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Keywords:

  • jumonji;
  • ARID transcription factor family;
  • cardiac development;
  • gene trap technology;
  • transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Cardiac development is a complex biological process requiring the integration of cell specification, differentiation, migration, proliferation, and morphogenesis. Although significant progress has been made recently in understanding the molecular basis of cardiac development, mechanisms of transcriptional control of cardiac development remain largely unknown. In search for the developmentally important genes, the jumonji gene (jmj) was identified by gene trap technology and characterized as a critical nuclear factor for mouse embryonic development. Jmj has been shown to play important roles in cardiovascular development, neural tube fusion process, hematopoiesis, and liver development in mouse embryos. The amino acid sequence of the JUMONJI protein (JMJ) reveals that JMJ belongs to the AT-rich interaction domain transcription factor family and more recently has been described as a member of the JMJ transcription factor family. Here, we review the roles of jmj in multiple organ development with a focus on cardiovascular development in mice. Developmental Dynamics 232:21–32, 2005. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Congenital heart defects are the most common form of human birth defects. Formation of the heart requires precise interactions among multiple embryonic cell types that induce or suppress a panel of tissue-specific and ubiquitously expressed genes. Understanding the genetic control of cardiac development has been significantly improved by the ability to generate targeted and random mutations in the mouse, which also provides a rich source of new information concerning the possible etiology of human cardiovascular anomalies. A gene trap approach is designed to identify novel, developmentally regulated genes in mouse embryos by random mutation (Friedrich and Soriano, 1991; Wurst et al., 1995). The gene trap technology provides a versatile strategy by which genes that are critical for development can be identified and characterized, while at the same time, corresponding mutants can be produced.

Previous studies have provided evidence that a more systematic approach directed at identifying genes important in cardiac formation may yield insight into the bases of congenital heart disease (Chen et al., 1994; Takeuchi et al., 1995, 1999; Lee et al., 2000). For example, Lyons et al. (1995) have harnessed the powerful genetics available in mice and performed a large-scale screen to search for genes that are activated during the critical periods of cardiogenesis (Baker et al., 1997). The promoterless gene trap vector, β-geo (Friedrich and Soriano, 1991), which contains the lacZ reporter and neomycin-resistance fusion gene (neoR) was randomly inserted into the embryonic stem cell genome by retroviral infection. From this screen, an ES cell clone that contains the jmj mutant allele by insertion of the gene trap vector was identified and jmj was described as a gene that is expressed in ES cell-derived cardiomyocytes (Baker et al., 1997). The jmj gene was also identified by a similar gene trap technology and characterized as a developmentally important gene (Takeuchi et al., 1995). However, morphological and physiological defects of jmj homozygous mutants seem to vary depending on the genetic background, which necessitates careful review of its developmental roles. First, we will briefly review current knowledge of the morphogenetic mechanisms controlling cardiac development followed by a discussion on the roles of the jmj gene in embryonic development with an emphasis on cardiac development.

CARDIAC DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

The heart is the first organ to form and function during organogenesis. The earliest functioning embryonic vertebrate heart is a linear tube, which forms in the human at approximately 3 weeks of gestation and in the mouse at approximately embryonic day (E) 8.0. The contractile cells of the tube constitute the myocardium and the lumen is lined by a single-cell layer of endocardium formed by a type of noncontractile endothelial cells similar to those lining blood vessels. The heart of higher vertebrates further undergoes a series of morphogenetic steps including looping, septation, trabeculation, and thickening of the ventricular walls to generate the four-chambered heart (Fig. 1).

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Figure 1. Diagram of normal heart development. Schematic representation of normal heart development, illustrating heart tube looping, valve formation, and chamber septation. LV, left ventricle; RV, right ventricle; A-V, atrioventricular; S-A, sinoatrial. This figure is adapted from Olson, 2004.

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Formation of a Mature Cardiac Chamber

The fetal ventricular chamber at E9.5 is a thin-walled and undivided structure. Formation of a mature ventricular chamber requires two distinct proliferative and morphogenic processes, both of which initiate around E9.5–E10.5. Proliferation of the myocardium on the inner (endocardial) side of the ventricular chamber wall and the alignment of these cells into bundles forms the trabecular layer of the myocardium (Moorman and Lamers, 1999). Proliferation of the myocardium on the outer (epicardial) side of the chamber wall, and the alignment of these cells along the circumferential axis of the heart forms the compact zone of the ventricular chamber wall. The epicardial lineage originates from a region adjacent to the septum transversum and migrates over the outer surface to ultimately (by E9.5–E10.5) surround the heart (Viragh and Challice, 1981). Subsequent interactions between these primary cell layers are responsible for generating the endothelium and smooth muscle of the coronary arteries, as well as the cardiac fibroblast lineage (Mikawa and Gourdie, 1996). The epicardial layer also plays important roles in myocardial cell growth in the compact zone, because the defects in the epicardium seem to cause a thin (hypoplastic) ventricular wall (Chen et al., 2002b; Olson and Schneider, 2003).

The normal formation of the cellularized endocardial cushion is a prerequisite for the septation, valve leaflet formation, and structural alignment of the four-chambered heart (Eisenberg and Markwald, 1995; Mjaatvedt et al., 1999). Substantial heart cushions form in two segments of the heart tube, the atrioventricular canal and conotruncus (Fig. 1). Within these two segments, some endothelial cells (Wunsch et al., 1994) undergo a transition to form mesenchyme that invades and populates the underlying expanded layer of extracellular matrix (cardiac jelly). The myocardium in these segments is highly secretory and is responsible for the localized expansion of the cushion by the addition of matrix molecules, which include laminin, collagen, fibronectin, and proteoglycans. After initial epithelial to mesenchymal transition, subpopulations of endocardial-derived mesenchyme continue to proliferate and/or differentiate.

