The Docking Protein FRS2{alpha} Is an Essential Component of Multiple Fibroblast Growth Factor Responses during Early Mouse Development

The docking protein FRS2 ${alpha}$ is a major mediator of fibroblast growthfactor (FGF) signaling. However, the physiological role of FRS2 ${alpha}$ in vivo remains unknown. In this report, we show that Frs2 ${alpha}$ -nullmouse embryos have a defect in anterior-posterior (A-P) axisformation and are developmentally retarded, resulting in embryoniclethality by embryonic day 8. We demonstrate that FRS2 ${alpha}$ is essentialfor the maintenance of self-renewing trophoblast stem (TS) cellsin response to FGF4 in the extraembryonic ectoderm (ExE) thatgives rise to tissues of the placenta. By analyzing chimericembryos, we found that FRS2 ${alpha}$ also plays a role in cell movementthrough the primitive streak during gastrulation. In addition,experiments are presented demonstrating that Bmp4 expressionin TS cells is controlled by mitogen-activated protein kinase-dependentFGF4 stimulation. Moreover, both the expression of Bmp4 in ExEand activation of Smad1/5 in epiblasts are reduced in Frs2 ${alpha}$ -nullembryos. These experiments underscore the critical role of FRS2 ${alpha}$ in mediating multiple processes during embryonic developmentand reveal a potential new link between FGF and Bmp4 signalingpathways in early embryogenesis.

INTRODUCTION

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Introduction

A great variety of cellular processes are controlled by thefibroblast growth factor (FGF) family of polypeptide growthfactors. FGFs mediate their pleiotropic responses by bindingto and activating a family of four receptor tyrosine kinases,designated FGF-receptor 1 to 4 (FGFR1 to -4) (14, 33). Geneticstudies have shown that FGFs and FGFRs mediate a variety ofcellular responses during embryonic development and in the adultorganism (14, 33). In the adult organism, FGF signaling playsa critical role in the control of the nervous system, bone homeostasis,tissue repair, wound healing, and tumor angiogenesis. In addition,FGF signaling plays a critical role in the early stages of embryogenesis(3, 7, 10, 13, 40, 51, 52). While Fgf4-null embryos become arrestedin their development around the time of implantation, Fgf8-and Fgfr1-null embryos become arrested later in developmentdue to gastrulation defects caused by impairment in cell movementfrom the primitive streak (6, 7, 10, 40, 52).

Many of the cellular responses to FGFs are mediated by the membrane-linkeddocking proteins FRS2 ${alpha}$ and FRS2ß (7, 10, 32, 38, 40,52). Both proteins contain myristyl anchors and phosphotyrosinebinding (PTB) domains in their N termini and multiple tyrosinephosphorylation sites in their C termini that serve as bindingsites for the adaptor protein Grb2 and the protein tyrosinephosphatase Shp2 (17, 21). Expression of Frs2 ${alpha}$ begins in theearly stages of development, whereas expression of Frs2ßbegins at mid-gestation, predominantly in tissues of neuronalorigin (15). The different expression patterns of Frs2 ${alpha}$ and Frs2ßsuggest specific, nonoverlapping roles for each docking protein.Using fibroblasts derived from Frs2 ${alpha}$ -null embryos that do notexpress FRS2ß, we have demonstrated that FRS2 ${alpha}$ playsan important role in FGF1 stimulation of both the Ras/mitogen-activatedprotein kinase (MAPK) cascade and the phosphatidylinositol 3-kinase(PI3K)/Akt cell survival pathway (16). However, the physiologicalrole of FRS2 ${alpha}$ in vivo remains unknown.

After implantation, a mouse embryo develops as a cup-shapedstructure, an egg cylinder, with two major layers, the insideectoderm and the outside visceral endoderm (VE) (4, 46) (seeFig. 7K). The ectoderm is composed of extraembryonic ectoderm(ExE) at the proximal side and epiblast at the distal side.It was proposed that FGF4 secreted from the epiblast mediatesthe proliferation of multipotent trophoblast stem (TS) cellsin the ExE (34, 43).

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FIG. 7. Expression of marker genes and activation of Smads in epiblast of Frs2 ${alpha}$ -null embryos at E6.0 to 6.5. Embryonic stage, genotype, and marker gene are indicated for each image. All embryos are oriented with anterior side to the left. Whole-mount antibody staining: bright-field images (A, C, and E) followed by corresponding fluorescent images (B, D, and F). Control samples without primary antibodies are shown in panels E and F. Whole-mount in situ hybridization (G to J). Scale bars, 100 µm. Multiple functions of FRS2 ${alpha}$ during early embryogenesis. (L) FRS2 ${alpha}$ is essential for FGF4 stimulation of maintenance of self-renewing TS cells in the ExE. At the egg cylinder stage, FGF4 produced in epiblast stimulates FGFRs expressed in ExE resulting in recruitment and tyrosine phosphorylation of FRS2 ${alpha}$ . Signaling pathways that are dependent on FRS2 ${alpha}$ are required for proliferation of self-renewing TS cells in ExE to develop trophoblast lineage. (K and M) A hypothetical connection between FGF and Bmp4 signaling mediated by FRS2 ${alpha}$ . FGF4 stimulation of FGFRs that are expressed in ExE leads to recruitment and tyrosine phosphorylation of FRS2 ${alpha}$ . As FRS2 ${alpha}$ is required for expression of Bmp4 in ExE, it is postulated that Bmp4 functions as a paracrine factor in ExE that contributes towards the induction and maintenance of Nodal expression in the proximal epiblast and towards the development of the epiblast. (N) FRS2 ${alpha}$ plays a role in cell movements through the primitive streak during gastrulation. FGF8 stimulation of FGFR1 and other FGFRs that may be expressed in the primitive streak (PS) and nascent mesoderm leads to recruitment and tyrosine phosphorylation of FRS2 ${alpha}$ . Signaling pathways that are dependent on FRS2 ${alpha}$ are required for cell movement through the primitive streak during gastrulation.

