Serum response factor is required for cell contact maintenance but dispensable for proliferation in visceral yolk sac endothelium
© Holtz and Misra; licensee BioMed Central Ltd. 2011
Received: 20 July 2010
Accepted: 14 March 2011
Published: 14 March 2011
Endothelial-specific knockout of the transcription factor serum response factor (SRF) results in embryonic lethality by mid-gestation. The associated phenotype exhibits vascular failure in embryos as well as visceral yolk sac (VYS) tissues. Previous data suggest that this vascular failure is caused by alterations in cell-cell and cell-matrix contacts. In the current study, we sought to more carefully address the role of SRF in endothelial function and cell contact interactions in VYS tissues.
Tie2-Cre recombinase-mediated knockout of SRF expression resulted in loss of detectable SRF from VYS mesoderm by E12.5. This loss was accompanied by decreased expression of smooth muscle alpha-actin as well as vascular endothelial cadherin and claudin 5, endothelial-specific components of adherens and tight junctions, respectively. Focal adhesion (FA) integrins alpha5 and beta1 were largely unchanged in contrast to loss of the FA-associated molecule vinculin. The integrin binding partner fibronectin-1 was also profoundly decreased in the extracellular matrix, indicating another aspect of impaired adhesive function and integrin signaling. Additionally, cells in SRF-null VYS mesoderm failed to reduce proliferation, suggesting not only that integrin-mediated contact inhibition is impaired but also that SRF protein is not required for proliferation in these cells.
Our data support a model in which SRF is critical in maintaining functional cell-cell and cell-matrix adhesion in endothelial cells. Furthermore, we provide evidence that supports a model in which loss of SRF protein results in a sustained proliferation defect due in part to failed integrin signaling.
Serum response factor (SRF) is a member of the MADS (MCM1, Agamous, Deficiens, SRF) family of nuclear transcription factors. SRF acts as a dimer to recognize the serum response element (SRE), a ten base pair AT-rich sequence (CC(AT)6 GG), also referred to as the CArG box [1, 2]. The SRE binding sequence is found in a diverse array of genes including cellular immediate early genes (IEGs), neuronal nuclear receptors, and cytoskeletal and contractile proteins. The specificity of SRF regulatory actions is context dependent and relies on combinatorial interactions between SRF and various accessory factors. The Elk-1 and SAP-1 Ets family members, which form nuclear complexes with SRF, are direct targets for mitogen activated kinase (MAPK) phosphorylation. Also, the myocardin family of SRF-interacting proteins (MRTFs) are important for regulating transcriptional targets associated with Rho-mediated actin polymerization .
SRF is a central regulator of myogenic gene expression, cell differentiation and function. It is robustly expressed in cells of myogenic lineage [4–6], and required for differentiation and development of skeletal myoblasts [7, 8], cardiomyocytes [9, 10] and smooth muscle cells (SMC) [1, 11, 12]. The expression and regulation of muscle cell contractile proteins depend on SRF transcriptional control [13, 14], and SRF has been shown to provide a direct link between alterations in actin dynamics and consequential changes in nuclear transcription (reviewed in . The G-actin associated protein MAL (a.k.a. myocardin-related transcription factor-4, MRTF-4) is released from monomeric actin upon Rho GTP-ase mediated actin polymerization . Once released, MAL translocates to the nucleus and interacts with SRF to mediate gene transcription of cytoskeletal apparatus proteins such as vinculin, actins, myosin, and focal adhesion (FA) molecules as well as SRF itself [16, 17].
SRF has also been implicated as an important regulator of numerous events during early development. Embryos globally lacking SRF are unable to generate the embryonic mesoderm germ layer and die during gastrulation . Tissue specific deletions of the Srf gene show it is essential for vascular SMC differentiation (reviewed in  and cardiogenesis [9, 19, 20]. SRF is also important for development of brain cells [21, 22], immune cells  and skin epithelium . A requirement for SRF in early vasculogenesis has been demonstrated by virtue of its importance as a regulator of SMC gene expression. In avian systems, SRF is required for differentiation of coronary SMC from progenitors within the proepicardium, a transient embryonic structure that contributes to coronary vasculogenesis [25, 26].
