FGFR1-Frs2/3 Signalling Maintains Sensory Progenitors during Inner Ear Hair Cell Formation

Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27Kip1, Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.


Introduction
The mammalian cochlea transduces sound using a dedicated sensory organ, the organ of Corti, which comprises of a highly ordered array of mechanosensory hair cells (HCs) and their associated support cells (SCs). The arrangement of cochlear HCs, 3 rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs), together with SCs results from a balance between specification, progenitor expansion and differentiation [1].
The first step in HC specification is the induction of a Sox2positive territory known as the sensory patch. Sox2 is critical for neurosensory precursor formation in the inner ear [2][3][4] and is induced by Notch signalling through its ligand Jagged (Jag)1 [5][6][7][8][9]. BMP signalling [10] then specifies the prosensory domain, the immediate precursors of the HCs and SCs, from within this Sox2positive sensory patch. At specification, the prosensory domain exits the cell cycle, expressing the cell cycle inhibitor p27 Kip1 as well as other prosensory domain markers. Importantly, the prosensory domain first becomes post-mitotic at the apical end of the cochlea from E12.5, spreading basally until E14.5 [11,12].
HCs and SCs are picked out from within the prosensory domain through Notch signalling from putative SCs, acting on Delta1 or Jag2 in potential HCs [5,[13][14][15]. This lateral inhibition ensures that only some of the cells of the prosensory domain retain the transcription factor Atoh1 [16,17]. Atoh1 is both necessary and sufficient for HC differentiation [18]. In contrast to the apical to basal wave of cell cycle exit of the prosensory domain, the wave of differentiation occurs basally at E14.5 extending apically at E17.5 [19].
In addition to the above, fibroblast growth factor (FGF) signalling has also been shown to be important in the development of the cochlear HC. Conditional deletion of Fgf receptor (Fgfr) 1, results in the loss of HCs [20]. This phenotype is observed to a lesser extent, when the proposed ligand for FGFR1, Fgf20, is deleted [21]. Ex vivo explant studies suggest that FGF signalling enhances Notch-Jag signalling after sensory patch induction [22]. However the in vivo significance of these observations and how they lead to the Fgfr1 deletion phenotype are not clear.
Fgf ligand binding causes the dimerization and activation of the canonical receptor tyrosine kinase [23]. Activation, generally by phosphorylation of particular tyrosine residues in the intracellular domain of the Fgf receptor, results in recruitment of adaptor proteins that are essential for the intracellular response to the extracellular signal. Each group of phosphorylated residues mediate distinct functions, for example phosphorylation of tyrosine 766 in FGFR1 serves as a potential binding site for phospholipase C-c (PLCc) [24]. Other adaptor proteins include Fgf Receptor Substrate (Frs) 2 or 3 (collectively termed Frs2/3) [25,26]. Frs2/3 recruitment and activation leads to the stimulation of multiple FGFR-dependent signaling pathways such as Ras/MAP kinase pathway, and the phosphatidylinositol-3-kinase (PI3K) pathway [27]. Studies into a mouse allele in which the Frs2/3 interaction motif has been deleted, reveal that Frs2/3 recruitment mediates aspects of FGFR1 signalling [28]. However, the necessity of these pathways in inner ear development had not been investigated.
In this study, we found that FGFR1 signalling through Frs2/3 is necessary for prosensory formation. Even in the absence of FGFR1-Frs2/3 signalling, the prosensory domain becomes post-mitotic, however the expression of prosensory markers is impaired. This results in fewer sensory precursors, giving rise to a reduction in HC numbers. We also found that the expression of Sox2 is transient, suggesting that the strength and duration of Sox2 expression, under the direct or indirect control of FGF-mediated MAP kinase activation, commits progenitors to sensory cell differentiation.