Emerging evidence indicates that the anterior-posterior (A-P)axis, the first overt manifestation of the body plan, is establishedwhen the mouse embryo is at the prestreak stage (4). Severalgenes, such as Hex, are expressed in distal tip VE, and othergenes, such as Brachyury, are expressed in the proximal epiblast,defining the proximal-distal (P-D) axis. Between embryonic day5.5 (E5.5) and E6.0, the distal VE tip cells move anteriorwardto give rise to the anterior visceral endoderm (AVE), and byE6.5 the proximal epiblast cells move and/or alter gene expressionpatterns in a posteriorward manner to form the primitive streak.These directional cell movements transform the P-D axis intothe A-P axis and can be traced by the shifts in the expressionpatterns of these genes (41). Signaling pathways stimulatedby Nodal, a member of the TGFß family of cytokines,appear to play a central role in the formation of the P-D axisand the conversion to the A-P axis (30, 48, 54). When the primitivestreak appears on the posterior side of the epiblast, gastrulationbegins to generate the embryonic and extraembryonic mesoderms.

In this report, we demonstrate that FRS2 ${alpha}$ deficiency resultsin impairment in A-P axis and primitive streak formation. Furthermore,FGF4 stimulation of TS cell self-renewal is impaired in Frs2 ${alpha}$ ^–^/^–embryos, causing impaired development of the trophobast lineage.Using chimeric embryos, we uncover a role of FRS2 ${alpha}$ in the controlof cell movement through the primitive streak during gastrulation.Finally, we describe a potential new link between FGF and Bmp4signaling during A-P axis formation in the developing mouseembryo.

MATERIALS AND METHODS

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Materials and Methods

Production of mutant embryos. The Frs2 ${alpha}$ ^–^/^– allele has been generated by replacing22 codons following the ATG codon in the first exon of Frs2 ${alpha}$ gene by a neomycin-resistant gene by homologous recombination(16). Full details about the targeting vector, the strategyfor disrupting the Frs2 ${alpha}$ gene, and the characterization of theFrs2 ${alpha}$ ^–^/^– allele are described in Hadari et al. (16).The Frs2 ${alpha}$ ^–^/^– allele was maintained on a (129sv/EVx Swiss Webster or 129sv/EV) background by intercrossing. Mutantswere identified either by their characteristic morphology orby PCR analysis. PCR amplification was carried out using theprimer pairs 5'-TCGTGCTTTACGGTATCGCCGC-3' and 5'-TTAGGTGTTTGCTGCACTCG-3'for the mutant allele and 5'-TTTTTGGAGACAGGGTTTCACTG-3' and5'-CGCACCTCTGTGCTTTGTAAG-3' for the wild-type allele. For genotypingof early-stage embryos and TS colonies, the cultured ectoplacentalcone, entire embryos or small amount of cells from TS colonieswere lysed and genotyped as described previously (20).

Histology and in situ hybridization. Embryos were embedded in plastic resin (JB-4; Polysciences,Inc.) and decidua were embedded in paraffin. Sections 5 to 8µm thick were used for histological analysis. RNA in situhybridization was performed as described previously (26), and[³³P]UTP was used instead of [³⁵S]UTP to label the probes. Whole-mountRNA in situ hybridization was performed as described previously(15).

Establishment and culture of TS cell lines. TS cell lines were obtained from the blastocysts as describedpreviously (43). TS cell lines were grown in 70% of conditionedmedium (EMFI-CM) prepared from the culture of embryonic mousefibroblasts (EMFI) as described previously (43) and 30% of theTS medium in the presence of FGF4 and heparin.

Immunoprecipitation and immunoblotting. Cells were starved overnight in 1% serum without EMFI-CM andstimulated with FGF1 (100 ng/ml) together with heparin (5 µg/ml)for 10 min at 37°C. Subsequently, the cells were lysed andsubjected to immunoprecipitation followed by immunoblottingas described previously (16). Rabbit polyclonal anti-FRS2 ${alpha}$ antibodieswere previously described (20). The other antibodies used inthis study are anti-Shp2 antibodies (Santa Cruz; SC-424), anti-phosphotyrosine(phospho) antibodies (PY20; BD Transduction Labs; 610000), anti-Grb2antibodies (BD Transduction Labs; 610111), anti-phospho-ERKantibodies (Cell Signaling; 9101), and anti-ERK antibodies (CellSignaling; 9102).

Whole-mount immunohistochemistry. Embryos were fixed in 4% paraformaldehyde at 4°C overnight.The fixed embryos were washed in 0.5% Nonidet P-40 in phosphate-bufferedsaline (PBS), blocked with PBSST (5% goat serum plus 0.1% TritonX-100 in PBS), and incubated with 1:350 anti-phospho ERK antibodies(Cell Signaling), 1:75 anti-phospho-Smad 1/5/8 antibodies (CellSignaling), 1:100 anti-ERK antibodies (Cell Signaling), and1:100 anti-Smad 1 antibodies (UBI) in PBSST. After washing withPBSST, the embryos were incubated with 1:350 biotin-conjugateddonkey anti-rabbit antibodies (Jackson) for staining with anti-phosphoERK antibodies, 1:1,000 fluorescein isothiocyanate (FITC)-conjugatedgoat anti-rabbit antibodies was used (ICN) for staining withanti-phospho-Smad 1/5/8 antibodies or anti-ERK antibodies, and1:1,000 rhodamine-conjugated goat anti-rabbit antibodies (ICN)was used for staining with anti-Smad 1/5/8 antibodies in PBSST;after incubation, the embryos were washed in PBSST. For stainingwith anti-phospho ERK antibodies, the embryos were further incubatedwith 1:1,000 CyX-streptavidin (Jackson) in PBSST and washedwith PBSST.

Northern blotting. A group of samples were prepared from the TS cells culturedin TS medium at the indicated time points after removal of FGF4,heparin, and EMFI-CM. The other group of samples were preparedfrom the TS cells that were cultured in TS medium containing1% fetal bovine serum (FBS) in place of 20% FBS for 8 h, withor without pretreatment with 50 µM PD98059 for 30 min,and stimulated with FGF4 (100 ng/ml) and heparin (1 µg/ml)for the indicated time. Northern blotting was performed as describedpreviously (43).

