Left-right (LR) asymmetry is an essential feature of bilateral animals. Studies in vertebrates show that LR asymmetry formation comprises three major steps: symmetry breaking, asymmetric gene expression, and LR morphogenesis. Although much progress has been made in the first two events, mechanisms underlying asymmetric morphogenesis remain largely unknown due to the complex developmental processes deployed by vertebrate organs. We here addressed this question by studying Pitx gene function in the basal chordate amphioxus whose asymmetric organogenesis, unlike that in vertebrates, occurs essentially in situ and does not rely on cell migration. Pitx null mutation in amphioxus causes loss of all left-sided organs and incomplete ectopic formation of all right-sided organs on the left side, whereas Pitx partial loss-of-function leads to milder phenotypes with only some LR organs lost or ectopically formed. At the N1 to N3 stages, Pitx expression is gradually expanded from the dorsal anterior domain to surrounding regions. This leads to activation of genes like Lhx3 and/or Prop1 and Pit, which are essential for left-side organs, and downregulation of genes like Hex and/or Nkx2.1 and FoxE4, which are required for right-side organs to form ectopically on the left side. In Pitx mutants, the left-side expressed genes are not activated, while the right-side genes fail to decrease expression on the left side. In contrast, in embryos overexpressing Pitx genes, the left-side genes are induced ectopically on the right side, and the right-side genes are inhibited. Several Pitx binding sites are identified in the upstream sequences of the left-side and right-side genes which are essential for activation of the former and repression of the latter by Pitx. Our results demonstrate that (1) Pitx is a major (although not the only) determinant of asymmetric morphogenesis in amphioxus, (2) the development of different LR organs have distinct requirements for Pitx activity, and (3) Pitx controls amphioxus LR morphogenesis probably through inducing left-side organs and inhibiting right-side organs directly. These findings show much more dependence of LR organogenesis on Pitx in amphioxus than in vertebrates. They also provide insight into the molecular developmental mechanism of some vertebrate LR organs like the lungs and atria, since they show a right-isomerism phenotype in Pitx2 knockout mice like right-sided organs in Pitx mutant amphioxus. Our results also explain why some organs like the adenohypophysis are asymmetrically located in amphioxus but symmetrically positioned in vertebrates.

中文翻译：

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Pitx 通过促进左侧发育和抑制右侧形成来控制文昌鱼不对称形态发生

左右 (LR) 不对称是双边动物的基本特征。脊椎动物研究表明，LR 不对称形成包括三个主要步骤：对称破坏、不对称基因表达和 LR 形态发生。尽管在前两个事件中取得了很大进展，但由于脊椎动物器官部署的复杂发育过程，不对称形态发生的机制仍然很大程度上未知。我们在这里通过研究基部脊索动物文昌鱼中的 Pitx 基因功能来解决这个问题，文昌鱼的不对称器官发生与脊椎动物不同，基本上发生在原位并且不依赖于细胞迁移。文昌鱼中的 Pitx 无效突变导致所有左侧器官的丧失和左侧所有右侧器官的不完全异位形成，而 Pitx 部分功能丧失导致更温和的表型，只有一些 LR 器官丢失或异位形成。在 N1 到 N3 阶段，Pitx 表达逐渐从背前域扩展到周围区域。这导致 Lhx3 和/或 Prop1 和 Pit 等基因的激活，这些基因对左侧器官至关重要，以及 Hex 和/或 Nkx2.1 和 FoxE4 等基因的下调，这些基因是右侧器官异位形成所必需的在左手边。在 Pitx 突变体中，左侧表达的基因没有被激活，而右侧的基因不能降低左侧的表达。相反，在过表达Pitx基因的胚胎中，左侧基因在右侧异位诱导，而右侧基因被抑制。在左侧和右侧基因的上游序列中鉴定了几个 Pitx 结合位点，这些位点对于 Pitx 激活前者和抑制后者至关重要。我们的研究结果表明（1）Pitx 是文昌鱼不对称形态发生的主要（尽管不是唯一）决定因素，（2）不同 LR 器官的发育对 Pitx 活性有不同的要求，（3）Pitx 可能控制文昌鱼 LR 形态发生通过诱导左侧器官直接抑制右侧器官。这些发现表明，文昌鱼的 LR 器官发生对 Pitx 的依赖性比脊椎动物要大得多。它们还提供了对一些脊椎动物 LR 器官（如肺和心房）的分子发育机制的深入了解，因为它们在 Pitx2 敲除小鼠中显示出右异构表型，如 Pitx 突变文昌鱼中的右侧器官。我们的研究结果也解释了为什么像腺垂体这样的器官不对称地位于文昌鱼中，而对称地位于脊椎动物中。