Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Dev Cell. 2008 Feb;14(2):298–311. doi: 10.1016/j.devcel.2007.11.018

Endocardial Brg1 Represses ADAMTS1 to Maintain the Microenvironment for Myocardial Morphogenesis

Kryn Stankunas 1, Calvin T Hang 1, Zhi-Yang Tsun 1, Hanying Chen 2, Nathan V Lee 3, Jiang I Wu 4, Ching Shang 1, J Henri Bayle 4,5, Weinian Shou 2, M Luisa Iruela-Arispe 3, Ching-Pin Chang 1,*
PMCID: PMC2274005  NIHMSID: NIHMS40729  PMID: 18267097

SUMMARY

Developing myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.

Keywords: Brg1, BAF complex, chromatin remodeling, primitive erythropoiesis, yolk sac vasculogenesis, endocardium, heart development, trabeculation, cardiac jelly, ADAMTS1, microenvironments

INTRODUCTION

Cardiac progenitor cells of mesoderm origin differentiate into both the myocardium and the endocardium that forms the outer and inner layers of the heart, respectively. As development proceeds, these two cell layers interact with each other to form specialized structures of the heart such as heart valves and myocardial trabeculae (reviewed in (Harvey, 2002)). Myocardial trabeculation is a key morphogenetic event in heart development that begins at embryonic day E9.0 in mice, when clusters of myocardial cells in the ventricles protrude into the extracellular matrix (cardiac jelly) separating the myocardium from the endocardium. These nascent trabeculae continue to expand, forming long, thin projections. Contiguous with this expansion, ventricular endocardial cells invaginate between the myocardial projections. By E14.5, the ventricular endocardium becomes tightly associated with the myocardial cells as the cardiac jelly dissipates. Finally, the trabeculae largely collapse to thicken the compact layer of myocardium (termed compaction of myocardium). Excessive trabeculation or a failure of the trabeculae to undergo compaction causes cardiomyopathy and heart failure in humans (Jenni et al., 1999). Despite its importance, very little is known about the genetic and molecular programs that initiate and terminate trabeculation.

Several studies implicate the importance of the exchange of intercellular signals between the endo- and myocardium during trabeculation. Neuregulin is produced by endocardial cells and acts through the myocardial receptors, ErbB2 and ErbB4, to drive myocardial trabeculation (Gassmann et al., 1995; Kramer et al., 1996; Lee et al., 1995). Conversely, vascular endothelial growth factor (VEGF) and angiopoietin signal from the myocardium to the endocardium to regulate trabeculation (Ferrara et al., 1996; Sato et al., 1995; Suri et al., 1996). For these signals to be transmitted appropriately, they must traverse the cardiac jelly lying between the endo- and myocardium. Interestingly, both neuregulin and VEGF signaling are modified by extracellular matrix components, suggesting that the establishment of the correct cardiac jelly complement is critical for appropriate receipt of signals between ventricular endo- and myocardium. Consistent with this idea, mice lacking either Hyaluronan synthase-2 (Has-2), an enzyme required for production of the mucopolysaccharide hyaluronan, or Versican, a chondroitin sulphate proteoglycan, do not develop trabeculae (Camenisch et al., 2000; Mjaatvedt et al., 1998; Yamamura et al., 1997). The decrease in ventricular cardiac jelly that is observed concomitant with the termination of trabeculation further raises the possibility that active degradation of the cardiac jelly participates in this process. This could be accomplished by a decrease in expression of cardiac jelly components or a change in expression or activity of extracellular matrix regulators. Included in the latter class are the hyaluronidases and members of the matrix metalloproteinase family, notably the aggrecanases that comprise the ADAMTS family (Reviewed in (Porter et al., 2005)). While both Has-2 and Versican, among other ECM components, and ADAMTS family members exhibit dynamic expression patterns during cardiac development (Camenisch et al., 2000; Henderson and Copp, 1998; Klewer et al., 2006; Thai and Iruela-Arispe, 2002), the mechanisms of their transcriptional regulation are unknown.

Alteration of chromatin structure provides an important source of gene regulation by determining the accessibility of DNA regulatory elements to transcription factors. Chromatin can be modified non-covalently by the rearrangement of nucleosomes catalyzed by ATP-dependent chromatin remodeling complexes to facilitate both transcriptional activation and repression (Dunaief et al., 1994). The Brg1-associated-factor (BAF) complex is a 10-subunit complex in which the ATPase component is encoded by one of two genes, Brg1 or Brm. Although both genes are broadly expressed, Brg1 null mice show peri-implantation lethality (Bultman et al., 2000) while Brm null mice are viable (Reyes et al., 1998). Recent reports suggest that BAF subunits are required for cardiac morphogenesis. BAF180, a specific subunit of the PBAF form of the BAF complex, regulates cardiac chamber maturation and is required for survival past mid-embryogenesis (Wang et al., 2004). RNA interference of BAF60c, which is expressed throughout the developing heart, results in severe heart defects including a single ventricle, a poorly developed outflow tract, and no trabeculation (Lickert et al., 2004). While expression of several myocardial transcripts is reduced in BAF60c knockdown embryos, the molecular basis of these phenotypes is unclear. It is also unknown whether the BAF complex is required in the endocardium, myocardium, or both for cardiac morphogenesis.

We use tissue specific deletion of Brg1 in both the endocardium and myocardium to study the roles of the BAF complex in each tissue during cardiogenesis. The absence of Brg1 by embryonic day E9.0 produces trabeculation defects when it is deleted from the endocardium but not the myocardium. Neither deletion causes widespread changes in transcription of several key regulators of cardiac development. Instead, the trabeculation defects in the absence of endocardial Brg1 result from derepression of a secreted matrix metalloproteinase, ADAMTS1, which normally only increases in expression later in development to prevent excessive trabeculation. The increase in ADAMTS1 causes premature breakdown of the cardiac jelly and termination of trabeculation, demonstrating that cardiac jelly regulation by endocardial cells coordinates maturation of ventricular myocardium. Further, our data indicates the BAF complex has a surprisingly specific role in the developing heart after the basic tissue layers are formed. More broadly, we demonstrate that chromatin remodeling can influence tissue morphogenesis through regulation of the microenvironment.

RESULTS

Endothelial Brg1 is Required for Yolk Sac Vasculogenesis and Primitive Erythropoiesis

To study the function of the BAF complex in endocardial cells, we specifically deleted the core Brg1 subunit throughout the endothelium by crossing mice carrying a loxP-flanked allele of Brg1 (Sumi-Ichinose et al., 1997) with Tie2Cre transgenic mice (Kisanuki et al., 2001). Brg1 was normally expressed in the nuclei of both myocardial and endocardial cells, as well as all surrounding tissues (Figure 1A). In contrast, Brg1 was undetectable in the endocardial cells, as marked by expression of the transcription factor NFATc1 (de la Pompa et al., 1998; Ranger et al., 1998), of Tie2Cre;Brg1F/F embryos but remained expressed in all other non-endothelial tissues (Figure 1B and S2). This deletion was first evident at E9.0 (data not shown). At E9.5, Tie2Cre;Brg1F/F embryos were of comparable size to littermate controls (Figure 1C). However, they appeared pale due to a decrease in circulating red blood cells caused by defects in yolk sac vasculogenesis and primitive hematopoiesis (Supplemental Text and Figure S1). In contrast, the basic pattern of the embryonic vasculature was intact in E9.5 Tie2Cre;Brg1F/F embryos. By E10.5, Tie2Cre;Brg1F/F embryos were considerably smaller than their littermates (Figure 1D) and none survived to E11.5.

Figure 1.

Figure 1

Open in a new tab

Endocardial Brg1 is required for trabeculation. (A and B) Paraffin sections from wildtype (A) and Tie2Cre;Brg1F/F (B) E9.5 embryos were double stained with an anti-Brg1 antibody (green) and an anti-NFATc1 antibody (cyan) to mark endocardial cells. The arrows point at endocardial cells. (C and D) Images of E9.5 and E10.5 wildtype and Tie2Cre;Brg1F/F embryos. A: atrium. V: ventricle. (E and F) Hematoxylin and eosin (H&E) stained paraffin sections showing E9.5 wildtype and Tie2Cre;Brg1F/F heart ventricles. Arrows denote ventricular endocardial cells. (G and H) E10.5 wildtype and Tie2Cre;Brg1F/F frozen heart sections are stained with antibodies against PECAM (green) and MF20 (cyan) to mark the endocardium and myocardium, respectively. (I and J) Morphometric analysis of trabecular thickness (I) and the ratio of trabecular area to compact wall length (J) by measurement of sections stained as in (G and H) from E9.25, E9.75, and E10.5 wildtype and Tie2Cre;Brg1F/F embryos. Error bars are one standard deviation. Three hearts were measured for each group and p values were calculated using two-tailed Student’s t-Tests.

