Title: HIF-1alpha is required for solid tumor formation and embryonic vascularization
Abstract: Article1 June 1998free access HIF-1α is required for solid tumor formation and embryonic vascularization Heather E. Ryan Heather E. Ryan Department of Biology, University of California, San Diego, La Jolla, CA, 92093-0366 USA Search for more papers by this author Jessica Lo Jessica Lo UCSD Cancer Center, University of California, San Diego, La Jolla, CA, 92093-0366 USA Search for more papers by this author Randall S. Johnson Corresponding Author Randall S. Johnson Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, 92093-0366 USA Search for more papers by this author Heather E. Ryan Heather E. Ryan Department of Biology, University of California, San Diego, La Jolla, CA, 92093-0366 USA Search for more papers by this author Jessica Lo Jessica Lo UCSD Cancer Center, University of California, San Diego, La Jolla, CA, 92093-0366 USA Search for more papers by this author Randall S. Johnson Corresponding Author Randall S. Johnson Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, 92093-0366 USA Search for more papers by this author Author Information Heather E. Ryan1, Jessica Lo3 and Randall S. Johnson 2 1Department of Biology, University of California, San Diego, La Jolla, CA, 92093-0366 USA 2Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, 92093-0366 USA 3UCSD Cancer Center, University of California, San Diego, La Jolla, CA, 92093-0366 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3005-3015https://doi.org/10.1093/emboj/17.11.3005 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcriptional response to lowered oxygen levels is mediated by the hypoxia-inducible transcription factor (HIF-1), a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) and the hypoxic response factor HIF-1α. To study the role of the transcriptional hypoxic response in vivo we have targeted the murine HIF-1α gene. Loss of HIF-1α in embryonic stem (ES) cells dramatically retards solid tumor growth; this is correlated with a reduced capacity to release the angiogenic factor vascular endothelial growth factor (VEGF) during hypoxia. HIF-1α null mutant embryos exhibit clear morphological differences by embryonic day (E) 8.0, and by E8.5 there is a complete lack of cephalic vascularization, a reduction in the number of somites, abnormal neural fold formation and a greatly increased degree of hypoxia (measured by the nitroimidazole EF5). These data demonstrate the essential role of HIF-1α in controlling both embryonic and tumorigenic responses to variations in microenvironmental oxygenation. Introduction Hypoxia causes a wide range of responses in an organism at both the systemic and cellular levels (Bunn and Poyton, 1996). Lowered environmental oxygen levels cause increased erythropoietin (EPO) production, which results in higher oxygen-carrying capacity in the blood (Blanchard et al., 1993). Decreased oxygen pressure in interstitial fluid results in a metabolic switch to glycolysis for energy production (Semenza et al., 1994). In addition, hypoxia causes secretion of vascular endothelial growth factor (VEGF) (Shweiki et al., 1992; Ladoux and Frelin, 1993; Banai et al., 1994; Ikeda et al., 1995; Levy et al., 1995; Liu et al., 1995; Pe'er et al., 1995; Shima et al., 1995; Stone et al., 1995), an angiogenic/permeability factor which acts to increase the immediate availability of oxygen from capillaries (through increased vascular permeability) as well as induce formation of new vessels. The hypoxic induction of angiogenesis is a hallmark of pathological processes such as wound healing and solid tumor formation; it is strongly correlated with the disrupted circulation and rapid growth characteristic of those states. In addition, hypoxic regions within tumors are resistant to radiotherapy and thus constitute an extremely important fraction of cancer cells from a clinical perspective (Brown and Giaccia, 1994; Dachs et al., 1997). These hypoxic areas of the tumor form a depot of cells resistant to p53-mediated apoptosis and are thus likely locations of selection for mutations in p53 prior to invasive growth and metastasis (Graeber et al., 1996). One component of