1 Department of Molecular and Cellular Biology,
2 Section of Cardiovascular Sciences,
3 Center for Cardiovascular Development,
4 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
5 Department of Pathology, Mie University, School of Medicine, Tsu, Mie, Japan
*Author for correspondence (e-mail: schwartz{at}bcm.tmc.edu)
Accepted 8 January 2002
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SUMMARY |
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Key words: Rho GTPases, Rho GDI, Cardiac morphogenesis, Cardiomyocyte proliferation, Mouse
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INTRODUCTION |
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Rho GTPase family proteins, which include RhoA, Rac1 and Cdc42, may have an important role in early mouse heart morphogenesis as they control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation and apoptosis (reviewed by Hall, 1994; Van Aelst and DSouza-Schorey, 1997
). Functional studies in Drosophila and Xenopus suggest that Rho GTPases control a variety of developmental processes associated with cell shape changes, cell adhesion and cell migration (Murphy and Montell, 1996
; Barrett et al., 1997
; Magie et al., 1999
; Strutt et al., 1997
; Wunnenberg-Stapleton et al., 1999
). However, their potential roles in mammalian development, including cardiac morphogenesis, are unknown. A recent study has shown that cardiac-specific overexpression of RhoA results in sinus and atrioventicular nodal dysfunction and contractile failure in adult transgenic mice (Sah et al., 1999
). In addition, cardiac-specific expression of constitutively active Rac1 has been shown to lead to either a lethal neonatal dilated cardiomyopathy or a resolving transient cardiac hypertrophy in juveniles (Sussman et al., 2000
). However, roles for RhoA and Rac1 in embryonic heart development were not addressed.
Rho GDP dissociation inhibitors (Rho GDIs), endogenous inhibitors of Rho GTPases, play an important role in regulating the biological activities of Rho GTPases (reviewed by Sasaki and Takai, 1998). Rho GTPases possess intrinsic GTPase activity and cycle between the inactive, cytoplasmic, GDP-bound and the active, membrane-associated, GTP-bound state. Rho GDIs possess at least two biochemical functions (Ueda et al., 1990
; Isomura et al., 1991
). First, they preferentially interact with the inactive, GDP-bound form of Rho family proteins, and prevent them from being converted to the active, GTP-bound form that is translocated to the membrane. Second, after the active GTP-bound form is converted to the inactive GDP-bound form at the membrane, Rho GDI forms a complex with it and translocates it to the cytosol. The Rho GDI family comprises at least three isoforms: Rho GDI
, ß and
. Rho GDI
is ubiquitously expressed (including heart) and binds to all of the Rho family proteins thus far examined, including RhoA, RhoB, Rac1, Rac2, and Cdc42 (Fukumoto et al., 1990
; Leonard et al., 1992
). Rho GDIß is expressed exclusively in hematopoietic tissues (Lelias et al., 1993
; Scherle et al., 1993
) and Rho GDI
is preferentially expressed in brain (Zalcman et al., 1996
; Adra et al., 1997
). The modes of activation and action of the Rho GTPases are quite different from those of Ras GTPases, since Rho GTPases are predominantly cytosolic GDP-bound and associated with Rho GDIs while Ras GTPases are constitutively located on the plasma membrane, and a Ras GDI has not yet been identified.
We employed a reverse genetic approach to explore the role of Rho family GTPases in murine cardiac development. Specific inhibition of Rho GTPases in cardiomyocytes was achieved by expressing Rho GDI using the cardiac-specific
-myosin heavy chain (
MHC) promoter, which is activated during early cardiogenesis (Subramaniam, 1991
). This approach was expected to disrupt signaling by all Rho GTPases concomitantly during heart tube looping, septation and chamber maturation. We observed that targeted inhibition of Rho GTPase signaling resulted in disruption of cardiac morphogenesis and reduced cardiomyocyte proliferation, thus elucidating a critical role for Rho family proteins in heart morphogenesis.
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MATERIALS AND METHODS |
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Whole mount in situ hybridization of mouse embryos
Whole mount in situ hybridization of mouse embryos was carried out as described previously (Yamada et al., 1999). The antisense probe for the transgene was generated from the polyadenylation sequences of the transgene (human G-CSF cDNA). No signal was observed with a sense probe derived from the same DNA fragment (data not shown). The cardiac
-actin antisense probe was generated from a cDNA fragment containing the 3'UTR (Wei et al., 1998
). Following color development, 10-µm sections were cut from paraffin-embedded whole mounts.
