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. 2001 Apr 2;20(7):1663-73.
doi: 10.1093/emboj/20.7.1663.

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice

J Larsson  1 , M J Goumans, L J Sjöstrand, M A van Rooijen, D Ward, P Levéen, X Xu, P ten Dijke, C L Mummery, S Karlsson
Affiliations

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice

J Larsson et al. EMBO J. .

Abstract

Deletion of the transforming growth factor beta1 (TGF-beta1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-beta more precisely, we inactivated the TGF-beta type I receptor (TbetaRI) gene by gene _targeting. Mice lacking TbetaRI 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 TbetaRI(-/-) yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TbetaRI(-/-) mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TbetaRI-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 TbetaRI is crucial for the function of TGF-beta 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-beta signaling via TbetaRI.

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Figures

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Fig. 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.
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Fig. 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).
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Fig. 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).
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Fig. 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.
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Fig. 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 additional 4 h to monitor DNA synthesis. The figure shows the mean [3H]thymidine incorporation of triplicate samples. (D) Cells were seeded in the absence or presence of TGF-β1. On day 3 and day 5 the total cell number per well was counted. Shown is a representative experiment giving the mean of triplicate samples.
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Fig. 6. Impaired migration of TβRI–/– endothelial cells. The migration assay was performed using a Boyden chamber and an 8 µm filter. Cells were added to the upper wells with or without FCS, and with or without TGF-β1. After 5 h, cells that had migrated to the lower wells were counted. The figure shows a representative experiment. Values are expressed as means of four wells.
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