Summary Requirements for FGF 3 and FGF 10 during inner ear formation

The formation of the vertebrate inner ear has been postulated to comprise several stages (Groves and Bronner-Fraser, 2000; Baker and Bronner-Fraser, 2001; Rinkwitz et al., 2001; Noramly and Grainger, 2002). Initially, a signal from the endomesoderm underlying the presumptive otic field initiates the induction of the otic placode. This process appears to be complemented by a signal originating from hindbrain neural tissue next to the developing placode to complete formation of the otic vesicle. Subsequently, the otic vesicle undergoes a series of morphogenetic processes, leading to the formation of distinct subcompartments, including the cochlea, the different parts of the vestibular system and the endolymphatic sac. Simultaneously, cellular differentiation leads to the generation of specialised cell types, including cochleovestibular neurons and sensory hair cells that permit correct functioning of the mature sensory organ. Owing to their gene expression patterns and various experimental manipulations, several members of the fibroblast growth factor (FGF) gene family, including FGF2, FGF3, FGF8, FGF10 and FGF19 have been implicated in different stages of inner ear formation (Baker and Bronner-Fraser, 2001; Rinkwitz et al., 2001; Noramly and Grainger, 2002). Among these, FGF3 in particular has been the earliest candidate postulated to play a role during early inner ear development. Initially, it was proposed on the basis of its expression pattern in the developing hindbrain next to the forming inner ear placode and vesicle in mice, thus consistent with a role in inner ear induction (Wilkinson et al., 1988). Furthermore, this early hindbrain expression pattern is fundamentally conserved between different vertebrate species including avians (Mahmood et al., 1995), amphibians (Lombardo et al., 1998a) and fish (Phillips et al., 2001). This idea gained further support by experiments in which antibodies and antisense oligonucleotides, presumably directed against FGF3, blocked otic vesicle formation in chicken explants (Represa et al., 1991), although later studies have questioned the conclusions that were drawn from these experiments (Mahmood et al., 1995). Further doubt for a role in otic vesicle formation was derived from the generation of Fgf3 mutant mice, where a Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both lossand gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.