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(Circulation Research. 2000;86:1024.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Cardiovascular Overexpression of Transforming Growth Factor-ß₁ Causes Abnormal Yolk Sac Vasculogenesis and Early Embryonic Death

Ramtin Agah, K. S. Srinivasa Prasad, Ruth Linnemann, Meri T. Firpo, Thomas Quertermous, David A. Dichek

From the Gladstone Institute of Cardiovascular Disease, San Francisco, Calif (R.A., R.L., D.A.D.); the Departments of Medicine (R.A., D.A.D.) and Obstetrics, Gynecology, and Reproductive Sciences (M.T.F.) and the Cardiovascular Research Institute (R.A., D.A.D.), University of California, San Francisco; and the Department of Medicine, Stanford University, Stanford, Calif (K.S.S.P., T.Q.).

Correspondence to David A. Dichek, MD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100. E-mail ddichek{at}gladstone.ucsf.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Transforming growth factor-ß₁ (TGF-ß₁) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-ß₁ expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-ß₁ in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-ß₁ is expressed from the SM22 promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22–ß-galactosidase transgene. SM22– ß-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22–TGF-ß₁ transgenic mice died before E11.5. SM22–TGF-ß₁ transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-ß₁ from the SM22 promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-ß₁ expression in adults may require a conditional transgenic approach. Moreover, because the SM22 promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22 promoter to drive expression of "vasculoactive" transgenes may be particularly likely to cause embryonic death.

Key Words: blood vessels • transgenic mice • morphogenesis • hematopoiesis

	Introduction

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Transforming growth factor-ß₁ (TGF-ß₁) is a multifunctional cytokine expressed in the embryonic and adult vasculature.^{1 2 3} Correlational studies suggest a role for altered TGF-ß₁ expression in important aspects of vascular development and disease, including vasculogenesis,¹ angiogenesis,⁴ atherosclerosis,⁵ and restenosis.⁶ Nevertheless, the precise role of TGF-ß₁ in these processes remains elusive. For example, several studies support an atherogenic role for vascular TGF-ß₁,^{3 7 8 9} whereas others support an atheroprotective role.^{10 11 12}

Direct experimental approaches to elucidate the role of TGF-ß₁ in the vasculature have included in vivo gene transfer of TGF-ß₁^{7 9} and infusion of TGF-ß₁ antagonists.^{8 13} These studies suggest that vascular TGF-ß₁ accelerates neointimal formation after acute arterial injury; however, the role of TGF-ß₁ in other aspects of arterial lesion development is less well defined. Targeted deletion of the TGF-ß₁ gene has not been a useful approach to delineate the function of TGF-ß₁ in the adult vasculature because of embryonic and early postnatal lethality in TGF-ß₁–null mice. A significant proportion of TGF-ß₁–null embryos have defective yolk sac vasculogenesis leading to embryonic death at approximately embryonic day (E) 10.5,¹⁴ whereas surviving embryos develop a lethal inflammatory disease.^{15 16}

Here, we report results of a transgenic approach to investigate the role of TGF-ß₁ in vascular development and disease. Vasculature-specific overexpression of TGF-ß₁ in transgenic mice (along with appropriate breeding and dietary manipulations) could help determine whether increased arterial wall TGF-ß₁ protected against or accelerated atherosclerosis. To achieve this goal, we generated mice in which a TGF-ß₁ transgene is expressed from the SM22 promoter, which can target gene expression to smooth muscle cells in large arteries of transgenic mice.^{17 18}

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of Transgenes
Two transgenes were constructed. The first contained a 1.3-kb constitutively active porcine TGF-ß₁ cDNA¹⁹ fused to a 2.8-kb fragment of the murine SM22 promoter¹⁷ (Figure 1A). The second transgene contained the same SM22 promoter fused to a "floxed" ß-galactosidase (ß-gal) spacer upstream of the TGF-ß₁ cDNA. The ß-gal transgene was used to identify the pattern of expression from the SM22 promoter. The loxP sites were included to allow use of this same construct (in experiments beyond the scope of the present study) to achieve conditional, spatially restricted expression of TGF-ß₁ after removal of the ß-gal gene by digestion with Cre recombinase.

