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J. Biol. Chem., Vol. 280, Issue 37, 32531-32538, September 16, 2005
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Conditional Mutagenesis of the Murine Serum Response Factor Gene Blocks Cardiogenesis and the Transcription of Downstream Gene Targets*
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From the
Center for Cardiovascular Development, Division of Cardiovascular Sciences, Departments of Molecular and Cellular Biology, ¶Medicine, and ||Surgery, Baylor College of Medicine, Houston, Texas, 77030, **Division of Cardiovascular Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania 19104, Institute of Cell Biology, Department of Molecular Biology, Tuebingen University, D-72704 Tuebingen, Germany, and the Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, Texas 77030
Received for publication, February 4, 2005 , and in revised form, May 18, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum response factor (SRF) homozygous-null embryos from our backcross of SRFLacZ/+ "knock-in" mice failed to gastrulate and form mesoderm, similar to the findings of an earlier study (Arsenian, S., Weinhold, B., Oelgeschlager, M., Ruther, U., and Nordheim, A. (1998) EMBO J. 17, 6289–6299). Our use of embryonic stem cells provided a model system that could be used to investigate the specification of multiple embryonic lineages, including cardiac myocytes. We observed the absence of myogenic
-actins, SM22, and myocardin expression and the failure to form beating cardiac myocytes in aggregated SRF null embryonic stem cells, whereas the appearance of transcription factors Nkx2–5 and GATA4 were unaffected. To study the role of SRF during heart organogenesis, we then performed cardiac-specific ablation of SRF by crossing the transgenic -myosin heavy chain Cre recombinase line with SRF LoxP-engineered mice. Cardiac-specific ablation of SRF resulted in embryonic lethality due to cardiac insufficiency during chamber maturation. Conditional ablation of SRF also reduced cell survival concomitant with increased apoptosis and reduced cellularity. Significant reductions in SRF (
95%), atrial naturetic factor (80%), and cardiac (60%), skeletal (90%), and smooth muscle (75%) -actin transcripts were also observed in the cardiac-conditional knock-out heart. This was consistent with the idea that SRF directs de novo cardiac and smooth muscle gene activities. Finally, quantitation of the knock-in LacZ reporter gene transcripts in the hearts of cardiac-conditional knock-out embryos revealed an 
30% reduction in gene activity, indicating SRF gene autoregulation during cardiogenesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum response factor (SRF)2 is a member of an ancient DNA binding protein family that shares a highly conserved DNA-binding/dimerization domain of 90 amino acids termed the MADS box (Refs. 1 and 2 reviewed in Refs. 3 and 4). SRF, yeast transcription factors MCM1 and ARG80, and plant proteins, such as Deficiens, all have a related MADS box. These MADS boxes have similar DNA binding specificities and dimerize to symmetrically contact the serum response element with a consensus sequence CC(A/T)6GG, also known as the CArG box. In vertebrates, SRF expression is enriched to tissues of mesodermal and neuroectodermal origins (5).
The appearance and diversification of nascent embryonic cardiac and smooth muscle cells require the combinatorial interactions of SRF with other transcription factors, enriched in the early progenitor cells (3, 4). Also, a feature of a large number of cardiac- and virtually all of the smooth muscle-expressed genes to date is their dependence upon CArG boxes (4). In fact, SRF expression is dependent on its promoter, which contains two CArG boxes and SP1 sites, indicating that SRF may modulate its own expression (6, 7). In contrast to the c-fos gene promoter, which contains a single high affinity CArG box with an adjacent Ets factor binding site, the promoters of many muscle-specific genes, including the skeletal, cardiac, and smooth muscle -actins, lack the Ets binding site (2, 8) but contain combinations of two or more CArG boxes that bind SRF in a highly cooperative manner (9, 10). Mutations that prevent SRF binding severely impair the expression of c-fos, as well as muscle-restricted genes. In addition, CArG boxes recruit SRF and cofactors (such as Nkx2.5 and GATA4) as transcriptosomes that strongly enhance SRF-DNA binding affinity, thus permitting the formation of higher order DNA binding complexes at relatively low SRF levels (11, 12). Similarly, CArG boxes recruit the cysteine-rich protein 2 LIM protein, which bridges SRF and GATA factors through interaction with the MADS I coil (13) and myocardin, which competes with Ets factors that interact with the MADS box II coil (14, 15). All of these myogenic cofactors greatly enhance SRF transactivation. Thus, the MADS box domain is likely to be an important regulatory nexus for the convergence of crucial cellular signals enabling SRF to recruit specific cofactors to their respective DNA binding sites and enhance transactivation of target genes.
