Oct 19, 1994 - hematopoietic system (Adams et al., 1992; Bustelo et al.,. 1993). vav ...... Pevney,L., Simon,M.C., Robertson,E., Klein,W.H., Tsai,S.F., D'Agati,V.,.
The EMBO Journal vol.14 no.1 pp.1-11, 1995
The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro Antanina Zmuidzinas1, Klaus-Dieter Fischer2, Sergio A.Lira, Lesley Forrester23, Sherri Bryant, Alan Bernstein24 and Mariano Barbacid5 Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA, 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital and 4Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G IX5, Canada 'Present address: EPG Research Foundation, Manhasset, NY 11030, USA 3Present address: AFRC Centre for Genome Research, University of Edinburgh, West Mains Road, Edinburgh, UK 5Corresponding author Communicated by J.Tooze
Previous studies have suggested that the vav protooncogene plays an important role in hematopoiesis. To study this further, we have ablated the vav protooncogene by homologous recombination in embryonic stem (ES) cells. Homozygous vav (-/-) ES clones differentiate normally in culture and generate cells of erythroid, myeloid and mast cell lineages. Mice heterozygous for the targeted vav allele do not display any obvious abnormalities. However, homozygous embryos die very early during development. Crosses of vav (+/-) heterozygous mice yield apparently normal vav (-/-) E3.5 embryos but not post-implantation embryos (¢'E7.5). Furthermore, homozygous vav (-/-) blastocysts do not hatch in vitro. These results indicate that vav is essential for an early developmental step(s) that precedes the onset of hematopoiesis. Consistent with the phenotypic analysis of vav (-/-) embryos, we have identified Vav immunoreactivity in the extra-embryonic trophoblastic cell layer but not in the inner embryonic cell mass of E3.5 preimplantation embryos or in the egg cylinder of E6.5 and E7.5 postimplantation embryos. These results suggest that the vav gene is essential for normal trophoblast development and for implantation of the developing embryo. Key words: embryoid bodies/gene targeting/hematopoiesis/
implantationNVav
Introduction The vav proto-oncogene encodes a cytoplasmic 95 kDa protein (Vav) which is primarily expressed in cells of hematopoietic origin (Katzav et al., 1989; Coppola et al., 1991). In situ hybridization analysis of fetal, newborn and adult mouse tissues has indicated that expression of vav parallels the onset and subsequent development of the hematopoietic system (Adams et al., 1992; Bustelo et al., 1993). vav transcripts are first detected in the developing K Oxford University Press
fetal liver at embryonic day 11.5 (El1.5), the main hematopoietic organ in development at this time. During diversification of hematopoietic activity in the embryo, vav expression is down-regulated in the liver and activated in the thymus and spleen. After birth, vav gene expression continues to be highly restricted to cells of the thymus, spleen, bone marrow and lymph nodes (Bustelo et al., 1993). Interestingly, the vav proto-oncogene also appears to be expressed in pluripotent embryonic stem (ES) cells, albeit at very low levels (Keller et al., 1993; Wulf et al., 1993). vav expression continues at low levels in embryoid bodies until the time of hematopoietic differentiation when there is a significant accumulation of vav transcripts (Wulf et al., 1993). However, vav does not appear to be expressed in the egg cylinder of early post-implantation embryos (E6.5 and E7.5) or in the yolk sac (or whole embryo) of E8.5 embryos, a time when there is hematopoietic activity, at least within the erythroid lineage (blood islands; Keller et al., 1993). These observations indicate that vav has two distinct temporal patterns of expression during development, one of which coincides with and another which precedes the onset of hematopoiesis. Accumulating evidence indicates that Vav plays an important role in at least some hematopoietic signaling pathways. The oncogenic properties of the Vav protein in rodent fibroblasts suggests a role in mitogenic signaling (Katzav et al., 1989, 1991; Coppola et al., 1991). In hematopoietic cells Vav becomes rapidly phosphorylated on tyrosine residues upon activation of a wide variety of receptors, including the T-cell receptor (Bustelo et al., 1992; Margolis et al., 1992), the mast cell IgE high affinity FceRI receptor (Margolis et al., 1992), the B-cell IgM antigen receptor (Bustelo and Barbacid, 1992) and the cKit (Alai et al., 1992) and Flk-2 tyrosine protein kinase receptors (Dosil et al., 1993). Vav also becomes phosphorylated on tyrosine residues upon interleukin (IL)-2, IL-3 and a-interferon treatment of hematopoietic cells (Dosil et al., 1993; Evans et al., 1993; Platanias and Sweet, 1994). Ectopic expression of Vav in mouse fibroblasts also results in tyrosyl phosphorylation by the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors (Bustelo et al., 1992; Margolis et al., 1992) upon activation by their respective ligands. In this case, Vav forms stable complexes with the receptors, an interaction presumably mediated by the binding of its SH2 domain to specific phosphotyrosine residues on the receptor. In hematopoietic cells, Vav has not been found to be associated with any of the above receptors or with known cytoplasmic tyrosine kinases which are presumably responsible for the phosphorylation of its tyrosine residues. Little is known regarding the putative downstream elements that mediate Vav signaling. Engagement of the B-cell IgM antigen receptor induces the association of Vav with Vap- 1, another tyrosine phosphorylated protein 1
A.Zmuidzinas et al.
