535Development 126, 535-546 (1999)Printed in Great Britain © The Company of Biologists Limited 1999DEV4056

BMP signaling plays a role in visceral endoderm differentiation and cavitation

in the early mouse embryo

Electra Coucouvanis* ,‡ and Gail R. Martin

Department of Anatomy and Program in Developmental Biology, School of Medicine, University of California, San Francisco, CA94143-0452, USA*Present address: Department of Cell Biology and Neuroanatomy, Institute of Human Genetics, University of Minnesota School of Medicine, 4-144 Jackson Hall, 321Church Street SE, Minneapolis, MN 55455, USA‡Author for correspondence (e-mail: electra@lenti.med.umn.edu)

Accepted 9 November 1998; published on WWW 7 January 1999

At E4.0 the inner cell mass of the mouse blastocyst consistsof a core of embryonic ectoderm cells surrounded by anouter layer of primitive (extraembryonic) endoderm, whichsubsequently gives rise to both visceral endoderm andparietal endoderm. Shortly after blastocyst implantation,the solid mass of ectoderm cells is converted by a processknown as cavitation into a pseudostratified columnarepithelium surrounding a central cavity. We havepreviously used two cell lines, which form embryoid bodiesthat do (PSA1) or do not (S2) cavitate, as an in vitro modelsystem for studying the mechanism of cavitation in theearly embryo. We provided evidence that cavitation is theresult of both programmed cell death and selective cellsurvival, and that the process depends on signals fromvisceral endoderm (Coucouvanis, E. and Martin, G. R.(1995) Cell 83, 279-287). Here we show that Bmp2 andBmp4are expressed in PSA1 embryoid bodies and embryosat the stages when visceral endoderm differentiation and

cavitation are occurring, and that blocking BMP signalingvia expression of a transgene encoding a dominant negativemutant form of BMP receptor IB inhibits expression of thevisceral endoderm marker, Hnf4, and prevents cavitationin PSA1 embryoid bodies. Furthermore, we show thataddition of BMP protein to cultures of S2 embryoid bodiesinduces expression of Hnf4and other visceral endodermmarkers and also cavitation. Taken together, these dataindicate that BMP signaling is both capable of promoting,and required for differentiation of, visceral endoderm andcavitation of embryoid bodies. Based on these and otherdata, we propose a model for the role of BMP signalingduring peri-implantation stages of mouse embryodevelopment.

Key words: BMP2, BMP4, BMP receptor, Cavitation, Embryoidbody, Programmed cell death, Visceral endoderm

SUMMARY

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INTRODUCTION

Morphogenesis of the peri-implantation mouse embrinvolves the transformation of the solid inner cell mass (ICMof the late blastocyst into a hollow structure known as the ecylinder. At the time of implantation (~E4.5), the ICM consisof an inner core of embryonic ectodermal cells, which will girise to all the cells of the embryo proper, surrounded byouter layer of extraembryonic endoderm. Shortly aftimplantation (~E5.0), a cavity (the proamniotic cavity) begito form in the core of the ICM and, by E6.0, the embryonectodermal cells that line the cavity have differentiated intopseudostratified columnar epithelium. The trophectoderderived extraembryonic ectoderm undergoes a similar procof cavitation at a slightly later stage. The process by whthese changes occur is known as cavitation, and this typmorphogenetic conversion also occurs during the formationother hollow (tubular) structures that arise from solprimordia, such as the ducts of various exocrine glandsearly postimplantation mouse development, cavitation prepa

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the embryo for gastrulation, the process by which the thregerm layers are formed.

Cavitation in the early embryo is difficult to study in vivobecause of the small size and relative inaccessibility of thembryo at this stage of development. However, mousembryonic stem (ES) or embryonal carcinoma (EC) cells thaform cavitating embryoid bodies in vitro serve as a usefumodel system for investigating the mechanism of cavitatio(Coucouvanis and Martin, 1995). Embryoid bodies areproduced when appropriate ES or EC cells are grown aaggregates in suspension. These aggregates spontaneoudifferentiate in culture in a manner that mimics many keyaspects of early mouse embryo development, includinendoderm differentiation and cavitation (Martin et al., 1977Coucouvanis and Martin, 1995). We have previously maduse of the PSA1 EC cell line to investigate the mechanism ocavitation. Our results demonstrated that cavitation iinitiated near the periphery of the embryoid body andproceeds inward. In addition, our data suggested thacavitation in embryoid bodies and embryos is the result o

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the interaction of two signals, one that is produced by depends on the presence of the outer endodermal layercauses the death of inner ectodermal cells, and one promotes survival of the single layer of columnar epithelcells that lines the cavity. In the present study, we soughidentify the molecule(s) that serves as the death signal ducavitation by determining whether proteins that habeen shown to cause cell death in other contexts responsible for the cell death that is observed duricavitation of embryoid bodies and the pregastrulation mouembryo.

Several members of the TGFβ superfamily of secretedsignaling molecules have been implicated in the controlcell death. For example, TGFβ1 can cause apoptosis wheadded to cultures of primary rabbit epithelial cells (Rotelet al., 1991) and Mullerian Inhibiting Substance (MIS) responsible for regression of the Mullerian duct in maembryos, a process that occurs by apoptosis (Price et1977). Bone Morphogenic Protein 4 (BMP4), a member the BMP subfamily of TGFβ-related proteins, has beenshown to cause apoptosis in several developmental settiFor example, in the embryonic chick hindbrain, Bmp4 isexpressed in subpopulations of cells in rhombomeres 3 5 that undergo apoptosis as part of their normdevelopmental program. When these rhombomeres explanted and cultured in vitro, Bmp4 expression isdownregulated and cell death does not occur. AdditionBMP4 protein to these explant cultures induces apoptotic cdeath (Graham et al., 1994). Similarly, in the chick limb buBmp4and Bmp2are expressed specifically in the interdigitaregions where PCD occurs (Lyons et al., 1990; Francis et 1994; Yokouchi et al., 1996), and application of beacontaining BMP4 protein to regions in which death does nnormally occur results in PCD (Ganan et al., 1996Moreover, interference with BMP signal transduction hbeen shown to prevent normal interdigital PCD (Yokoucet al., 1996; Zou and Niswander, 1996). Theobservations prompted us to explore the possibility thBMP4 and related proteins might play a role in promotinthe PCD that occurs during cavitation of embryoid bodiand the early mouse embryo. Our results indicate tsignaling by members of the BMP family is capable promoting and is required for both differentiation of VE ancavitation. Based on these data, we have developed a mfor possible functions of BMP signaling in pregastrulatiomouse development.

