Endothelial-specific ablation of Serum Response Factor causes hemorrhaging, yolk sac vascular failure, and embryonic lethality
© Holtz and Misra; licensee BioMed Central Ltd. 2008
Received: 25 July 2007
Accepted: 20 June 2008
Published: 20 June 2008
Serum response factor (SRF), a member of the MADS box family of nuclear transcription factors, plays an important role in cardiovascular development and function. Numerous studies demonstrate a central role for SRF in regulating smooth and cardiac muscle cell gene expression. Consistent with this, loss of SRF function blocks differentiation of coronary vascular smooth muscle cells from proepicardial precursors, indicating SRF is necessary for coronary vasculogenesis. The role of SRF in endothelial cell contribution during early vascular development, however, has not been addressed. To investigate this, we generated transgenic mice lacking expression of SRF in endothelial cells. Mice expressing Cre recombinase (Tie2Cre+) under Tie2 promoter control were bred to mice homozygous for Srf alleles containing loxP recombination sites within the Srf gene (Srff/f). Tie2 is a tyrosine kinase receptor expressed predominantly on endothelial cells that mediates signalling during different stages of blood vessel remodelling. Resulting embryos were harvested at specific ages for observation of physical condition and analysis of genotype.
Tie2Cre+/-Srff/fembryos appeared to develop normally compared to wild-type littermates until embryonic day 10.5 (E10.5). Beginning at E11.5, Tie2Cre+/-Srff/fembryos exhibited cerebrovascular hemorrhaging and severely disrupted vascular networks within the yolk sac. Hemorrhaging in mutant embryos became more generalized with age, and by E14.5, most Tie2Cre+/-Srff/fembryos observed were nonviable and grossly necrotic. Hearts of mutant embryos were smaller relative to overall body weight compared to wild-type littermates. Immunohistochemical analysis revealed the presence of vascular endothelial cells; however, vessels failed to undergo appropriate remodelling. Initial analysis by electron microscopy suggested a lack of appropriate cell-cell contacts between endothelial cells. Consistent with this, disrupted E-cadherin staining patterns were observed in mutant embryos.
These results provide the first in vivo evidence in support of a role for SRF in endothelial cell function and strongly suggest SRF is required for appropriate vascular remodelling.
Serum response factor (SRF) is a nuclear transcription factor of the MADS (MCM1, Agamous, Deficiens, SRF) box family. SRF interacts as a dimer with DNA at the serum response element (SRE), a 10 base pair AT-rich sequence [CC(AT)6GG] also known as the CArG box (for review see [1, 2]). The SRE sequence is present in a wide variety of genes, including those encoding for immediate early proteins (e.g. Fos, Jun, HSP70), neuronal nuclear receptors (e.g. Nurr1, Nur77) and numerous contractile and cytoskeletal proteins (eg. actins, myosins). Expression of SRF is essential in early development as SRF-null embryos die during gastrulation . SRF is required for development of mesoderm . The expression pattern of genes under SRF control is regulated in a combinatorial fashion by SRF's ability to interact with a variety of accessory factors such as Elk-1 and SAP-1 to regulate expression of genes involved in cell growth and proliferation, and myocardin and related family factors to control myogenic gene expression (for review see ).
SRF plays a critical role in myogenesis. Early studies established the expression of SRF in cells of the myogenic lineage [5–7], and SRF has been shown to be required for appropriate skeletal, cardiac and smooth muscle cell growth and differentiation [8, 9]. SRF gene knockout studies from our laboratory indicate that disruption of SRF in cardiomyocytes leads to severe defects in the contractile apparatus, including Z-disc and stress fiber formation, as well as mislocalization and/or attenuation of sarcomeric protein expression . Consistent with these observations, in vivo cardiovascular-specific knockout of SRF results in embryonic death via cardiovascular failure due to perturbations in normal muscle cytoarchitecture and contractile assembly [11–13]. Numerous studies suggest that disordered myogenic gene expression is due to defective SRF-mediated regulation of the smooth muscle cell-specific regulatory factor myocardin [14–16].
SRF is a key regulator in development of the coronary vasculature. Coronary vasculature is derived from a transient embryonic structure termed the proepicardium (PE) [17–19]. The PE is characterized as a small, grape cluster-like aggregate of cells developing at embryonic day 9 (E9) from an extension of the septum transversum, lying beneath the developing heart tube . Cells of the PE undergo an epithelial-to-mesenchymal transition (EMT) and migrate over the surface of the heart. Some cells form the primitive epicardium, and others appear to continue to infiltrate into the subepicardial space. Once dispersed into the heart tissue, these subepicardial mesenchymal cells (SEMC) differentiate into the vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC) that comprise the coronary vasculature . Previous work in our laboratory has demonstrated expression of SRF within cells of the PE and newly formed coronary vessels . Landerholm and colleagues  have shown that inhibitory SRF constructs block differentiation of avian PE to VSMC, indicating a central role for SRF in differentiation of PE-derived cells to VSMC.