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Fig. 1. Targeting of the mouse vav locus by homologous recombination in ES cells. (A) Schematic representation of the strategy utilized to target the vav locus. Thick horizontal lines represent vav genomic sequences incorporated into the targeting vector pAZIO. Those sequences incorporated into the pAZlO targeting vector are indicated by dashed vertical lines. Exons are represented by thick vertical lines ('a-k'). pAZ10 has a 300 bp deletion of vav genomic DNA spanning 180 nucleotides of exons 'c-e' and the intervening introns (see text). Putative regions of recombination between the endogenous vav locus and pAZ1O DNA are indicated by crossed dashed lines. The PGKI-neo cassette (neo) and the HSV-thymidine kinase cassette (tk) are indicated by open and shaded boxes, respectively. The arrows inside the boxes indicate the direction of transcription. Cleavage sites for restriction endonucleases include Eco47III (E), EcoRI (R), HindIII (H), KpnI (K), PstI (P) and XbaI (X). The XhoI (Xh) cleavage site used to linearize pAZ1O is indicated in brackets. The 1.6 kbp region amplified by PCR to identify G418R-GancR ES cell clones carrying homologous recombination is indicated by a small open box with arrowheads (PCR). The 600 bp long probe (probe A) used to identify the diagnostic 4.9 kb HindIII and 8.8 kb KpnI DNA fragments and the 1.2 kbp long probe (probe B) used to identify the diagnostic 11 kbp HindIII DNA fragment are indicated by black boxes. (B) Southern blot analysis of vav-targeted ES cells. Genomic DNA was extracted from PCR-positive G41 8R-GancR ES cells clones B 142-34, B 142-313 and B 142-321, as well as from untransfected D3 ES cells, digested with HindIII or KpnI and subjected to Southern blot analysis using probe A. The migration of the wild-type (14 kbp HindIII and 7 kbp KpnI DNA fragments) and targeted (4.9 kbp HindIll and 8.8 kbp KpnI DNA fragments) vav alleles is indicated by arrows. (C) Southern blot analysis of mice derived from heterozygous vav (+/-) matings. Genomic DNA was extracted from tail biopsies of four (lanes 1-4) mice and from untransfected D3 ES cells, digested with HindIII and submitted to Southern blot analysis using probes A or B. Lanes 1 and 2, vav (+/-) heterozygous mice; lanes 3 and 4, vav (+/+) wild-type mice. The migration of the wild-type (14 kbp DNA fragment) and targeted (11.0 and 4.9 kbp DNA fragments) vav alleles is indicated by arrows.
of unknown function (Bustelo and Barbacid, 1992). Vav also becomes associated with a novel high molecular weight GTP binding protein (X.R.Bustelo et al., unpublished observations) and with ribonucleoprotein K, a poly(C)-specific RNA binding protein (Bustelo et al., submitted for publication; Hobert et al., 1994). A potential clue to the nature of the signaling element(s) downstream from Vav has been provided by the identification of a region of significant homology to the product of the Dbl proto-oncogene, a molecule that has GDP/GTP exchange
2
activity for CDC42 (a member of the Rho/Rac subfamily of small GTP binding proteins; Adams et al., 1992). A number of investigators have examined whether Vav has similar GDP/GTP exchange activity. Gulbins et al. (1993, 1 994a,b,c) have reported that Vav is a GDP/GTP exchange factor for Ras, although these observations have not been confirmed by others (Bustelo et al., 1994; Khosravi-Far et al., 1994). The restricted pattern of vav gene expression, along with its putative signaling role in hematopoietic cells,
Phenotype of vav (-/-) mice and ES cells
have led us to investigate the physiological role of vav by genetic analysis. In this study we report the generation of ES cells carrying an inactivated vav allele. ES cell clones homozygous for this mutation retain the ability to differentiate in vitro into embryoid bodies that contain hematopoietic cells of erythroid, myeloid and mast cell lineages. Mice heterozygous for this mutation also appear normal. However, homozygous vav mutations result in early lethality at the time of blastocyst implantation. Consistent with this early lethality, Vav protein is expressed in trophoblast cells, the first lineage to differentiate in the mammalian embryo. These results define the vav protooncogene as an essential gene for embryonic development at a step(s) that precedes the onset of hematopoiesis.
Results Targeting the mouse vav locus A mouse genomic library was screened with a vav cDNA probe as indicated in Materials and methods. One of the recombinant phages was found to contain 14.2 kbp of vav genomic DNA which encompassed six exons spanning nucleotides 193-780 of the pMB24 mouse vav cDNA clone described by Coppola et al. (1991) (Figure lA). Since the exon/intron structure of the vav locus has not been fully established, the mapped exons will be referred to as exons 'a-k' (Figure lA). To disrupt the endogenous vav gene in ES cells, a targeting vector, designated pAZ 10, was generated as described in Material and methods. pAZ1O contains 9.65 kbp of vav genomic sequences encompassing exons 'a-f' (nucleotides 193-780 of pMB24), except for a 300 bp EcoRI DNA fragment that was replaced by a phosphoglycerate kinase 1 (PGK-1)neo cassette (McBurney et al., 1991) inserted in the opposite transcriptional orientation (Figure 1 A). The deleted nucleotides include the 3' 22 bp of exon 'c', the entire exon 'd' and at least 30 bp of exon 'e'. These sequences encode amino acid residues 135-195 of the Vav protein, a region that encompasses its entire acidic domain (Coppola et al., 1991). A thymidine kinase cassette used for negative selection of cells carrying nonhomologous recombinational events (Monsour et al., 1988) was inserted 5' of the long arm of pAZIO (Figure lA). pAZ1O DNA was transfected into ES cells (D3 clone; Doetschman et al., 1987) by electroporation; potential recombinants were selected in the presence of G418 and gancyclovir (Gan). A total of 900 G418R/GanR doubleresistant D3 clones were screened by a PCR-aided strategy (Joyner et al., 1989) for homologous recombination events at the vav locus. Six PCR-positive D3 clones were analyzed by genomic Southern blot analysis to confirm that a targeting event had taken place. Four of these colonies exhibited the predicted 4.9 kbp HindIll (14 kbp in wildtype D3 DNA) and 8.8 kbp KpnI (7.0 kbp in wild-type D3 DNA) DNA fragments when probed with a DNA fragment derived from vav genomic sequences not included in pAZ1O (Figure lA). Figure lB depicts the results obtained with two representative clones B142-34 and B 142-313. These observation indicate that our targeting frequency was 1 in 225 G418R/GanR double-resistant D3 clones.