MATERIALS AND METHODS

Cell cultureAll cell lines were cultured and embryoid bodies were producedpreviously described (Martin et al., 1977; Coucouvanis and Mar1995). Embryoid bodies were cultured in the presence of BMproteins as follows: 12-20 embryoid bodies were selected frbulk culture dishes after 2 or 3 days of suspension culture transferred to a 100 µl drop of culture medium to whichrecombinant human BMP2, BMP4 or BMP7 (generously providby Genetics Institute, Cambridge, MA) had been added to a ficoncentration of 1 to 5 ng/µl. The drops were incubated undeparaffin oil (Fluka, Ronkonkoma, NY) for 3-4 days, during whictime the medium and BMP protein were replaced every 24-48 ho

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Dominant negative Bmpr-Ib transgeneWe constructed an expression vector in which we placed a dominanegative mutant form of the mouse Bmpr-Ibgene (Zou andNiswander, 1996; kindly provided by Dr L. Niswander, MemorialSloan Kettering Institute, New York) under the control of the mousPhosphoglycerate kinase (Pgk1) promoter (Tybulewicz et al., 1991),upstream of an internal ribosome entry site (IRES) sequen(Mountford et al., 1994) followed by the selectable reporter gene β-geo (Friedrich and Soriano, 1991). The IRES-β-geo cassette waskindly provided by Dr A. Smith (University of Edinburgh). Theresulting Pgk-dnBmpr-Ib-IRES-β-geo construct was introduced intoPSA1 cells by electroporation as previously described by Hébert al. (1994). After 12-14 days of culture in selection medium(containing up to 400 µg/ml G418), resistant clones were isolatedand analyzed for lacZexpression by X-GAL staining. Clones chosenfor further analysis were routinely tested for continued expressioof the transgene by staining with X-GAL.

Histological analysis and in situ hybridizationCavitation of embryoid bodies was assayed by histological analysof samples embedded in glycol methacrylate (GMA; PolyscienceWarrington, PA) as previously described (Coucouvanis and Martin1995). X-GAL staining to assay for β-GAL activity was performedessentially as described by Sanes et al. (1986) prior to GMembedding. In situ hybridization analysis was performed oembryos obtained by mating random-bred CD1 mice obtained froCharles River Laboratories (Hollister, CA). The day on which avaginal plug was detected was considered day 0 of gestatioThere was considerable variation in developmental stage within aamong litters. Embryos were fixed within the uterine deciduadehydrated and embedded in paraffin and sectioned. Embryobodies to be analyzed by in situ hybridization were harvested various stages of culture and were similarly fixed, embedded asectioned.

In situ hybridization was performed as previously describe(Frohman et al., 1990) with the following modifications: probes werlabeled with 33P, sections were not dehydrated prior to hybridizationa 3 hour prehybridization step was always included, hybridization waperformed at 65-68°C for all probes, and the high-stringency waswas for 2 hours at 65°C. Probes used included mouse Bmp2and Bmp4(Wozney et al., 1988), Bmp7(Furuta et al., 1997), Hnf4(Duncan etal., 1994) and Bmpr-Ib (Alk6; Zou and Niswander, 1996). Hybridizedslides were dipped in NTB3 emulsion (Eastman Kodak, RochesteNY) and exposed for 14-30 days. For each probe, no specihybridization was observed with a sense strand control (data nshown).

RT-PCR analysisAn RT-PCR analysis of gene expression was performed oembryoid bodies harvested after culture in drops as described aboor on an equivalent number of embryoid bodies isolated from bucultures. Total RNA was harvested from embryoid bodies using thRNAeasy kit (Qiagen, Valencia, CA) and cDNA was prepared usinthe Superscript system (Gibco BRL, Gaithersburg, MD). ThescDNAs provided templates for PCRs using the primer pairdescribed by Duncan et al. (1997) for the specific amplification oHepatocyte nuclear factor 4 (Hnf4), α-fetoprotein (Afp) andTransthyretin (Ttr) sequences. To control for the relative amount ocDNA used in each PCR assay, we also used primers that amplβ- and γ-cytoskeletal actin sequences (Hoppe et al., 1992), whicare expressed ubiquitously at relatively constant levels. Thconditions of the PCRs were the same for all primer pairs (annealitemperature 63°C, 28 cycles) except Ttr (annealing temperature60°C, 30 cycles).

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RESULTS

BMP gene expression in embryoid bodies and peri-implantation embryosAs a first step in determining whether BMP signaling might responsible for the cell death that occurs in the coursecavitation, we sought to determine whether the functionarelated BMP genes, Bmp4, Bmp2, and Bmp7, are expressed inembryoid bodies and embryos prior to and/or during cavitatioEmbryoid bodies were produced by seeding undifferentiaPSA1 cells at high density in tissue culture dishes, and thdetaching the resulting cell aggregates after three days transferring them to suspension culture. During the next 1days of culture >95% of the aggregates differentiate, giving rfirst to simple embryoid bodies with an outer layer oendoderm surrounding a solid core of ectodermal cells and tto cavitated embryoid bodies in which many of the inner cehave undergone apoptosis and the remaining ones hdifferentiated into a single layer of columnar epithelial cesurrounding a central cavity. An RNA in situ hybridizatioanalysis using probes for Bmp2, Bmp4 and Bmp7 wasperformed on sections of embryoid bodies harvested successive days of development beginning with the daydetachment (D+0).

We detected Bmp4RNA in PSA1 cell aggregates on D+0and D+1 (Fig. 1A,A′ and data not shown). At these stages, taggregates consisted primarily of undifferentiated ectodermcells, although by D+1 some of the embryoid bodies had beto form a morphologically distinct layer of endoderm on thouter surface (Fig. 1A). Bmp4expression was always found tobe specific to the ectodermal core cells (Fig. 1A′ and data notshown). On D+3, when some of the embryoid bodies hcavitated and others had not, Bmp4RNA was not detected inthe cavitated embryoid bodies but was detected in ectodermal cells of most non-cavitated embryoid bodies (dnot shown). By D+4, when most of the embryoid bodies hcavitated (Fig. 1B), Bmp4RNA was no longer detected (Fig1B′). These data indicate that Bmp4is expressed inundifferentiated ectodermal cells and that expression of gene is downregulated during the process of cavitation.