Generation of chimeric embryos. In the case of 2N <-> 4N chimeras 4- or 8-cell stage embryos(2N) derived from Frs2 ${alpha}$ ^–^/^– intercross matings wereaggregated with 4-cell stage tetraploid (4N) ICR embryos. TetraploidICR embryos were produced by the electrofusion of embryos atthe 2-cell stage as described previously (34). Chimeric embryoswhich developed into blastocysts within 2 days in culture weretransferred to the uterus of pseudopregnant females.

To isolate Frs2 ${alpha}$ ^–^/^– ES cell lines marked with a ubiquitousexpressed Lac Z marker, Frs2 ${alpha}$ ^–^/+ mice (129sv/EV background)were crossed with ROSA26 mice. Two independent Frs2 ${alpha}$ ^–^/^–ES cell lines were established as described previously. In thecase of Frs2 ${alpha}$ ^–^/^– ES <-> 2N chimeras, blastocystsderived from ICR wild-type matings were injected with Frs2 ${alpha}$ ^–^/^–ES cells marked with Lac Z.

Bmp4 promoter assay. A genomic fragment containing mouse Bmp4 gene was as describedpreviously (22). The Bmp4 1B promoter and intron 2 promoterused in these studies were as described previously (45). TheBmp4 1A promoter includes nucleotides 34 to 2478 of GenBankaccession no. L47480, and the fragment in the 5'-flanking regionof the Bmp4 gene includes nucleotides 53 to 1424 of GenBankaccession no. L47480. The sequence of thymidine kinase (TK)minimal promoter was as described previously (25). These fragmentswere ligated into the firefly luciferase vector (PicaGene Basicvector 2).

TS cells were starved without FGF4, heparin, and EMFI-CM for12 h and transiently transfected with Lipofectamine 2000 (Invitrogen)in accordance with the manufacturer's instructions. Each wellreceived 0.475 µg of firefly luciferase vector (PicaGeneBasic vector 2; TOYO Ink Inc.) carrying fragments of the genomeof mouse Bmp4 gene and 0.1 µg of PicaGene SeaPansy Controlvector (pRL-TK) as an internal control. The transfected cellswere stimulated with FGF4 (25 ng/ml) and heparin (1 µg/ml)for 24 h and harvested. The transcriptional activity was analyzedwith Dual Luciferase Plus Kit (Promega) in accordance with themanufacture's instructions.

RESULTS

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Results

To define the phenotype of Frs2 ${alpha}$ -null embryos, we performed ahistological examination of embryos from Frs2 ${alpha}$ ^–^/+ intercrosses.While embryos were present in most decidua at E5.75, a few deciduahad degenerating embryos or empty implantation sites, indicatingthat a population of mutant embryos likely died immediatelyafter implantation (5/42 decidua, 11.9%) (data not shown). Atthe onset of gastrulation (E6.5), some empty decidua with implantationsites were detected (4/38 decidua, 10.5%), but the remainingcontained embryos with normal morphology. Between E6.75 andE7.5, a mesodermal layer develops between the ectoderm and VEin wild-type embryos, resulting in the formation of three cavitiesbounded by the amnion and/or chorion (Fig. 1E, G, and I). AtE7.0, surviving mutant embryos could be unambiguously identifiedby their small size (around 30% smaller than wild-type embryos)(Fig. 1A and B). Frs2 ${alpha}$ -null embryos fail to develop a primitivestreak but appeared to have mesoderm-like cells (Fig. 1F, H, and J).In some mutant embryos, a thickened visceral endodermwas present at the distal region of the egg cylinder (Fig. 1H).By E7.5, Frs2 ${alpha}$ -null embryos are significantly retarded in theirdevelopment (Fig. 1J). Primitive streak remained deficient,failing to extend (Fig. 1F, H, and J). By E8.0, a few mutantembryos were still present, but they were severely underdevelopedand gradually died (Fig. 1D). Among 79 dissected embryos, 10mutants (12.7%) were found at E6.75, 24 mutants survived among154 embryos (15.6%) at E7.5, and only 1 mutant out of 90 examinedembryos was found at E8.5 (1.1%). Thus, Frs2 ${alpha}$ -null embryos havea variable phenotype, with approximately half of the mutantsdying shortly after implantation, and the remaining half survivingthrough E7.5 with growth and primitive streak defects.

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FIG. 1. Morphology and histology of Frs2 ${alpha}$ -null embryos and expression of Frs2 ${alpha}$ at E6.5. Embryonic stage and genotype are indicated for each image. Gross morphology of wild type and mutant embryos (A to D). All embryos are oriented with anterior side to the left. Histological sections of wild-type and mutant embryos (E to J). Frs2 ${alpha}$ mRNA expression in a wild-type embryo at E6.5; bright-field (K) and dark-field (L) images. Hf, head fold; ep, epiblast; exm, extraembryonic mesoderm; ps, primitive streak; ve, visceral endoderm; m, mesodermal cells; ch, chorion; am, amnion; exe, extraembryonic ectoderm. Scale bars, 200 µm.

Since Frs2 ${alpha}$ -null embryos exhibit lethality at early developmentalstages, we analyzed the expression of Frs2 ${alpha}$ at E5.5 and E6.5and found that Frs2 ${alpha}$ is expressed throughout the embryo, withparticularly strong expression in the ExE (Fig. 1L and datanot shown).

Frs2 ${alpha}$ -null embryos fail to properly establish the A-P axis. To understand the early mutant phenotype, we examined a panelof molecular markers, using 4 to 5 embryos per probe. Firstwe analyzed the expression of several marker genes diagnosticfor proper primitive streak assembly and function between E6.5and E7.5. While Brachyury is expressed in cells ingressing intothe primitive streak in wild-type embryos (Fig. 2A and M) (49),the expression of Brachyury is confined to a proximal band inthe embryonic region of mutant littermates (Fig. 2B and N).Normally, Lim1 is strongly detected in nascent mesodermal cellsmoving out of the streak as well as in AVE (Fig. 2C) (39). Incontrast, expression of Lim1, like that of Brachyury, is detectedin the proximal region of the mutant embryos (Fig. 2D). Fgf4and Fgf8 also mark the primitive streak in the wild-type embryos(Fig. 2E and G) (9, 29). However, in the mutant littermatesat E7.5, Fgf8 is expressed proximally at a reduced level, whileFgf4 is expressed throughout the epiblast (Fig. 2F and H). Theseresults indicate that Frs2 ${alpha}$ -null embryos fail to assemble a properlyelongating primitive streak in the posterior region; instead,markers of the streak retain a proximal position, one normallyfound in E5.5 to E6.0 wild-type embryos.