the hypoxic response is transcriptional activation, which is mediated by binding of the hypoxia-inducible transcription factor, HIF-1, to an 8 bp hypoxia response element (HRE), an enhancer found in genes subject to transcriptional regulation by oxygen pressure (Semenza et al., 1991; Semenza and Wang, 1992; Wang and Semenza, 1993a,b). The binding moiety of HIF-1 is a heterodimer of the helix–loop–helix (HLH)/PAS proteins HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT) (Wang and Semenza, 1995; Wang et al., 1995). HIF-1α is the hypoxically responsive component of this complex, while ARNT is expressed constitutively and dimerizes with a number of other HLH proteins, including the PAS proteins Per, Sim, and the aryl hydrocarbon receptor (AhR) (Swanson and Bradfield, 1993; Rowlands and Gustafsson, 1997). A recently cloned homolog of ARNT, ARNT2, has also been shown to participate in heterodimeric DNA binding with AhR and Sim, but its expression is limited to the neural tube (Hirose et al., 1996). Targeted null mutations of the ARNT and the AhR genes have widely varying phenotypes in mice. The ARNT mutation causes a mid-gestation retardation of growth first evident at embryonic day (E) 9.5 (Maltepe et al., 1997), whereas the AhR knockout mice exhibit a high degree of post-natal mortality and greatly diminished tolerance of xenobiotic compounds (Fernandez-Salguero et al., 1995; Schmidt et al., 1996). Due to ARNT's role in processes other than hypoxic induction, we chose to create a null mutation at the HIF-1α locus via homologous recombination in mouse embryonic stem (ES) cells (Papaioannou and Johnson, 1992) in order to investigate the role of the hypoxic response in vivo. ES cells lacking HIF-1α are unable to up-regulate several HIF-1 target genes under hypoxic or low glucose conditions, with the most severe effect seen on several enzymes in the glycolytic pathway. Even more striking is the finding that HIF-1α null ES cells form teratocarcinomas one quarter the size of tumors derived from wild-type ES cells. In addition, HIF-1α null embryos display abnormal neural development beginning at E8.0. There is a failure of the neural tube to close, and this failure correlates with the absence of cephalic vascularization, loss of neural tube expression of at least one glycolytic enzyme (phosphoglycerate kinase) and greatly increased neuroectodermal hypoxia and apoptosis. Results Generation of HIF-1α-deficient ES cells The HIF-1α gene was disrupted by two different vectors: one resulting in a null mutation through a replacement of the HLH domain of HIF-1α with a neomycin resistance cassette. The second was a ‘knock-in’ vector, which produces an in-frame fusion to the lacZ gene followed by a loxP-flanked neomycin resistance gene (Figure 1A). The neomycin resistance gene in the second targeting vector was removed through the transient transfection of a cre expression vector; this was done to prevent interference of the neomycin resistance gene with HIF-1α-driven lacZ expression. After successful targeting of the locus with both vectors, HIF-1α null ES cells were generated by high G418 selection (Mortensen et al., 1992) and analyzed by Southern and Northern blot analyses (Figure 1B and C). To further confirm that these ES cells contain no functional HIF-1α, we performed electromobility shift assays on wild-type and null cells exposed to hypoxia (Figure 1D). Wild-type ES cells demonstrate a mobility shift under hypoxic conditions while null cells have lost the ability to bind the HRE in a hypoxia-specific manner (Figure 1D). This verifies that functional HIF-1 has been abolished in the HIF-1α null cells. Figure 1.Targeted disruption of the mouse HIF-1α gene. (A) Part of the wild-type HIF-1α locus, the two targeting vectors used and the targeted locus. The HIF-1α gene was disrupted by replacement of the HLH with either a neomycin resistance gene alone (pMC1neo) or an in-frame fusion of the lacZ gene followed by a loxP-flanked pMC1neo. B, BamHI; Bg, BglII; E, EcoRI; P, PstI; X, XbaI. Homologous recombination at the HIF-1α locus introduces a new BamHI site. This results in a 5.5 kb targeted band versus a 13 kb endogenous band upon digestion of genomic