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Fig. 5. Hematopoietic gene expression in vav (-/-) embryoid bodies. Total cellular RNA was isolated from embryoid bodies derived from vav (+/+) D3 and vav (-/-) B143-321-8 ES cells after 0, 6, 9 and 15 days of in vitro differentiation, as well as from bone marrowderived mast cells. These RNAs were submitted to RT-PCR analysis for the expression of vav, c-kit, IL-3R, GATA-1, PH1 and jmaj globin genes as described in Materials and methods. HPRT expression served as a positive control. Molecular weight standards (Std) were those described in the legend to Figure 2.
and adult f-globin genes in the vav (-/-) embryoid bodies suggests that Vav is not essential for either primitive or definitive erythropoiesis in vitro. The absence of any apparent differences in the expression pattern of any of the genes analyzed here adds further support to the concept that vav is not required in culture for the normal development of hematopoietic cells within embryoid bodies.
Genotype
Newborn mice
E7.5-E12.5 embryos
Preimplantation embryos (E3.5)
(+/+) (+/-) (-/-)
101/430 (23%) 329/430 (77%) 0/430 (0%)
10/67 (15%) 57/67 (85%) 0/67 (0%)
9/64 (14%) 48/64 (75%) 7/64 (11%)
Generation of mice heterozygous for a disrupted vav allele To study the role of the vav gene in vivo, we generated strains of mice carrying the targeted vav allele. Heterozygous vav (+/-) ES clones were injected into C57BL/6J blastocysts and transferred into the uteri of pseudopregnant CD-1 female mice. Two of the clones (B142-313 and B142-332) generated chimeric mice (ranging from 30 to 90% color chimerism) as judged by the presence of agouti coat color. One male chimera (derived from clone B 142332) yielded germline transmission of its targeted vav allele. The resulting vav (+I-) heterozygous mice appeared normal and were indistinguishable from their wild-type litter mates. Fluorescence-activated cell sorting analysis of their hematopoietic cell compartment utilizing a panel of antibodies reacting to hematopoietic-specific cell surface markers (i.e. CD4, CD8, CD3, IgM, B220, Mac-1, Thyl.2) showed a wild-type distribution (data not shown). vav homozygosity results in early embryonic lethality Heterozygous vav (+/-) mice were intercrossed and tail biopsies of their progeny assayed by Southern blot analysis (Figure IC). None of a total of 430 offspring mice were homozygous for the mutated vav allele (Table I). These results indicate that the vav gene fulfils an essential function during embryonic development. In an effort to determine the embryonic stage that requires vav gene function, we genotyped embryos obtained from heterozygous matings at mid-gestation stages E7.5-EI2.5. As summarized in Table I, no homozygous vav (-/-) E7.5-E12.5 embryos were found among the 64 embryos analyzed [16 vav (-/-) embryos were expected if disruption of the vav locus had no effect on viability at these embryonic stages]. Heterozygous embryos were phenotypically indistinguishable from those containing both vav alleles. The genotypic analysis of viable (born) mice derived from heterozygous vav (+I-) crosses (n = 430) revealed a slight distortion (77 versus the expected 66%) of the expected Mendelian distribution in favor of the heterozygous phenotype (Table I). The significance of this observation is not known. The absence of post-implantation vav (-/-) embryos suggested that the vav gene may be essential for an earlier developmental stage such as blastocyst formation or implantation. To address this possibility, E3.5 blastocysts were isolated from the uteri of heterozygous vav (+/-) females mated with vav (+I-) males. A total of 64 blastocysts were isolated and found to be phenotypically indistinguishable from one another. These blastocysts were genotyped by a combination of PCR-aided amplification step followed by Southern blot analysis of the amplified
5
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Fig. 6. PCR analysis of blastocysts derived from matings of heterozygous vav (+/-) mice. (A) Schematic diagram illustrating the strategy utilized for PCR-aided amplification of wild-type and targeted vav alleles. PCR bands specific to the wild-type and targeted vav alleles. Symbols were those described in the legend to Figure IA. A diagnostic 300 bp DNA fragment unique to the wild-type allele was generated with primers corresponding to exons 'c' and 'e' missing in the targeted allele. A diagnostic 150 bp DNA fragment unique to the wild type allele was generated with primers corresponding to sequences within the PGK-1 promoter of the PGK-neo (neo) cassette and to the remaining sequences in the partially deleted 'Ae' exon. (B) DNA from preimplantation blastocysts (E3.5) was assayed by PCR for the presence of wild-type (upper panel) and targeted (lower panel) vav alleles as described in Materials and methods. Amplification products were submitted to Southern blot analysis using as probes internal oligonucleotide DNA probes. Lanes 1-8, individual blastocysts; lane 9, genomic DNA from a heterozygous vav (+/-) mouse. The genotypes of the individual blastocysts are indicated at the bottom. The sizes of the PCR-amplified DNA fragments are indicated by arrows.