SRF and other members of the MADS-box family have also been shown to regulate cell growth and proliferation in numerous cell types, including rat embryonic fibroblasts , myoblasts , and gut and liver tissues . While the precise mechanism by which SRF controls proliferation is not known, it has been demonstrated that activated MAPK phosphorylates a nuclear complex containing SRF and Ets/TCFs to induce expression of the IEG c-fos [30, 31]. Therefore, it is likely that SRF is at least critical for MAPK-mediated cellular proliferation where it acts to mediate cellular IEG expression and enable the G0 to G1 cell cycle transition [30–33]. SRF is also important for proper cellular adhesion. In particular, several proteins associated with integrin-fibronectin signaling at FA are known SRF-target genes; among them are integrins α1, α5, α9, β1, talin 1, vinculin, and syndecans 2 and 4 . Matrix metalloproteinase 9 (MMP9) is also potentially regulated by SRF . MMP9, together with MMP2, is responsible for degradation of fibronectin and other ECM proteins, suggesting SRF plays a role in the modulation of extracellular matrix (ECM) deposition and maintenance as well.
While SRF has been established as a critical regulator of myogenic cells, relatively little is known about the role of SRF in vascular endothelial cells (VEC). Chai and colleagues  showed that SRF is required for appropriate vascular endothelial growth factor (VEGF)-dependent signaling in endothelial cells in vitro, suggesting a role for SRF in VEC function. More recent in vivo studies from our laboratory and others demonstrate that SRF plays a critical role in endothelial cell function during early vascular development in the mouse [35, 36]. Knockout of SRF expression in an endothelial specific manner by either Tie2-Cre  or Tie1-Cre  -mediated genomic recombination resulted in death by embryonic day 13-14.5 (E13-E14.5). TIE2 is a tyrosine kinase receptor expressed specifically on endothelial cells where it acts to mediate angiopoietin signaling . Both studies suggest that the defect in SRF-null VECs stems from dysfunctional cell-cell and cell-ECM contacts. Lack of appropriate cell contacts could also lead to inappropriate vascular permeability (e.g. ions, solutes) as well as gross vascular damage, causing vascular failure and formation of microthrombi , which are especially notable in the heart and body of VEC-specific SRF-null embryos . We also observed severe disruption of vascular integrity in visceral yolk sac (VYS) tissues.
In the current study, we sought to characterize the severe disruption of VYS vascular tissues observed in VEC-specific SRF-null embryos. Mice homozygous for a floxed Srf gene, Srf f/f  were bred to mice expressing Tie2-promoter driven Cre recombinase . The resulting Tie2-Cre +/0 Srf f/f mutant genotype resulted in embryonic lethality by E13.5. Analysis of Tie2-Cre construct expression in mid-gestation VYS revealed widespread contributions to VYS mesoderm tissue from early progenitors. Adhesion molecule organization, cell-cell and cell-matrix contacts, and various junction complexes as well as actin dynamics associated with intracellular signaling are disrupted throughout the VYS mesoderm following the Tie2-Cre-mediated loss of SRF. These results are consistent with previous studies suggesting SRF plays a role in controling expression of adhesion molecules and is involved with cell-matrix associated signaling cascades. Additionally, we find that SRF-null VYS mesoderm cells continue to proliferate while wild-type tissues with unimpaired adhesive contacts do not. These data suggest that perturbed signaling through cell-cell and cell-matrix contacts results in a loss of adhesion-dependent growth arrest. These data also suggest that a non-SRF dependent mechanism such as the Jak/STAT pathway may be responsible for proliferation in VYS mesoderm. Consequently, SRF appears to be vital for the formation and maintenance of adhesive contacts but dispensable for proliferation during angiogenic remodeling and vascular plexus maturation.
Tie2-Cre-mediated loss of SRF is specific to VYS mesoderm endothelial cells and is complete by E12.5
Together the data presented in Figures 1 and 2 demonstrate that SRF protein is progressively lost from E10.5 through E12.5, being largely or completely lost by E12.5 in VYS mesoderm tissues of Tie2Cre +/0 ·Srf f/f embryos. Complete loss of SRF protein is preceded by detectable alterations in SRF-dependent proteins such as ACTA2.
Alterations in cell contacts contribute to VYS failure
SRF has been demonstrated to be an important regulator of cell shape, integrity, migration, and adhesion (reviewed in . Cytoskeletal elements rely on interactions with the plasma membrane to transduce extracellular signals generated by receptor ligands and ECM proteins. We previously showed ultrastructural evidence that loss of SRF in VYS tissues resulted in a lack of cell-cell adhesion contacts and disrupted ECM deposition . Using electron microscopy analysis of E12.5 VYS from Tie2Cre +/0 ·Srf f/f embryos, we found cells in VYS mesoderm lacked appropriate cell-cell junctions compared to wild-type littermate tissues. We also observed deficient ECM deposition between mesoderm and endoderm layers in these same tissues. Our observations are consistent with other studies that point to a role for SRF in regulation of adhesion molecules. Embryonic stem cells lacking SRF are unable to form FAs or bind appropriately with different ECM components . Furthermore, expression of FA molecules such as vinculin and tropomyosin is regulated by SRF and MRTFs through a Rho/MAL-dependent mechanism . In the current study, we investigated the cause of the observed loss of tissue integrity in VYS tissues, focusing on the role of SRF in: 1) cell-cell contacts, 2) cell-matrix contacts, and 3) ECM deposition.
