Retinoid signaling in inner ear development: A "Goldilocks" phenomenon.

Retinoic acid (RA) is a biologically active derivative of vitamin A that is indispensable for inner ear development. The normal function of RA is achieved only at optimal homeostatic concentrations, with an excess or deficiency in RA leading to inner ear dysmorphogenesis. We present an overview of the role of RA in the developing mammalian inner ear, discussing both how and when RA may act to critically control a program of inner ear development. Molecular mechanisms of otic teratogenicity involving two members of the fibroblast growth factor family, FGF3 and FGF10, and their downstream targets, Dlx5 and Dlx6, are examined under conditions of both RA excess and deficiency. We term the effect of too little or too much RA on FGF/Dlx signaling a Goldilocks phenomenon. We demonstrate that in each case (RA excess, RA deficiency), RA can directly affect FGF3/FGF10 signaling within the otic epithelium, leading to downregulated expression of these essential signaling molecules, which in turn, leads to diminution in Dlx5/Dlx6 expression. Non-cell autonomous affects of the otic epithelium subsequently occur, altering transforming growth factor-beta (TGFß) expression in the neighboring periotic mesenchyme and serving as a putative explanation for RA-mediated otic capsule defects. We conclude that RA coordinates inner ear morphogenesis by controlling an FGF/Dlx signaling cascade, whose perturbation by deviations in local retinoid concentrations can lead to inner ear dysmorphogenesis.

The application of regular expression-based pattern matching to profiling the developmental factors that contribute to the development of the inner ear.

The biomedical literature has always played a critical role in the development of hypotheses to test, experimental design, and the analysis of study results. Yet, the ever-expanding body of biomedical literature is starting to present new challenges, in which locating pertinent literature from among the millions of published research articles is often a challenging task. A regular expression-based pattern matching method has been developed to profile the various gene and protein factors that may play a role in various tissues contained within an organism. This methodology has been demonstrated through the profiling of the various factors that are involved in the development of the inner ear, and is shown to be both effective and accurate.

Coordinated molecular control of otic capsule differentiation: functional role of Wnt5a signaling and opposition by sfrp3 activity.

Wnt proteins constitute one of the major families of secreted ligands that function in developmental signaling, however, little is known of the role of Wnt5a during inner ear development. It is hypothesized that Wnt5a acts as a mediator of chondrogenesis in the developing otic capsule, a cartilaginous structure that surrounds the developing inner ear and presages the formation of the endochondral bony labyrinth. We report the pattern of expression of Wnt5a protein and mRNA in the developing mouse inner ear using immunohistochemistry, whole-mount in situ hybridization and RT-PCR, and the ability of exogenous Wnt5a to stimulate otic capsule chondrogenesis when added to high-density cultures of periotic mesenchyme containing otic epithelium (periotic mesenchyme + otic epithelium), a well-established model of otic capsule formation. We show that in the presence of secreted frizzled related protein 3 (sfrp3), a Wnt antagonist expressed in the developing inner ear, or Wnt5a-specific antisense oligonucleotide, which diminishes endogenous Wnt5a, otic capsule chondrogenesis is suppressed in culture. We determined by histological analysis and aggrecan immunoreactivity that chondrogenic differentiation is disturbed in Wnt5a null embryos, and provide evidence that the periotic mesenchyme + otic epithelium harvested from Wnt5a null mice is compromised in its ability to differentiate into cartilage when interacted in culture. We propose a model whereby sfrp3 and Wnt5a act antagonistically to ensure appropriate patterns of chondrogenesis and provide coordinated control of otic capsule formation. Our findings support Wnt5a and sfrp3 as regulators of otic capsule formation in the developing mouse inner ear.

Retinoic acid-induced inner ear teratogenesis caused by defective Fgf3/Fgf10-dependent Dlx5 signaling.

BACKGROUND: Retinoic acid (RA) is essential for inner ear development. However, exposure to excess RA at a critical period leads to inner ear defects. These defects are associated with disruption in epithelial-mesenchymal interactions. METHODS: This study investigates the role of Dlx5 in the epithelial-mesenchymal interactions that guide otic capsule chondrogenesis, as well as the effect of excess in utero RA exposure on Dlx5 expression in the developing mouse inner ear. Control of Dlx5 by Fgf3 and Fgf10 under excess RA conditions is investigated by examining the developmental window during which Fgf3 and Fgf10 are altered by in utero RA exposure and by testing the ability of Fgf3 and Fgf10 to mitigate the reduction in chondrogenesis and Dlx5 expression mediated by RA in high-density cultures of periotic mesenchyme containing otic epithelium, a model of epithelial-mesenchymal interactions in which chondrogenic differentiation of periotic mesenchyme ensues in response to induction by otic epithelium. RESULTS: Dlx5 deletion alters expression of TGFbeta(1), important for otic capsule chondrogenesis, in the developing inner ear and compromises the ability of cultured periotic mesenchyme containing otic epithelium, harvested from Dlx5 null embryos, to differentiate into cartilage when compared with control cultures. Downregulation in Dlx5 ensues as a consequence of in utero RA exposure in association with inner ear dysmorphogenesis. This change in Dlx5 is noted at embryonic day 10.5 (E10.5), but not at E9.5, suggesting that Dlx5 is not a direct RA target. Before Dlx5 downregulation, Fgf3 and Fgf10 expression is modified in the inner ear by excess RA, with the ability of exogenous Fgf3 and Fgf10 to rescue chondrogenesis and Dlx5 expression in RA-treated cultures of periotic mesenchyme containing otic epithelium supporting these fibroblast growth factors (FGFs) as intermediary genes by which RA mediates its effects. CONCLUSIONS: Disruption in an Fgf3, -10/Dlx5 signaling cascade is operant in molecular mechanisms of inner ear teratogenesis by excess RA.