Brg1 is Required in the Endocardium for Trabeculation

We examined hematoxylin and eosin-stained (H&E) sections of Tie2Cre;Brg1F/F embryos between E9.5 and E10.5 and found defects in trabeculation of the heart ventricles. Normally, nascent myocardial trabeculae are present by E9.5, forming discrete outgrowths of myocardial cells migrating into the cardiac jelly. In Tie2Cre;Brg1F/F embryos, there was only limited formation of trabeculae at E9.5 (Figures 1E and 1F). By E10.5, when trabeculae were normally extensive, Tie2Cre;Brg1F/F embryos had very sparse trabeculation (Figure S2). We double stained E10.5 embryos for MF20, a myocardial marker, and PECAM, an endocardial marker, to assess the organization of the two cell layers (Figures 1G and 1H). Although both cell types were present in Tie2Cre;Brg1F/F embryos, PECAM-positive endocardial cells did not invaginate around MF20 positive cardiomyocytes outgrowing from the compact layer, consistent with histological observations.

We quantified the extent of the trabeculation defect by morphometric measurements of the thickness of individual trabeculae, the total area of trabecular myocardium, and the length of the compact wall in anti-PECAM and anti-MF20 double stained sections. The trabecular thickness was significantly reduced at both E9.75 and E10.5 in Tie2Cre;Brg1F/F embryos (Figures 1I and S2). At E10.5, the compact layer was also significantly thinner than control embryos (Figure S2). We measured the total degree of trabecular atrophy by normalizing trabecular area to compact wall length. This analysis showed a significant decrease in the extent of trabeculation as well as the size of each trabecular sheet at E9.75 and E10.5 in Tie2Cre;Brg1F/F embryos (Figure 1J). These results demonstrate that Brg1 has critical roles in the endocardium to regulate trabecular development.

Myocardial Brg1 is Not Required for Trabeculation between E9.5 and E10.5

To investigate the possibility that Brg1 is also required in ventricular myocardium for trabeculation, we deleted Brg1 using the myocardial-expressed SM22αCre transgene (Holtwick et al., 2002; Umans et al., 2007), Figure S3). Less than 5% of ventricular myocardial cells in E9.5 SM22αCre;Brg1F/F embryos expressed detectable levels of Brg1, with no evidence of deletion in endocardial cells (Figure 2A and 2B and S3). In contrast to Tie2Cre;Brg1F/F embryos, SM22αCre;Brg1F/F embryos had no gross defects in embryonic development up to E10.5 (Figure 2C and 2D). H&E staining of sections through the hearts of these embryos demonstrated that trabeculation was normal (Figure 2E and 2F). Therefore, myocardial BAF complexes do not regulate trabeculation between the time Brg1 was deleted in these mice (between E8.5 and E9.5) and E10.5.

Figure 2.

Figure 2

Open in a new tab

Myocardial Brg1 does not have major effects on cardiac morphogenesis between E9.0 and E10.5. (A and B) E9.5 wildtype and SM22αCre;Brg1F/F heart sections are double immunostained for Brg1 (green) and smooth muscle actin (SMactin) to mark the myocardium (red). Nuclei (blue) are stained using Hoechst. Arrows indicate ventricular endocardial cells. (C and D) Gross images of E10.5 wildtype and SM22αCre;Brg1F/F embryos (E and F) H&E stained heart sections of E9.5 wildtype and SM22αCre;Brg1F/F embryos. A: atrium. V: ventricle.

Deletion of Brg1 After E9.0 Does Not Have Widespread Effects on Cardiac Gene Expression

To understand the molecular basis for the myocardial trabeculation defects in Tie2Cre;Brg1F/F embryos, we examined the expression of myocardial genes important for this process. The expression of Nkx2.5, which is required for trabeculation (Lyons et al., 1995), and NPPA was decreased in BAF60c RNAi embryos (Lickert et al., 2004). However, both Nkx2.5 and NPPA were expressed normally in the myocardium of both Tie2Cre;Brg1F/F (Figure 3A to 3D) and SM22αCre;Brg1F/F embryos (Figure S3). The defects in Nkx2.5 and NPPA expression seen in the BAF60c RNAi mice may therefore reflect a requirement for the BAF complex prior to the onset of Brg1 deletion using Tie2Cre or SM22αCre. BMP10, which is also required for trabeculation (Chen et al., 2004) and whose expression is regulated by endocardial signals (H. C and W. S. unpublished observations), was also expressed normally in Tie2Cre;Brg1F/F embryos (Figure 3E and 3F). Additional myocardial proteins, such as MF-20 and Actinin (Figures 1G and 1H and S4) or transcripts, including GATA4, ErbB2, Hey1, MEF2C, VEGF, MEST and Irx4 were also expressed normally in Tie2Cre;Brg1F/F embryos (Figures S5 and S6). Therefore, signals originating from the endocardium required for maturation of ventricular myocardium are unaffected by the absence of endocardial Brg1.

Figure 3.

Figure 3

Open in a new tab

Myocardial and endocardial transcripts known to regulate trabeculation are expressed normally in Tie2Cre;Brg1F/F embryos. (A to D, G to L) In situ hybridizations on paraffin sections of E9.5 wildtype and Tie2Cre;Brg1F/F embryonic hearts for the indicated transcripts (stained brown with DAB). The blue counter stain is hematoxylin. (E and F) E9.5 wildtype and Tie2Cre;Brg1F/F embryos are whole-mount stained for BMP10 transcripts (blue). Arrows indicate ventricular endocardial cells. A: atrium. V: ventricle.

Next, we used RNA in situ hybridization to survey the expression of endocardial transcripts in Tie2Cre;Brg1F/F embryos to determine if Brg1 was required for their transcription. We examined neuregulin, Tie1, Tie2, and VEGFR2 because each of these signaling molecules and/or their receptor/ligand partners have been shown to be required for trabeculation (Ferrara et al., 1996; Meyer and Birchmeier, 1995; Meyer et al., 1997; Sato et al., 1993; Sato et al., 1995; Suri et al., 1996). However, they were all expressed normally in E9.5 Tie2Cre;Brg1F/F embryos (Figures 3G to 3L and S5). Recently, the Notch pathway has been shown to be required for trabeculation by regulating expression of Neuregulin (indirectly via EphrinB2) and BMP10 (Grego-Bessa et al., 2007). Normal expression of these transcripts and the Notch target Hey1 in Tie2Cre;Brg1F/F embryos (Figure S5) suggests the Notch pathway is not perturbed by the absence of endocardial Brg1. These results indicate that endocardial cells maintained their differentiated state and did not have widespread changes in transcription in the absence of Brg1. Rates of apoptosis and cellular proliferation were also not significantly changed in E9.5 Tie2Cre;Brg1F/F embryos (Figure S4). Together, these results suggest that the BAF complex has a restricted role in regulating the function rather than differentiation of endocardial cells to control trabecular morphogenesis.

Endocardial Brg1 Is Required for Establishment of Ventricular Cardiac Jelly

Several extracellular matrix (ECM) components, notably hyaluronan and Versican, are required for trabeculation (Camenisch et al., 2000; Yamamura et al., 1997). Interestingly, histological analysis of Tie2Cre;Brg1F/F embryos showed an apparent decrease in the amount of ECM, termed the cardiac jelly, separating the ventricular endocardium from the myocardium (Figures 1E and 1F). We stained E9.5 heart sections with Alcian blue, pH 2.5 to detect the presence of acidic mucopolysaccharides, including hyaluronan and sulfated and carboxylated proteoglycans such as Versican. In Tie2Cre;Brg1F/F embryos, there was a clear decrease in the amount of Alcian-blue positive material in the ventricular cardiac jelly (Figure 4A and 4B). We examined Tie2Cre;Brg1F/F embryos for the presence of hyaluronan by staining sections with biotinylated hyaluronan binding protein. Hyaluronan was found throughout the cardiac jelly, but was nearly absent in the ventricular cardiac jelly of Tie2Cre;Brg1F/F embryos (Figure 4C and 4D). Similarly, Versican protein levels were reduced in the ECM of the ventricles (Figures 4E and 4F). Nevertheless, transcription of Hyaluronan synthase-2 (Has-2), and UDP-glucose-dehydrogenase (UGDH), two enzymes required for hyaluronan production, and Versican persisted in the ventricles of Tie2Cre;Brg1F/F embryos (Figures 4I to 4L and S5). Perlecan, a heparan sulphate proteoglycan required for heart development (Costell et al., 2002) was also transcribed normally (Figure S5), although Perlecan protein levels were also diminished in ventricular cardiac jelly by E9.75 (Figure 4G and 4H). Interestingly, and as previously reported (Henderson and Copp, 1998), Versican was expressed exclusively in myocardium while Has-2 transcripts were found at higher levels in ventricular myocardium compared to the neighboring endocardium. These results are consistent with the observation that the myocardium is the primary source of cardiac jelly components (Manasek et al., 1973). The normal transcription of Has-2, UGDH, Versican, and Perlecan suggest that a change in expression of a modulator of ECM rather than the components themselves accounts for the ECM deficiency in Tie2Cre;Brg1F/F embryos.

