mEomes indicative of an increase in protein turnover as the is associated with mesoderm differentiation from the trophoblast develops. Expression of Cdx2 and mEomes, epiblast at gastrulation and induction of genes regulating which are abundant in trophoblast stem cells of the mesoderm migration (Showell et al., 2004). Sutherland extraembyronic ectoderm, is downregulated during differ- (2003) points out that both trophoblast and pre-meso- entiation to other phenotypes, including the invasive dermal epiblast cells express mEomes and subsequently secondary TGCs (Tanaka et al., 1998). A similar mecha- undergo an epithelial to mesenchymal transition. Expres- nism operates during primary TGC differentiation in sion patterns obtained by microarray-based gene tran- blastocysts. As blastocysts become adhesion competent scription profiling show a sudden accumulation of Cdx2 and initiate outgrowth, strong nuclear expression of Cdx2 and mEomes between the 16-cell and blastocyst stages observed at earlier stages is downregulated (Fig. 2). The (Wang et al., 2004), suggesting that these regulatory genes half-life of proteins like Cdx2 that are transcribed during are activated during blastocyst formation. It is thought that blastocyst formation could regulate trophoblast differ- their expression in trophectoderm cells confers the ability entiation. Cdx2 and mEomes have been ascribed roles in to differentiate into trophoblast. Implantation rescue of maintaining the undifferentiated trophoblast phenotype, Cdx2 null embryos by aggregation with wild type although no experimental evidence is available to support tetraploid blastomeres demonstrates that trophoblast failure this hypothesis. Without more information that specifically is directly responsible for the implantation defect in addresses the role of protein turnover in the ontogeny of mutant blastocysts (Chawengsaksophak et al., 2004). trophoblast adhesion competence, this concept remains The putative functions of Cdx2 and mEomes and their speculative. patterns of expression support the prediction of a- The oligosaccharide moieties of glycoproteins and amanitin experiments that transcription during blastocyst proteoglycans have a vital function in blastocyst attach- formation is requisite for trophoblast adhesive and ment to the uterine epithelium (Carson, 2002; Kimber, migratory activities (Schindler and Sherman, 1981; 2000), and could impact adhesion to the ECM. Genes Schultz et al., 1997). expressed during blastocyst formation may require post- translational modification by asparagine (N)- or serine (O)- Post-translational regulation linked oligosaccharides before they are fully functional. Integrins that mediate trophoblast adhesion to the ECM are Presumably, genes activated during blastocyst forma- among the proteins that require N-glycosylation (Bellis, tion, together with other embryonic gene products, 2004). Indeed, inhibition of N-glycosylation by treatment perform the work of transforming trophoblast cells to with tunicamycin at the mid-blastocyst stage (see Table 2) the invasive phenotype. Since the embryonic program inhibits trophoblast outgrowth in vitro (Armant et al., operates without further need for transcription or protein 1986b; Surani, 1979). During the blastocyst stage, there is synthesis, post-translational mechanisms must temporally a sharp increase in the activity of UDP-GlcNAc: dolichol regulate peri-implantation development. During this period phosphate N-acetylglucosamine-1-phosphate transferase of 48–72 h, the apical surface of the trophoblast cell (GPT), a rate-limiting enzyme in the assembly of N-linked undergoes sequential modifications that direct blastocyst oligosaccharide precursors bound to dolichol (Armant et hatching from the zona pellucida, attachment competence, al., 1986b). Elevation of GPT enzymatic activity at the adhesion to ECM, and invasive activity. Protein turnover, blastocyst stage is unimpeded by a-amanitin, consistent glycosylation, and trafficking are among the metabolic with the ability of trophoblast cells to differentiate processes that could direct trophoblast differentiation independently of de novo mRNA synthesis. Targeted during this period in the absence of further genetic deletion of the GPT gene results in peri-implantation instruction. lethality and generates trophoblast cells incapable of 268 D.R. Armant / Developmental Biology 280 (2005) 260–280

Fig. 2. Downregulation of the Cdx2 transcription factor during primary TGC differentiation. Immunofluorescence was conducted according to published procedures (Liu et al., 2004) using 10 Ag/ml of a mouse monoclonal antibody against the Cdx2 protein (BioGenex, San Ramon, CA) in fixed, permeablilized blastocysts (A–D) or outgrowing trophoblast cells (E–F). For blastocysts, 1-Am optical sections were obtained by fluorescence microscopy and deconvolution, as previously described (Liu et al., 2004). The deconvolved images were then recombined to produce a montage of the entire embryo. Mouse embryos were obtained at the early blastocyst stage on E3.5 (A), cultured to the mid-blastocyst stage approximately 24 h before attaining adhesion competence (B), cultured to the adhesion competent stage (C,D) or cultured on fibronectin-coated plates for 24 h after becoming adhesion competent (E,F). Nuclear localization of Cdx2 is evident in trophoblast cells before blastocysts become adhesion competent (A,B). However, the protein was dramatically downregulated in blastocysts capable of adhesion and outgrowth. A collapsed, adhesion competent blastocyst shown in bright field (C) is devoid of labeled Cdx2 (D). Giant nuclei (arrowheads in E) are abundant in outgrowing trophoblast cells, but none are labeled by the antibody (F). Scale bars, 30 Am, shown for blastocysts in D and for outgrowth in F. survival in culture (Marek et al., 1999), establishing a Treatment with brefeldin A between GD6 and GD7 delays requirement for N-glycosylation during implantation. The blastocyst differentiation, reducing fibronectin-binding time required for protein processing by glycosylation in the activity on GD7 (Schultz et al., 1997). The effectiveness endoplasmic reticulum and Golgi apparatus could influ- of the drug during the period that immediately precedes ence the rate of development to the adhesive blastocyst the onset of trophoblast adhesion (Table 2) indicates that stage. protein trafficking directly regulates blastocyst adhesion Gene products required for cell adhesion must be competence. Moreover, the fibronectin-binding integrin, processed, transported to the plasma membrane, and a5h1, translocates to the apical plasma membrane of assembled into functional multiprotein complexes. There- trophoblast cells on GD7 coincident with the appearance fore, protein trafficking constitutes another level of of adhesion competence (Schultz et al., 1997). Linkage intrinsic regulation during trophoblast differentiation. between insertion of a5h1 into the apical domain of the Brefeldin A interferes in vivo with intracellular protein plasma membrane and trophoblast adhesiveness is sup- trafficking to block secretion and membrane transfer ported by experiments that jointly shift a5h1 trafficking between intracellular organelles (Klausner et al., 1992). and the onset of fibronectin-binding activity from GD7 to D.R. Armant / Developmental Biology 280 (2005) 260–280 269

GD6 through treatments that accelerate in vitro blastocyst fibronectin are achieved by GD6 (Wang et al., 2000). This development (Wang et al., 1998, 1999, 2000). mEomes experiment demonstrates that the uterus provides an optimal knockout blastocysts, which are incompetent to outgrow, environment for development of the free-floating blastocyst. express a5h1(Russ et al., 2000), but it is not known A similar acceleration of trophoblast differentiation is whether the protein trafficks normally to the apical surface achieved when early blastocysts (E3.5) are cultured in of trophoblast cells. As discussed below, the trafficking of medium supplemented with hormones (Wang et al., 1998), additional integrins may be required for trophoblast endocannabinoids (Wang et al., 1999), phospholipids (Liu adhesion to fibronectin. and Armant, 2004), or growth factors (Wang et al., 2000) The intrinsic developmental program of the primary produced by the uterus during the peri-implantation period. trophoblast utilizes mRNAs synthesized during cavitation A large number of molecules produced within the uterus are and proteins that are translated shortly after blastocyst capable of accelerating preimplantation development (Arm- formation, but probably not fully functional until they are ant et al., 2000; Cavagna and Mantese, 2003; Dey et al., processed and transported to their sites of action. Outside of 2004), but discussion here will be limited to a few examples the uterus, completion of the entire developmental process representing the above listed categories. These bioactive from the time of gene activation to attaining adhesion agonists account for at least some of the benefit derived competence takes 3–4 days (summarized in Table 1). from the uterine environment. However, the coordinate regulation of blastocyst and uterine Signaling pathways induced by bioactive factors pro- development to achieve strong adhesion between tropho- duced in the receptive uterus activate downstream bio- blast cells and the endometrium requires the intervention of chemical steps in the intrinsic developmental program of the extrinsic factors that modify and accelerate the embryonic blastocyst. Paracrine and autocrine signaling during the pre- developmental program. and peri-implantation periods supports embryogenesis, in part, through the generation of intracellular Ca2+ transients that operate downstream of at least three important agonists: Extrinsic factors that regulate trophoblast adhesion calcitonin, lysophosphatidic acid (LPA), and HB-EGF (Liu and Armant, 2004; Wang et al., 1998, 2000). Receptors for Advanced blastocyst development is dependent on ex- each protein are expressed by the mouse blastocyst. trinsic factors provided by the maternal reproductive tract. Calcitonin and LPA bind to G protein-coupled receptors Uterine growth factors alter the rate of blastocyst develop- that mobilize Ca2+ from intracellular stores through the ment to synchronize the embryo with the bwindowQ of inositol 1,4,5-trisphosphate (IP3) pathway (Contos et al., uterine receptivity for implantation. Several endogenous 2000; Goldring et al., 1993). HB-EGF also increases agonists that accelerate blastocyst development have been intracellular Ca2+ levels, but through influx mediated by identified and their impact on intracellular signaling to N-type voltage-gated Ca2+ channels in the plasma mem- regulate trophoblast differentiation has been described brane (Wang et al., 2000). Downstream