The Role of Alternative Splicing of Fgf8 in Mouse Development

描述（由申请人提供）：成纤维细胞生长因子8（Fgf 8）在控制各种发育过程中起关键作用。Fgf 8结合并激活跨膜受体酪氨酸激酶（FGFR 1 -4）。已知两种主要的信号传导途径Ras-ERK和PI 3 K-Akt介导Fgf 8信号传导。人和小鼠中的单个Fgf 8基因通过选择性剪接产生可变N-末端的多种同种型。Fgf 8剪接变体的生物学意义仍然没有答案。使用发育中的中脑和后脑作为模型系统，我们已经证明了两种FGF 8亚型FGF 8a和FGF 8b的不同诱导活性。我们最近发现，小鼠中的所有八种Fgf 8同种型根据其与Fgf 8a和Fgf 8 B相似的活性分为两个功能组，a样（a、c、e和g）和b样（B、d、f和h）。待检验的总体假设是Fgf 8的功能由这两组Fgf 8同种型的不同活性协调。我们进一步假设Fgf 8的选择性剪接是调节Fgf 8信号传导的水平和范围的重要机制。聚焦于Fgf 8在鸡和小鼠中脑和小脑发育中的功能，我们建议剖析Fgf 8a样和Fgf 8b样亚型的功能作用，以及这些亚型的不同活动及其相互作用的机制。首先，为了确定不同Fgf 8同种型在发育中的中脑/后脑中的组合活性，我们将使用功能获得方法，并检查单个或组合Fgf 8同种型在鸡胚中的活性。其次，为了确定Fgf 8a样和Fgf 8b样亚型对正常发育的相对贡献，我们将使用功能丧失方法，并确定发育期间对Fgf 8a样和Fgf 8b样亚型的需求。第三，为了确定Fgf 8亚型的不同功能的分子机制，我们将研究Fgf 8a和Fgf 8b在脑外植体中的作用范围、扩散和功能特性。此外，为了确定Fgf 8a和Fgf 8b在中脑/后脑中的不同功能背后的机制，我们将检查Fgfr 1和Fgfr 2亚型在该脑区域中的表达。最后，我们将研究ERK和AKT的激活响应于前两个目标中产生的鸡和小鼠胚胎中Fgf 8a样或Fgf 8b样亚型的改变。这些研究将为发育过程中Fgf 8功能的调节机制提供新的见解，这对预防人类发育障碍具有重要意义。FGF 8表达的特异性增加与各种人类癌症的发生和进展有关。因此，我们提出的研究应该阐明Fgf 8剪接在病理条件下的异常调节，包括人类肿瘤形成。

DESCRIPTION (provided by applicant): Fibroblast growth factor 8 (Fgf8) plays crucial roles in controlling various developmental processes. Fgf8 binds and activates transmembrane receptor tyrosine kinases (FGFR1-4). Two major signaling pathways, Ras-ERK and PI3K-Akt, are known to mediate Fgf8 signaling. The single Fgf8 gene in both tiuman and mouse produces multiple isoforms of variable N-termini by alternative splicing. The biological significance of Fgf8 splice variants remains unanswered. Using the developing midbrain and hindbrain as a model system, we have demonstrated the distinct inductive activities of two Fgf8 isoforms, Fgf8a and Fgf8b. We recently discovered that all eight Fgf8 isoforms in mice fall into two functional groups, a-like (a, c, e and g) and b-like (b, d, f and h), based on their similar activities to Fgf8a and Fgf8b. The overall hypothesis to be tested is that the function of Fgf8 is orchestrated by the different activities of these two groups of Fgf8 isoforms. We postulate further that the alternative splicing of Fgf8 is an important mechanism in regulating both the levels and range of Fgf8 signaling. Focusing on Fgf8 function in development of the midbrain and cerebellum in the chick and mouse, we propose to dissect the functional roles of Fgf8a-like and Fgf8b-like isoforms, and the mechanisms underlying distinct activities of these isoforms and their mutual interactions. First, to determine the combinatorial activities of different Fgf8 isoforms in the developing mid/hindbrain, we will use a gain-of-function approach, and examine the activity of individual or combinatorial Fgf8 isoforms in chick embryos. Second, to ascertain the relative contributions of Fgf8a-like and Fgf8b-like isoforms to normal development, we will use a loss-of-function approach, and determine the requirements for Fgf8a-like and Fgf8b-like isoforms during development. Third, to determine the molecular mechanism underlying the distinct functions of Fgf8 isoforms, we will examine the range of action, diffusion and functional properties of Fgf8a and Fgf8b in brain explants. Furthermore, to determine the mechanism behind the different functions of Fgf8a and Fgf8b in the mid/hindbrain, we will examine expression of subtypes of Fgfrl and Fgfr2 in this brain region. Finally, we will examine activation of ERK and AKT in response to alterations of Fgf8a-like or Fgf8b-like isoforms in chick and mouse embryos generated in the first two aims. These studies will provide new insights into the mechanisms for the regulation of Fgf8 function during development, which should have important implications for preventing human developmental disorders. Specific increase of FGF8 expression has been implicated in the onset and progression of various human cancers. Therefore, our proposed studies should shed light on abnormal regulation of Fgf8 splicing on pathological conditions, including tumor formation in human.