AN AUTOCRINE FUNCTION FOR TRANSFORMING-GROWTH-FACTOR (TGF)-BETA-3 IN THE TRANSFORMATION OF ATRIOVENTRICULAR-CANAL ENDOCARDIUM INTO MESENCHYME DURING CHICK HEART DEVELOPMENT

Transformation of atrioventricular canal endocardium into invasive mes enchyme is a critical antecedent of cardiac septation and valvulogenes is. Previous studies by Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1 510-1520, 1991) showed that treatment of atrioventricular canal endoca rdial and myocardial cocultures with TGF beta 3 antisense oligodeoxynu cleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGF beta 3 antisense olig os in this transformation bioassay, and (ii) more clearly define the m echanism of TGF beta 3 function in atrioventricular canal mesenchyme f ormation. In situ hybridization and immunohistochemistry showed little or no TGF beta 3 mRNA or protein in the atrioventricular canal myocar dium or endocardium prior to mesenchyme formation (stage 14; paraforma ldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were posi tive for both TGF beta 3 mRNA and protein. In culture bioassays, atrio ventricular canal endocardial monolayers pretreated with antisense pho sphorothioate oligodeoxynucleotides to TGF beta 3 did not transform in to invasive mesenchyme in response to cardiocyte conditioned medium: t he subsequent addition of exogenous TGF beta 3 protein relieved this i nhibition. Control cultures without pretreatment or those receiving mi ssense oligos generated similar numbers of invasive mesenchyme in resp onse to cardiocyte conditioned medium. Direct addition of TGF beta 3 p rotein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associ ations and stimulated cellular hypertrophy, but did not engender invas ive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGF beta 2 protein, ei ther alone or in combination with TGF beta 3. The results of this stud y indicate that: (i) atrioventricular canal endocardium expresses TGF beta 3 in response to a myocardially derived signal other than TGF bet a 3, (ii) atrioventricular canal endocardial TGF beta S functions in a n autocrine fashion to elicit selected characteristics necessary for c ushion tissue formation, and (iii) TGF beta 3 alone or in combination with TGF beta 2 is insufficient to transform atrioventricular canal en docardium into invasive mesenchyme in culture. (C) 1998 Academic Press .