Requirement of a novel gene, Xin, in cardiac morphogenesis

A novel gene, Xin, from chick (cXin) and mouse (mXin) embryonic hearts, may be required for cardiac morphogenesis and looping, Both cloned cDNAs have a single open reading frame, encoding proteins with 2,562 and 1,677 amino a cids for cXin and mXin, respectively. The derived amino acid sequences shar e 46% similarity The overall domain structures of the predicted cXin and mX in proteins, including proline-rich regions, 16 amino acid repeats, DNA-bin ding domains, SH3-binding motifs and nuclear localization signals, are high ly conserved, Northern blot analyses detect a single message of 8.9 and 5.8 kilo base (kb) from both cardiac and skeletal muscle of chick and mouse, r espectively. In situ hybridization reveals that the cXin gene is specifical ly expressed in cardiac progenitor cells of chick embryos as early as stage 8, prior to heart tube formation, cXin continues to be expressed in the my ocardium of developing hearts. By stage 15, cXin expression is also detecte d in the myotomes of developing somites, Immunofluorescence microscopy reve als that the mXin protein is colocalized with N-cadherin and connexin-43 in the intercalated discs of adult mouse hearts. Incubation of stage 6 chick embryos with cXin antisense oligonucleotides results in abnormal cardiac mo rphogenesis and an alteration of cardiac looping. The myocardium of the aff ected hearts becomes thickened and tends to form multiple invaginations int o the heart cavity, This abnormal cellular process may account in part for the abnormal looping. cXin expression can be induced by bone morphogenetic protein (BMP) in explants of anterior medial mesoendoderm from stage 6 chic k embryos, a tissue that is normally non-cardiogenic, This induction occurs following the BMP-mediated induction of two cardiac-restricted transcripti on factors, Nkx2.5 and MEF2C, Furthermore, either MEF2C or Nkx2.5 can trans activate a luciferase reporter driven by the mXin promoter in mouse fibrobl asts, These results suggest that Xin may participate in a BMP-Nkx2.5-MEF2C pathway to control cardiac morphogenesis and looping.