Differential expression and functional analysis of Pitx2 isoforms in regulation of heart looping in the chick

Pitx2, a bicoid-related homeobox gene, plays a crucial role in the left-rig ht axis determination and dextral looping of the vertebrate developing hear t. We have examined the differential expression and function of two Pitx2 i soforms (Pitx2a and Pitx2c) that differ in the region 5' to the homeodomain , in early chick embryogenesis. Northern blot and RT-PCR analyses indicated the existence of Pitx2a and Pitx2c but not Pitx2b in the developing chick embryos. In situ hybridization demonstrated a restricted expression of Pitx 2c in the left lateral plate mesoderm (LPM), left half of heart tube and he ad mesoderm, but its absence in the extraembryonic tissues where vasculogen esis occurs. RT-PCR experiments revealed that Pitx2a is absent in the left LPM, but is present in the head and extra-embryonic mesoderm, However, ecto pic expression of either Pitx2c or Pitx2a via retroviral infection to the r ight LMP equally randomized heart looping direction. Mapping of the transcr iptional activation function to the C terminus that is identical in both is oforms explained the similar results obtained by the gain-of-function appro ach. In contrast, elimination of Pitx2c expression from the left LMP by ant isense oligonucleotide resulted in a randomization of heart looping, while treatment of embryos with antisense oligonucleotide specific to Pitx2a fail ed to generate similar effect. We further constructed RCAS retroviral vecto rs expressing dominant negative Pitx2 isoforms in which the C-terminal tran scriptional activation domain was replaced by the repressor domain of the D rosophila Engrailed protein (En(r)), Ectopic expression of Pitx2c-En(r), bu t not Pitx2a-En(r), to the left LPM randomized the heart looping. The resul ts thus demonstrate that Pitx2c plays a crucial role in the left-right axis determination and rightward heart looping during chick embryogenesis.