A role for SRF in differentiation and function of VSMC is well established, however, little is known about the role of SRF in VEC [5, 23]. Using primary HUVEC cultures, Chai and colleagues  have shown that vascular endothelial growth factor (VEGF) signalling pathways require SRF. Endothelial cells in which SRF has been knocked down fail to respond to intercellular VEGF signalling and show abolished VEGF-induced in vitro angiogenesis, impaired endothelial cell migration and proliferation, and inhibited VEGF-induced actin polymerization and immediate early gene expression.
These studies indicate that SRF likely plays an important role in endothelial cell function. The in vivo role of endothelial SRF, however, has not been addressed. Therefore, in the current study we investigated the role of SRF in VEC function by carrying out endothelial cell-specific ablation of the SRF gene. Mice homozygous for Srf-Lox P (Srff/f) alleles were bred to mice expressing the Cre recombinase protein under Tie2 promoter control (Tie2Cre+/-). Tie2 is a vascular endothelial-specific tyrosine kinase receptor for the angiopoietin family of vascular remodelling factors [25, 26]. The onset of Tie2Cre expression is concurrent with the appearance of endothelium in E7.5 embryos and continues in adult tissues . In embryos expressing the mutant genotype Tie2Cre+/-Srff/f, we observed hemorrhaging by E11.5 and near complete lethality by E14.5. Examination of cardiac morphology revealed smaller hearts as well as mild ventricular-septal defects in mutant embryos as compared to wild-type littermates. Immunohistochemical analysis suggests VEC properly differentiated and assembled blood vessels. However, the appearance of blood pools and segmented blood vessels observed in embryos and yolk sacs of E11.5–E13.5 embryos indicated disrupted vascular networks consistent with a defect in vascular remodelling. Further analysis of yolk sac tissues by electron microscopy demonstrated a lack of desmosomal-type junction complexes as well as a paucity of collagen matrix between the endodermal and mesodermal cell layers. E-cadherin protein detected by immunofluorescence showed a dramatic decrease in signal along the apical brush-border of endothelial cells in the extraembryonic endodermal layer in yolk sac. These results provide the first in vivo evidence in support of a role for SRF in endothelial cell function and strongly suggest SRF is required for appropriate vascular remodelling.
Conditional ablation of SRF protein in endothelium causes hemorrhaging in developing embryos
Endothelial-specific disruption of the Srfgene during development causes embryonic lethality
Embryos with mutant genotype Tie2·Cre+/-Srff/fdie by E14.5
Hearts of Tie2Cre+/-Srf f/f mutant embryos are smaller
Hearts of mutant embryos appeared smaller than those of wild-type littermates. To assess this observation, whole body and isolated heart weights from E12.5 and E13.5 embryos were used to calculate heart weights as a percentage of overall body weight (Fig. 3E). The decrease in apparent size of mutant hearts appeared as early as E12.5 (compare Fig. 3A with 3B); however, differences in whole body and heart weights at this age did not reach statistical significance (1.5 ± 0.4 vs. 1.08 ± 0.5, wild-type vs. mutant respectively; p = 0.054). Normalized heart weights from E13.5 mutant embryos were reduced (1.4 ± 0.39% vs. 1.04 ± 0.17%, wild-type vs. mutant respectively; p = 0.021). Furthermore, immunofluorescent analysis of phosphorylated histone H3 in E11.5 embryos revealed an apparent reduction in the number of endocardial endothelial cells undergoing proliferation (see Additional file 1); no difference in apoptotic activity was detected (cleaved caspase 3 immunofluorescent analysis; data not shown). In contrast, the ratio of cardiomyocytes to total cells was not different between wild-type and mutant heart tissues (MF-20-immunolabelled cells as a percentage of total DAPI-positive nuclei; data not shown), suggesting that the heart size differences noted are not due to a selective loss of cardiomyocytes. E14.5 embryos with mutant genotype were grossly necrotic and unsuitable for analysis.