Figure 4.

Figure 4

Open in a new tab

Tie2Cre;Brg1F/F embryos have defects in ECM formation in ventricular cardiac jelly. (A and B) Alcian Blue staining of paraffin sections through ventricles of E9.5 wildtype and Tie2Cre;Brg1F/F embryos. The sections are counterstained with nuclear fast red. (C and D) Hyaluronan (HA) staining using biotinylated-HABP of wildtype and Tie2Cre;Brg1F/F ventricle sections. (E and F) Anti-Versican antibody staining of ventricle-containing sections of wildtype and Tie2Cre;Brg1F/F hearts. (G and H) Antibody staining of Perlecan in left ventricle sections of E9.75 wildtype and Tie2Cre;Brg1F/F embryos. In situ hybridizations for Has-2 (I and J) and Versican (K and L) transcripts on E9.5 wildtype and Tie2Cre;Brg1F/F heart sections. The staining in (C to L) was developed with DAB (brown) and counterstained with hematoxylin. Arrows indicate ventricular endocardial cells. V: ventricle. AVC: atrioventricular canal.

ADAMTS1 is Derepressed in the Absence of Endocardial Brg1

We employed a candidate gene approach to identify transcripts whose misregulation in Tie2Cre;Brg1F/F embryos could cause the observed ECM defects (Figures S5 and S6). In our screen, we included Hyal1, Hyal2, and Hyal3, the three hyaluronidases whose potential upregulation could account for a decrease in HA content. We also examined members of the matrix metalloproteinase family MMP1, MMP2, MMP3, MMP9, and MMP13, each of which has been shown to degrade Versican or related proteoglycans (Reviewed in (Flannery, 2006)). Finally, we checked expression of ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS8, ADAMTS9, and ADAMTS18, their proposed co-factor, Fibulin-1 (Lee et al., 2005), and their inhibitor TIMP3 (Kashiwagi et al., 2001). The ADAMTS family consists of 19 genes of the metalloproteinase superfamily, whose substrates include aggrecans such as Versican and Perlecan (reviewed in (Porter et al., 2005)) and which have been implicated in cardiac development (reviewed in (Manso et al., 2006)). ADAMTS1, 4, 5, 8, 9, and 18 were chosen for analysis because they have been described as Versicanases and/or have been shown to express in endothelial cells (A. Connolly, unpublished observations). In addition, each of ADAMTS1, ADAMTS9, and Fibulin-1 have been reported to regulate endocardial cushion development (Kern et al., 2006). Because TIMP3 is expressed in the developing endocardium (Brauer and Cai, 2002), we also examined whether its expression was decreased in Tie2Cre;Brg1F/F embryos.

Among the 19 screened ECM-related transcripts, only ADAMTS1 exhibited a clear change in expression in Tie2Cre;Brg1F/F embryos (Figures S5 and S6). In these embryos, ADAMTS1 was expressed at high levels in ventricular endocardial cells relative to littermate controls while maintaining a low level of expression in the myocardium (Figures 5A and 5B). To confirm the observed increase in ADAMTS1 seen by in situ hybridization, we performed RT-PCR analyses on mRNA from the hearts of E9.5 embryos. Using quantitative RT-PCR and normalization to HPRT, a ubiquitously expressed transcript, we found that ADAMTS1 transcripts were present at 2.8x higher levels in the mutant embryos (Figure 5C). A collection of other myocardial and endocardial transcripts were not significantly changed in expression, consistent with our in situ hybridization experiments. An increase in ADAMTS1 expression could account for the subsequent decrease in Versican levels and, indirectly, in the HA content of the ventricular cardiac jelly as Versican contains HA-binding domains that may stabilize HA levels (LeBaron et al., 1992). Consistent with augmented ADAMTS1 activity, increased staining using an antibody that recognizes a specific cleavage product of Versican (Neo-Versican) arising from ADAMTS activity was observed in E9.25 Tie2Cre;Brg1F/F embryos prior to further dissipation of Versican levels by E9.5 (Figure 5D and 5E).

Figure 5.

Figure 5

Open in a new tab

ADAMTS1 is derepressed in the absence of endocardial Brg1. (A and B) RNA in situ hybridization for ADAMTS1 transcripts (brown) on E9.5 wildtype and Tie2Cre;Brg1F/F ventricle sections. (C) Quantitative RT-PCR (qRT-PCR) for ADAMTS1 and endocardial transcripts (Tie2, VEGFR2, NFATc1), myocardial transcripts (Nkx2.5, MEF2C, GATA4, Tbx5), or ECM-related transcripts (Versican, Has-2, UGDH) normalized to HPRT expression using RNA from dissected hearts of E9.5 wildtype and Tie2Cre;Brg1F/F embryos. Error bars are one standard deviation. P<0.002 (n=3 wildtype and mutant) of a non-significant change in ADAMTS1 in Tie2Cre;Brg1F/F hearts by a two-tailed Student’s t-Test. (D and E) Antibody staining for a Versican cleavage product (Neo-Versican) produced in ventricle sections of E9.25 wildtype and Tie2Cre;Brg1F/F embryos. Arrows denote subendocardial cardiac jelly. (F) qRT-PCR for ADAMTS1 and Brg1 transcripts normalized to HPRT on RNA from cultured primary human umbilical vein endothelial cells transduced with a control lentivirus or one expressing small hairpin RNA (shRNA) directed against Brg1 transcripts. Experiment #1 used a different shRNA sequence from Experiments #2 and #3. Each experimental set represents independent experiments that varied in their extent of RNA interference of Brg1.

We examined whether repression of ADAMTS1 by Brg1 was a primary effect of Brg1 in endocardial cells using loss-of-function studies of Brg1 in cultured cells. We used human umbilical vein endothelial cells (HUVECs), which express ADAMTS1 (Norata et al., 2004), as a heterologous source of primary cells. We reduced expression of Brg1 using RNA interference and assayed changes in ADAMTS1 transcript levels. Lentiviral mediated expression of two separate small hairpin RNAs targeting Brg1 produced an increase in ADAMTS1 expression proportionate to the degree of Brg1 transcript knockdown (Figure 5F). The most robust knockdown, to 21% of normal Brg1 levels, resulted in a 3 fold increase in endogenous ADAMTS1 transcripts. This result supports our in vivo observations that loss of endocardial Brg1 is the primary cause of the increase in endocardial ADAMTS1 expression and demonstrates that similar repression of ADAMTS1 by Brg1 occurs in additional endothelial cell types.

Brg1 Directly Associates With the ADAMTS1 Locus in Endothelial Cells

The upregulation of ADAMTS1 in HUVECs and endocardial cells in the absence of Brg1 could represent a secondary effect of misexpression of ADAMTS1 transcriptional regulators or could be accounted for by a direct requirement for the BAF complex in ADAMTS1 repression. To determine whether endogenous Brg1 directly binds the ADAMTS1 locus, we performed chromatin immunoprecipitation assays and quantitative PCR using HUVECs as a source of material. We aligned the mouse, dog, rat, and human genomes to identify conserved regions in the ADAMTS1 locus that may act as sites for Brg1 association (Figure 6A). Primer pairs within each of these regions were designed for amplification of DNA from immunoprecipitated chromatin. Antibodies directed against Brg1 precipitated more chromatin fragments from the immediate promoter area (3.7x, p<0.002) and a conserved stretch in the first intron (4.6x, p<0.03) relative to a control anti-GFP antibody (Figure 6B). Limited or no enrichment was found for an upstream candidate regulatory site (1.4x, p<0.13) or an unconserved region approximately 30 kb upstream from the promoter (Figure 6B and data not shown). These results support a model in which the BAF complex directly binds the ADAMTS1 locus near the immediate promoter region to maintain its repression in developing ventricular endocardial cells.

Figure 6.

Figure 6

Open in a new tab

The ADAMTS1 promoter is directly repressed by Brg1. (A) An alignment of the ADAMTS1 locus between the mouse and dog, human, and rat genomes, respectively. Increased conservation is shown by the height of the colored curves. Blue regions are translated portions of exons, yellow is 5′ untranslated region, and red indicates candidate regulatory regions. Three conserved regions, labeled 1, 2 and 3, were selected for further analysis. (B) Quantitative PCR analysis of anti-Brg1 immunoprecipitated chromatin from regions 1, 2, and 3 in human umbilical vein cells normalized to an anti-GFP control antibody. Error bars represent one standard deviation and p values were determined by two-tailed Student’s t-Tests. (C) Episome-based reporter assays using the indicated ADAMTS1 candidate regulatory regions cloned in luciferase expression plasmids co-transfected in SW13 cells with either a control or Brg1-expressing plasmid to restore BAF complexes to the cells. The luciferase activity is graphed relative to a co-transfected constitutive Renilla luciferase control and error bars represent one standard deviation between 3 independent transfections. P values were calculated using unpaired two-tailed Student’s t-Tests.

