Role of Pitx3 signaling cascades in the regulation of Xenopus eye, laterality and somite development

我们首先研究了有助于眼睛正常发育和维护的基因网络，特别是调节基因Pitx3在晶状体形成中的作用。我们的兴趣扩展到内脏器官不对称和脊椎发育，因为很明显，Pitx3在这些方面也起到了作用。这种基因与老鼠和人类的眼睛和神经缺陷有关。这种基因在眼睛和眼睛基因网络中的作用几乎相同，这意味着在青蛙等生物身上的实验对理解我们这样的哺乳动物的发育有很大好处。例如，青蛙胚胎在外部发育(没有子宫阻碍观察和实验)，它们可以很容易地通过手术或基因操作，而对两栖动物长达一个世纪的眼睛研究开创并澄清了我们对人类眼睛发育的理解。*在我们的研究中，我们以前展示了它如何在晶状体的形成中发挥主要作用，晶状体的形成反过来又对视网膜的形成至关重要。青蛙Pitx3缺乏活动，没有晶状体形成，视网膜发育受到损害或受到阻碍。然后，我们开发了一种新的程序来检查这种基因是如何调节构成眼睛的基因网络中的其他基因的。我们的系统以极高的灵敏度在逐个细胞的基础上揭示基因调控的变化：我们发现了一些新的和重要的角色，这些角色受到Pitx3的调控，但在人眼发育中尚未得到很好的理解。*bitx3功能的一个奇怪特征是，它可以以一种与上下文相关的方式打开或关闭目标基因。我们的建议是寻找可以解释这种灵活性的Pitx3交互合作伙伴。此外，通过对Pitx3相互作用的伙伴(互动组)进行分类，我们可能会了解：它如何影响脊椎前体和心脏的发育；人类和青蛙在伴侣的可用性和时机上是否存在差异；以及我们可能会从哺乳动物的近亲中识别新的伴侣。例如，由于Pitx3的突变效应之一是导致晶状体白内障，并且Pitx3激活了至少一个基因，该基因提供了构成和定义晶状体透明度的大部分蛋白质(β-晶体蛋白基因)，因此我们提出的确定相互作用的伙伴的建议将提高我们的理解，并揭示一些导致白内障形成的机制。Pitx3活性的另一个特征是，在青蛙中，它在确定心脏、胃肠道等一些内脏器官的不对称性方面发挥作用。Pitx3功能障碍会导致器官放置反转，例如心脏、血管和肠道的图案化缺陷。由于相关基因在人类器官不对称中起作用，我们在青蛙中寻找相互作用的伴侣有可能为人类先天性心脏缺陷的研究提供新的途径。

We started by studying the network of genes that contribute to normal eye development and maintenance, specifically the role of a regulatory gene, pitx3, in the formation of lens. Our interests expanded to visceral organ asymmetry and vertebral development when it became apparent that pitx3 plays a role in these aspects of development too. This gene is linked to eye and neurological deficits in mice and humans. The preservation of nearly identical roles for this gene in eye, and in eye gene networks, means that experiments in organisms such as frog offer big benefits to understanding development in mammals such as ourselves. For example, frog embryos develop externally (no uterus to get in the way of observation and experimentation), they can be easily manipulated surgically or genetically, and the century-long eye studies in amphibians have pioneered and clarified our understanding of how eyes develop in human. ******In our studies of pitx3, we previously showed how it plays a primary role in the formation of lens, which is in turn critical to formation of the retina. The absence of frog pitx3 activity, no lens forms, and retinal development is either impaired or prevented. We then developed a new procedure to examine how this gene regulates others in the network of genes that build an eye. Our system works with exquisite sensitivity to reveal gene regulatory changes on a cell-by-cell basis: we identified some new and important players that are regulated by pitx3, but that are not yet well understood in human eye development. ******A curious feature of pitx3 function is that it can turn target genes on or off in a context-dependent manner. Our proposal is to look for pitx3-interacting partners that might explain this flexibility. Moreover, by cataloging the interacting partners (interactome) of pitx3, we might come to understand: how it affects development of vertebral precursors and heart; whether there are differences in partner availability and timing between humans and frogs; and we might identify new partners with mammalian relatives. For example, since one of the mutational effects of pitx3 is to produce lens cataracts, and pitx3 activates at least one of the genes that provides the bulk of protein constituting and defining the clarity of lens (a beta-crystallin gene), our proposal to identify interacting partners will improve our understanding and reveal some of the mechanisms that underly cataract formation. An additional feature of pitx3 activity is that in frog, it plays a role in defining the asymmetry of some of the visceral organs such as heart, gastro-enteric tract etc. Pitx3 dysfunction results in inversion of organ placement, e.g.; patterning defects of heart, vasculature, and gut. Since related genes play a role in organ asymmetry in human, our search for interacting partners in frog has potential to illuminate new avenues for investigation of human congenital heart defects.