of Ca2+, both (reviewed in Armant et al., 2000). As embryonic and uterine calmodulin and protein kinase C play important roles in the tissues interact more intimately, additional synchronization is acquisition of adhesion competence by mouse blastocysts achieved between trophoblast cells and the molecular milieu (Armant et al., 2000). The cortical reaction in oocytes of the endometrium. For example, the local ECM compo- (Abbott and Ducibella, 2001) and exocytosis in a number of sition may vary as trophoblast cells begin to implant and cell types follows the activation of calmodulin (Chamberlain invade, requiring adjustments in cell surface receptors. et al., 1995; Peters and Mayer, 1998) and protein kinase C Evidence of the fine tuning that occurs on the surface of (Chen et al., 1999; Lan et al., 2001; Quetglas et al., 2000) the trophoblast during adhesion to fibronectin (reviewed in downstream of intracellular Ca2+ transients (Guo et al., Wang and Armant, 2002) suggests a mechanism that 1996; Kiraly-Borri et al., 1996). In blastocysts treated with mobilizes receptors with ligand specificity appropriate for calcitonin or HB-EGF, the induction of a Ca2+ transient on the composition of the ECM engaged by the cells. GD4 or GD5 leads to precocious trafficking of a5 to the apical surface of the trophoblast on GD6, implying that Uterine products that accelerate the intrinsic program reactions downstream of cytosolic free Ca2+ control protein trafficking required for blastocyst adhesion to fibronectin Blastocysts cultured in serum-free medium beginning on (Wang et al., 1998, 2000). Indeed, direct pharmacological GD4 are adhesion competent by GD7 (Armant et al., 1986a; induction of a Ca2+ transient at the early blastocyst stage Schultz et al., 1997; Yelian et al., 1995), about 24 h later using ethanol accelerates the rate of trophoblast outgrowth than when they would have adhered to the endometrial and doubles implantation rates after transferring embryos ECM had they been allowed to develop in vivo (Blanken- back to the uterus (Stachecki et al., 1994). ship and Given, 1992). With prolonged exposure to the In humans, autocrine and paracrine roles for calcitonin maternal environment prior to in vitro culture (harvesting and HB-EGF are prominent during implantation and blastocysts on E4.5/GD5), a5h1 trafficking to the apical placentation. Both are expressed by the uterine epithelium surface of trophoblast cells and blastocyst adhesion to during the bwindowQ of implantation (Kumar et al., 1998; 270 D.R. Armant / Developmental Biology 280 (2005) 260–280

Leach et al., 1999a; Yoo et al., 1997)andHB-EGF sulfate proteoglycans in the ECM. Juxtacrine signaling to stimulates human blastocyst development in vitro (Chobo- the trophoblast by HB-EGF during the attachment reaction tova et al., 2002; Martin et al., 1998). In the placenta, stimulates integrin trafficking required in the final invasive extravillous trophoblast cells express high levels of preparation for adhesion to the ECM and invasion of HB-EGF (Leach et al., 1999a) that can alter integrin the endometrium. expression in cultured cytotrophoblast cells to increase their In addition to its expression in the uterus, HB-EGF is invasive motility (Leach et al., 2004). Additionally, HB- induced in embryonic tissues during activation of dormant EGF is a survival factor (Iwamoto and Mekada, 2000) that blastocysts (Hamatani et al., 2004b). Embryonic HB-EGF could moderate assaults by the maternal immune system and can potentially stimulate uterine differentiation to the other stresses encountered in the reproductive tract. It is receptive state (Paria et al., 2001). We now have evidence notable that aberrantly low levels of HB-EGF are found in that autocrine signaling by HB-EGF in trophoblast cells, placentas of women with pre-eclampsia, a disorder asso- as depicted in Fig. 3, could integrate diverse paracrine ciated with poor trophoblast invasion and survival (Leach et signals arising from the endometrium. For example, the al., 2002). Ca2+-dependent acceleration of blastocyst development by The endocannabinoid, anandamide, accelerates blasto- LPA requires HB-EGF, based on the inhibitory effects of cyst development in a bimodal fashion, becoming inhibitory antibodies against HB-EGF or its receptors, ErbB1 and at higher concentrations (Wang et al., 1999). Anandamide is ErbB4 (Liu and Armant, 2004). HB-EGF trafficks from present at implantation sites, but its levels are much higher the cytoplasm to the surface of trophoblast cells in in the interimplantation region (Schmid et al., 1997), cultured blastocysts treated on GD5 with LPA (Fig. 3A). suggesting a role in embryo spacing within the rodent G protein-coupled receptors can transactivate ErbB1 or uterus. Interestingly, at low, stimulatory levels, anandamide ErbB4 through ectodomain shedding of HB-EGF (Umata has no effect on intracellular Ca2+ levels, but at high et al., 2001). Transactivation occurs downstream to either concentrations that are detrimental to embryonic develop- intracellular Ca2+ or protein kinase C activation (Dethlef- ment anandamide prevents influx of Ca2+ through voltage- sen et al., 1998; Dong and Wiley, 2000). The LPA- gated channels (Wang et al., 2003). Furthermore, low, induced trafficking of HB-EGF to the apical surface of stimulatory concentrations of anandamide activate mitogen- trophoblast cells is blocked by chelation of intracellular activated protein kinase (MAPK). Whether the Ca2+- Ca2+ with BAPTA-AM or by interrupting the IP3 pathway dependent acceleration of blastocyst development involves (Liu and Armant, 2004), supporting our hypothesis that LPA MAPK is not presently known. However, pharmacological initiates Ca2+-dependent trafficking of HB-EGF to the cell elevation of intracellular Ca2+ levels in blastocysts increases surface where it is shed by metalloproteinases (Fig. 3B) and expression of c-Myc (Leach et al., 1999b), a MAPK binds its receptors in an autocrine loop (Fig. 3C). Intra- substrate that is required for preimplantation development cellular Ca2+ signaling is also required downstream of HB- (Paria et al., 1992). Invasiveness of cultured human EGF/ErbB binding (Fig. 3C) for integrin trafficking and cytotrophoblast cells stimulated by the urokinase plasmi- precocious blastocyst differentiation (Wang et al., 2000). nogen activator receptor is dependent on MAPK activation Blastocyst exposure to high, detrimental concentrations of downstream of an intracellular Ca2+ transient (Liu et al., anandamide interferes with Ca2+ entry through voltage- 2003). Therefore, MAPK could be a target of anandamide, gated channels (Wang et al., 2003), perhaps by interrupting as well as agonists that stimulate trophoblast differentiation autocrine HB-EGF signaling (Fig. 3C). It is plausible that through intracellular Ca2+ signaling. intracellular Ca2+ mobilization by other endogenous agonists of G protein-coupled receptors, including calcitonin Integration of multiple uterine signals and histamine (Goldring et al., 1993; Hill et al., 1997; Paria et al., 1998), operate similarly through the transactivation of Temporal and spatial availability within the uterus ErbB receptors by HB-EGF, accounting for their similar varies among paracrine-acting factors that influence biological activities. blastocyst development, as indicated in Fig. 1.LPA, calcitonin, histamine, and several other bioactive agents Fibronectin engagement alters blastocyst adhesion to are secreted into the uterine fluid as the blastocyst exits fibronectin the oviduct on E3.5 (Armant et al., 2000). However, HB- EGF is produced on E4.5 by murine uterine epithelial The positioning of fibronectin receptors on the surface of cells solely at the site of blastocyst apposition (Das et al., the blastocyst is requisite, but not necessarily sufficient, for 1994). This transmembrane peptide can operate locally strong adhesion to fibronectin. The integrin aVh3is through juxtacrine signaling to receptors on adjacent available on the apical surface of trophoblast cells by trophoblast cells (Raab et al., 1996). The ectodomain of GD4 (Sutherland et al., 1993), but FN-120-coated micro- HB-EGF is secreted through metalloproteinase-mediated spheres will not adhere to intact blastocysts until GD7 cleavage (Iwamoto and Mekada, 2000), but may function (Schultz and Armant, 1995). Another fibronectin-binding as a proximal signal due to its strong binding to heparan integrin, a5h1, translocates to the apical plasma membrane D.R. Armant / Developmental Biology 280 (2005) 260–280 271 of trophoblast cells between GD6 and GD7 (Schultz et al., a5h1; evidence that they contribute an essential element of 1997). Although the timing of a5h1 insertion suggests that the adhesion mechanism. this is required for adhesion to fibronectin, the fibronectin- In the presence of brefeldin A, fibronectin fails to binding activity of blastocysts remains extremely low until upregulate fibronectin-binding activity of adhesion compe- they are exposed to fibronectin for at least 1 h (Schultz and tent blastocysts (Table 2), revealing a requirement for Armant, 1995). Therefore, trafficking of aVh3 and a5h1to additional protein trafficking after a5h1 insertion in the the surface of trophoblast cells is, in itself, insufficient for apical cell surface (Schultz and Armant, 1995). Competitive strong adhesion to fibronectin. However, after ligand- inhibition experiments indicate no increase in receptor mediated upregulation by fibronectin, fibronectin-binding affinity (IC50) upon exposure to fibronectin, suggesting that activity is inhibited by antibodies that recognize aVh3or some fully active receptors are present before exposure, but the majority of receptors are inactive. Hypotheses that might account for the increased adhesive activity include, (1) the number of fibronectin receptors on the apical cell surface is increased, (2) additional integrin subtypes are recruited to the apical surface, and (3) fibronectin receptors must be reorganized with other proteins into adhesion complexes. Blastocysts treated with fibronectin show no readily apparent change in the expression of a5h1oraVh3on their surface (Schultz et al., 1997), suggesting that the first hypothesis does not fully account for the altered adhesiveness. However, the second hypothesis is supported by the discovery of a third fibronectin-binding integrin that trafficks to the apical surface of the trophoblast following exposure to fibronectin on GD7 (Rout et al., 2004). The third hypothesis could prove valid, as well, since the cytoplasmic domains of integrins must interact with cytoskeletal and signaling components to promote adhesion (Sastry and Burridge, 2000). Indeed, disruption of the actin cytoskeleton during exposure to fibronectin prevents the upregulation of fibronectin activity (Schultz and Armant, 1995).