We further tested this concept using luciferase reporter assays with regions from the ADAMTS1 locus. We prepared four different constructs that contained either a 2.0 kb immediate promoter fragment or a longer 3.2 kb region that included the second conserved region not bound by Brg1 each with or without the conserved intron region cloned downstream of luciferase. Importantly, the reporter constructs were cloned into Epstein-Barr Virus derived pREP4 episomal vectors that contain the virus replication origin and express nuclear antigen EBNA-1. These vectors become chromatinized when transfected into mammalian cells and therefore are informative when studying the effects of chromatin remodeling complexes (Liu et al., 2001). We transfected each of the four reporter plasmids into SW13 adenocarcinoma cells, which lack both Brg1 and its homologue Brm (Muchardt and Yaniv, 1993) and therefore lack functional BAF complexes, in the presence or absence of a co-transfected Brg1-expressing plasmid. Expressing Brg1 in SW13 cells restores BAF complex function. Lysates from each of the transfection experiments contained between 2.8 and 4.5 fold lower luciferase levels when Brg1 was restored to the cells (Figure 6C). These results further support a model of direct repression of ADAMTS1 by Brg1. The site of action is likely through the immediate promoter region (Region 2) of the ADAMTS1 locus, as each of the reporters behaved similarly despite having only that region in common. The results also demonstrate that Brg1 activity, separate from Brm, is necessary and sufficient for repression of ADAMTS1.

ADAMTS1 Derepression Terminates Trabeculation

To investigate whether the increase in ADAMTS1 expression underlies the ECM defects in Tie2Cre;Brg1F/F embryos, we treated cultured embryos (Chang et al., 2004) with GM6001, a broad spectrum inhibitor of matrix metalloproteinases including ADAMTS1 (Shimada et al., 2004). Wildtype embryos harvested at E9.0 and treated with GM6001 for 36 hours developed relatively normally (Figures 7A, 7B, 7E, and 7F). Mock-treated Tie2Cre;Brg1F/F embryos had severe defects in ventricle development, even more so than seen in utero at E10.5. They had an increase in compact layer thickness, no trabeculation, and a near complete absence of Alcian blue staining in the sub-endocardial space (Figure 7G). Tie2Cre;Brg1F/F embryos treated with GM6001 remained growth-retarded and pale, being unchanged in gross appearance (Figures 7D). However, they contained Alcian blue-positive material in ventricular cardiac jelly and exhibited a significant recovery of trabeculation (Figure 7H and 7M). GM6001-treated Tie2Cre;Brg1F/F embryos also recovered expression of Versican and hyaluronan in the ventricular cardiac jelly to levels seen in wildtype embryos (Figures 7I to 7L, 7N and 7O). These results indicate that the increase in ADAMTS1 in Tie2Cre;Brg1F/F embryos is responsible for the collapse of the ECM through proteolysis of Versican and the subsequent failure of trabeculation. A separate broad-spectrum MMP inhibitor, minocycline (Paemen et al., 1996), which does not inhibit ADAMTS1 (Rodriguez-Manzaneque et al., 2002), did not rescue trabeculation or cardiac jelly in cultured Tie2Cre;Brg1F/F embryos (Figure S7). Together with the absence of changes in expression of other matrix proteinases (Figures S5 and S6), this finding indicates that an increase in ADAMTS1 alone largely accounts for the defective trabeculation in Tie2Cre;Brg1F/F embryos.

Figure 7.

Figure 7

Open in a new tab

Derepression of ADAMTS1 underlies the trabeculation defects in Tie2Cre;Brg1F/F embryos. (A to D) Whole mount images of wildtype and Tie2Cre;Brg1F/F embryos grown in culture for 36 hours beginning at E9.0 in the presence or absence of 20 μM GM6001, an ADAMTS1 inhibitor. (E to H) Heart ventricle sections of the embryos shown in (A to D) are stained with Alcian blue to mark mucopolysaccharide components of the cardiac jelly and counterstained using nuclear fast red. (I to L) Anti-Versican antibody staining in the ventricular cardiac jelly of the same cultured wildtype and Tie2Cre;Brg1F/F embryos control-treated or treated with GM6001. The sections are counterstained with hematoxylin. (M) The mean ratio of trabecular to outside length of the left ventricle measured on H&E stained sections is graphed for wildtype and Tie2Cre;Brg1F/F embryos cultured with or without the matrix proteinase inhibitor GM6001 for 36 hours beginning at E9.0. Error bars represent one standard deviation. n=3 Wildtype/ND, Wildtype/GM6001, and Tie2Cre;Brg1F/F/ND groups, n=8 Tie2Cre;Brg1F/F/GM6001. P values were determined using Student’s t-Tests. (N, O) Biotinylated-HABP stained heart sections of Tie2Cre;Brg1F/F embryos cultured from E9.0 for 36 hours in the presence or absence of GM6001 to mark hyaluronan content (in brown). The sections are counterstained with hematoxylin (blue).

Since GM6001 treatment did not rescue the hematopoietic defects in Tie2Cre;Brg1F/F embryos, as shown by the absence of red blood cells in sections of the cultured embryos (Figure 7L), ADAMTS1 derepression does not cause all the phenotypes of Tie2Cre;Brg1F/F embryos. Consistent with this observation, ADAMTS1 was not derepressed in other endothelial cells in Tie2Cre;Brg1F/F embryos (Figure S8). This result indicates that Brg1 may have an active and specific role in maintaining ADAMTS1 repression in specific types of endothelial cells, including endocardial cells, rather than a general requirement for establishing the ADAMTS1 transcriptional ground state. This observation raises the question why does ADAMTS1 need to be repressed in developing ventricular endocardial cells? Interestingly, cardiac expression of ADAMTS1 was low in wildtype embryos until E12.5, when it became highly expressed in the endocardium (marked by Tie1 expression) and persisted through E14.5 (Figures 8A to 8F). Consistent with this observation, the amount of cardiac jelly diminished at E12.5, and was essentially absent by E14.5, when the ventricular endocardium and myocardium were in continuous, direct contact (Figures 8G to 8I). Furthermore, the ADAMTS1 substrate, Versican, dramatically decreased in expression in the ventricular cardiac jelly at E12.5, and is essentially absent by E14.5 (Figures 8J to 8L). Since myocardial trabeculation terminates during the period from E12.5 to E14.5, the increase in ADAMTS1 expression and subsequent dissolution of the cardiac jelly might be required to prevent excessive trabeculation. We tested this idea by examining the phenotype of mice lacking ADAMTS1. While ADAMTS1-/- mice exhibited approximately 50% embryonic lethality, they were grossly normal at E13.5 (Figure 8M and 8N). At E13.5, the ventricles of ADAMTS1-/- embryos had increased trabecular muscle (Figure 8O and 8P). We quantitated the extent of myocardial dysmorphogenesis by morphometric measurements of the left ventricles of 3 ADAMTS1+/- and 5 ADAMTS1-/- E13.5 embryos. ADAMTS1-/- embryos had a statistically significant increase in the ratio of trabecular area to total ventricle area (0.85 +/- 0.07 to 0.58 +/- 0.11, p<0.04) (Figure 8Q). There was no change in the number of trabeculae normalized to the ventricle length (0.04 +/- 0.007 to 0.04 +/- 0.005, p<0.83), trabeculae width (14.1 +/- 1.2 to 13.7 +/- 0.6, arbitrary units, p<0.67), or compact layer thickness (31.7 +/- 2.8 to 33.0 +/- 3.2, arbitrary units, p<0.57) (Figure S9). These findings suggest that the normal upregulation of ADAMTS1 at E12.5 terminates trabeculation by reconfiguring the extracellular matrix (Figure 8R). The transition from repression to activation of ADAMTS1 ensures the proper microenvironment is present for initial support of trabecular growth and then its termination once sufficient trabeculation has occurred.

Figure 8.

Figure 8

Open in a new tab

An increase in expression of ADAMTS1 in ventricular endocardium terminates myocardial trabeculation by breaking down cardiac jelly. (A to F) In situ hybridizations on ventricle sections of wildtype embryos of the indicated stages for ADAMTS1 (A to C) and Tie1 (D to F) transcripts. Transcripts are stained brown and the slides are counterstained with hematoxylin. Arrows indicate ventricular endocardial cells. (G to I) Alcian blue stained sections of wildtype embryonic hearts of the indicated stages. The sections are counterstained with nuclear fast red. (J to L) Sections from E10.5, E12.5, and E14.5 wildtype ventricles are stained with anti-Versican antibodies (brown) and counterstained with hematoxylin. (M and N) Gross images of E13.5 ADAMTS1+/- and littermate ADAMTS1-/- embryos. (O and P) H&E stained sections of the ventricles of an E13.5 ADAMTS1+/- and littermate ADAMTS1-/- embryos. (Q) The mean ratio of trabecular/ventricle area in E13.5 ADAMTS1+/- and littermate ADAMTS1-/- hearts from measurements of H&E stained left ventricle sections (n=3 ADAMTS1+/-, n=5 ADAMTS1-/-, error bars are one standard deviation, p<0.04 by two-sample two-tailed Student’s t-Test). (R) A working model of myocardial trabeculation. Brg1 represses ADAMTS1 in endocardial cells to allow myocardial cells to produce an extracellular environment that supports trabeculation. By E12.5, BAF-complex mediated repression of ADAMTS1 is relieved (by an unknown mechanism, “X”) dissipating the cardiac jelly and preventing excessive trabeculation. In Tie2Cre;Brg1F/F embryos, ADAMTS1 is derepressed, causing the cardiac jelly to prematurely break down and disrupting myocardial morphogenesis.

