SEARCH

SEARCH BY CITATION

Keywords:

  • heart development;
  • cardiac conduction system;
  • muscle segment homeobox genes;
  • Msx1;
  • Msx2

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Msx1 and Msx2 are essential for the development of many organs. In the heart, they act redundantly in development of the cardiac cushions. Additionally, Msx2 is expressed in the developing conduction system. However, the exact expression of Msx1 has not been established. We show that Msx1 is expressed in the cardiac cushions, but not in the myocardium. In Msx2-null mice, Msx1 is not ectopically expressed in the myocardium. The absence of myocardial defects in the Msx2 knock-out can therefore not be attributed to a redundant action of Msx1 in the myocardium. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Msx1 and Msx2 are members of the highly conserved Nk-like family of homeodomain transcription factors, and their expression is associated with epithelio-mesenchymal interactions at many sites in vertebrate embryos, such as the limb buds, craniofacial regions, and tooth buds (Hill et al., 1989; Robert et al., 1989). Knock-out experiments have shown that Msx1-null mutations provoke defects in craniofacial development, cleft palate, inner ear malformations, and tooth agenesis (Satokata and Maas, 1994; Houzelstein et al., 1997), whereas Msx2-null mice display abnormal tooth development, defects in bone growth and abnormal development of the cerebellum (Satokata et al., 2000). Additionally, Msx1–Msx2 compound mutant mice die in late gestation with severe craniofacial abnormalities (Bei and Maas, 1998; Satokata et al., 2000), limbs defects (Lallemand et al., 2005) and heart defects (Ishii et al., 2005), indicating a redundant role for Msx1 and Msx2 during several developmental processes.

During chicken and mouse heart development, Msx2 is expressed in the myocardium of the atrioventricular canal and outflow tract, and shows a close association with the developing conduction system (MacKenzie et al., 1992; Chan-Thomas et al., 1993; Tanaka et al., 1999; Abdelwahid et al., 2001; Thomas et al., 2001). The myocardium of the developing conduction system is prevented from differentiating to chamber myocardium, (Christoffels et al., 2000), a feature which can be attributed, in part, to the repressive action of the T-box transcription factors Tbx2 and Tbx3 (Habets et al., 2002; Christoffels et al., 2004; Hoogaars et al., 2007). However, other factors are likely to be involved in this process. Recently, we showed that Tbx2 and Tbx3 can interact with Msx1 and Msx2 and cooperate in the regulation of Gja1 expression, the Connexin43 encoding gene that marks the differentiating chamber myocardium (Boogerd et al., 2008). Msx2 is also expressed in the mesenchyme and endocardium of the cushions (MacKenzie et al., 1992; Tanaka et al., 1999; Abdelwahid et al., 2001). Msx1 has been shown to be expressed in the cushions as well (Robert et al., 1989), but its exact expression pattern in the developing mouse heart has not been adequately determined to date.

The fact that the hearts in Msx1 and Msx2 knockout mice develop normally indicates the existence of a redundant mechanism during heart development (Satokata and Maas, 1994; Satokata et al., 2000; Kwang et al., 2002). Indeed, in the absence of both Msx1 and Msx2, mice hearts develop a range of outflow tract alignment defects including double outlet right ventricle, overriding aorta and pulmonary stenosis (Chen et al., 2007), as well as hypoplastic atrioventricular cushions and deformed atrioventricular valves (Chen et al., 2008). Notably, myocardial patterning in the atrioventricular canal is also affected as noted by the decreased expression of Tbx2 as well as the ectopic expression of the chamber marker Nppa in the atrioventricular myocardium. These findings have contributed to the model that Msx1 and Msx2 act downstream of BMP's during epithelial mesenchymal transition in cushion morphogenesis (Ma et al., 2005; Chen et al., 2008). However, the mechanism underlying myocardial mis-patterning in the Msx1–Msx2 double knock-out is not fully understood.