Yolk sac vasculature fails in Tie2Cre+/-Srf f/f mutant embryos
SRF protein is localized within endothelial cells
SRF protein is decreased in VEC from Tie2Cre+/-Srf f/f embryos
Since the Tie2Cre construct has been shown to be expressed in endocardial endothelial cells, we analyzed embryonic ventricular tissue for the presence or absence of SRF protein using single-plane confocal images as described above (see Additional file 2). We found a higher number of endocardial endothelial cells exhibiting SRF-positive immunofluorescence in wild-type as compared to mutant tissues. No significant change in expression of SRF protein was observed between wild-type or mutant embryos in the epicardium (Additional file 2). Furthermore, to verify that SRF protein expression in cardiomyocytes was not grossly affected by loss of SRF in endothelial cells, we analyzed SRF protein expression in embryonic heart tissues by immunodetection; however, no differences were noted (Additional file 2).
Early coronary vessel formation appears normal in mutant embryos
Yolk sac tissues from Tie2Cre+/-Srf f/f embryos retain VEC despite fragmentation of blood vessels
Furthermore, cross-sectional examination of Tie2Cre+/-Srff/fyolk sac tissues revealed delamination of the endodermal and mesodermal components of the yolk sac layers (Fig. 9C and 9D). Tissues immunolabelled by the VEC marker PECAM-1 showed separation of the endodermal and mesodermal tissues (compare Fig. 9C and 9D, asterisks). Also, some areas of laminar separation were observed to contain blood cells, suggesting these areas derived from either blood vessels or blood islands. Quantitation of the linear length of laminar separation in cross-sections shows that yolk sac tissues from mutant animals have a greater degree of laminar separation than wild-type littermates (see Fig. 9E; 16.3 ± 10.8% vs. 62.0 ± 10.4% wild-type vs. mutant; p = 0.003).
Mutant yolk sac tissues lack cell-cell junctions and intra-laminar collagen deposition
E-Cadherin is decreased in Tie2Cre+/-Srf f/f embryonic yolk sac tissues
Numerous studies have shown that SRF is a critical transcriptional regulator of genes important for VSMC differentiation and development, and therefore a key regulator of vascular development. Consistent with this, SRF is required for differentiation of PE-derived precursors to VSMC during coronary vascular development . We have previously shown, however, that SRF is expressed in the PE prior to detectable expression of VSMC markers , raising the possibility that SRF may play a role in differentiation of PE-derived coronary VEC as well. To begin to address this idea, we have sought to investigate the role of SRF in endothelial cells in vivo, and whether endothelial SRF is required for normal vascular development. Towards that end, we crossed mice expressing Cre recombinase under the VEC-specific Tie2-derived promoter with mice carrying floxed Srf alleles. Our data demonstrate that SRF plays a crucial role in appropriate VEC function beginning early in embryonic development.
Mutant embryos lacking SRF expression in VEC die by E14.5. Examination of heart structure reveals that hearts of Tie2Cre+/-Srff/fembryos undergo looping and chamber formation; however, hearts of mutant embryos are measurably smaller with increasing gestational age compared to wild-type littermates. We did not detect a difference in the percentage of cardiomyocytes in heart tissue between wild-type and mutant embryos despite noting a small decrease in cardiomyocyte proliferation. Analysis of apoptotic activity by cleaved caspase 3 immunoreactivity also showed no discernible difference between wild-type and mutant embryos. Based on the well-documented specificity of the Tie2Cre driver, it is unlikely that the small decrease in cardiomyocyte proliferation observed is due to direct Cre-mediated excision of the Srf allele in cardiomyocytes . One possible explanation for the reduction in cardiomyocyte proliferation in mutant embryos is that there is a defect in signalling to cardiomyocytes. In support of this idea, a variety of studies have demonstrated the importance of reciprocal signalling pathways between endocardial endothelial cells and the developing myocardium (for review see ). This raises the possibility that impaired signalling from SRF-null endocardial endothelial cells is contributing to reduced cardiomyocyte proliferation and the overall decrease in heart size observed in Tie2Cre+/-Srff/fembryos. The apparent reduction in endocardial cell proliferation we observed in mutant embryos further suggests that loss of SRF in endothelial cells results in impaired endocardial endothelial cell function; however, which endothelial signalling pathways are impaired in SRF-null cells remains to be determined.
Endocardial cushion tissues are present; however, septal tissues appear poorly formed in mutant embryos, and in some cases exhibit a failure to form ventricular septa. Nevertheless, in utero lethality lacking gross defects in heart structure supports the idea of vascular failure as a cause of death. Lethal vessel malfunction may be occurring within the atrial tree, embryonic or extra-embryonic vasculature, or yolk sac vasculature. Consistent with this, mutant embryos exhibit hemorrhaging within the head, abdomen, and limbs as early as E12.5, a time when blood vessel remodelling is occurring. Yolk sac vasculature is also disrupted, suggesting a common mechanism of vessel malfunction in both embryonic and extra-embryonic tissues.
