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

我们从研究促进正常眼睛发育和维护的基因网络开始，特别是一个调节基因pitx3在晶状体形成中的作用。当发现pitx3基因在内脏器官不对称和椎体发育中也起作用时，我们的兴趣扩展到了这些方面。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几乎扮演着相同的角色，这意味着在青蛙等生物身上进行的实验为理解人类等哺乳动物的发育提供了巨大的好处。例如，青蛙胚胎是外部发育的（没有子宫妨碍观察和实验），它们可以很容易地通过手术或基因操作，而且对两栖动物长达一个世纪的眼睛研究开创了我们对人类眼睛发育方式的理解。******在我们对pitx3的研究中，我们之前展示了它是如何在晶状体的形成中起主要作用的，而晶状体反过来又对视网膜的形成至关重要。缺乏蛙核活动，没有晶状体形成，视网膜发育受损或受阻。然后，我们开发了一种新的程序来研究这个基因如何调节构成眼睛的基因网络中的其他基因。我们的系统在揭示细胞间基因调控变化方面非常敏感：我们发现了一些由pitx3调控的新的重要参与者，但在人类眼睛发育中尚未得到很好的理解。****** pitx3功能的一个奇怪的特征是，它可以以一种依赖于上下文的方式打开或关闭目标基因。我们的建议是寻找可能解释这种灵活性的pitx3相互作用的伙伴。此外，通过对pitx3的相互作用伙伴（interactome）进行编目，我们可能会了解：它如何影响椎体前体和心脏的发育；人类和青蛙在伴侣的可获得性和时间上是否存在差异；我们可能会与哺乳动物的亲戚寻找新的伴侣。例如，由于pitx3的突变效应之一是产生晶状体白内障，并且pitx3激活了至少一个提供构成晶状体清晰度的蛋白质的基因（β -晶体蛋白基因），我们提出的确定相互作用伙伴的建议将提高我们的理解并揭示白内障形成的一些机制。pitx3活性的另一个特征是，在青蛙中，它在确定一些内脏器官（如心脏、胃肠道等）的不对称性方面起着作用。Pitx3功能障碍导致器官位置倒置，例如；心脏、脉管系统和肠道的模式缺陷。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伙伴可能为人类先天性心脏缺陷的研究开辟新的途径。

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.