DNA products (see Materials and methods). Figure 6 outlines the strategy utilized and depicts a representative experiment. As summarized in Table I, genotyping of individual blastocysts demonstrated the presence of all three possible genotypes, including vav (-/-) blastocysts at close to expected Mendelian frequencies. These results indicate that the vav gene is not essential for embryonic development up to the blastocyst stage (E3.5).
Vav is essential for blastocyst hatching in vitro To determine whether vav (-/-) pre-implantation embryos were able to proliferate and develop normally, an additional 60 E3.5 blastocysts derived from heterozygous vav (+I-) crosses were isolated and individually cultured in vitro. Normal embryos hatch from the zona pellucida and attach to extracellular matrix-coated plates within 24-36 h by a process that involves outgrowth of
6
their trophoblast cell layer. After 5 days in culture, we observed that nine blastocysts (15%) failed to hatch. Genotypic analysis of these blastocysts revealed that seven of them were vav (-/-). Similar results were obtained in a second experiment in which all cultured blastocysts were genotyped. Out of 109 cultured blastocysts, only nine failed to hatch. Of these nine blastocysts, eight were vav (-/-) homozygous. In contrast, all of the 100 blastocysts that hatched normally were either vav (+/-) or wild-type. These results indicate that the vav gene is essential for in vitro hatching of mouse blastocysts, a step that mimics some of the events required during implantation of the embryo in vivo (Rossant, 1986).
Previous studies have indicated that vav is expressed in hematopoietic cells of mid-gestation (and older) embryos (Adams et al., 1992; Bustelo et al., 1993) but not in the egg cylinder of E6.5 and E7.5 embryos or in the yolk sac or whole E8.5 embryos (Keller et al., 1993). However, the genetic data described above suggest that vav might also be expressed in pre-implantation embryos. To examine this possibility, we isolated E3.5 blastocysts from superovulated C57BL/6J females and allowed them to hatch from the- zona pellucida. Whole blastocysts cytospun or cryostat-sectioned onto glass slides were subjected to immunocytochemical analysis using Vav-specific antibodies (Bustelo et al., 1993). As illustrated in Figure 7A, Vav immunoreactivity was readily detected in the exterior trophectodermal cell layer of these embryos. Immune staining of blastocyst cross-sections confirmed that the anti-Vav immunoreactivity was localized to the outer trophoblast cell layer, as little if any Vav immunoreactivity could be observed in the inner cell mass (Figure 7C). Control experiments, in which embryos were incubated with either anti-Vav antibodies preincubated with an excess (10-fold) of immunizing antigen (Figure 7B) or with preimmune sera (not shown), failed to show significant immunoreactivity. vav expression in early post-implantation embryos The mouse trophectodermal cell layer differentiates at the late blastocyst stage to form the invasive trophoblast layer that mediates implantation of the embryo into the uterine wall (Copp, 1978). To determine whether Vav is expressed in these cells after the implantation process, we performed immunocytochemical studies in mouse embryos at various stages of post-implantation development (E6.5 and E7.5). At this time, the inner cell mass of the preimplantation embryo is transformed into a three-layer structure consisting of ectoderm, endoderm and a middle layer of mesoderm. As illustrated in Figure 8A, E6.5 embryos display strong Vav staining in the trophoblast-derived cells which invade the maternal tissue. These trophoblastic cells enlarge, forming primary and secondary giant cells that migrate irregularly from the ectoplacental cone, the site of maternal to embryo nutrient flow, which later becomes the placental structure (Snell and Stevens, 1966; Gardner et al., 1973; Copp, 1978). A similar pattern of Vav expression was observed in E7.5 embryos (Figure 8C and D). Strong anti-Vav immunoreactivity was detected in migrating trophoblasts,
Phenotype of
vav
(-/-) mice and ES cells
that vav has a biphasic pattern of expression during embryogenesis which includes the trophoblastic cell layer of the pre- and post-implantation embryo and the fetal hematopoietic precursors in older, mid-gestation embryos (Adams et al., 1992; Bustelo et al., 1993). The early lethality of vav (-/-) embryos suggests that vav is required for the generation and/or proper function of the trophoblastic cells that invade the maternal stroma of the uterine wall to anchor the embryo during the implantation process.