The homologous recombinant knock-out of the murine SRF gene causes early lethality prior to the formation of mesoderm (16), which prevents the elucidation of the functions of SRF in directing cardiac gene expression. Our complementary approach was to first use SRF null embryonic stem (ES) cells, because differentiating ES cells provide an in vitro embryo model system that can be used to investigate the function of genes involved in the developmental specification of multiple embryonic lineages. Usually, a beating myocardium appears in an ES cell induced to form embryoid bodies (17). In SRF null ES cells, we observed the absence of myogenic -actins, SM22, and myocardin expression and the failure to form beating cardiac myocytes. Furthermore, to study the role of SRF during heart organogenesis, we then performed cardiac-specific ablation of SRF by crossing the transgenic -myosin heavy chain Cre recombinase line with SRF LoxP-engineered mice. Although our study also supports and extends the recent observation that ablation of SRF in the embryonic heart disrupts cardiac sarcomerogenesis, reduced downstream target gene expression, and reduced cell survival in SRF cardiac-conditional knock-out embryos (18), we did not observe the nonspecific repression of many non-SRF targets, as reported in conditional deletion with a relatively late expressing 
MHC-Cre transgene (19). Finally, our study revealed a novel role for SRF in driving its own gene autoregulatory loop during cardiogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of SRF Cardiac-conditional Null Embryos and Genotyping—The SRF LacZ "knock-in" mouse (SRFLacZ/+) was generated, as described previously (20). In-frame replacement of the SRF coding sequence, from ATG to most of the second exon, by insertion of bacterial
-galactosidase generated an SRF null allele. LoxP sites have been inserted into the SRF gene by homologous recombination and generated into mouse lines by Nordheim and colleagues (21). Cardiomyocyte-specific Cre recombinase transgenic mice (-MyHC-Cre) were used to mediate cardiac myocytic Cre/LoxP recombination (21). All mice used in this study were crossed into the C57/B6 background. SRF LacZ knock-in mice (SRFLacZ/+) were bred with -MyHC-Cre mice to obtain double transgenic mice (SRFLacZ/+:-MyHC-Cre). Breeding between double positive mice (SRFLacZ/+:-MyHC-Cre) and SRF LoxP-engineered (SRFLoxP/+ or SRFLoxP/LoxP) mice generated SRF cardiac-conditional knock-out progeny (SRFLacZ/LoxP:-MyHC-Cre, also referred to as CKO mutant mice). Genotyping was performed by PCR analysis of tail DNA for adult mice and yolk sac DNA for embryos. The SRF LacZ knock-in allele was genotyped using primers: srfpCARG2.2, 5'-GGGCTCGCCATATAAGGAGCGGCC-3'; msrfEX1, 5'-GGCTCCGGTACCTTCTTCATGAT-3'; and GALbsu361, 5'-ATCGGCCTCAGGAAGATCG-3', which produced a DNA fragment at 950 bp for wild type and 720 bp for LacZ knock-in allele. The presence of -MyHC-Cre was genotyped using primers: MHC, 5'-ATGACAGACAGATCCCTCCTATCTCC-3'; and Cre3', 5'-CTCATCACTCGTTGCATCGAC-3' with a DNA band at 280 bp. Mice containing the SRF LoxP-engineered allele were genotyped as described previously (4).
Whole Mount X-Gal Staining—Detailed Cre recombinase activity was examined by visualizing the -galactosidase activity of staged embryos collected from crosses between -MyHC-Cre transgenic mice and R26R reporter mice (22) (The Jackson Laboratory, Bar Harbor, ME). X-Gal staining was performed according to Hogan et al. (23). Staining was carried out at 37 °C for 3 h, unless otherwise noted. -Galactosidase activity in SRF LacZ knock-in embryos and SRF cardiac-conditional knock-outs were detected in the same way. After photography, selected embryos were dehydrated in serial ethanol solutions, cleared in xylene, embedded in paraffin, and sectioned at 7 µm and nuclei were counter-stained with nuclear fast red using standard histological techniques. In addition, primer R26R-F, 5'-CGCCATCCCGCATCTGACCAC-3'; and R26R-R, 5'-CCGCTCTGCTACCTGCGCC-3' were used to genotype the Rosa26R allele.