The platelet-associated integrin aIIbb3 strengthens trophoblast adhesion to fibronectin

Preimplantation embryos express mRNA encoding the alpha subunit of the integrin, aIIbh3(Schultz et al., 1997), which is associated primarily with cells of the megakar- yocytic lineage (Ginsberg et al., 1993; Phillips et al., 1988). In platelets, aIIbh3 binds fibrinogen during thrombus formation, although its recognition of fibronectin and

Fig. 3. Transactivation of ErbB receptors by G protein-coupled receptors in trophoblast cells. (A) LPA, and other agonists of G protein-coupled receptors, mobilizes intracellular Ca2+ through the IP3 receptor (IP3-R) after activating phospholipase C (PLC). (B) The increased Ca2+ concen- tration induces trafficking of HB-EGF to the plasma membrane, where it is cleaved by metalloproteinases (MP). (C) HB-EGF is then able to bind its receptor in an autocrine loop. Experimental evidence points to Erb4 as the primary receptor for HB-EGF (Paria et al., 1999; Wang et al., 2000). Intracellular Ca2+ levels are again raised, this time by influx through N-type Ca2+ channels (N). A recent report by Wang et al. (2003) suggests that voltage-gated channels might be inhibited in the presence of high concentrations of anandamide (AN), blocking this autocrine pathway. Protein kinase C (PKC) and calmodulin (CAM) operate downstream of Ca2+ to accelerate a5h1 trafficking to the plasma membrane. As a result, blastocysts are adhesion competent approximately 1 day earlier than in unstimulated embryos (Wang et al., 2000). 272 D.R. Armant / Developmental Biology 280 (2005) 260–280 several other RGD-containing proteins, including vitronec- with a mechanism involving their insertion into the plasma tin, von Willebrand factor, and thrombospondin, is probably membrane. Thus, a strong correlation exists between vesicle also physiologically relevant for hemostasis (Savage et al., translocation, aIIb trafficking, and the upregulation of 1996). Semi-quantitative assessment of aIIb mRNA during fibronectin-binding activity. The content of the intracellular preimplantation development reveals an apparent fivefold vesicles is unknown, but it is tempting to speculate that they increase in gene expression during blastocyst formation that deliver to the apical plasma membrane aIIb and other is blocked by a-amanitin (Rout et al., 2004), suggesting that proteins required for strong adhesion. The studies by Rout et aIIb is among the gene products transcribed during al. (2004) and Wang et al. (2002) suggest that aIIb cavitation that later direct trophoblast outgrowth (Table 2). trafficking is a key step in establishing strong adhesion Immunofluorescence microscopy reveals aIIb in focal between trophoblast cells and fibronectin (Figs. 4A,B). adhesions of trophoblast cells outgrowing on fibronectin Examination of embryo implantation sites reveals aIIb (Rout et al., 2004; Yelian et al., 1995), as expected of an localized in primary TGCs of the parietal yolk sac adhering integrin that mediates adhesion to fibronectin. Most to the Reichert’s membrane on E5.5 (Rout et al., 2004). By interesting was the observation that aIIb remains intra- E6.5, aIIb also appears in migrating secondary TGCs as cellular after aV and a5 have translocated to the apical they differentiate near the margins of the EPC. During surface of trophoblast cells on GD7; however, upon implantation, aIIbh3-mediated adhesion could initially exposure to fibronectin, there is a significant increase in anchor mural trophoblast cells to the uterine wall, and aIIb at the blastocyst surface (Rout et al., 2004). In support may later function in secondary TGC migratory activity of its role in trophoblast adhesion, a function-blocking (Fig. 4C). antibody against the aIIb subunit inhibits fibronectin-binding activity (Rout et al., 2004). Cooperative activities of fibronectin receptors The trafficking of aIIb to the apical surface of the trophoblast coincides with the second period of sensitivity As mural trophoblast cells acquire adhesive activity, to brefeldin A (Table 2). As the blastocyst becomes some division of labor can be discerned among the adhesion competent, intracellular vesicles accumulate in fibronectin-binding integrins. According to antibody inhib- mural trophoblast cells; however, they are absent after ition studies (Rout et al., 2004; Schultz and Armant, 1995), exposure to fibronectin (Wang et al., 2002). Brefeldin A contributions from three fibronectin-binding integrins, inhibits vesicle depletion induced by fibronectin, consistent aVh3, aIIbh3, and a5h1, are required to achieve strong

Fig. 4. Integrin trafficking during trophoblast development. (A) In the early blastocyst, aVh3 (white circles) is present at the apical surface of non-adhesive trophoblast cells, whereas a5h1 (red circles) and aIIb subunits (black circles) are localized either intracellularly or at the basal cell surface. (B) In adhesion competent blastocysts, a5h1 has been inserted into the apical plasma membrane of trophoblast cells. Integrin ligation by fibronectin (FN), particularly its binding to a5h1, initiates intracellular signaling (*) required for trafficking of aIIb subunits to the apical surface. Once aIIbh3 is available, fibronectin-binding activity is strengthened. (C) During early postimplantation development, aIIbh3 is the predominant fibronectin-binding integrin of trophoblast cells. It is highly expressed in primary TGCs adhering to the Reichert’s membrane (left) and in secondary TGCs migrating at the periphery of the EPC. In vitro analysis of migrating trophoblast cells suggests a model (right) in which the h3 integrins are primarily responsible for motility. They appear in focal adhesions near the leading edge of cells, where they presumably attach the plasma membrane to ECM. Focal adhesions are disassembled in the middle and at the trailing edge of cells to allow forward movement. D.R. Armant / Developmental Biology 280 (2005) 260–280 273 adhesion to fibronectin and promote cell migration. The outgrowth. Perhaps there is a point at which the embryo contribution of additional integrins that recognize fibronec- commits to an adhesive mechanism incorporating the tin, in particular a8h1 and other aV integrins, has yet to be available integrin genes. The use of RNA interference examined. Culturing blastocysts on fibrinogen, which is a (Svoboda et al., 2001) to knock down integrin transcripts poor substrate for a5h1, in the presence of function- might have advantages over gene knockouts as an approach blocking antibodies against h3 to eliminate aVh3 and to understanding how integrins are recruited for specific aIIbh3 activity completely abolishes trophoblast outgrowth functions, although these experiments are complicated (Rout et al., 2004), suggesting that these three integrins somewhat by the disparity between the periods of integrin function as the principal receptors for fibronectin. synthesis and function in peri-implantation embryos. Trophoblast outgrowth on fibronectin is inhibited almost completely by an antibody against h3 or a combination of Cellular recognition of ECM components antibodies against aV and aIIb, whereas inhibition of a5h1 has no significant effect on migration. From this result, it Trophoblast adhesion to the ECM in vivo is complex, appears that the h3 integrins are primarily responsible for considering that the cells initially adhere to the epithelial migratory activity. Anti-aIIb antibody disrupts focal adhe- basement membrane, migrate throughout the decidua and sions of trophoblast cells, displacing them from the finally invade uterine blood vessels. Invasion would be migrating edge of the cell. The redistribution of focal facilitated by an efficient mechanism to recruit adhesive adhesions to the center of cells could convert them from receptors as trophoblast cells encounter various ECM migratory to stationary. While there has been much attention components. The ligand-mediated upregulation of tropho- paid to the promotion of cell invasion by aVh3(Chatterjee blast adhesion to fibronectin could operate as a fibronectin and Chatterjee, 2001; Huang et al., 2000; Leavesley et al., sensor that activates its cognate receptors. Together with 1992; Marshall and Hart, 1996), more studies are needed to sensors for other ECM components, invading trophoblast understand the role of aIIbh3 in trophoblast migration. It cells could rapidly adapt within a heterogeneous uterine has come to light in recent years that aIIb expression is not ECM. restricted to the megakaryocyte lineage, and that aIIbh3 The ECM-dependent selectivity of integrin activation can serves a critical function in the motility of highly invasive be demonstrated by measuring fibronectin-binding activity tumors (Chen et al., 1997; Trikha et al., 2002). The after exposing blastocysts to either fibronectin or laminin. documented activity of aVh3andaIIbh3 in other invasive Only fibronectin upregulates fibronectin-binding activity cell types is in keeping with their essential function in (Wang et al., 2002). Conversely, laminin, but not fibronec- trophoblast migration. tin, can upregulate laminin binding activity (Schultz and Ligation of a5h1 influences the initiation of trophoblast Armant, unpublished). These results support the idea that outgrowth (Rout et al., 2004). Fibrinogen, which binds only contact with different components of the ECM activates the h3 integrins, supports trophoblast outgrowth after a appropriate receptors at the surface of trophoblast cells. This substantial delay and upregulates the fibronectin-binding example of physiological fine-tuning during trophoblast activity of intact blastocysts at a rate three times slower than differentiation may reflect an increased reliance after fibronectin. Mice lacking the h1 gene produce primary blastocyst formation on environmental cues over a rigid TGCs that adhere to and outgrow on fibronectin (Stephens developmental