In this study, we try to shed light on the roles of Msx1 and Msx2 in the development of the conduction system myocardium by examining in detail their expression patterns in wildtype and Msx2-null mice. We show that Msx1 is expressed in the mesenchyme and endocardium of the cardiac cushions, but not in the myocardium of the outflow tract or atrioventricular canal. Moreover, in an Msx2-null background, Msx1 is not ectopically expressed in the myocardium. These findings suggest that myocardial patterning defects in the Msx1–Msx2 compound mutant mice are due to an interaction between the atrioventricular cushion tissue and the myocardium. Still, the role of myocardial Msx2 expression in the cardiac conduction system remains the subject of ongoing investigations.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Msx2−/− mice were described earlier (Satokata et al., 2000), mutant phenotypes were analyzed in a mixed BALB/c-Black6-FVB background. In situ hybridization was performed as described earlier on 10-μm thick sections (Moorman et al., 2001). A 500 bp EcoNI-SphI fragment of the 3′-untranslated region (UTR) of mouse Msx1 (NM_010835; bp 1161–1697) was used as a template for the Msx1 antisense probe. Msx2 probe, directed against a part of the coding sequence 3′ of the homeodomain plus part of the 3′-UTR (pHox8) has been described before (Monaghan et al., 1991). Sections were photographed on a Leica Axiophot microscope.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

To gain understanding into the roles of Msx1 and Msx2 during conduction system development, we analyzed the cardiac expression patterns of Msx1 and Msx2 during mouse heart development. At embryonic day 10.5 (E10.5) and E11.5, Msx2 is expressed in both the myocardium and the cushions of the atrioventricular canal and outflow tract (Fig. 1a) (Tanaka et al., 1999; Abdelwahid et al., 2001). Msx1 is expressed in the mesenchyme and endocardium of the cardiac cushions (Fig. 1a). Msx1 expression was not detected in the myocardium of the atrioventricular canal, the outflow tract or the cardiac chambers. In addition, we observed Msx1 expression in previously recognized expression domains including the limb buds, neural tube, mandibular, and maxillary component of the first branchial arch, as well as the anterior pituitary or Rathke's pouch (Supporting Information Fig. 1) (Hill et al., 1989; Robert et al., 1989; MacKenzie et al., 1991, 1992). Expression in the atrioventricular cushions was also described in the first publication by Robert et al. (1989). However, more recent articles describing Msx1–Msx2 compound mutant hearts seem to indicate that Msx1 is also expressed in the myocardium of the outflow tract and right ventricle (Ishii et al., 2005; Chen et al., 2007). Our study shows that Msx1 is clearly not expressed in the myocardium, a discrepancy which may find its origin in the probes that were used to detect Msx1. For this study, we made use of probes directed against the 3′-UTR of the Msx1 messenger, which is specific for Msx1. The probes used in the studies by Chen et al. and Ishii et al. make use of the entire Msx1 coding region, including the highly conserved homeodomain, therefore it cannot be excluded that cross hybridization with other homeodomain containing messengers has occurred.

thumbnail image

Figure 1. In situ hybridization on embryonic mouse heart tissue. (A) Saggital sections showing that at E10.5 and E11.5, Msx1 and Msx2 expression in the heart is detected in the mesenchyme and endocardium of the cardiac cushions (arrowheads). Msx2 is also expressed in the myocardium (arrows), in which Msx1 expression was not detected (red arrows). (B) and (C) Transversal sections of E11.5 heart tissue of wildtype and Msx2-null hearts, showing the expression of Msx1 and Msx2 in the cushions (arrowheads), and expression on Msx2 in the myocardium of the atrioventricular canal and outflow tract (arrows), in which Msx1 expression is not detected (red arrows). Note that Msx2 mRNA, which is detected in Msx2-null mice encodes a truncated nonfunctional protein (Satokata et al., 2000). cTnI marks all myocardium. Size bars: 0.2 mm. Abbreviations: avc, atrioventricular canal; v, ventricle; oft, outflow tract; ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle.

