Directionality of Heart Looping: Effects of Pitx2c Misexpression on Flectin Asymmetry and Midline Structures

Abstract

A critical regulatory laterality gene expressed in the left side of the straight heart tube during development is Pitx2, which when mutated in humans underlies Rieger's Syndrome. Previously reported results have indicated that, when using gain-of-function and loss-of function approaches of the chick cPitx2c isoform, this results in randomization of heart looping. To determine whether Pitx2c misexpression affects downstream morphogenesis by altering the expression of specific proteins in the myocardium during looping, after experimental manipulations, we analyzed immunohistochemically for the extracellular matrix molecule flectin that normally is expressed predominantly in the left lateral plate mesoderm (LPM) and left side of the straight chick heart tube before and during looping. We show here that the left-side predominance of flectin is due to a delay in the timing of expression in one heart field vs the other. Experimental results indicate that misexpression of Pitx2c in the heart fields using antisense or retroviral delivery perturbs the normal temporal pattern of flectin expression in the left LPM relative to the right: abnormally leftward looping hearts show predominate right-sided flectin expression in the dorsal mesocardial regions around the foregut ventral midline. Additionally, Pitx2c misexpression affects the positioning of the developing foregut to more lateral areas, either on the right or left side of the embryonic midline. The position of the heart with respect to the embryo midline is defined by the position of the foregut. Incubating embryos in the presence of flectin antibody caused randomization of heart looping or no looping.