Transcriptional Control of Heart Development

Significant advances have been made recently in understanding the role of transcription factors during heart development on the basis of their spatial and temporal expression patterns or their phenotypic effects when they are functionally inactivated in flies or mice. Transcription factors playing critical roles in early cardiac morphogenesis include the following: Nkx2.5, the homeodomain protein (Lyons et al., 1995); MEF2C, a MADS box factor (Lin et al., 1997); GATA-4, a zinc finger domain protein (Kuo et al., 1997; Molkentin et al., 1997); and HAND1 and HAND2, the basic helix-loop-helix (bHLH) factors (Srivastava et al., 1995; also see review articles for other nuclear factors, Olson and Srivastava, 1996; Cripps and Olson, 2002; Olson and Schneider, 2003; Olson, 2004). It is interesting that there are cooperative interactions among the cardiac-restricted transcription factors Nkx2.5, Tbx5, GATA4, MEF2, and serum response factor to activate cardiac-specific genes (Chen and Schwartz, 1996; Durocher et al., 1997; Lee et al., 1997, 1998; Belaguli et al., 2000). However, none of these factors alone or in combination can induce endogenous cardiac gene expression in cultured cells. In addition, despite their apparent importance in early cardiac development, deletion of the genes encoding Nkx2.5, Tbx5, MEF2, and GATA4 in mice does not prevent specification or differentiation of cardiomyocytes (Lyons et al., 1995; Kuo et al., 1997; Molkentin et al., 1997; Bruneau et al., 2001). These data suggest that there may be unidentified factors that are cardiogenic determinants. Recently, myocardin has been reported to function as a master regulator of smooth muscle genes (Chen et al., 2002a) and to activate transcription of cardiac genes (Wang et al., 2001).

Identification of molecular pathways involved in normal heart development led to the discovery of the genetic basis for human congenital heart disease. For example, congenital heart disease characterized by septal defects and abnormal atrioventricular conduction is caused by mutations in the transcription factor Nkx2.5 (Schott et al., 1998). Holt–Oram syndrome characterized by upper limb malformations and cardiac septation defects is caused by mutations in the Tbx5 gene (Basson et al., 1997; Bruneau et al., 1999; Hiroi et al., 2001). More recently, human cardiac septal defects were linked to mutations of GATA4 (Garg et al., 2003). Therefore, we are entering a new exciting era of identifying genetic etiologies of human heart diseases.

MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

JMJ encodes a nuclear factor that belongs to an AT-rich interaction domain (ARID) transcription factor family (Takeuchi et al., 1995; Lee et al., 2000; see Fig. 2). The ARID motif was identified initially from the analysis of four regulatory factors—mouse Bright (B-cell regulator of IgH transcription; Herrscher et al., 1995), human modulator recognition factors MRF-1 and MRF-2 (Huang et al., 1996; Whitson et al., 1999), and Drosophila melanogaster Dead ringer (DRI; Gregory et al., 1996; Iwahara and Clubb, 1999), which were isolated by their abilities to bind DNA in vitro. Members of the ARID family are found in fungi, plants, and animals. They play essential roles in transcriptional regulation of embryonic development, cell cycle control, and chromatin remodeling. The ARID motif is highly conserved evolutionarily in eukaryotes (Kortschak et al., 2000). Recently, two separate homology regions (jmjN and jmjC) common to members of the JMJ family of eukaryotic transcription factors have been described (Balciunas and Ronne, 2000). On the basis of significant sequence similarity, jmjC domains were identified in more than 100 eukaryotic and bacterial sequences (Clissold and Ponting, 2001). These include human hairless mutated in individuals with alopecia universalis, retinoblastoma-binding protein 2 (RBP2), and several putative chromatin-associated proteins (Defeo-Jones et al., 1991; Fattaey et al., 1993; Ahmad et al., 1998; Clissold and Ponting, 2001) such as a novel yeast protein, Epe-1, that destabilizes heterochromatization through its jmjC domain (Ayoub et al., 2003).

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Figure 2. Schematic diagram of the structure of JUMONJI protein (JMJ). The amino acid sequence of JMJ reveals the jmjN and jmjC domains and AT-rich interaction domain (ARID) motif as indicated. Structural and functional analyses by Kim et al. (2003) showed functional domains including the domains for nuclear localization signal (NLS), transcriptional repression (TR), and DNA binding (DBD) that mediates binding to DNA sequence (AT-rich) as well as protein–protein interaction.

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Some ARID proteins exhibit sequence-specific DNA binding such as Bright, DRI, and MRF2 (Herrscher et al., 1995; Gregory et al., 1996; Whitson et al., 1999), whereas others do not, such as OSA and RBP1 (Treisman et al., 1997; Vazquez et al., 1999). Although some ARID factors are transcriptional activators such as yeast SWI1 (Peterson and Herskowitz, 1992), other ARID proteins are involved in gene repression mechanisms. Dri, for example, has been shown to be required for dorsal-dependent repression (Valentine et al., 1998). DRI binds adjacent to Dorsal-binding sites in the zerknult (zen) enhancer permitting the efficient binding of the Groucho co-repressor to DRI and Dorsal, converting Dorsal from its normal activity as an activator to a repressor. Repression of transcription is also an important function of the RBP1 protein (Fattaey et al., 1993). Overexpression of RBP1 inhibited E2F-dependent gene expression and suppressed cell growth (Lai et al., 1999). RBP1 appears to mediate repression partly by binding histone deacetylases (HDAC; Lai et al., 1999). The role of the ARID in the function of RBP1 is yet to be determined.

