Article2 April 2001free access Abnormal angiogenesis but intact hematopoietic potential in TGF-β type I receptor-deficient mice Jonas Larsson Jonas Larsson Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Marie-José Goumans Marie-José Goumans Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Lottie Jansson Sjöstrand Lottie Jansson Sjöstrand Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Marga A. van Rooijen Marga A. van Rooijen Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands Search for more papers by this author Dorien Ward Dorien Ward Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands Search for more papers by this author Per Levéen Per Levéen Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Xiufeng Xu Xiufeng Xu Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Peter ten Dijke Peter ten Dijke Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Christine L. Mummery Christine L. Mummery Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands Search for more papers by this author Stefan Karlsson Corresponding Author Stefan Karlsson Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Jonas Larsson Jonas Larsson Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Marie-José Goumans Marie-José Goumans Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Lottie Jansson Sjöstrand Lottie Jansson Sjöstrand Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Marga A. van Rooijen Marga A. van Rooijen Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands Search for more papers by this author Dorien Ward Dorien Ward Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands Search for more papers by this author Per Levéen Per Levéen Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Xiufeng Xu Xiufeng Xu Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Peter ten Dijke Peter ten Dijke Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Christine L. Mummery Christine L. Mummery Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands Search for more papers by this author Stefan Karlsson Corresponding Author Stefan Karlsson Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden Search for more papers by this author Author Information Jonas Larsson1, Marie-José Goumans2, Lottie Jansson Sjöstrand1, Marga A. van Rooijen3, Dorien Ward3, Per Levéen1, Xiufeng Xu1, Peter ten Dijke2, Christine L. Mummery3 and Stefan Karlsson 1 1Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Lund, Sweden 2Netherlands Cancer Institute, Amsterdam, The Netherlands 3Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1663-1673https://doi.org/10.1093/emboj/20.7.1663 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Deletion of the transforming growth factor β1 (TGF-β1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-β more precisely, we inactivated the TGF-β type I receptor (TβRI) gene by gene targeting. Mice lacking TβRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TβRI−/− yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TβRI−/− mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TβRI-deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TβRI is crucial for the function of TGF-β during vascular development and can not be compensated for by the activin receptor-like kinase-1 (ALK-1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF-β signaling via TβRI. Introduction Transforming growth factor β (TGF-β) belongs to a large superfamily of structurally related polypeptides that includes the activins, bone morphogenetic proteins (BMPs) and the growth differentiation factors (GDFs) (Massagué, 1998). TGF-β regulates biological processes as diverse as embryonic development, cell proliferation, differentiation, migration and survival, extracellular matrix (ECM) production and immunomodulation (Roberts and Sporn, 1990; McCartney-Francis and Wahl, 1994). TGF-β is also a very important mediator of vascular development and hematopoiesis, and is generally regarded as having inhibitory effects on endothelial cells and hematopoietic progenitors (Pepper, 1997; Fortunel et al., 2000). However, the actions of TGF-β are complex and largely dependent on the context of individual target cells (Sporn and Roberts, 1988); in most in vitro assays, TGF-β acts as an inhibitor of endothelial proliferation and migration (Muller et al., 1987; Merwin et al., 1991) and a potent stimulant of ECM accumulation (Ignotz et al., 1987). There are three isoforms of TGF-β in mammals (TGF-β1, 2 and 3). Mice lacking TGF-β1 exhibit two different phenotypes. Approximately 50% of the mice die