View larger version (7K): [in this window] [in a new window]   Figure 1. Constructs for generation of transgenic mice. A, A 2.8-kb fragment of the murine SM22 promoter fused to a cDNA encoding a constitutively active form of TGF-ß1. B, A 2.8-kb SM22 fragment was fused to a nucleus-targeted LacZ gene (nLacZ) surrounded by loxP restriction sites and placed upstream of the TGF-ß1 cDNA.

Generation and Screening of Transgenic Mice
Transgenic mice (C57BL6/JxSJL hybrids) were generated by standard methods.²⁰ In some cases, pregnant mothers were killed by cervical dislocation, and the embryos and yolk sacs were removed surgically. The tissues were either fixed and embedded in paraffin or processed for DNA extraction without fixation.

Mice were genotyped both by Southern blotting of unamplified DNA²¹ and by agarose-gel analysis of DNA amplified by polymerase chain reaction. For Southern blotting, DNA was prepared from postnatal tail tissue, whole embryos, or whole yolk sacs. For polymerase chain reaction–based genotyping, DNA was extracted from 3 to 5 sections 10 µm thick cut from paraffin-embedded embryos or yolk sacs.²²

Northern Analysis
Total RNA was extracted from descending aortas with RNAzol (Tel-Test), separated by formaldehyde–agarose gel electrophoresis,²¹ blotted, and hybridized to cDNA probes for TGF-ß₁ and GAPDH.²³

Histological Analysis
The cross-sectional areas of yolk sac blood islands were measured in 2 E8.5 transgenic and 2 E8.5 nontransgenic littermate embryos with a computer-assisted image-analysis system (Image 1/AT, Universal Imaging Corp).

TGF-ß₁ Immunostaining
Expression of TGF-ß₁ was detected with the LC1-30 antibody.²⁴

Detection of ß-gal Expression
Expression of ß-gal in embryos and yolk sacs was detected by X-gal staining of fixed tissues.²⁵

Yolk Sac Hematopoietic Precursor Colony Assays
Yolk sacs from E9 embryos were processed as described for detection of precursors.²⁶ Colonies were counted 1 week after plating and scored as granulocyte/macrophage (CFU-GM) or erythroid (BFU-E).

Statistics
Frequencies of transgenic mice were evaluated with the ² test. Other statistical analyses were performed with the unpaired t test. Statistical significance was set at P<0.05. Data are expressed as mean±SD.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Expression of TGF-ß₁ From the SM22 Promoter Causes Embryonic Death
We genotyped 56 postnatal mice from 10 litters of potential SM22–TGF-ß₁ founders. Only 1 mouse carried the SM22–TGF-ß₁ transgene (1.8%). Because the overall frequency of generating transgenic founder mice in our facility is 10% to 20%, we suspected that the SM22–TGF-ß₁ transgene was embryonically lethal and that the single founder survived because of lack of expression. Indeed, Northern analysis of the aorta of the single founder did not reveal a TGF-ß₁ transcript.

We performed additional microinjections and genotyped 227 embryos at E7.5 to E12.5 (Table 1). Transgenic embryos were present at the anticipated frequency (11% to 19%) at early time points; however, no transgenic embryos were detected after E10.5.

View this table: [in this window] [in a new window]   Table 1. Frequency of Transgenesis After Injection of SM22–TGF-ß1 Transgene

SM22 Promoter Directs Tissue-Specific Transgene Expression
To exclude nonspecific toxicity as a cause of embryonic death and to confirm that the 2.8-kb SM22 sequence directed transgene expression in the anticipated pattern,^{17 18} we injected zygotes with a construct in which the SM22 promoter drives expression of the ß-gal marker gene (Figure 1B). We genotyped 69 postnatal mice from 17 litters of potential SM22–ß-gal founders. Nine transgenic mice were identified (13%; P=0.03 versus frequency of live-born transgenic mice from SM22–TGF-ß₁ injections). We bred 2 of the SM22–ß-gal founders and examined the pattern of ß-gal expression in midterm embryos. Embryos from 5 litters (2 to 3 per line) harvested between E8.5 and E12 revealed ß-gal expression in the anticipated tissue-specific pattern: embryonic heart, blood vessels, and somites and yolk sac blood vessels (Figure 2A through 2C).