In vitro analyses on structural and functional relationships revealed that the JMJ protein contains three functional domains (Fig. 2); a nuclear localization signal domain (NLS, amino acids [aa] 1-130), a transcriptional repression domain (TR, aa 131-222), and a DNA binding domain (DBD, aa 528-798; Kim et al., 2003). Thus, JMJ appears to function as a transcriptional repressor by means of binding to AT-rich DNA sequences, which contain a 5′-(T)4-6, −TATT, or −TAAT motif (Kim et al., 2003). However, endogenous target genes of JMJ and molecular mechanisms by which JMJ regulates expression of target genes remain largely unknown until recently. Toyoda et al. (2003) reported that JMJ might regulate cell cycle by repressing the cyclin D1 promoter (see below for details). JMJ also appears to repress expression of the ANF gene by interacting with cardiac transcription factors that activate ANF expression such as Nkx2.5 and GATA4 (Kim et al., in press; see below for details). Therefore, it is possible that JMJ has multiple target genes and uses different regulatory mechanisms, depending on the tissue and promoter context.

EXPRESSION PATTERNS OF THE jmj GENE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Examination of developmental expression patterns of jmj would provide a necessary foundation for functional studies in developing embryos. Jmj expression pattern in mouse embryos, especially in the heart, was examined by in situ hybridization on paraffin-embedded tissue sections (Fig. 3; Lee et al., 2000). Jmj transcripts were expressed at a higher level in the neural ectoderm and decidual tissue than in embryonic mesoderm and endoderm in parasagittal sections of E7.5 embryos (Fig. 3A). Jmj transcripts were expressed at E8.0 in the myocardium (Fig. 3C) as delineated by the specific Nkx2.5 expression hybridized to a serial section (Fig. 3E), which is one of the earliest cardiac marker genes (Komuro and Izumo, 1993; Lyons et al., 1995). Expression continued throughout embryonic heart development in the myocardium with higher mRNA levels in the outflow tract, ventricular septum, and the ventricular wall, including the compact layer and trabeculae than in the atrial wall (Fig. 3F,G,I). Expression was elevated in the spinal cord, dorsal root ganglia, thymus, limb bud, and brown fat as the embryo develops (Fig. 3F,G,I). Jmj continues to be expressed in the adult heart detected by in situ hybridization, β-galactosidase (β-gal) staining, and Northern blot analyses at somewhat reduced levels (data not shown). When expression of jmj in adult mouse tissues was examined by Northern blot analysis, a single band of 5.4 kb was observed in all tissues. Jmj was expressed at higher levels in the heart, brain, thymus, and skeletal muscle than in the lung, liver, and spleen (Lee et al., 2000).

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Figure 3. Developmental expression pattern of jmj between embryonic day (E) 7.5 and E16.0. Frontal (C–E), transverse (F–H), or sagittal sections (A,B,I,J) of normal mouse embryos were in situ hybridized using an antisense jmj cRNA probe. A,B: E7.5. CE: E8.0. F: E13.5. G,H: E15.0. I,J: E16.0. The sense cRNA probe used as a negative control (B,D,H,J) did not hybridize to any tissues, indicating the specificity of the antisense probe. a, atrium; bf, brown fat; dg, dorsal root ganglia; d, maternal deciduum; ee, extraembryonic endoderm; en, endoderm; ht, heart; lg, lung; lv, liver; me, mesoderm; ot, outflow tract; ne, neural ectoderm; v, ventricle; sp, spinal cord; ty, thymus. This figure is reprinted from Lee et al. (2000). Scale bars = 200 μm in A (applies to A–E), 400 μm in F (applies to F–J).

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Expression patterns of jmj in embryos and brains (E14.5–postnatal day 14) have been described by lacZ expression whose pattern reflected the expression of the endogenous gene (Fig. 4; Takeuchi et al., 1995, 1999; Lee et al., 2000). Strong expression of jmj in the midbrain–hindbrain boundary (E8.0–E16.5) and the dorsal root ganglia neurons was observed throughout development. Expression in other tissues was seen such as in the primitive pharynx, the bulbus cordis of the heart, and around the posterior neuropore in the tail (Takeuchi et al., 1995) in addition to limb bud, liver primordium, foregut, and primitive left ventricle (Fig. 4A–C; Lee et al., 2000). Expression in the cerebellum was first detected at E14.5 in its lateral regions and spread to the whole cerebellum at E16.5 (Takeuchi et al., 1995). The transverse sections of embryos from E11.5 to E16.0 showed continued lacZ expression in the myocardium of heterozygous embryos during this period (Fig. 4D–F; Lee et al., 2000).