program. Recent studies of trophoblast et al., 1995), consistent with the ability of fibrinogen to adhesion to fibronectin have begun to shed light on the support outgrowth. While the h3 integrins can operate in the biochemical basis of ECM recognition, which incorporates absence of a5h1, antibody inhibition demonstrates that an autocrine signaling mechanism. a5h1 most efficiently initiates the pathway that establishes strong adhesion (Rout et al., 2004). Although none of these fibronectin-binding integrins are Autocrine signaling in trophoblast cells contacting indispensable for trophoblast outgrowth or implantation fibronectin (Hynes, 1996), the accumulated evidence indicates that the default pathway in wild type trophoblast cells relies on The ability of integrins to mediate intracellular signaling a5h1 signaling to upregulate adhesion, aIIbh3 to mediate upon ligation by ECM components (Kornberg et al., 1991) strong adhesion (Figs. 4A,B), and the h3 integrins for cell has been known for over a decade to regulate cell growth, motility (Fig. 4C). The survival and invasive integrity of adhesion, motility, and differentiation (Aplin et al., 1998; primary TGCs in mutant mice lacking fibronectin or its Burridge and Chrzanowska-Wodnicka, 1996; Hynes, 1992). integrin receptors suggests that functional redundancies in Autocrine signaling involving integrins includes both the the developmental program ensure the success of this initiation and transduction of intracellular signals. Integrin critical step. It is interesting that trophoblast cells can ligation produces boutside-in signalingQ through integrin manage a bwork aroundQ when an integrin gene is deleted, clustering and association with cytoplasmic proteins that but they are considerably less agile when inhibitory initiate intracellular biochemical cascades (Fig. 5A). Signals antibodies are introduced immediately prior to blastocyst originating within cells can activate cytoplasmic proteins 274 D.R. Armant / Developmental Biology 280 (2005) 260–280

increases binding activity to the same extent as fibronectin (Wang et al., 2002). Integrins become clustered in the plasma membrane upon ligation by fibronectin, which recruits protein tyrosine kinases, their substrates and other signaling proteins to associate in focal adhesion complexes with cytoskeletal elements of the cortical cytoplasm (Miyamoto et al., 1995). Focal adhesion complexes mediate adhesion at the basal surface of cells in culture. Occupied integrins can also aggregate with signaling and cytoskeletal proteins at the apical cell surface, as demonstrated by Miyamoto et al. (1995). The same process could occur at the apical surface of trophoblast cells in blastocysts exposed to fibronectin in vitro or contacting fibronectin in the endometrial ECM during implantation (see Fig. 5). In addition to protein tyrosine phosphorylation (Korn- berg et al., 1991), early reports demonstrated that integrin ligation could elevate intracellular levels of Ca2+ in lymphocytes (Pardi et al., 1989), neutrophils (Ng-Sikorski et al., 1991), platelets (Smith et al., 1991), and endothelial cells (Schwartz, 1993). Ligation of fibronectin receptors on the surface of mouse blastocysts induces an immediate increase in intracellular Ca2+ that is sustained over a 30- to 60-min period (Wang et al., 2002). Suppression of Ca2+ signaling by pretreating embryos with the intracellular Ca2+ chelator BAPTA-AM inhibits the upregulation of fibronectin-binding activity by FN-120. Moreover, pharmacological elevation of intracellular Ca2+ upregulates fibronectin-binding activity and induces vesicle depletion in the mural trophoblast. These experiments show that Ca2+ signaling induced by integrin ligation is both necessary and sufficient to strengthen trophoblast adhesion to fibronectin. Integrins elevate intracellular Ca2+ levels either through Fig. 5. Hypothetical model of autocrine signaling in adhesion competent 2+ trophoblast cells exposed to fibronectin. (A) With ligation of a5h1by influx or release from intracellular Ca stores (McNamee et fibronectin (FN), cytoplasmic proteins associate with integrin aggregates at al., 1993; Sjaastad et al., 1996; Somogyi et al., 1994; Wu et the plasma membrane. Protein tyrosine kinases (PTK) phosphorylate PLC- al., 1998). In adrenal glomerulosa cells, fibronectin ligation g within this complex, leading to the production of IP3 and diacylglycerol of a5h1oraVh3 elevates intracellular Ca2+ by a 2+ (DAG). IP3 binds to the IP3 receptor (IP3-R), releasing stored Ca into the mechanism involving release from intracellular stores cytoplasm. During development to the adhesion competent stage, intracellular vesicles accumulate in mural trophoblast cells. It is hypothesized through the IP3 receptor (Campbell et al., 2003). Phospho- that the vesicles contain aIIbh3 and other proteins that promote cell lipase C-g (PLC-g) is among the cytoplasmic proteins that adhesion. (B) With the elevation of intracellular Ca2+, calmodulin (CAM) is colocalize with clustered integrins (Miyamoto et al., 1995). activated. Depending on the isoform, PKC may be activated by DAG or Its phosphorylation by protein tyrosine kinases that also 2+ Ca . Signaling by both proteins is required for the upregulation of associate with aggregated integrins (e.g., Src, FAK) could fibronectin-binding activity. Ca2+ signaling is needed for the depletion (apparent trafficking, solid arrows from PKC and CAM) of the intracellular activate PLC-g to produce IP3 (discussed in Wang and vesicles. CAM and PKC could also activate aIIbh3 after its trafficking to Armant, 2002). In mouse blastocysts, the ligand-mediated the apical plasma membrane (dashed arrows). Presumably, aIIbh3is upregulation of fibronectin-binding activity and associated integrated into a nascent adhesion complex linked to elements of the actin Ca2+ transients are blocked by antagonists of protein cytoskeleton, which strengthens adhesion to fibronectin. tyrosine kinases, PLC and the IP3 receptor, but not by inhibition of Ca2+ influx or voltage-gated Ca2+ channels that associate with integrins to modulate their adhesive (Wang, Mayernik, Kilburn, and Armant, in submission). function, a process termed binside-out signalingQ (Fig. 5B). This information suggests a mechanism of outside-in Ligand-mediated upregulation of fibronectin-binding activ- integrin signaling (Fig. 5A). In this scheme, integrin ligation ity in trophoblast cells is an example of integrin autocrine by fibronectin activates associated protein tyrosine kinases signaling. and PLC-g to generate IP3, which activates IP3 receptors The participation of integrins in the upregulation of that release stored Ca2+ into the cytoplasm. fibronectin-binding activity can be demonstrated by directly Integrin downstream signaling leads to inside-out ligating a5h1oraVh3 with appropriate antibodies, which signaling, as depicted in Fig. 5B. As in the Ca2+-dependent D.R. Armant / Developmental Biology 280 (2005) 260–280 275 acceleration of blastocyst differentiation by growth factors, blastocyst development to the adhesive stage. Transit fibronectin-induced upregulation of adhesion requires both through key steps in differentiation is initiated by the calmodulin and protein kinase C activities (Wang et al., activity of Ca2+-dependent proteins, including calmodulin 2002). Calmodulin is directly activated by Ca2+ (Lu and and protein kinase C, that trigger the delivery of proteins Means, 1993) and calmodulin-dependent proteins can alter needed for cell adhesion to the apical cell domain. The integrin binding activity (Pomies et al., 1995). Protein regulation of trophoblast adhesion independently of gene kinase C is recruited to integrins clustered by fibronectin activation enables cells to rapidly adapt to variations in (Miyamoto et al., 1995) where it could be activated by the local environment. As trophoblast cells migrate diacylglycerol or Ca2+ mobilized through the IP3 receptor. through a heterogeneous ECM, adhesion is maintained Once active, protein kinase C could upregulate integrin by switching integrin receptors. The integrin switching binding activity. Protein kinase C-a activates aIIbh3 hypothesis (Damsky et al., 1994) suggests that trophoblast during platelet aggregation (Tabuchi et al., 2003), suggest- cells recognize the molecular composition of the local ing the intriguing idea that a similar process occurs in the ECM and modify their cell surfaces to optimally interact trophoblast. Additionally, both calmodulin and protein with it for purposes of both embryo anchorage and cell kinase C regulate exocytosis and protein trafficking (Chen invasion. et al., 1999). Calmodulin is required for essential steps in Evidence discussed here suggests that trophoblast the fusion of secretory vesicles with the plasma membrane adhesion is orchestrated through a series of trafficking (Quetglas et al., 2000; Peters and Mayer, 1998). Delivery of events that position receptors at the embryo surface for proteins to the plasma membrane is stimulated by the subsequent signaling, which initiates further trafficking activity of protein kinase C (Lan et al., 2001), particularly (see Fig. 1B). Paracrine signaling by secreted uterine the h isoform (Long et al., 2001; Marinari et al., 2003; agonists (e.g., LPA, calcitonin) advance blastocyst devel- Nechushtan et al., 2000). The protein kinase C isoform that opment, perhaps in part through protein trafficking. regulates fibronectin-binding activity in trophoblast cells Trafficking of ErbB4 to the surface of early blastocysts has not yet been identified, nor have the critical proteins (E4.0) is required before HB-EGF becomes an effective downstream of it or calmodulin. Several isoforms of protein agonist (Wang et al., 2000). Ca2+ influx downstream of kinase C are expressed by the early blastocyst stage and HB-EGF binding to ErbB4 drives the insertion of a5h1 most appear to accumulate in the