Regulation of bone growth by hetero-oligomerization of Cx43 and Cx40.8

描述（由申请人提供）：连接蛋白基因突变导致人类疾病，表明间隙连接通讯是正常组织稳态和功能所必需的。 例如，人CX43的错义突变导致骨骼畸形和与常染色体显性眼齿指发育不良（ODDD）相关的其他多效性表型。 目前还不清楚错义突变如何导致疾病表型。 一种可能性是突变以“显性负性”方式起作用，有效地改变或抑制Cx43间隙连接的正常偶联性质。 然而，很难使用哺乳动物系统将特定错义等位基因的功能与特定表型直接相关。 斑马鱼cx43基因突变导致短鳍表型，其特征是骨鳍条段长度缺陷和细胞增殖水平降低。 使用相对简单的系统，斑马鱼鳍的生长，它已经有可能相关的鳍长，节长和细胞增殖的缺陷，使用三个错义等位基因的斑马鱼cx43。 斑马鱼的基因组也包含一个类似cx43的基因，称为cx40.8。 有趣的是，在鳍再生过程中，Cx40.8在与Cx43相同的细胞中表达。 与Cx43不同，Cx40.8似乎定位于高尔基体，而不是主要在质膜。 然而，Cx40.8的敲低导致细胞增殖和节段长度的缺陷与Cx43功能降低相似，表明Cx40.8在调节鳍生长中起着积极的作用。 一种可能性是Cx40.8与Cx43寡聚并直接改变Cx43间隙连接的活性。 因此，斑马鱼Cx43和Cx40.8可能代表了一个天然的系统来检查Cx43异源寡聚化及其对间隙连接组装和耦合特性的影响。 本申请的具体目的1和2是确定Cx40.8是否与Cx43在体内物理相互作用，以及Cx40.8/Cx43间隙连接是否表现出不同的电耦合特性。 这种相互作用和调节的证据可能揭示了调节间隙连接偶联的内源性机制。 具体目标3是确定允许Cx40.8保留在高尔基体中的分子机制。 事实上，保留可以发育调节，使得在某些条件下Cx40.8被排除在间隙连接之外，而在不同条件下Cx40.8被允许与Cx43异源寡聚化。 这些目标的结果将提供新的见解Cx43错义等位基因如何导致ODDD疾病表型的潜在机制。 在人类中，CX43中的错义等位基因与眼齿指发育不良相关。 这种疾病是常染色体显性遗传，表明野生型和错义形式的蛋白质的相互作用有助于疾病相关的表型。 相关性：本申请旨在评估斑马鱼cx43和斑马鱼cx43样蛋白cx40.8的相互作用。 我们的目标是揭示密切相关的连接蛋白蛋白的共表达是否可以在体外改变间隙连接特性，并在体内改变cx43相关的表型。

DESCRIPTION (Provided by Applicant): Mutations in connexin genes lead to human disease, suggesting that gap junctional communication is required for normal tissue homoeostasis and function. For example, missense mutations in human CX43 cause skeletal malformations and other pleiotropic phenotypes associated with the autosomal dominant oculodentodigital dysplasia (ODDD). It is unclear how missense mutations might cause disease phenotypes. One possibility is that the mutations act in a "dominant-negative" manner, effectively modifying or inhibiting the normal coupling properties of the Cx43 gap junctions. However, it has been difficult to correlate directly the function of particular missense alleles with particular phenotypes using mammalian systems. Mutations in zebrafish cx43 cause the short fin phenotype, characterized by defects in the length of bony fin ray segments and reduced levels of cell proliferation. Using the relatively simple system of zebrafish fin growth, it has been possible to correlate defects in fin length, segment length, and cell proliferation using three missense alleles of zebrafish cx43. The zebrafish genome also contains a cx43-like gene called cx40.8. Interestingly, Cx40.8 is expressed in the same cells as Cx43 during fin regeneration. Unlike Cx43, Cx40.8 appears to localize to the Golgi apparatus rather than primarily at the plasma membrane. Yet, knock-down of Cx40.8 causes similar defects in cell proliferation and segment length as reduced Cx43 function, indicating that Cx40.8 plays an active role in regulating fin growth. One possibility is that Cx40.8 oligomerizes with Cx43 and directly modifies the activity of Cx43 gap junctions. Thus, zebrafish Cx43 and Cx40.8 might represent a natural system to examine Cx43 hetero-oligomerization and its effects on gap junction assembly and coupling properties. Specific Aims 1 and 2 of this application are to determine if Cx40.8 physically interacts with Cx43 in vivo, and if Cx40.8/Cx43 gap junctions exhibit distinct electrical coupling properties. Evidence for this interaction and regulation may reveal an endogenous mechanism that regulates gap junctional coupling. Specific Aim 3 is to identify the molecular mechanism that allows Cx40.8 to be retained in the Golgi. Indeed, retention may be developmentally regulated so that under certain conditions Cx40.8 is excluded from gap junctions, while under different conditions Cx40.8 is permitted to hetero-oligomerize with Cx43. Results from these aims will provide novel insights into the underlying mechanism of how Cx43 missense alleles cause ODDD disease phenotypes. In humans, missense alleles in CX43 are associated with oculodentodigital dysplasia. This disease is transmitted as autosomal dominant, indicating that the interaction of both wild-type and missense forms of the proteins contributes to the disease-related phenotypes. RELEVANCE: This application seeks to evaluate the interactions of zebrafish cx43 and a zebrafish cx43-like protein, cx40.8. The goal is to reveal if the co-expression of closely related connexin proteins can modify gap junctional properties in vitro, and cx43-related phenotypes in vivo.