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FIG. 2. Expression of marker genes in Frs2 ${alpha}$ -null embryos at gastrulation stages. Embryonic stage, genotype, and marker gene are indicated for each image. All embryos are oriented with anterior side to the left. Scale bars, 100 µm.

We next examined the expression of anterior-visceral endoderm(AVE) marker genes to assess whether anterior identity is properlyspecified in the mutant embryos. By E6.0, AVE expression ofHex, Cerberus-related 1 (Cer-1), and Hesx1 is confined to theanterior aspect of wild-type embryos (5, 19, 44), analogousto the pattern shown in Fig. 2I and K. However, even at E6.5,the expression of these genes still has a distal location inFrs2 ${alpha}$ ^–^/^– embryos (arrowheads in Fig. 2J and L anddata not shown). At the onset of gastrulation, Otx2 is normallyexpressed throughout the epiblast and overlying VE; by E7.25its expression is inhibited posteriorly (Fig. 2O and data notshown) (1). Otx2 exhibits a similar pattern of expression inwild-type and mutant embryos at the onset of gastrulation. However,by E7.25, as shown in Fig. 2P, Otx2 is still broadly expressedin the mutants, with transcripts detected in the posterior aswell as anterior embryonic regions. Thus, although AVE and posteriormarker genes are expressed in Frs2 ${alpha}$ -null embryos, they retain,respectively, their initial distal and proximal positions andfail to acquire the expected anterior and posterior locationsfound in wild-type embryos.

These experiments show impairment of A-P axis formation in Frs2 ${alpha}$ -nullembryos. Since Frs2 ${alpha}$ -null embryos surviving to E6.5 are similarin size to wild-type embryos (Fig. 2I to N and Fig. 3), Frs2 ${alpha}$ deficiency may first block A-P axis formation in these embryosprior to affecting embryonic growth. Furthermore, since nascentmesodermal cells are produced in mutant embryos (Fig. 1F, H, and J),the block in A-P axis formation does not result froma general developmental arrest at E5.5 to E6.0.

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FIG. 3. Activation of Erk and expression of marker genes in ExE of Frs2 ${alpha}$ -null embryos at E5.5 and E6.0. Embryonic stage, genotype, and marker gene are indicated for each image. Whole-mount antibody staining: bright-field images (A, C, E, G, and I) followed by corresponding fluorescent images (B, D, F, H, and J). Control samples without primary antibodies are shown in panels E and F. Scale bars, 100 µm.

Reduced MAPK activation in the ExE of Frs2 ${alpha}$ -null embryos. To further elucidate the defect in Frs2 ${alpha}$ -null embryos, we analyzedembryos at the pregastrulation stages, before any gross morphologicalor histological abnormalities are apparent. We analyzed MAPKactivation in wild-type and mutant embryos by immunohistochemistry,utilizing antibodies that specifically recognize the activatedform of MAPK (anti-phospho Erk). We checked at least 4 littersof embryos including 5 mutants. Consistent with a previous report(8), strong activation of MAPK is found in a rim of distal ExEcells adjacent to the epiblast in wild-type and heterozygousembryos (Fig. 3B and D). In contrast, only weak activation ofMAPK is observed in mutant embryos (Fig. 3H and J). The reductionin MAPK activation is not caused by impaired development ofthe ExE and epiblast in egg cylinder stage mutants, as a similaramount and distribution of nonphosphorylated MAPK was foundin wild-type and mutant embryos, as revealed by immunostainingwith anti-MAPK antibodies (data not shown). These results demonstratethat FRS2 ${alpha}$ plays a critical role in MAPK activation in the ExE.

TS cell viability requires FRS2 ${alpha}$ -mediated FGF4 signaling. To further explore the potential impairment of ExE caused byFRS2 ${alpha}$ deficiency, we tried to establish TS cell lines from theFrs2 ${alpha}$ -null blastocysts. TS cell lines can be isolated from blastocystsby culturing them in the presence of FGF4 (43). Since all presumptivemutants had implantation sites, they should survive beyond theblastocyst stage. We isolated 19 blastocysts from Frs2 ${alpha}$ ^–^/+intercrosses and cultured them individually in the presenceof FGF4. All blastocysts became attached to the dish and werecultured for 4 days. After one passage, colonies of TS cellswith a tight epithelial morphology appeared (Fig. 4A, upperpanel). In 14 of the 19 dishes, TS cells developed well andcontinued to proliferate after more than 5 passages. However,TS colonies failed to develop in the remaining 5 dishes (Table1). PCR analysis revealed that these five dishes were seededwith presumptive Frs2 ${alpha}$ ^–^/^– blastocysts (Table 1 andFig. 4A, lower panel). These experiments demonstrate that FRS2 ${alpha}$ is required for maintenance of TS cells in response to FGF4.

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FIG. 4. Activation of signaling pathways through FRS2 ${alpha}$ in TS cells in response to FGF4 stimulation. (A) Morphology of a wild-type TS colony (upper panel) and PCR genotyping (lower panel). Each line indicates individual TS colonies. The genotypes indicated in lanes 1 and 3 were heterozygotes, and that indicated in lane 2 was wild type. (B) TS cells were starved in 0.5% serum overnight, stimulated with FGF4 (100 ng/ml) and heparin (5 µg/ml) for 10 min, lysed, and subjected to immunoprecipitation (IP) as indicated. The samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted (IB) with several antibodies as indicated.