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Fig. 7. Vav expression in preimplantation blastocysts. E3.5 blastocysts were cultured in vitro until hatching from the zona pellucida and either (A and B) cytospun or (C) cryostat-sectioned at 8 gm and then mounted onto polycationic slides. (A) Whole and (C) sectioned late blastocysts immunostained with the bound fraction of affinity-purified anti-Vav antibodies raised against the SH2 domain. (B) Whole late blastocyst immunostained with the same antiserum preincubated with an excess (10-fold) of immunizing antigen. Immunoreactivity was detected using the avidin-biotin peroxidase complex system and counter-stained with hematoxylin. Arrows indicate the inner cell mass (ICM) and trophectoderm-derived cells (T). Magnification X400.
particularly in those cells located at the ectoplacental cone (Figure 8C). The primitive extra-embryonic endoderm cells, which are derived from the inner cell mass and bear a characteristic columnar and cuboidal shape, also display detectable anti-Vav immunoreactivity (Figure 8C and D). A light immunostaining relative to samples treated with control antisera was also detected in the maternal decidua (results not shown). This Vav immunoreactivity is likely to be due to infiltrating hematopoietic cells. Indeed, vav gene expression has been observed previously by PCR analysis of RNA derived from decidua tissue of E6.5 embryos (Keller et al., 1993). These observations reveal
Accumulating evidence indicates that the product of the vav proto-oncogene may play an important role in hematopoiesis. First, the Vav protein contains a series of structural motifs shared by a variety of molecules involved in the signal transduction processes (Puil and Pawson, 1992). Moreover, the Vav protein becomes rapidly phosphorylated on tyrosine residues, a landmark of many signaling events, upon activation of a variety of hematopoietic cell surface receptors. Direct proof of the involvement of the Vav protein in signal transduction was provided by early experiments demonstrating that its overexpression and/or mutation results in the malignant transformation of at least rodent fibroblasts in culture. Finally, the exquisite specificity of the pattern of expression of the vav protooncogene within hematopoietic tissues of embryonic and adult mice, suggests a specialized function for the Vav protein in some unique aspects of hematopoietic signaling. In our studies we have disrupted the mouse vav locus by homologous recombination in ES cells in an effort to understand, the contribution of the Vav protein to the ontogeny and/or function of the mammalian hematopoietic system. In a previous report, expression of antisense vav transcripts in ES cells abrogated the development of hematopoietic cells in a dose-dependent fashion (Wulf et al., 1993). However, our experiments using vav-deficient ES cells do not support these observations. vav (-/-) ES cells differentiate into erythroid, macrophage and mast cells with efficiencies comparable with those observed with the parental vav (+/-) and vav (+/+) ES cells. These differentiated vav (-I-) cells express normal levels of c-kit, GATk- 1, IL-3R and both embryonic and adult globin genes. At present, we do not have an explanation for the results reported by Wulf et al. (1993). However, it is likely that overexpression of antisense vav transcripts is altering ES cells in ways other than blocking vav gene function. These observations should raise a note of caution regarding the use of antisense technologies over the more definitive, albeit more laborious, gene targeting approaches. Our results strongly suggest that the vav proto-oncogene is not required for hematopoietic development, at least within the erythroid, myeloid and mast cell lineages. It is possible, however, that the vav gene might be required for the production of critical cytokines that in vitro can be provided by factors in the media. It is also possible that vav (-/-) hematopoietic cells have functional abnormalities not revealed by the in vitro developmental studies reported here. Finally, it is possible that the vav gene plays a role in other hematopoietic cell types such as those derived from the lymphoid lineage. The generation
7
A.Zmuidzinas et aL
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Fig. 8. Vav expression in post-implantation embryos. E6.5 (A and B) and E7.5 (C and D) embryos were isolated within their decidua, cryostatsectioned at 8 urn and subjected to immunocytochemical staining as indicated in the legend to Figure 7. Adjacent sagittal sections of an E6.5 embryo stained with either (A) affinity-purified anti-Vav antibodies or (B) control antiserum. (C and D) Sagittal sections of an E7.5 embryo stained with affinity-punfied anti-Vav antibodies. The ectoplacental cone (Ec), trophoblast cells (T) and extra-embryonic endoderm (En) are indicated by arrows. Magnification x200 (A, B and D) and xl100 (C).
of chimeric animals with mice defective in the RAG genes should address this possibility. Mice homozygous for the targeted vav allele are not viable. Surprisingly, the defect in these animals occurs earlier than the onset of hematopoiesis, indicating that the vav proto-oncogene is essential for a developmental step unrelated to its putative role in hematopoietic cells. vav (-/-) embryos survive 3.5 days of gestation but not 7.5 days, suggesting that the Vav protein plays a critical role during either the implantation process and/or one of the subsequent developmental steps that take place immediately after implantation. Previous studies using RT-PCR have indicated that the vav proto-oncogene is expressed at very low levels in undifferentiated ES cells (Keller et al., 1993; Wulf et al., 1993). The generation of homozygous vav (-/-) ES clones indicates that the Vav protein is not required for the proliferation of these multipotent precursor cells. In vitro differentiation of ES cells into embryoid bodies results in a short-term decrease (24 h) in the levels of vav expression, followed by a moderate increase during the next 48 h, a stage in which embryonic bodies contain an average of 40-80 cells (Keller et al., 1993). At this stage, the first hematopoietic precursors can be detected, albeit at very low levels. By day 6 of culture, the level of hematopoietic precursors
