Csx : A murine homeobox - containing gene specifically expressed in the developing heart ( cardiac development / transcription factor / tissue - specific gene expression / embryonic stem cell / evolutionary conservation )

The molecular control of the differentiation process depends in part on lineage-restricted transcription factors that regulate expression of tissue-specific genes. Although signicant progress has been made in molecular understanding of skeletal muscle differentiation, no information is available concerning the genes involved in development ofthe heart, the first organ to form in vertebrate embryos. Many vertebrate homeobox-containing genes have been shown to be expressed in broad regions of the mouse embryo, but no expression of a homeobox gene has been found in the most anterior region of the early embryo, the heart primordium. We report here on the cloning of a murine homeobox cDNA, Csx (cardiac-specific homeobox). The Csx homeodomain sequence is divergent from those of the Hox class genes but is related to that of Drosophia msh-2 (NK-4), which plays a key role in Drosophia heart formation. Csx is conserved in evolution and Csx homologs exist in all vertebrates examined. Transcripts of Csx are detected from the presomite stage (7.5 days postcoitum), when mesoderm differentiates into promyocardium. Csx expression is restricted in the myocardial cells from 8.5 days postcoitum through adult. Csx is not expressed in skeletal or smooth muscle or any other tissues examined. Expression of Csx precedes that of cardiac-specific genes in embryonic stem cells differentiating into beating myocardial cells in vitro. Although physiological function of Csx is yet to be determined, the temporal and spacial pattern of Csx expression raises a possibility that Csxmay play a critical role in the differentiation of cardiac cells. Concerted activation ofregulatory genes plays a fundamental role in determining the temporal and spacial patterns of embryonic development. Lineage-restricted transcription factors that regulate tissue-specific genes are especially important for tissue differentiation. The isolation and extensive characterization of the MyoD gene family have brought significant progress to the molecular understanding of skeletal muscle differentiation (1). Although many genes expressed in skeletal muscle are also expressed in cardiac muscle, the MyoD gene family is not expressed in the heart. To date, no cardiac-specific helix-loop-helix-type gene (like the MyoD family) has been isolated, despite intensive efforts by many laboratories. This raises the possibility that there might be very divergent MyoD-like genes expressed in the heart or, alternatively, that differentiation of cardiac muscle may be controlled by transcription factors other than the helix-loop-helix type. Much progress has been made in unraveling the regulatory events of differentiation process of Drosophila (2). Among the genes that govern development of Drosophila, the sequential activation of homeotic and segmentation genes controls the identity, polarity, and number ofbody segments (3). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Many such genes, including the Antennapedia (Antp), Engrailed (En), and Paired (Prd) families, contain a characteristic 180-bp sequence motif called the homeobox (4). Homeodomain-containing proteins act as sequence-specific transcription factors that transregulate the expression of other genes (5). Many homologs of invertebrate homeodomain proteins have been isolated in mammals, including mice and humans (4). The best-studied vertebrate homeoboxcontaining genes are Antp-like Hox genes, which exist in four major clusters in the mouse genome (6). Each cluster exhibits intriguing similarities with the complement of genes within the fly Antp and Bithorax clusters, not only in homeodomain sequences but also in the temporal order of activation, anterior boundary of expression during embryogenesis, and possible role in segmentation (4). Extensive data of ectopic expression and null mutation of Hox genes suggest that mammalian Hox-type homeobox genes might function similarly to the invertebrate homologs during development (7-9). A homeobox gene whose expression is restricted to specific cell lineages would be of particular interest as a candidate for a "cell-type commitment" gene. Some members of the POU gene family, a class of homeobox-containing genes, are expressed in developing central nervous system and show restricted expression patterns in the adult brain (10). One of the POU family genes, Pit-i, which is expressed only in the anterior pituitary gland, is necessary for formation of pituitary cells and activates pituitary-specific genes such as the growth hormone and prolactin genes (11). Recently, the Drosophila homeobox-containing gene msh-2 (NK4) has been shown to be expressed in the developing dorsal vessel, an insect equivalent of the vertebrate heart (12). Mutations in msh-2 (NK4) gene do not affect mesoderm invagination or dorsal spreading but result in loss of heart formation in embryo (12). This suggests that msh-2 (NK4) plays a critical role in Drosophila heart development. Because the genes that play key roles in cell differentiation are likely to be conserved in evolution, we searched for a mammalian homolog of msh-2 (NK4), which may play a critical role in vertebrate heart development. Here, we report on the isolation and characterization of a murine gene whose homeodomain sequencet has similarity to that of msh-2 (NK4) but differs significantly from that of any Hox class genes. This gene is expressed from the time of heart differentiation and its cardiac-specific expression continues from early embryo through adult. To indicate this restricted expression, this cDNA is denoted Csx, for cardiac-specific homeobox. Expression of Csx precedes that of cardiacAbbreviations: ES, embryonic stem; RT-PCR, reverse transcription PCR; p.c., postcoitum; aMCH, a-myosin heavy chain; EB, embryoid body. *To whom reprint requests should be addressed at: Molecular Medicine Division, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. L20300).