To address potential changes in expression of adhesion molecules and ECM in SRF-null VYS mesoderm in more detail, we generated endothelial-specific SRF-null embryos using the breeding scheme described above and harvested Tie2Cre +/0 ·Srf f/f and wild-type littermate embryos at E12.5 for analysis. We focused our attention on tissues of this age since the vascular phenotype observed was most consistent by this developmental timepoint. We examined the EC-specific adhesion molecule VE-Cad as a measure of appropriate cell-cell contact. VE-Cad is a VEC-specific transmembrane adhesion protein associated with adherens junctions (AJ) . It forms homodimeric complexes between adjacent cells and is expressed in all VECs upon committed differentiation. Tie2Cre +/0 ·Srf f/f VYS mesoderm tissues display decreased VE-Cad immunoreactivity compared to wild-type tissues (see Figure 1C & 1D). This result is consistent with our previous observation of contact deficiencies at an ultrastructural level between endothelial cells.
Taken together, these data provide evidence that the loss of SRF in VYS mesoderm results in disruption of cell-cell and cell-ECM contacts. Decreased VE-Cad protein indicates that intercellular adhesion between EC is disordered. Cell-ECM adhesion is also disturbed due to a significant lack of ECM-associated FN1 despite apparently unaffected levels of α5. Furthermore, the observed decrease in VCL protein suggests that VCL-dependent intracellular signaling cascades may be perturbed.
Gene expression analysis shows SRF-null VYS tissue is contact deficient
SRF is not required for proliferation of VYS mesoderm cells
To assess the possibility that SRF-null cells were not being detected due to apoptotic loss, we also assayed SRF-null and wild-type littermate VYS tissues using terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL). Tie2Cre +/0 ·Srf f/f VYS tissues stained using TUNEL did not show any consistent differences in the level of apoptosis between wild-type and SRF-null tissues, (see Additional File 4), strongly suggesting that the vascular failure observed in SRF null embryos is not due simply to loss of cells in developing VYS.
Examination of the Tie2-Cre-mediated knockdown of SRF in VYS mesoderm has provided evidence to support a significant role for SRF in endothelial cell function. Previous work demonstrated that endothelial-specific ablation of SRF protein in mice resulted in loss of vascular integrity and function within VYS and ultimately caused embryonic death by mid-gestation . Our current studies suggest that failure of SRF-null VYS tissues and subsequent vascular failure of embryos is due to disrupted junction complexes and junction-related signaling molecules in endothelial cells of the VYS mesoderm. Our observation that loss of SRF leads to alterations in cell contact proteins is consistent with previous reports in other cell types. Embryonic stem cells lacking SRF are unable to form FA or bind appropriately with extracellular matrices [43, 64]. Studies in epithelial cells have verified a need for SRF in establishing cell-cell contacts in developing epidermis . Various studies suggest that this effect is dependent at least in part on SRF's ability to regulate actin dynamics, including those necessary for formation and maintenance of adhesive contacts. Actin filaments link with FA via integrin-associated proteins such as α-actinin, talin, and VCL, providing adhesion-related cellular input and subsequent modulation of cell shape, motility, survival and proliferation . Our results demonstrating a lack of VCL in SRF-null tissues strongly suggest that FAs are unable to connect with the actin cytoskeleton appropriately, resulting in impaired FA-dependent signaling.