Embryo Collection and in Situ Hybridization—Embryos were collected and fixed in 4% paraformaldehyde overnight at 4 °C. For whole mount in situ hybridization, post-fixed embryos were briefly washed in diethylpyrocarbonate (DEPC)-treated standard phosphate-buffered saline, and dehydrated in serial methanol. Whole mount in situ hybridization was performed as previously described (24). For in situ hybridization on sections, fixed embryos were washed in DEPC-treated phosphate-buffered saline and handled as stated above. Sagittal and transverse sections (7 µm) were collected onto PL-100 poly-L-lysine slides (CEL Associates, Inc., Pearland, Texas). Two to three sections were placed on each slide and hybridized with cRNA probes according to Yamada et al. (25). cRNA probes were prepared according to the digoxigenin labeling kit (Roche Applied Science). Detailed probe information for cardiac -actin, SRF, ANF, and Nkx2.5 is available upon request.
Histology, Immunohistochemistry, and Imaging—Serial transverse sections pre-incubated in phosphate-buffered saline containing 0.3% Triton X-100 for 30 min and then in phosphate-buffered saline containing 2% bovine serum albumin, 0.1% Triton X-100, and 2% serum were treated with primary antibodies at 4 °C overnight. Primary antibodies were anti-SRF (at 1:200 dilution; Santa Cruz Biotechnology), anti-MHC (MF20) (2), and monoclonal anti-sarcomeric -actin (at 1:100 dilution; Sigma). After washing, secondary antibodies were incubated at room temperature for 1 h. Fluorescent-tagged secondary antibodies were applied at 1:200 dilutions (Molecular Probes). After additional washes, the sections were mounted in Vectashield (Vector Laboratory). Images were captured using the Axio imager system and deconvolution microscopy (Zeiss, Inc.).
TUNEL Assay—Apoptosis was monitored using a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay kit ApopTag Plus (Chemicon, Inc.) and detected with fluorescein conjugate. Staining was performed on transverse sections counter-stained for sarcomeric MHC, with MF20 antibodies and for nuclei with 4',6-diamidino-2-phenylindole. The TUNEL-positive signal from the nuclei in MF20-positive myocytes in sectioned ventricles were quantitated to determine the apoptosis ratio in developing ventricular myocytes.
Quantitative and Semiquantitative Reverse Transcription (RT)-PCR— Embryonic hearts dissected free of other tissues were stored at -80 °C. Embryonic yolk sacs were used to extract DNA for genotyping. Thereafter, four equivalent hearts were pooled together for total RNA extraction using the RNeasy mini-kit (Qiagen). First strand cDNA was synthesized using SuperScript III (Invitrogen) following the manufacturer's instructions. Quantitative RT-PCR analysis for GAPDH, SRF, cardiac -actin, smooth muscle -actin, skeletal muscle -actin, ANF, and Nkx2.5 was performed with Taqman probes in an Applied Biosystems Prism 7700. GAPDH served as a loading control. Wild-type controls were given a value of 1.0. Relative expression levels were determined semiquantitatively using two sets of primers against bacterial -galactosidase: 5'-LACZAFGGCGTTACCCAACTTAATCG-3', 5'-LACZARACGACGACAGTATCGGCCTC-3'; and 5'-LACZBFGTCGTCCCCTCAAACTGGCAGATGC-3', 5'-LACZBRTTCGGCGCTCCACAGTTTCGGGTTTTC-3'.
Statistical Analysis—RT-PCR and TUNEL data were expressed as mean ± S.D. The Student's t test was applied for data comparison, and the p value was assigned as 0.05 or 0.01 being significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Direct SRF Targets,
-Actins, and SM22 Are Down-regulated in SRF Null ES Cells—Murine ES cells afforded us a model system to investigate the function of genes involved with the developmental specification of multiple embryonic lineages. Usually within a week, cystic embryoid bodies containing fluid-filled cavities form, and beating myocardia appear in embryoid bodies approximately 2 weeks after initiation of aggregate formation (17). Aggregation of SRF null ES cells failed to form embryoid bodies, as previously observed (26). Fig. 1 shows the absence of SRF transcripts in SRF null ES cells. A number of genes expressed during embryogenesis were unaffected by the loss of SRF, such as the growth factor morphogens, Tgf2, Wnt5a, and BMP4 and the transcription factors Nkx2–5, GATA3, GATA4, GATA5, GATA6, and Smad2. However, we observed reduced transcription of Hhex and the absence of myocardin, both factors important for cardiovascular development. Also, the lack of expression of cardiac, skeletal, and smooth muscle -actins and SM22 transcripts was consistent with the observation that SRF directs de novo cardiac and smooth muscle gene activity. However, it may be argued that the absence of SRF may still indirectly affect cardiogenesis in ES cells at essential steps during and/or prior to mesoderm specification. Therefore, to understand the biological function of SRF as a presumed regulator of cardiac muscle gene expression, it was important to conditionally ablate the SRF gene in the developing heart, which bypassed stages of early gastrulation.