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TABLE 1. Isolation of TS cell lines derived from blastocysts obtained by heterozygous intercrossing^a

To verify that FRS2 ${alpha}$ mediates FGF4 signaling in TS cells, weanalyzed tyrosine phosphorylation of FRS2 ${alpha}$ and FRS2 ${alpha}$ complex formationwith Grb2 and Shp2 in response to FGF4 stimulation. Lysatesfrom unstimulated or FGF4-stimulated wild-type TS cells weresubjected to immunoprecipitation with anti-FRS2 ${alpha}$ or anti-Shp2antibodies, followed by immunoblotting with anti-phosphotyrosine,anti-Shp2, or anti-Grb2 antibodies (Fig. 4B). The experimentpresented in Fig. 4B shows that FGF4 stimulation of TS cellsinduced tyrosine phosphorylation of FRS2 ${alpha}$ as well as complexformation between FRS2 ${alpha}$ and Grb2 and Shp2. Immunoblotting withanti-phospho-Erk antibodies shows that MAPK was activated uponFGF4 stimulation of these cells (Fig. 4B). Taken together, theseresults argue that a FRS2 ${alpha}$ deficiency in egg cylinder stage mutantembryos impairs the development and differentiation of the ExEby preventing FGF4 signaling in TS cells.

Analysis of chimeric embryos reveals a primary defect in extraembryonic tissues of Frs2 ${alpha}$ -null embryos and a role for Frs2 ${alpha}$ in cell movement through the primitive streak. If the primary defect in the Frs2 ${alpha}$ -null embryos resides in ExE,then the block in A-P axis formation should be rescued in chimerasconstructed by aggregation of Frs2 ${alpha}$ ^–^/^– diploid embryoswith wild-type tetraploid embryos (Frs2 ${alpha}$ ^–^/^– 2N <->Frs2 ${alpha}$ ^+/+ 4N). The tetraploid cells will exclusively colonizethe extraembryonic tissues, namely the ExE and visceral andparietal endoderms, while diploid cells will colonize both theepiblast and extraembryonic tissues (28). Four- to eight-cell-stagediploid embryos from Frs2 ${alpha}$ ^+/^– intercrosses were aggregatedwith wild-type tetraploid embryos at the four-cell stage, culturedto the blastocyst stage, and then transplanted to foster mothersfor development to E8.5. We recovered 8 heterozygous, 6 wild-type,and 2 homozygous embryos. All chimeras derived from diploidFrs2 ${alpha}$ ^+/+ or Frs2 ${alpha}$ ^+/^– embryonic cells had well-developedhead folds, 4 to 7 somites, and an allantois (Fig. 5A). Chimerasderived from Frs2 ${alpha}$ ^–^/^– diploid embryonic cells developedhead folds, but these had an abnormal shape (Fig. 5B). Furthermore,these chimeras did not develop somites or an allantois. Althoughthe Frs2 ${alpha}$ ^–^/^– 2N<-> wild-type 4N chimeras didnot complete gastrulation, they did morphologically establishan A-P axis. Thus, the block to A-P axis formation in the Frs2 ${alpha}$ -nullembryos appears to reside in the extraembryonic tissues includingthe ExE, the major site of Frs2 ${alpha}$ expression, but not in the epiblast.

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FIG. 5. Rescue of A-P patterning defects in Frs2 ${alpha}$ ^–^/^– <-> wild-type 4N chimeric embryos and the presence of gastrulation defects in Frs2 ${alpha}$ ^–^/^– ES cell <-> wild-type 2N chimeric embryos. Chimeric embryos recovered at E8.5 derived from Frs2 ${alpha}$ ^–^/+ (A) or Frs2 ${alpha}$ ^–^/^– (B) diploid and wild-type tetraploid morula aggregations. Chimeric embryos constructed by injection of Frs2 ${alpha}$ ^–^/^– ES cells into 2N wild-type blastocysts were stained with LacZ at E7.5 (C to G). Transverse sections (F) and sagittal sections (D and E) of chimeric embryos. The sections in D and F were obtained from the embryos shown in C and G, respectively. Hf, head fold; al, allantois; ps, primitive streak; am, amnion; ep, epiblast; em, embryonic mesoderm.

The abnormal head folds and the absence of somites and allantoisin the Frs2 ${alpha}$ ^–^/^– 2N <-> wild-type 4N chimerasindicate that FRS2 ${alpha}$ likely plays a role in gastrulation as well.To examine the role of Frs2 ${alpha}$ in gastrulation, chimeras were constructedby injecting Frs2 ${alpha}$ ^–^/^– ES cells, constitutively expressingLacZ, into wild-type diploid blastocysts. In such chimeras,ES cells exclusively colonize the epiblast and its derivatives,whereas cells from the host blastocysts will colonize both theepiblast and extraembryonic tissues (42). From embryos recoveredat E7.5, we obtained chimeras exhibiting a wide range of Frs2 ${alpha}$ ^–^/^–ES cell contribution. In several chimeras with a low contributionof donor ES cells, LacZ (+) Frs2 ${alpha}$ ^–^/^– cells were oftenfound in the primitive streak (Fig. 5C). Although many mutantcells were present in the epiblast and the ectodermal layerof the amnion, few mutant cells were found in embryonic mesoderm(Fig. 5D to F). Transverse sections of the chimeras with a highcontribution of mutant cells revealed the presence of a well-definedprimitive streak at E7.5 (Fig. 5F and G). Thus, as observedin the tetraploid chimeras, wild-type extraembryonic tissues,i.e., the ExE, can rescue A-P axis formation in a largely Frs2 ${alpha}$ ^–^/^–epiblast. The transverse sections also show an accumulationof a large number of mutant cells within the streak, suggestingthat Frs2 ${alpha}$ ^–^/^– cells are impaired in their abilityto move through the streak (Fig. 5F and G). Consistent witha requirement for Frs2 ${alpha}$ in cells traversing the primitive streak,Frs2 ${alpha}$ expression was detected in the primitive streak of E7.5embryos by in situ hybridization (data not shown).

