Ductin's Role in Craniofacial Patterning and Development

Ductin 在颅面图案形成和发育中的作用

• 批准号: 7120574
• 负责人: DANY SPENCER ADAMS
• 金额: $ 9.23万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-08 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7120574
• 关键词: Xenopus; acidity /alkalinity; adenosinetriphosphatase; biophysics; bone development; cell cell interaction; chick embryo; craniofacial; developmental genetics; enzyme activity; gap junctions; gene expression; histogenesis; hydrogen channel; immunocytochemistry; in situ hybridization; ion transport; nonmammalian vertebrate embryology; protein structure function; proteolipids; species difference; zebrafish

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.

描述（由申请人提供）：最近的实验提供了强有力的证据，证明蛋白质导管蛋白在颅面发育中发挥着重要作用。 Ductin，也称为 V-ATP 酶的 16kD 亚基 c，在质子跨膜转运中发挥作用，从细胞质进入囊泡或细胞外空间；导管蛋白也可能单独或与连接蛋白一起形成间隙连接。因此，导管蛋白在穿过细胞内、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，非洲爪蟾胚胎中野生型导管蛋白的错误表达会导致颅面畸形，表达显性失活导管蛋白或注射抗导管蛋白抗体也会导致颅面畸形。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非洲爪蟾卷曲 3 蛋白错误表达引起的表型非常相似。我们已经知道，导管蛋白在 Sonic Hedgehog 信号通路的上游发挥作用；我们的新结果提出了一个令人着迷的前景，即在脊椎动物头部的形态发生过程中，导管蛋白和离子流也在 Wnt 卷曲级联的上游。我们建议探索以下假设： 基于导管蛋白的离子流是颅面形态发生的早期步骤，它通过 V-ATP 酶依赖的质子泵和细胞间间隙连接通讯来调节信号级联。使用原位杂交、免疫细胞化学以及体内 pH 值和荧光标记蛋白监测的成像将用于表征导管蛋白的空间和时间表达模式，并监测导管蛋白抑制和错误表达的影响。使用野生型和显性失活导管蛋白构建体进行功能丧失和获得的实验将使我们能够研究导管蛋白与下游遗传途径的关系。这项工作将产生有关发育事件的生物物理控制（例如分泌信号的离子控制）的未充分研究的重要且新颖的信息，从而为正常形态发生提供独特且有价值的见解，并提出治疗和预防出生缺陷的新方法。