Development 119, 1175-1186 (1993) 1175 Printed in Great Britain © The Company of Biologists Limited 1993

Developmental regulation of expression at the time of implantation in the mouse embryo

A. E. Sutherland1,*, P. G. Calarco2 and C. H. Damsky1,2 Departments of 1Stomatology and 2Anatomy, University of California San Francisco, San Francisco, CA 94143, USA *To whom correspondence should be addressed at: HSW 604, 513 Parnassus Ave., University of California, San Francisco, San Francisco, CA 94143-0512, USA

SUMMARY

The trophectoderm layer of the mouse blastocyst differ- be induced by contact with matrix. At E7.5, three of the entiates at the late blastocyst stage to form the invasive temporally regulated ( 1, 6A, 7), all of which trophoblast that mediates implantation of the embryo can form receptors for laminin, were detected only in the into the uterine wall. The first sign that trophoblast cells ectoplacental cone (differentiating trophoblast), and have developed an invasion-specific cell behavior may thus play specific roles in trophoblast adhesion appears about 10-15 hours after the embryo hatches and/or differentiation. from the zona pellucida, when the quiescent, non- Because laminin expression is upregulated in decidu- adherent trophectoderm cells initiate protrusive activity alized uterine stroma in response to the implanting and become adhesive to . Our embryo, we examined trophoblast-laminin interactions, previous findings that trophoblast outgrowth on extra- using laminin fragments and integrin antibodies to cellular-matrix-coated substrata involves the integrin determine which integrin receptors were involved. Tro- family of adhesion receptors (Sutherland, A. E., Calarco, phoblast cells attached and spread on both the E8 and P. G. and Damsky, C. H., 1988, J. Cell Biol. 106, 1331- P1 fragments of laminin; however, the P1 binding site 1348), suggested that the onset of trophoblast adhesive was cryptic in intact laminin. Interaction with P1 was and migratory behavior at the time of implantation may RGD- and v 3-dependent, whereas outgrowth on E8 be due to changes in expression or distribution of was RGD-independent and not inhibited by antibodies integrin receptors. We have thus examined the mRNA to the laminin receptor 6 1, suggesting that 7 1 is the and protein expression of individual integrin subunits major trophoblast integrin E8 receptor. In contrast, during pre- and periimplantation development (E0- migration of parietal endoderm cells on laminin was E7.5). A basic repertoire of integrins, including receptors blocked by antibodies to 6, demonstrating that these for fibronectin ( 5 1), laminin ( 6B 1) and vitronectin two contemporaneous migratory cell populations have ( v 3), was expressed continuously throughout this different modes of interaction with laminin. We conclude period, whereas the expression of five other integrin that developmental regulation of integrin expression subunits was developmentally regulated. The mRNA for appears to have functional significance for trophoblast three of these ( 2, 6A and 7) was first detected in the invasion of the laminin-rich uterine stroma. late blastocyst, coincident with endoderm differentiation and development of attachment competence. The mRNA for another ( 1) was not detected until after trophoblast Key words: cell-matrix interactions, implantation, integrins, outgrowth had begun, suggesting that its expression may laminin, mouse embryo, trophoblast