DISCUSSION

The trabeculation defects in Tie2Cre;Brg1F/F embryos are caused by derepression of ADAMTS1, which encodes a matrix metalloproteinase that degrades cardiac jelly and prevents trabeculation. ADAMTS1 is a secreted matrix metalloproteinase of aggrecans, including Versican (Kuno et al., 2000; Rodriguez-Manzaneque et al., 2002). Although ADAMTS1 deficient mice have not been previously examined for cardiac abnormalities, ADAMTS1 has been implicated in cleaving Versican within the endocardial cushion cardiac jelly to facilitate the remodeling of the cushions into atrial and ventricular septa as well as the valve leaflets (Kern et al., 2006). Versican contains hyaluronan-binding domains (LeBaron et al., 1992) which may be important for establishment of an elaborate glycosaminoglycan-containing network within the cardiac jelly. Therefore, inactivation of Versican by the observed upregulation of ADAMTS1 in Tie2Cre;Brg1F/F embryos could facilitate the degradation of hyaluronan, which then directly leads to collapse of ventricular cardiac jelly. This notion is supported by studies showing that addition of hyaluronidase to whole rat embryo cultures causes collapse of ventricular cardiac jelly and leads to trabeculation defects (Baldwin and Solursh, 1989).

There are several ways how cardiac jelly composition could influence myocardial trabeculation. During trabeculation, both myocardial and endocardial cells must undergo extensive cellular movements to form the long thin projections protruding into the ventricular cavity. Therefore, establishment of an appropriate extracellular milieu that facilitates changes in cell shape, adhesion, or migration may be critical for trabeculation. Second, ECM components could regulate trabeculation by providing a permissive environment for diffusion or post-translational modification of signaling molecules involved in trabeculation, including Notch (Grego-Bessa et al., 2007), Neuregulin (Meyer and Birchmeier, 1995; Meyer et al., 1997), EphrinB2 (Gerety and Anderson, 2002), VEGF (Ferrara et al., 1996), and angiopoietin (Sato et al., 1993; Sato et al., 1995; Suri et al., 1996). In particular, VEGFR2 signaling in embryonic stem cells is promoted by heparan sulphate proteoglycans produced by adjacent cells (Jakobsson et al., 2006) and heparan sulphate modulates VEGF gradients (Ruhrberg et al., 2002). Third, certain components of the ECM are active signaling molecules themselves or can directly modify the ability of signaling molecules to act on their receptors. For example, HA binds to the CD44 and LYVE-1 cell receptors, and activates intracellular signaling (Aruffo et al., 1990; Banerji et al., 1999). HA is also a co-factor for neuregulin signaling (Bourguignon et al., 1997; Camenisch et al., 2002). Therefore, regulated changes in the microenvironment of the heart ventricle during development could facilitate a developmental transition to terminate cardiac trabeculation independent of the expression of the signaling factors involved.

A specific human congenital cardiomyopathy, termed spongy myocardium, hypertrabeculation, or non-compaction of the myocardium, reflects developmental defects during the trabeculation process. The genetic lesions underlying this disease remain obscure. Hearts of these patients exhibit excessive trabeculation and develop congestive heart failure with the only treatment being a heart transplant. Our observation that premature expression of ADAMTS1 in the absence of endocardial Brg1 prevents trabeculation suggests that the normal increase in endocardial ADAMTS1 expression during development is essential to prevent hypertrabeculation. Interestingly, overexpression of a non-cleavable form of Versican in embryonic chick hearts causes a thickening of the right ventricle and possibly hypertrabeculation (Kern et al., 2007). Here, we show that ADAMTS1-/- mice, a genotype which is approximately 50% embryonically lethal (Mittaz et al., 2004; Shindo et al., 2000), develop excessive trabeculation. This result supports a model in which Brg1 represses expression of ADAMTS1 until developmental cues alleviate the repression around E12.5 to facilitate the termination of trabeculation (Figure 8R). The increase in ADAMTS1 prevents excessive trabeculation by breaking down ventricular cardiac jelly and altering the microenvironment that supports cellular movements and/or signaling events. Subsequently, the heart develops the thick compact layer that provides the extensive muscular power required in a mature heart. ADAMTS1-/- embryos that escape embryonic lethality and survive to adulthood do not have apparent non-compaction of the ventricles (data not shown). Therefore, additional cardiac jelly degrading enzymes may cooperate with ADAMTS1 in a partially redundant fashion to terminate trabeculation. Candidates include additional ADAMTS proteins or members of the MMP family, some of which are expressed in the developing heart (Figures S5 and S6 and (Ratajska and Cleutjens, 2002)) and whose substrates also include proteoglycans (Reviewed in (Flannery, 2006). A detailed understanding of the roles of cardiac jelly regulators during heart development may help identify candidate genes mutated in families with congenital cardiomyopathy and eventually lead to novel treatments for this disease.

Activities of chromatin remodeling enzymes are commonly associated with transcriptional activation given their ability to loosen chromatin to facilitate the access of transcription factors and transcriptional machinery. Therefore, although a number of genes have been described as being repressed by the BAF complex (reviewed in (de la Serna et al., 2006)), it is surprising to find that a critical function of the BAF complex in developing endocardial cells is as a site-specific transcriptional repressor. While Brg1 directly associates with the ADAMTS1 locus, it remains to be determined how the BAF complex is recruited and how it mediates repression. Since Brg1 remains ubiquitously expressed throughout heart development (Figure S10), changes in Brg1 levels do not account for the normal derepression of ADAMTS1 and therefore recruitment factors likely provide active regulation of the locus. Alternatively, a change in activity of the BAF complex on ADAMTS1 may lie in a developmental switch in the combinatorial assembly of homologous BAF subunits, which can produce differential BAF complex activities in specific cell types (Lessard et al., 2007).

Our findings demonstrate an unexpectedly specific role for the BAF complex in maintaining transcriptional repression of ADAMTS1 in developing endocardial cells to establish the appropriate microenvironment for trabeculation of the ventricles. The connection between the BAF complex and regulation of the microenvironment in the developing heart ventricle raises an intriguing possibility that chromatin-based regulation may be a common source for the establishment of extracellular environments. Recent reports indicate an analogous relationship to that between Brg1 and ADAMTS1 exists between the histone deacetylase HDAC7 and MMP10 in the developing vasculature (Chang et al., 2006) and between HDACs and ECM components during worm development (Whetstine et al., 2005). Furthermore, Brg1 has been implicated in regulating the expression of MMP2 and MMP9 in cultured cells (Ma et al., 2004; Ma et al., 2004). Chromatin regulation may provide a source for signaling specificity by modifying the ability of intercellular signals to be transmitted in a complex environment. This premise may have broad implications given the important roles of chromatin remodeling and the microenvironment in both normal and pathologic settings (Anderson et al., 2006; Bhowmick et al., 2004; Roberts and Orkin, 2004) of tissue growth and differentiation.

EXPERIMENTAL PROCEDURES

Mouse husbandry

All mouse strains were maintained in outbred backgrounds. The targeted null allele of Brg1, Brg1- (Bultman et al., 2000), the loxP allele of Brg1, Brg1F (Indra et al., 2005), Tie2Cre (Kisanuki et al., 2001), Sm22αCre (Holtwick et al., 2002), Rosa26RLacZ (Soriano, 1999), and ADAMTS1- (Lee et al., 2006) strains are described. For tissue specific deletions, males of genotype Tie2Cre;Brg1F/+ were crossed with Brg1F/F females. The date of observation of a vaginal plug was set as embryonic day E0.5, which was confirmed by ultrasonography prior to sacrificing pregnant mice (Chang et al., 2003) and examination of embryo morphology and somite number. Identical styles of crosses were used with Sm22αCre deletion of Brg1.

Immunostaining

Antibody staining of paraffin sections used standard methods, following protocols detailed in the Supplemental Experimental Procedures. The following antibodies were used: anti-Brg1 (G7) mouse monoclonal (Santa Cruz Biotechnology), anti-Brg1 rabbit polyclonal (Chemicon), anti-Brg1 (J1) rabbit polyclonal, anti-NFATc1 (7A6) (BD Biosciences), anti-smooth muscle actin (Sigma), anti-Actinin (Sigma), anti-phospho-Histone H3 (Millipore), anti-Versican (Millipore), anti-Perlecan (SPM255) (Santa Cruz Biotechnology), and anti-Neo-Versican (anti-Versican V0/V1 Neo) (Affinity Bioreagents). TUNEL staining was performed using an in situ cell detection kit (Roche).

