DIFFERENTIAL DISPLAY OF mRNAs FROM THE ATRIOVENTRICULAR REGION OF DEVELOPING CHICKEN HEARTS AT STAGES 15 AND 21

Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242-1324

Received 11/15/95; Accepted 11/28/95; On-line 1/1/96

The molecular mechanisms for cardiac morphogenesis are not completely understood. In chicken development, it has been shown that presumptive cardiac forming cells migrate anteriorly and medially, and then the right and left regions of these cells (lateral plate mesoderm) fuse at the midline to generate the primitive heart tube at stage 10 which starts to beat almost immediately (1). The cell lineage studies of the development of zebrafish embryos have led to the identification of cardiac forming regions in the early blastula (2). However, little is known about specific genes involved in the early cardiac morphogenesis except for several structural genes, such as ventricular myosin heavy chain (3 ), atrial myosin heavy chain (4), cardiac troponin I (5) and vascular smooth muscle alpha-actin ( 6), as well as few regulatory genes, such as MEF2 (7) and Drosophila homeobox gene tinman (8) and its mouse homologue Csx/Nkx-2.5 (9,10). In an effort to identify genes that are differentially expressed in embryonic hearts at the time of the formation of endocardial cushions, the progenitors of the valves and membranous septa, we have employed the differential mRNA display method (11) in this study. Since the differential display is highly sensitive, and likely to detect a large number differentially expressed genes, we have focused on comparing differences of mRNA expression between stage 15 and stage 21 of the atrioventricular (AV) canal regions of developing chicken hearts. It has been demonstrated that the epithelial-mesenchymal cell transformation occurring between stage 15 and 21 may be a key process during the AV cushion morphogenesis (12). Furthermore, this cell transformation appears to be induced by local stimuli derived from the adjacent myocardium (13-15). Thus, genes which are differentially expressed in the AV canal regions of stages 15 and 21 embryonic hearts may be important for the formation of the cardiac valves and septum. The cloned gene fragments we isolated using differential display were sequenced and compared against sequences in the GenBank database. In addition, cloned cDNA fragments were used as probes in whole-mount in situ hybridization. In total 14 differentially expressed fragments were cloned. Here, we report three examples of such clones: 15H16 for cardiac phospholamban, E13 for skeleton alpha-tropomyosin and 21C for a novel gene with a message size of 9.5 Kb.