An Autocrine Function for Transforming Growth Factor (TGF)-β3 in the Transformation of Atrioventricular Canal Endocardium into Mesenchyme during Chick Heart Development

Abstract

Transformation of atrioventricular canal endocardium into invasive mesenchyme is a critical antecedent of cardiac septation and valvulogenesis. Previous studies by Pottset al.(Proc. Natl. Acad. Sci. USA88, 1510–1520, 1991) showed that treatment of atrioventricular canal endocardial and myocardial cocultures with TGFβ3 antisense oligodeoxynucleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGFβ3 antisense oligos in this transformation bioassay, and (ii) more clearly define the mechanism of TGFβ3 function in atrioventricular canal mesenchyme formation.In situhybridization and immunohistochemistry showed little or no TGFβ3 mRNA or protein in the atrioventricular canal myocardium or endocardium prior to mesenchyme formation (stage 14; paraformaldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were positive for both TGFβ3 mRNA and protein. In culture bioassays, atrioventricular canal endocardial monolayers pretreated with antisense phosphorothioate oligodeoxynucleotides to TGFβ3 did not transform into invasive mesenchyme in response to cardiocyte conditioned medium: the subsequent addition of exogenous TGFβ3 protein relieved this inhibition. Control cultures without pretreatment or those receiving missense oligos generated similar numbers of invasive mesenchyme in response to cardiocyte conditioned medium. Direct addition of TGFβ3 protein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associations and stimulated cellular hypertrophy, but did not engender invasive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGFβ2 protein, either alone or in combination with TGFβ3. The results of this study indicate that: (i) atrioventricular canal endocardium expresses TGFβ3 in response to a myocardially derived signal other than TGFβ3, (ii) atrioventricular canal endocardial TGFβ3 functions in an autocrine fashion to elicit selected characteristics necessary for cushion tissue formation, and (iii) TGFβ3 alone or in combination with TGFβ2 is insufficient to transform atrioventricular canal endocardium into invasive mesenchyme in culture.