Histology

Paraffin sections were prepared as described in the immunostaining section. Following re-hydration, the slides were consecutively stained with hematoxylin and eosin (Polysciences), dehydrated and mounted in Permount (Sigma) prior to imaging. Alcian blue staining was performed using a 1% solution of Alcian blue, pH 2.5 in acetic acid followed by counter-staining with Nuclear Fast Red (Vector Laboratories). Hyaluronan was stained using biotinylated-hyaluronan binding protein (Calbiochem) at 4 μg/mL, HRP-conjugated streptavidin (DAKO) and DAB chromogenic development. The sections were counterstained with hematoxylin.

Trabeculation Quantification

We used a dual fluorescence confocal image system similar to one previously described (Chen et al., 2004) to visualize, quantitate and compare the development of trabecular myocardium in Tie2Cre;Brg1F/F and littermate control embryos. Frozen sections of the embryos were stained with anti-PECAM (BD Biosciences) and anti-MF20 (Developmental Studies Hybridoma Bank) antibodies (described further in Supplemental Material). The images were captured using LaserSharp software and analyzed with Metamorph (version 6.1). We measured the thickness of trabecular ridges/sheets (μm), thickness of compact wall (μm), and area of trabecular myocardium normalized by the length of compact wall (μm2/μm). For the ADAMTS1 null and heterozygous control mice, a modification of the above protocol was used for morphometric measurements of the hearts. We performed the quantification using images of H&E stained paraffin section analyzed with Nikon Advanced Elements software. We measured the total number of trabeculae (where the trabecular layer meets the compact layer) normalized to ventricle length, the average thickness of the trabeculae, the average thickness of the compact layer, and the total area of the trabecular sheet normalized to the area of the ventricle (in pixel units). These latter ratios do not represent absolute ratios as the normalization measurements were performed using images taken at a different magnification.

Whole mount in situ hybridization

In situ hybridization assays were based on the protocol described in (Wilkinson and Nieto, 1993) with details and modifications described in Supplemental Experimental Procedures. The plasmid template for the BMP10 in situ probe is described (Chen et al., 2004).

In situ hybridization on paraffin sections

Digoxigenin-labelled antisense transcripts were synthesized using plasmid templates (Roche). Plasmids for synthesizing the probes are from the following sources: VEGF-A from Douglas Hanahan (Christofori et al., 1995), Neuregulin from Carmen Birchmeier (Meyer et al., 1997) Tie2 from Kari Alitalo (Korhonen et al., 1994), Has-2 from Todd Camenisch (Camenisch et al., 2000), Tie1, VEGFR2, Nkx2.5, GATA4, ErbB2, Hey1, MEF2C, NPPA, UGDH, Versican, Perlecan, Irx4, MEST, TIMP3, Hyal1, Hyal2, Hyal3, ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS8, ADAMTS9, ADAMTS18, MMP1, MMP2, MMP3, MMP9, MMP13, and Fibulin1 from Open Biosystems. All plasmids were confirmed by sequencing. Method details for probe hybridization and visualization are included in the Supplemental Experimental Procedures.

RT-PCR and quantitative RT-PCR

Whole hearts were dissected from E9.5 embryos. The hearts were disrupted in Trizol (Invitrogen) and RNA was purified using standard methods. 150 ng of total RNA from each sample was used as a template to prepare cDNA using the Superscript III reverse transcription system (Invitrogen). In the case of HUVECs, RNA was isolated using a Trizol-based method and 500 ng of RNA was used for cDNA synthesis. Quantitative PCR to measure transcript content in the individual samples was performed using primers and methods described in the Supplemental Experimental Procedures.

Lentivirus Production and RNA Interference

Lentivirus were produced in 293T cells following protocols of the Didier Trono lab by co- transfecting them with packaging and envelope plasmids and pGIPZ transfer vectors for shRNA expression (Open Biosystems). Two separate plasmids expressing shRNAs targeting human Brg1 were used while the control pGIPZ vector contains a non-silencing shRNA. Two days post-transfection of the 293T cells, the media was isolated, filtered, and concentrated using a 100k MW cutoff centrifugal filter unit (Millipore). This viral concentrate was used for transduction of HUVECs. The cells were cultured for 5 days, at which point most of the cells expressed GFP, indicating a high rate of infection. RNA was prepared using Trizol extractions and then used for cDNA synthesis. Expression levels were determined using quantitative PCR as described in the previous section.

Chromatin immunoprecipitations

Human umbilical vein cells were cultured to approximately 75% confluency on six 15 cm plates and harvested using reagents and methods from a chromatin immunoprecipitation kit (Upstate) with modifications and details described in Supplemental Experimental Procedures. Chromatin was prepared and immunoprecipitated using the J1 anti-Brg1 antibody (Wang et al., 1996) and an anti-GFP (Invitrogen) antibody as an isotype-matched negative control. Following washes, DNA was retrieved and used for quantitative PCR using primers and methods described in Supplemental Experimental Procedures.

Reporter Assays

Reporter plasmids were constructed by cloning PCR amplified region of the mouse ADAMTS1 locus into the episomal pREP4-Luc plasmid (Liu et al., 2001). These vectors, along with an episomal renilla-luciferase plasmid (pREP7-RL) to normalize for transfection efficiency and a plasmid expressing human Brg1 or a matching empty vector plasmid were transfected into SW13 cells using lipofectamine 2000 (Invitrogen). The cells were cultured for two days and harvested for luciferase assay using the dual luciferase assay kit (Promega).

Whole embryo culture

Whole embryo cultures were performed as described (Chang et al., 2004). Embryos were isolated intact in their yolk sacs at E9.0 and cultured for 36 hours in the presence of either 20 μM GM6001 (Calbiochem), 20 μM Minocycline (Sigma), or an equivalent amount of DMSO alone. The yolk sacs were used for genotyping and the embryos harvested for histological analysis.