INTRODUCTION cells that encloses the blastocoel cavity and the inner cell mass of the preimplantation blastocyst. After an initial period of cleavage and blastocyst formation, Conversion of the epithelial trophectoderm to invasive mammalian embryos must make contact with an external trophoblast begins at the late blastocyst stage, and comprises source of nutrients in order to grow and continue their devel- both a change in adhesive behavior and the onset of motility opment. The mouse embryo accomplishes this by implant- (Chavez, 1984; Bevilacqua and Abrahamsohn, 1988). Tro- ing itself into the stroma of the uterus and forming contacts phoblast differentiation and invasion can be modelled in with the maternal blood supply that lead to establishment of vitro, using embryos cultured on substrata of extracellular a hemochorial placenta (Schlafke and Enders, 1975; Enders, matrix molecules (Jenkinson and Wilson, 1973; Armant et 1976). Implantation of the embryo is mediated by a spe- al., 1986; Sutherland et al., 1988). The ability to attach to cialized population of cells, the trophoblast cells, which such substrata is acquired during trophoblast differentiation; arise from the trophectoderm, the outer layer of epithelial early blastocysts cultured under these conditions do not 1176 A. E. Sutherland, P. G. Calarco and C. H. Damsky adhere and spread (Chavez, 1984), whereas late blastocysts sera and the rat monoclonal antibody AIIB2 against human b1, will form an embryo outgrowth, consisting of a monolayer which does not recognize the mouse protein (Hall et al., 1990). of spreading trophoblast cells with the inner cell mass Materials remaining on top. Time-lapse observations of embryo Mouse laminin and Mito+ were purchased from Collaborative outgrowth cultures show that the first signs of outgrowth are Research (Bedford, MA). Mouse fibronectin and the hexapeptides detected about 10-15 hours after the embryo hatches from Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) and Gly-Arg-Gly-Glu-Ser- the zona pellucida, when the previously quiescent trophec- Pro (GRGESP) were purchased from Telios (La Jolla, CA). toderm cells exhibit an abrupt onset of protrusive activity Sepharose CL-4B and protein A-Sepharose were from Pharmacia followed by attachment and spreading on the substratum (Piscataway, NJ); Expre35S35S protein labelling mixture and (Sutherland et al., 1988). En3Hance were purchased from New England Nuclear-Dupont Trophoblast interactions with extracellular matrix (Boston, MA). Bovine serum albumin (BSA), , N-octyl-b- molecules depend largely on the integrin family of adhesion D-glucopyranoside, pancreatin, Tween 20 and the protease receptors, as broad spectrum anti-integrin antibodies will inhibitors phenylmethylsulfonylfluoride (PMSF), leupeptin and pepstatin were from SigmaChemicals (St Louis, MO). Acrylamide block trophoblast attachment and outgrowth on fibronectin, gel electrophoresis reagents were purchased from BioRad laminin or collagen IV-coated substrata (Sutherland et al., (Richmond, CA). Fetal bovine serum (FBS) was from the UCSF 1988). The changes in trophoblast adhesive and migratory Cell Culture Facility. Prestained protein molecular weight behavior that occur at the time of implantation may therefore standards were obtained from Bethesda Research Laboratories 3 stem from changes in the expression or distribution of (Gaithersburg, MD), and included (Mr 205´ 10 ), phos- 3 3 integrin receptors. To address this possibility, we examined phorylase b (Mr 97.4´ 10 ), bovine serum albumin (Mr 68´ 10 ), 3 3 the temporal and spatial expression patterns of integrin ovalbumin (Mr 43´ 10 ) and carbonic anhydrase (Mr 29´ 10 ). subunits, and tested the function of several integrins in Embryo culture attaching to laminin, an extracellular matrix molecule Preimplantation embryos were flushed at the 2-cell stage from the known to be a major component of the uterine environment. oviducts of superovulated female ICR mice (12 weeks old; Harlan We found that, whereas five integrin subunits were Sprague-Dawley Inc.) and cultured through the preimplantation expressed throughout preimplantation and early postim- stages of development as described previously (Sutherland et al., plantation stages, four other subunits were first expressed at 1988). Late blastocysts were obtained by culturing embryos at the the time of implantation, three of which appeared to be tro- early blastocyst stage (3 days in culture from the 2-cell stage) for phoblast-specific. Expression of the regulated subunits an additional 48 hours in serum-free Eagle’s medium (Spindle and appears to have functional significance for implantation, as Pedersen, 1973; Spindle, 1980) supplemented with insulin (10 trophoblast interactions with laminin, whose expression in mg/ml), BSA (4 mg/ml), and Mito+ (0.1%). Embryo outgrowths for use in polymerase chain reaction, immunoprecipitation and the uterine stroma is upregulated in response to the implant- immunofluorescence experiments were obtained by culturing late ing blastocyst (Wewer et al., 1986; Glasser et al., 1987; blastocysts for an additional 24 hours in Eagle’s medium supple- Senior et al., 1988), are not mediated by integrin receptors mented with 10% FBS. Ectoplacental cone (EPC) explants were continuously expressed prior to implantation. obtained and cultured as described previously (Sutherland et al., 1991). Embryo outgrowth on defined substrata MATERIALS AND METHODS Antibody and peptide dilutions were prepared in CMRL medium 1066 (Gibco, Grand Island, NY) supplemented with 0.05 mg/ml Antibodies sodium pyruvate, 4 mg/ml BSA, and 1% Nutridoma-HU. All media Polyclonal rabbit antisera to cytoplasmic domain peptides of a 1, were filter-sterilized, using syringe-tip filters (Millipore, HV) of a 2, and a 5 subunits were the generous gift of Dr Louis Reichardt 0.45 mm pore size, before addition to the substrata. Endoderm (UCSF). Polyclonal rabbit antiserum to a cytoplasmic domain outgrowth assays were done using Eagle’s medium supplemented peptide of a 3 was the gift of Dr Richard Hynes (Massachusetts with Mito+ and 2% FBS, as these were the only conditions under Institute of Technology, Cambridge, MA). Polyclonal rabbit which parietal endoderm cells were produced reliably in vitro. antiserum against a vb3, was the gift of Dr James Gailit and Dr Substrata [laminin (25 mg/ml), fibronectin (20 mg/ml), laminin Erkki Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, fragments E1¢, E3, E4, E8, P1¢and rG (each at 25 mg/ml)] were CA). Polyclonal antiserum to a 6 and the rat monoclonal antibody prepared as described previously (Sutherland et al., 1988), and GoH3 against mouse a 6 were gifts of Dr Arnoud Sonnenberg (The experimental media were added and equilibrated for 30 minutes at Netherlands Cancer Institute, Amsterdam). The rat monoclonal 37°C. Late blastocysts were then added (5-15 embryos per drop) antibody 346-11A against mouse b4 was the gift of Dr Steve and cultured at 37°C for 72-96 hours. Trophoblast outgrowth was Kennel (Oak Ridge National Laboratory, Oak Ridge, TN). The rat assayed at 72 hours as described previously (Sutherland et al., monoclonal antibody 5H10 against mouse a 5 was purchased from 1988). Endoderm outgrowth was assayed visually at 96 hours by Pharmingen Inc. (San Diego, CA). Laminin fragments E1¢, E3, E4, examining the embryos for the presence and distribution of small E8 and P1¢, and a recombinant form of the laminin A chain COOH- parietal endoderm cells. Each treatment was used in duplicate in at terminal globular domain (rG), as well as antibodies against these least three independent experiments. fragments were the generous gift of Dr Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ). Each anti- Radiolabelling of embryos fragment antiserum had been affinity-purified over a column of the Preimplantation embryos (4-cell to blastocyst stages) were immunizing fragment, then cross-absorbed over columns of each incubated for 6-8 hours in medium containing 75 mCi/ml of of the other fragments. The specificity of each anti-fragment Expre35S35S protein labelling mixture, lysed in 100 ml OGB [0.2 antibody had then been confirmed by Western blotting and com- M N-octyl-b-D-glucopyranoside in PBS, containing the protease petition ELISA experiments. Controls included nonimmune rabbit inhibitors PMSF (2 mM), pepstatin (1 mg/ml) and leupeptin (1 Integrins in early mouse embryos 1177