DNA with BamHI and hybridization to a 5′ external probe. (B) Southern blot analysis of genomic DNA showing correct targeting of the HIF-1α allele. Genomic DNA was isolated from R1 ES cells (Nagy and Rossant, 1992), digested with BamHI and hybridized with the 5′ probe. HIF-1α −/− cells were generated via high G418 selection (Mortensen et al., 1992). (C) Northern blot analysis of total RNA. Thirty μg of total RNA was loaded per lane and hybridized with a 345 bp HIF-1α cDNA probe. (D) Electromobility shift assay on normoxic and hypoxic (1%O2 for 5 h) nuclear extracts from wild-type and HIF-1α null ES cells. Two binding sites were used: the 18 bp HRE from the EPO gene (Semenza and Wang, 1992) and the 24 bp HRE from the VEGF gene (Forsythe et al., 1996). Lanes 1, 2, 5, 6, 9 and 10 contain extracts incubated with the EPO HRE. Lanes 3, 4, 7, 8, 11 and 12 contain extracts incubated with the VEGF HRE. Wild-type ES cells exhibit a shift under hypoxia with either binding site (lanes 2 and 4, arrow). HIF-1α null ES cells do not show a shift under hypoxia with either binding site (lanes 6, 8, 10 and 12). Download figure Download PowerPoint HIF-1α is essential for up-regulation of a wide range of hypoxically responsive genes The targeted ES cells allowed us to compare mRNA levels of hypoxia-responsive genes in mutant cell lines with those in wild-type cells under hypoxia (Figure 2A) and glucose deprivation (Figure 2B). In addition, we quantitated the levels of target gene expression in order to determine the exact role of HIF-1α-dependent gene regulation during hypoxia (Figure 2C). The most dramatic effects of the null mutation were seen in glycolytic enzymes, and reveal that HIF-1α is essential for constitutive expression as well as hypoxic induction of phosphoglycerate kinase-1 (PGK) and lactate dehydrogenase A (LDH). Aldolase A (ALDA) is unaffected at the 0 time point and so is not dependent on HIF-1α for basal regulation, but both it and the glucose transporter-1 (GLUT1) gene exhibit a greatly reduced capacity for hypoxic and hypoglycemic induction in the null cells. Figure 2.Northern blot analysis of several HIF-1 target genes from cells cultured under hypoxia (A) or low glucose (B) conditions. Thirty μg of total RNA was loaded per lane and hybridized to cDNA probes from the following genes: PGK, ALDA, LDH, PK, GLUT1, VEGF, EPO, ARNT and HIF-2α. Equal loading was monitored by ethidium bromide staining of 28S rRNA. (C) Phosphorimage analysis of HIF-1 target genes from cells cultured under hypoxic conditions. Ten μg of total RNA was loaded per dot, hybridized to cDNA probes and quantified with a phosphorimager and ImageQuaNT software (both Molecular Dynamics). Download figure Download PowerPoint VEGF is both hypoxically responsive and essential for normal embryonic development (Carmeliet et al., 1996; Ferrara et al., 1996). Despite complete loss of binding to the VEGF HRE in the HIF-1α null cells (Figure 1D), the hypoxic response of the VEGF gene is only partially eliminated (Figure 2A and C), although quantitation of the signal (Figure 2C) shows that the remaining hypoxic response is marginal and transient. The remaining response is probably due to hypoxically induced stabilization of the VEGF mRNA, which is mediated independently through an element in the 3′-untranslated region (3′ UTR) of the VEGF transcript (Levy et al., 1995, 1996; Liu et al., 1995; Damert et al., 1997). The partial reduction in the hypoxic response of VEGF at the mRNA level was roughly equivalent to that at the protein level at later time points, as assayed by secretion of VEGF from hypoxically induced HIF-1α null cells (Figure 4B), although there is a more severe reduction in the protein level seen at the final point (72 h). This probably reflects accumulation of VEGF in the conditioned medium dictated by the differential rates depicted in the assay for mRNA levels. Figure 3.Loss of HIF-1α decreases tumor mass. (A) Analysis of tumor mass from wild-type and HIF-1α null teratocarcinomas after 21–22 days of growth. Statistical analysis was performed using Statview (Abacus Software). (B) VEGF-specific ELISA results demonstrate decreased VEGF production during hypoxia in HIF-1α