Yu X, St Amand T R, Wang S, Li G, Zhang Y, Hu Y P, Nguyen L, Qiu M S, Chen Y P
Department of Cell and Molecular Biology, Molecular and Cell Biology Graduate Program and Center for Bioenvironmetal Research, Tulane University, New Orleans, LA 70118, USA.
Development. 2001 Mar;128(6):1005-13. doi: 10.1242/dev.128.6.1005.
Pitx2, a bicoid-related homeobox gene, plays a crucial role in the left-right axis determination and dextral looping of the vertebrate developing heart. We have examined the differential expression and function of two Pitx2 isoforms (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 Pitx2c in the left lateral plate mesoderm (LPM), left half of heart tube and head mesoderm, but its absence in the extra-embryonic tissues where vasculogenesis occurs. RT-PCR experiments revealed that Pitx2a is absent in the left LPM, but is present in the head and extra-embryonic mesoderm. However, ectopic expression of either Pitx2c or Pitx2a via retroviral infection to the right LMP equally randomized heart looping direction. Mapping of the transcriptional activation function to the C terminus that is identical in both isoforms explained the similar results obtained by the gain-of-function approach. In contrast, elimination of Pitx2c expression from the left LMP by antisense oligonucleotide resulted in a randomization of heart looping, while treatment of embryos with antisense oligonucleotide specific to Pitx2a failed to generate similar effect. We further constructed RCAS retroviral vectors expressing dominant negative Pitx2 isoforms in which the C-terminal transcriptional activation domain was replaced by the repressor domain of the Drosophila Engrailed protein (En(r)). Ectopic expression of Pitx2c-En(r), but not Pitx2a-En(r), to the left LPM randomized the heart looping. The results thus demonstrate that Pitx2c plays a crucial role in the left-right axis determination and rightward heart looping during chick embryogenesis.
Pitx2是一种与双尾相关的同源框基因，在脊椎动物发育中心脏的左右轴确定和右旋环化过程中发挥着关键作用。我们研究了在鸡早期胚胎发生过程中，两种在同源异型域5'端区域不同的Pitx2亚型（Pitx2a和Pitx2c）的差异表达和功能。Northern印迹和RT-PCR分析表明，在发育中的鸡胚胎中存在Pitx2a和Pitx2c，但不存在Pitx2b。原位杂交显示Pitx2c在左侧侧板中胚层（LPM）、心脏管左半部分和头部中胚层中表达受限，但在发生血管生成的胚外组织中不表达。RT-PCR实验表明，Pitx2a在左侧LPM中不存在，但存在于头部和胚外中胚层中。然而，通过逆转录病毒感染右侧LMP异位表达Pitx2c或Pitx2a均能使心脏环化方向随机化。将转录激活功能定位到两种亚型中相同的C末端，解释了通过功能获得方法获得的相似结果。相反，用反义寡核苷酸消除左侧LMP中的Pitx2c表达会导致心脏环化随机化，而用针对Pitx2a的反义寡核苷酸处理胚胎未能产生类似效果。我们进一步构建了表达显性负性Pitx2亚型的RCAS逆转录病毒载体，其中C末端转录激活结构域被果蝇Engrailed蛋白（En(r)）的阻遏结构域取代。向左侧LPM异位表达Pitx2c-En(r)，而不是Pitx2a-En(r)，会使心脏环化随机化。因此，结果表明Pitx2c在鸡胚胎发生过程中的左右轴确定和心脏向右环化中起关键作用。
