Differential requirements for FGF3, FGF8 and FGF10 during inner ear development

FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development.     

Fgf8 and Fgf3 are required for zebrafish ear placode induction,maintenance and inner ear patterning

The vertebrate inner ear develops from initially 'simple' ectodermal placode and vesicle stages into the complex three-dimensional structure which is necessary for the senses of hearing and equilibrium. Although the main morphological events in vertebrate inner ear development are known, the genetic mechanisms controlling them are scarcely understood. Previous studies have suggested that the otic placode is induced by signals from the chordamesoderm and the hindbrain, notably by fibroblast growth factors (Fgfs) and Wnt proteins. Here we study the role of Fgf8 as a bona-fide hindbrain-derived signal that acts in conjunction with Fgf3 during placode induction, maintenance and otic vesicle patterning. Acerebellar (ace) is a mutant in the fgf8 gene that results in a non-functional Fgf8 product. Homozygous mutants for acerebellar (ace) have smaller ears that typically have only one otolith, abnormal semi-circular canals, and behavioral defects. Using gene expression markers for the otic placode, we find that ace/fgf8 and Fgf-signaling are required for normal otic placode formation and maintenance. Conversely, misexpression of fgf8 or Fgf8-coated beads implanted into the vicinity of the otic placode can increase ear size and marker gene expression, although competence to respond to the induction appears restricted. Cell transplantation experiments and expression analysis suggest that Fgf8 is required in the hindbrain in the rhombomere 4-6 area to restore normal placode development in ace mutants, in close neighbourhood to the forming placode, but not in mesodermal tissues. Fgf3 and Fgf8 are expressed in hindbrain rhombomere 4 during the stages that are critical for placode induction. Joint inactivation of Fgf3 and Fgf8 by mutation or antisense-morpholino injection causes failure of placode formation and results in ear-less embryos, mimicking the phenotype we observe after pharmacological inhibition of Fgf-signaling. Fgf8 and Fgf3 together therefore act during induction and differentiation of the ear placode. In addition to the early requirement for Fgf signaling, the abnormal differentiation of inner ear structures and mechanosensory hair cells in ace mutants, pharmacological inhibition of Fgf signaling, and the expression of fgf8 and fgf3 in the otic vesicle demonstrate independent Fgf function(s) during later development of the otic vesicle and lateral line organ. We furthermore addressed a potential role of endomesomerm by studying mzoep mutant embryos that are depleted of head endomesodermal tissue, including chordamesoderm, due to a lack of Nodal-pathway signaling. In these embryos, early placode induction proceeds largely normally, but the ear placode extends abnormally to midline levels at later stages, suggesting a role for the midline in restricting placode development to dorsolateral levels. We suggest a model of zebrafish inner ear development with several discrete steps that utilize sequential Fgf signals during otic placode induction and vesicle patterning.     

Fgf3 and Fgf10 are required for mouse otic placode induction

The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation, but its absence affects only the later stages of otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3 and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation.     

Tissue-specific requirements for FGF8 during early inner ear development

Several members of the FGF gene family have been shown to intervene from various tissue sources to direct otic placode induction and otic vesicle formation. In this study we define the roles of FGF8, found in different expression domains during this process, in mice and chickens. By conditional inactivation of Fgf8 in distinct tissue compartments we demonstrate that Fgf8 is required in the mesoderm and endoderm during early inner ear development. In the chicken embryo, overexpression of Fgf8 from various tissue sources during otic specification leads to a loss of otic tissue. In contrast ectopic overexpression of Fgf10, a major player during murine otic induction, does not influence otic vesicle formation in chicken embryos but results in the formation of ectopic structures with a non-otic character. This study underlines the crucial role of a defined Fgf8 expression pattern controlling inner ear formation in vertebrates.     