FGFR1 signalling through Frs2/3 recruitment is required for normal cochlear hair cell development
To determine gross morphology, the inner ear from Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs at E14.5 were examined first by paint-filling [29]. The cochlear duct of the conditional mutant (Six1enh21-Cre::Fgfr1 flox/flox ) was shorter than control ( Figure 1A and B). Fgfr1 DFrs/DFrs also exhibited a truncated cochlear duct although the phenotype was milder than that of the conditional mutant ( Figure 1C). No significant difference in the formation of vestibular components was observed.
A requirement for FGFR1 function in cochlear HC development had been previously shown [20], however the mechanisms used remained unknown. We asked when FGFR1 signalling was acting during HC development, by exploiting the difference in the timing activation of of two different Cre driver lines. To first verify Cre activity, we crossed these lines with a Rosa26-flox-STOP-flox-EYFP reporter, in which the expression of EYFP is initiated after the Cre-mediated excision of the STOP, transcription terminator sequence. Six1enh21-Cre activity can be detected as early as E9.5 specifically in whole otic epithelium (Figure 2A-C and G). In contrast, Emx2-Cre activity cannot be detected at E9.5, but is active at E12.5, with EYFP labelled in the almost all putative sensory organs except three semicircular ampullae (Figure 2D-G and data not shown). Quantitative PCR for the deleted portion of Fgfr1 confirmed the temporal activity of the two Cre lines ( Figure 2H). Fgfr1 levels in the cochlear rudiment were reduced to approximately 20% of normal in Six1enh21-Cre::Fgfr1 flox/flox from E10.5. In contrast, Fgfr1 levels in Emx2-Cre::Fgfr1 flox/flox cochleae were close to wild-type levels at E10.5, falling to 60% at E12.5 and 20% by E14.5. We thus used these lines to examine the cochlear phenotypes when Fgfr1 deletion occurred at around E9.5 to 10.5 (using Six1enh21-Cre) or at around E12.5 (using Emx2-Cre).
To investigate HC phenotype, whole-mount cochlear samples from E18.5 mice were dissected and immunostained for Myo7a. Control, wild-type, cochleae showed the typical arrangement of three rows of OHCs and one row of IHCs along the entire length of the cochlea ( Figure 3A, B). In Six1enh21-Cre::Fgfr1 flox/flox the arrangement of HCs was altered, with those in the apical third of the cochlea more severly affected ( Figure 3C-E). Here the rows of HCs were discontinuous, and arranged in islands. Typically, OHCs were missing, although isolated OHCs could be found basally. The cochlear phenotype of Emx2-Cre::Fgfr1 flox/flox inner ears was milder ( Figure 3F-H). Basally, OHC loss was less pronounced with the outer-most row most severely affected ( Figure 3F). Further apically, the HC row became discontinuous, and islands that were present were made up of IHCs and OHCs, with occasional additional IHCs observed ( Figure 3G). HCs were more sparsely distributed in the apical-most part of the cochlea ( Figure 3H).

Author Summary
The ability of our brain to perceive sound depends on its conversion into electrical impulses within the cochlea of the inner ear. The cochlea has dedicated specialized cells, called inner ear hair cells, which register sound energy. Environmental effects, genetic disorders or just the passage of time can damage these cells, and the damage impairs our ability to hear. If we could understand how these cells develop, we might be able to exploit this knowledge to generate new hair cells. In this study we address an old problem: how do signals from the fibroblast growth factor (FGF) family control hair cell number? We used mice in which one of the receptors for FGF (Fgfr1) is mutated and found that the expression of a stem cell protein, Sox2 is not maintained. Sox2 generally acts to keep precursors in the cochlea in a pre-hair cell state. However, in mutant mice Sox2 expression is transient, diminishing the ability of precursors to commit to a hair cell fate. These findings suggest that it may be possible to amplify the number of hair cell progenitors in culture by tuning FGF activity, providing a route to replace damaged inner ear hair cells. respectively) ( Figure 3M). The significant difference was also determined when comparing Six1enh21-Cre::Fgfr1 flox/flox and Emx2-Cre::Fgfr1 flox/flox cochleae (p,0.05). Given the differences in the timing of the two Cre drivers (Figure 2), these results suggest that FGFR1 signalling commences prior to E12.5.