Supplementary Material

01

ACKNOWLEDGEMENTS

G. Crabtree for supporting the initiation of the project; L. Pang, V. Devasthali, G. Krampitz, L. Chen and K. Zhang for technical assistance; M. Winslow for performing the flow cytometry; P. Chambon for providing Brg1F mice; M. Rabinovitch for providing SM22αCre mice; B. Zhou, C. Birchmeier, K. Alitalo, and T. Camenisch for providing plasmids; K. Zhao for the episomal-based reporter plasmids, S. Stewart for lentiviral reagents, L. Ho for antibodies, T. Quertermous for advice and support; A. Connolly, V. Devasthali and members of the Chang laboratories for suggestions. This work was supported by grants from the American Heart Association (AHA), Children’s Heart Foundation, March of Dimes Birth Defects Foundation and the National Institutes of Health (NIH) to C-P.C. K.S. is supported by an AHA Postdoctoral Fellowship, C.H. by a Stanford Graduate Fellowship, Z-Y.T by an AHA Undergraduate Research Award, and C.S. by a Ruth L. Kirschstein National Research Service Award Fellowship.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  1. Anderson AR, Weaver AM, Cummings PT, Quaranta V. Tumor morphology and phenotypic evolution driven by selective pressure from the microenvironment. Cell. 2006;127:905–915. doi: 10.1016/j.cell.2006.09.042. [DOI] [PubMed] [Google Scholar]
  2. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell. 1990;61:1303–1313. doi: 10.1016/0092-8674(90)90694-a. [DOI] [PubMed] [Google Scholar]
  3. Baldwin HS, Solursh M. Degradation of hyaluronic acid does not prevent looping of the mammalian heart in situ. Dev Biol. 1989;136:555–559. doi: 10.1016/0012-1606(89)90281-9. [DOI] [PubMed] [Google Scholar]
  4. Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, Jones M, Jackson DG. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol. 1999;144:789–801. doi: 10.1083/jcb.144.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature. 2004;432:332–337. doi: 10.1038/nature03096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC. Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation. J Biol Chem. 1997;272:27913–27918. doi: 10.1074/jbc.272.44.27913. [DOI] [PubMed] [Google Scholar]
  7. Brauer PR, Cai DH. Expression of tissue inhibitor of metalloproteinases (TIMPs) during early cardiac development. Mech Dev. 2002;113:175–179. doi: 10.1016/s0925-4773(02)00016-3. [DOI] [PubMed] [Google Scholar]
  8. Bultman S, Gebuhr T, Yee D, La Mantia C, Nicholson J, Gilliam A, Randazzo F, Metzger D, Chambon P, Crabtree G, Magnuson T. A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol Cell. 2000;6:1287–1295. doi: 10.1016/s1097-2765(00)00127-1. [DOI] [PubMed] [Google Scholar]
  9. Camenisch TD, Schroeder JA, Bradley J, Klewer SE, McDonald JA. Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors. Nat Med. 2002;8:850–855. doi: 10.1038/nm742. [DOI] [PubMed] [Google Scholar]
  10. Camenisch TD, Spicer AP, Brehm-Gibson T, Biesterfeldt J, Augustine ML, Calabro A, Jr., Kubalak S, Klewer SE, McDonald JA. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest. 2000;106:349–360. doi: 10.1172/JCI10272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chang CP, Chen L, Crabtree GR. Sonographic staging of the developmental status of mouse embryos in utero. Genesis. 2003;36:7–11. doi: 10.1002/gene.10186. [DOI] [PubMed] [Google Scholar]
  12. Chang CP, Neilson JR, Bayle JH, Gestwicki JE, Kuo A, Stankunas K, Graef IA, Crabtree GR. A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis. Cell. 2004;118:649–663. doi: 10.1016/j.cell.2004.08.010. [DOI] [PubMed] [Google Scholar]
  13. Chang S, Young BD, Li S, Qi X, Richardson JA, Olson EN. Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell. 2006;126:321–334. doi: 10.1016/j.cell.2006.05.040. [DOI] [PubMed] [Google Scholar]
  14. Chen H, Shi S, Acosta L, Li W, Lu J, Bao S, Chen Z, Yang Z, Schneider MD, Chien KR, et al. BMP10 is essential for maintaining cardiac growth during murine cardiogenesis. Development. 2004;131:2219–2231. doi: 10.1242/dev.01094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Christofori G, Naik P, Hanahan D. Vascular endothelial growth factor and its receptors, flt-1 and flk-1, are expressed in normal pancreatic islets and throughout islet cell tumorigenesis. Mol Endocrinol. 1995;9:1760–1770. doi: 10.1210/mend.9.12.8614412. [DOI] [PubMed] [Google Scholar]
  16. Costell M, Carmona R, Gustafsson E, Gonzalez-Iriarte M, Fassler R, Munoz-Chapuli R. Hyperplastic conotruncal endocardial cushions and transposition of great arteries in perlecan-null mice. Circ Res. 2002;91:158–164. doi: 10.1161/01.res.0000026056.81424.da. [DOI] [PubMed] [Google Scholar]
  17. de la Pompa JL, Timmerman LA, Takimoto H, Yoshida H, Elia AJ, Samper E, Potter J, Wakeham A, Marengere L, Langille BL, et al. Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum. Nature. 1998;392:182–186. doi: 10.1038/32419. [DOI] [PubMed] [Google Scholar]
  18. de la Serna IL, Ohkawa Y, Imbalzano AN. Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers. Nat Rev Genet. 2006;7:461–473. doi: 10.1038/nrg1882. [DOI] [PubMed] [Google Scholar]
  19. Dunaief JL, Strober BE, Guha S, Khavari PA, Alin K, Luban J, Begemann M, Crabtree GR, Goff SP. The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest. Cell. 1994;79:119–130. doi: 10.1016/0092-8674(94)90405-7. [DOI] [PubMed] [Google Scholar]
  20. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439–442. doi: 10.1038/380439a0. [DOI] [PubMed] [Google Scholar]
  21. Flannery CR. MMPs and ADAMTSs: functional studies. Front Biosci. 2006;11:544–569. doi: 10.2741/1818. [DOI] [PubMed] [Google Scholar]
  22. Gassmann M, Casagranda F, Orioli D, Simon H, Lai C, Klein R, Lemke G. Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature. 1995;378:390–394. doi: 10.1038/378390a0. [DOI] [PubMed] [Google Scholar]
  23. Gerety SS, Anderson DJ. Cardiovascular ephrinB2 function is essential for embryonic angiogenesis. Development. 2002;129:1397–1410. doi: 10.1242/dev.129.6.1397. [DOI] [PubMed] [Google Scholar]
  24. Grego-Bessa J, Luna-Zurita L, Del Monte G, Bolos V, Melgar P, Arandilla A, Garratt AN, Zang H, Mukouyama YS, Chen H, et al. Notch signaling is essential for ventricular chamber development. Dev Cell. 2007;12:415–429. doi: 10.1016/j.devcel.2006.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harvey RP. Patterning the vertebrate heart. Nat Rev Genet. 2002;3:544–556. doi: 10.1038/nrg843. [DOI] [PubMed] [Google Scholar]
  26. Henderson DJ, Copp AJ. Versican expression is associated with chamber specification, septation, and valvulogenesis in the developing mouse heart. Circ Res. 1998;83:523–532. doi: 10.1161/01.res.83.5.523. [DOI] [PubMed] [Google Scholar]
  27. Holtwick R, Gotthardt M, Skryabin B, Steinmetz M, Potthast R, Zetsche B, Hammer RE, Herz J, Kuhn M. Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure. Proc Natl Acad Sci U S A. 2002;99:7142–7147. doi: 10.1073/pnas.102650499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Indra AK, Dupe V, Bornert JM, Messaddeq N, Yaniv M, Mark M, Chambon P, Metzger D. Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation. Development. 2005;132:4533–4544. doi: 10.1242/dev.02019. [DOI] [PubMed] [Google Scholar]
  29. Jakobsson L, Kreuger J, Holmborn K, Lundin L, Eriksson I, Kjellen L, Claesson-Welsh L. Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis. Dev Cell. 2006;10:625–634. doi: 10.1016/j.devcel.2006.03.009. [DOI] [PubMed] [Google Scholar]
  30. Jenni R, Rojas J, Oechslin E. Isolated noncompaction of the myocardium. N Engl J Med. 1999;340:966–967. doi: 10.1056/NEJM199903253401215. [DOI] [PubMed] [Google Scholar]
  31. Kashiwagi M, Tortorella M, Nagase H, Brew K. TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-TS5) J Biol Chem. 2001;276:12501–12504. doi: 10.1074/jbc.C000848200. [DOI] [PubMed] [Google Scholar]
  32. Kern CB, Norris RA, Thompson RP, Argraves WS, Fairey SE, Reyes L, Hoffman S, Markwald RR, Mjaatvedt CH. Versican proteolysis mediates myocardial regression during outflow tract development. Dev Dyn. 2007;236:671–683. doi: 10.1002/dvdy.21059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kern CB, Twal WO, Mjaatvedt CH, Fairey SE, Toole BP, Iruela-Arispe ML, Argraves WS. Proteolytic cleavage of versican during cardiac cushion morphogenesis. Dev Dyn. 2006;235:2238–2247. doi: 10.1002/dvdy.20838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol. 2001;230:230–242. doi: 10.1006/dbio.2000.0106. [DOI] [PubMed] [Google Scholar]
  35. Klewer SE, Yatskievych T, Pogreba K, Stevens MV, Antin PB, Camenisch TD. Has2 expression in heart forming regions is independent of BMP signaling. Gene Expr Patterns. 2006 doi: 10.1016/j.modgep.2005.11.005. [DOI] [PubMed] [Google Scholar]
  36. Korhonen J, Polvi A, Partanen J, Alitalo K. The mouse tie receptor tyrosine kinase gene: expression during embryonic angiogenesis. Oncogene. 1994;9:395–403. [PubMed] [Google Scholar]
  37. Kramer R, Bucay N, Kane DJ, Martin LE, Tarpley JE, Theill LE. Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development. Proc Natl Acad Sci U S A. 1996;93:4833–4838. doi: 10.1073/pnas.93.10.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kuno K, Okada Y, Kawashima H, Nakamura H, Miyasaka M, Ohno H, Matsushima K. ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan. FEBS Lett. 2000;478:241–245. doi: 10.1016/s0014-5793(00)01854-8. [DOI] [PubMed] [Google Scholar]