View larger version (88K): [in this window] [in a new window]   Figure 2. Use of the SM22 promoter to express ß-gal and TGF-ß1 in transgenic mice. A through C, Embryos derived from mating of a mouse transgenic for the SM22-nLacZ construct were collected at E9.0 and stained for detection of ß-gal expression. ß-gal expression is present in the embryonic heart, blood vessels, and somites and along the yolk sac vasculature. Inset (C) shows the everted yolk sac, in which the vasculature is more easily seen. D through I, Embryos harvested at E8.5 from pseudopregnant mothers that had been implanted with zygotes injected with the SM22–TGF-ß1 transgene. D, A transgenic yolk sac has several enlarged blood islands (arrows); no such islands are present in a nontransgenic yolk sac (E). F, Transgenic embryo is growth-retarded compared with a nontransgenic littermate (I). G, Transgenic blood island is dilated, highly cellular, and lacks septation. H, Nontransgenic blood islands are smaller, divided by septations (arrows), and have fewer cells per island.

SM22–TGF-ß₁ Transgenic Embryos Have Cellular, Dilated Yolk Sac Blood Islands and Are Resorbed in Midgestation
To identify a morphological phenotype associated with embryonic lethality in the SM22–TGF-ß₁ transgenic mice, we examined 73 of the E8.5 to E9.5 embryos. Most embryos appeared to be well developed; however, several were grossly abnormal. The most remarkable abnormality was the presence of large red areas on the yolk sac (apparently enlarged blood islands: Figure 2D; compare with Figure 2E). Yolk sacs with this appearance typically surrounded a growth-retarded embryo (Figure 2F; compare with Figure 2I).

To establish a phenotype-genotype correlation based on macroscopic features, we classified each of the 73 concepti as having a grossly abnormal embryo with either a normal or abnormal yolk sac, a grossly abnormal yolk sac with either a normal or abnormal embryo, or a grossly normal embryo and yolk sac. The embryos were genotyped, and the genotypes were correlated with the 3 phenotypes (Table 2). An abnormal embryo was present in 8 of 11 transgenic (73% sensitivity) and 17 of 62 of nontransgenic (62% specificity) concepti. In contrast, abnormal yolk sacs were found in 8 of 11 transgenic (73% sensitivity) but only 3 of 62 nontransgenic (95% specificity) embryos. Thus, applying a phenotypic criterion associating a grossly abnormal yolk sac at E8.5 to E9.5 with transgenesis correctly genotyped 67 of 73 (92%) of the embryos. In contrast, use of a grossly abnormal embryo as a criterion to predict genotype was correct in only 53 of 73 cases (73%). These data suggested that the transgene caused a primary yolk sac abnormality.

View this table: [in this window] [in a new window]   Table 2. Association of Phenotype With Genotype in E8.5 to E9.5 Concepti

Histological examination of transgenic yolk sacs revealed enlarged blood islands containing apparently normal hematopoietic cells (Figure 2G; compare with Figure 2H). The blood islands of transgenic mice were nearly 10 times larger in cross-sectional area than those of nontransgenic littermate yolk sacs (7500±2400 versus 990±170 µm²; n=5 each; P<0.01), suggesting a defect in organization of the primordial vascular cells that surround the blood islands. In contrast, blood vessels in the transgenic embryo proper appeared normal and were not dilated (not shown). Because of the well-described role of TGF-ß₁ in cardiac development,²⁷ we examined all transgenic hearts to identify potential abnormalities of cardiac chamber size, position, wall thickness, and trabeculation. Consistent with the general state of the transgenic embryos, we observed evidence of developmental delay and early resorption. However, we did not consistently identify specific cardiac abnormalities.

At E9.0, the SM22–TGF-ß₁ Transgene Is Expressed Primarily in the Heart
Although the data obtained with the SM22– ß-gal transgene (Figure 2A through 2C) provided indirect evidence that the SM22–TGF-ß₁ transgene was expressed in an appropriate tissue-specific pattern, we investigated this issue more directly by staining transgenic E9.0 embryos for active TGF-ß₁ protein. Intense positive staining for active TGF-ß₁ was present almost exclusively in the heart (Figure 3A). Faint positive staining was also present in the yolk sac vasculature. We did not observe staining in the somites, which could be a result of the sensitivity of the assay, the nature of TGF-ß₁ as a secreted rather than an intracellular protein, or the relatively late onset of SM22 expression in somites (E9.5) compared with the heart and vasculature (E8.0 to E8.5).^{17 18} Notably, nontransgenic, age-matched embryos also expressed TGF-ß₁ predominantly in the heart, although this staining was less intense than in the transgenic embryos (Figure 3B and 3C). These data confirm appropriate tissue-specific expression of TGF-ß₁ from the SM22 promoter.