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Myocyte-enriched SRF Expression during Cardiogenesis—We then evaluated the expression of SRF by immunofluorescence staining in the hearts of mouse embryos staged at 9.5 dpc (Fig. 2, A–D, SRF red stain) and 10.5 dpc (Fig. 2, E–H, SRF green stain). At 9.5 dpc, SRF was predominantly localized to nuclei in myocytes, as demonstrated by the positive co-staining of SRF with muscle MHC protein with the MF20 antibody. At 10.5 dpc, SRF was detected in atrial and ventricular myocytes. In the trabeculation region, a few non-myocyte-appearing cells had detectable SRF signals. Also, an in situ hybridization assay showed SRF transcripts were expressed at relatively higher levels in myocytes in the myocardium than in the endocardium (Fig. 2I).
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We recently generated a line of LacZ knock-in mice (SRFLacZ/+) that was used as a reporter gene to follow endogenous SRF gene transcription activity (20). These mice were engineered to have the bacterial
-galactosidase coding sequence with a nuclear localization signal inserted into the translation start site of SRF. -galactosidase staining was performed on SRFLacZ/+ embryos, which reproduced endogenous SRF expression in embryonic muscle tissues (20) and in early mesoderm (Fig. 3A). SRFLacZ/+ haploid embryos allowed the expression of the mesoderm marker T(Brachyury), as shown by in situ hybridization in Fig. 3B. The homozygous SRFLacZ/LacZ embryo, generated from a backcross of SRFLacZ/+ to itself, displayed a severe gastrulation defect and lost T(Brachyury) expression at 7.0 dpc (Fig. 3C). Thus, our SRF LacZ knock-in allele appeared equivalent to a SRF null allele, as previously described (16).
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Myocyte-specific Recombination Mediated by
-MyHC-Cre—Cardiac myocyte lineage-restricted Cre/LoxP recombination was mediated by -MyHC-Cre recombinase. To guide our functional analysis and phenotyping, using -MyHC-Cre mice as the deleter line, Cre recombinase activity was profiled by crossing with R26R reporter mice (27). As shown, at 8.5 dpc, the earliest discrete X-gal stain was detected in the heart tube prior to looping (8 pairs of somites) (Fig. 4B). By 9.5 dpc (Fig. 4, C–E), enriched X-gal stain was observed in the developing heart, thus indicating specific -MyHC-Cre-mediated Cre/LoxP recombination in the cardiac myocyte lineage. By 10.5 dpc, fairly uniform staining was observed (Fig. 4, F–H). As shown in cardiac tissue sections at 10.5 dpc, the X-gal stain was restricted to myocytes and not found within the endocardial cushion or the adjacent endothelial lining (Fig. 4, I–K).
Poor Heart Development and Pericardial Effusion in SRF Cardiac-conditional Knock-out Embryos (SRFLacZ/LoxP:-MyHC-Cre)—We assumed that if SRF participates in the developmental expression of direct gene targets, such as the -actins, then cardiac morphogenesis would be severely affected by cardiac-specific SRF deletion. As shown in the schematic diagram (Fig. 5, top panel), the breeding of SRFLacZ/+:-MyHC-Cre mice with SRFLoxP/LoxP or SRF+/LoxP mice did not result in the birth of any SRF cardiac CKO mice (SRFLacZ/LoxP:-MyHC-Cre). All of the other genotypic combinations were present at Mendelian ratios and were able to produce progeny with no obvious phenotypic abnormalities. Genotyping of embryos at various gestational stages recovered mutant embryos and others at the expected ratios at 9.5–10.5 dpc. Beyond 12.5 dpc, no viable mutant embryos were found.