Support cell development is perturbed in the absence of Frs2/3-mediated FGFR1 signalling
The decision by sensory precursors to generate either HCs or SCs is controlled by Notch-Delta cell-cell signalling [5,[13][14][15]. Therefore, one possible mechanism of FGFR1 action is in modifying the action of Notch and Delta in this choice. We thus investigated whether SC formation was disrupted in the absence of Frs2/3-mediated FGFR1 signalling. We crossed Fgfr1 DFrs/DFrs onto an Atoh1-GFP reporter line to reveal HCs. At E18.5, Prox1 is strongly expressed in the Deiter's cells and in the pillar cells [31]. In the control, Fgfr1 DFrs/+ cochlea, Prox1-labeled 5 rows of cells ( Figure 5A). In mutant Fgfr1 DFrs/DFrs cochlea, only two to three rows of Prox1-labelled cells were detected and were confined within sensory islands ( Figure 5B). In whole mount view of Fgfr1 DFrs/+ cochlea, p75 expression was apparent in the inner pillar cells that are found along the length of the cochlear duct ( Figure 5C). In Fgfr1 DFrs/DFrs cochlea, p75 staining was only found in the sensory cell islands highlighted by Atoh1-GFP and not found in the intervening spaces ( Figure 5D). Within severely affected region, the row of p75-positive cells was mostly present lateral to the one row of HCs, suggesting that these islands were exclusively IHCs.
The other SC marker at this stage, Sox2, was also only found within the sensory islands ( Figure 5E and F). Section analysis revealed that Sox2 is expressed in SCs, in both control organ of Corti ( Figure 5G) and in sections taken through the level of the islands in Fgfr1 DFrs/DFrs cochlea ( Figure 5H). In sections taken through the gaps in between the islands, we could only detect weak Sox2 expression in the Kölliker's organ, a region medial to lateral compartment ( Figure 5I). Combined, these results suggest that the FGFR1-Frs2/3 signalling axis also affects the formation of SCs, and is thus acting upstream of HC/SC decision mediated by Notch-Delta signalling.

FGFR1/Frs2/3 interaction is not essential for FGFR1mediated cell cycle regulation during inner ear development
Precursors of auditory HCs and SCs form from a domain known as the prosensory domain [1]. This region emerges from within the Sox2-positive sensory patch between E12.5 and E14.5, depending on the exact position within the cochlea. It is initially characterised by the cessation of mitosis, forming the zone of nonproliferating cells (ZNPC), as well as the expression of a cell cycle inhibitor, p27 Kip1 . Subsequently, the ZNPC expresses specific markers of the prosensory domain such as Hey2 and Hes5. It had been previously shown that a conditional deletion of Fgfr1 regulates proliferation in the cochlea [20]. We thus asked if cell cycle regulation within the cochlea was mediated by FGFR1mediated Frs2/3 activity.
As well as the prosensory domain, the Sox2-positive sensory patch also forms Kölliker's organ and the outer sulcus. We thus asked if Fgfr1 mutation also affected these structures. Cells in Kölliker's organ normally express Fgf10 and Jag1. In both Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs mutants, the spatial expression of Jag1 ( Figure 7G-I) and Fgf10 ( Figure 7J-L) was unchanged. However, quantitative PCR revealed a down-regulation of Fgf10 expression, although Jag1 did not show any significant difference ( Figure 7P). The spatial pattern of Bmp4, a marker for the outer sulcus located lateral to prosensory domain, was also unchanged in Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs mutants ( Figure 7M-O). Quantitation revealed up-regulation of Bmp4 only in Six1enh21-Cre::Fgfr1 flox/flox mutant but not in Fgfr1 DFrs/DFrs ( Figure 7P). These results indicate that although cell cycle exit, an aspect of prosensory domain induction, occured normally, the induction of genes marking the prosensory domain is impaired in the absence of Frs2/3-mediated FGFR1 signalling. This signalling also contributes to the up-regulation of Fgf10 in Kölliker's organ. However, FGFR1 signalling independently of Frs2/3 recruitment, may negatively regulate Bmp4 expression in the outer sulcus.