at midgestation due to defects in yolk sac vasculogenesis and hematopoiesis (Dickson et al., 1995). The other embryos survive beyond birth, possibly through maternal transfer of TGF-β1 (Letterio et al., 1994), but then develop a severe multifocal inflammatory disorder and die at 3–4 weeks of age (Shull et al., 1992; Kulkarni et al., 1993). TGF-β2 null mice have many developmental defects and die perinatally due to congenital cyanosis (Sanford et al., 1997), while TGF-β3 null mice have delayed lung development and die shortly after birth (Kaartinen et al., 1995; Proetzel et al., 1995). Members of the TGF-β family signal through a heteromeric complex consisting of two types of transmembrane serine/threonine kinases known as type I and type II receptors. Upon ligand binding, type II receptors recruit and transphosphorylate type I receptors, subsequently activating specific intracellular Smad transcription factors (Massagué, 1998). TβRI, also termed activin receptor-like kinase 5 (ALK-5), is a widely expressed type I receptor for TGF-β (Franzén et al., 1993), which can induce the phosphorylation of Smad2 and Smad3. Evidence that TGF-β signaling is of importance for development of the vascular system and hematopoiesis derives from the comparison of mouse embryos lacking either the ligand, TGF-β1 or TβRII. Like TGF-β1 null embryos, TβRII-deficient embryos die at midgestation as a result of defects in yolk sac vasculogenesis and hematopoiesis (Dickson et al., 1995; Oshima et al., 1996). Accumulating data suggest that there is an additional type I receptor for TGF-β in the vascular system. In human umbilical vein endothelial cells, TGF-β has been shown to bind to the endothelial-specific ALK-1 in a heteromeric complex with TβRII (Attisano et al., 1993; ten Dijke et al., 1994; Roelen et al., 1997; Lux et al., 1999; Oh et al., 2000), as well as the more usual TβRI–TβRII complex. In addition, ALK-1-deficient mice die around midgestation, exhibiting severe vascular abnormalities (Oh et al., 2000) reminiscent of mice lacking TGF-β1 or TβRII (Dickson et al., 1995; Oshima et al., 1996). Interestingly, in contrast to TβRI, ALK-1 activates Smad1 and Smad5 (Oh et al., 2000). Thus, TGF-β may signal in endothelial cells through two pathways: one via TβRI, which results in phosphorylation of Smad2 and Smad3, and the other via ALK-1, with the potential to phosphorylate Smad1 and Smad5. In addition to the TGF-β serine/threonine kinase receptors, TGF-β binds to an accessory receptor in endothelial cells, termed endoglin. Interestingly, mutations of endoglin and ALK-1, but not TβRI and TβRII, have been linked to a vascular disorder known as hereditary hemorrhagic telangiectasia (McAllister et al., 1994; Johnson et al., 1996). In a previous study we sought to examine in more detail the mechanisms through which TGF-β regulates vascular development in the yolk sac (Goumans et al., 1999). By overexpressing either a full-length or a truncated, dominant-negative TβRII in chimeric embryos we demonstrated an important role for TGF-β in maintaining yolk sac integrity (Goumans et al., 1999). Since interfering with TβRII will inhibit all TGF-β signaling, both downstream of TβRI as well as ALK-1, we dissected further the function of TGF-β signaling in yolk sac hematopoiesis and vascular development by generating mice deficient in TβRI. The defects seen in the TGF-β1 and TβRII null embryos suggested a role for TGF-β as an essential regulator of yolk sac hematopoiesis (Dickson et al., 1995; Oshima et al., 1996). We report here that mice lacking TβRI die at around E10.5 due to defects in vascular development of the yolk sac. Surprisingly, the hematopoietic potential was not deficient in these mice; on the contrary, we observed the presence of a normal number of CFU-GM and CFU-mix myeloid progenitor colonies and a large increase in the number of erythroid colonies from mutant yolk sacs compared with controls. Endothelial cells isolated from TβRI-deficient mice exhibited impaired fibronectin production and migration properties that most likely contribute to the abnormal vessel formation. Interestingly, all of the parameters commonly associated with TGF-β signaling and its biological responses were completely blocked in the endothelial cells lacking TβRI, suggesting that TβRI is in fact essential for TGF-β signaling in endothelial cells, despite the presence of ALK-1. Thus, our mouse model has provided important new insights into TGF-β signal transduction and has identified apparently independent mechanisms