Analysis of SRFLacZ/LoxP:-MyHC-Cre embryos revealed cardiac-restricted developmental defects. SRF cardiac CKO embryos were first discernible at 10.25 dpc by the appearance of dilated pericardial walls (Fig. 5, A and B). At 10.5 dpc, mutant hearts exhibited less constriction in the atrial ventricular canal (Fig. 5, C and D) and poorly developed intraventricular groove (Fig. 5, E and F). At 10.5 dpc, several mutant embryos were arrested at heart-looping stage (not shown), thus suggesting that SRF could alter early morphological movement, given the observed mosaic Cre activity at early stages (Fig. 4B). At 11.5 dpc, SRF cardiac-conditional knock-out embryos lost rhythmic beating and displayed severe pericardial effusion (Fig. 5, G and H). Examination of transverse sections revealed the appearance of a hypoplastic ventricular wall in the mutant. At 10.5 dpc, the breadth of the mutant ventricular wall compact layer was greatly reduced. Furthermore, cellular wall expansion, as shown by trabeculation, was blocked, in comparison with control SRF+/LoxP embryos (Fig. 5, I–L). The intraventricular septation was less developed in the mutant. In the outflow tract region of the mutant heart, endothelium appeared comparable with controls (Fig. 5, I and J).
Reduced Cardiac Gene Expression in SRF Cardiac-conditional Knock-out Embryos—In situ hybridization assays were performed on sectioned 10.5 dpc embryos from control (Fig. 6A; a, b, e, f, i, and j under SRFLacZ/LoxP) and from SRF cardiac CKO embryos (Fig. 6A; c, d, g, h, k, and l). Cardiac -actin gene transcripts were down-regulated in mutant ventricles at 10.25 dpc (data not shown) and 10.5 dpc in comparison to the robust expression found in control sections (Fig. 6A; a and b versus c and d). Down-regulated ANF expression was found in 10.5 dpc mutant hearts (Fig. 6A, e and f versus g and h). No obvious reduction in Nkx2–5 transcription was detected in comparison to controls (Fig. 6A, i and j versus k and l).
Significant reduction in SRF (95%), ANF (80%), and cardiac (60%), skeletal (90%) and smooth muscle (75%) -actin gene transcripts were observed in the SRF cardiac-conditional knockouts, as assayed by quantitative PCR (Fig. 6B). However, although a recent report suggests that Nkx2–5 might also be an SRF target (19), on the basis that we and Miano et al. (18) failed to observe a significant decrement in Nkx2–5 transcripts in embryonic cardiac tissue from SRF cardiac-conditional knock-out hearts (Fig. 6, A and B) and in SRF null ES cells (Fig. 1), Nkx2–5 is not a direct target of SRF.
SRF, a Stable Cardiac Transcription Factor—Given that -MyHC-Cre activity could be detected as early as 8.5 dpc (Fig. 4B), X-gal staining reached homogeneity in heart tubes by 9.5 dpc, and our breeding strategy required only one recombination event/cell to disrupt SRF at the genomic level, we were surprised that morphological defects or changes in several SRF direct gene targets were not detected by 9.5 dpc. To examine SRF protein depletion in SRF-conditional knock-out mutant hearts, immunofluorescence staining against SRF was performed on transverse sections of mutant hearts. At 9.75 dpc, in the ventricular wall of an SRF cardiac-conditional knock-out, a fraction of the developing myocytes gave a positive SRF stain in nuclei, whereas some other myocytes had weaker SRF signals compared with a non-primary antibody control (not shown). However, by 10.5 dpc, the vast majority of mutant ventricular chamber myocytes ( 90%) were negative for SRF stain (Fig. 6C). This observation suggests that SRF may have a relatively long half-life in embryonic myocytes, as found in fibroblasts of at least 12 h (28).
Reduction in sarcomeric actin transcripts directed our attention toward the examination of sarcomere organization in the hearts of the SRF cardiac-conditional knock-out embryos. Immunolabeling of sarcomeric MHC by the MF20 antibody revealed disrupted sarcomere organization in mutant hearts (Fig. 7, A–D) in comparison to striated structures detected both in the trabeculae and ventricular wall regions of the control. Three pairs of mutant and control embryonic hearts examined under laser confocal microscopy agreed well with the electron microscopic observations made by Miano et al. (18).