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Figure 4. Expression pattern of jmj by β-gal staining. AC: Whole-mount β-gal staining of embryonic day (E) 9.5 heterozygotes (A) and homozygotes embryos (B,C at a higher magnification) showed wide expression of the JUMONJI protein (JMJ) -βgeo fusion protein, including the heart. DF: β-gal staining was performed on transverse sections of E11.5, E14.0, and E16.0 heterozygous embryonic hearts (D, E, and F, respectively). An arrowhead in A indicates the mid–hind brain boundary. The arrows in D, E, and F indicate the ventricular septum. ht, heart; lp, liver primordium; fg, foregut; lb, limb bud; bc, bulbus cordis; plv, primitive left ventricle. This figure is reprinted from Lee et al., 2000. Scale bars in D–F = 200 μm.

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The human JMJ protein shows 90 % identity to the mouse JMJ (Berge-Lefranc et al., 1996). Human jmj mRNAs are expressed in similar patterns to those of the mouse, although jmj expression in the human heart was not known. Therefore, jmj is expressed in multiple organs but its expression seems to be tightly regulated in a temporal and spatial manner during development.

ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

JMJ is expressed throughout all the cardiac developmental stages from the precardiac mesoderm to adult hearts (Lee et al., 2000). Severe defects have been reported in jmj homozygous mouse embryos, although variable phenotypes were described depending on the genetic background of the mice (Takeuchi et al., 1995, 1999; Lee et al., 2000). The mutant embryos with a pure C3H/He genetic background died around E11.5 and showed a swollen bulbus cordis and neural tube defects at approximately E7.5 (Takeuchi et al., 1999). Histological examination of tissue sections revealed that the left ventricle of mutant embryos were filled with cells of the trabeculae at E10.5, whereas normal embryos evidenced developing trabeculae in the ventricle (Fig. 5). Examination of the ultrastructure by transmission electron microscopy revealed that myocytes in the trabeculae of control embryos at E10.5 adhere tightly to one another and often show long myofilaments. In contrast, many spaces are apparent between the cells in the trabeculae of mutant embryos and the myofilaments are absent in many cells and appear short and disoriented. Therefore, the cause of lethality in jmj mutants with a C3H/He background may be the heart defect where the left ventricle is filled with trabecular myocytes, which would prevent blood in the ventricular lumen from circulating. However, the identity of these cells that fill the left ventricle has not been examined by immunohistochemical staining. It would be critical to perform immunohistochemical studies using widely used antibodies recognizing cardiomyocytes such as anti-cardiac troponin I, cardiac α-actinin, or sarcomeric myosin heavy chain.

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Figure 5. Cardiac defects of early stage embryos. Hematoxylin and eosin staining of embryonic heart section of wild-type and jmj mutant at embryonic day 10.5. Note that the trabecular layer of the mutant is abnormally hyperplastic. Adapted from Takeuchi et al. (1999). Scale bars = 50 μm.

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This phenotype observed in the jmj mutant heart with a C3H/He background is rather unique because hyperproliferation was observed only in the trabeculae of the left ventricle. Such a hyperproliferation phenotype has not been reported with any of the knockout mice to our knowledge. Toyoda et al. (2003) subsequently reported increased cell density and mitosis in trabecular cardiomyocytes of mutants at E10.5 compared with those in wild-type. Their analyses on lacZ expression showed that expression of jmj began in the trabecular layer of the ventricles at E10.5 when the mitotic index in the trabecular layer started to decrease. Jmj expression was subsequently observed in the compact layer at E12.5 (Takeuchi et al., 1999; Toyoda et al., 2003) when the mitotic index of the compact layer began to decrease, suggesting JMJ normally suppresses cell growth in the heart. These mutants at E10.5 showed lower expression levels of cardiac-specific genes in the trabecular layer by in situ hybridization, such as MLC 2V, MLC 2A, α-MHC, and cardiac α-actin, compared with wild-types, suggesting impaired differentiation of trabecular cardiomyocytes (Takeuchi et al., 1999). However, in mutants with a BALB/c background, no apparent heart defects were observed but instead hypoplasia of the liver was reported (Motoyama et al., 1997), indicating that the hyperplasia phenotypes in the mutant hearts with a C3H/He background are highly dependent on the genetic background (Ohno et al., 2004).

ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Homozygous jmj mutant embryos with a mixed genetic background C57BL/6 x 129/Svj were independently generated by a gene trap method (Lee et al., 2000). When these mutants were examined by hematoxylin and eosin (H&E) staining, severe heart defects that mimic human congenital heart defects were observed with full penetrance (Fig. 6; Lee et al., 2000). All homozygous embryos with this background were alive in the uterus and often exhibited edema but died soon after birth. Jmj heterozygotes did not show any discernible phenotypic abnormalities.

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Figure 6. Cardiac defects of late stage embryos. Hematoxylin and eosin staining of jmj homozygous mutants. All are transverse sections except that M in embryonic day (E) 19.0 is sagittal. A,B: At E11.0, homozygous mutant heart (B) is morphologically normal compared with wild-type (A). CF: Mutant hearts at E13.0 (E,F) have DORV and VSD (arrow in E) compared with wild-type (C,D). D and F are images of C and E at higher magnification, respectively. GJ: At E16.0, compared with wild-type (G,H), the mutant heart (I,J) exhibits a thin outer compact zone (arrow in J) and VSD (arrowhead in I). H and J are a higher magnification G and I, respectively. There are many red blood cells between the trabecular cells of the ventricle in J. KN: At E19.0, mutant hearts show DORV (M, N) and the thin ventricular wall (L) compared with the wild-type (K). V, common ventricular chamber; At, common atrial chamber; RV, right ventricle; LV, left ventricle; RA, right atria; A, aorta; P, pulmonary trunk; +/+, wild-type; −/−, homozygote. Adapted from Lee et al. (2000). Scale bars in A, B, M, and N = 400 μm.