apical or junctional plasma into the apical plasma membrane, enabling integrin signal- membranes of the trophectoderm (Eckert et al., 2004; ing (Wang et al., 2000). Once the embryo contacts Pauken and Capco, 2000). These or other protein kinase C fibronectin within the endometrial ECM, integrin signaling isoforms that are expressed by the late blastocyst stage initiates the Ca2+-dependent events that translocate aIIb to could mediate the inside-out signaling pathway. During the apical surface and strengthen adhesion. Signaling at peri-implantation development, it appears that the second each of these steps is dependent on a previous trafficking messenger Ca2+, operating through established downstream event, providing a glimpse of the developmental program pathways, orchestrates trophoblast adhesive differentiation that guides the conversion of primary trophoblast cells to by initiating the sequential trafficking of critical proteins an adhesion competent phenotype. into the plasma membrane (Figs. 3 and 5). Whether other Many unanswered questions remain regarding the aspects of trophoblast phenotype are impacted by this appropriateness of generalizing processes observed during process remains to be determined. the upregulation of fibronectin-binding activity to other ECM components or to later stages of trophoblast development. Functions unrelated to trophoblast adhesion and Concluding remarks outgrowth could depend on de novo gene expression. It would be interesting to know whether intracellular signaling The developing blastocyst has proven to be an initiated by integrin ligation differentially regulates embry- informative model for investigating the ontogeny of cell onic gene expression. While the emergence of trophoblast adhesion during mammalian embryogenesis. Reminiscent subtypes during placentation is certainly regulated by of the early zygote, the intrinsic developmental program specific genes (Cross, 2000; Rossant and Cross, 2001), of primary trophoblast cells is directed by transcripts or regulation by extrinsic factors operating at the post- proteins synthesized at an earlier stage (Schultz et al., transcriptional level remains an open and intriguing 1997). Post-translational processing and trafficking of possibility. The paradigm represented by fibronectin- gene products, rather than biosynthesis, determines the mediated adhesion in the implanting blastocyst suggests onset of trophoblast adhesiveness in preparation for how positional information could be translated to direct the blastocyst implantation. One caveat of this paradigm is rapid emergence of trophoblast subtype populations during a lack of information about the integrity of other pattern formation within the developing placenta. The embryonic functions, such as proliferation and endoredu- innovation of new technologies for investigating tropho- pication, in embryos blocked for transcription or trans- blast stem cells in vitro (Gerami-Naini et al., 2004; Tanaka lation. Ca2+ signaling plays a major role in advancing et al., 1998; Xu et al., 2002) presents an opportunity to 276 D.R. Armant / Developmental Biology 280 (2005) 260–280 examine the role of extrinsic factors on trophoblast differ- Burridge, K., Chrzanowska-Wodnicka, M., 1996. Focal adhesions, con- entiation into its various subtypes. tractility, and signaling. Annu. Rev. Cell Dev. Biol. 12, 463–518. Burrows, T.D., King, A., Loke, Y.W., 1993. Expression of integrins by human trophoblast and differential adhesion to laminin or fibronectin. Hum. Reprod. 8, 475–484. Acknowledgments Cammarata, P.R., Oakford, L., Cantu-Crouch, D., Wordinger, R., 1987. Attachment of blastocysts to lens capsule: a model system for Studies by the author that were discussed in this review trophoblast–epithelial cell interaction on a natural basement membrane. were supported by National Institutes of Health grants Cell Tissue Res. 250, 633–640. Campbell, S., Otis, M., Cote, M., Gallo-Payet, N., Payet, M.D., 2003. HD36764 and AA12057. The author thanks Drs. Stephen Connection between integrins and cell activation in rat adrenal Krawetz and Daniel Rappolee for helpful discussions and glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of Brian A. Kilburn for technical assistance with mouse the p42/p44(mapk) pathway and intracellular calcium. Endocrinology embryo culture and Cdx2 protein immunofluorescence 144, 1486–1495. microscopy. Carson, D.D., 2002. The glycobiology of implantation. Front Biosci. 7, d1535–d1544. Carson, D.D., Tang, J.P., Gay, S., 1988. Collagens support embryo attachment and outgrowth in vitro: effects of the Arg-Gly-Asp References sequence. Dev. Biol. 127, 368–375. Carson, D.D., Bagchi, I., Dey, S.K., Enders, A.C., Fazleabas, A.T., Abbott, A.L., Ducibella, T., 2001. Calcium and the control of mammalian Lessey, B.A., Yoshinaga, K., 2000. Embryo implantation. Dev. Biol. cortical granule exocytosis. Front Biosci. 6, D792–D806. 223, 217–237. Adamson, S.L., Lu, Y., Whiteley, K.J., Holmyard, D., Hemberger, M., Cavagna, M., Mantese, J.C., 2003. Biomarkers of endometrial receptivity— Pfarrer, C., Cross, J.C., 2002. Interactions between trophoblast cells and A review. Placenta 24, S39–S47 (Suppl B). the maternal and fetal circulation in the mouse placenta. Dev. Biol. 250, Chai, N., Patel, Y., Jacobson, K., McMahon, J., McMahon, A., Rappolee, 358–373. D.A., 1998. FGF is an essential regulator of the fifth cell division in Aplin, A.E., Howe, A., Alahari, S.K., Juliano, R.L., 1998. Signal preimplantation mouse embryos. Dev. Biol. 198, 105–115. transduction and signal modulation by cell adhesion receptors: the role Chamberlain, L.H., Roth, D., Morgan, A., Burgoyne, R.D., 1995. Distinct of integrins, cadherins, immunoglobulin–cell adhesion molecules, and effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and selectins. Pharm. Rev. 50, 197–263. triggering of regulated exocytosis. J. Cell Biol. 130, 1063–1070. Aplin, J.D., Haigh, T., Jones, C.J., Church, H.J., Vicovac, L., 1999. Chatterjee, N., Chatterjee, A., 2001. Role of alphavbeta3 integrin Development of cytotrophoblast columns from explanted first-trimester receptor in the invasive potential of human cervical cancer (SiHa) cells. human placental villi: role of fibronectin and integrin alpha 5 beta 1. J. Environ. Pathol., Toxicol. Oncol. 20, 211–221. Biol. Reprod. 60, 828–838. Chawengsaksophak, K., James, R., Hammond, V.E., Kontgen, F., Beck, F., Armant, D.R., 1991. Cell interactions with laminin and its proteolytic 1997. Homeosis and intestinal tumours in Cdx2 mutant mice. Nature fragments during outgrowth of mouse primary trophoblast cells. Biol. 386, 84–87. Reprod. 45, 664–672. Chawengsaksophak, K., de Graaff, W., Rossant, J., Deschamps, J., Beck, F., Armant, D.R., Kameda, S., 1994. Mouse trophoblast cell invasion of 2004. Cdx2 is essential for axial elongation in mouse development. extracellular matrix purified from endometrial tissue: a model for peri- Proc. Natl. Acad. Sci. U. S. A. 101, 7641–7645. implantation development. J. Exp. Zool. 269, 146–156. Chen, Y.Q., Trikha, M., Gao, X., Bazaz, R., Porter, A.T., Timar, J., Honn, Armant, D.R., Kaplan, H.A., Lennarz, W.J., 1986a. Fibronectin and laminin K.V., 1997. Ectopic expression of platelet integrin alphaIIb beta3 in promote in vitro attachment and outgrowth of mouse blastocysts. Dev. tumor cells from various species and histological origin. Int. J. Cancer Biol. 116, 519–523. 72, 642–648. Armant, D.R., Kaplan, H.A., Lennarz, W.J., 1986b. N-linked glyco- Chen, Y.A., Duvvuri, V., Schulman, H., Scheller, R.H., 1999. Calmodulin protein biosynthesis in the developing mouse embryo. Dev. Biol. and protein kinase C increase Ca(2+)-stimulated secretion by modulat- 113, 228–237. ing membrane-attached exocytic machinery. J. Biol. Chem. 274, Armant, D.R., Kaplan, H.A., Mover, H., Lennarz, W.J., 1986c. The effect 26469–26476. of hexapeptides on attachment and outgrowth of mouse blastocysts Chobotova, K., Spyropoulou, I., Carver, J., Manek, S., Heath, J.K., Gullick, cultured in vitro: evidence for the involvement of the cell recognition W.J., Barlow, D.H., Sargent, I.L., Mardon, H.J., 2002. Heparin-binding tripeptide Arg-Gly-Asp. Proc. Natl. Acad. Sci. U. S. A. 83, 6751–6755. epidermal growth factor and its receptor ErbB4 mediate implantation of Armant, D.R., Wang, J., Liu, Z., 2000. Intracellular signaling in the the human blastocyst. Mech. Dev. 119, 137–144. developing blastocyst as a consequence of the maternal–embryonic Collins, J.E., Fleming, T.P., 1995. Epithelial differentiation in the mouse dialogue. Semin. Reprod. Med. 18, 273–287. preimplantation embryo: making adhesive cell contacts for the first Babiarz, B., Romagnano, L., Afonso, S., Kurila, G., 1996. Localization and time. Trends Biochem. Sci. 20, 307–312. expression of fibronectin during mouse decidualization in vitro: Contos, J.J., Ishii, I., Chun, J., 2000. Lysophosphatidic acid receptors. Mol. mechanisms of cell:matrix interactions. Dev. Dyn. 206, 330–342. Pharmacol. 58, 1188–1196. Beck, F., Erler, T., Russell, A., James, R., 1995. Expression of Cdx-2 in the Cross, J.C., 2000. Genetic insights into trophoblast differentiation and mouse embryo and placenta: possible role in patterning of the extra- placental morphogenesis. Semin. Cell Dev. Biol. 11, 105–113. embryonic membranes. Dev. Dyn. 204, 219–227. Cross, J.C., Werb, Z., Fisher, S.J., 1994. Implantation and the placenta: key Bellis, S.L., 2004. Variant glycosylation: an underappreciated regulatory pieces of the development puzzle. Science 266, 1508–1518. mechanism for beta1 integrins. Biochim. Biophys. Acta 1663, 52–60. Damsky, C.H., Librach, C., Lim, K.H., Fitzgerald, M.L., McMaster, M.T., Blake, E.J., Schindler, J., Sherman, M.I., 1982. Protein synthetic re- Janatpour, M., Zhou, Y., Logan, S.K., Fisher, S.J., 1994. Integrin quirements for the outgrowth of trophoblast cells from mouse blasto- switching regulates normal trophoblast invasion. Development 120, cysts. J. Exp. Zool. 224, 401–408. 3657–3666. Blankenship, T.N., Given, R.L., 1992. Penetration of the uterine epithelial Das, S.K., Wang, X.N., Paria, B.C., Damm, D., Abraham, J.A., basement membrane during blastocyst implantation in the mouse. Anat. Klagsbrun, M., Andrews, G.K., Dey, S.K., 1994. Heparin-binding Rec. 233, 196–204. EGF-like growth factor gene is induced in the mouse uterus D.R. Armant / Developmental Biology 280 (2005) 260–280 277

