We identified a novel gene family in vertebrates which is preferentially expressed in developing and adult striated muscle. Three genes of the Popeye (POP) family were detected in human and mouse and two in chicken. Chromosomal mapping indicates that Pop1 and Pop3 genes are clustered on mouse chromosome 10, whereas Pop2 maps to mouse chromosome 16. We found evidence that POP1 and POP3 in chicken may also be linked and multiple transcript isoforms are generated from this locus. The POP genes encode proteins with three potential transmembrane domains that are conserved in all family members. Individual POP genes exhibit specific expression patterns during development and postnatally. Chicken POP3 and mouse Pop1 are first preferentially expressed in atrium and later also in the subepicardial compact layer of the ventricles. Chicken POP1 and mouse Pop2 are expressed in the entire heart except the outflow tract. All three Pop genes are expressed in heart and skeletal muscle of the adult mouse and lower in lung. Pop1 and Pop2 expression is upregulated in uterus of pregnant mice. Like the mouse genes, human POP genes are predominantly expressed in skeletal and cardiac muscle. The strong conservation of POP genes during evolution and their preferential expression in heart and skeletal muscle suggest that these novel proteins may have an important function in these tissues in vertebrates.

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.

Popeye (Pop) genes are a novel gene family encoding putative transmembrane proteins predominantly present in striated and smooth muscle cells. In this study, a null mutation of Pop1 was generated by replacing the first coding exon of the Pop1 gene with the lacZ reporter gene. Homozygous mice lacking Pop1 were fertile and had a normal life span without any apparent phenotype. LacZ staining of tissues of heterozygous and homozygous Pop1-LacZ mice revealed strong expression in embryonic and fetal hearts. Pop1-LacZ was also expressed in the myotome and in myogenic progenitor cells within the limb and in smooth muscle cells of various organs. In the heart, Pop1-LacZ activity was downregulated postnatally in heterozygous mice but not in homozygous mice. Administration of the ␤-adrenergic agonist isoproterenol led to a rapid increase in Pop1-LacZ activity in heterozygotes without induction at the transcriptional level, suggesting stabilization of the protein. No difference, however, was observed between homozygous and heterozygous mice in the ability to develop cardiac hypertrophy in response to isoproterenol. The capacity to regenerate skeletal muscle was tested after cardiotoxin injection into the hind limbs of hetero-and homozygous mice. In activated satellite cells of both genotypes, rapid activation of Pop1-LacZ expression was observed. In heterozygous animals, LacZ activity was only transiently elevated in muscle precursor cells undergoing fusion and in newly formed myotubes. In homozygotes, persistence of LacZ expression and a retarded ability to regenerate skeletal muscle were apparent, suggesting that Pop1 plays a role in muscle regeneration.Popeye (Pop) genes represent a novel gene family encoding proteins with three putative transmembrane domains and no other recognizable domain of known structure (2). In mammals, three family members, Pop1 to -3, have been identified that are differentially expressed in heart and skeletal muscles. At the mRNA level, Pop1 and Pop3 are expressed in both muscle types while Pop2 is expressed predominantly in cardiac muscle (2). In the chick, several Pop1 and Pop3 transcripts have been isolated that are presumably generated by alternative splicing from a single gene (2). In contrast, in mammals, the different Pop transcripts are transcribed from individual genes. In situ hybridization of embryonic chicken and mouse hearts revealed that Pop genes are expressed in the myocardial layer and not in the endocardium or epicardium (2). Pop1 was independently isolated by others and referred to as bves (17). In contrast to the expression pattern observed by in situ hybridization, immunohistochemistry analysis employing antibodies raised against synthetically made portions of the chicken bves protein revealed expression in the proepicardial organ and later in the epicardium and the developing coronary vascular system (17, 25). The reason for the contradictory descriptions of mRNA and protein localization of Pop1/bves is not known. Recently, it was reported that Pop1/bves encodes the protot...

In Drosophila induction of the homeobox gene tinman and subsequent heart formation are dependent on dpp signaling from overlying ectoderm. In order to define vertebrate heart-inducing signals we screened for dpp-homologues expressed in HH stage 4 chicken embryos. The majority of transcripts were found to be BMP-2 among several other members of the BMP family. From embryonic HH stage 4 onwards cardiogenic mesoderm appeared to be in close contact to BMP-2 expressing cells which initially were present in lateral mesoderm and subsequently after headfold formation in the pharyngeal endoderm. In order to assess the role of BMP-2 for heart formation, gastrulating chick embryos in New culture were implanted with BMP-2 producing cells. BMP-2 implantation resulted in ectopic cardiac mesoderm specification. BMP-2 was able to induce Nkx2-5 expression ectopically within the anterior head domain, while GATA-4 was also induced more caudally. Cardiogenic induction by BMP-2, however remained incomplete, since neither Nkx2-8 nor the cardiac-restricted structural gene VMHC-1 became ectopically induced. BMP-2 expressing cells implanted adjacent to paraxial mesoderm resulted in impaired somite formation and blocked the expression of marker genes, such as paraxis, Pax-3, and the forkhead gene cFKH-1. These results suggest that BMP-2 is part of the complex of cardiogenic signals and is involved in the patterning of early mesoderm similar to the role of dpp in Drosophila.

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