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At E11.0, wild-type (Fig. 6A) and homozygous mutant (Fig. 6B) hearts were histologically indistinguishable. Both the outflow tract and atrioventricular endocardial cushions had formed normally and were correctly positioned. The trabeculated wall of the common ventricular chamber of the heart, and the atrial wall of both the wild-type and homozygous mutant hearts appeared to be of equal thickness. At E13.0, the homozygous mutant embryos (Fig. 6E,F) exhibited the outflow tract defect double-outlet right ventricle (DORV) and a prominent interventricular septal defect (VSD) compared with wild-type (Fig. 6C,D). The ventricular septum is normally completed by E13.0, when the muscular part of the septum is fused to the inferior endocardial cushion, dividing the ventricle into two chambers (McFadden and Olson, 2002; Olson, 2004). In the wild-type hearts, the aorta exits the left ventricle and the pulmonary trunk exits the right ventricle. However, in the homozygous mutant embryo (Fig. 6E), both the aorta and pulmonary vessels exit the right ventricle. Note that the trabeculated walls of the left and right ventricular chambers appeared to be of equal thickness between the wild-type and mutant (Fig. 6D,F).

At E16.0, the mutant hearts (Fig. 6I,J) exhibited an abnormally thin trabeculated wall of both the left and right ventricular chambers in addition to VSD (indicated by arrowhead in Fig. 6I) compared with the wild-type hearts (Fig. 6G,H). Higher-power images of the ventricular walls indicated that the abnormally thinned myocardium in homozygous mutants was due to a deficiency of the outer compact zone (indicated by arrow in Fig. 6J), as the trabeculation is still evident. Proliferation of myocytes at the ventricular epicardial surface (known as the compact zone) from E12.5 onward generates the thickened outer wall and contributes to the muscular interventricular septum that separates these two chambers. The compact zone is composed of replicating cardiomyoblasts, whereas the forming trabeculae consist of nonreplicating, more fully differentiated cardiomyocytes. At E19.0, the outer compact zone of homozygous mutant hearts (Fig. 6L) was absent compared with wild-type (Fig. 6K), and histological examination suggested this affected coronary vessel formation, as there was a significant reduction in the number of coronary vessels. The homozygous embryos exhibited DORV in the sagittal (Fig. 6M) and transverse section (Fig. 6N) of the hearts. No significant increase in the number of apoptotic cells was observed in the mutant ventricular myocardium and ventricular septum from E12.0 to E17.0 compared with those in wild-type (unpublished data by Y. Lee). Therefore, the thin myocardium and VSD may have resulted from reduced cell proliferation in the myocardium.

All cardiac defects observed in these jmj mutants mimic human congenital heart defects. VSD is the most common form of congenital heart defects. The ventricular septum is formed by two processes. The trabeculae condense at the interventricular groove, which denotes the future boundary of the developing right and left ventricular chambers. In addition, the medial walls of the expanding ventricles fuse together and grow inward, forming the major muscular portion of the septum. The poor development of the muscular ventricular septum in jmj homozygous embryos is most likely accounted for by the lack of ventricular wall expansion. A subsequent lack of fusion of the endocardial cushions to the basal portion of this incomplete septum may result in the observed VSD. VSD may also result from impaired development or failure of precise positioning of the endocardial cushion. Numerous genes contributing to VSD when mutated have been reported, including CHF/Hey2 and Tbx5 (Bruneau et al., 2001; Sakata et al., 2002). Any perturbation that affects normal cardiac looping often results in a DORV phenotype (Hogers et al., 1997). For example, altered expression of Pitx2, which regulates asymmetric morphogenesis of body organs, including heart, guts, and lung, correlates with DORV (Logan et al., 1998; Campione et al., 2001; Franco and Campione, 2003). The perturbation in repositioning and remodeling processes during looping may lead to DORV in homozygous jmj mutants. The neural crest cells provide progenitors of smooth muscle cells in the aortic and pulmonary trunks, but not those of the coronary arteries (Gourdie et al., 1995; Noden et al., 1995), where they participate in outflow tract septation in the heart. Disruption of neural crest cells can result in a spectrum of outflow tract defects, including persistent truncus arteriosus, aortic arch anomalies, perhaps DORV, and tetralogy of Fallot where the aorta overrides the ventricular septum and communicates with the right and left ventricles. Many mutations and experimental manipulations result in DORV. Thus, it really should not be characterized as a neural crest-related defect without sufficient evidence that this is indeed the case.