We detected profound alterations in VCL and ACTA2 protein patterns and mRNA expression levels in SRF-null VYS tissues. Expression of VCL and other FA molecules such as tropomyosin is regulated by SRF and MRTFs through a Rho/MAL-dependent mechanism . Furthermore, the Vcl gene has a predicted SRE binding element , raising the likelihood that SRF directly controls Vcl expression in VYS tissues. Disrupted Vcl expression causes perturbations in integrin-actin signaling as well as decreased FAK signaling . Acta2 is regulated by SRF, and its loss likely contributes to the defect generated by disrupted VCL, compounding the mis-regulation of SRF-dependent actin dynamics. Our study did not detect significant disruption of Itga5 expression despite the presence of a predicted SRE in the Itga5 gene , perhaps indicating that Itga5 is not under SRF regulatory control in VYS tissues. Additionally, there is no evidence to suggest Fn1 is under direct regulation by SRF. However, we observed substantial loss of FN1 in ECM of SRF-null VYS tissues, suggesting defects in either the transmission of extracellular signaling or in ECM maintenance. VYS endoderm signals to VYS mesoderm through a retinoic acid-transforming growth factor β1 (TGFβ1) dependent pathway , and chimaeric mice lacking functional TGFβ receptors show deficient deposition of FN1 between the VYS endoderm and mesoderm layers . Our observed loss of FN1 protein was accompanied by a slight decrease in Tgfb1 expression (data not shown), highlighting the requirement of this signaling cascade for proper VYS development and subsequent vascular integrity. Furthermore, FN1 deposition and maintenance depends on appropriate FA signaling. The lack of intracellular VCL we observed in SRF-null VYS tissues suggests that impaired FA signaling contributes to the loss of extracellular FN1 . Our observation of decreased VE-Cad and CLDN5 proteins suggests that AJ and TJ functions are also impaired. VE-Cad associated with AJ has been demonstrated to mediate contact inhibition of EC proliferation . CLDN5 has not been shown to act on cell cycle progression; however, expression of CLDN5 is at least in part regulated by VE-Cad , and suggests that TJ act along with AJ to stabilize EC intracellular adhesion.
Given results presented here that SRF-null VYS endothelial cells continue to proliferate, it seems likely that these cells do not require SRF to proliferate even in wild-type tissues. Numerous cell types have demonstrated a dependence on SRF-driven IEG expression for proliferation, including fibroblasts  and cells of myogenic lineage . However, few cell types are known to proliferate without SRF, among them mouse embryonic stem cells . Recently, Koegel and colleagues  found that hyperproliferative skin cells down-regulate SRF expression, suggesting that there may be multiple cell types that use alternative non-SRF dependent pathways to regulate proliferation. They also observed defects in both cell-cell and cell-matrix adhesive contacts, suggesting that these phenomena are likely interrelated. Furthermore, recent studies of SRF-null hematopoietic stem cells (HSC) demonstrate that they also exhibit cell-cell and cell-matrix adhesion failure, that this failure is due to impaired integrin-related signaling, and that HSC proliferation is not negatively affected by the loss of SRF . These observations are consistent with our model where SRF acts as a modulator of cell-cell and cell-matrix adhesion molecules that indirectly affect proliferation due to a loss of adhesion-mediated inhibition. Down-regulation of SRF in this context allows modulation of adhesive integrity and associated actin-dependent junction maintenance necessary for cellular proliferation and migration; however, SRF-independent proliferation of cells within the VYS mesoderm that is required for angiogenic remodeling remains functionally intact.
Our study provides evidence to suggest that the role of SRF in proliferation is balanced with its role in actin-dependent junction dynamics. It remains to be determined which junction proteins are affected through direct SRF-mediated transcriptional regulation and which may be dependent on SRF-related actin dynamics for proper function. The intimacy between the actin cytoskeleton and membrane bound adhesion molecules provides a broad reach for SRF to respond to signaling that initiates at the plasma membrane. Our results demonstrating that loss of cellular adhesive contacts results in impaired proliferative control is consistent with previous studies; however, we show for the first time evidence that VYS mesoderm endothelial cells do not require SRF for proliferation. Further study will be necessary to determine which proliferative pathway dominates in these cells.