mg/ml)], and stored at - 80°C until use. Hatched blastocysts and extension (72°C, 1 minute); and (3) 35 cycles of denaturation EPC explants were labelled for 18 hours in Eagle’s medium lacking (94°C, 1 minute), annealing (60°C, 1 minute) and extension (72°C, methionine and cysteine (serum-free in the case of the late blasto- 2 minutes). These steps were followed by a final extension (72°C, cysts, containing 10% FBS in the case of EPC explants) and con- 15 minutes) and cooling to 4°C until use. For a 1 and a 2 primers, taining 100 mCi/ml Expre35S35S, then lysed in 100-200 ml OGB the process was identical except that annealing temperatures were and frozen at - 80°C until use. lowered by 10°C. For a 2, double amplification was performed; first the 2316-3540 primers were used, then 10% of each of these Immunoprecipitation samples underwent the second amplification, in which the 3226- Preimplantation embryo lysates were pooled to obtain samples 3453 primers were used. The amplified products were separated on containing the equivalent of 1200-1500 embryos and lysates from 4% agarose gels using 1 kb DNA ladder (BRL) for size compari- 5-8 EPC explants were pooled. These pooled samples were then son. Primers for a 7 were 5¢-CCAGGACCTGGCCATCCGTG-3¢ subjected to sequential immunoprecipitation. The lysates were and 5¢-CTATCCTTGCGCAGAATGAC-3¢ and were the gift of spun in a refrigerated microfuge for 15 minutes, preabsorbed twice Drs. Barry Ziober and Randall Kramer (Ziober et al., in prepara- on pellets of Sepharose CL-4B and incubated with primary tion). The 2316-3540 primers to a 2 were the gift of Drs John antibody overnight at 4°C. The samples were then added to 50 ml Adelman and Clayton Buck. The identities of the amplified pellets of protein A-Sepharose beads for one hour, absorbed twice fragments were confirmed by restriction analysis, and controls for more on 50 ml pellets of protein A-Sepharose (30 minutes each), genomic DNA contamination included DNAse treatment of after which the next primary antibody was added to the lysates and mRNA samples prior to reverse transcription, and PCR amplifica- incubated overnight. The protein A-Sepharose absorption protocol tion of mRNA alone. Amplification was repeated on cDNA from was repeated for each antibody used in the experiment, and each at least three independent isolates of embryo mRNA to insure reli- lysate was treated with 3-5 different primary antibodies. The first ability of the data. Since variation was seen between individual and third sets of protein A-Sepharose beads for each antibody were cDNA isolates for a given primer pair, each new preparation of washed as described previously (Sutherland et al., 1988), resus- embryo cDNA was characterized using both b- primers pended in nonreducing sample buffer and frozen at - 80°C until (Rappolee et al., 1988) and integrin b1 primers to provide refer- use. When all immunoprecipitations in the series were finished, the ences for comparing results from other primer sets. samples were run on 7% nonreducing SDS-polyacrylamide gels. Unfertilized eggs and embryos at four different stages of preim- The gels were fixed, treated with En3Hance, dried and exposed to plantation development were used: 2-cell, 8-cell, early blastocyst Kodak XAR-5 X-ray film. (32-40 cells, cavitation has just begun) and late blastocyst (hatched from the zona pellucida, with a layer of differentiated primitive Reverse transcription-polymerase chain reaction (RT- endoderm on the inner cell mass). To examine integrin expression PCR) assays in postimplantation embryos, two samples were used: (1) embryo Poly(A)+ RNA was isolated using the Micro-Fast Track kit (Invit- outgrowths, which contain differentiated trophoblast cells, rogen), from which single-stranded cDNA was synthesized using primitive endoderm and primitive ectoderm, and (2) E7.5 embryos, the cDNA Cycle kit (Invitrogen) and random hexamer primers. The which are at the mid- to late gastrula stage. Two regions of the E7.5 cDNA was ethanol precipitated and resuspended in 30 ml of sterile embryo were further isolated for mRNA preparation: the water and 3 ml was used per 50 ml PCR reaction. The reaction embryonic region (dissected at the level of the amnion) and the mixture contained 0.01 M Tris (pH 8.3), 0.05 M KCl, 2.5 mM EPC. Trophoblast giant cells and decidual cells were removed from MgCl2, 0.1 mg/ml gelatin, 0.7 mM each of dATP, dGTP, dCTP the EPC by manual dissection, leaving the core of undifferentiated and dTTP, 2.8 mM oligonucleotide primers and 0.5 U Taq 1 poly- trophoblast stem cells, which were then cultured in vitro for 4 days merase (AmpliTaq, Perkin-Elmer-Cetus Corp., Emeryville, CA). to obtain monolayers of differentiated trophoblast giant cells The primers used are listed in Table 1. For b1, a 3, a 5, a 6 and a 7 (Johnson and Rossant, 1981). Identical results were obtained in primers, PCR amplification was performed using a three-step initial experiments using freshly dissected EPC (used without process: (1) initial denaturation (4 minutes, 94°C); (2) five cycles removing trophoblast giant cells) and cultured explants, so cultured of denaturation (94°C, 1 minute), annealing (65°C, 1 minute), and explants were used in all further experiments in order to obtain