Although much less common, human newborns have been reported to be born with the thin-walled ventricle (Pignatelli et al., 2003). There are possible explanations for the thin-walled ventricle, for example, a failure in intrinsic proliferative signaling pathways involving numerous cell cycle components and a reduction in coronary vasculature, which may result in an insufficient nutrient supply for cardiomyocyte growth. In addition, the epicardium provides a mitotic signal to the compact cell layer during later stages of development. Mutant mice lacking retinoic acid (RA) signaling or erythropoietin (epo)/epo receptor display heart growth defects ascribed to the absence of epicardium-derived signaling (Sucov et al., 1994; Chen et al., 1998; Wu et al., 1999). It is interesting that multiple heart defects observed in the jmj mutant hearts are often found together. For example, disruption of FOG-2, a cofactor of GATA, causes a wide range of cardiac abnormalities, including VSD and ventricular compact layer hypoplasia, tetralogy of Fallot malformation and impaired coronary vasculature in mice (Tevosian et al., 2000). Mice lacking AP-2α, helix-span-helix family of transcription factors, prominently expressed in cardiac neural crest, exhibit cardiac outflow tract defect, DORV with VSD (Brewer et al., 2002). Targeted disruption of transforming growth factor-β2 (TGFβ2) exhibit several heart defects including VSD and DORV, with craniofacial, eye developmental, inner ear and urogenital defects, similar to those of bone morphogenetic protein-7 or vitamin A–deficient mice (Dudley et al., 1995; Sanford et al., 1997; Bartram et al., 2001). Vitamin A deficiency in embryos leads to a variety of heart defects. The major defects in retinoid X receptor (RXR)α null mutation are hypoplastic development of ventricular walls with VSD (Sucov et al., 1994). Abnormalities in retinoic acid receptor (RAR) double mutant mice include DORV, VSD, and malformations of the great vessels (Mendelsohn et al., 1994). The complex interplay between TGFβ and RA signaling has been described by showing that the addition of RA may induce or repress the expression of TGFβ, depending on the cell type examined (Sanford et al., 1997). Given the widespread developmental expression of TGFβ receptors, RAR, RXR, and JMJ in addition to the similarity in mutant phenotypes, it is conceivable that JMJ may share a common molecular pathway with these signaling pathways in developing hearts.

REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

There is growing evidence that JMJ plays an important role in regulating cardiac-specific gene expression, which leads to normal cardiovascular development (Takeuchi et al., 1999; Lee et al., 2000; Kim et al., in press). The pattern of sarcomeric protein expression is tightly regulated in a tissue- and developmental stage-specific manner and is responsive to hormonal, physiological, and pathological stimuli (for a review, see Wang and Stockdale, 1999). The expression of multiple cardiac genes such as α-MHC, α-cardiac actin, MLC 2v, and MLC 2a was decreased in jmj mutant hearts at E10.5 (Takeuchi et al., 1999). Normal regulation of the cardiac gene expression of ANF, α-MHC, and MLC2v in later stage embryos was disrupted in the hearts of jmj homozygotes at E17.0 (Lee et al., 2000).

One of the candidate downstream target genes is the ANF gene, which shows a cardiac-specific expression pattern throughout development. ANF expression is normally down-regulated in the ventricle near birth but re-expressed in hypertrophic hearts. In situ hybridization showed that ANF was expressed at a higher level in the mutant ventricle than wild-type at E17.0, when ANF expression in the ventricle normally declines (Lee et al., 2000). Consistent with this phenomenon, cotransfection assays indicate that JMJ represses ANF gene activation in primary-cultured cardiomyocytes (Kim et al., in press). We and others have reported previously that ANF expression is synergistically activated by Nkx2.5 and GATA4 in vitro (Durocher et al., 1997; Lee et al., 1998). JMJ represses activation of the ANF reporter gene by Nkx2.5 and GATA4. Of interest, JMJ physically interacts with Nkx2.5 or GATA4 in vitro and in vivo, suggesting that these physical interactions lead to the repression of Nkx2.5 or GATA4 activity (Kim et al., in press). Because the expression level of jmj seems to be increased in the ventricle as it develops (Lee et al., 2000; Toyoda et al., 2003), it is plausible that increasing amounts of JMJ in the normal ventricle results in repression of ANF expression near birth.

Recent evidence suggests that transcriptional repressors play important roles in regulating the expression of cardiac muscle genes. The MEF2 activities are essential for early normal cardiac development (Lin et al., 1997). The MEF2 activities are inhibited by twist, a bHLH protein that is specifically expressed in the mesoderm (Spicer et al., 1996), activated Notch (Wilson-Rawls et al., 1999) and HDAC, a general transcriptional repressor (Zhang et al., 2002; Czubryt et al., 2003). Regulation of ANF expression is controlled primarily at the transcriptional level. The transcription factors involved in activation of ANF expression include Nkx2.5, GATA4, Pitx2, and Tbx5 (Durocher et al., 1996, 1997; Lee et al., 1998; Hiroi et al., 2001; Ganga et al., 2003), which bind to their cis-elements in the ANF enhancer region. In contrast, mechanisms that repress ANF expression have not been well characterized, except for the observation that Tbx2 inhibits ANF expression in the atrioventricular canal (Yamagishi et al., 2001). Other nuclear factors that repress ANF expression have been reported such as HOP (Shin et al., 2002) and FOG-2 (Svensson et al., 1999; Tevosian et al., 1999). Further studies on regulation of the putative target genes by JMJ may reveal its regulatory mechanism on cardiac-specific gene expression and, therefore, heart development.

CELL CYCLE REGULATION BY JMJ

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Possible roles of JMJ in cell proliferation have been suggested. Lee et al. (2000) reported that jmj mutant embryonic hearts with a mixed background C57BL/6 × 129/Svj showed a VSD and thin ventricular wall of later stage embryos by E16.0, likely caused by deficiency of the compact cell layer. The phenotypes suggest that JMJ is involved in stimulating cell growth in the myocardium at later stages of development. The JMJ mutants also showed decreased total cell number in the liver with a BALB/c as well as C57BL6J and DBA/2J background (Motoyama et al., 1997; Anzai et al., 2003), and hematopoietic cells in embryonic thymus and spleen of jmj mutants decreased (Motoyama et al., 1997). In contrast, jmj mutant embryos with a C3H/He background exhibited hyperplasia in the trabeculae of the left ventricle at E10.5 (Takeuchi et al., 1999). The mutant embryos die at E11.5 possibly due to overgrowth of cells in the trabecular layer, which fill the left ventricles, suggesting that JMJ suppresses cell proliferation in the trabecular layer in a C3H/He background. Although little is known about such opposite effects of JMJ on regulation of cell growth in the distinct cell layers of the embryonic heart, these results suggest that JMJ may be required for spatiotemporally regulated cell proliferation in developing hearts. Alternatively, function of JMJ may be greatly influenced by modifier genes that differ in different genetic backgrounds.