We also assayed for Bmp4expression in embryoid bodiesproduced by S2 EC cells. The S2 EC cell line was derived frthe same teratocarcinoma as the PSA1 cell line, but embryoid bodies differ from PSA1 embryoid bodies in the tyof endoderm that they form (they develop parietal endode(PE), whereas the endodermal layer in PSA1 embryoid bodcontains a mixture of visceral endoderm (VE) and PE, with Vpredominating) and S2 embryoid bodies fail to cavitate (F1C,D; see Martin et al., 1977). Interestingly, Bmp4RNA wasnot detected in S2 embryoid bodies at any of the stagesdevelopment examined (D+ 0 to D+ 5; Fig. 1C′, D′ and datanot shown).

In contrast to the results obtained for Bmp4, Bmp2RNA wasnot detected in ectodermal core cells of PSA1 or S2 embrybodies. However, Bmp2RNA was detected at low levels in theendoderm of PSA1 and S2 embryoid bodies, at all stagestheir development (Fig. 1E,E′,F,F′ and data not shown). Bmp7RNA was not detected in either undifferentiated PSA1 or cells, or in the embryoid bodies that they produce (data shown).

To determine whether the expression patterns observed

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Bmp4, Bmp2 andBmp7 in embryoid bodies reflect their normalpatterns of expression in the pregastrulation embryo, wperformed a gene expression analysis in precavitation apostcavitation stage mouse embryos. The undifferentiataggregates of PSA1 cells and the endoderm-containiprecavitation PSAl embryoid bodies in which Bmp4expressionwas detected most closely resemble the ICM of the early (E3and late (E4.5) blastocyst, respectively, whereas the cavitaembryoid bodies in which Bmp4expression was not detectedmost closely resemble the embryonic portion of the E5conceptus (see Fig. 2A). When we assayed for Bmp4expression in the embryo, Bmp4RNA was detected in ICMcells and the polar trophectoderm of the E3.5 blastocyst (F2B,B′). At E4.5, after the blastocyst has implanted in thuterus, Bmp4RNA was also detected in the ICM and polatrophectoderm, but not in the primitive endoderm celcovering the blastocoelic surface of the ICM (data not shownA subset of the E4.5 embryos examined were clearly modevelopmentally advanced, in that extraembryonic ectode(which develops from the polar trophectoderm of thblastocyst) was evident. In such embryos, Bmp4 expressionwas detected in the extraembryonic ectoderm but not the ICderived embryonic ectoderm (data not shown). By E5.5, tembryonic ectoderm has undergone cavitation, and Bmp4 RNAwas not detected in those cells, although it was still detecin the cells of the as yet uncavitated extraembryonic ectode(Fig. 2C,C′). The latter undergoes cavitation at a slightly latestage than the embryonic ectoderm. By E6.5, when cavitatof the extraembryonic ectoderm is complete, Bmp4 RNAbecomes restricted to a circumferential band of extraembryoectoderm just proximal to the boundary between the embryoand extraembryonic regions of the conceptus (Fig. 2D,D′; seeWaldrip et al., 1998). When we performed assays for Bmp2andBmp7expression, we found that Bmp2RNA was detectable atlow levels in the endoderm of embryos at E5.0 to E6.5 (Fi2E,E′,F,F′), whereas Bmp7RNA was not detected in theconceptus, but was detected at high levels in maternal decidtissue surrounding it (Fig. 2G,G′). Expression of Bmp2 andBmp4was also detected in uterine tissue, but in cells locatat a considerable distance from the conceptus (data not show

Taken together, these results indicate that the expresspatterns of these BMP genes are similar in PSA1 embryobodies and normal embryos in vivo, and that PSA1 cetherefore provide a valid model system in which to studcavitation in the peri-implantation embryo. The data on BMgene expression are consistent with a role for these molecuin cavitation-associated programmed cell death. MoreovBmp4 RNA is detected in the ectoderm of PSA1 embryoibodies and embryos prior to cavitation, and is not detectedS2 embryoid bodies, which do not cavitate.

Blocking BMP signaling prevents cavitation of PSA1embryoid bodiesTo determine whether interfering with BMP signalingprevents cavitation, we examined the effects of expressingdominant negative (dn) mutant BMP receptor transgene embryoid bodies that normally cavitate. The strategy that wused is based on current knowledge of the mechanismBMP signaling, which occurs through heterodimericomplexes of two different types of serine/threonine kinareceptors (type I and type II). Upon ligand binding to a typ

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Fig. 1. Expression of Bmp2and Bmp4inembryoid bodies. In situ hybridizationusing antisense probes to detect Bmp4(A,A′ -D,D′) and Bmp2(E,E′,F,F′) RNAin embryoid bodies harvested after either1 day (A,C) or 4 days (B,D-F) ofsuspension culture. (A-F) Bright-fieldimages, (A′-F′) corresponding dark-fieldviews. (A,B,E) Embryoid bodies(cavitating) produced by the PSA1 cellline; (C,D,F) embryoid bodies (non-cavitating) produced by the S2 cell line.Abbreviations: ecto, ectoderm; endo,endoderm. Radioautographic exposuretime: (A′) 16 days; (B′,C′) 20 days; (D′)24 days; (E′,F′) 14 days.

II receptor, a type I receptor is recruited to the complex aits kinase domain is activated, leading to signal transductthrough intracellular proteins known as SMADs (reviewed bBaker and Harland, 1997; Heldin et al., 1997). The BMP tyII receptor (BMPR-II) can bind BMP2, BMP4 or BMP7, anthen associate with and activate signaling through anythree type I receptors: BMPR-IA, BMPR-IB or ActR-I(reviewed by Massague and Weis-Garcia, 1996). One wayinhibit signaling by these three BMP ligands is to expresshigh levels a mutant transgene that encodes a non-functioBMPR-IB protein. This can result in the recruitment of aligand-bound BMPR-II receptors into non-functionacomplexes and thus prevent them from interacting with aactivating any wild-type BMPR-IA, BMPR-IB or ActR-Iproteins that may be present.

