Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping.

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping.

Directionality of heart looping: effects of Pitx2c misexpression on flectin asymmetry and midline structures.

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.