Mice and genotyping
Tie2-Cre +/0 and Srf f/f mice have been described previously [39, 40]. The ROSA26R-eYFP +/+ (stock #006148) and ROSA26R-βGal +/+ (stock #003310) reporter mouse strains were purchased from the Jackson Laboratory. Embryos were generated by timed mating, designating noon on the day a vaginal plug was observed as embryonic day 0.5 (E0.5). Genotyping was performed using standard protocols utilizing genomic DNA isolated from embryonic yolk sac, amnion or tail tissue. Primers used were: Tie2-Cre +/0 fwd 5'-GTTCGCAAGAACCTGATGGACA-3' and rvs 5'-CTAGAGCCTGTTTTGCACGTTTC-3'; Srf f/f fwd 5'-TGCTTACTGGAAAGCTCATGG-3' and rvs 5-'TGCTGGTTTGGCATCAACT. ROSA26R-eYFP mice were genotyped according to protocols from Jackson using the following three primers: WT-fwd 5'-GGAGCGGGAGAAATGGATATG-3', eYFP fwd 5'-AAGACCGCGAAGAGTTTGTC-3', and rvs 5'-AAAGTCGCTCTGAGTTGTTAT-3'. ROSA26R-βGal +/+ mice were genotyped according to protocols from Jackson using the following three primers: F150 fwd 5'-GGCTTAAAGGCTAACCTGATGTG-3', Tg rvs 5'-GCGAAGAGTTTGTCCTCAACC-3', and WT-rvs 5'-GGAGCGGGAGAAATGGATATG-3'. Animals designated for BrdU incorporation studies were administered BrdU Labeling Reagent by intraperitoneal injection (dose 1 mL/100 g body weight; Invitrogen 00-0103) and harvested for tissues after 2-4 hours. All procedures were in compliance with the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
Histology and imaging
Embryos were harvested from timed pregnant females, and yolk sac tissues were dissected and fixed in either 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) or zinc formalin (Richard-Allan Scientific). Samples were processed for paraffin embedding, and sections (5-7 μm) were used for immunofluorescence histochemistry as described [36, 73]. Reagents used were: anti-serum response factor (SRF; Protein-Tech custom); anti-smooth muscle α-actin (ACTA2; Sigma, clone 1A4); anti-integrin α5 (ITGA5; Millipore, AB1921); anti-fibronectin (FN1; Millipore, AB2033); anti-vinculin (VCL; abcam, ab18058); anti-BrdU (Invitrogen, 03-3900); anti-Histone H3, phosphorylated form (PhH3; Millipore 06-570); anti-vascular endothelial cadherin (CDH5, a.k.a. VE-Cad; abcam 33168); anti-epithelial cadherin (CDH1, a.k.a. E-Cad; BD Biosciences 610181). eYFP protein was detected using anti-GFP antibody (Invitrogen, A10262). Anti-ACTA2 antibody clone 1A4 detects α-actin found in several cell types, and was not used as a specific SMC label. DNA/nuclei were counterstained with DAPI. Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) was performed on zinc formalin fixed paraffin-embedded 5 μm sections using the DeadEnd Colorimetric Apoptosis Detection System (Promega); DNase-treated wild-type VYS tissue was used as a positive control according to detection system protocol. Tissues harvested for detection of Tie2-Cre-associated β-galactosidase activity were fixed in 4% paraformaldehyde and LacZ stained under standard protocols. Digital image capture was performed using a Nikon Eclipse 80i microscope equipped with Nikon Digital Sight DS-2MBW monochrome and DS-F1 color cameras and NIS Elements-D imaging software. Tri-color image merge and post-processing was done in Adobe Photoshop CS4.
Thin sections (5 μm) of embryonic yolk sac from wild-type and Tie2Cre +/0 ·Srf f/f embryos were stained for designated markers and imaged for cell counting. Positive nuclear staining was scored only in cells of the mesoderm layer of the visceral yolk sac. Between 300-500 nuclei over 5 visual fields (1 per section; 200× magnification) were counted for each embryo, with a total of 3 embryos per treatment group; embryos were taken from 2-3 different litters harvested on different days. Tissues were stained for SRF protein to verify loss of expression in BrdU treated tissues. Anti-BrdU antibody was used to detect incorporation into nuclear material of proliferating cells; PhH3 was used to label cells undergoing mitosis. Detection of ACTA2 protein was used to verify loss of Srf expression in PhH3 stained tissues due to incompatibility between anti-PhH3 and anti-SRF antibodies. DAPI was used as counter-stain and to identify all visible nuclei. Final cell counts of cells staining positive for SRF, BrdU or PhH3 were expressed as a percentage of total number of DAPI-positive nuclei counted.
Real-time quantitative PCR (qPCR) and analysis
Embryos were harvested from timed pregnant females, and yolk sac tissues were dissected and processed for RNA (Qiagen RNeasy Mini Kit) and cDNA template (SABiosciences RT2 First Strand Kit C-03) using published protocols. Samples were probed using SYBR Green with ROX reference (SABiosciences RT2 Real-Time SYBR Green/Rox Master Mix PA-012) using 100 nm primer oligos under published protocols in a Stratagene Mx3005P Real-Time PCR System. Wild type and Tie2Cre +/0 ·Srf f/f whole VYS tissues (n = 3 each) were assayed separately using 100 ng template per reaction. Differences between wild type and SRF null tissues were calculated using the 2^ΔΔCt method based on the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR (Applied Biosystems) with all genes normalized to Gapdh expression. Data is presented as fold change with positive SEM only owing to the asymmetrical nature of confidence intervals in exponential fold change calculations. See Additional File 5 for real-time qPCR primer sequences.
We thank Mr. Kurt Kolander for assistance with graphic design. This work was supported by grant HL084636 to R.P.M. from the National Institutes of Health.
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