FGFR1 is necessary for Sox2 maintenance during sensory patch formation
The expression of Sox2 in the sensory patch is known to be critical in the formation of prosensory domain and subsequent HC formation; mutation or reduction in Sox2 expression affects their development in a dose-dependant fashion [2]. Furthermore, FGF signalling has been shown to be sufficient for Sox2 expression [22]. We thus hypothesised that the HC phenotype observed in Fgfr1 mutants were, in part, due to alterations in Sox2 expression. Initially, Sox2 is expressed in the neuronal and sensory precursors in the otocyst at E10.5. Between E12.5 to E14.5, Sox2 expression in the cochlear duct is detected in the thickened epithelial cells that mark the site of the prosensory domain [32]. By E18.5, Sox2 is confined to the SCs of the organ of Corti [4].
Sox2 was initially expressed at comparable levels between control, heterozygous, inner ears and Six1enh21-Cre::Fgfr1 flox/flox mutants at E10.5 ( Figure 8A and B). By E11.5, expression in Six1enh21-Cre::Fgfr1 flox/flox inner ears was decreased ( Figure 8D), although in Fgfr1 DFrs/DFrs expression levels were equivalent to those in control inner ears ( Figure 8C and E). By E12.5, decreased expression of Sox2 in the cochlea of both Fgfr1 mutant lines was apparent ( Figure 8F-H), although Sox2 expression in the saccule was unchanged. To quantify this decrease, we measured Sox2 protein levels in E12.5 mouse cochlea. Levels were reduced by approximately 78% in Six1enh21-Cre::Fgfr1 flox/flox to the levels found in Six1enh21-Cre::Fgfr1 flox/+ , while a 55% decrease was observed in Fgfr1 DFrs/DFrs when compared to heterozygous controls ( Figure 9A). This down-regulation was confirmed by immunostaining whole cochleae with Sox2 antibody (Figure 9B and C). To exclude the possibility that the early Sox2 down-regulation occurred due to accelerated prosensory domain development, we used BrdU uptake to indicate its formation. At E12.5, even though Sox2 is downregulated in the cochlear rudiment of Six1enh21-Cre::Fgfr1 flox/flox , BrdU-positive cells can still be detected ( Figure 9D and E), indicating that Sox2 down-regulation occured prior to prosensory domain formation. Furthermore, the down-regulation is not a result of cell survival: No difference in cell death was observed between controls and Six1enh21-Cre::Fgfr1 flox/flox cochleae using an antibody against activated caspase-3 to detect apoptotic cells (data not shown).
Sox2 expression in the sensory patch is induced by activation of the Notch receptor by its ligand Jag1 [6,14]. Expression analysis of Jag1 in Six1enh21-Cre::Fgfr1 flox/flox mutant revealed that its expression pattern is unchanged ( Figure 11A and B), suggesting that FGFR1 signalling affects Sox2 expression independent of any affect on Jag1 regulation. Taken together, we suggest that FGFR1-Frs2/3 signalling is required for Sox2 maintenance in sensory progenitors.