Download figure to PowerPoint

In the absence of the closely related protein Nkx2.5, Msx2 is ectopically expressed throughout the myocardium of the cardiac chambers (Tanaka et al., 1999). To establish whether a similar mechanism might induce ectopic Msx1 expression in the Msx2-null mice, we established the expression pattern of Msx1 in Msx2-null mice. As shown in Fig. 1b,c, Msx1 is not expressed in the myocardium of the atrioventricular canal and outflow tract of Msx2−/− mice. The myocardium of the atrioventricular canal and outflow tract does not express chamber myocardial markers Cx43 and Nppa (Chen et al., 2008). Therefore, we conclude that myocardial expression of Msx genes is not essential for normal heart development.

Whereas the redundant action of Msx1 has always been used as an argument to explain that Msx2-null mice develop normal hearts, we show that Msx1 is not expressed in the myocardium of wildtype or Msx2 knock-out mice. Therefore, we conclude that the function of Msx2 in the myocardium is either dispensable, or is masked by the action of another redundant factor. The question that then arises is what causes the myocardial patterning defects in the Msx1–Msx2 double null mice (Chen et al., 2008), which differ only from Msx2 knock-outs in that they additionally lack Msx1 expression in the cushions. In the atrioventricular canal, BMP2 activates myocardial Tbx2 and Msx2 expression, as well as Msx1 expression in the cushions (Fig. 2) (Ma et al., 2005; Singh et al., 2009). In the absence of Msx1 and Msx2, Tbx2 expression is lost, which is likely to lead to the observed ectopic ANF expression (Habets et al., 2002; Chen et al., 2008). We propose a model in which a non cell-autonomous signal downstream of Msx1 and Msx2 in the cushions, is necessary for the expression of Tbx2 in the myocardium. This signal, designated “y” in Fig. 2, may induce Tbx2 expression directly, or indirectly, for instance via BMP2 induction (Ma et al., 2005; Singh et al., 2009). The role of Msx2 in the conduction system myocardium still needs to be addressed in more detail.

thumbnail image

Figure 2. Proposed Msx1 and Msx2 function in the atrioventricular canal. Msx1 and Msx2 expression in the cushions is necessary for expression of Tbx2 in the atrioventricular myocardium, via an unknown non cell-autonomous signal (y), that activates Tbx2 expression directly, or indirectly, for instance via BMP2. Msx2 expression in the myocardium seems to be dispensable for myocardial patterning.