Table 1. Primers used for RT-PCR assays Sequence Sequence Fragment Subunit Forward primer Location Reverse primer Location size (bp) Reference a 1 5¢-CCTGTACTGTAC 3459-3482 5¢-GTGCTCTTATGA 3688-3711 252 Ignatius et al., 1990 (rat) CCAATTGGATGG-3¢ AAGTCGGTTTCC-3¢ a 2 5¢-TGCTGGTTGAAA 3226-3249 5¢-TATAACTCCTGTT 3430-3453 227 Takada and Hemler, 1989 (human) GACGTTCACATG-3¢ GGTACTTCGGC-3¢ 5¢-GCGTGTGG 2316-2330 5¢-AGAAGCCG 3526-3540 1225 Takada and Hemler, 1989 ACATCAG-3¢ AGCTTCC-3¢ a 3 5¢-ATTGACTCAGAG 3223-3252* 5¢-TACTTGGGCATA 3872-3891* 660 (A) Tamura et al., 1991 (mouse) CTGGTGGAGGAG-3¢ ATCCGGTAGTAG-3¢ 516 (B) a 5 5¢-CTGCAGCTGCAT 880-903 5¢-GAAGCCGAGCTT 1132-1155 275 Holers et al., 1989 (mouse) TTCCGAGTCTGG-3¢ GTAGAGGACGTA-3¢ a 6 5¢-GAGGAATATTCCA 3032-3055 5¢-GGAATGCTGTCA 3407-3430 398 (A) Cooper et al., 1991 (mouse) AACTGAACTAC-3¢ TCGTACCTAGAG-3¢ 268 (B) b1 5¢-GTGACCCATTGC 2062-2086 5¢-GTCATGAATTATCA 2253-2279 217 Holers et al., 1989 (mouse) AAGGAGAAGGA-3¢ TTAAAAGTTTCCA-3¢

*Numbers correspond to those in the published hamster sequence (Tamura et al., 1991; Tsuji et al., 1990) 1178 A. E. Sutherland, P. G. Calarco and C. H. Damsky more material. Comparison of these two regions of the E7.5 embryo allowed us to determine relative expression of different integrin subunits in embryonic and extraembryonic tissues. Immunofluorescence Live embryos between the 2-cell and late morula stages were incubated 45 minutes at 37°C in medium containing primary antibody, rinsed three times, incubated 20 minutes at 37°C in medium containing secondary antibody, rinsed three times in medium and twice in PBS, then mounted on slides in PBS and viewed under epifluorescence. The zona pellucida gave high levels of background in live embryos at the 8-cell and later stages, so it was removed prior to staining by incubating the embryos in 0.002% chymotrypsin for 2 minutes, followed by two rinses in medium and manual removal. Embryos were allowed to recover for 4 hours prior to antibody incubation. Other methods of zona removal (pronase treatment, acid Tyrode’s buffer treatment) led to loss of staining. Surface distribution of integrins on blastocysts was examined using fixed, rather than live, embryos as the amount of manipula- tion in the procedure resulted in a high rate of blastocoel collapse. Embryos were fixed for 15 minutes at room temperature in fresh 2% paraformaldehyde in PBS, rinsed twice in PBS, once in 0.15 M glycine in PBS, incubated 1 hour in PBS containing 0.1% BSA (PBS-BSA), and then in primary antibody diluted in PBS-BSA for 1 hour. Embryos were then rinsed three times in PBS-BSA, incubated for 30 minutes in rhodamine-conjugated secondary antibody diluted 1:100 in PBS-BSA, given three final rinses in PBS-BSA, mounted on slides and viewed under epifluorescence. Parallel samples of early blastocysts were fixed in fresh 2% paraformaldehyde in PBS and permeabilized with 0.2% Triton X- Fig. 1. RT-PCR amplification of embryo cDNA showing 100 in PBS prior to staining to examine integrin distribution in continuous expression of integrin b1, a 5 and a 6 subunits. Each inaccessible parts of the embryo. These embryos were viewed on was detectable in mRNA from unfertilized eggs (UE), 2-cell a Nikon Microphot equipped with a confocal scanning laser embryos (2C), 8-cell embryos (8C), early blastocysts (EB), and (Biorad). Embryo outgrowths were grown on circular glass cover- late blastocysts (LB). The last lane (0) shows RT-PCR done slips, fixed with cold methanol, rinsed with PBS and then stained without added cDNA. Positions of DNA size markers are as described previously (Sutherland et al., 1988). indicated to the right of the figure.

only detectable in embryo outgrowths and cultured EPC RESULTS (Fig. 2), suggesting that there may be posttranscriptional regulation of a 3 expression. mRNA for a 2 and a 7 was first Integrin expression is regulated at the late detected at the late blastocyst stage, at the time of endoderm blastocyst stage segregation and just prior to implantation (Fig. 3). Both were Integrin expression during the early development of the expressed in embryo outgrowths and in E7.5 EPC explants mouse embryo was examined using the reverse transcrip- (which consist of trophoblast derivatives), whereas only a 2 tion-polymerase chain reaction (RT-PCR; Rappolee et al., was expressed in the E7.5 embryonic region, suggesting that 1988) to determine the mRNA profiles, and immunoprecip- a 7 may be trophoblast-specific at this time. The a 1 subunit itation of metabolically labelled proteins to determine the was not detectable in preimplantation embryos, including corresponding protein profiles. We found two general the late blastocyst, at either the protein or the message level patterns; continuous expression throughout the stages (Fig. 2), but a 1 mRNA was present in embryo outgrowths examined and regulated expression beginning at the time of after 24 hours of culture (Fig. 3), and is thus an early implantation. Three integrin subunits were detected at both response to conditions that permit outgrowth. a 1 was also mRNA and protein levels throughout preimplantation devel- expressed in the E7.5 EPC, (Figs 2, 3), but was undetectable opment; mRNA for a 5, a 6 and b1 was detectable in unfer- in the embryonic region (Fig. 3), suggesting that its tilized eggs and in embryos of all preimplantation stages expression, like that of a 7, may be trophoblast-specific at (Fig. 1), and sequential immunoprecipitation from lysates of this time. 35 S-labeled embryos confirmed that the a 5b1 and a 6b1 het- The mRNAs for a 6 and a 3 each have two splicing erodimers were expressed throughout preimplantation variants that code for protein isoforms with different cyto- development. Another heterodimer, a vb3, was also detected plasmic domains (a 6A and B, and a 3A and B; Hogervorst in immunoprecipitates from all stages examined (4-cell, 8- et al., 1991; Tamura et al., 1991). To examine the develop- cell, morula, early blastocyst, late blastocyst; Fig. 2). mental expression of these variants, we performed RT-PCR In contrast, the expression of four other subunits was using primers which, in each case, bracket the segment developmentally regulated. Messenger RNA for a 3 was spliced out of the B form (see Table 1 and references detectable at the 8-cell stage (Fig. 3), but the protein was therein). The two forms of a 6 mRNA give rise to amplifi- Integrins in early mouse embryos 1179

