Ductin's Role in Craniofacial Patterning and Development

描述（由申请人提供）：最近的实验提供了强有力的证据，表明蛋白质导管蛋白在颅面发育中起着重要作用。导管蛋白，也称为V-ATP酶的16 kD亚基c，在质子跨膜运输出细胞质并进入囊泡或细胞外空间中起作用;导管蛋白也可能单独或与连接蛋白形成间隙连接。因此，导管蛋白在跨细胞内、细胞间和细胞外边界的离子通量中具有重要作用。我们最近发现，在非洲爪蟾胚胎中错误表达野生型导管蛋白会导致颅面畸形，表达显性阴性导管蛋白或注射抗导管蛋白抗体也会导致颅面畸形。处理过的胚胎的表型，包括畸形的眼睛、耳朵和头骨，看起来非常像注射抗连接蛋白抗体和非洲爪蟾卷曲蛋白-3蛋白的错误表达引起的表型。我们已经知道，导管蛋白的功能上游的声波刺猬信号通路，我们的新结果提出了迷人的前景，导管蛋白和离子通量也上游的Wnt卷曲级联在脊椎动物头部的形态发生。我们建议探讨的假设， 基于导管蛋白的离子流是颅面形态发生的早期步骤，其通过V-ATP酶依赖性质子泵和细胞-细胞间隙连接通讯调节信号级联。成像使用原位杂交，免疫细胞化学，并在体内监测pH值和荧光标记的蛋白质将被用来表征导管蛋白的空间和时间的表达模式，并监测导管蛋白抑制和错误表达的影响。使用野生型和显性阴性导管蛋白构建体的功能丧失和获得实验将使我们能够研究导管蛋白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颖的信息，例如，离子控制的分泌信号，从而提供了独特的和有价值的洞察正常形态发生，并提出了新的方法来治疗和预防出生缺陷。

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.