Ductin's Role in Craniofacial Patterning and Development

描述(申请人提供)：最近的实验提供了强有力的证据表明，蛋白质Ductin在头面部发育中起着重要作用。Ductin，也被称为V-ATPase的16kD亚单位c，在质子跨膜转运出细胞质和进入囊泡或细胞外间隙中起作用；Ductin也可能单独或与连接蛋白形成缝隙连接。因此，Ductin在跨越细胞内、细胞间和细胞外边界的离子流动中发挥着重要作用。我们最近发现，在非洲爪哇胚胎中错误表达野生型ductin会导致头面部异常，表达显性阴性ductin或注射抗ductin抗体也是如此。经过处理的胚胎的表型，包括畸形的眼睛、耳朵和头骨，看起来与注射抗连接蛋白抗体和错误表达非洲爪哇卷曲-3蛋白引起的表型非常相似。我们已经知道Ductin在Sonic Hedgehog信号通路的上游发挥作用；我们的新结果提出了一个有趣的前景，即在脊椎动物头部的形态发生过程中，Ductin和离子通量也位于WNT卷发级联的上游。我们建议探索这样一个假设： 基于Ductin的离子通量是颅面形态发生的早期步骤，通过V-ATPase依赖的质子泵和细胞-细胞缝隙连接通讯来调节信号级联反应。利用原位杂交成像、免疫细胞化学以及体内pH和荧光标记蛋白质的监测将被用来描述Ductin的空间和时间表达模式，并监测Ductin抑制和错误表达的影响。使用野生型和显性负向ductin结构的功能实验的得失将使我们能够研究ductin与下游遗传途径的关系。这项工作将产生关于发育事件的生物物理控制现象的重要和新的信息，例如，离子控制分泌信号，从而为正常的形态发生提供独特和有价值的见解，并提出治疗和预防出生缺陷的新方法。

DESCRIPTION (provided by applicant): Recent experiments provide strong evidence that the protein ductin plays an important role in craniofacial development. Ductin, also known as the 16kD subunit c of V-ATPase, functions in transmembrane transport of protons out of the cytoplasm and into vesicles or the extracellular space; ductin can probably also form gap junctions either alone or with connexins. Thus, ductin has important roles in ion flux across intra-, inter-, and extra-cellular boundaries. We recently discovered that misexpressing wildtype ductin in Xenopus embryos leads to craniofacial abnormalities, as does expressing a dominant negative ductin or injecting antiductin antibodies. The phenotypes of treated embryos, including malformed eyes, ears, and skull, look remarkably like those caused by injection of anti-connexin antibodies and misexpression of the Xenopus frizzled-3 protein. We already know that ductin functions upstream of the Sonic hedgehog signaling pathway; our new results raise the fascinating prospect that ductin and ion flux are also upstream of the Wnt-frizzled cascade during morphogenesis of the vertebrate head. We propose to explore the hypothesis that ductin-based ion flux is an early step in craniofacial morphogenesis that regulates signaling cascades via both V-ATPase dependent proton pumping and cell-cell gap junctional communication. Imaging using in situ hybridization, immuncytochemistry, and in vivo monitoring of pH and fluorescently labeled proteins will be used to characterize ductin's spatial and temporal expression pattern and to monitor the effects of ductin inhibition and misexpression. Loss and gain of function experiments using wildtype and dominant negative ductin constructs will allow us to study the relationship of ductin to downstream genetic pathways. This work will yield important and novel information about the under-studied phenomenon of biophysical control of developmental events, e.g., ion control of secreted signals, thus providing unique and valuable insight into normal morphogenesis and suggesting novel approaches to treating and preventing birth defects.