ERK phosphorylation is repressed in the inner ear of FGFR1 signalling mutants
Frs2/3-mediated FGFR1 signalling is transduced by a number of downstream pathways. We investigated which were activated during Sox2 maintenance in the sensory patch. The MAP kinase pathway is one of the key mediators of receptor tyrosine kinase signalling, and is activated through Frs2/3 recruitment to FGFR1 [25]. To determine if this pathway was activated in the inner ear, we used antibodies specific for the di-phosphorylated form of Erk1 and Erk2 (dpERK), an indicator of MAPK activity [33], to investigate the spatiotemporal activation of this pathway in the inner ear.
At later stages of sensory cell development, FGF8 signalling mediated through FGFR3 is thought to play a role in the specification of pillar and Deiter's cells [34,35]. To verify the specificity of the FGFR1 signalling mutants, we asked if ERK phosphorylation was affected at these later stages. We found no obvious difference in dpErk localization to the cells of E14.5 Six1enh21-Cre::Fgfr1 flox/+ heterozygous and Six1enh21-Cre::Fgfr1 flox/ flox homozygous inner ears ( Figure 12G and H), where nascent pillar cells IHCs are present. Thus, inhibition of signalling by FGFR1 specifically affects early ERK phosphorylation at E10.5 and E11.5, but does not affect later activation at E14.5 by other FGF receptors.

Discussion
Formation of cochlear HCs takes place progressively, with the potential of a group of Sox2-positive precursor cells, known as the sensory patch, becoming gradually restricted under the influence of a number of signalling molecules. Our observations suggest that FGFR1 signalling, acting through the adaptor Frs2/3, is responsible for sensory progenitor maintenance, partly through the maintenance of early Sox2 expression, and that in its absence, down-regulation of Sox2 results in a reduction in the number of HCs. However, despite the reduction of early Sox2 expression, subsequent patterning of the sensory patch into the precursor domain of the sensory cells, the prosensory domain, is only partially affected. Surprisingly, even though prosensory domain markers such as p27 Kip1 and Hey2 are dramatically downregulated in both the conditional Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs allele, a ZNPC is still established normally, and on schedule, showing the normal apical to basal progression. This also implies that the cell cycle inhibitor p27 Kip1 is required redundantly for sensory progenitors to exit the cell cycle exit. In the mouse, the cell cycle inhibitor p19 Ink4d is also found in the sensory progenitors, and is known to act redundantly with p27 Kip1 [36].