PSA1 cells were transfected with an expression vector (Fig. 3A) that includes a mutated mouse Bmpr-Ib gene with apoint mutation in the region that encodes the ATP-bindidomain of the BMPR-IB kinase. This mutation has been shoto abolish kinase activity of the receptor protein and rendeinactive in a signaling assay (Zou and Niswander, 1996). Whexpressed in the chick limb bud in ovo, this dominant negatmutant receptor gene interferes with BMP signaling aprevents PCD (Zou and Niswander, 1996). To provide a meof monitoring expression of the transgene, we placed internal ribosome entry site (IRES) downstream of the stcodon in the mutant Bmpr-Ib gene, followed by the β-georeporter gene. This configuration results in production ofsingle, dicistronic message encoding both the domin

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negative receptor and β-geogenes (Mountford et al., 1994). β-geo is a fusion gene between lacZ and neoR. Its expression canbe detected by staining for β-galactosidase (β-GAL) activitywith X-GAL, and provides a selectable marker (neomyciresistance) to facilitate the isolation of cells that express tdominant negative receptor transgene (Friedrich and Sorian1991).

We isolated numerous stably transfected neoR stem cellclones and assayed the undifferentiated cells for β-GALactivity. From these clones, we selected nine for furtheanalysis, five that were resistant to the levels of G418 used selection, but did not produce enough β-geo to displaydetectable β-GAL activity, and four that produced β-GALactivity at high levels. We then produced embryoid bodies frothese nine clones and assayed for cavitation after 4-6 dayssuspension culture. Whereas the embryoid bodies formed the five β-GAL-negative clones cavitated normally (Fig. 3Band data not shown), those produced by the four clones tstained strongly for β-GAL activity failed to undergocavitation. Although occasional embryoid bodies derived fromthe four positive clones displayed both a loss of β-GAL activityand evidence of cavitation, we never observed cavitation embryoid bodies that were strongly positive for β-GAL activity(Fig. 3C and data not shown). To confirm that the high leveof β-GAL activity observed reflected expression of thednBmpr-Ib gene, we performed an in situ hybridizationanalysis using a probe for Bmpr-Ib, which detects both mutatransgene and endogenous wild-type RNA. In the embryobodies that expressed high levels of β-GAL activity, we

539BMP signaling and cavitation

Fig. 2. Expression of BMP genes in peri-implantation embryos. (A) Schematic diagrams of the mouse embryo at E3.5 through E6.5, illustratingthe development of the extraembryonic endoderm and the process of cavitation leading to the formation of the proamniotic cavity. (B-G) Hybridization of antisense probes for Bmp4 (B,B′-D,D′), Bmp2 (E,E′,F,F′), or Bmp7 (G,G′) to sections through embryos at the stagesindicated. (B-G) bright-field images; (B′-G′) corresponding dark-field views. (C,D,F) Sagittal sections; (E) the embryonic portion of theconceptus sectioned in a plane that is midway between transverse and sagittal. The proamniotic cavity is present in this embryo but is notevident due to the oblique plane of section. The plane of section in G is transverse, but slightly oblique. Abbreviations: EE, embryonicectoderm; ExE, extraembryonic ectoderm; ICM, inner cell mass; PE, parietal endoderm; PrE, primitive endoderm; TE, trophectoderm; VE,visceral endoderm. Radioautographic exposure time: (B′,C′,E′) 24 days; (D′,F′) 30 days; (G′) 20 days.

Fig. 3. Expression of a dominant negativemutant Bmpr-Ib transgene prevents cavitation.(A) Schematic diagram of the construct usedto express a dominant negative mutant formof BMP receptor IB in PSA1 cells. Thepromoter from the mouse Pgk1gene (orangebox) drives expression of the coding sequenceof the mouse Bmpr-Ibgene (purple box),which contains a point mutation in the ATP-binding domain. Downstream of the mutantBmpr-Ibgene is an internal ribosome entrysite (IRES; yellow box) followed by the β-geofusion gene (blue box). (B,C) Embryoidbodies produced by clonal lines isolatedfollowing stable transfection of PSA1 cellswith the construct shown in A. The embryoidbodies, which were fixed and stained with X-GAL after 4 days in suspension culture, wereproduced by (B) a clonal line in whichexpression of the transgene is low or absent (as indicated by the lack of blue stain); note that these embryoid bodies cavitate normally, althoughat the stage shown they have not yet completed cavitation, or (C) a clonal line that expresses high levels of the construct; note that theseembryoid bodies fail to cavitate (<5% of embryoid bodies displayed evidence of cavitation).

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detected Bmpr-Ib RNA at much higher levels than in theembryoid bodies formed by control (non-transfected) PScells (data not shown).

Interestingly, two of the four clones that initially producehigh levels of β-GAL activity displayed a substantial decreasin transgene expression in successive passages (althoughunder selection), as evidenced by low or undetectable levof β-GAL activity, which was paralleled by a dramatidecrease in Bmpr-Ib expression as assayed by in sithybridization. Embryoid bodies produced by these lines afthis decrease in transgene expression appeared to regained the ability to cavitate normally. These data indicthat the initial failure to cavitate seen in the embryoid bodproduced by these lines was reversible. Importantly, embryoid bodies produced by the two clones that continuto express the transgene at high levels, line A2 and line cavitation failed to occur (see below). Taken together, thresults demonstrate a close correlation between high levednBmpr-Ib transgene expression and a lack of cavitationembryoid bodies.

Cavitation of S2 embryoid bodies is induced by BMPproteinsThe data described above indicate that BMP signalingrequired for cavitation. Moreover, the observation that embryoid bodies (which fail to cavitate) do not express Bmp4raises the possibility that lack of BMP4 is responsible for tlack of cavitation in S2 embryoid bodies. To test thhypothesis, we examined the effect of recombinant BMprotein on S2 embryoid bodies. In each experiment (n=8), wecultured 12-20 embryoid bodies with 1 to 5 ng/µl BMP4 andan equal number in control medium. By analyzing sersections of these samples, we found that in contrast to conS2 embryoid bodies, which never exhibited any featurescavitation (Fig. 4A and data not shown), 20-60% of BMPtreated S2 embryoid bodies in each experiment contained or more small cavities located near the periphery of tembryoid body, in which apoptotic cells were apparent, andwhich the cells surrounding these cavities had formedcolumnar epithelium (Fig. 4B). However, the complecavitation typical of PSA1 embryoid bodies (see Fig. 1Bwhich initiates near the periphery and progresses inward uthe core cells are completely gone, was not observed in anthe BMP4-treated S2 embryoid bodies, even when they wcultured in the presence of BMP4 for 6-8 days (data nshown). Thus addition of BMP4 induces cavitation near tperiphery of S2 embryoid bodies.