null cells when compared with wild-type cells. (C) Mean vessel density in wild-type and null tumors. (D) CD31 staining of a wild-type and a HIF-1α null tumor. Counterstain is Mayer's hematoxylin. (E) TUNEL assay on a wild-type and a HIF-1α null tumor. Magnification for (D) is 100× and for (E) is 200×. Download figure Download PowerPoint HIF-2α is a recently cloned HIF-1α homolog which can also form heterodimers with the ARNT protein (Ema et al., 1997; Hogenesch et al., 1997; Tian et al., 1997). However, no evidence for the presence of HIF-2α–ARNT dimers is seen at the level of DNA binding in HIF-1α null ES cells (Figure 1D). Basal expression of the HIF-2α gene transcript was up almost 50% in HIF-1α null cells (Figure 2C), indicating a possible auto-regulation of the component members of this complex. As a number of other investigators have reported, we saw total transcript levels of HIF-2α, HIF-1α and ARNT decline over time in hypoxic conditions; this has been reported as evidence of increased protein stability as a primary mechansim of HIF-1 induction. Reduced tumor mass and increased apoptosis in HIF-1α-deficient teratocarcinomas ES cells have the capacity to form teratocarcinomas when injected into syngeneic or immunocompromised mice. We exploited this property to determine the effect of the HIF-1α null mutation on solid tumor formation. We found that the null cells are greatly compromised in their ability to form tumors (Figure 4A). Interestingly, the retardation in the formation of the tumors is not evident until later time points; in the first 9 days of tumor growth, the HIF-1α null tumors have approximately the same mass as wild-type tumors (Figure 4A). It is only in the second and third week of tumor growth that differences in size become apparent, finally resulting in a 75% reduction in the size of the null tumors in comparison with the wild-type after 3 weeks. This is correlated with a 40% reduction in tumor vessel density (Figure 4C). This is also demonstrated in the histology shown in Figure 4D, where both vessel density and vessel morphology are clearly altered in the HIF-1α null tumors. This reduction in tumor volume as tumor mass increases could also conceivably be due to differences in cell growth rates, especially within the hypoxic and typically inefficiently vascularized tumor microenvironment. As can be seen in Figure 3A and B, cell growth rates of the wild-type and null cells in culture are virtually identical in low glucose and hypoxic conditions. This indicates that the difference in tumor size is probably due to an alteration in the tumor microenvironment of HIF-1α null tumors, and is not simply a cell autonomous defect. Figure 4.Cell viability curves under low glucose (A) and hypoxia (B). Cells were cultured for the indicated times, stained with trypan blue and viable cells were counted. Download figure Download PowerPoint In order to determine what factors might be causing the reduced tumor volume, we analyzed the tumors for increased hypoxia, as measured by the immunofluorescently detectable marker EF5. Although this marker detected profound differences in the oxygenation of HIF-1α null embryos (see below), there were no reproducible differences in tumor binding of the marker (data not shown). This may be due to the characteristic heterogeneity of individual teratocarcinomas, which are prone to widely varying levels of internal differentiation as they expand. One factor contibuting to the reduced mass of the null tumors is a clear increase in the amount of apoptosis in the non-necrotic, undifferentiated sections of the tumor. As can be seen in Figure 4E, tumor apoptosis is increased in these non-necrotic regions of nested germ cells. This was difficult to quantify in the teratocarcinomas as a whole, however, due to the large variations in cell density, degree of necrosis and differentiation of the tumors. Further characterization of apoptotic frequency and tumor expansion will require a more uniform tumor type to be derived from HIF-1α null cell lines. One mechanism for this will certainly involve the generation of mice carrying inducible HIF-1α alleles from which differentiated tissue can be derived and