Download figure to PowerPoint

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

The authors thank Dr. Robert Maxson for the kind gift of Msx2 knock-out mice, and Corrie de Gier-de Vries and Quinn Gunst for their technical assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information
  • Abdelwahid E, Rice D, Pelliniemi LJ, Jokinen E. 2001. Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart. Cell Tissue Res 305: 6778.
  • Bei M, Maas R. 1998. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development 125: 43254333.
  • Boogerd KJ, Wong LYE, Christoffels VM, Klarenbeek M, Ruijter JM, Moorman AFM, Barnett P. 2008. Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of connexin 43. Cardiovasc Res 78: 485493.
  • Chan-Thomas PS, Thompson RP, Robert B, Yacoub MH, Barton PJR. 1993. Expression of homeobox genes Msx-1 (Hox-7) and Msx-2 (Hox-8) during cardiac development in the chick. Dev Dyn 197: 203216.
  • Chen YH, Ishii M, Sucov HM, Maxson RE, Jr. 2008. Msx1 and Msx2 are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium. BMC Dev Biol 8: 75.
  • Chen YH, Ishii M, Sun J, Sucov HM, Maxson RE, Jr. 2007. Msx1 and Msx2 regulate survival of secondary heart field precursors and post-migratory proliferation of cardiac neural crest in the outflow tract. Dev Biol 308: 421437.
  • Christoffels VM, Habets PEMH, Franco D, Campione M, de Jong F, Lamers WH, Bao ZZ, Palmer S, Biben C, Harvey RP, Moorman AFM. 2000. Chamber formation and morphogenesis in the developing mammalian heart. Dev Biol 223: 266278.
  • Christoffels VM, Hoogaars WMH, Tessari A, Clout DEW, Moorman AFM, Campione M. 2004. T-box transcription factor Tbx2 represses differentiation and formation of the cardiac chambers. Dev Dyn 229: 763770.
  • Habets PEMH, Moorman AFM, Clout DEW, van Roon MA, Lingbeek M, Lohuizen M, Campione M, Christoffels VM. 2002. Cooperative action of Tbx2 and Nkx2.5 inhibits ANF expression in the atrioventricular canal: implications for cardiac chamber formation. Genes Dev 16: 12341246.
  • Hill RE, Jones PF, Rees AR, Sime CM, Justice MJ, Copeland NG, Jenkins NA, Graham E, Davidson DR. 1989. A new family of mouse homeo box-containing genes: molecular structure, chromosomal location, and developmental expression of Hox-7.1. Genes Dev 3: 2637.
  • Hoogaars WMH, Barnett P, Moorman AFM, Christoffels VM. 2007. T-box factors determine cardiac design. Cell Mol Life Sci 64: 646660.
  • Houzelstein D, Cohen A, Buckingham ME, Robert B. 1997. Insertional mutation of the mouse Msx1 homeobox gene by an nlacZ reporter gene. Mech Dev 65: 123133.
  • Ishii M, Han J, Yen HY, Sucov HM, Chai Y, Maxson RE, Jr. 2005. Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest. Development 132: 49374950.
  • Kwang SJ, Brugger SM, Lazik A, Merrill AE, Wu LY, Liu YH, Ishii M, Sangiorgi FO, Rauchman M, Sucov HM, Maas RL, Maxson RE, Jr. 2002. Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest. Development 129: 527538.
  • Lallemand Y, Nicola MA, Ramos C, Bach A, Cloment CS, Robert B. 2005. Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development. Development 132: 30033014.
  • Ma L, Lu MF, Schwartz RJ, Martin JF. 2005. Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning. Development 132: 56015611.
  • MacKenzie A, Ferguson MW, Sharpe PT. 1992. Expression patterns of the homeobox gene, Hox-8, in the mouse embryo suggest a role in specifying tooth initiation and shape. Development 115: 403420.
  • MacKenzie A, Leeming GL, Jowett AK, Ferguson MW, Sharpe PT. 1991. The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro. Development 111: 269285.
  • Monaghan AP, Davidson DR, Sime C, Graham E, Baldock R, Bhattacharya SS, Hill RE. 1991. The Msh-like homeobox genes define domains in the developing vertebrate eye. Development 112: 10531061.
  • Moorman AFM, Houweling AC, de Boer PAJ, Christoffels VM. 2001. Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol. J Histochem Cytochem 49: 18.
  • Robert B, Sassoon D, Jacq B, Gehring W, Buckingham M. 1989. Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J 8: 91100.
  • Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang Z, Maxson R, Maas R. 2000. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet 24: 391395.
  • Satokata I, Maas R. 1994. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet 6: 348356.
  • Singh R, Horsthuis T, Farin HF, Grieskamp T, Norden J, Petry M, Wakker V, Moorman AF, Christoffels VM, Kispert A. 2009. Tbx20 interacts with smads to confine Tbx2 expression to the atrioventricular canal. Circ Res 105: 442452.
  • Tanaka M, Chen Z, Bartunkova S, Yamasaki N, Izumo S. 1999. The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development 126: 12691280.
  • Thomas PS, Kasahara H, Edmonson AM, Izumo S, Yacoub MH, Barton PJR, Gourdie RG. 2001. Elevated expression of Nkx-2.5 in developing myocardial conduction cells. Anat Rec 263: 307313.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
AR_21112_sm_suppinfofig1.tif7458KSupplementary figure 1. Fig. 1. In situ hybridization for Msx1 and Msx2 on sagital sections of E11.5 mouse embryos. Msx1 and Msx2 are expressed in the maxillary (mx) and mandibulary (md) part of the first branchial arch, thoracic body wall (tb), the hindlimb bud (hl), the neural tube (nt) and specific parts of the developing brain (arrows). Msx1 is also expressed in the developing anterior pituitary (ap).

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.