View larger version (84K): [in this window] [in a new window]   Figure 3. Expression of TGF-ß1 protein in SM22–TGF-ß1 transgenic and nontransgenic embryos at E9.0. A, Immunostaining of a transgenic embryo reveals TGF-ß1 expression in the developing heart (box), which is also shown at higher magnification. Faint positive staining in the enlarged yolk sac blood island (arrowhead) is also present. B, Nontransgenic embryo also expresses TGF-ß1 primarily in the heart (box and C). Staining appears less intense than in the transgenic embryo.

SM22–TGF-ß₁ Transgene Does Not Alter the Frequency of Yolk Sac Clonogenic Progenitors
Because TGF-ß₁ has been implicated in the development of hematopoietic lineages¹⁴ and because the blood islands appeared highly cellular, we hypothesized that blood cell development might be altered in the SM22–TGF-ß₁ mice. We harvested embryos at E9.0 and tested the ability of the transgenic and nontransgenic yolk sac cells to form CFU-C and BFU-E. The yield of cells from collagenase-digested transgenic (n=5) and nontransgenic (n=12) yolk sacs was identical (3.3±1.0x10⁴ versus 3.7±1.4x10⁴). The CFU-C and BFU-E colony counts were also not significantly different (Figure 4).

View larger version (20K): [in this window] [in a new window]   Figure 4. Frequency of clonogenic hematopoietic precursors in nontransgenic (n=5) and SM22–TGF-ß1 transgenic (n=12) yolk sacs at E9.0. Myeloid (CFU-GM) and erythroid (BFU-E) colonies were counted 1 week after plating of yolk sac cells onto medium containing growth factors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used the SM22 promoter to drive expression, in transgenic mice, of either the ß-gal marker gene or an active TGF-ß₁ cDNA. Our major findings were that (1) transgene expression was present in the anticipated pattern: primarily in the heart at E9.0, but also in the embryonic and yolk sac vasculature; (2) expression of active TGF-ß₁ disrupted yolk sac vasculogenesis and caused embryonic lethality before E11.5; and (3) there was no detectable effect of TGF-ß₁ expression on cardiogenesis or hematopoietic precursor differentiation.

Establishing a precise mechanism for embryonic lethality is often difficult,²⁸ and the present case is no exception. Embryonic death at E8 to E11 is often associated with yolk sac abnormalities, including failed yolk sac hematopoiesis,²⁹ yolk sac hemorrhage,^{30 31} and deficient yolk sac angiogenesis.^{32 33 34} Before establishment of the chorioallantoic placenta at E10 to E11, the embryo is absolutely dependent on the yolk sac for nourishment. An insult that disturbs the ability of the yolk sac to perform this function will be lethal. Our conclusion that death of the SM22–TGF-ß₁ embryos by E10.5 is due to a primary yolk sac abnormality is consistent with the temporospatial pattern of SM22 expression: onset at E8.0 with expression in the yolk sac vasculature (Reference ¹⁸ and Figure 2C). This conclusion is supported by the high sensitivity and specificity of a gross yolk sac abnormality in predicting the embryonic genotypes (Table 2).

Localization of the lethal developmental defect to the yolk sac vasculature is far easier than pinpointing the precise cause of yolk sac failure. There was no evidence for hematopoietic failure in the transgenic mice. In addition, hematopoietic failure is an unlikely cause of embryonic deterioration as early as E9.0, because embryos at this stage do not yet depend on circulating blood cells. It is possible that yolk sac failure is due to impaired nutrient diffusion resulting from loss of total yolk sac vessel surface area (a small number of large yolk sac vessels has less total surface than a large number of small yolk sac vessels; compare Figure 2G and 2H). Alternatively, in view of the well-established ability of TGF-ß₁ to increase expression of matrix proteins, an increase in perivascular extracellular matrix in transgenic yolk sacs might account for decreased diffusion or transport of nutrients into the yolk sac vessels. We did not find histological evidence of increased yolk sac extracellular matrix with either hematoxylin/eosin (Figure 2G) or Movat’s pentachrome staining (not shown); however, this analysis might miss subtle abnormalities.