transformed, and we currently are pursuing this strategy to define the role of HIF-1 in tumorigenesis more clearly. Loss of HIF-1α results in disorganized yolk sac vascularization HIF-1α heterozygotes were bred to each other in order to generate HIF-1α null homozygous mice. Null homozygous mice were isolated at mid-gestation and analyzed for gross morphological defects. One obvious difference at this stage (E9.5) is the greatly disturbed vascularization of the embryonic yolk sac. The yolk sacs from mutant embryos exhibit a complete lack of vascular organization when compared with wild-type embryos (Figure 5C). There is no organized branching of the vasculature, although there are fully formed vessels and red blood cells. This demonstrates that HIF-1α is essential for the proper formation of this vascular network, and implies that oxygen levels function as a key determinant of capillary arrangement and organization in this tissue. Figure 5.Gross morphological characterization, whole-mount immunohistochemistry and lacZ staining of HIF-1α null embryos obtained from heterozygous crosses. (A) E8.5 littermates, at which point the embryos are approximately the same size. (B) E9.5 littermates, displaying a substantial size difference. (C) Yolk sacs from E9.5 littermates. The HIF-1α null yolk sac shows disorganized vascularization compared with the wild-type. (D) E8.5 littermates stained for the endothelial cell-specific marker CD31 demonstrate absence of neural vascularization (arrows) and incomplete somitic vascularization. (E) Closer view of the neural folds of an E8.5 HIF-1α null embryo stained for CD31 (arrow). (F, G and H) Staining of heterozygous embryos carrying an in-frame lacZ fusion at the HIF-1α locus. A distinct pattern of somitic expression is seen in (F), an E8.5 embryo. At E8.0 (G) we see intense staining in the neural folds (arrows) while at E8.5 (H) the expression appears to be localized to a layer of cells lining the inner neural ectoderm (arrow). Download figure Download PowerPoint Abnormal neural development and cephalic vascularization in HIF-1α mutant mice HIF-1α null embryos were clearly discernible in utero beginning at E8.0, when the null mutant embryos are smaller and have reduced and somewhat convoluted neural folds. E8.0 is also the stage in which we see high expression of the HIF-1α–lacZ knock-in fusion in the cephalic region (Figure 5G). At E8.5, embryonic expression of the HIF-1α–lacZ fusion in the neural fold is localized to the innermost layer of cells (arrow, Figure 5H). Loss of HIF-1α expression at this stage and in this location is correlated with the aberrantly formed and incompletely closed neural fold seen in null mutants (Figure 5A). In addition, the HIF-1α null mutants have a reduced number of somites (Figure 5A), another site of a distinct pattern of HIF-1α–lacZ expression (Figure 5F). At E9.5, the null mutant embryos (Figure 5B) are much smaller than their wild-type littermates; interestingly, the neural folds at this stage are still not closed, but neural vesicles have begun to form on either side of the head, resulting in paired balloon-like structures in the fore-, mid- and hindbrain regions of the embryo. Because loss of HIF-1α has a profound effect on VEGF expression and VEGF plays an essential role in embryonic development, we examined the vascularization pattern of HIF-1α null embryos. In embryos stained for the endothelial cell marker CD31 (Figure 5D and E) the requirement for HIF-1α in embryonic vascularization is clear. The neural folds have a very small number of capillaries and no vascular network, and the intersomitic vasculature is interrupted. This result indicates that HIF-1 is an essential regulator of cephalic vascularization. Despite the abnormalities in capillary network formation seen in the null embryos, the dorsal aorta is present but reduced in size, and the heart is almost normally sized and was found beating in both E8.5 and E9.5 embryos (Figure 5A and B). Increased hypoxia and apoptosis in HIF-1α null embryos In order to determine whether the loss of HIF-1α had any effect on levels of embryonic oxygenation, we undertook a study of wild-type and null embryos using the nitroimidazole