cation products that differ in size by 130 bp, and the two either pre- or postimplantation embryos up to E7.5, as RT- forms of a 3 mRNA to products that differ by 144 bp. We PCR generated only the 660 bp fragment (Fig. 3). found that a 6B was detectable in mRNA from unfertilized These results show that integrin receptors for fibronectin eggs and at all stages of preimplantation development (Fig. (a 5b1), laminin (a 6Bb1) and vitronectin (a vb3) are 4). In contrast, a 6A was expressed at a relatively lower level expressed throughout preimplantation development, and are in unfertilized eggs and variably in mRNA from 2-cell present long before the trophectoderm can attach and spread embryos, but was then undetectable until the late blastocyst stage (Fig. 4). Both variants were detectable in embryo out- growths and E7.5 EPC explants, whereas the isolated E7.5 embryonic regions expressed only a 6B (Fig. 4). Thus the expression of a 6A is developmentally regulated and may be trophoblast-specific at E7.5. The presence of both variants in the unfertilized egg and 2-cell embryo probably repre- sents maternal message, which normally undergoes degra- dation at the 2-cell stage (Flach et al., 1982; Clegg and Piko, 1983; Giebelhaus et al., 1983). In contrast to the pattern of splicing changes seen for a 6, only a 3A was expressed in

Fig. 2. Immunoprecipitation of integrins from preimplantation embryos (A) and EPC explants (B) labelled with [35S]methionine/cysteine. (A) Immunoprecipitation from labelled lysates of preimplantation embryos using antibodies to the Fig. 3. RT-PCR amplification of embryo cDNA showing integrins a 1 (lane 1), a 2 (lane 2), a 3 (lane 3), a 5 (lane 4), a 6 (lane developmentally regulated expression of a 1, a 2, a 3 and a 7 5), a vb3 (lane 6) and b4 (lane 7). The anti-a vb3 results pictured subunits. Amplification was performed on samples of 2-cell here were from 8-cell embryos; all others were from late embryos (2C), 8-cell embryos (8C), early blastocysts (EB), late blastocysts. Results similar to those illustrated here were obtained blastocysts (LB), embryo outgrowths (OG), ectoplacental cone for all preimplantation stages examined. (B) Immunoprecipitation explants (EPC), and isolated embryonic regions (E) of E7.5 from labelled lysates of EPC explants cultured 5 days, using embryos. The last lane (0) shows RT-PCR done without added antibodies to the integrins a 1 (lane 1), a 2 (lane 2), a 3 (lane 3), a 5 cDNA. The results shown were obtained by using each set of - 3 (lane 4), a 6 (lane 5), and a vb3 (lane 6). Mr standards (´ 10 ) are primers on the same preparation of embryo cDNA, and b1 primers indicated to the right of each panel, and the positions of the were used as a positive control for each cDNA sample. Positions various subunits are indicated to the left. of DNA size markers are indicated to the right of the figure. 1180 A. E. Sutherland, P. G. Calarco and C. H. Damsky