We also tested the ability of BMP2, BMP7 and a variety related and unrelated signaling molecules to induce cavitaof S2 embryoid bodies. Both BMP2 and BMP7 had the saeffect as BMP4. At the same concentrations used for BMBMP2 (n=5 experiments) and BMP7 (n=2 experiments)treatment caused the appearance of small cavities lined columnar epithelium and containing apoptotic cells in 20-60of the embryoid bodies in each experiment (data not showIn experiments in which all three proteins were compardirectly, no differences in their effects were observed wrespect to number of embryoid bodies affected or extentcavitation. In contrast, addition of human Activin (provided bNational Hormone and Pituitary Program, National InstituteDiabetes and Digestive and Kidney Diseases, and found to

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active in Xenopus animal cap assays [Dr T. Musci, UCSpersonal communication]), human TGFβ1 (kindly provided byDrs. A. Erlebacher and R. Derynk, UCSF), human IGF(Sigma, St. Louis, MO), human FGF4 (kindly provided byGenetics Institute, Cambridge, MA), or human EGF (R&DSystems, Minneapolis, MN) at 5 ng/µl had no effect on S2embryoid bodies (data not shown). These results demonstrthat the ability to induce cavitation in S2 embryoid bodies displayed by certain BMPs but not other types of signalinmolecules (including the TGFβ superfamily members Activinand TGFβ1).

BMP signaling promotes the differentiation ofvisceral endodermIn the protein addition experiments described above, we nothat the outer endodermal cells of S2 embryoid bodies treawith BMPs differed from those of control S2 embryoid bodiesSpecifically, endoderm of untreated S2 embryoid bodieconsists of individual cells that do not appear to be organizinto a cohesive epithelium (Fig. 4A). In contrast, endoderm othe surface of BMP-treated S2 embryoid bodies is more highorganized into an epithelial layer and the cells appear to more tightly adherent to one another. In addition, thendodermal cells of BMP-treated S2 embryoid bodies contanumerous vacuoles that are not evident in the endodermuntreated embryoid bodies (Fig. 4B and data not shown). Othe whole, we noted that the endoderm of BMP4-treated embryoid bodies strongly resembles the VE of cavitatinembryoid bodies and normal embryos, rather than the PE fouon the surface of untreated S2 embryoid bodies.

To determine whether these BMP-induced morphologicchanges are accompanied by changes in endoderm gexpression, we used an RT-PCR assay to test for texpression of the VE-specific molecular markers Hnf4(Duncan et al., 1994), Afp (Dziadek and Adamson, 1978) andTtr (Makover et al., 1989). Hnf4has been shown to be specificto VE as early as E4.5 (Duncan et al., 1994), whereas AfpandTtr are markers for VE at later stages of developme(Dziadek and Adamson, 1978; Makover et al., 1989Expression of all three genes was detected in PSA1 but control S2 embryoid bodies (Fig. 4C), consistent with thconclusion from previous studies that the endodermal layof S2 embryoid bodies does not normally contain VE cel(Martin et al., 1977). Similar results were obtainedirrespective of whether the embryoid bodies were cultured small drops or in bulk culture (data not shown). In contrato the lack of VE gene expression observed in control Scultures, expression of these three genes was detected inembryoid bodies treated with BMP4 (Fig. 4C).

To confirm that the gene expression detected in BMP-treatS2 embryoid bodies is specific to the endoderm, we alexamined expression of Hnf4by in situ hybridization. Hnf4RNA was detected in the VE of embryos at E4.5- E6.5 (Fi4D,D′ and data not shown; see also Duncan et al., 1994) aalso in the endoderm of PSA1 embryoid bodies prior to anduring cavitation (Fig. 4E,E′ and data not shown). Expressionof Hnf4 is not homogeneous throughout the endoderm of PSAembryoid bodies, presumably due to the presence of PE cintermingled with the VE cells (Martin et al., 1977). Inagreement with the results of the RT-PCR assay describabove, Hnf4 RNA was not detected in control S2 embryoid

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bodies at any of the stages assayed (Fig. 4F,F′ and data notshown). However, Hnf4 RNA was detected in the outerendodermal cell layer of S2 embryoid bodies that had betreated with BMP4 or BMP2 (Fig. 4G,G′ and data not shown).In some cases, the region in which Hnf4 RNA was mostabundant coincided with areas of underlying ectoderm which cavitation was occurring (see inset in Fig. 4G′). Thesedata indicate that one effect of BMP proteins on S2 cells ispromote the differentiation of VE.

Additional evidence in support of this conclusion came froanalysis of the embryoid bodies produced by one of the PScell lines expressing the dnBmpr-Ibtransgene (line B4), whichfail to cavitate. β-GAL activity was detected at high levels inthe endoderm of those embryoid bodies (Fig. 5A), and Hnf4expression in the endoderm was substantially reduced or ab(Fig. 5B′). In contrast, Hnf4 RNA is strongly expressedthroughout the endoderm of control PSA1 embryoid bod(compare Fig. 5B′ with Fig. 4E′). Thus, expression of thedominant negative receptor transgene at high levels in endoderm of PSA1 embryoid bodies appears to greadiminish the expression of at least one marker of Vsuggesting that BMP signaling in embryoid bodies is requirfor VE differentiation. Taken together, these results provievidence that BMP signaling is both capable of promoting arequired for the differentiation of VE.

Cavitation may also require BMP signal reception inthe ectodermThe data described above, indicating that BMPs promdifferentiation of VE, raise the possibility that this is their onfunction in cavitation and that once differentiated, the VE cethen produce some other (non-BMP) signal that is modirectly involved in initiating the cavitation process. If thiwere the case, then the presence of VE should be the requirement for cavitation to occur, and an embryoid boexpressing high levels of the dnBmpr-Ib transgene throughouthe ectoderm should still cavitate, as long as the endoderlayer is negative for dnBmpr-Ib expression so that VEdifferentiation can occur. However, the results of an analyof the embryoid bodies formed by one of the dominant negamutant receptor transgene-expressing PSAl cell lines leadto propose that BMP signaling may also play a more direct rin cavitation.