hypoxia marker EF5 (Lee et al., 1996). This small molecule forms adducts to macromolecules in reducing microenvironments and thus can be used to detect hypoxic regions in tissues (Lord et al., 1993; Lee et al., 1996). This is accomplished through detection of these adducts by EF5-specific monoclonal antibody binding, as shown in Figure 6C. There was a striking increase in the binding of EF5 in the null mutant embryos. This binding was seen in most embryonic structures, but was highest in the neural ectoderm and somites. The increase in binding was quantified by CCD measurement of pixel illumination per embryo section and revealed a 10.5-fold increase in bound antibody in a null embryo versus its wild-type littermate. Figure 6.Analysis of PGK expression in embryos and characterization of hypoxia and apoptosis in E8.5 embryos. (A) Whole mount in situs of wild-type and HIF-1α null embryos showing intense expression of PGK in the neural tube region and complete loss of this expression in null embryos (arrows). (B) Vibratome sections of a wild-type and null embryo stained for PGK expression, demonstrating the neural-specific pattern of expression (arrows) in the wild-type embryo and lack of such expression in the null embryo (arrow). (C) EF5 staining of frozen sections from wild-type and null embryos. When exposure time is held constant for the null and wild-type embryos, it becomes clear that there is a significant difference in the level of hypoxia in the null embryo. (D) Whole mount TUNEL assay on wild-type and null embryos. There are a few scattered apoptotic cells in the head of the wild-type embryo while the neural folds of the null embryo are littered with darkly staining apoptotic cells (close up, arrows). Download figure Download PowerPoint We wished to determine whether the increase in embryonic hypoxia was reflected in an increase in apoptotic cell death. To assay this, we measured apoptosis in wild-type and null embryos by TUNEL assay, as shown in Figure 6D. As can be seen, there is a significant increase in apoptotic cell number in the interior of the null mutant neural fold, correlated with increased hypoxia in this area and an absence of normal vascularization. Loss of PGK expression in mutant embryos The neural fold is clearly the site of significant levels of HIF-1α expression, and we wished to correlate this with expression of HIF-1 target genes. Due to the significant role HIF-1α plays in normoxic and hypoxic PGK expression, as evidenced by the Northern blot data (Figure 2A and C), we chose to examine its regulation in the embryo. In situ data (Figure 6A and B) reveal that PGK is also largely HIF-1α dependent in the embryo as loss of HIF-1α completely ablates PGK expression. The expression of PGK is highest in the margins of the neural fold (Figure 6A and B, arrows), implying that glycolytic activity may contribute to the normal development of this tissue, and that its absence is contributing to the abnormal neural fold formations seen in HIF-1α null mutants. Discussion Hypoxia has been proposed to regulate many physiological and pathological processes. In order to understand better the significance of the hypoxic response in such processes, we have created a null mutation at the HIF-1α locus, generating both null ES cells and null mutant mice. In the null ES cells, we have completely abolished HIF-1 binding to both the EPO HRE and the VEGF HRE, as assayed by electromobility shift assays. This result is interesting in that it indicates that HIF-1α is the primary dimerization partner of ARNT in binding both EPO HRE and VEGF HRE sequences. Also, at least in ES cells, HIF-2α is not involved in binding the VEGF HRE under hypoxia, as has been proposed by others (Ema et al., 1997). Northern blot analysis, quantitation and, in the case of PGK, in situ hybridization reveal that HIF-1α is essential for the basal expression of two genes, LDH and PGK, and the hypoxic and hypoglycemic induction of a number of other, more disparate genes. In the case of PGK, LDH and ALDA, the hypoxically induced increase in message is completely abolished in the HIF-1α null cells. The profound effect on these genes indicates that HIF-1α plays a key role