The effects of JMJ on cell proliferation suggest that the cell cycle regulators are target genes of JMJ. Among many cell cycle components, cyclin D1 was suggested to mediate the antiproliferating effect of JMJ by analyzing jmj mutant hearts with a C3H/He background (Toyoda et al., 2003). Reverse transcriptase-polymerase chain reaction analysis indicated that cyclin D1 expression but not cyclin D2 or D3 was elevated in jmj mutant hearts compared with those in wild-type at E10.5. This hyperproliferation phenotype of the trabecular cell layer in jmj mutants was rescued when JMJ was overexpressed or cyclin D1 was inactivated in transgenic mice. In addition, cotransfection assays showed that JMJ repressed the transcriptional activity of the cyclin D1 promoter reporter gene in COS-7 cells. Further analyses are required to determine whether JMJ binds to the cyclin D1 enhancer region directly or by means of interaction with other factors that bound to this enhancer region. Because jmj mutants with other genetic backgrounds showed different heart defects, mechanisms of the cyclin D1 repression by JMJ would warrant further investigation. Indeed, jmj mutant embryos with a BALB/c background showed neither hyperproliferation nor elevated cyclin D1 level in the heart (Ohno et al., 2004). Mutants with a mixed genetic background C57BL/6 x 129/Svj also exhibited normal trabecular formation and the normal compact layer until E13.0 followed by thinning of the ventricular wall by E16.0 (Lee et al., 2000). Based on heart defects in jmj mutants, further investigation of underlying mechanism of JMJ in regulating cell cycle and cardiac-specific gene expression would provide valuable insights into understanding the pathogenesis of human congenital heart diseases.

ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

By using gene trap technology, Takeuchi's group mutated jmj that appears to cause an improper neural tube closure in homozygotes with a mixed background between 129/Ola and BALB/cA (Takeuchi et al., 1995). Some, but not all, showed a defect in neural tube closure in the midbrain region, and all died between E10.5 and E15.5. They called the gene jumonji, because the morphology produced by the normal neural groove and abnormal grooves resembles a cross (jumonji is literally translated as cruciform in Japanese). When the phenotypes of homozygous jmj mutants of the pure BALB/cA background were analyzed (Motoyama et al., 1997), almost all homozygotes died around E15.5–E16.5 with edema, enlargement of the pericardial cavity and hemorrhaging in the back at E13.5. In all homozygotes, hypoplasia of the liver, thymus, and spleen was reported with full penetrance, but no neural tube defects were observed. The number of liver cells in homozygotes was markedly low at all stages examined compared with controls, possibly due to necrosis and an absence of cell proliferation in the liver of homozygous embryos at E13.5. Jmj was expressed in at least four cell types in the fetal mouse liver from E12.5–E15.5 by β-gal staining; large hematopoietic cells that are megakaryocytes, small hematopoietic cells, fibroblastic cells, and endothelial cells, but not in hepatocytes at this stage of the liver. In mutant embryos, the number of megakaryocytes progenitors was increased in the fetal liver, yolk sac, and peripheral blood, possibly due to delayed growth arrest in the progeny (Kitajima et al., 1999). The thymic and splenic primordia of heterozygous embryos showed accumulation of many prethymocytes, whereas no accumulation was seen in the primordia of homozygotes at E13.5. It has been reported that the same phenotypes were observed for mutant mice with C57BL/6J and DBA/2J backgrounds as for those with a BALB/cA background.

In peripheral blood of jmj mutant embryos with a BALB/cA background, the number of fetal liver-derived definitive erythrocytes but not yolk sac-derived primitive erythrocytes was markedly reduced, suggesting anemia (Kitajima et al., 1999). Because hematopoietic stem cells in the mutant fetal liver could reconstitute the hematopoietic system of lethally irradiated recipients, an organ-specific environmental defect may induce the impaired hematopoiesis in the fetal liver of jmj mutants. The role of JMJ in hepatocyte differentiation at later stages of development was studied using an in vitro culture system (Anzai et al., 2003). The number of hepatocytes was reduced in mutants with a BALB/c background at E12.5–E14.5 but not at E11.5 compared with wild-type mice. In the fetal liver, weak expression of jmj began in the hepatocytes from E14.5–E18.0 by β-gal staining and the expression increased along with hepatic development. In vitro culture systems showed that proliferation of mutant hepatocytes was comparable to controls but the maturation was impaired, suggesting that JMJ plays a pivotal role in development of the mid-fetal liver to the neonatal liver.

DISCUSSION AND PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Although the molecular roles of JMJ in normal organ development requires further investigation, phenotypic analyses of jmj mutants suggest that JMJ exhibits a dual role in the developing heart. At the earlier stage (E10.5), JMJ seems to repress cell growth in the ventricular trabeculae (Fig 5; Takeuchi et al., 1999), while JMJ appears to facilitate cell growth in the compact layer of the ventricle at the later stage (E16.0; Fig. 6; Lee et al., 2000). Likewise, JMJ appears to support cell growth in the liver at the early stage (Motoyama et al., 1997) but mediates differentiation, not growth, of hepatocytes at a later stage (Anzai et al., 2003).