8
increases considerably to as many as 0.5% of the total cells present in the embryoid bodies. Meanwhile, the number of undifferentiated ES cells (as determined by their ability to generate secondary embryoid bodies) decreases to almost undetectable levels. At this time, the levels of vav gene expression remain low and they do not increase until days 9-15 of culture, a period that coincides with the onset of hematopoiesis in the embryoid bodies (Keller et al., 1993; Wulf et al., 1993). In vivo, however, vav gene expression could be detected in the decidua but not in the egg cylinder of early postimplantation embryos (E6.5 and E7.5 embryos). Likewise, there is no detectable vav expression in the yolk sac or whole embryos at E8.5 (Keller et al., 1993). These observations indicate that the vav proto-oncogene has a biphasic pattern of expression during embryogenesis. First, vav is expressed during the early stages of embryonic development that precede hematopoiesis. Later, vav is turned off during or immediately after implantation to appear again in mid-gestation embryos in a fashion concomitant with the development of the fetal and adult hematopoietic system. The early lethality of vav (-/-) embryos prompted us to look more closely at the sites of Vav expression around the time of implantation. Immunocytochemical analysis
Phenotype of vav (-/-) mice and ES cells
with two distinct anti-Vav antibodies of cultured blastocysts corresponding to an E3.5 developmental stage showed significant Vav expression in the extra-embryonic cell layer, but not within the inner embryonic cell mass. These observations support the RT-PCR results that indicated the presence of low, but detectable, expression of vav transcripts in embryoid bodies. More importantly, Vav immunoreactivity was exclusively localized to the external trophoblastic cell layer of post-implantation E6.5 and E7.5 embryos. In agreement with the results of Keller et al. (1993), no Vav expression could be observed in the inner embryonic structures (egg cylinder). These observations, along with the early lethality observed for vav (-/-) blastocysts, suggest that Vav is necessary for the formation of trophoblasts and the trophectoderm, a structure absolutely required for proper embryo implantation and survival of the fetus. In support of this conclusion, all vav (-/-) blastocysts failed to hatch under in vitro culture conditions, further suggesting a requirement for this gene in at least one of the early stages of the implantation process. The trophoblast cell lineage is derived from the trophectoderm of the preimplantation blastocyst and is the first lineage to differentiate during mammalian embryogenesis (Rossant, 1986). Further studies will be required to ascertain the precise role of the vav proto-oncogene in the development of the trophoblast cell lineage and to determine how the absence of Vav signaling affects this earliest known stage of embryogenesis.
Materials and methods Construction of the vav targeting vector An NIH-3T3 mouse genomic DNA library was screened with a probe spanning nucleotides 25-404 of pMB24, a plasmid containing a fulllength mouse vav cDNA clone (Coppola et al., 1991). One of the recombinant phages, Xvavl, contained a 14.2 kb DNA insert encompassing six exons (designated 'a-f') corresponding to nucleotides 193780 of pMB24 (Coppola et al., 1991). The targeting vector, pAZIO (see below), contains 9.65 kbp of vav genomic DNA sequences, including a 8.1 kbp long arm and a 1.55 kbp short arm flanking a bacterial neo gene driven by a phosphoglycerate kinase promoter (PGK-1/neo; McBurney et al., 1991) and a thymidine (tk) cassette located 5' of the long arm (Monsour et al., 1988; Figure IA). Both PGK-l/neo and tk cassettes were placed in the opposite transcriptional orientation with respect to vav (Figure IA). pAZIO lacks 300 bp of vav genomic sequences which encode nucleotides 403-583 of pMB24 (Coppola et al., 1991). To generate pAZI0, an 8.6 kbp Eco47III DNA fragment of Xvavl was subcloned into the SmaI site of pBluescript and digested with XbaI to liberate an 8.1 kbp XbaI DNA fragment containing the 5' 22 bp of exon 'c' and the 20 bp SmaI-XbaI linker of pBluescript. This 8.1 kbp XbaI fragment was subcloned into the XbaI site of pKS-NT, a vector containing the PGK-l/neo and tk cassettes (Wurst and Joyner, 1993), to generate pAZ9. A 2.8 kbp Eco47III-NotI fragment of Xvavl (NotI is located in the kvavl polylinker) was subcloned into the SmaI-NotI sites of pBluescript to generate pAZ4. A 1.55 kbp PstI insert of pAZ4 was liberated, Sall linkers added and the resulting DNA fragment subcloned into the Sall site of pBluescript to generate pAZ7. Digestion of pAZ7 with HindIll and XhoI liberated a 1.55 kbp DNA fragment that contains at its 5' end a 34 bp polylinker sequence followed by the 3' half of exon 'e' and the entire exon 'f'. This 1.55 kbp HindIII-XhoI DNA fragment was ligated into HindIll and XhoI-digested pAZ9 to generate the targeting vector pAZ10 (Figure IA). Transfection and analysis of targeted ES cells Cell culture and electroporation of D3 ES cells (Doetschman et al., 1987) were performed essentially as described (Wurst and Joyner, 1993). Pools of 10 G418R/GancR ES transformants were screened for homologous recombination events by PCR analysis as described