Line A2 was initially found to produce embryoid bodiewith high levels of β-GAL activity throughout, but afternumerous passages it was found that the embryoid boproduced by these cells displayed high levels ofβ-GALactivity in the ectodermal cells, but little or no activity in thendoderm (Fig. 5C). Because the endodermal cells expresslevels of the dominant negative receptor transgene, BMsignaling in those cells should not be blocked. Therefore, thshould be able to respond to endogenous BMPs and prodVE. Indeed, we found that Hnf4 RNA could be detected inmany (but not all) of the endodermal cells of line A2 embryobodies, suggesting that VE had formed (Fig. 5D,D′).Importantly, despite the apparent presence of VE, the embrybodies failed to cavitate (Fig. 5C,D). These data suggest tin the presence of VE, cavitation does not occur unleectodermal cells are capable of BMP signal reception and tare consistent with a requirement for BMP signaling in that ctype, as well as in the endoderm, for normal cavitation.

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This study was initiated to explore the possibility that BMPmight serve as the signal for the programmed cell death thaa key feature of the cavitation that occurs in embryoid bodieand the early postimplantation mouse embryo. Although thresults of our experiments have not resolved the question whether BMPs function as the death signal per se, they provide strong evidence that BMP signaling is required focavitation. Specifically, we have shown that Bmp2and Bmp4are expressed in a temporally and spatially restricted pattethat is consistent with a role for these factors in the cavitatioof both PSA1 embryoid bodies and normal embryos, and whave demonstrated that interfering with BMP signaling viexpression of a dominant negative mutant receptor transgeabolishes cavitation of PSA1 embryoid bodies. Furthermorwe showed that treatment of S2 embryoid bodies, whicnormally fail to cavitate, with BMP2, BMP4 or BMP7 inducescavitation near the periphery of the embryoid bodies. Analysof the expression of molecular markers of VE in both PSAembryoid bodies that express a dominant negative mutant BMreceptor transgene and S2 embryoid bodies treated with BMrevealed that BMP signaling is required for differentiation oVE in embryoid bodies.

Based on these results, and on accumulated evidendemonstrating that embryoid bodies formed by certain EC aES cell lines provide a valid model system in which to studthe mechanisms of normal early mouse embryogenesis (seeexample, Coucouvanis and Martin, 1995; Duncan et al., 199Choi et al., 1998), we propose a model for peri-implantatiomouse development in which BMP signaling is required tpromote differentiation of primitive endoderm to VE andcavitation of the ectoderm (Fig. 6). According to this modeBMP2 and BMP4 are functionally redundant with respect tthese two proposed activities. Thus, as depicted in Fig. during endoderm development, BMP4 produced in thectoderm or BMP2 produced in the endoderm can promote Vdifferentiation. Likewise, during cavitation, BMP2 produced inthe endoderm or BMP4 produced in the ectoderm can act the ectodermal core cells, possibly in conjunction with anothmolecule(s) produced by VE, to cause the cell death acolumnar epithelial differentiation that constitute cavitationThis proposed functional redundancy is a key aspect of omodel and is consistent with the observation that embryhomozygous for a null allele of either Bmp2or Bmp4cavitatenormally (Winnier et al., 1995; Zhang and Bradley, 1996).

BMP signaling and the differentiation of visceralendodermIn the normal embryo, VE derives from the extraembryoniendodermal lineage that is first established at ~E4.0, when cein the outer layer of the ICM of the blastocyst delaminate tform the primitive endoderm; the remaining inner cells of thICM form the embryonic ectoderm. Primitive endoderm cellthat maintain contact with the ectoderm differentiate into VEwhereas those that migrate away from the ICM along the innsurface of the trophectoderm form parietal endoderm(Nadijcka and Hillman, 1974; Enders et al., 1978; see Fig. 2AAlthough it has been established through studies of chimeembryos that an initial precursor cell population (primitiveendoderm) gives rise to both VE and PE (Gardner, 1982), t

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Fig. 4.S2 embryoid bodies treated with BMP4 undergo partialcavitation and express markers of visceral endoderm. (A,B) Sectiothrough S2 embryoid bodies harvested after 4 days of culture incontrol medium (A) or in medium with BMP4 at 5 ng/µl (B). Notethe cavities surrounded by a columnar epithelium (marked withasterisks) at the periphery of the embryoid body. Arrows point tovacuoles in endodermal cells. (C) Expression analysis of VE markgenes by RT-PCR. RNA was isolated from PSA1 or S2 embryoidbodies cultured in control medium, or S2 embryoid bodies culturein medium containing BMP4 (3 ng/µl). Following reversetranscription, the cDNA templates were assayed by PCR usingprimer pairs specific for Hnf4, Ttr, Afp and Actin. Control samplesincluded templates prepared in the absence of reverse transcriptausing RNA isolated from S2 embryoid bodies cultured in mediumcontaining BMP4, as well as PSA1 and S2 embryoid bodies cultuin control medium (−RT lane and data not shown). (D,E) Hnf4expression in an E5.5 embryo (D) and in PSA1 embryoid bodies (In the embryoid bodies shown, cavitation is occurring normally bunot yet complete in most of them. (F,G) Bright-field views of S2embryoid bodies assayed by in situ hybridization for Hnf4RNAfollowing culture in control (F) or BMP4-containing (G) medium.(D′-G′) Τhe dark-field views corresponding to D-G. The scale barsD and E illustrate the relative sizes of the E5.5 embryo and embrybodies. Radioautographic exposure time: (D′) 28 days; (E′-G′) 21days.

Fig. 5. Dominant negative BMP receptor expression prevents VEdifferentiation. (A) Assay by X-GAL staining for expression of thednBmpr-Ib transgene in embryoid bodies formed by clonal line B4.Note that expression is detected at high levels predominantly in theendoderm and peripheral ectoderm, and at lower levels throughoutthe ectodermal core. (B,B′) Hnf4expression in B4 embryoid bodies.(C) X-GAL assay for transgene expression in embryoid bodiesformed by clonal line A2. Note that expression is detectedpredominantly in the ectoderm. (D,D′) Hnf4expression in A2embryoid bodies. Note the negative correlation between expressionof the dnBmpr-Ib transgene in the endoderm and the level of Hnf4expression. Radioautographic exposure time: (B′,D′) 26 days.

precise timing, as well as the nature of the transition froprimitive endoderm to VE or PE is poorly understood. Tavailable evidence suggests that interactions betwendodermal cells and embryonic ectoderm or trophectodecells promote the development of VE or PE, respective(Hogan et al., 1981; Hogan and Tilly, 1981). Several stud

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have recently demonstrated that VE plays an essential rolepatterning the anterior embryonic ectoderm (Thomas anBeddington, 1996; Varlet et al., 1997; Rhinn et al., 1998), analso that embryos with defective VE fail to gastrulate normall(Duncan et al., 1994; Sirard et al., 1998; Waldrip et al., 1998Despite the importance of VE in early development, thmechanisms governing its formation and differentiation remalargely unknown. There is evidence from genetic studiesuggesting that BMP signaling may be involved in VEdevelopment. In embryos homozygous for a null allele oBmpr-Ia, the VE appears morphologically different from wild-type VE at E7.0 (Mishina et al., 1995). Moreover, embryos anembryoid bodies homozygous for a null allele of Smad4, a genethat encodes a component that appears to be common toTGFβ-related signal transduction pathways (including theBMP pathway), display abnormalities in VE morphology andgene expression (Sirard et al., 1998).