  39. LeBaron RG, Zimmermann DR, Ruoslahti E. Hyaluronate binding properties of versican. J Biol Chem. 1992;267:10003–10010. [PubMed] [Google Scholar]
  40. Lee KF, Simon H, Chen H, Bates B, Hung MC, Hauser C. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature. 1995;378:394–398. doi: 10.1038/378394a0. [DOI] [PubMed] [Google Scholar]
  41. Lee NV, Rodriguez-Manzaneque JC, Thai SN, Twal WO, Luque A, Lyons KM, Argraves WS, Iruela-Arispe ML. Fibulin-1 acts as a cofactor for the matrix metalloprotease ADAMTS-1. J Biol Chem. 2005;280:34796–34804. doi: 10.1074/jbc.M506980200. [DOI] [PubMed] [Google Scholar]
  42. Lee NV, Sato M, Annis DS, Loo JA, Wu L, Mosher DF, Iruela-Arispe ML. ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2. Embo J. 2006;25:5270–5283. doi: 10.1038/sj.emboj.7601400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Lessard J, Wu JI, Ranish JA, Wan M, Winslow MM, Staahl BT, Wu H, Aebersold R, Graef IA, Crabtree GR. An essential switch in subunit composition of a chromatin remodeling complex during neural development. Neuron. 2007;55:201–215. doi: 10.1016/j.neuron.2007.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lickert H, Takeuchi JK, Von Both I, Walls JR, McAuliffe F, Adamson SL, Henkelman RM, Wrana JL, Rossant J, Bruneau BG. Baf60c is essential for function of BAF chromatin remodelling complexes in heart development. Nature. 2004;432:107–112. doi: 10.1038/nature03071. [DOI] [PubMed] [Google Scholar]
  45. Liu R, Liu H, Chen X, Kirby M, Brown PO, Zhao K. Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell. 2001;106:309–318. doi: 10.1016/s0092-8674(01)00446-9. [DOI] [PubMed] [Google Scholar]
  46. Lyons I, Parsons LM, Hartley L, Li R, Andrews JE, Robb L, Harvey RP. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev. 1995;9:1654–1666. doi: 10.1101/gad.9.13.1654. [DOI] [PubMed] [Google Scholar]
  47. Ma Z, Chang MJ, Shah R, Adamski J, Zhao X, Benveniste EN. Brg-1 is required for maximal transcription of the human matrix metalloproteinase-2 gene. J Biol Chem. 2004;279:46326–46334. doi: 10.1074/jbc.M405438200. [DOI] [PubMed] [Google Scholar]
  48. Ma Z, Shah RC, Chang MJ, Benveniste EN. Coordination of cell signaling, chromatin remodeling, histone modifications, and regulator recruitment in human matrix metalloproteinase 9 gene transcription. Mol Cell Biol. 2004;24:5496–5509. doi: 10.1128/MCB.24.12.5496-5509.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Manasek FJ, Reid M, Vinson W, Seyer J, Johnson R. Glycosaminoglycan synthesis by the early embryonic chick heart. Dev Biol. 1973;35:332–348. doi: 10.1016/0012-1606(73)90028-6. [DOI] [PubMed] [Google Scholar]
  50. Manso AM, Elsherif L, Kang SM, Ross RS. Integrins, membrane-type matrix metalloproteinases and ADAMs: potential implications for cardiac remodeling. Cardiovasc Res. 2006;69:574–584. doi: 10.1016/j.cardiores.2005.09.004. [DOI] [PubMed] [Google Scholar]
  51. Meyer D, Birchmeier C. Multiple essential functions of neuregulin in development. Nature. 1995;378:386–390. doi: 10.1038/378386a0. [DOI] [PubMed] [Google Scholar]
  52. Meyer D, Yamaai T, Garratt A, Riethmacher-Sonnenberg E, Kane D, Theill LE, Birchmeier C. Isoform-specific expression and function of neuregulin. Development. 1997;124:3575–3586. doi: 10.1242/dev.124.18.3575. [DOI] [PubMed] [Google Scholar]
  53. Mittaz L, Russell DL, Wilson T, Brasted M, Tkalcevic J, Salamonsen LA, Hertzog PJ, Pritchard MA. Adamts-1 is essential for the development and function of the urogenital system. Biol Reprod. 2004;70:1096–1105. doi: 10.1095/biolreprod.103.023911. [DOI] [PubMed] [Google Scholar]
  54. Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR. The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev Biol. 1998;202:56–66. doi: 10.1006/dbio.1998.9001. [DOI] [PubMed] [Google Scholar]
  55. Muchardt C, Yaniv M. A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor. Embo J. 1993;12:4279–4290. doi: 10.1002/j.1460-2075.1993.tb06112.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Norata GD, Bjork H, Hamsten A, Catapano AL, Eriksson P. High-density lipoprotein subfraction 3 decreases ADAMTS-1 expression induced by lipopolysaccharide and tumor necrosis factor-alpha in human endothelial cells. Matrix Biol. 2004;22:557–560. doi: 10.1016/j.matbio.2003.11.003. [DOI] [PubMed] [Google Scholar]
  57. Paemen L, Martens E, Norga K, Masure S, Roets E, Hoogmartens J, Opdenakker G. The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors. Biochem Pharmacol. 1996;52:105–111. doi: 10.1016/0006-2952(96)00168-2. [DOI] [PubMed] [Google Scholar]
  58. Porter S, Clark IM, Kevorkian L, Edwards DR. The ADAMTS metalloproteinases. Biochem J. 2005;386:15–27. doi: 10.1042/BJ20040424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ranger AM, Grusby MJ, Hodge MR, Gravallese EM, de la Brousse FC, Hoey T, Mickanin C, Baldwin HS, Glimcher LH. The transcription factor NF-ATc is essential for cardiac valve formation. Nature. 1998;392:186–190. doi: 10.1038/32426. [DOI] [PubMed] [Google Scholar]
  60. Ratajska A, Cleutjens JP. Embryogenesis of the rat heart: the expression of collagenases. Basic Res Cardiol. 2002;97:189–197. doi: 10.1007/s003950200011. [DOI] [PubMed] [Google Scholar]
  61. Reyes JC, Barra J, Muchardt C, Camus A, Babinet C, Yaniv M. Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha) Embo J. 1998;17:6979–6991. doi: 10.1093/emboj/17.23.6979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Roberts CW, Orkin SH. The SWI/SNF complex--chromatin and cancer. Nat Rev Cancer. 2004;4:133–142. doi: 10.1038/nrc1273. [DOI] [PubMed] [Google Scholar]
  63. Rodriguez-Manzaneque JC, Westling J, Thai SN, Luque A, Knauper V, Murphy G, Sandy JD, Iruela-Arispe ML. ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochem Biophys Res Commun. 2002;293:501–508. doi: 10.1016/S0006-291X(02)00254-1. [DOI] [PubMed] [Google Scholar]
  64. Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, Fujisawa H, Betsholtz C, Shima DT. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev. 2002;16:2684–2698. doi: 10.1101/gad.242002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Sato TN, Qin Y, Kozak CA, Audus KL. Tie-1 and tie-2 define another class of putative receptor tyrosine kinase genes expressed in early embryonic vascular system. Proc Natl Acad Sci U S A. 1993;90:9355–9358. doi: 10.1073/pnas.90.20.9355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maguire M, Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature. 1995;376:70–74. doi: 10.1038/376070a0. [DOI] [PubMed] [Google Scholar]
  67. Shimada M, Nishibori M, Yamashita Y, Ito J, Mori T, Richards JS. Down-regulated expression of A disintegrin and metalloproteinase with thrombospondin-like repeats-1 by progesterone receptor antagonist is associated with impaired expansion of porcine cumulus-oocyte complexes. Endocrinology. 2004;145:4603–4614. doi: 10.1210/en.2004-0542. [DOI] [PubMed] [Google Scholar]
  68. Shindo T, Kurihara H, Kuno K, Yokoyama H, Wada T, Kurihara Y, Imai T, Wang Y, Ogata M, Nishimatsu H, et al. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J Clin Invest. 2000;105:1345–1352. doi: 10.1172/JCI8635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71. doi: 10.1038/5007. [DOI] [PubMed] [Google Scholar]
  70. Sumi-Ichinose C, Ichinose H, Metzger D, Chambon P. SNF2beta-BRG1 is essential for the viability of F9 murine embryonal carcinoma cells. Mol Cell Biol. 1997;17:5976–5986. doi: 10.1128/mcb.17.10.5976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996;87:1171–1180. doi: 10.1016/s0092-8674(00)81813-9. [DOI] [PubMed] [Google Scholar]
  72. Thai SN, Iruela-Arispe ML. Expression of ADAMTS1 during murine development. Mech Dev. 2002;115:181–185. doi: 10.1016/s0925-4773(02)00115-6. [DOI] [PubMed] [Google Scholar]
  73. Umans L, Cox L, Tjwa M, Bito V, Vermeire L, Laperre K, Sipido K, Moons L, Huylebroeck D, Zwijsen A. Inactivation of Smad5 in endothelial cells and smooth muscle cells demonstrates that Smad5 is required for cardiac homeostasis. Am J Pathol. 2007;170:1460–1472. doi: 10.2353/ajpath.2007.060839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Wang W, Cote J, Xue Y, Zhou S, Khavari PA, Biggar SR, Muchardt C, Kalpana GV, Goff SP, Yaniv M, et al. Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. Embo J. 1996;15:5370–5382. [PMC free article] [PubMed] [Google Scholar]
  75. Wang Z, Zhai W, Richardson JA, Olson EN, Meneses JJ, Firpo MT, Kang C, Skarnes WC, Tjian R. Polybromo protein BAF180 functions in mammalian cardiac chamber maturation. Genes Dev. 2004;18:3106–3116. doi: 10.1101/gad.1238104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Whetstine JR, Ceron J, Ladd B, Dufourcq P, Reinke V, Shi Y. Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase. Mol Cell. 2005;18:483–490. doi: 10.1016/j.molcel.2005.04.006. [DOI] [PubMed] [Google Scholar]
  77. Wilkinson DG, Nieto MA. Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 1993;225:361–373. doi: 10.1016/0076-6879(93)25025-w. [DOI] [PubMed] [Google Scholar]
  78. Yamamura H, Zhang M, Markwald RR, Mjaatvedt CH. A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse. Dev Biol. 1997;186:58–72. doi: 10.1006/dbio.1997.8559. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS40729-supplement-01.pdf (6.4MB, pdf)

RESOURCES