temporally by the blastocyst solely at the site of its apposition: a Guo, X., Przywara, D.A., Wakade, T.D., Wakade, A.R., 1996. possible ligand for interaction with blastocyst EGF-receptor in Exocytosis coupled to mobilization of intracellular calcium by implantation. Development 120, 1071–1083. muscarine and caffeine in rat chromaffin cells. J. Neurochem. 67, Denker, H.W., 1993. Implantation: a cell biological paradox. J. Exp. Zool. 155–162. 266, 541–558. Gwatkin, R.B., 1966. Defined media and development of mammalian eggs Dethlefsen, S.M., Raab, G., Moses, M.A., Adam, R.M., Klagsbrun, M., in vitro. Ann. N.Y. Acad. Sci. 139, 79–90. Freeman, M.R., 1998. Extracellular calcium influx stimulates metal- Gwatkin, R.B., Meckley, P.E., 1966. Chromosomes of the mouse blastocyst loproteinase cleavage and secretion of heparin-binding EGF-like following its attachment and outgrowth in vitro. Ann. Med. Exp. Biol. growth factor independently of protein kinase C. J. Cell Biochem. Fenn. 44, 125–127. 69, 143–153. Hamatani, T., Carter, M.G., Sharov, A.A., Ko, M.S., 2004a. Dynamics of Dey, S.K., Lim, H., Das, S.K., Reese, J., Paria, B.C., Daikoku, T., Wang, global gene expression changes during mouse preimplantation develop- H., 2004. Molecular cues to implantation. Endocr. Rev. 25, 341–373. ment. Dev. Cell 6, 117–131. Dickson, A.D., 1963. Trophoblastic giant cell transformation of mouse Hamatani, T., Daikoku, T., Wang, H., Matsumoto, H., Carter, M.G., Ko, blastocysts. J. Reprod. Fertil. 6, 465–466. M.S., Dey, S.K., 2004b. Global gene expression analysis identifies Dong, J., Wiley, H.S., 2000. Trafficking and proteolytic release of molecular pathways distinguishing blastocyst dormancy and activation. epidermal growth factor receptor ligands are modulated by their Proc. Natl. Acad. Sci. U. S. A. 101, 10326–10331. membrane-anchoring domains. J. Biol. Chem. 275, 557–564. Hancock, S.N., Agulnik, S.I., Silver, L.M., Papaioannou, V.E., 1999. Downs, K.M., 2002. Early placental ontogeny in the mouse. Placenta 23, Mapping and expression analysis of the mouse ortholog of Xenopus 116–131. Eomesodermin. Mech. Dev. 81, 205–208. Earl, U., Estlin, C., Bulmer, J.N., 1990. Fibronectin and laminin in the early Hierck, B.P., Thorsteinsdottir, S., Niessen, C.M., Freund, E., Iperen, L.V., human placenta. Placenta 11, 223–231. Feyen, A., Hogervorst, F., Poelmann, R.E., Mummery, C.L., Sonnen- Eckert, J.J., McCallum, A., Mears, A., Rumsby, M.G., Cameron, I.T., berg, A., 1993. Variants of the alpha-6 beta-1 laminin receptor in early Fleming, T.P., 2004. Specific PKC isoforms regulate blastocoel murine development: distribution, molecular cloning and chromosomal formation during mouse preimplantation development. Dev. Biol. localization of the mouse integrin alpha-6 subunit. Cell Adhes. 274, 384–401. Commun. 1, 33–53. Enders, A.C., Chavez, D.J., Schlafke, S., 1981. Comparison of implantation Hill, S.J., Ganellin, C.R., Timmerman, H., Schwartz, J.C., Shankley, N.P., in utero and in vitro. In: Glasser, S.R., Bullock, D.W. (Eds.), Cellular Young, J.M., Schunack, W., Levi, R., Haas, H.L., 1997. International and Molecular Aspects of Implantation. Plenum Press, New York, NY, union of pharmacology: XIII. Classification of histamine receptors. pp. 365–382. Pharmacol. Rev. 49, 253–278. Evans, J.P., Schultz, R.M., Kopf, G.S., 1995. Identification and localization Huang, S., Stupack, D., Liu, A., Cheresh, D., Nemerow, G.R., 2000. Cell of integrin subunits in oocytes and eggs of the mouse. Mol. Reprod. growth and matrix invasion of EBV-immortalized human B lympho- Dev. 40, 211–220. cytes is regulated by expression of alpha(v) integrins. Oncogene 19, Faria, T.N., Deb, S., Kwok, S.C., Talamantes, F., Soares, M.J., 1990. 1915–1923. Ontogeny of placental lactogen-I and placental lactogen-II expression in Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell the developing rat placenta. Dev. Biol. 141, 279–291. adhesion. Cell 69, 11–25. Farrar, J.D., Carson, D.D., 1992. Differential temporal and spatial ex- Hynes, R.O., 1996. Targeted mutations in cell adhesion genes: what have pression of mRNA encoding extracellular matrix components in decidua we learned from them? Dev. Biol. 180, 402–412. during the peri-implantation period. Biol. Reprod. 46, 1095–1108. Hynes, R.O., 2002. Integrins: bidirectional, allosteric signaling machines. Feinberg, R.F., Kliman, H.J., Lockwood, C.J., 1991. Is oncofetal Cell 110, 673–687. fibronectin a trophoblast glue for human implantation? Am. J. Pathol. Irving, J.A., Lysiak, J.J., Graham, C.H., Hearn, S., Han, V.K., Lala, P.K., 138, 537–543. 1995. Characteristics of trophoblast cells migrating from first Fusi, F.M., Vignali, M., Gailit, J., Bronson, R.A., 1993. Mammalian oocytes trimester chorionic villus explants and propagated in culture. Placenta exhibit specific recognition of the RGD (Arg-Gly-Asp) tripeptide and 16, 413–433. express oolemmal integrins. Mol. Reprod. Dev. 36, 212–219. Iwamoto, R., Mekada, E., 2000. Heparin-binding EGF-like growth Gardner, R.L., 2000. Flow of cells from polar to mural trophectoderm is factor: a juxtacrine growth factor. Cytokine Growth Factor Rev. 11, polarized in the mouse blastocyst. Hum. Reprod. 15, 694–701. 335–344. Gardner, R.L., Papaioannou, V.E., Barton, S.C., 1973. Origin of the Jackson, P.K., Eldridge, A.G., Freed, E., Furstenthal, L., Hsu, J.Y., Kaiser, ectoplacental cone and secondary giant cells in mouse blastocysts B.K., Reimann, J.D., 2000. The lore of the RINGs: substrate reconstituted from isolated trophoblast and inner cell mass. J. Embryol. recognition and catalysis by ubiquitin ligases. Trends Cell Biol. 10, Exp. Morphol. 30, 561–572. 429–439. Gerami-Naini, B., Dovzhenko, O.V., Durning, M., Wegner, F.H., Jenkinson, E.J., Wilson, I.B., 1973. In vitro studies on the control of Thomson, J.A., Golos, T.G., 2004. Trophoblast differentiation in trophoblast outgrowth in the mouse. J. Embryol. Exp. Morphol. 30, embryoid bodies derived from human embryonic stem cells. Endo- 21–30. crinology 145, 1517–1524. Khidir, M.A., Stachecki, J.J., Krawetz, S.A., Armant, D.R., 1995. Rapid Giancotti, F.G., Ruoslahti, E., 1999. Integrin signaling. Science 285, inhibition of mRNA synthesis during preimplantation embryo develop- 1028–1032. ment: vital permeabilization by lysolecithin potentiates the action of a- Ginsberg, M.H., Xiaoping, D., O’Toole, T.E., Loftus, J.C., Plow, E.F., amanitin. Exp. Cell 219, 619–623. 1993. Platelet integrins. Thromb. Haemostasis 70, 87–93. Kidder, G.M., McLachlin, J.R., 1985. Timing of transcription and protein Glass, R.H., Spindle, A.I., Pedersen, R.A., 1976. Differential inhibition of synthesis underlying morphogenesis in preimplantation mouse trophoblast outgrowth and inner cell mass growth by actinomycin D in embryos. Dev. Biol. 112, 265–275. cultured mouse embryos. J. Reprod. Fertil. 48, 443–445. Kimber, S.J., 2000. Molecular interactions at the maternal–embryonic Glasser, S.R., Lampelo, S., Munir, M.I., Julian, J., 1987. Expression of interface during the early phase of implantation. Semin. Reprod. Med. desmin, laminin and fibronectin during in situ differentiation (decidu- 18, 237–253. alization) of rat uterine stromal cells. Differentiation 35, 132–142. Kimber, S.J., Spanswick, C., 2000. Blastocyst implantation: the adhesion Goldring, S.R., Gorn, A.H., Yamin, M., Krane, S.M., Wang, J.T., 1993. cascade. Semin. Cell Dev. Biol. 11, 77–92. Characterization of the structural and functional properties of cloned Kiraly-Borri, C.E., Morgan, A., Burgoyne, R.D., Weller, U., Wollheim, calcitonin receptor cDNAs. Horm. Metab. Res. 25, 477–480. C.B., Lang, J., 1996. Soluble N-ethylmaleimide-sensitive-factor 278 D.R. Armant / Developmental Biology 280 (2005) 260–280

attachment protein and N-ethylmaleimide-insensitive factors are Lu, K.P., Means, A.R., 1993. Regulation of the cell cycle by calcium and required for Ca2+-stimulated exocytosis of insulin. Biochem. J. 314, calmodulin. Endocr. Rev. 14, 40–58. 199–203. Marek, K.W., Vijay, I.K., Marth, J.D., 1999. A recessive deletion in the Kirby, D.R., Potts, D.M., Wilson, I.B., 1967. On the orientation of the GlcNAc-1-phosphotransferase gene results in peri-implantation embry- implanting blastocyst. J. Embryol. Exp. Morphol. 17, 527–532. onic lethality. Glycobiology 9, 1263–1271. Kisalus, L.L., Herr, J.C., Little, C.D., 1987. Immunolocalization of Marinari, U.M., Nitti, M., Pronzato, M.A., Domenicotti, C., 2003. Role of extracellular matrix proteins and collagen synthesis in first-trimester PKC-dependent pathways in HNE-induced cell protein transport and human decidua. Anat. Rec. 218, 402–415. secretion. Mol. Aspects Med. 24, 205–211. Klaffky, E., Williams, R., Yao, C.C., Ziober, B., Kramer, R., Sutherland, Marshall, J.F., Hart, I.R., 1996. The role of alpha v-integrins in tumour A., 2001. Trophoblast-specific expression and function of the integrin progression and metastasis. Semin. Cancer Biol. 7, 129–138. alpha 7 subunit in the peri-implantation mouse embryo. Dev. Biol. Martin, K.L., Barlow, D.H., Sargent, I.L., 1998. Heparin-binding epidermal 239, 161–175. growth factor significantly improves human blastocyst development Klausner, R.D., Donaldson, J.G., Lippincott-Schwartz, J., 1992. Brefeldin and hatching in serum-free medium. Hum. Reprod. 