Here we provide evidence that BMP signaling is bothcapable of promoting and is required for the differentiation oVE. We have shown that addition of BMP4, which is normallyproduced in the ectoderm, to S2 embryoid bodies (which fato express Bmp4) induces morphological and molecularchanges in the endodermal cells indicative of VEdifferentiation, including the expression of Hnf4, a marker forVE in the embryo as early as E4.5. Furthermore, we hashown that interfering with BMP signaling in the endoderm oPSA1 embryoid bodies apparently inhibits the expression Hnf4. Because our studies demonstrated that BMP2 and BMhave similar effects on S2 embryoid bodies, we hypothesiz

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Fig. 6. A model illustrating proposed roles of BMP signaling indevelopment of peri-implantation embryos and embryoid bodies.Schematic diagram depicting the proposed role of BMP signaling VE differentiation and cavitation. The top row shows sectionsthrough the endoderm and ectoderm of a precavitation embryo orembryoid body. BMP4 produced in the ectoderm acts on theprimitive endoderm to promote VE differentiation. BMP2 producedin the endoderm may also play a role in the process. The bottom rshows similar sections immediately prior to and after cavitation.BMP4 produced in the ectoderm and/or BMP2 produced in theendoderm act(s) on the ectoderm (perhaps in conjunction with othVE-derived signals) to promote PCD of the inner cells anddifferentiation of the outer layer of columnar cells.

that BMP2, which is normally produced in the endoderm, malso promote VE differentiation. Consistent with our modewe have found that Bmp4 is expressed in the ectoderm oembryoid bodies and the ICM of embryos prior to, during, afor a short time after primitive endoderm formation, and migtherefore influence Hnf4 expression in the nascent VEHowever, it is unclear whether Bmp2is expressed in primitiveendoderm or nascent VE prior to Hnf4, in part because thelevels of Bmp2detected are low. If Bmp2is first expressed afterHnf4expression commences, then BMP2 would be unlikelypromote early stages of VE differentiation under normcircumstances, but might do so when other BMP signals absent, as for example in Bmp4-deficient embryos.

One finding that appears to be inconsistent with thypothesis that VE can be induced by BMP2 produced in endoderm is that the endodermal cells of S2 embryoid bodexpress Bmp2RNA yet they morphologically resemble PE ando not express Hnf4. One possible explanation for this appareinconsistency is that the Bmp2RNA produced by S2 embryoidbodies does not produce functional BMP2 protein, atherefore S2 embryoid bodies, which do not express Bmp4, failto form VE due to the lack of a BMP signal. To address th

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issue experimentally, we attempted to determine directwhether BMP2 protein is produced in S2 embryoid bodieusing anti-BMP2 antibodies. Unfortunately, the antibodieavailable proved unsuitable for such studies. We also compathe coding sequences of PCR-amplified Bmp2cDNAs derivedfrom S2 and PSA1 cells, but found no differences betweethem (data not shown). Another possibility is that functionaBMP2 protein is produced in S2 endoderm, but some aspecBMP signal reception is abnormal in S2 cells, resulting in BMsignal transduction when large amounts of protein (such as amounts that were added to the culture medium) are presebut not in response to lower, endogenous levels of BMprotein.

The role of BMP signaling in cavitationOur results demonstrate that the PCD and columnar epithedifferentiation of ectodermal cells that constitute cavitation adependent on BMP signaling. However, they do not excludthe possibility that this is an indirect effect, and the requiremefor BMP signaling might be met by the BMP-mediated effecton endoderm discussed above. According to this model, Vdifferentiation in response to BMP signaling would result inthe production of other molecules required for cavitation. Sucmolecules would presumably be produced by early rather thlate stage VE, since cavitation is normal in Hnf4−/− or Smad4−/− embryos, in which early but not mature VE isapparently formed (Chen et al., 1994; Duncan et al., 199Sirard et al., 1998).

Although it is possible that BMP signals are necessary onfor differentiation of VE, and VE subsequently causecavitation via a BMP-independent mechanism, our dasuggest that BMP signaling may also play a more direct roin cavitation. Our observation that embryoid bodies formed bline A2 (which express the dominant negative mutant recepttransgene at high levels exclusively in the ectoderm) fail cavitate is consistent with a requirement for downstreacomponents of BMP signaling in ectodermal cells in order focavitation to occur, thus suggesting that BMP might be thdeath signal per se. It should be noted, however, that a dirrole for BMP signaling in the PCD and/or differentiation of thecolumnar epithelial cells does not preclude a possibrequirement for some other molecule(s) produced by VE thworks in conjunction with a BMP signal (either VE-derivedBMP2 or ectoderm-derived BMP4) to promote cavitation.

There have been two reports demonstrating that BMPinduces cell death in P19 EC cells. Glozak and Rogers (199found that, in combination with retinoic acid (RA), BMP4caused apoptosis in aggregates or monolayer cultures of Pcells. In their assay, BMP4 alone had only a minimal effect ocell death. In contrast to those findings, Marazzi et al. (199reported that addition of BMP4 (with or without RA) increasecell death in aggregates, but not monolayers, of P19 cells. Trelevance of these results to our study is unclear, since Paggregates neither form an outer layer of endoderm nundergo cavitation.