Decreased expression of Bmp4 and Nodal and reduced Smad activation in Frs2 ${alpha}$ ^–^/^– embryos. We next examined the expression of Bmp4 and Eomesodermin (Eomes)transcripts, both of which are normally expressed in ExE atthe egg cylinder stage, E6.0 (23, 35) (Fig. 3K and M). The experimentpresented in Fig. 3K to O shows that the expression of Bmp4and Eomes was decreased in mutant embryos. Therefore, we nextasked whether FGF4 regulates Bmp4 expression in cultured TScells. We found that following FGF4 deprivation, Bmp4 expressionwas markedly downregulated, reaching basal levels within 12h. Under the same conditions of FGF4 deprivation, Fgfr2 expressionwas gradually decreased but detected at the significant levelsafter 36 h (Fig. 6A). On the other hand, Bmp4 expression wasrapidly and strongly induced in response to FGF4 stimulation(Fig. 6B). In addition, Bmp4 expression was reduced when, priorto FGF4 stimulation, TS cells were pretreated with the specificMEK (MAPK kinase [MAPKK]) inhibitor PD98059 to inhibit activationof MEK-MAP kinase cascade (Fig. 6B). Thus, expression of Bmp4appears tightly regulated by FGF4 in TS cells. As FRS2 ${alpha}$ is acritical mediator of the FGF-induced MAP kinase response inthe ExE (Fig. 3B to J), this finding is consistent with a rolefor FRS2 ${alpha}$ in FGF4 stimulation of Bmp4 expression in TS cells.

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FIG. 6. FGF4 stimulation of Bmp4 expression in TS cells and transcriptional activity of Bmp4 promoters. (A) Total RNA was prepared from the TS cells at the indicated time points after deprivation of FGF4, heparin, and EMFI-CM. (B) TS cells were starved in 2% serum for 8 h with or without pretreatment with 50 µM the MEK inhibitors PD98059 for 30 min, stimulated with FGF4 (100 ng/ml) and heparin (5 µg/ml) for the indicated time points, and lysed for extraction of total RNA. (A and B) The RNA samples were subjected to Northern blotting with probes specific for Bmp4 or Fgfr2. The 5'-flanking region of the mouse Bmp4 gene confers FGF4-induced activation of transcription. (C) Schematic representation of the mouse Bmp4 gene and constructs used for measuring transcriptional activity. Hatched boxes indicate a luciferase reporter gene. An open circle indicates the TK minimal promoter. (D) The results of transcriptional activity measurements obtained from at least three separate transfections. TK indicates a construct carrying only the TK minimal promoter fused to the luciferase reporter gene.

To further explore the mechanism of FGF4-induced expressionof Bmp4 in TS cells, we examined Bmp4 promoter activity. Themouse Bmp4 gene possesses three known promoters that are utilizedpredominantly in a cell type-specific manner (designated 1A,1B, and intron 2 in Fig. 6C) (12, 22, 45). To measure FGF4-inducedactivity of these promoters in TS cells, we ligated either a2.5-kb fragment of the 5'-flanking region containing the 1Apromoter (construct 1A), intron 1 containing the 1B promoter(construct 1B), or intron 2 containing the intron 2 promoter(construct 2) to a promoterless luciferase reporter gene. Wefound increased transcriptional activity with construct 2, butall the constructs, including construct 2, showed no significantdifference in activity in response to FGF4 stimulation. Thisfinding suggests that the intron 2 promoter, but not the 1Aor 1B promoter, is utilized in TS cells. To determine whetheran FGF4-responsive regulatory region is located in an upstream5'-flanking sequence, we ligated a 1.4-kb fragment of the upstream5'-flanking region (construct 5') to the luciferase reportergene containing the thymidine kinase (TK) minimal promoter (Fig.6C). The results presented in Fig. 6D show that construct 5'gave an approximately 3.5-fold increase in transcription inresponse to FGF4 stimulation. This finding suggests that anFGF4-responsive element resides in the sequence of the upstream5'-flanking region (Fig. 6C). We found that FGF4 stimulationof the Bmp4 promoter activity is blocked by the MEK inhibitorPD98059 (N. Gotoh, unpublished data), suggesting that ERK activityis required for stimulation of Bmp4 transactivation. In addition,several consensus motifs for transactivating growth factorsthat are known to be activated by MAP kinase, such as AP-1,were found in the FGF4-responsive element of the Bmp4 gene.

Bmp4 belongs to a family of tumor growth factor ß-likeligands that activate Smad 1, 5, and 8, two of which, Smad1and Smad5, are expressed at the pregastrulation stage of mouseembryos (27). To explore the role of FRS2 ${alpha}$ in Bmp4 signaling,we examined the activation of Smads 1 and 5 in embryos by immunohistochemistryusing anti-phospho-Smad 1, 5, and 8 antibodies. We analyzedembryos at the prestreak and early-streak stages, before grossmorphological or histological abnormalities are apparent inFrs2 ${alpha}$ -null embryos. In wild-type embryos, Smads 1 and 5 werestrongly activated in a proximal to distal gradient within theepiblast (18) (Fig. 7B). In contrast, activation of Smads 1and 5 was weak in FRS2 ${alpha}$ -deficient embryos (Fig. 7D), while asimilar amount and distribution of Smad1/5 was found in wild-typeand mutant embryos (data not shown). These results are consistentwith Bmp4, expressed on the distal rim of ExE, activating Bmpreceptors in the epiblast and triggering the phosphorylationof Smads 1 and 5. In FRS2 ${alpha}$ -deficient embryos, the decreased levelsof Bmp4 expression would then lead to reduced Smad 1 and 5 phosphorylationin mutant embryos.

We also compared the expression of Nodal and its cofactor Criptoin mutant and wild-type embryos. Nodal and Cripto transcriptsare localized to the posterior proximal epiblast of E6.5 wild-typeembryos (Fig. 7G and I) (11, 47, 48). Decreased levels of Nodaland Cripto expression were observed in Frs2 ${alpha}$ mutants; furthermoreboth transcripts were not localized to the posterior proximalepiblast but are rather symmetrically distributed (Fig. 7H and J).These experiments raise the possibility that the primarydefect in FRS2 ${alpha}$ -deficient ExE may contribute secondarily to aberrantexpression of Nodal and Cripto in the epiblast.