through which TGF-β regulates vascularization and hematopoiesis during development. Results Loss of TβRI results in embryonic lethality To study the function of TβRI in vivo, a targeting vector for removing exon 3 through Cre/loxP recombination was designed (Figure 1A). Since exon 3 spans the transmembrane domain and the so-called GS domain that is essential for TβRI activation by TβRII (Wieser et al., 1995), it was predicted that deletion of this exon would generate a null allele. After homologous recombination and subsequent Cre transfection, two successfully targeted and Cre-deleted embryonic stem (ES) cell clones were injected into C57BL/6 blastocysts and germline transmission of the null mutation was obtained (Figure 1A–C). Endothelial cells isolated from homozygous mutant embryos were analyzed for receptor expression in a binding assay with [125I]TGF-β1 to confirm that TβRI was absent. Antisera specific for TβRI or TβRII immunoprecipitated a TβRI–TβRII heteromeric complex in the wild-type or heterozygous endothelial cells, but in the homozygous null cells only TβRII was precipitated (Figure 1D). TβRI mRNA levels were determined in homozygous mutant endothelial cells using northern blot analysis. TβRI mRNA was undetectable using a full-length TβRI cDNA probe, indicating that the exon 3 deletion generates an unstable mRNA (Figure 1E). In order to determine whether very low levels of mutated TβRI mRNA might generate a soluble TβRI receptor that could exert a dominant-negative effect, conditioned medium from endothelial cells derived from homozygous mutant embryos was tested for interference with TGF-β signaling in a sensitive TGF-β transcriptional read-out assay (Dennler et al., 1998). Conditioned medium (5 days) from the mutant endothelial cells had no effect on the induction of the reporter gene by TGF-β in HepG2 cells (data not shown). Thus, the possibility of a dominant-negative effect in the mouse through the TβRI mutant allele appears very unlikely. Figure 1.Targeting of the TβRI gene. (A) The TβRI wild-type locus, the targeting vector, the targeted allele and the Cre-loxP recombined allele. The targeting vector was generated by inserting a loxP-flanked neomycin (neo) cassette in the Bsu36 site upstream of exon 3 and a single loxP in the NheI site downstream. A thymidine kinase (tk) gene was placed at one end of the construct for negative selection against random integration. Transient expression of the Cre enzyme allowed excision of exon 3 and the neo gene in the targeted allele. Exons are indicated by filled boxes and loxP sites by filled arrows. PCR primers for screening are indicated by open arrows and numbers: 1, RI5′; 2, loxdown; 3, lnl3′; 4, lnl5′; 5, llox3′. Restriction enzymes: Bs, Bsu36; EI, EcoRI; EV, EcoRV; K, KpnI; N, NotI; Nh, NheI. (B) PCR screening for homologous recombination of the targeting vector using the 5′ external primer RI5′ (1) and the vector-specific primer loxdown (2). (C) Genotyping by PCR of an E9.5 litter from a heterozygous intercrossing. The three primers lnl3′ (3), lnl5′ (4) and llox3′ (5) generate specific bands for the wild-type (0.15 kb, primers 3 and 4) and the deleted alleles (0.35 kb, primers 3 and 5). (D) Analysis of TGF-β receptor expression on endothelial cells. Endothelial cells, isolated from wild-type, heterozygous or TβRI−/− embryos were grown to confluency, affinity labeled with [125I]TGF-β1, followed by cross-linking. Cell lysates were subjected to immunoprecipitation using antisera against TβRI or TβRII. (E) Northern blot analysis of total RNA from endothelial cells derived from wild-type and homozygous mutated embryos hybridized with a full-length TβRI cDNA probe. GAPDH mRNA was measured to control for equal loading. Download figure Download PowerPoint Mice heterozygous for the mutated TβRI allele (TβRI+/−) were fertile and appeared phenotypically normal during development and beyond, also supporting the view that the exon 3 deletion does not generate a soluble TβRI receptor that could exert a dominant-negative effect. Genotyping offspring from heterozygous intercrosses showed only heterozygous and wild-type pups, indicating that TβRI homozygous mutants died during embryogenesis. To determine when death had taken place, embryos from heterozygous intercrosses were analyzed at different gestational stages. At E8.5, homozygous embryos were indistinguishable from their littermates. At E9.5, all TβRI mutants were alive, but had developed severe abnormalities. A majority of the mutant embryos recovered by E10.5 