FGF signalling regulates Sox2 maintenance in the sensory patch
The regulation of Sox2 by FGF signaling has been characterized in a number of other systems, for example during foregut development [37], retinal pigmented epithelia [38], the lens placode [39] and in the differentiation of osteoblasts [40]. We show that in the cochlear precursor, FGF signalling maintains Sox2 expression.  The reduction of Sox2 is not as a consequence of reduced proliferation (and hence reduced numbers) of Sox2-positive cells. While the number of proliferating cells in Six1enh21-Cre::Fgfr1 flox/flox cochlea is reduced, the numbers in the Fgfr1 DFrs/DFrs allele are not. Despite this difference, Sox2 levels are reduced in both mutants at E12.5 and E14.5, suggesting that during cochlear HC formation one role for FGFR1 signaling is in the maintenance of Sox2 expression. Further support for the regulation of Sox2 by FGFR1 signaling comes from the correspondence of HC loss seen in Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs cochlea with other mutants. Sensory cell loss is more prominent apically in the cochlea, with the phenotype becoming milder basally. Such phenotypes are similar to knockouts or hypomorphic alleles of Jag1 and Sox2 [2,6], suggesting their involvement in a gene network with Fgfr1. Indeed further support for this molecular network comes from explant studies that show that exogenous application of FGF20 can overcome Notch-Jagged-mediated inhibition of Sox2 [22]. One caveat is that it is unclear whether the regulation of Sox2 maintenance by FGFR1 signalling is direct or indirect, through the upstream regulation of other factors important in Sox2 maintenance. It is clear that further studies are necessary to determine the exact mechanism by which FGFR1 signalling regulates Sox2.
At least two roles for Sox2 have been described during the formation of the cochlear sensory cells. The above-mentioned network, apparent from E10.5 to E12.5, maintains the competence of precursor cells to form sensory progenitors. This is supported by the analysis of the cochlear phenotype of mutant mice with little or no Sox2. These mutants show reduced, or absent HCs in the cochlea [2]. A later role for Sox2, from around E15, has been proposed. Here, Sox2 maintains SC fate, and preventing ectopic HC formation through the repression of Atoh1 [4]. This is suggested by hypomorphic alleles where the reduction of Sox2 is not as severe. Here, HC number is increased [2,4]. Our results suggest that these two activities are separable, with FGFR1 signalling maintaining sensory commitment, partly through Sox2 regulation.
The question remains, how does decreased Sox2 as a result of reduced FGFR1 signalling translate into reduced sensory cells in the cochlea? Sox2 expression as well as other prosensory markers expressed in prosensory domain were down-regulated in both Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs mutants, whereas only Six1enh21-Cre::Fgfr1 flox/flox mutants showed defect in cell proliferation. Moreover, both mutants showed similar effects on the formation of HCs. We thus conclude that early cell cycle exit provides, at most, a minor contribution to the disruption of prosensory formation, and hence cochlear HC development in FGFR1 signalling mutants. Instead, it is possible that the level or duration of Sox2 expression determines the commitment or competence to form HCs. A number of studies have described the quantitative requirement for Sox2 in other systems such as in the retinal progenitors [41], anterior foregut [37] and in taste buds [42]. Indeed, over-expression studies have suggested this is also the case in HC [4]. One possible mechanism, through which the duration of Sox2 expression in progenitors and precursors may be translated into effects on commitment and differentiation, is suggested from work on the effects of Sox2 binding to target gene enhancers in other systems [43,44]. Here silenced genes, important for cell type differentiation, are pre-bound with Sox2. Pre-binding is thought to be associated with the generation of local epigenetic changes [44] or is required for successive binding of co-operative factors [43], important in gene activation, priming the genes for activation. Consistent with this is data showing Sox2 binding sites in the Atoh1, a gene that is responsible for sensory cell differentiation in the inner ear [45]. Similarly, we suggest that one function of maintained early Sox2 expression, controlled by FGFR1 signalling, is to prime prosensory genes, such as Atoh1, for subsequent activation and thus control the differentiation of the sensory cells.
The disruption of the transition from Sox2-positive sensory progenitors to prosensory precursors also provides an explanation for the discontinuous ''island'' phenotype of HCs in the cochlea of FGFR1 signalling mutants. Convergent extension movements that  partially drive cochlear extension normally distribute sensory precursors over the length of the organ of Corti [46,47]. However the fewer numbers of precursors in FGFR1 signalling mutants cannot be evenly dispersed. The apical to basal difference in the distribution of the sensory cells in these mutants may suggest directionality for these rearrangements.