Elevated Apoptosis in SRF Cardiac-conditional Knock-out Hearts— SRF cardiac-conditional knock-out embryos lost rhythmic beating and were developmentally arrested at 10.5–11.5 dpc. Cardiac ventricular walls in these embryos displayed poor trabeculation and thinning of the ventricular compact layer (Figs. 5 and 6), suggesting that SRF might determine the cardiomyocyte number by regulating cell replication and/or cell survival. Because Nordheim and colleagues (29) show that the SRF null mutation does not effect cell replication in ES cells, we asked whether loss of SRF could affect cardiomyocyte survival. TUNEL assays were performed to evaluate apoptosis. As shown (Fig. 7, E–H), TUNEL signals were detected in the endocardial cushion, endothelial lining, cardiac myocytes, and the epicardium. Only TUNEL signals in the nuclei of ventricular myocytes, as indicated in Fig. 7G, were assessed to determine a myocyte apoptosis ratio. At 10.25 dpc, a slightly elevated TUNEL ratio was detected in SRF cardiac-conditional knockout. It was noticed that, at this stage, the number of MF20-positive ventricular myocytes was already severely reduced. At 10.5 dpc, an 2-fold increase in TUNEL ratio was identified over SRF+/LoxP:-MyHC-Cre controls. The TUNEL total signal from the neural tube was evaluated as a nonspecific tissue control to the embryonic hearts. In SRF-conditional knock-out mutants, the neural tube TUNEL signals were not statistically different from the control (p value of 0.229).
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Autoregulation of SRF Gene Transcription Revealed by Reduced Reporter
-Galactosidase Expression after Depletion of SRF Protein in Cardiac Myocytes—Generation of the SRFLacZ/+ haploid mice gave us the opportunity to determine whether SRF gene transcription was under a positive autoregulatory loop in vivo. We examined -galactosidase expression by semiquantitative RT-PCR using specific oligonucleotide probes with RNA extracted from heart, as shown in Fig. 8. We observed strong attenuation of the SRF-driven -galactosidase transcripts in SRF-conditional knock-out mutant hearts. To confirm this result, we also performed X-gal staining on mutant heart, whereas the tails served as internal controls. As shown in (Fig. 8), LacZ was reduced by 30% in SRF-conditional knock-out mutant ventricle compared with the SRFLacZ/+ control, whereas no significant differences in -galactosidase staining or transcript levels were detected in the presomitic tail mesenchyme. Thus, our study revealed a novel role for SRF in driving its own gene activity during cardiogenesis. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study provided strong evidence that SRF is required for normal cardiogenesis and the transcriptional activity of itself and other direct SRF targets, such as the
-actin contractile protein genes. Previously, the homologous recombinant knockout of the murine SRF gene demonstrated a severe block to mesoderm formation during mouse embryogenesis (16). These very early lethal SRF-deficient embryos, which appear to have normal cell replication, also have severe gastrulation defects and never form the cardiovascular system (16). In fact, SRF null embryos consisted of misfolded ectoderm and endoderm cell layers and did not form primitive streak or any detectable mesoderm. SRF null mice impeded the appearance of SRF-regulated c-fos and muscle -actin gene activities. Our LacZ knock-in mice (SRFLacZ/+), backcrossed to generate SRFLacZ/LacZ embryos, also yielded homozygous null embryos that were gastrulation-defective and lost T(Brachyury) expression. Also, the SRFLacZ/+ knock-in mice show highly restricted expression in early embryonic cardiac and skeletal muscle (20).
SRF inactivation in the developing mouse heart through a conditional knock-out strategy revealed the requirement for SRF in cardiogenesis, as mutant embryos die at 11.5 dpc because of cardiac insufficiency. However, SRF null ES cells are viable and readily proliferate, but they demonstrate severe cytoskeletal defects seen as defective ES cell migration, adhesion, and spreading (26, 29). SRF null ES cells also have reduced expression of the SRF targets, namely cardiac, skeletal, and smooth muscle -actins and SM22, compared with haploid-insufficient ES cells. The expression of the developmental morphogen BMP4 and the transcription factors Nkx2–5 and GATA4 were not changed in SRF null ES cells. SRF cardiac-conditional mutants did not show a significant reduction in Nkx2–5 expression, a finding opposite to that shown by Parlakian et al. (19), who use the latter expressing -MyHC-Cre.