DISCUSSION

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Discussion

In this report we demonstrate that FRS2 ${alpha}$ plays a critical rolein several steps during mouse embryogenesis.

FRS2 ${alpha}$ is essential for maintenance of TS cells in the ExE. Experiments are presented demonstrating that FRS2 ${alpha}$ is essentialfor FGF4-dependent maintenance of self-renewing TS cells. Inwild-type embryos, pronounced MAPK activation was detected inthe ExE region, where TS cells reside. However, marked reductionof MAPK activation was observed in FRS2 ${alpha}$ deficient mutant embryos(Fig. 3A to J). While FGF4 stimulation leads to the formationof wild-type TS colonies, we have shown that Frs2 ${alpha}$ ^–/–TS colonies that appear after the first passage die within fivepassages. This experiment shows that the integrity of FRS2 ${alpha}$ isrequired for FGF4 stimulation of TS colonies and that signalingpathways that are dependent upon FRS2 ${alpha}$ are required for the survivaland/or self-renewal of TS cells. The loss of function of TScells in the ExE may be the main cause of death of mutant embryos.

Blastocyst stage embryos are composed of three different celltypes: inner cell mass (ICM), primitive endoderm, and trophectoderm(46). After implantation, the ICM forms the epiblast and theprimitive endoderm differentiates into the visceral and parietalendoderm layers of the yolk sac. Meanwhile, the polar trophectodermoverlying the ICM develops into ExE, ectoplacental cone, andsecondary giant cells, all precursors to the trophoblast tissues,the fetal portion of the placenta (34). It has been proposedthat the preimplantation ICM and the postimplantation epiblastproduce FGF4, which maintains self-renewal of TS cells in theoverlying polar trophoectoderm and ExE, respectively (34, 43).As TS cells move away from the source of FGF4 in the ICM/epiblast,they progressively differentiate into ectoplacental cone andtrophoblast giant cells. Similarly in culture, TS cells canself-renew in the presence of FGF4, but they differentiate intopolyploid giant cells upon FGF4 deprivation. The developmentalarrest of Fgf4-null embryos at the peri-implantation stage likelyresults from a failure to generate differentiated trophoblasttissues due to the loss of TS cells (13). In fact, some of theFrs2 ${alpha}$ -null embryos died around implantation. Other mutants werenot phenotypically discernible prior to the onset of gastrulation,but the majority of these generally had degenerated by E8.5.The more advanced Frs2 ${alpha}$ mutants were observed in crosses betweenmice of a mixed genetic background (129sv/EV and Swiss Webster);thus, the variability in viability may reflect the presenceof modifier loci. Such loci may promote the transient productionand/or survival of TS cells, potentially through FRS2 ${alpha}$ -independentintracellular signaling pathways.

Because Fgf4 and several Fgfr isoforms are expressed in Frs2 ${alpha}$ -nullembryos, FRS2 ${alpha}$ -independent signaling pathways could still operate.FRS2 ${alpha}$ promotes MAPK activation through complex formation withShp2 and Grb2, enabling tyrosine phosphorylation of Shp2 byactivated FGFRs (Fig. 4B). Although FRS2 ${alpha}$ plays a role in MAPKstimulation, a diminished and transient FGF1-induced MAPK responsecan be detected in Frs2 ${alpha}$ ^–^/^– mouse embryonic fibroblasts(MEFs) (16). Furthermore, it was shown that Shp2 can mediate,at least partially, MAPK activation even in the absence of bothof its tyrosine phosphorylation sites (2). Moreover, FGF1 stimulationof tyrosine phosphorylation of Shc, resulting in Ras/MAPK stimulation,may provide an alternative route for FGF1-induced MAPK stimulationin Frs2 ${alpha}$ ^–^/^– MEFs. FGF1-stimulation of phospholipaseC- ${gamma}$ (PLC ${gamma}$ ) activation, followed by Ca⁺² release and protein kinase-C(PKC) stimulation, are preserved in Frs2 ${alpha}$ ^–^/^– MEFs.Both PLC ${gamma}$ and Shc are expressed in TS cells (data not shown),and therefore these signaling proteins may support partial FGF-signalingin Frs2 ${alpha}$ -null embryos.

FRS2 ${alpha}$ plays a role in cell movement through the primitive streak during gastrulation. To examine the role of Frs2 ${alpha}$ during gastrulation, we constructedchimeras from Frs2 ${alpha}$ ^–^/^– ES cells and diploid wild-typeblastocysts. Similar to cellular defects present in Fgfr1- orFgf8-null embryos (7, 10, 40, 52), the FRS2 ${alpha}$ -deficient cellsare impaired in their ability to exit the primitive streak.This experiment suggests that, in addition to the requirementof FRS2 ${alpha}$ in FGF4-signaling in ExE, the docking protein also functionsas an intracellular mediator of signaling pathways, stimulatedby FGF8 and FGFR1 during gastrulation (Fig. 7N). It should benoted that Shp2, one of the effectors of FRS2 ${alpha}$ , has been shownto play a role in cell migration through primitive streak (37).

A potential FRS2 ${alpha}$ -mediated link between FGF, Bmp4, and Nodal signaling pathways. We have shown that the expression of Bmp4 is decreased in Frs2 ${alpha}$ ^–^/^–embryos and that FGF4 stimulation leads to enhanced expressionof Bmp4 in TS cells (Fig. 3N and O and 6B). Moreover, the expressionof Bmp4 is rapidly down-regulated after removal of FGF4 fromTS cells (Fig. 6A). Although correlative and indirect, thesefindings suggest that FGF4 signaling contributes to Bmp4 generegulation in ExE. Indeed, it appears that a FGF4 responsiveelement is located in the 5'-flanking region of the mouse Bmp4gene (Fig. 6C and D).