FGFR1 signalling and inner ear hair cell development
Several studies have proposed FGF20 as the FGFR1 ligand during mouse cochlear development [21,48]. Indeed there is good correlation of the phenotype between Fgf20 2/2 mutants and Emx2-Cre::Fgfr1 flox/flox described in this study; both have moderate reduction in the number of OHC, and IHC remains unaffected. In addition, their prosensory domain formation is largely unaffected. In contrast, there are important differences between Fgf20 nulls and both Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs mutants. In these more severe Fgfr1 mutants, HC number is more severely reduced and IHC are also affected. Analysis of Fgf20 nulls revealed a function for Fgf20 in HC differentiation since undifferentiated Sox2-positive cells between sensory islands have been reported [21]. In Fgfr1 DFrs/DFrs mutant cochleae, however, there are no Sox2positive cells detected in the lateral compartment among the HC islands. Furthermore, and in contrast to Fgf20 2/2 mutant cochlea, Sox2 is down-regulated in both Six1enh21-Cre::Fgfr1 flox/flox and Fgfr1 DFrs/DFrs mutants from E12.5 to at least E14.5, and prosensory domain formation is disrupted. Our use of the two Cre drivers suggests a reason for this discrepancy. We propose that the FGFR1 has at least two distinct functions in auditory HC development. An early role, prior to E13.5, is in the maintenance of prosensory function, in part through the regulation of Sox2, and in the development of IHC. A later role, in OHC development, is demonstrated by the use of Emx2-Cre, which only reaches the same level of driver activity as Six1enh21-Cre at E14.5. Here, Sox2 expression in prosensory domain is not severely affected despite significant reduction in OHC numbers. This suggests that a second Fgf ligand, operating either earlier or in combination with Fgf20, is required for the maintenance of Sox2. Although Fgf20 is expressed in the sensory patches from E10.5 to E14.5 [21,22], it is likely that prosensory development, but not OHC development, could be compensated by the second ligand in Fgf20 2/2 mutant cochlea. A number of Fgf ligands are expressed in the inner ear at these stages of development. Fgf3, -4, -5, -9, -10, -16, as well as Fgf20 are all detected in the mammalian inner ear at early stages [21,[48][49][50][51][52][53][54]. Receptor specificity can be used to narrow down the likely early ligand for FGFR1. It is known that mutation of the Fgfr1-IIIb isoform does not affect inner ear development, thus it is likely that the Fgfr1-IIIc isoform is operating in the sensory epithelium [20]. Of these 7 ligands, FGF4, -5, -9, -16, and FGF20 can bind and signal through FGFR1-IIIc [55,56], suggesting that one or more of these FGF molecules may act with FGF20 to maintain early Sox2 expression.
FGF signalling triggers a downstream response, transducing external cues into an internal response. We find that in the absence of Fgfr1, or Frs2/3-mediated FGFR1 signalling, MAP kinase phosphorylation is attenuated, suggesting that this pathway is necessary for sensory progenitor maintenance. The similarity of the Six1enh21-Cre::Fgfr1 flox/flox phenotype with that of Fgfr1 DFrs/DFrs suggests that adaptor proteins Frs2/3 transduce the FGF signal during sensory progenitor maintenance. However there is an important difference between the two mutants. The defect in proliferation seen in Six1enh21-Cre::Fgfr1 flox/flox (and previously in Foxg1-Cre::Fgfr1 flox/flox [20]) is rescued in Fgfr1 DFrs/DFrs . This suggests the involvement of another downstream pathway in control proliferation in the cochlea. Indeed, the recovery of cell cycle impairment in Fgfr1 DFrs/DFrs is consistent with previous findings that cell lines obtained from Fgfr1 DFrs/DFrs are still capable of proliferating [28]. It is likely that other binding partners of FGFR1, such as Grb14, Crk, and Shc, which are known to regulate FGFR1-dependent cell proliferation may respond to mitogenic stimulation in the developing cochlea [57][58][59]. In contrast to the Fgfr1 DFrs/DFrs , which lacks the Frs2/3 interaction motif on FGFR1, mice carrying a point mutation in tyrosine at position 766, Fgfr1 Y766F/Y766F mice, showed no defect in inner ear development. Previous reports have suggested that Y766 phosphorylation may act to negatively regulate FGFR1 activity [30]. It is likely that FGFR1 activity is up-regulated in the inner ear of Fgfr1 Y766F/Y766F mutants. Given that previous studies have suggested that exogenous FGF ligands do not result in an obvious phenotype in the normal mouse cochlea [21], our observation of a normal cochlea in Fgfr1 Y766F/Y766F mice is not unreasonable.
Our analysis of a mutant of Frs2 in which its subsequent binding to Shp2 is impaired (Frs2a/2F) revealed a very early defect in inner ear development, with the inner ear arrested at the otocyst stage (unpublished observations). This phenotype is more reminiscent of the Fgfr2(IIIb) mutant, which is thought to mediate signalling from Fgf3 and Fgf10 during inner ear induction [60]. This indicates that Frs2/3-mediated FGFR signalling, like FGF signalling itself, is reiteratively employed during inner ear formation.

Ethics statement
Experiments were conducted and mice were housed, in accordance with local (RIKEN CDB) and national guidelines for animal experiments.