previously (Joyner et al., 1989). Briefly, - 104 cells were lysed by freezing and thawing in de-ionized water and proteinase K treated for 90 min at 50°C. Half of the sample was used in a PCR which contained 10 ng/ml of each amplimer, 200 mM dNTPs, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 4.0 mM MgCl2 and 2.5 U Taq polymerase (Perkin Elmer). The 5' primer (5'-AAGCGCCTCCCCTACCCGGTA-3') corresponds to the PGK- 1 promoter (Adra et al., 1987), whereas the 3' primer (5'-TGGTCTCCATGTCTTGAGGCTTAAGG-3') corresponds to vav sequences downstream of the targeting vector pAZ10. PCR-aided amplification was carried out at 94°C for 1 min (first cycle 2 min), 65°C for 2 min and 72°C for 3 min for 40 cycles. Aliquots of the PCRamplified sample were analyzed by electrophoresis on 1.0% agarose gels. Gels were soaked for 30 min in 0.5 M NaOH containing 1.5 M NaCl, DNA fragments blotted for 3 h onto GeneScreen membranes (Dupont), cross-linked to the membrane by UV light and incubated with a 32P-labeled probe derived from the 1.55 kbp PstI DNA insert of pAZ4 in hybridization buffer [0.5 M sodium phosphate (pH 7.0), 0.5% SDS, 15% formamide, 1 mM EDTA and 10 mg/ml bovine serum albumin] for 3 h at 60°C. Hybridized filters were washed twice for 30 min with 150 mM sodium phosphate buffer (pH 7.0) containing 0.1% SDS, airdried and exposed to Kodak X-OMAT film at -70°C with an intensifying screen. PCR-positive clones were subsequently confirmed by genomic Southern hybridization. Briefly, DNA (15 jg) was digested with restriction endonucleases, electrophoresed on 0.7% agarose gels, transferred to nitrocellulose filters and hybridized with two different vav 32P-labeled genomic DNA probes, including a 0.6 kbp EcoRI-SalI DNA fragment derived from pAZ5 and located immediately downstream of the short arm of pAZIO (probe A) and a 1.2 kbp EcoRI-XbaI DNA fragment from pAZ6 located within the long arm of pAZIO (probe B) (Figure IA). Targeted ES cell clones were trypsinized, washed in PBS and kept on ice. Approximately 10-15 cells were injected into C57BL/6J blastocysts (Joyner et al., 1989). Surviving blastocysts were transferred into the uterus of pseudopregnant CDI females. The resulting chimeras were bred onto a C57BU6J background.
Isolation and differentiation of homozygous vav (-/-) ES cells vav (-/-) ES cells were selected by cultivation of vav (+/-) ES cells in high doses of G418 (Mortensen et al., 1992). Briefly, SX 106 vav (+/-) ES cells (clones B142-313 and B142-321) were plated on gelatincoated 100 mm dishes in the presence of 1.0 or 1.5 mg/ml G418 and LIF. After 2 weeks in culture, small colonies composed of undifferentiated ES cells were transferred to 24-well plates and genotyped by either Southern blot or PCR analyis. In vitro differentiation assays were performed in methyl cellulose medium as described previously (Keller et al., 1993). At 2 days prior to the initiation of differentiation, freshly thawed ES cells were passaged onto gelatinized tissue culture plates. After trypsinization, single-cell suspensions were preplated for 20 min and unattached cells cultured in the absence of LIF in 0.9% methyl cellulose (Terry Fox Laboratories, Vancouver, Canada) in IMDM (Gibco) supplemented with 20% fetal calf serum (FCS; Hyclone), huIL- 1 (0.65 ng/ml; Immunex), muIL-3 (0.65 ng/ml; Immunex), muEpo (2.5 U/mI; Boehringer) and monothioglycerol (4.5X 10-4 M). Depending on their plating efficiencies, 10002000 ES cells were plated in a final volume of 1.5 ml in 35 mm non-tissue culture dishes. Cultures were maintained in a humidified 5%
CO2 atmosphere at 37°C. Colonies were scored by light microscopy for visibly hemoglobinized erythroid cells at days 12 and 20. Macrophages were identified by morphology. The presence of hematopoietic cells was further verified by Wright Giemsa stain. ES cell-derived mast cells were obtained as described by Wiles and Keller (1991). Briefly, three to five colonies grown in methyl cellulose were transferred at day 15 of differentiation into single wells of a 96well plate with IMDM, 5% FCS and 1% rIL-3 containing conditioned medium (Karasuyama and Melchers, 1988). After 2 weeks in culture, suspension cells were expanded in IMDM supplemented with 1% FCS, 0.05% BSA, 5 mg/ml insulin (Sigma), 5 mg/ml transferrin (Sigma), 2 mg/ml concanavalin A (Sigma) and 1 % rIL-3. Mast cells were identified by alcian blue/safranin staining. RT-PCR Total RNA was isolated from either undifferentiated ES cells or differentiation cultures as described previously (Chomczynski and Sacchi, 1987). 1 jig of RNA was reversed transcribed in a volume of 20 ,ul in the presence of oligo(dT) as primer for cDNAs by using a Perkin Elmer RT-PCR kit as recommended by the manufacturer. Amplifications were carried out at 94°C for 1 min, 50 or 55°C for 1 min and 72°C for 3 min
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A.Zmuidzinas et aL. for 30 cycles (40 cycles in the case of vav transcripts). vav primers included those corresponding to nucleotides 412-431 (5'-GACGAAGATATTTACAGTGG-3') and 583-564 (5'-GCTTATCATACTCTGTCATC-3') of pMB24 (Coppola et al., 1991). The primers for the PHI globin, 1maJ globin, GATA-1, IL-3R, c-kit and HPRT genes were chosen as described by Keller et al. (1993). The use of equal amounts of RNA for each RT-PCR was verified by comparing the expression of the housekeeping gene HPRT between all preparations. As controls, PCR amplifications were also performed without a preceding RT reaction to rule out contamination by genomic DNA. The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide and photographs were taken with a still video system for gel documentation (Professional Diagnostic Inc., Toronto, Canada).
