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.     

Dysmorphogenesis of the inner ear: disruption of extracellular matrix (ECM) formation by an L-proline analog in otic explants

L-azetidine-2-carboxylic acid (LACA), a l-proline analog, disrupts collagen secretion by cells and prevents normal morphogenesis of in vitro developing organ rudiments. Otic explants derived from 10.5-through 14-day-old mouse embryos were continuously exposed to LACA in the nutrient medium at concentrations of 75, 150, and 300 micrograms/ml. LACA disrupted normal in vitro otic morphogenesis in inner ears explanted from embryos of 10.5 through 13 days' gestation. Development of 14-day-old otic explants were not affected by LACA at the concentrations tested. There was a direct correlation between the embryonic age of the explant when exposed to LACA, and the severity of otic dysmorphogenesis. The younger explants (10.5-to 12-day-old) developed abnormalities of both vestibular and auditory structures, but with increasing embryonic age of the explants (12-to 13.5-day-old) abnormalities were confined more to the auditory portion of the inner ear. Disruption of collagen secretion of connective tissue cells of the otic explants are a major teratogenic action of LACA on inner ear development. Disrupted collagen secretion alters otic extracellular matrix production, which in turn affects the tissue interactions that regulate the progressive expression of otic morphogenesis and differentiation.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

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.     

Transforming growth factor beta 1 is an epithelial-derived signal peptide that influences otic capsule formation.

Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work indicates that many growth factors are distributed in the early embryo in vivo in a temporal-spatial pattern that correlates with sites of ongoing morphogenetic events. We report here that the localization of transforming growth factor beta 1 (TGF-beta 1) in both epithelial and mesenchymal tissues of the mouse inner ear between 10 and 16 days of embryonic development (E10-E16). In addition, utilizing a high-density culture system as an in vitro model of otic capsule chondrogenesis, we show that modulation of chondrogenesis by TGF-beta 1 in cultured mouse periotic mesenchyme mimics the in vitro effects of otic epithelium on the expression of chondrogenic potential. We provide evidence of a causal relationship of this growth factor to otic capsule formation in situ by demonstrating that the actual sequence of chondrogenic events that occur in the developing embryo is reproduced in culture by the addition of exogenous TGF-beta 1 peptide. Furthermore, in cultures of mesenchyme containing otic epithelium, we demonstrate the localization of endogenous TGF-beta 1, first within the epithelial tissue and later within both the epithelium and its surrounding periotic mesenchyme, contrasted to an absence of endogenous TGF-beta 1 in cultures of mesenchyme alone. Our results suggest that TGF-beta 1 is one of the signal molecules that mediate the effects of otic epithelium in influencing the formation of the cartilaginous otic capsule.     

Epithelial control of periotic mesenchyme chondrogenesis

Morphogenesis of the cartilaginous otic capsule is directed by interactions between the epithelial anlage of the membranous labyrinth (otocyst) and its associated periotic mesenchyme. Utilizing a developmental series of high-density (micromass) cultures of periotic mesenchyme to model capsule chondrogenesis, we have shown that the early influence of otic epithelium in cultures of 10.5- or 14-gestation day (gd) periotic mesenchyme results in initiation or suppression of chondrogenesis, respectively. Furthermore, we have shown that introduction of otic epithelium at two distinct times during in vitro development to cultures of 10.5-gd mesenchyme cells results first in an initiation and then in an inhibition of their chondrogenic response. These influences of epithelial tissue on chondrogenic differentiation by periotic mesenchyme are not tissue specific but are characterized by temporal selectivity. The ability of otic epithelium to influence chondrogenesis and the competence of the periotic mesenchyme to respond to its signals are dependent upon the developmental stage of both tissues. This study provides conclusive evidence that otic epithelium acts as a developmental "switch" during otic capsule morphogenesis, signaling first the turning on and then the turning off of chondrogenic programs in the responding cephalic mesenchyme.     

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.     

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.     

In vitro development of the embryonic mouse inner ear following exposure to trypsin

Trypsin has been shown to disrupt normal in vitro morphogenesis of embryonic organ rudiments. Otic tissues derived from 11-, 12-, and 13-day-old mouse embryos were exposed to either Ca++- and Mg++-free PBS or 0.25% trypsin dissolved in Ca++- and Mg++-free PBS prior to explanation into organ culture. Trypsin treatment of otic explants disrupted the expression of the normal pattern of inner-ear development in vitro. There was a direct correlation between the embryonic age at time of exposure to trypsin and the severity of dysmorphogenesis of the inner ear. The younger explants showed abnormalities of both vestibular and auditory structures, whereas with increasing embryonic age, abnormalities were confined more to the auditory portion of the inner ear. The results suggest that integrity of the otocyst basal lamina and epitheliomesenchymal tissue interactions are important factors in early otic development. It is postulated that the major effect of trypsin on inner-ear morphogenesis is through disruption of these factors, which may act to regulate the progressive expression of early otic development.     

Innervation of the rat pineal gland by nerve fibres originating in the sphenopalatine, otic and trigeminal ganglia. A retrograde in vivo neuronal tracing study.

The innervation of the rat pineal gland from the sphenopalatine, otic, superior cervical and trigeminal ganglia was investigated in animals by use of in vivo retrograde tracings. A solution of 2% Fluorogold was iontophoretically injected into the superficial pineal gland in a series of Wistar rats. After a survival time of 4-10 days, the animals were fixed by perfusion and the brains, sphenopalatine, otic, superior cervical and trigeminal ganglia were investigated with a fluorescence microscope. Many retrogradely labelled perikarya were found in the superior cervical ganglia, but a smaller number of neurones were also labelled in the sphenopalatine, otic and trigeminal ganglia. Injections of the tracer into the subarachnoidal space were used as the control for unspecific uptake and transport of the tracer. The input to the pineal gland from the parasympathetic sphenopalatine and otic ganglia might be involved in the regulation of the annual rhythms of the pineal gland. The projections from the sensory trigeminal ganglion could be involved in the control of the blood flow of the gland.     

Developmental expression of the TGF beta s in the mouse cochlea.

Mice with targeted disruption of the TGF beta 2 gene display defects in epithelial-mesenchymal tissue interactions in several tissues including the developing cochlea. Specifically, the region of the spiral limbus and the overlying interdental cells, structures putatively involved in endolymphatic fluid homeostasis, display morphogenetic abnormalities. These findings prompted us to explore the pre-natal and post-natal expression of all three mammalian TGF beta genes in the developing mouse inner ear. TGF beta 2 mRNA expression was identified throughout the cochlear epithelium at all of the developmental stages examined. TGF beta 3 mRNA expression was identified in the mesenchymal tissues of the cochlea surrounding the otic epithelium. We found no evidence for compensation by the other two TGF beta isoforms in the cochleas of the TGF beta 2 mutants.     

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.