13, 1645–1652. A: insights into the control of membrane traffic and organelle structure. McNamee, H.P., Ingber, D.E., Schwartz, M.A., 1993. Adhesion to J. Cell Biol. 116, 1071–1080. fibronectin stimulates inositol lipid synthesis and enhances PDGF- Kornberg, L.J., Earp, H.S., Turner, C.E., Prockop, C., Juliano, R.L., 1991. induced inositol lipid breakdown. J. Cell Biol. 121, 673–678. Signal transduction by integrins: increased protein tyrosine phosphor- Miner, J.H., Li, C., Mudd, J.L., Go, G., Sutherland, A.E., 2004. Composi- ylation caused by clustering of beta 1 integrins. Proc. Natl. Acad. Sci. tional and structural requirements for laminin and basement membranes U. S. A. 88, 8392–8396. during mouse embryo implantation and gastrulation. Development 131, Kumar, S., Zhu, L.J., Polihronis, M., Cameron, S.T., Baird, D.T., Schatz, F., 2247–2256. Dua, A., Ying, Y.K., Bagchi, M.K., Bagchi, I.C., 1998. Progesterone Miyamoto, S., Teramoto, H., Coso, O.A., Gutkind, J.S., Burbelo, P.D., induces calcitonin gene expression in human endometrium within the Akiyama, S.K., Yamada, K.M., 1995. Integrin function: molecular putative window of implantation. J. Clin. Endocrinol. Metab. 83, hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131, 4443–4450. 791–805. Kunath, T., Strumpf, D., Rossant, J., 2004. Early trophoblast determi- Nechushtan, H., Leitges, M., Cohen, C., Kay, G., Razin, E., 2000. nation and stem cell maintenance in the mouse—A review. Placenta Inhibition of degranulation and interleukin-6 production in mast 25, S32–S38 (Suppl A). cells derived from mice deficient in protein kinase Cbeta. Blood 95, Lan, J.Y., Skeberdis, V.A., Jover, T., Grooms, S.Y., Lin, Y., Araneda, R.C., 1752–1757. Zheng, X., Bennett, M.V., Zukin, R.S., 2001. Protein kinase C Ng-Sikorski, J., Andersson, R., Patarroyo, M., Andersson, T., 1991. modulates NMDA receptor trafficking and gating. Nat. Neurosci. 4, Calcium signaling capacity of the CD11b/CD18 integrin on human 382–390. neutrophils. Exp. Cell Res. 195, 504–508. Leach, R.E., Khalifa, R., Ramirez, N.D., Das, S.K., Wang, J., Dey, S.K., Pardi, R., Bender, J.R., Dettori, C., Giannazza, E., Engleman, E.G., 1989. Romero, R., Armant, D.R., 1999a. Multiple roles for heparin-binding Heterogeneous distribution and transmembrane signaling properties of epidermal growth factor-like growth factor are suggested by its cell lymphocyte function-associated antigen (LFA-1) in human lymphocyte specific expression during the human endometrial cycle and early subsets. J. Immunol. 143, 3157–3166. placentation. J. Clin. Endocrinol. Metab. 84, 3355–3363. Paria, B.C., Dey, S.K., Andrews, G.K., 1992. Antisense c-myc effects on Leach, R.E., Rout, U.K., Schultz, J.F., Saunders, D.E., Armant, D.R., preimplantation mouse embryo development. Proc. Natl. Acad. Sci. 1999b. Ethanol elevates c-Myc levels in cultured mouse preimplanta- U. S. A. 89, 10051–10055. tion embryos. Alcohol. Clin. Exp. Res. 23, 778–784. Paria, B.C., Huet-Hudson, Y.M., Dey, S.K., 1993. Blastocyst’s state of Leach, R.E., Romero, R., Kim, Y.M., Chaiworapongsa, T., Kilburn, B.A., activity determines the bwindowQ of implantation in the receptive Das, S.K., Dey, S.K., Johnson, A., Qureshi, F., Jacques, S., Armant, mouse uterus. Proc. Natl. Acad. Sci. U. S. A. 90, 10159–10162. D.R., 2002. Pre-eclampsia and expression of heparin-binding EGF-like Paria, B.C., Das, N., Das, S.K., Zhao, X., Dileepan, K.N., Dey, S.K., 1998. growth factor. Lancet 360, 1215–1219. Histidine decarboxylase gene in the mouse uterus is regulated by Leach, R.E., Kilburn, B.A., Wang, J., Liu, Z., Romero, R., Armant, D.R., progesterone and correlates with uterine differentiation for blastocyst 2004. Heparin-binding EGF-like growth factor regulates human implantation. Endocrinology 139, 3958–3966. extravillous cytotrophoblast development during conversion to the Paria, B.C., Elenius, K., Klagsburn, M., Dey, S.K., 1999. Heparin-binding invasive phenotype. Dev. Biol. 266, 223–237. EGF-like growth factor interacts with mouse blastocysts independently Leavesley, D.I., Ferguson, G.D., Wayner, E.A., Cheresh, D.A., 1992. of ErbB1: a possible role for herparan sulfate proteoglycans and ErbB4 Requirement of the integrin beta 3 subunit for carcinoma cell in blastocyst implantation. Development 126, 1997–2005. spreading or migration on vitronectin and fibrinogen. J. Cell Biol. Paria, B.C., Ma, W., Tan, J., Raja, S., Das, S.K., Dey, S.K., Hogan, B.L., 117, 1101–1107. 2001. Cellular and molecular responses of the uterus to embryo Leivo, I., Vaheri, A., Timpl, R., Wartiovaara, J., 1980. Appearance and implantation can be elicited by locally applied growth factors. Proc. distribution of collagens and laminin in the early mouse embryo. Dev. Natl. Acad. Sci. U. S. A. 98, 1047–1052. Biol. 76, 100–114. Parr, E.L., Tung, H.N., Parr, M.B., 1987. Apoptosis as the mode of uterine Liu, Z., Armant, D.R., 2004. Lysophosphatidic acid regulates murine epithelial cell death during embryo implantation in mice and rats. Biol. blastocyst development by transactivation of receptors for heparin- Reprod. 36, 211–225. binding EGF-like growth factor. Exp. Cell Res. 296, 317–326. Pauken, C.M., Capco, D.G., 2000. The expression and stage-specific Liu, J., Chakraborty, C., Graham, C.H., Barbin, Y.P., Dixon, S.J., Lala, P.K., localization of protein kinase C isotypes during mouse preimplantation 2003. Noncatalytic domain of uPA stimulates human extravillous development. Dev. Biol. 223, 411–421. trophoblast migration by using phospholipase C, phosphatidylinositol Peters, C., Mayer, A., 1998. Ca2+/calmodulin signals the completion 3-kinase and mitogen-activated protein kinase. Exp. Cell Res. 286, of docking and triggers a late step of vacuole fusion. Nature 396, 138–151. 575–580. Lohnes, D., 2003. The Cdx1 homeodomain protein: an integrator of Phillips, D.R., Charo, I.F., Parise, L.V., Fitzgerald, L.A., 1988. The platelet posterior signaling in the mouse. BioEssays 25, 971–980. membrane glycoprotein IIb–IIIa complex. Blood 71, 831–843. Long, A., Kelleher, D., Lynch, S., Volkov, Y., 2001. Cutting edge: pro- Pijnenborg, R., Robertson, W.B., Brosens, I., Dixon, G., 1981. Review tein kinase C beta expression is critical for export of Il-2 from T cells. article: trophoblast invasion and the establishment of haemochorial J. Immunol. 167, 636–640. placentation in man and laboratory animals. Placenta 2, 71–91. D.R. Armant / Developmental Biology 280 (2005) 260–280 279

Plow, E.F., Haas, T.A., Zhang, L., Loftus, J., Smith, J.W., 2000. Ligand trafficking regulates adhesion to fibronectin during differentiation of binding to integrins. J. Biol. Chem. 275, 21785–21788. mouse peri-implantation blastocysts. Dev. Genet. 21, 31–43. Pomies, P., Frachet, P., Block, M.R., 1995. Control of the alpha 5 beta 1 Schwartz, M.A., 1993. Spreading of human endothelial cells on fibronectin integrin/fibronectin interaction in vitro by the serine/threonine protein or vitronectin triggers elevation of intracellular free calcium. J. Cell phosphatase calcineurin. Biochemistry 34, 5104–5112. Biol. 120, 1003–1010. Psychoyos, A., 1973. Endocrine control of egg implantation. In: Greep, Sherman, M.I., Atienza-Samols, S.B., 1978. In vitro studies on the surface R.O., Astood, E.G., Geiger, S.R. (Eds.), Handbook of Physiology. adhesiveness of mouse blastocysts. In: Ludwig, H., Tauber, P.F. (Eds.), American Physiology Society, Washington, DC, pp. 187–215. Human fertilization. Georg Thieme Publishers, Stuttgart, Germany, Quetglas, S., Leveque, C., Miquelis, R., Sato, K., Seagar, M., 2000. Ca2+- pp. 179–183. dependent regulation of synaptic SNARE complex assembly via a Shiokawa, S., Yoshimura, Y., Sawa, H., Nagamatsu, S., Hanashi, H., Sakai, calmodulin-and phospholipid-binding domain of synaptobrevin. Proc. K., Ando, M., Nakamura, Y., 1999. Functional role of arg-gly-asp Natl. Acad. Sci. U. S. A. 97, 9695–9700. (RGD)-binding sites on beta1 integrin in embryo implantation using Raab, G., Kover, K., Paria, B.C., Dey, S.K., Ezzell, R.M., Klagsbrun, M., mouse blastocysts and human decidua. Biol. Reprod. 60, 1468–1474. 1996. Mouse preimplantation blastocysts adhere to cells expressing the Showell, C., Binder, O., Conlon, F.L., 2004. T-box genes in early transmembrane form of heparin-binding EGF-like growth factor. embryogenesis. Dev. Dyn. 229, 201–218. Development 122, 637–645. Sjaastad, M.D., Lewis, R.S., Nelson, W.J., 1996. Mechanisms of Red-Horse, K., Zhou, Y., Genbacev, O., Prakobphol, A., Foulk, R., integrin-mediated calcium signaling in MDCK cells: regulation of McMaster, M., Fisher, S.J., 2004. Trophoblast differentiation during adhesion by IP3- and store-independent calcium influx. Mol. Biol. embryo implantation and formation of the maternal–fetal interface. Cell 7, 1025–1041. J. Clin. Invest. 114, 744–754. Slater, M., Murphy, C.R., 1999. Temporal changes in the expression of Redline, R.W., Lu, C.Y., 1989. Localization of fetal major histocompati- platelet-derived growth factor and fibronectin in the uterine epithelium bility complex antigens and maternal leukocytes in murine placenta, during early pregnancy. Anat. Rec. 255, 1–6. Implications for maternal–fetal immunological relationship. Lab. Invest. Smith, J.B., Dangelmaier, C., Selak, M.A., Daniel, J.L., 1991. Facile 61, 27–36. platelet adhesion to collagen requires metabolic energy and actin Richa, J., Damsky, C.H., Buck, C.A., Knowles, B.B., Solter, D., 1985. Cell polymerization and evokes intracellular free calcium mobilization. surface glycoproteins mediate compaction, trophoblast attachment, and J. Cell. Biochem. 47, 54–61. endoderm formation during early mouse development. Dev. Biol. 108, Somogyi, L., Lasic, Z., Vukicevic, S., Banfic, H., 1994. Collagen type IV 513–521. stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via Rider, V., Carlone, D.L., Witrock, D., Cai, C., Oliver, N., 1992. Uterine activation of phospholipase C. Biochem. J. 299, 603–611. fibronectin mRNA content and localization are modulated during Spindle, A.I., Pedersen, R.A., 1973. Hatching, attachment, and outgrowth implantation. Dev. Dyn. 195, 1–14. of mouse blastocysts in vitro: fixed nitrogen requirements. J. Exp. Zool. Rossant, J., Cross, J.C., 2001. Placental development: lessons from mouse 186, 305–318. mutants. Nat. Rev., Genet. 