were dead and no live homozygous mutants were recovered after E11.5 (Table I), suggesting that TβRI−/− embryos die at around E10.5. Table 1. Genotype analysis of embryos isolated from TβRI+/− intercrosses Age +/+ +/− −/− Total E8.5 7 (7) 13 (13) 7 (7) 27 E9.5 29 (28) 55 (55) 22 (22) 77 E10.5 8 (8) 14 (14) 7 (2) 29 E11.5 9 (9) 20 (20) 8 (0) 37 Post-natal 60 136 0 196 Numbers in parentheses indicate live embryos. TβRI−/− embryos have abnormal vascular development At E9.5, the yolk sacs of TβRI−/− embryos were markedly anemic and lacked a distinct branching network of vessels. This became even more pronounced by E10.5 (Figure 2A). Some embryos exhibited areas of accumulated red blood cells in the yolk sac and hemorrhagic spots on the amnion, indicating leakiness or dilation of the vitelline vessels (Figure 2B). We also detected accumulated blood cells in the vitelline vessels connecting the yolk sac and the embryo proper, suggesting that circulation would be impaired. A circulation defect, causing elevated intracardial pressure, would also explain the pericardial effusion observed in most mutants (Figure 2A). All TβRI−/− embryos were developmentally retarded compared with their littermates at E9.5. They were reduced in size, rarely developing beyond 20 somites, and some embryos had not turned or were still in the process of turning (Figure 2B and C). Histological sections of wild-type and mutant yolk sacs revealed that vessel-like structures were indeed formed in mutants, but they appeared enlarged and fragile and contained few red blood cells compared with controls (Figure 2D and E). Staining E9.5 sections with anti-smooth muscle cell actin antibody showed that smooth muscle cells had started to surround the endothelial-lined vessels of the yolk sac in wild-type and TβRI+/− embryos (Figure 2D), while this was clearly not the case in the TβRI−/− mutants at this stage (Figure 2E), or even 1 day later (not shown). These results are compatible with expression of TβRI mRNA at E7.5, the time at which or just before the precursors of endothelial and hematopoietic cells develop (Figure 2J), as well as in the yolk sac mesoderm 1 day later (Goumans et al., 1999). Sections through the placenta demonstrated a defect in vascularization in TβRI−/− embryos, with embryonic vessels apparently unable to sprout properly into the labyrinthine layer (Figure 2G). The vasculature derived from the extra-embryonic tissue of controls had clearly infiltrated the maternal tissue at E9.5 (Figure 2F), the allantoic vessels were dilated and surrounded by smooth muscle cells (Figure 2F); this allows intermingling of fetal and maternal blood cells. Both endothelial cells and smooth muscle cells of the placenta express TβRI (data not shown). Allantoic vessels in the placentae of the TβRI−/− embryos, on the other hand, were significantly smaller, and smooth muscle cells did not invade the deeper-lying labyrinthine layers through the ectoplacental plate (open arrowheads in Figure 2G), which appeared to act as a barrier precluding mixing of fetal and maternal blood. Intermingling of embryonic and maternal vessels in this layer is necessary for proper exchange of nutrients between the two circulations, but is probably not the cause of death since embryos are not dependent on placental exchange at E9.5 (Cross et al., 1994) when the mutants already have severe defects. These defects included abnormalities in the neural tube, which was kinked (not shown) and, in histological sections, dilated and thin (Figure 2H and I), particularly ventrally. Again, in situ hybridization showed that TβRI is expressed in the neural tube at E8.5 (Figure 2K), just before the phenotype becomes evident, and is coincident ventrally with expression of TβRII, described previously (Wang et al., 1995). This makes it likely that the neural tube defects are a direct result of the lack of TβRI expression in cells of the neural tube and are not an indirect effect, secondary to impaired yolk sac circulation. Impairment of circulation at this stage can result in hypoxia causing dilation of internal cavities in the embryo (Iyer et al., 1998), including that of the neural tube and heart sac, somewhat similar to that observed here. Figure 2.TβRI−/− embryos exhibit severe defects in vascular development. (A) Gross morphology of whole-mount yolk sacs in mutant embryos compared with heterozygous littermates. Note the enlarged pericardial sac (arrowhead). (B) Defects in TβRI−/− embryos. Some embryos have not turned (upper left). Asterisk, accumulated