Initial vessel formation in the heart begins at approximately E11.5 and is completed with establishment of mature coronary arteries by E16.5 . Early coronary vasculogenesis appears to occur normally in embryos lacking SRF in VEC as shown by the presence of PECAM-1-positive vessels in hearts from both wild-type and mutant E12.5 embryos. Nevertheless, mutant embryos die by E14.5, leaving the issue of SRF's role in coronary vasculature remodelling and maturation unclear. Determining the precise requirement for SRF in VEC specification is also complex since the Tie2Cre construct begins expression presumably after hemangioblast specification. Examination of blood smears from E12.5 embryos reveals no grossly observable differences between wild-type and mutant samples (data not shown). This may suggest that early VEC progenitors in the yolk sac are not influenced by excision of the Srf gene; however since the SRF protein has a relatively long half-life , we cannot rule out the possibility that residual SRF protein levels remain sufficient to sustain specification and differentiation of early VEC. These questions are better suited to investigation using in vitro model systems of VEC specification and differentiation, and are the object of ongoing studies.
While it is evident that SRF protein levels are reduced in VEC of mutant embryos, detectable SRF protein in both wild-type and Tie2Cre+/-Srff/fembryos suggests a significant degree of mosaicism. Also, the stringency of our method for quantitation of SRF protein in VEC may result in an underestimation of the number of cells displaying detectable SRF protein. We have observed varying degrees of Srf-floxed allele excision efficiency based on the Cre recombinase driver, as well as possible variability from cell to cell. This results in a mosaic expression pattern between individual cells; however, the phenotypic effects of the excision still manifest in the tissue as a whole. Nevertheless, it is likely that more complete ablation may result in a more severe phenotype.
Yolk sac vascular networks undergo extensive remodelling during the course of embryogenesis. In our study, yolk sac vascular networks appear to form normally, echoing what was observed in initial coronary vessel development. However, yolk sac vascular networks became severely disrupted by E12.5, indicating a failure of the blood vessels to remodel and mature. Disintegration of yolk sac vasculature was accompanied by separation of endodermal and mesodermal cell layers. Further analysis revealed a lack of desmosomal junctions and intralaminar collagen as well as dramatic loss of E-cadherin in cells of the endodermal layer. Anchoring-type desmosomal junctions are plentiful in tissues subject to persistent mechanical stress, such as yolk sac, skin, and heart. Proper junction formation relies on appropriate expression of cytoskeletal and cell surface anchoring proteins such as actins, cadherins, and integrins. Recent work in our laboratory has shown that SRF-null cardiomyocytes have decreased levels of a number of genes associated with cell adhesion and cytoskeletal function including integrins β1 and α9, tight junction protein ZO-1, and protocadherins 7 and 18 , raising the possibility that the same genes may be affected in SRF-null endothelial cells, resulting in deficient cell-cell contacts.
Previous research has shown cadherins are also required for pericyte-endothelial interactions during angiogenic sprouting . Furthermore, VE-cadherin, a major component of adherens junctions between endothelial cells, has been shown to promote angiogenesis . Consistent with these findings, putative SRF-binding sites have been identified in the VE-cadherin-2/protocadherin12 , integrin-β1, integrin-β1 binding protein 2, protocadherin7 and protocadherin18 genes . These observations suggest the involvement of SRF in regulation of cell-cell contacts; however as of yet, no experimental studies have addressed whether SRF is a significant player in these interactions, or whether SRF is a regulator of these genes in VEC.
Our results also raise the possibility that SRF may play a role in periendothelial cell/pericyte recruitment and vessel stabilization via involvement in angiopoietin (Ang) signalling. PECAM staining of yolk sac from Ang1-null mice shows a loss of vascular integrity similar to that seen in our study . Ang1, a member of the angiopoietin family of ligands , activates the Tie2 receptor by binding to it and inducing its tyrosine phosphorylation. Ang1 is generated by non-endothelial cells, such as VSMC and other vascular pericytes recruited to vessels. Early in development between E9 to E11, Ang1 is found most prominently in the heart myocardium surrounding the endocardium. Later, it becomes more widely distributed, most often detected in the mesenchyme surrounding developing vessels and in close association with endothelial cells. Ang1-dependent vessel stabilization occurs by supporting reciprocal interactions between the vascular endothelium, pericytes, and surrounding extracellular matrix and mesenchyme . Tie2 mediates Ang1 signalling via several known signalling pathways, including the Akt, RhoA/Rac1, MAPK, and ERK1/2 pathways . Since SRF is a known mediator of all these pathways [36–39], this suggests that a role for SRF in Tie-2 mediated angiogenic remodelling may also lie as a downstream effector in the Ang1/2 signalling cascade in endothelial cells.