One potential argument against a role for BMP signaling either the early stages of VE differentiation or cavitation ibased on the assumption that SMAD4 is required for all formof TGFβ signaling, and the finding that early markers of VEdifferentiation are expressed and cavitation occurs in Smad4null mutant embryos (Sirard et al., 1998). However, there

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mounting evidence that SMAD4 may not be absoluterequired, either because there might be additional SMADsyet unidentified) that can functionally substitute for it, obecause there might exist TGFβfamily signaling mechanismsin which SMAD4 function is not required. For example, in thpresence of wild-type extraembryonic tissues, developmenSmad4null mutant embryos appears relatively normal unE9.0 (Sirard et al., 1998), but it seems unlikely that signalby all TGFβ family members in the embryo proper idispensable until that stage (see Varlet et al., 1997). Thusfavor the view that the ability of Smad4mutant embryos tocavitate normally is a manifestation of functional redundanof this type of SMAD in the BMP signal transduction pathwa

One interesting question is why addition of BMPs to Scultures causes cavitation only at the periphery of the embrybodies. One possible explanation of this observation is baon the assumption that BMP protein added to the cultmedium is not able to diffuse very far into the core of the embryoid bodies. Thus, in S2 embryoid bodies, which do expressBmp4in the ectoderm and may not produce functionBMP2 in the endoderm, the added BMP might be availaonly to cells in the periphery of the embryoid bodies. If thisthe case, it raises the possibility that the effective rangeBMP2 produced in the endoderm of PSA1 embryoid bodmay also be limited, and that cavitation occurs throughothose embryoid bodies because BMP2 activity complemented by BMP4 produced throughout the ectodeIn the normal mouse embryo, BMP2 produced in the endodemay be sufficient for complete cavitation of the embryonectoderm because at the stage when cavitation occurs,embryo is only 6-8 cell widths in diameter.

Functional redundancy of BMPs during peri-implantation developmentAn important feature of the model discussed above is concept that BMPs produced at peri-implantation stagesdevelopment are redundant with respect to their propofunctions in VE differentiation and cavitation. There substantial evidence from other types of studies for functiooverlap among members of the BMP family. For exampBMP2 and BMP4 both promote apoptosis in chick lemesenchyme cells in culture (Yokouchi et al., 1996) and bfactors can induce a dorsomedial phenotype when addetelencephalic neuroectoderm explants (Furuta et al., 199Consistent with those results, we observed identical effectscavitation of S2 embryoid bodies with BMP2, BMP4 anBMP7. Such functional overlap is presumably related to treported ability of these different ligands to interact with thsame receptor, BMPR-II, which has been shown to expressed in the late blastocyst and from E6.0 onwards (Roet al., 1997). The BMPR-II-ligand complex can associate wand activate signaling via any of three type I receptors: BMPIA, BMPR-IB or ActR-I (reviewed by Massague and WeisGarcia, 1996). If multiple receptor interactions and cexpression occur in the precavitation embryo, these miprovide an explanation for the observation that embryhomozygous for a null allele of Bmpr-Ia cavitate normally(Mishina et al., 1995).

Limited data are available on the expression of BMP typreceptors during peri-implantation mouse development. Bmpr-Ia has been detected by RT-PCR assays at the late blasto

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stage, and again at E7.0, but not at E6.0; other stages betwlate blastocyst and E6.0 were not examined (Roelen et 1997). However, the observation that embryos homozygous a null allele of Bmpr-Iaappear normal at E5.5 but display aphenotype by E7.0 (Mishina et al., 1995) suggests that BmIa is expressed between E4.5 and E7.0. Bmpr-Ibexpression hasalso been detected by RT-PCR assays at the early morula stbut not in the compacted morula or blastocyst. Furthermore,was the case for Bmpr-Ia, Bmpr-Ibexpression was not detectedat E6.0, but was detected at E7.0 (Roelen et al., 1997).apparent conflict with those data, we detected Bmpr-Ibexpression by in situ hybridization at low levels throughout thembryo at E5.5 and E6.5 (data not shown). Actr-Iexpressionhas also been detected by RT-PCR assays at the blastostage (Roelen et al., 1997) and at E6.0 and E6.5 (Roelen et1994).

We have shown that Bmp2, Bmp4and Bmp7are expressedin temporally and spatially restricted patterns that aconsistent with a role for the products of these genes promoting VE differentiation and cavitation. Indeed, our dademonstrate that Bmp2and Bmp4are expressed earlier in themouse embryo than has been previously reported (Jones e1991; Lyons et al., 1995; Winnier et al., 1995). However, sinembryos homozygous for null alleles of either Bmp2or Bmp4are reported to develop normally up to approximately E7.5 aE6.0, respectively, it appears that neither gene alone is requfor normal peri-implantation development and cavitatio(Winnier et al., 1995; Zhang and Bradley, 1996). In accord wiour model, we speculate that functional redundancy betweBMP2 and BMP4, or perhaps rescue by maternally derivBMP7, accounts for the lack of a cavitation phenotype in tmutant embryos. Furthermore, we would predict that Bmp2/4double homozygous null embryos and embryoid bodies woufail to cavitate in vitro, although that might not be the case vivo, where maternal BMP7 might complement the lack oBMP2 and BMP4 in the double mutant embryos.

Concluding remarksThe results of our previous studies led us to propose tcavitation depended on VE as the source of a signal that caudeath of cells in the ectoderm, and that a concurrent survisignal prevented the death of any cells adhering to tbasement membrane separating the ectoderm from endoderm (Coucouvanis and Martin, 1995). In the presestudy, we have provided further evidence for a connectibetween the presence of VE and the occurrence of cavitatand we have shown that BMP signaling promotedifferentiation of VE. We also identified BMPs as possiblmediators of PCD during cavitation. Further studies will brequired to determine whether their role in cavitation indirect, via an effect on the differentiation of VE, or whethethey function as the death signal per se. In addition, appropriexperiments with mutant embryos or embryoid bodies shouhelp to determine the extent to which BMP2, BMP4 anperhaps BMP7 are able to functionally substitute for onanother during cavitation of embryos in vivo.

We thank Linda Prentice, Allison Gannon, Millicent Embry, anJudy Mak for excellent technical assistance, Carmen Tam technical advice, Tom Musci for providing data on animal cap assaand Jeff Wrana for helpful discussion. We thank Brigid Hoga

545BMP signaling and cavitation

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Stephen Duncan, Lee Niswander and Genetics Institute for providprobes. We are also grateful to our laboratory colleagues for critreadings of the manuscript. E. C. was the recipient of an individNational Research Service Award from the NIH. This work wsupported by grant DB-156 from the American Cancer Society toR. M.

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