At the late blastocyst stage, surface staining for a 6, a 5 and a vb3 gave the same results as in earlier blastocysts: staining for a vb3 was detected on the external surface of the embryo, whereas no surface staining for either a 6 or a 5 was seen (Fig. 6). It is thus unlikely that redistribution of existing integrin receptors accounts for the onset of trophoblast adhesive behavior at the late blastocyst stage. These results also suggest that a 6b1 and a 5b1 may not mediate initial adhesion of trophoblast cells to laminin or fibronectin, respectively. In fact, both a 5 and a 6 were predominantly expressed in the inner cell mass in embryo outgrowths, and not in the spread trophoblast cells (Fig. 7). By contrast, a vb3 was expressed in both the inner cell mass and in the spread trophoblast, and was localized to focal contacts in tro- Fig. 4. RT-PCR amplification showing developmental regulation phoblast cells (Fig. 7). Thus, of the integrin receptors that of integrin a 6 mRNA splicing. RT-PCR was performed using are expressed continuously during early development, only primers that span the 130 bp region in a 6 mRNA that undergoes a b appears to be in a position to mediate primary tro- alternative splicing (Tamura et al., 1991), on cDNA preparations v 3 from unfertilized eggs (UE), 2-cell embryos (2C), 8-cell embryos, phoblast adhesion and migration. (8C), early blastocysts (EB), late blastocysts (LB), and cultured In EPC explants, staining for a 6 was confined to the less EPC explants (EPC) and isolated embryonic regions (E) from differentiated cells in the center of the explant; no staining E7.5 embryos. was seen in the trophoblast giant cells at the periphery of the explant (Fig. 7). This result suggests that a 6 expression by trophoblast is reduced with differentiation, explaining the paradox between the lack of detectable a 6 on trophoblast on any of these substrata. To determine whether redistribu- giant cells in embryo outgrowths and the presence of both tion of integrins might contribute to the acquisition of tro- a 6 mRNA and protein in EPC explants (Figs 1, 2). In phoblast adhesivity, we performed immunolocalization contrast, a 5 and a vb3 were detectable on the surface of all experiments using antibodies to a 6, a 5 and a vb3. Staining cells in the EPC explant, and a vb3 was localized to focal for a 6 and a vb3, but not a 5, was detected on the surface of adhesions in trophoblast giant cells (Fig. 7). the embryo from the 2-cell to the late morula/early blasto- cyst stage (32 cells) (Fig. 5). After blastocoel formation, Molecular nature of trophoblast-laminin only a vb3 was detected on the surface of the embryo while interactions both a 6b1 and a 5b1 were localized internally (Fig. 6). a 6b1 The results described above suggest that integrin-mediated was present at the cell surface of all cells of the blastocyst, trophoblast-laminin interactions are likely to involve newly except the external surface of the trophectoderm (Fig. 6). synthesized receptors. All five of the integrin subunits The distribution of a 5b1 at this stage was similar, although showing regulated expression can form laminin receptors, with a greater amount of intracellular staining (Fig. 6). and three appear to be trophoblast-specific at E7.5. Laminin

Fig. 5. Surface expression of integrins as detected by immunolocalization. Live embryos at the 2-cell, 8-cell and 32-cell (mor) stages were stained with mAb GoH3 against the a 6 subunit (a 6; A-C), mAb 5H10 against the a 5 subunit (a 5; D-F), a polyclonal antiserum to a vb3 (a vb3; G-I) or normal rabbit serum (NRS; J-L). Scale bar, 50 mm. Integrins in early mouse embryos 1181

Fig. 6. Immunolocalization of integrins at early and late blastocyst stages. Early blastocysts (A,B,D,E,G,H,J,K) and late blastocysts (C,F,I,L) were stained with mAb GoH3 against the a 6 subunit (a 6; A-C), mAb 5H10 against the a 5 subunit (a 5; D, E, F), a polyclonal antiserum against a vb3 (a vb3; G-I), or normal rabbit serum (NRS; J-L). Scale bar, 50 mm. is a major component of the decidualized uterine stroma into To determine which binding sites are recognized in intact which the trophoblasts invade; in fact, stromal fibroblasts laminin, outgrowth assays were performed using heat express laminin only during implantation, while decreasing denatured laminin as a substratum. Heat treatment (80°C, 10 their production of fibronectin (Wewer et al., 1986; Glasser minutes) of intact laminin denatures the E8 binding site, et al., 1987; Senior et al., 1988). We therefore examined tro- leaving E1/P1 sites intact (Goodman et al., 1991). Embryos phoblast-laminin interactions more closely to determine did not form outgrowths on heat-treated laminin (D-Ln; Fig. which receptor(s) might be involved. 8B), although a number of cultured cell types could attach Outgrowth experiments were performed using laminin and spread on this substratum (not shown). This suggests and laminin fragments (Fig. 8A) as substrata, in the presence that only the E8 binding site is involved in trophoblast or absence of antibodies to the laminin fragments. Embryos recognition of intact laminin, and that the P1¢binding site is formed outgrowths on intact laminin and two of its cryptic. fragments, P1¢and E8 (Fig. 8B). A small amount of attach- There are two known integrin receptors for the P1¢ ment was seen to E1¢and to a recombinant form of the A domain, the a 1b1 heterodimer, which mediates Arg-Gly- chain COOH-terminal globular domain, which contains the Asp (RGD)-independent interactions (Forsberg et al., 1990; putative binding site for the a 3b1 integrin (Gehlsen et al., Rossino et al., 1990) and the a vb3 heterodimer, which 1992). No trophoblast outgrowth was detected on either of mediates RGD-dependent interactions (Sonnenberg et al., these substrata, or on substrata of E3 or E4. Outgrowth on 1990; Aumailley et al., 1991). Outgrowth on P1¢ was E8 and P1¢ was specific, as assessed using substratum- inhibited by both GRGDSP peptides and polyclonal specific antibodies (not shown). antiserum to a vb3, but not by the control peptide GRGESP, 1182 A. E. Sutherland, P. G. Calarco and C. H. Damsky

Fig. 7. Immunolocalization of integrins in 48 hour embryo outgrowths and in 72 hour EPC explants. Embryos (OG) and explants (EPC) cultured in serum- containing medium were fixed and stained with mAb GoH3 against the a 6 subunit (a 6; A-C), mAb 5H10 against the a 5 subunit (a 5; D-F), a polyclonal antiserum against a vb3 (a vb3; G-I), or normal rabbit serum (NRS; J-L). icm, inner cell mass; tb, trophoblast; g, giant cells. The expression of a 6 in EPC explants was restricted to less-differentiated cells in the center of the explant (cells above arrowheads in C). Arrowheads mark migrating parietal endoderm cells in A and B, and focal contacts in H and I. Scale bars, 50 mm.