in the induction of a glycolytic response in tissues under hypoxic stress. We also see a decreased hypoxic response of GLUT1 in the null cells. This further indicates the importance of hypoxic transcriptional induction in cellular metabolism. It also underscores the importance of glycolysis during hypoxic stress; curiously, we saw no differential effect between the mutants and wild-type cells during low glucose or hypoxic cell conditions. This may be the result of the somewhat unusual characteristics of ES cell growth, however, and be unrelated to the role of HIF-1α in the adult organism. VEGF is another HIF-1 target gene that is affected by the loss of HIF-1α. Quantitation of VEGF mRNA indicates that the hypoxic regulation of the gene is severely affected in HIF-1α null cells. This is not surprising given the considerable evidence that VEGF is regulated by hypoxia-induced transcriptional activation, although it has also been shown to be strongly regulated by increased mRNA stability (Finkenzeller et al., 1995; Ikeda et al., 1995; Levy et al., 1995, 1996; Shima et al., 1995). It is surprising, given this other important mode of transcript regulation, that we see such a marked decrease in both the mRNA levels and the secreted protein. It is also striking that the kinetics of VEGF expression in response to hypoxia change considerably in the HIF-1α null cells: there is a slight increase in RNA and secreted protein levels at 4 h of hypoxia, and only a very gradual up-regulation of protein expression (and no increase in RNA levels) after extended incubation in a hypoxic environment. This result indicates that the primary means of up-regulating VEGF expression may be via transcriptional induction of expression, and that increased mRNA stability plays a more minor role than has been proposed previously (Ikeda et al., 1995; Levy et al., 1995, 1996; Shima et al., 1995) Gel shift data indicate that there is no redundancy in HIF-1 components in ES cells, as there is no compensation for the loss of HIF-1α at the level of DNA binding. The HIF-1α homolog HIF-2α is expressed in vivo primarily by endothelial cells (Ema et al., 1997; Hogenesch et al., 1997; Tian et al., 1997), and there may be tissue-specific activation of the gene necessary for DNA binding during hypoxia. Thus there may be functional redundancy in some tissue types, although we see no evidence for this at the level of ES cells or embryos. Further analysis of possible tissue-specific redundancy will require the creation of HIF-1α conditional knockouts and analysis of tissues individually targeted for the loss of HIF-1α. The role of hypoxia in tumors, from inducing angiogenesis to selecting for p53 null cells, has been well studied (Shweiki et al., 1992; Potgens et al., 1995; Forsythe et al., 1996; Damert et al., 1997; Gassmann et al., 1997). Using the HIF-1α null ES cells as a tool, we were able to characterize the contribution that the transcriptional hypoxic response makes to tumor growth. Teratocarcinomas lacking HIF-1α exhibited a substantial decrease in tumor mass when compared with wild-type teratocarcinomas (Figure 4A). This result indicates that an intact HIF-1 response is essential to sustain rapid growth in the tumor microenvironment. Our results in cell culture, where no significant difference in hypoxic cell survival between wild-type and null cells was seen, suggests that the role of HIF-1 in expansion of a solid tumor mass is non-cell autonomous. The role of VEGF as a paracrine regulator of angiogenesis makes it a logical candidate effector of the tumor phenotype. Indeed, when VEGF protein levels were assayed under hypoxic conditions, we found a 4-fold mean decrease in secreted protein in the null cells. This striking difference between wild-type and null cells indicates that angiogenic insufficiency may be one cause of the decreased tumor mass. This finding is bolstered further by the decrease in vessel numbers found in the null tumors, clearly tying hypoxic response through HIF-1α to tumor vascularization. One exciting finding of this work is the demonstration that HIF-1α, and by extension the transcriptional response to hypoxia, plays an essential role in embryonic dev