Overgrowth of trabecular cells in jmj mutant embryonic hearts at the early stage may be attributed to a defective signaling between endocardial and myocardial cells. Signals from endocardial cells such as neuregulin, through its cognate tyrosine kinase receptors (erbB2 and erbB4) expressed by myocytes are required for initiating trabeculation. Knockout mice lacking ErbB2, ErbB4, or neuregulin-1 die at midgestation from cardiac growth defects characterized by the absence of trabeculae (Gassmann et al., 1995; Lee et al., 1995). This abnormality can be ascribed to the lack of paracrine signaling between the endocardium and myocardium. Similarly, it is plausible that the thin ventricular wall in jmj mutant embryonic hearts at the later stage may be due to a defective signaling between epicardial and myocardial cells. This effect of the epicardium on myocardial growth can be observed in VCAM-1 and possibly in α4-integrin mutant mice (Gurtner et al., 1995; Kwee et al., 1995; Yang et al., 1995), where a physical interaction between these two molecules is normally responsible for adherence of the epicardium to the myocardium. In addition, when formation of the epicardium in chick embryos was partially blocked by inserting a piece of eggshell membrane in front of the proepicardial organ, patches of absent epicardium with the myocardium underneath becoming hypoplastic were observed (Gittenberger-de Groot et al., 2000). It is interesting that the thin-walled hypoplastic ventricle in RXRα knockout mice appears to be due to a defective signal originating from the epicardium, not due to cardiomyocyte defects (Chen et al., 1998; Tran and Sucov, 1998; Chen et al., 2002b).

Although the jmj gene plays a critical role in mouse embryonic development, the reported phenotypes vary, depending on the genetic background and perhaps due to slightly different insertion sites of the gene trap vectors used by two groups (Takeuchi et al., 1995; Lee et al., 2000). The genetic background seems to affect the phenotype of the jmj mutants, suggesting the presence of modifier gene(s) or cofactor(s). This phenomenon of changes in phenotype due to different genetic backgrounds has been well-documented for other factors such as retinoblastoma-related genes (Lee et al., 1996; LeCouter et al., 1998a, b), keratin 8 (Baribault et al., 1994), or EGF receptor (Sibilia and Wagner, 1995). The gene trap insertion site may also cause the differences in phenotypes, which presumably depends on specific aspects of long-range gene organization and cis-regulatory elements of unidentified genes located nearby. Although both gene trap vectors were inserted into the same intron, the precise insertion site was different. The insertion site appears to affect the expression pattern of jmj, because jmj expression was abolished in our homozygous mutant embryo body with a mixed C57BL/6 x 129/Svj background, but the wild-type jmj mRNA was expressed at a normal level in all nervous tissues of mutants (Lee et al., 2000). As a consequence, these homozygotes would be protected from the nervous system defects. A striking case of such neighboring effects emerged from the targeted inactivation of the myogenic bHLH gene, MRF4. The phenotypes of MRF4 null mice were very different, ranging from complete viability to complete lethality of homozygotes (Olson and Srivastava, 1996). Regardless of the varying phenotypes, all jmj homozygous mutants die between E10.5 and E15.0 or upon birth, indicating the critical roles of JMJ in embryo development.

To unravel the logic of congenital heart disease and normal cardiac development, it is necessary to move beyond simply cataloging genes that are associated with heart defects. To tackle the biological complexity of cardiogenesis, cre-loxP systems are now considered as model systems for understanding the interplay of diverse signaling pathways from distinct cell lineages during organogenesis. For example, the ventricular myocardium phenotypes in jmj mutant mice may reflect a primary deficiency of JMJ in the ventricular myocyte lineage. However, it cannot be assumed that the spatial requirement for JMJ in embryonic development is indeed localized within cardiomyocytes, because JMJ is expressed widely in various tissues during embryonic development. Thus, it is possible that another lineage might influence myocardium expansion in the ventricular wall as well as appropriate cushion and outflow tract development. This issue can be directly investigated by generating embryos that harbor a ventricular myocyte-restricted deletion of JMJ at the earliest stage of cardiac chamber specification.

Regulation of the cardiac-specific gene expression appears to be dependent on combinatorial associations between cardiac-specific and ubiquitous transcription factors. The precise regulation of temporal and spatial expression of tissue-specific genes may require interaction among transactivators and repressors. Likewise, JMJ may interact with cofactor(s) to regulate the target gene expression in a spatial and temporal manner during development. Therefore, identifying cofactor(s) and downstream target genes of JMJ remain an important area of investigation for the immediate future.

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  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CARDIAC DEVELOPMENT
  5. MOLECULAR FUNCTIONS AND STRUCTURE OF JMJ
  6. EXPRESSION PATTERNS OF THE jmj GENE
  7. ROLES OF JMJ IN EARLY CARDIAC DEVELOPMENT
  8. ROLES OF JMJ IN LATE STAGE CARDIOVASCULAR DEVELOPMENT
  9. REGULATION OF CARDIAC-SPECIFIC GENE EXPRESSION BY JMJ
  10. CELL CYCLE REGULATION BY JMJ
  11. ROLES OF JMJ IN OTHER ORGAN DEVELOPMENT
  12. DISCUSSION AND PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES
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