Cell counting and measurement of cochlear length
For cochlear and vestibular HC counting, E16.5-E18.5 samples stained with Myo7a or expressing Atoh1-GFP were used since most Fgfr1 mutants die before birth. Inner and outer HC were distinguished by location and morphology as described previously [21]. Group of single row of HCs was regarded as IHCs since they were located medial side of p75-expressing pillar cells. Relative cochlear length was measured using ImageJ software. For evaluation of HC number per length, we counted more than 300 mm regions of the base, middle, and apex of the cochlea and normalized counts to 100 mm (more than n = 4 in each HC type) as described [21]. For Sox2-positive cell counting, cross sections from E14.5 samples were made and middle turn of cochlear duct stained with anti-Sox2 antibody was chosen.

RNA in situ hybridization
RNA in situ hybridization on cryo-sections was performed as previously described [69].

Paint-filling
The gross anatomy of bony labyrinths at E14.5 was visualized by paint-filling as previously described [29]. Briefly, decapitated heads were fixed in Bodian's fixative over night. Specimens were subsequently immersed in a graded ethanol series to dehydrate, and cleared in a 2:1 mixture of benzyl benzoate and benzoic acid (BABB). The inner ears were visualized by injection of 1% white paint in BABB into the common crus.

Bromo-deoxyuridine incorporation assay
BrdU (100 mg/g body weight) was injected into pregnant mice intra-peritoneally at E10.5-E14.5. BrdU injected mice were sacrificed 2 hours after injection, and then fixed in 4% PFA. BrdU-positive cells were counted in at least four cross sections of the cochlear apical (at E12.5) or middle (at E14.5) turn. For E14.5 samples, only BrdU-labelled cells in Kölliker's organ were counted. Data shown are mean 6 SD. P-values were calculated using unpaired t-test, to determine the significance of the difference between experimental and control samples.

Quantitative reverse transcription PCR
Whole otocyst or cochlear epithelial cells were dissected from embryos at E10.5-E14.5 (at least n = 2 in each sample). Enzymatic treatment was conducted to remove mesenchyme [70]. Total RNA from pure otic epithelial cells was extracted using the RNAqueous-Micro kit (#AM1931, Ambion, Austin, TX) and then reverse-transcribed using First Strand cDNA Synthesis Kit for RT-PCR (#11483188001, Roche, Indianapolis, IN). Synthesized cDNA and primer sets were mixed with Power SYBR Green PCR Master Mix (#4367669, Applied Biosystems, Warrington, UK), and real-time quantitative PCR was performed using an ABI Prism 7900 Sequence Detection System (Applied Biosystems). All reactions were carried out in duplicate. The relative amount of mRNA was calculated by standard curve method, and normalized to that of 36B4 mRNA [71]. P-values were calculated using unpaired t-test, to determine the significance of the difference between experimental and control samples. Immunoblotting E12.5 or E14.5 cochlear epithelial cells, purified from underlying mesenchymal cells were lysed in a buffered solution, consisting of SDS, salt, phosphatase inhibitor, and proteinase inhibitor. A mixture of lysate, sample buffer, and 2-mercaptoethanol, were boiled at 98uC for 2 min and separated on a SuperSep Ace gel (Wako), and subsequently transferred into PVDF membrane (GE Healthcare). The following antibodies were used: rabbit anti-Akt antibody (1: 1000) (#9272, Cell Signalling Technology), anti-Sox2 (1: 1000) (#AB5603, Millipore, Temecula, CA), rabbit anti-Phospho Akt antibody (1: 1000) (#9271, Cell Signalling Technology), and anti-Actin antibody (1: 10000) (MBL). Horseradish peroxidase-linked anti-rabbit IgGs were used as secondary antibodies (1:10,000) (GE Healthcare) and revealed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) according to the manufacturer's instruction. ImageJ software was used to compare the relative Sox2 protein amount between control and Fgfr1 mutants.