RT-PCR analysis
To compare endogenous gene expression in transgenic hearts versus nontransgenic hearts, RNA samples were prepared from a pool of ten hearts from transgenic or nontransgenic E9.5 embryos of the H2 founder using TRIZOL (Gibco-BRL, Gaithersburg, Maryland). Two RNA samples were analyzed for each group (transgenic versus nontransgenic). First strand cDNA synthesis was carried out with the SuperScrit Preamplification System (Gibco-BRL) in a volume of 50 µl using 2.5 µg of RNA and 125 ng of oligo(dT)12-18. Three reverse transcription products from each RNA sample were pooled. PCR reaction was then carried out with 2 µl of first strand cDNA and one set of specific primers. For each primer set, two or three cycle-numbers were tested to be certain that PCR product accumulates within a linear range. GAPDH (Gapd; 16 to 19 cycles) was amplified as a control marker with primers as described (Charng et al., 1998). Other genes were amplified, each with a specific primer set: p21 (19 to 22 cycles),
MHC (16 to 19 cycles) and MLC2V (16 to 19 cycles) (Charng et al., 1998
); Raldh2 (Aldh1a2; 28 to 30 cycles) (Ulven et al., 2000
); atrial natriuretic factor (ANF/Nppa) (16 to 19 cycles) (Xu et al., 1999
); Mef2c (22 to 25 cycles) (Martin et al., 1993
); Gata4 (22 to 25 cycles) (Xu et al., 1999
); cardiac
-actin (19 to 22 cycles), forward 5'-TGAGATGTCTCTCTCTTA-3', reverse 5'-CGTACAATGACTGATGAG-3'; dHAND (Hand2; 19 to 22 cycles), forward 5'-TACAGTATGGCCCTGTCCTA-3', reverse 5'-TCCAGGGCCCAGACGTGCTG-3'; eHAND (Hand1; 23 to 26 cycles), forward 5'-CCGGCGAGAAGAGGATTAAA-3', reverse 5'-TCAAATGACATTGCACGTGC-3'; Nkx2-5 (19 to 22 cycles), forward 5'-TTGGCGTCGGGGACTTGAAC-3', reverse 5'-AGGCTACGTCAATAAAGTGG-3'; Srf (22 to 25 cycles), forward 5'- GCTGGGAGCAGCAGCAACCT-3', reverse 5'-CCCGTCTCTTTGGCTGGAGT-3'; cyclin A (Ccna2; 19 to 22 cycles), forward 5'-TAAGCCTTGTCTTGTGGACC-3', reverse 5'-CAGGTGGCAGCACCAATGTT-3'.
Proliferation and apoptosis assays
E9.5 embryos were separated from their yolk sacs, which were used for determining their genotype as described above. Embryos were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned (7 µm). The sections containing cardiac structures were selected for immunohistochemical analysis. Proliferation was assayed by examining histone H3 phosphorylation as a marker of mitosis, using a rabbit polyclonal antibody raised against the Ser10 phosphopeptide of histone H3 (Upstate Biotechnology, Lake Placid, NY) (Wei, Y. et al., 1998). Sections were deparaffinized, rehydrated, and incubated with 0.05% trypsin for 15 minutes at 37°C. Sections were then exposed to anti-phosphohistone H3 antibody (5 µg/ml), followed by incubation with fluorescein-conjugated goat anti-rabbit IgG (Molecular Probes) at 1:500 dilution. After several washes, sections were counterstained with DAPI (Molecular Probes) and mounted with Vectashield (Vector Laboratories). The identical overlapping images were used to quantify the number of phosphohistone H3-positive cells as a percentage of total number of cells (nuclei) within the subepicardial cell layer. The data are expressed as mean ± s.e.m. Students t-test was used for data comparison, using a significance level of P<0.05. Apoptosis was assayed using the ApopTag kit according to the manufacturers instructions (Intergen Company). Sections were also counterstained with DAPI (Molecular Probes) and mounted with Vectashield.