The Frs2 ${alpha}$ -null embryos show defects in transformating the P-Daxis of the egg cylinder into the A-P axis of the gastrula (Fig.1F, H, and J and 2). As the P-D to A-P rotation always occursbefore the appearance of nascent mesoderm in wild-type embryos,the detection of mesoderm-like cells in Frs2 ${alpha}$ mutants, suggeststhat they develop beyond the time the A-P axis formation. Thus,FRS2 ${alpha}$ dependent FGF-signaling may also contribute towards theestablishment of the A-P axis.

The Nodal signaling pathway plays a central role in a numberof developmental events in the early postimplantation embryo:the establishment of the P-D axis, rotation of the AVE and organizationof the A-P axis, mesoderm and endoderm formation, and left-right(L-R) patterning. It has been shown that Nodal expression isinitially induced in the proximal epiblast by an unknown stimulus,thought to be produced at approximately E5.0 by the ExE (31,48). A Nodal signaling cascade is then amplified by a positivefeedback loop that involves its own enhancer sequence, termedASE, and an enhancer binding factor FoxH1 (6, 31, 36). Nodalexpression is propagated throughout the epiblast, and then Nodalacts on the overlying distal VE to induce the expression ofgenes required for AVE formation, rotation, and function. Oncethe AVE is specified on the anterior side of the egg cylinder,expression of Nodal becomes confined to the proximal posteriorside of epiblast to promote mesoderm and endoderm productionby the primitive streak. Nodal acts in a dosage-dependent mannerto execute these different steps in embryonic patterning (24,30). In the absence of Nodal, patterning along the P-D axisdoes not occur and no AVE precursor forms at the distal tipof the egg cylinder (6). Low doses of Nodal signaling, achievedthrough hypomorphic alleles, induce the AVE distally but donot support its anterior migration. At slightly higher, butstill subnormal levels of Nodal, the embryos properly completegastrulation but fail to correctly establish the L-R axis.

It is noteworthy that the absence of AVE rotation in Frs2 ${alpha}$ -nullembryos strikingly resembles the phenotype observed in embryosexpressing hypomorphic alleles of Nodal (23). Other mutationsthought to reduce the level of Nodal signaling also disruptgastrulation by obstructing rotation of the AVE. These includemutants lacking Cripto, a positive cofactor for Nodal and mutantsdeficient in FoxH1 (11, 30, 53).

The decreased expression of Nodal in the Frs2 ${alpha}$ -null mutants atE6.5 raises the possibility that the absence of AVE rotationmay result from insufficient levels of Nodal signaling. In thisstudy, we showed the phenotype of Frs2 ${alpha}$ -null embryos in a mixedgenetic (129sv/Ev x Swiss Webster) background. We also analyzedFrs2 ${alpha}$ -null mutants in a 129sv/EV inbred background and foundthat the mutant embryos showed reduced levels of expressionof Brachyury and Cer-1 and very little levels of expressionof Nodal at E6.0 to 6.5, suggestive of a defect in the P-D axisformation (data not shown). This phenotype is more severe thanthose described in this study, in association with more reducedlevels of expression of Nodal.

Bmp4-null embryos have a variety of phenotypes, underscoringthe multiple roles of Bmp4 during embryogenesis. Bmp4^–^/^–embryos on the C57BL/6 background are developmentally retardedat E7.5; mutant embryos lack an organized primitive streak andproduce only a small amount of extraembryonic mesoderm (50).This phenotype is similar to that of Frs2 ${alpha}$ -null embryos, hintingof a possible functional link between FRS2 ${alpha}$ and Bmp4 signalingin pre- and early-streak-stage embryos. We analyzed expressionof Cer-1, Hex, and Nodal in the Bmp4^–^/^– embryosand found that these were expressed at very low levels, similarto those of the Nodal-null embryos or Frs2 ${alpha}$ -null embryos in a129sv/EV background (data not shown) (6).

On the basis of these findings, it appears that FRS2 ${alpha}$ plays arole in FGF4 stimulation of Bmp4 expression in ExE. Acting asa paracrine factor, Bmp4 may, accordingly, contribute to theinduction and maintenance of Nodal expression in the proximalepiblast and development of epiblast (Fig. 7K and M). Bmp4 mightbe an initial stimulus to induce expression of Nodal in theproximal epiblast.

In summary, the experiments presented in this report demonstratethat loss of FRS2 ${alpha}$ impairs at least two critical processes inthe development of extraembryonic and embryonic tissues in theearly postimplantation stage mouse embryo. We demonstrate thatFRS2 ${alpha}$ is essential for FGF4-dependent maintenance of self-renewingTS cells in the ExE (Fig. 7L) and that FRS2 ${alpha}$ may play a criticalrole in cell movement through the primitive streak during gastrulation(Fig. 7N). Our experiments also suggest a potential new linkbetween FGF signaling and Bmp4 and Nodal signaling pathwaysto promote A-P axis formation (Fig. 7K and M).

ACKNOWLEDGMENTS

We thank X. Sun and G. R. Martin for helping in the sectioningof embryos and for valuable advice. We are thankful to L. Corsonfor the protocol of whole-mount immunohistochemistry with anti-phosphoERK antibodies. We thank A. Auerbach and staff members in TransgenicAnimal Facility of NYU for making chimeric embryos. We are gratefulto J. Rossant for critical readings of the manuscript and providingthe probes, to V. P. Eswarakumar for critical readings of themanuscript, and to G. R. Martin, A. L. Joyner, and M. M. Shenfor providing the probes.

N.G. was a recipient of Long Term Fellowship of Human FrontierScience Organization and Research Fellowship of Uehara MemorialFoundation during the early stages of the study. During thelate stages of the study, this work was supported by Grant-in-Aidfor Scientific Research from the Ministry of Education, Science,Sports, and Culture, Japan, to N.G. and M.S. Joseph Schlessingeris supported by NIH grants RO1-AR051448-01 and RO1-AR051886-01and funds from the Ludwig Institute for Cancer Research.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: (203) 785-7395. Fax: (203) 785-3879. E-mail: joseph.schlessinger{at}yale.edu.

Present address: NoviTech Metagenome Research Institute, Ho372-3,Yachimata-shi, 289-1115, Japan.

REFERENCES

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