Our results are consistent with several studies suggesting that TGF-ß₁, acting by paracrine or autocrine mechanisms,³⁵ is a critical mediator of yolk sac vasculogenesis beginning at E8.5 to E9.5. By this time, both TGF-ß₁ and its high-affinity receptor (the type II TGF-ß receptor) are expressed in the yolk sac vasculature.^{1 14 36 37} Moreover, at least 3 induced mutations that disrupt TGF-ß₁ signaling are lethal at E9.5 to E11.5 and are accompanied by abnormalities in yolk sac vasculogenesis.^{14 36 38} The vascular defects in these mutant embryos (which are deficient in TGF-ß₁,¹⁴ the type II TGF-ß receptor,³⁶ or the TGF-ß–binding protein endoglin³⁸ ) include abnormal morphogenesis without evidence of impaired vascular cell differentiation. Similarly, in the present study, the yolk sac endothelium appeared to differentiate normally (on the basis of observations made on hematoxylin/eosin-stained sections and positive immunostaining for the endothelial marker Flk-1; not shown). However, transgenic yolk sacs exhibited aberrant vascular morphogenesis. It is of interest that targeted overexpression of TGF-ß₁ in the lung (in which the type II TGF-ß receptor is expressed at a high level)³⁹ also causes prenatal lethality.⁴⁰ In contrast, numerous other TGF-ß₁–transgenic embryos develop normally, including those with expression targeted to the liver, pancreas, and mammary gland.^{41 42 43} Forced, nonphysiological autocrine or paracrine activation of TGF-ß₁ signaling may explain lethality due to expression in the lung and yolk sac and suggests that overexpression of active TGF-ß₁ in a highly responsive tissue undergoing rapid morphogenesis is poorly tolerated.

To the best of our knowledge, this study is the first to report phenotypic consequences of expression of a biologically active transgene directed by the SM22 promoter. Previous reports, limited to expression of marker genes, showed that fragments of the SM22 promoter direct transgene expression specifically to smooth muscle cells of large arteries in late-term and postnatal mice.^{17 18} This pattern of transgene expression is far more vasculature-specific than that obtained with other smooth muscle–specific promoters, which also drive expression in extravascular tissues such as the intestine and urinary tract.⁴⁴ It is therefore logical to use the SM22 promoter to test hypotheses involving increases in gene expression in the artery wall. Our results suggest that the SM22 promoter may be useful for investigating yolk sac vascular development. However, this initial report sets a cautionary tone regarding use of SM22 to drive transgene expression in the adult vasculature. The SM22 promoter is expressed in the yolk sac at a time that the embryo is absolutely dependent on the yolk sac vasculature. Transgenes (such as TGF-ß₁) that are potent regulators of the blood vessel phenotype risk causing early embryonic death if expressed from the SM22 promoter. We are currently pursuing conditional transgenic strategies,⁴⁵ which should allow a TGF-ß₁ transgene to be silent during development but active during adult life. Conditional transgenic strategies, although technically challenging, seem to be required for investigations of the consequences of increased vascular TGF-ß₁ expression on adult arterial diseases, including atherosclerosis.

	Acknowledgments

 
This work was supported in part by grant HL-61860 (National Heart, Lung, and Blood Institute) and by an award from the Howard Hughes Medical Institute Research Resources Program at the University of California, San Francisco (to Dr Dichek). Dr Agah was supported by a National Research Service Award from the National Institutes of Health (T32-HL-07731). Dr Dichek is an Established Investigator of the American Heart Association. Plasmid pSM2735-lacZ was a kind gift of Dr Joseph Miano (University of Wisconsin, Madison). Plasmid pPK9a and the LC1-30 antibody were provided by Drs Anita Roberts and Kathleen Flanders (National Cancer Institute, Bethesda, Md). We thank Dale Newland for technical assistance, John C.W. Carroll and Neile Shea for help with graphics, Stephen Gonzalez and Chris Goodfellow for photography, and Gary Howard and Stephen Ordway for editorial advice.

Received February 28, 2000; accepted March 27, 2000.

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