suggesting that a vb3 is the major trophoblast P1¢receptor periphery of the outgrowths and did not migrate further on (Fig. 8C). the laminin substratum (Fig. 9). GoH3 did not affect PE Trophoblast outgrowth on intact laminin and E8 was migration on fibronectin substrata (Fig. 9). a 6b1 distribution unaffected by mAb GoH3, which blocks a 6b1 interactions in embryo outgrowths is consistent with these results; GoH3 with laminin (Fig. 8C). Of the two known integrin receptors stained the inner cell mass and migrating PE and not the tro- for the E8 region (a 6b1, Hall et al., 1990; Sonnenberg et al., phoblast cells (Fig. 7). Thus a 6 expression is decreased in 1990; and a 7b1, Kramer et al., 1991), a 6b1 is thus unlikely differentiated trophoblast cells but remains prominent in to be the sole or primary trophoblast integrin receptor for parietal endoderm, where it is the primary receptor for laminin. There are currently no antibodies that block the adhesion and migration on laminin. function of a 7b1 in the mouse, so the role of this integrin in trophoblast-laminin interactions could not be tested directly. DISCUSSION Another migratory cell population in the embryo, parietal endoderm cells, do appear to use a 6b1 as their sole or During the periimplantation development of the mouse dominant laminin receptor. Embryo outgrowths cultured 4 embryo, cell-matrix interactions are likely to be particularly to 5 days on fibronectin or laminin produced a population important for trophoblast differentiation and invasion during of small migratory cells that resembled parietal endoderm implantation. To analyze further the specific roles of (PE) morphologically and expressed several PE markers integrin-mediated cell-matrix interactions in trophoblast (laminin, collagen type IV, cytokeratin and vimentin; invasion and in other early developmental events, we have Behrendtsen et al., 1992; data not shown), suggesting that characterized the patterns of expression of several integrin they represent the in vitro equivalent of parietal endoderm. subunits and have investigated the role of individual While trophoblast outgrowth on laminin was unaffected by integrins in the interactions between trophoblast and the the presence of mAb GoH3, PE cells aggregated on the extracellular matrix (ECM) component laminin. Integrins in early mouse embryos 1183

A basic repertoire of integrins is expanded when A extraembryonic lineages differentiate Our results demonstrate that in the mouse, a basic repertoire of integrin heterodimers (a 5b1, a 6Bb1, a vb3) is expressed by the embryo throughout early development, whereas five other b1-associated a subunits (a 1, a 2, a 3, a 6A, a 7) show developmentally regulated expression (Fig. 10). Thus the embryo always has the potential to interact with fibronectin (a 5b1, a vb3), laminin (a 6Bb1, a vb3) and vitronectin (a vb3) as well as with other a vb3 receptor ligands, notably throm- bospondin. The later expression of additional a subunits allows greater diversity of cell-ECM interactions within the embryo and between the embryo and uterine tissues, by providing additional receptors for laminin (a 1b1, a 2b1, a 3b1, a 6Ab1, a 7b1) and fibronectin (a 3b1) and collagens B (a 1b1, a 2b1, a 3b1). The major point of regulation of integrin expression is the late blastocyst stage. There is little change in integrin expression during preimplantation stages, and none that cor- relates with any of the major developmental events of this period (compaction, blastocoel formation). a 3 mRNA is detected during early preimplantation stages but the protein is detectable only in embryo outgrowths or postimplantation embryos. Expression of a 2, a 6A and a 7 mRNA begins at the late blastocyst stage, before implantation, whereas a 1 expression begins with embryo outgrowth or implantation (Fig. 10). Expression patterns in the E7.5 embryo suggest that at least three subunits (a 1, a 6A and a 7) are specific to extraembryonic tissues, reflecting the fact that trophoblast and endoderm cells have the most extensive interactions C with ECM at this time, and may therefore require the greatest diversity of receptors. The variety of matrix receptors (e.g. integrins, cell surface galactosyltransferases and proteoglycans; Bayna et al., 1988; Sutherland et al., 1991) and ligands (fibronectin, laminin, collagen type IV, and thrombospondin; Wartio- vaara et al., 1978; Cooper and MacQueen, 1981; O’Shea et al., 1990; Thorsteinsdottir, 1992) expressed in the preim- plantation period begs the question of the role of cell-matrix interactions in early development. They are likely to be important for basement membrane formation on the inner surface of the trophectoderm and between the primitive ectoderm and primitive endoderm of the late blastocyst, but there is little evidence that integrin-mediated interactions are involved in other aspects of preimplantation development. Anti-integrin antiserum has no effect on preimplantation Fig. 8. (A) Diagram of laminin (Ln), showing the locations of , whereas antibodies to the cell-cell adhesion the various proteolytic fragments. Adapted from Yurchenko molecule E-cadherin interfere with both compaction and and Schittny, 1990. (B) Embryo outgrowth on substrata of primitive endoderm segregation (Damsky et al., 1983; Richa Ln, Ln fragments (E1¢, E3, E4, E8 and P1¢, and a recombinant et al., 1985). Anti-integrin antiserum does block trophoblast form of the A chain globular domain [rG]), and heat outgrowth and parietal endoderm migration in vitro, sug- denatured Ln (D-Ln). (C) Embryo outgrowth on substrata of gesting that integrin-mediated cell-matrix interactions are Ln, E8 and P1¢in the presence of antibodies against a 6 critical to the function of extraembryonic cell types (Richa (GoH3) or a vb3 (Anti-VnR), or in the presence of hexapeptides GRGDSP or GRGESP. Control medium et al., 1985; Sutherland et al., 1988). (Control) contained nonimmune rabbit serum at the same Integrin-mediated trophoblast-matrix interactions concentration as the anti-VnR antiserum. The effect of mAb GoH3 on attachment to P1¢was not determined (N.D.). For may involve newly expressed receptors each treatment in B and C, outgrowth was assayed by Previous results have shown that integrin receptors play an determining the percentage of embryos with trophoblast cells important role in trophoblast outgrowth on fibronectin, spreading on the substratum. Values represent the mean ± s.d. laminin and collagen type IV, as a pan-integrin antibody will of at least three experiments. completely block outgrowth on these substrata (Sutherland 1184 A. E. Sutherland, P. G. Calarco and C. H. Damsky