blood in vitilline vessels connecting the yolk sac and the embryo proper. Arrowheads, leakage of red blood cells and dilated vascular structures. (C) By E9.5 mutant embryos are severely growth retarded. (D–G) Immunohistochemical staining for smooth muscle cell actin in transverse sections of mutant embryos compared with heterozygous littermates. (D) Section through the yolk sac of a heterozygote at E9.5. Endothelial cells are either in close apposition with the visceral endoderm or contain red blood cells within the developing vessel. Smooth muscle cells, stained brown, begin to differentiate from mesenchyme and contribute to the structure of the vessel wall. (E) Section through the yolk sac of a mutant at E9.5. The vessels are lined with endothelial cells but are dilated, and have very few red blood cells and no smooth muscle cells. (F) Section through chorioallantoic region of the placenta of a heterozygote at E9.5 showing smooth muscle cells surrounding the dilated allantoic blood vessels derived from extra-embryonic tissue and their invasion of the labyrinthine part of the placenta (maternal part). This allows intermingling of the fetal (large arrowheads) and maternal (small arrow) red blood cells. (G) Allantoic blood vessels in the placenta of a mutant at E9.5 are much smaller, there are fewer smooth muscle cells and there is no evidence for their invasion of the maternal labyrinthine layer through the ectoplacental plate. The limit of ectoplacental plate is indicated by the row of open arrowheads in (G). (H and I) Histological sections of the neural tube at the level of the heart in a heterozygote (H) and homozygote (I) at E8.5. (J) Expression of TβRI at E7.5 in the extra-embryonic part of the conceptus. (K) Expression of TβRI in the rostral neural tube at the level of the heart at E8.5. Abbreviations: av, allantoic blood vessel; b, branchial arch; ch, chorion; D, dorsal; e, endothelial cell; ep, ectoplacental plate; h, heart; nt, neural tube; pl, placenta; rbc, red blood cell; smc, smooth muscle cells; V, ventral; ve, visceral endoderm; xm, extra-embryonic mesoderm; xn, extra-embryonic endoderm. Scale bars, 1 mm (A–C), 50 μm (D–E), 100 μm (F–K). Download figure Download PowerPoint Taken together, our observations suggest that the impaired yolk sac circulation is the primary cause of death in TβRI−/− embryos. The presence of vessels in the yolk sacs of the mutant embryos demonstrates that the formation of a primary capillary plexus in situ (vasculogenesis) (Risau, 1997) does occur, although the resulting vasculature is defective. However, the lack of vessel sprouting in the placenta indicates that branching from pre-existing vessels (angiogenesis) is inhibited. Progenitors from TβRI−/− yolk sacs have increased erythroid colony-forming ability The phenotype of the TβRI−/− embryos is similar to the TGF-β1−/− embryos, where defects in vascular development and hematopoiesis were reported (Dickson et al., 1995). The very low numbers of red blood cells detected in the yolk sac vessels of TβRI−/− embryos (Figure 2E) prompted us to examine further hematopoietic function in these embryos. This is also of interest since TGF-β is thought to play an important role in regulating proliferation of hematopoietic progenitors (Keller et al., 1992; Sitnicka et al., 1996; Fortunel et al., 2000). We therefore determined whether hematopoietic progenitors developed and/or were present in the yolk sacs of mutant embryos. We tested the ability of cell suspensions from mutant and control yolk sacs to form hematopoietic colonies in an in vitro clonogenic assay (Wong et al., 1986). No significant difference was observed in the ability to form myeloid colonies (CFU-GM and CFU-mix), whereas a large increase in the number of pure erythroid colonies (CFU-Ery) was observed in mutants compared with controls (Figure 3). This suggests that the lack of blood cells in TβRI−/− yolk sacs is not due to an intrinsic defect in hematopoiesis, but rather that the yolk sac environment does not permit proper differentiation and maturation of hematopoietic precursors and their progeny. Figure 3.Increased erythroid and normal myeloid potential in precursors from TβRI−/− yolk sacs. Yolk sacs were collected from E9.5 embryos, dispersed, and equal numbers of cells plated in methylcellulose supplemented with growth factors. Colonies were scored 6 days later. Data from three different experiments including seven litters and 11 mutant embryos. CFU, colony forming unit; Ery, erythroid; Mix, erythroid-myeloid; GM, myeloid (granulocyte–macrophage). Download figure Download PowerPoint TβRI mediates TGF-β signaling in endothelial cells To gain more insight into the mechanism underlying the phenotype, we isolated and immortalized endothelial cells and fibroblasts from embryos of heterozygous crossings. TβRI could not be detected in homozygous mutant endothelial cells (Figure 1D). We used a Smad3/Smad4 binding element (so-called CAGA) coupled to a luciferase reporter as a TGF-β transcriptional read-out assay (Dennler et al., 1998). As shown in Figure 4A, activation of this reporter is dependent on the presence of at least one functional TβRI allele in both fibroblasts and endothelial cells. The transcriptional activation of the CAGA-reporter was completely restored by re-introducing the TβRI in the knockout endothelial cells. Consistent with the lack of transcriptional activation upon TGF-β stimulation due to lack of Smad phosphorylation by the TβRI, a band of phosphorylated Smad2 in TGF-β-stimulated wild-type endothelial cells was not observed in homozygous mutant cells (Figure 4B). Thus, all signaling responses measured here were completely blocked in TβRI null endothelial cells. Figure 4.TβRI mediates TGF-β signaling in endothelial cells. (A) TGF-β-induced transcriptional activation of (CAGA)12 luciferase reporter is impaired in TβRI mutant cells. Cells were transfected and stimulated for 16 h without (light) or with (dark) TGF-β1, followed by the measurement of the luciferase activity in the cell lysates. In the case of the TβRI-deficient endothelial cells, an expression plasmid for TβRI was co-transfected (+ALK-5) to demonstrate that introduction of exogenous TβRI is sufficient to restore the TGF-β-induced transcriptional activity. (B) TGF-β-induced Smad phosphorylation is impaired in TβRI−/− endothelial cells. Endothelial cells, isolated from either TβRI knockout embryos or wild-type littermates, were serum starved and stimulated with TGF-β1 for 1 h. Samples were transferred and incubated with an antiserum against phosphorylated Smad2 (PS-2). Equal levels of Smad2 protein in each lane are shown. The asterisk indicates a non-specific band. Download figure Download PowerPoint No TGF-β induced fibronectin production in TβRI−/− endothelial cells One important target gene for TGF-β is fibronectin, an ECM molecule that is present in large amounts between the visceral endoderm and extra-embryonic mesoderm layers of the visceral yolk sac. The ECM components are critical for stabilizing the developing vasculature. The composition of the ECM also plays an important role in the regulation of endothelial precursors, and determines whether these cells will proliferate and migrate or whether they will differentiate and form a vascular network (Baldwin, 1996). We therefore determined the capacity of the endothelial cells to produce fibronectin. Upon TGF-β stimulation, fibronectin production was enhanced in the heterozygous cell line but no increase was seen in the TβRI−/− endothelial cells (Figure 5A). Histological sections of embryos were stained with an anti-fibronectin antibody to determine whether fibronectin production was reduced in vivo. Figure 5B indeed shows a reduction in fibronectin deposited in the yolk sac in a representative section through a TβRI−/− embryo, compared with that in a heterozygote. This indicates that there is reduced fibronectin production in the mutant embryos, which could affect vessel formation both at the level of vasculogenesis, by reduced ECM support to the vascular plexus, and at the level of angiogenesis, through dysregulation of endothelial precursors. These results also suggest a crucial role for TβRI in TGF-β-mediated regulation of ECM deposition by endothelial cells in the visceral yolk sac. Figure 5.No TGF-β-mediated fibronectin synthesis and growth inhibition in TβRI−/− endothelial cells. (A) Endothelial cells were serum starved and stimulated with TGF-β1. Samples were electrophoresed, transferred and incubated with an anti-fibronectin antibody (FN). (B) Representative histological sections of yolk sacs from heterozygous (+/−, top panel) and TβRI−/− (−/−, bottom panel) embryos were stained with an anti-fibronectin antibody. Arrowheads indicate discontinuities in fibronectin deposition under the visceral endoderm around the vessels in the mutant embryo. Abbreviations (e, rbc and ve) are as in the legend to Figure 2. The scale bar is 50 μm. (C) Endothelial cells were treated with 5 ng/ml TGF-β1 for 24 h and [3H]thymidine was added for an additiona