Fgf3 is required for dorsal patterning and morphogenesis of the inner ear epithelium

The inner ear, which contains sensory organs specialized for hearing and balance, develops from an ectodermal placode that invaginates lateral to hindbrain rhombomeres (r) 5-6 to form the otic vesicle. Under the influence of signals from intra- and extraotic sources, the vesicle is molecularly patterned and undergoes morphogenesis and cell-type differentiation to acquire its distinct functional compartments. We show in mouse that Fgf3, which is expressed in the hindbrain from otic induction through endolymphatic duct outgrowth, and in the prospective neurosensory domain of the otic epithelium as morphogenesis initiates, is required for both auditory and vestibular function. We provide new morphologic data on otic dysmorphogenesis in Fgf3 mutants, which show a range of malformations similar to those of Mafb (Kreisler), Hoxa1 and Gbx2 mutants, the most common phenotype being failure of endolymphatic duct and common crus formation, accompanied by epithelial dilatation and reduced cochlear coiling. The malformations have close parallels with those seen in hearing-impaired patients. The morphologic data, together with an analysis of changes in the molecular patterning of Fgf3 mutant otic vesicles, and comparisons with other mutations affecting otic morphogenesis, allow placement of Fgf3 between hindbrain-expressed Hoxa1 and Mafb, and otic vesicle-expressed Gbx2, in the genetic cascade initiated by WNT signaling that leads to dorsal otic patterning and endolymphatic duct formation. Finally, we show that Fgf3 prevents ventral expansion of r5-6 neurectodermal Wnt3a, serving to focus inductive WNT signals on the dorsal otic vesicle and highlighting a new example of cross-talk between the two signaling systems.     

Induction of otic structures by canonical Wnt signalling in medaka

The Wnt family of signalling proteins is known to participate in multiple developmental decisions during embryogenesis. We misexpressed Wnt1 in medaka embryos and observed anterior truncations, similar to those described for ectopic activation of canonical Wnt signalling in other species. Interestingly, when we induced a heat-shock Wnt1 transgenic line exactly at 30% epiboly, we observed multiple ectopic otic vesicles in the truncated embryos. The vesicles then fused, forming a single large ear structure. These “cyclopic ears” filled the complete anterior region of the embryos. The ectopic induction of otic development can be explained by the juxtaposition of hindbrain tissue with anterior ectoderm. Fibroblast growth factor (Fgf) ligands are thought to mediate the otic-inducing properties of the hindbrain. However, signals different from Fgf3 and Fgf8 are necessary to explain the formation of the ectopic ear structures, suggesting that Wnt signalling is involved in the otic induction process in medaka.     

Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus

Induction of the otic placode, the rudiment of the inner ear, is believed to depend on signals derived from surrounding tissues, the head mesoderm and the prospective hindbrain. Here we report the first attempt to define the specific contribution of the neuroectoderm to this inductive process in Xenopus. To this end we tested the ability of segments of the neural plate (NP), isolated from different axial levels, to induce the otic marker Pax8 when recombined with blastula stage animal caps. We found that one single domain of the NP, corresponding to the prospective anterior hindbrain, had Pax8-inducing activity in this assay. Surprisingly, more than half of these recombinants formed otic vesicle-like structures. Lineage tracing experiments indicate that these vesicle-like structures are entirely derived from the animal cap and express several pan-otic markers. Pax8 activation in these recombinants requires active Fgf and canonical Wnt signaling, as interference with either pathway blocks Pax8 induction. Furthermore, we demonstrate that Fgf and canonical Wnt signaling cooperate to activate Pax8 expression in isolated animal caps. We propose that in the absence of mesoderm cues the combined activity of hindbrain-derived Wnt and Fgf signals specifies the otic placode in Xenopus, and promotes its morphogenesis into an otocyst.     