Immunoblot analysis of Rho GDI and subcellular fractionation and immunoblot analysis of Rho family proteins
Protein samples were prepared from a pool of 10 hearts from E9.5-12.5 embryos or from a single heart of adult transgenic or nontransgenic mice. Hearts were quickly removed, washed, minced into small pieces then frozen in liquid nitrogen and stored at 70°C. The tissue fragments were thawed and disrupted with a Polytron homogenizer at 4°C in lysis buffer (Wei et al., 1998). The debris was pelleted with a 400 g centrifugation for 10 minutes. The supernatant (homogenate) was used for western blot analysis of Myc-Rho GDI
(transgene), endogenous Rho GDI
or Rho GTPases. Proteins (50 µg) were electrophoresed on a 12% SDS-polyacrylamide gel, transferred to Immobilon membranes (Millipore), and probed with a mouse anti-Myc antibody (Oncogene Research Product), a rabbit anti-Rho GDI antibody (Santa Cruz Biotechnology), a mouse anti-RhoA antibody (Santa Cruz Biotechnology), a mouse anti-Rac1 antibody (Upstate Biotechnology) or a rabbit anti-Cdc42 antibody (Santa Cruz Biotechnology). After incubating with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody, blots were visualized by the enhanced chemiluminescence system (Amersham).
To separate cytosolic and membrane fractions, the tissue fragments were thawed and disrupted with a Polytron homogenizer at 4°C in lysis buffer without Triton X-100. The debris was pelleted with a 400 g centrifugation for 10 minutes, and the supernatant was further centrifuged at 100,000 g for 30 minutes at 4°C. The supernatant was saved as the cytosolic fraction. The pellet was suspended in lysis buffer with 0.1% Triton X-100 and saved as the membrane fraction. Proteins (50 µg) from each fraction were then analyzed by western blotting as described above.
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RESULTS |
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To determine if the reduced cell number was due to altered proliferation of the myocytes, tissue sections from three nontransgenic and three transgenic littermates at E9.5 were stained for phosphorylated histone H3 on Ser10, an established marker for chromosome condensation during mitotic prophase in animal cells (Wei, Y. et al., 1998). In normal embryos at E9.5, the myocardium is subdivided into a peripheral compact zone and an inner trabecular zone. The rate of cell division decreases from the periphery to the inner zone. In transgenic embryos, the inner trabecular zone was absent. The transgenic heart sections displayed significant reduction in the number of phosphorylated histone H3 cells versus nontransgenic heart sections (Fig. 7A,B) and the vast majority of the phosphprylated histone H3 cells were located immediately beneath the epicardium (the subepicardial cell layer) in both nontransgenic and transgenic embryos (Fig. 7C). In tissue sections from nontransgenic embryos, 4.7±1.2% of subepicardial cell nuclei were stained positive, while 1.9±0.5% subepicardial cell nuclei were positive in the transgenic embryos (Fig. 7E). In addition, histone H3 phosphorylation in other tissues of transgenic embryos was markedly more abundant than in the heart (Fig. 7B). These observations indicated that decreased cell proliferation caused hypocellularity in transgenic hearts.
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DISCUSSION |
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A role for Rho family proteins in cell cycle control has been extensively studied using in vitro culture systems, where Rho proteins were required for G1 cell cycle progression (reviewed by Van Aelst and DSouza-Schorey, 1997). It is still unclear whether the effect of Rho proteins on cell proliferation is due to their effects on the actin cytoskeleton or to more direct effects on gene transcription. During normal cardiac development, progressive withdrawal of cardiac myocytes from the cell cycle is associated with up-regulation of p21 and down-regulation of cyclin A (Parker et al., 1995
; Yoshizumi et al., 1995
) (reviewed by MacLellan and Schneider, 2000
). Our study indicated that Rho family proteins were required for cardiac cell proliferation and were involved in repressing p21 expression and in inducing cyclin A expression during looping and ventricular maturation. Interestingly, up-regulation of p21 was also observed in the transgenic hearts expressing a constitutively active mutation of ALK5, a type I TGFß receptor, and both looping morphogenesis and chamber maturation were disrupted in activated ALK5 transgenic hearts at E9.5 (Charng et al., 1998
). Thus, cell cycle regulators appear to be the common targets of Rho and TGFß signaling pathways in cardiac morphogenesis. A hypoplastic ventricular chamber was also a characteristic defect of mouse null mutants for RXR
(Sucov et al., 1994
; Kastner et al., 1994
), N-myc (Moens et al., 1993
), WT-1 (Kreidberg et al., 1993
), TEF-1 (Chen et al., 1994
), gp130 (Yoshida et al., 1996
) or bARK1 (Jaber et al., 1996
) (lethal at E13-16). However, looping morphogenesis was not disrupted in these mutant mice, suggesting that Rho family proteins regulate cardiac morphogenesis via an independent regulatory pathway.