slides were air-dried for 15-30 min, fixed at -20°C for 5 min in a 5% acetic acid/95% ethanol solution and washed extensively in PBS. For immunostaining, cells were incubated overnight at 4°C with affinitypurified anti-Vav antibodies including those raised against GST-VavSH2 (Ab #261) and MBP-VavDH (Dbl homology domain) (Ab #302) fusion proteins. As negative controls, we utilized either the non-bound fraction of the affinity-purified antisera, the above anti-Vav antibodies preincubated with an excess (10-fold) of immunizing antigen or preimmune sera. The specificity of these affinity-purified antibodies (1:1000 dilution for Ab #261 and 1:10 000 dilution for Ab #302) was confirmed by immunohistochemical analysis of wild-type NIH-3T3 cells and NIH3T3 cells expressing the Vav protein (B36-212 cells; Bustelo et al., 1992). Immunoreactivity was developed using Histostain-SP kit (Zymed Laboratories) as suggested by the manufacturer.
Western blot analysis 5X 106 embryoid body-derived mast cells were lysed in lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X100, 1.5 mM MgCl2, 1 mM EGTA, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM PMSF, 200 mM sodium orthovanadate and 10 mM sodium fluoride], as described previously (Reith et al., 1991). Cleared supernatants were subjected to immunoprecipitation with anti-Vav rabbit polyclonal antibody raised against a synthetic peptide corresponding to residues 577-590 of the mouse Vav protein (Bustelo et al., 1992). After incubation with protein A-Sepharose, the precipitates were fractionated on a 7.5% SDS-PAGE, transferred to a nitrocellulose paper and incubated with a rabbit polyclonal antibody generated against the cysteine-rich region of mouse Vav protein (Bustelo et al., 1992). Proteins were visualized with an ICL Western blotting kit (Amersham) according to the instructions of the manufacturer.
Blastocyst cultures For in vitro blastocyst culture experiments, female C57BL/6J mice (34 weeks old) were superovulated by injection with 5 IU of pregnant mare's serum gonadotropin (Sigma) followed after 48 h by an injection of 5 IU of human chorionic gonadotropin (Sigma), and were caged with C57BL/6J males. Embryos were collected as 3.5 day post-coitum blastocysts or at the 2-cell stage (48 h post hCG injection). They were cultured in vitro in groups of 15-30 in Whitten's medium under silicon oil at 37°C in an atmosphere of 5% CO2 and 95% air (Wurst and Joyner, 1993) to the hatched blastocyst stage. Individual blastocysts were collected, suspended in 50 ,l of distilled water and boiled for 10 min to lyse cells and denature the DNA. Two PCR primer combinations were used to simultaneously detect the wild-type (vav+) and targeted (vav-) vav alleles. The vav+ primers included vav intron sequences located 3' of exon 'c' (5'-ATTAGGACCTGATGGGTGCAGCTT-3') and nucleotides 455-474 pMB24 corresponding to exon 'e' (5'-GTCCTCGTCTTCCTCTGCGG-3'). These primers amplify a 300 bp DNA fragment located in the wild-type vav locus but absent in pAZIO and therefore deleted during the targeting event. The vav- primers include nucleotides -498 to -518 of the PGK-1 promoter (5'-AAGCGCCTCCCCTACCCGGTA-3') and nucleotides 625-649 of pMB24 corresponding to exon 'e' (5'-GATGGAGCCCAGTGTGTCTGTATA-3'). These primers amplify a 150 bp DNA fragment unique to the vavtargeted allele. PCRs were run on the whole blastocyst lysate in a volume of 100 p1 containing 5 puM each of the vav+ and vav- amplimers, 200 pM dNTPs, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCl2 and 2.5 U Taq polymerase (Perkin Elmer). PCR cycling was carried out at 94°C for 1 min (first cycle 2 min), 55°C for 1 min and 72°C for 10 s for 40 cycles. To amplify the signal further, a second PCR round at the same reaction conditions was performed on a 1 ,ul aliquot of the first PCR utilizing either vav+ or vav- primers. PCR products were visualized on 2.5% ethidium bromide-stained agarose gels. To confirm the presence of amplified vav sequences, DNA was transferred to filters and hybridized to 2P-labeled oligonucleotide probes internal to the wild-type (5'-GTGAGACACAGTGGAGACTG-3') and targeted (5'-GGATCTCCCGCAGGCAGC-3') PCR products.
Immunocytochemistry In vitro blastocyst cultures were terminated at the blastocyst stage after hatching. Zona pellucida-free embryos were washed in 3% BSA in PBS and spun onto polycationic slides (Superfrost/Plus; Fischer Scientific) at 600 g for 10 min in a cytocentrifuge. For embryo outgrowths, blastocysts were cultured in vitro on gelatin-coated dishes in ES medium. After 56 days, blastocyst outgrowths were removed using a drawn out pipette, trypsinized as described by Robertson (1987) and cultured individually for another 2-3 weeks in the absence of feeder cells. Embryo tissue was cryostat-sectioned at 8 mm and mounted onto polycationic slides. The
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Acknowledgements We would like to thank X.R.Bustelo for helpful discussions throughout this work and A.Lewin and M.Swerdel for excellent technical assistance. K.-D.F. is supported by a Fellowship from the Human Frontiers Science Program. Work in A.B.'s laboratory is supported by grants from the Medical Research Council of Canada and Bristol-Myers Squibb. A.B. is an International Research Scholar of the Howard Hughes Medical Institute.
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Received on September 6, 1994; revised on October 19, 1994
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