A direct role for Fgf but not Wnt in otic placode induction

Induction of the otic placode, which gives rise to all tissues comprising the inner ear, is a fundamental aspect of vertebrate development. A number of studies indicate that fibroblast growth factor (Fgf), especially Fgf3, is necessary and sufficient for otic induction. However, an alternative model proposes that Fgf must cooperate with Wnt8 to induce otic differentiation. Using a genetic approach in zebrafish, we tested the roles of Fgf3, Fgf8 and Wnt8. We demonstrate that localized misexpression of either Fgf3 or Fgf8 is sufficient to induce ectopic otic placodes and vesicles, even in embryos lacking Wnt8. Wnt8 is expressed in the hindbrain around the time of otic induction, but loss of Wnt8 merely delays expression of preotic markers and otic vesicles form eventually. The delay in otic induction correlates closely with delayed expression of fgf3 and fgf8 in the hindbrain. Localized misexpression of Wnt8 is insufficient to induce ectopic otic tissue. By contrast, global misexpression of Wnt8 causes development of supernumerary placodes/vesicles, but this reflects posteriorization of the neural plate and consequent expansion of the hindbrain expression domains of Fgf3 and Fgf8. Embryos that misexpress Wnt8 globally but are depleted for Fgf3 and Fgf8 produce no otic tissue. Finally, cells in the preotic ectoderm express Fgf (but not Wnt) reporter genes. Thus, preotic cells respond directly to Fgf but not Wnt8. We propose that Wnt8 serves to regulate timely expression of Fgf3 and Fgf8 in the hindbrain, and that Fgf from the hindbrain then acts directly on preplacodal cells to induce otic differentiation.     

Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. In this study, we determined fgf3 expression in relation to otic development in the zebrafish and used antisense morpholino oligonucleotides to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode. fgf3 is co-expressed with fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. We propose that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction.     

Sprouty1 and Sprouty2 limit both the size of the otic placode and hindbrain Wnt8a by antagonizing FGF signaling

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. Here we report that in Spry1, Spry2 compound mutant embryos (Spry1^−/−; Spry2^−/− embryos), the otic placode is increased in size. We demonstrate that the otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1^−/−; Spry2^−/− embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. We demonstrate that both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1^−/−; Spry2^−/− embryos by reducing the gene dosage of Fgf10. Our results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center.     

RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.     

Fgf and Hh signalling act on a symmetrical pre-pattern to specify anterior and posterior identity in the zebrafish otic placode and vesicle

Specification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20 somite (early otic vesicle) stages. We now show that Fgf signalling is both necessary and sufficient for anterior otic specification during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia(-/-) (fgf3(-/-)) mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear.     

Analysis of mouse kreisler mutants reveals new roles of hindbrain-derived signals in the establishment of the otic neurogenic domain

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3−/−Fgf10−/− double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis.     

The role of Paraxial Protocadherin in Xenopus otic placode development

Vertebrate inner ear develops from its rudiment, otic placode, which later forms otic vesicle and gives rise to tissues comprising the entire inner ear. Although several signaling molecules have been identified as candidates responsible for inner ear specification and patterning, many details remain elusive. Here, we report that Paraxial Protocadherin (PAPC) is required for otic vesicle formation in Xenopus embryos. PAPC is expressed strictly in presumptive otic placode and later in otic vesicle during inner ear morphogenesis. Knockdown of PAPC by dominant-negative PAPC results in the failure of otic vesicle formation and the loss of early inner ear markers Sox9 and Tbx2, suggesting the requirement of PAPC in the early stage of otic vesicle development. However, PAPC alone is not sufficient to induce otic placode formation.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b

Background  

The inner ear arises from a specialized set of cells, the otic placode, that forms at the lateral edge of the neural plate adjacent to the hindbrain. Previous studies indicated that fibroblast growth factors (Fgfs) are required for otic induction; in zebrafish, loss of both Fgf3 and Fgf8 results in total ablation of otic tissue. Furthermore, gain-of-function studies suggested that Fgf signaling is not only necessary but also sufficient for otic induction, although the amount of induced ectopic otic tissue reported after misexpression of fgf3 or fgf8 varies among different studies. We previously suggested that Foxi1 and Dlx3b may provide competence to form the ear because loss of both foxi1 and dlx3b results in ablation of all otic tissue even in the presence of a fully functional Fgf signaling pathway.