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.