In addition to inhibition of cell proliferation, increased expression of Rho GDI most likely interrupted other morphogenic processes involved in heart tube looping. It is believed that looping of the embryonic heart is not a consequence of growth per se, but rather an intrinsic morphogenic property of the heart tube, partly linked to the left-right asymmetric expression of signaling molecules and extracellular matrix proteins (reviewed by Kathiriya and Srivastava, 2000
). Rho family proteins may be involved in cell shape changes and cell adhesion by regulating interactions between cardiomyocytes and asymmetrically-expressed extracellular matrix proteins during heart looping. The heart tube looping defect of Rho GDI
transgenic embryos was also similar to those of mouse embryos with null mutations for Nkx2.5 (Lyon et al., 1995
), MEF2C (Lin et al., 1997
), dHAND (Srivastava et al., 1997
), eHAND (Riley et al., 1998
) or Raldh2 (Niederreither et al., 2001
). We observed that the expression level of these cardiogenic factors was not dramatically affected in Rho GDI
transgenic hearts (up to 29%), and the heterozygous knockout embryos of each of these factors were previously shown to be phenotypically normal. Therefore, the interruption of looping in Rho GDI
transgenic embryos cannot be ascribed merely to the level of the expression of these factors. However, although the reduced expression of each of the factors was not sufficient to induce developmental arrest, the combined reduction in their expression levels may contribute, in part, to the morphogenic defects observed in Rho GDI
transgenic embryos.
In addition to the changes in cell cycle regulators, expression of Raldh2, MLC2V and dHAND was down-regulated by more than 20% in Rho GDI transgenic hearts. It remains unclear whether Rho family proteins directly regulate transcription of these genes, or whether such down-regulation is secondary to the morphogenic defects of the hearts. However, RhoA regulates SRF-dependent gene activity in cultured fibroblasts (Hill et al., 1995
), skeletal myoblasts (Wei et al., 1998
), smooth muscle cells (Mack et al., 2001
) and terminally differentiated cardiomyocytes (Wei et al., 2001a
). We have also observed that inhibition of Rho kinases, downstream effectors of RhoA, before the onset of cardiomyocyte differentiation caused precocious activation of the cardiac
-actin gene in the bilateral cardiogenic regions of cultured chick embryos (Wei et al., 2001b
). However, expression of these cardiac genes was affected only minimally in the Rho GDI
transgenic hearts, in which inhibition of Rho signaling occurred after the onset of cardiomyocyte differentiation. It is thus possible that RhoA-dependent signaling to SRF contributes to the regulation of cardiac genes in a stage-dependent manner: possibly Rho signaling may inhibit cardiomyocyte differentiation in precardiac cells during proliferation and migration, but they subsequently enhance cardiac gene expression in terminally differentiated cardiomyocytes which have an organized sarcomeric structure.
Our study also suggested that Rho GDI expression must be under tight control during cardiac development as increasing its expression to four times normal levels caused severe defective cardiac morphogenesis (M2 homozygotes). However, a basal level of RhoGDI
is not required for early heart development as mice lacking Rho GDI
were initially viable and normal in appearance, but showed progressive impairment of kidneys and reproductive organs in adult mice (Togawa et al., 1999
). Interestingly, although the heterozygotes of middle copy lines (M1, M2) had no detectable early embryonic phenotype, they developed an adult cardiac phenotype such as cardiac hypertrophy associated with abnormal cardiac functions (L. W., unpublished observations), most likely due to increased transgene expression in the adult hearts compared with embryonic hearts (6-fold versus 1.5-fold relative to the endogenous level).
It is also important to note that Rho GDI expression level was unchanged during cardiac development and increased expression of Rho GDI
had no effect on the expression of the endogenous gene. In contrast, increased expression of Rho GDI
markedly up-regulated expression of RhoA, Rac1 and Cdc42, most likely due to inhibition of Rho GTPase activities, which in turn induced their gene expression through a negative feedback regulatory mechanism. These observations suggested that the regulation of the ratio of Rho family proteins to their endogenous inhibitor is an important mechanism controlling the activities of Rho family proteins and this ratio is regulated through modulating the expression level of Rho family proteins while the level of Rho GDI
remains stable. Further studies will be required to determine the involvement of each protein of the Rho family and the roles of downstream effectors of Rho family proteins in cardiac morphogenesis, and how Rho family members regulate their own gene expression.
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ACKNOWLEDGMENTS |
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