2, 538–548. Stachecki, J.J., Yelian, F.D., Leach, R.E., Armant, D.R., 1994. Mouse Rout, U.K., Krawetz, S.A., Armant, D.R., 1997. Ethanol-induced intra- blastocyst outgrowth and implantation rates following exposure to cellular calcium mobilization rapidly alters gene expression in the ethanol or A23187 during culture in vitro. J. Reprod. Fertil. 101, mouse blastocyst. Cell Calcium 22, 463–474. 611–617. Rout, U.K., Wang, J., Paria, B.C., Armant, D.R., 2004. alpha5beta1, Stephens, L.E., Sutherland, A.E., Klimanskaya, I.V., Andrieux, A., alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordin- Meneses, J., Pedersen, R.A., Damsky, C.H., 1995. Deletion of beta 1 ately regulate adhesion and migration of differentiating mouse integrins in mice results in inner cell mass failure and peri-implantation trophoblast cells. Dev. Biol. 268, 135–151. lethality. Genes Dev. 9, 1883–1895. Ruoslahti, E., 1996. RGD and other recognition sequences for integrins. Surani, M.A., 1979. Glycoprotein synthesis and inhibition of glycosylation Annu. Rev. Cell Dev. Biol. 12, 697–715. by tunicamycin in preimplantation mouse embryos: compaction and Russ, A.P., Wattler, S., Colledge, W.H., Aparicio, S.A., Carlton, M.B., trophoblast adhesion. Cell 18, 217–227. Pearce, J.J., Barton, S.C., Surani, M.A., Ryan, K., Nehls, M.C., Wilson, Sutherland, A., 2003. Mechanisms of implantation in the mouse: differ- V., Evans, M.J., 2000. Eomesodermin is required for mouse trophoblast entiation and functional importance of trophoblast giant cell behavior. development and mesoderm formation. Nature 404, 95–99. Dev. Biol. 258, 241–251. Sastry, S.K., Burridge, K., 2000. Focal adhesions: a nexus for intracellular Sutherland, A.E., Calarco, P.G., Damsky, C.H., 1993. Developmental signaling and cytoskeletal dynamics. Exp. Cell Res. 261, 25–36. regulation of integrin expression at the time of implantation in the Savage, B., Saldivar, E., Ruggeri, Z.M., 1996. Initiation of platelet adhesion mouse embryo. Development 119, 1175–1186. by arrest onto fibrinogen or translocation on von Willebrand factor. Cell Sutovsky, P., Motlik, J., Neuber, E., Pavlok, A., Schatten, G., Palecek, J., 84, 289–297. Hyttel, P., Adebayo, O.T., Adwan, K., Alberio, R., Bagis, H., Bataineh, Schindler, J., Sherman, M.I., 1981. Effect of a-amanatin on programming Z., Bjerregaard, B., Bodo, S., Bryja, V., Carrington, M., Couf, M., de la, of mouse blastocyst development. Dev. Biol. 84, 332–340. F.R., Diblik, J., Esner, M., Forejt, J., Fulka Jr., J., Geussova, G., Gjorret, Schlafke, S., Enders, A.C., 1975. Cellular basis of interaction between J.O., Libik, M., Hampl, A., Hassane, M.S., Houshmand, M., Hozak, P., trophoblast and uterus at implantation. Biol. Reprod. 12, 41–65. Jezova, M., Kania, G., Kanka, J., Kandil, O.M., Kishimoto, T., Klima, Schlafke, S., Welsh, A.O., Enders, A.C., 1985. Penetration of the basal J., Kohoutek, J., Kopska, T., Kubelka, M., Lapathitis, G., Laurincik, J., lamina of the uterine luminal epithelium during implantation in the rat. Lefevre, B., Mihalik, J., Novakova, M., Oko, R., Omelka, R., Owiny, Anat. Rec. 212, 47–56. D., Pachernik, J., Pacholikova, J., Peknicova, J., Pesty, A., Ponya, Z., Schmid, P.C., Paria, B.C., Krebsbach, R.J., Schmid, H.H., Dey, S.K., 1997. Preclikova, H., Sloskova, A., Svoboda, P., Strejcek, F., Toth, S., Tepla, Changes in anandamide levels in mouse uterus are associated with O., Valdivia, M., Vodicka, P., Zudova, D., 2001. Accumulation of the uterine receptivity for embryo implantation. Proc. Natl. Acad. Sci. proteolytic marker peptide ubiquitin in the trophoblast of mammalian U. S. A. 94, 4188–4192. blastocysts. Cloning Stem Cells 3, 157–161. Schultz, J.F., Armant, D.R., 1995. Beta1-and beta3-class integrins mediate Svoboda, P., Stein, P., Schultz, R.M., 2001. RNAi in mouse oocytes and fibronectin binding activity at the surface of developing mouse peri- preimplantation embryos: effectiveness of hairpin dsRNA. Biochem. implantation blastocysts. Regulation by ligand-induced mobilization of Biophys. Res. Commun. 287, 1099–1104. stored receptor. J. Biol. Chem. 270, 11522–11531. Tabuchi, A., Yoshioka, A., Higashi, T., Shirakawa, R., Nishioka, H., Kita, Schultz, J.F., Mayernik, L., Rout, U.K., Armant, D.R., 1997. Integrin T., Horiuchi, H., 2003. Direct demonstration of involvement of protein 280 D.R. Armant / Developmental Biology 280 (2005) 260–280

kinase Calpha in the Ca2+-induced platelet aggregation. J. Biol. Chem. Watson, A.J., Barcroft, L.C., 2001. Regulation of blastocyst formation. 278, 26374–26379. Front. Biosci. 6, D708–D730. Tanaka, S., Kunath, T., Hadjantonakis, A.K., Nagy, A., Rossant, J., 1998. Welsh, A.O., Enders, A.C., 1991. Chorioallantoic placenta formation in the Promotion of trophoblast stem cell proliferation by FGF4. Science 282, rat: I. Luminal epithelial cell death and extracellular matrix modifica- 2072–2075. tions in the mesometrial region of implantation chambers. Am. J. Anat. Tarone, G., Russo, M.A., Hirsch, E., Odorisio, T., Altruda, F., Silengo, L., 192, 215–231. Siracusa, G., 1993. Expression of beta 1 integrin complexes on the Wewer, U.M., Damjanov, A., Weiss, J., Liotta, L.A., Damjanov, I., 1986. surface of unfertilized mouse oocyte. Development 117, 1369–1375. Mouse endometrial stromal cells produce basement-membrane compo- Trikha, M., Timar, J., Zacharek, A., Nemeth, J.A., Cai, Y., Dome, B., nents. Differentiation 32, 49–58. Somlai, B., Raso, E., Ladanyi, A., Honn, K.V., 2002. Role for beta3 Wordinger, R.J., McGrath, M., 1979. The collagen substratum influences in integrins in human melanoma growth and survival. Int. J. Cancer 101, vitro hatching and attachment of the mouse blastocyst in a serumless 156–167. medium. Experientia 35, 1683–1684. Umata, T., Hirata, M., Takahashi, T., Ryu, F., Shida, S., Takahashi, Y., Wordinger, R.J., Brun-Zinkernagel, A.M., Jackson, T., 1991. An ultra- Tsuneoka, M., Miura, Y., Masuda, M., Horiguchi, Y., Mekada, E., 2001. structural study of in-vitro interaction of guinea-pig and mouse A dual signaling cascade that regulates the ectodomain shedding of blastocysts with extracellular matrices. J. Reprod. Fertil. 93, 585–597. heparin-binding epidermal growth factor-like growth factor. J. Biol. Wu, T.C., Wan, Y.J., Chung, A.E., Damjanov, I., 1983. Immunohistochem- Chem. 276, 30475–30482. ical localization of entactin and laminin in mouse embryos and fetuses. Wang, J., Armant, D.R., 2002. Integrin-mediated adhesion and signaling Dev. Biol. 100, 496–505. during blastocyst implantation. Cells Tissues Organs 172, 190–201. Wu, X., Mogford, J.E., Platts, S.H., Davis, G.E., Meininger, G.A., Davis, Wang, J., Rout, U.K., Bagchi, I.C., Armant, D.R., 1998. Expression of M.J., 1998. Modulation of calcium current in arteriolar smooth muscle calcitonin receptors in mouse preimplantation embryos and their by alphav beta3 and alpha5 beta1 integrin ligands. J. Cell Biol. 143, function in the regulation of blastocyst differentiation by calcitonin. 241–252. Development 125, 4293–4302. Xu, R.H., Chen, X., Li, D.S., Li, R., Addicks, G.C., Glennon, C., Zwaka, Wang, J., Paria, B.C., Dey, S.K., Armant, D.R., 1999. Stage-specific T.P., Thomson, J.A., 2002. BMP4 initiates human embryonic stem cell excitation of cannabinoid receptor exhibits differential effects on mouse differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264. embryonic development. Biol. Reprod. 60, 839–844. Yang, J.T., Rayburn, H., Hynes, R.O., 1995. Cell adhesion events mediated Wang, J., Mayernik, L., Schultz, J.F., Armant, D.R., 2000. Acceleration of by alpha 4 integrins are essential in placental and cardiac development. trophoblast differentiation by heparin-binding EGF-like growth factor Development 121, 549–560. is dependent on the stage-specific activation of calcium influx by Yelian, F.D., Edgeworth, N.A., Dong, L.J., Chung, A.E., Armant, D.R., ErbB receptors in developing mouse blastocysts. Development 127, 1993. Recombinant entactin promotes mouse primary trophoblast cell 33–44. adhesion and migration through the Arg-Gly-Asp (RGD) recognition Wang, J., Mayernik, L., Armant, D.R., 2002. Integrin signaling regulates sequence. J. Cell Biol. 121, 923–929. blastocyst adhesion to fibronectin at implantation: intracellular calcium Yelian, F.D., Yang, Y., Hirata, J.D., Schultz, J.F., Armant, D.R., 1995. transients and vesicle trafficking in primary trophoblast cells. Dev. Biol. Molecular interactions between fibronectin and integrins during mouse 245, 270–279. blastocyst outgrowth. Mol. Reprod. Dev. 41, 435–448. Wang, H., Matsumoto, H., Guo, Y., Paria, B.C., Roberts, R.L., Dey, S.K., Yoo, H.J., Barlow, D.H., Mardon, H.J., 1997. Temporal and spatial 2003. Differential G protein-coupled cannabinoid receptor signaling by regulation of expression of heparin-binding epidermal growth factor- anandamide directs blastocyst activation for implantation. Proc. Natl. like growth factor in the human endometrium: a possible role in Acad. Sci. U. S. A. 100, 14914–14919. blastocyst implantation. Dev. Genet. 21, 102–108. Wang, Q.T., Piotrowska, K., Ciemerych, M.A., Milenkovic, L., Scott, M.P., Zhang, X., Shu, M.A., Harvey, M.B., Schultz, G.A., 1996. Regulation of Davis, R.W., Zernicka-Goetz, M., 2004. A genome-wide study of gene urokinase plasminogen activator production in implanting mouse activity reveals developmental signaling pathways in the preimplanta- embryo: effect of embryo interaction with extracellular matrix. Biol. tion mouse embryo. Dev. Cell 6, 133–144. Reprod. 54, 1052–1058. Wartiovaara, J., Leivo, I., Vaheri, A., 1979. Expression of the cell surface- Ziomek, C.A., Johnson, M.H., Handyside, A.H., 1982. The devel- associated glycoprotein, fibronectin, in the early mouse embryo. Dev. opmental potential of mouse 16-cell blastomeres. J. Exp. Zool. 221, Biol. 69, 247–257. 345–355.