et al., 1988). Of the integrin receptors expressed during preimplantation stages, a 5b1 and a 6b1 are both localized to the interior of the embryo at the late blastocyst stage, while a vb3 is also on the external trophectoderm surface at this time. As a vb3 can recognize many, but not all, of the substrata on which embryos can form outgrowths, the ability of trophoblast cells to interact with ECM components may be acquired as a result of either a matrix contact-induced redistribution of the existing receptors, the availability of new receptors, or both. Trophoblast outgrowth on laminin is likely to involve newly expressed integrin receptors, as neither a 6b1 nor a vb3, laminin receptors expressed prior to implantation, appeared to play a role in trophoblast outgrowth on intact laminin substrata. Results described here (Fig. 8), and by Armant (1991), show that trophoblast cells interact with intact laminin via site(s) in the E8 region. Of the two known integrin receptors for the E8 region (a 6b1, a 7b1), we were able to rule out a 6b1 as a likely trophoblast laminin receptor because outgrowth on laminin and E8 was not perturbed by the function-blocking anti-a 6 antibody GoH3. In addition, expression of a 6 appeared to be downregulated in migrating trophoblast giant cells (Fig. 7). The a 7b1 complex is thus Fig. 9. Embryo outgrowths on laminin and fibronectin in the the most likely candidate to mediate integrin-dependent tro- phoblast-laminin interactions. Other evidence supports the presence of function-perturbing antibodies to integrin a 6. Embryos were cultured on laminin (A,B) and fibronectin (C,D) in role of a 7b1 as a trophoblast laminin receptor: mRNA for the presence of normal rabbit serum (Control; A,C) or of mAb a 7 is first expressed just before implantation and it appears GoH3 (Anti-a 6 (GoH3); B,D). Trophoblast cells are indicated by to be trophoblast-specific in the early postimplantation arrowheads, and the smaller endoderm cells by asterisks. Scale period. Our data suggest that trophoblast and endoderm bar, 25 mm. lineages use different strategies to interact with laminin, as

Fig. 10. Diagram summarizing the pattern of integrin subunit expression seen in periimplantation mouse embryos Integrins in early mouse embryos 1185 parietal endoderm cell migration on laminin was inhibited systems suggest that the a 1b1 may play an important role in by GoH3, indicating that a 6b1 is a major laminin receptor trophoblast invasion. for these cells. Other non-integrin E8 receptors could also In summary, we have found that the expression of integrin be involved in trophoblast-laminin interactions. Cell-surface receptors is developmentally regulated in mouse embryos, galactosyltransferase has been shown to play a role in with a basic repertoire that is continuously expressed and secondary giant cell outgrowth from EPC explants additional receptors that are expressed when extraembryonic (Romagnano and Babiarz, 1990), but it may not have the lineages differentiate. We have identified two major sites of same function in primary giant cell migration from embryo adhesive interactions between trophoblast and laminin (E8 outgrowths (Armant, 1991). Non-integrin proteins that and P1¢) and the integrin receptor for the P1¢site (a vb3). recognize a laminin A chain region containing the amino However, it is likely that integrin-mediated interactions with acid sequence IKVAV (reviewed by Mecham, 1991) could laminin and with other ECM components will have effects also play a role; there is currently no information about their on trophoblast behavior and function beyond those involved expression or function in early mouse embryos. in attachment and migration uncovered thus far. The use of Trophoblast cells can also recognize the P1¢region when in vitro culture models of trophoblast invasion in conjunc- it is presented as a fragment. The cryptic nature of this inter- tion with antisense, antibody or gene-targeting approaches action is consistent with previous results documenting a should enable direct testing of these ideas in the future. cryptic RGD-dependent cell binding site in the cross region of laminin which is exposed by proteolysis (Nurcombe et We thank Drs Clayton Buck, Jonathan Edelman, James Gailit, al., 1989; Aumailley et al., 1991). Laminin and the P1¢ Richard Hynes, Steve Kennel, Randy Kramer, Lou Reichardt, fragment have been shown to have distinct effects on cell Erkki Rouslahti, Arnoud Sonnenberg and Barry Ziober for gifts of behavior; laminin promotes, but P1¢ inhibits, melanoma antibodies and PCR primers, and Peter Yurchenco for the gifts of metastasis to the lung (Terranova et al., 1984), and P1¢more laminin fragments and anti-fragment antibodies, all of which made effectively stimulates collagenase activity (Turpeenniemi- these experiments possible. We also thank Drs Caroline Alexander, Carol Burdsal, Ray Keller, Zena Werb and Debbie Zimmerman for Hujanen et al., 1986; Sang et al., 1991). The significance of critical comments on the manuscript and many helpful discussions, trophoblast interactions with the cryptic P1¢site is unknown, and Evangeline Leash for editorial review. Expert technical assis- but a role for such interactions in implantation cannot be tance was provided by Margaret Siebert and Mercedes Joaquin. ruled out. Trophoblast invasion is accompanied by consid- These studies were supported by grants P01 26732 and CA 42032 erable matrix degradation (Glass et al., 1983; Behrendtsen from the National Institutes of Health, and by a National Insitutes et al., 1992), which may result in the production of laminin of Health National Research Service Award 5 T32 ES07106 from fragments with RGD-dependent cell binding activity. the National Institute of Environmental Health. 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