The loss of -actin gene expression may have contributed to disorganized sarcomere assembly in SRF myocytic knock-out embryonic heart. Between 10.5–11.5 dpc, myocardial SRF-deleted embryos lost rhythmic beating, probably caused by defective sarcomere organization. Although cellular replication in the SRF cardiac-conditional knock-out embryonic heart was not affected, mutants displayed a thinner ventricular compact layer. We observed apoptosis in the heart 0.5–1 day prior to two recent observations (18, 19), which may be due to the fact that our conditional knockout required ablation of only a haploid copy of the SRFLacZ/LoxP gene locus, whereas their knock-outs required ablation of diploid SRFLoxP/LoxP copies with either MHC-Cre (19) or SM22-Cre (18).
Interestingly, SRF null ES cells demonstrated an increase in apoptosis due to the involvement of SRF in modulating expression of the cell survival genes Bcl-2 and Bcl-xl (33). We observed an approximate doubling in the number of TUNEL-positive cells undergoing apoptosis in the SRF null myocardium. Also, the number of MF20-positive ventricular myocytes was severely reduced. Apoptosis is associated with the activation of serial caspases, shown to lead to SRF cleavage (34, 35). This event generates truncated SRF that functions as a dominant negative transcription factor during human heart failure (36). Thus, such inhibitory SRF species may also be responsible for the partial suppression of cardiac-specific gene transcription and the reduction of the number of cardiomyocyte numbers in the conditional SRF null hearts.
SRF is thought to function as a master regulatory platform that directs gene expression programs through combinatorial interactions with other transcription co-factors (2–4). The recruitment of Nkx2–5 and GATA4 to multiple serum response elements allowed for the formation of higher ordered -actin promoter DNA binding complexes, which led us to a model of SRF physical association with these transfactors (12). Thus, both in SRF null ES cells and myocardium-conditional ablation of SRF, the loss of -actins and SM22 expression was because of the selective ablation of SRF and not due to the reduction of its co-accessory factors Nkx2–5 and/or GATA4.
However, myocardin, a co-regulator of smooth muscle gene activity, is highly dependent upon SRF (14). The loss of myocardin, as shown here in Fig. 1 and shown by Miano et al. (18), could have also contributed to the potent block to smooth -actin and Sm22 gene activity. Myocardin has a CArG box binding site (4) and may be a target of SRF. In addition, Selvaraj and Prywes (32) have identified a subset of genes that are MKL (myocardin-like factor, also known as MAL, MRTF-A and -B, and BSAC)-dependent by expression profiling using a cell line that expresses a dominant negative MKL1. This approach identified SRF as one of the key target genes in which activation was MKL-dependent.
Gene autoregulation serves as one of the mechanisms to ensure tissue-restricted expression, such as the positive autoregulation of the MyoD gene (30) and the negative feedback of Nkx2–5 (31). Previous analysis of SRF gene activity shows that two high affinity serum response elements in the core promoter were required for the SRF expression in fibroblasts (6) and skeletal muscle cells (7). The SRFLacZ allele allowed us to evaluate its gene activity by assaying the relative levels of galactosidase transcripts, in the presence and or absence of a haploid level of SRF protein. Selective reduction of -galactosidase RNA in the myocardium was observed in SRF cardiac-conditional knock-out heart but was unaffected in the presomitic tail mesenchyme, a region in which -MyHC-Cre activity was absent (Fig. 8B). Thus, the loss of myocardin, a potent SRF cofactor, might have also contributed to the autoregulatory down-regulation of SRF gene activity.
Finally a recent study from Olson and colleagues (37) reports that the conditional knock-out of SRF in somitogenesis also has a profound effect on the expression of SRF skeletal myogenic gene targets. Combined, these studies and ours indicate that SRF plays an early and obligatory role in directing cardiac, skeletal, and smooth muscle cell differentiation, possibly by also regulating its own expression.
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FOOTNOTES |
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* This project was supported by Grants P01 HL49953 and R01HL79628-01 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7710; Fax: 713-677-7784; E-mail: rschwartz{at}ibt.tamhsc.edu.
2 The abbreviations used are: SRF, serum response factor; ES, embryonic stem; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; dpc, days post-coitus; MHC, myosin heavy chain; ANF, atrial naturetic factor.
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ACKNOWLEDGMENTS |
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We are grateful to Claire Haueter for assistance on imaging. We thank David Zhang for his excellent technical support. Thanks to Dr. Karen Niederreither and Ben Johnson for their thoughtful discussion and critical reading of the manuscript.
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