The results presented here unequivocally establish SRF as a critical regulator of endothelial cell function in vivo and indicate that appropriate SRF protein expression is required for remodelling of vascular networks. However, these results do not directly address the role of SRF in endothelial specification and differentiation since the Tie2Cre construct is expressed in differentiated endothelial cells. Our results suggest that disruption of anchoring-type proteins may play a significant role in the observed vascular failure, although the precise underlying molecular mechanisms remain to be addressed. Development of reagents that allow functional analyses of SRF loss of in endothelial precursors as well as vessel maturation will be required to more fully address these questions.
Our study provides the first in vivo experimental evidence of a role for SRF in VEC function during embryonic development. Mouse embryos lacking SRF expression in endothelial cells die mid-gestation due to apparent vascular insufficiency. Initial analysis suggests a lethal malfunction in angiogenic remodelling and vessel maintenance. The extent to which this malfunction is due to failed reciprocal signalling between VEC and surrounding mesenchyme or perhaps incomplete vascular pericyte recruitment remains to be determined.
Mice and Genotyping
The Tie2Cre and Srff/ftransgenic mouse lines have been previously described [27, 40]. Previous work has demonstrated that expression of the Tie2Cre gene begins early at E7.5; expression has been noted in all endothelial and endocardial tissues as well as some hematopoietic tissues . Embryos were generated by timed mating, designating embryonic day 0.5 (E0.5) as noon on the day a vaginal plug was observed. Genotyping was performed under standard protocols using genomic DNA isolated from embryonic yolk sac, amnionic membrane, or tail tissue. Primers used were: Tie2Cre fwd 5'-GTTCGCAAGAACCTGATGGACA-3' and rev 5'-CTAGAGCCTGTTTTGCACGTTC-3'; Srff/ffwd 5'-TGCTTACTGGAAAGCTCATGG-3' and rev 5'-TGCTGGTTTGGmCATCAACT-3'; HPRT fwd 5'-AGCGCAAGTTGAATCTGC-3' and rev 5'-AGCGACAATCTACCAGAG-3'. All procedures were in compliance with the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
Quantitation of embryonic total body and isolated heart weights
Embryos were harvested from timed pregnant females and placed individually on a clean glass slide for weight determination; excess buffer fluid was absorbed by wicking with tissues prior to weighing. After whole body weight was recorded, the heart was dissected out and weighed separately. Amnionic membrane was reserved for genotypic analysis.
Histology, Immunohistochemistry, and Microscopy
Embryos were harvested from timed pregnant females, photographed, and then fixed in either 4% paraformaldehyde/PBS or Tris-buffered zinc fixative (0.1 M Tris pH7.4 with 3.2 mM calcium acetate, 22.8 mM zinc acetate, and 36.7 mM zinc chloride). Yolk sac tissues selected for whole mount immunostaining were processed as described . After staining, tissues were photographed and further processed for paraffin-embedding. All other samples were processed for paraffin-embedding, and subsequent sections (7 μm) were used for either hematoxylin & eosin staining, alcian blue staining, immunohistochemistry or confocal microscopy as described [42, 43]. Reagents utilized were: anti-E-cadherin (BD Bioscience, clone 36); anti-sarcomeric myosin (University of Iowa Developmental Studies Hybridoma Bank, clone MF-20); anti-PECAM-1 (BD Biosciences, clone MEC13.3); anti-phospho-Histone H3 (Millipore, Ser-10); anti-SRF (ProteinTech custom). Digital image capture was performed using a Nikon SM2100 microscope with a Nikon CoolPix995 camera (whole mount), Nikon Eclipse TE300 microscope with a SpotII digital camera (tissue sections), or a Leica TCS SP2 laser scanning confocal microscope imaging system (confocal imaging). For electron microscopy, embryos were harvested from timed pregnant females and fixed in glutaraldehyde/sodium arsenate buffer. Subsequent processing and image collection were performed by the Electron Microscopy Core Facility at the Medical College of Wisconsin.
We thank Dr. Mark Majesky and Dr. Simon Conway for valuable discussions, Dr. Alexandra Lerch-Gaggl and the Bryant Imaging Core Facility at MCW for assistance with confocal imagery, and Mr. Clive Wells of the Electron Microscopy Core Facility at MCW for assistance and expertise in electron microscopy. This work was supported by grants HL67272 and HL084636 to R.P.M. from the National Institutes of Health; and a grant from Sophia Wolf Quadracci Memorial Fund for Stem Cell Research to R.P.M.
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