Negative regulation of otic capsule chondrogenesis: it can make you Smad.

The transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta1 and bone morphogenetic protein (BmP2, BmP4), participates in the regulation of the developing cartilaginous otic capsule, which prefigures the endochondral bony labyrinth of the inner ear. This study investigates Smad-6 and -7, downstream components of the TGF-beta/BMP signaling pathway, in otic capsule chondrogenic control, and supports a function for these inhibitory Smads as negative regulators of capsule chondrogenesis. The importance of otic capsule chondrogenic control and implications of Smad signaling for otosclerosis, a disease affecting the endochondral bony labyrinth, are indicated.

Oral ethanol potentiates the loss of outer hair cells in cisplatin-exposed rats.

OBJECTIVE: The aim of this study was to examine the effect of oral ethanol on cisplatin ototoxicity. STUDY DESIGN AND SETTING: Twenty-seven-week-old, female Fisher 344 rats were divided into 4 experimental groups. The animals were administered per os (PO) saline (group 1), PO ethanol (group 2), PO saline with intraperitoneal (IP) cisplatin (group 3), or PO ethanol with IP cisplatin (group 4). After 3 days, scanning electron microscopy and counts of outer auditory hair cells were performed. RESULTS: A 2-fold increase in outer hair cell loss was obtained in the basal cochlear turn of rats receiving concomitant cisplatin and ethanol compared with animals receiving cisplatin and saline. No hair cell loss was observed in the middle cochlear turn of any experimental group. CONCLUSION: Our findings support potentiation of ototoxicity when cisplatin is combined with oral ethanol. SIGNIFICANCE: Contraindications for alcohol use in cancer patients receiving cisplatin are implicated.

Oxymetazoline solutions inhibit middle ear pathogens and are not ototoxic.

OBJECTIVES: This study was performed to explore the antimicrobial activity of two commercially available oxymetazoline hydrochloride preparations against the common pathogens of otitis media and to demonstrate the lack of ototoxicity of these agents and of United States Pharmacopeia (USP) oxymetazoline in a standard animal model. METHODS: Disc diffusion assays and minimum inhibitory concentration studies against American Type Culture Collection reference strains of common middle ear pathogens were used to evaluate the antimicrobial activity of oxymetazoline solutions and fluoroquinolone drops, and outer hair cell counts were performed on scanning electron micrographs of guinea pig basal cochlear segments after chronic exposure to oxymetazoline solutions and positive and negative controls. RESULTS: Oxymetazoline nasal spray and eyedrops had activity against all species tested except Haemophilus influenzae and Pseudomonas aeruginosa. The USP oxymetazoline had limited antimicrobial activity. Oxymetazoline nasal spray, oxymetazoline eyedrops, and USP oxymetazoline had ototoxicity profiles indistinguishable from that of the saline solution control. CONCLUSIONS: Commercially available oxymetazoline solutions are active against several of the common pathogens of otitis media. This antimicrobial activity is not due to oxymetazoline, and is more likely due to preservatives present in the solutions. The solutions tested are not ototoxic to guinea pig outer hair cells. Oxymetazoline solutions are potential substitutes for broad-spectrum antibiotic drops after tympanostomy tube placement.

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

Transforming growth factor-beta1 signaling participates in the physiological and pathological regulation of mouse inner ear development by all-trans retinoic acid.

BACKGROUND: Retinoic acid (RA) is a vitamin A derivative that participates in patterning and regulation of inner ear development. Either excess RA or RA deficiency during a critical stage of inner ear development can produce teratogenic effects. Previous studies have shown that in utero exposure of the developing mouse inner ear to a high dose of all-trans RA (atRA) results in severe malformations of the inner ear that are associated with diminished levels of endogenous transforming growth factor-beta1 (TGF-beta(1)) protein. METHODS: In this study, the effects of a teratogenic level of atRA on levels and patterns of expression of TGFbeta receptor II (TGFbetaRII) and Smad2, a downstream component of the TGFbeta signal transduction pathway, are investigated in the developing mouse inner ear. The expression pattern of endogenous RA receptor alpha (RARalpha) and the ability of an RARalpha(1)-specific antisense oligonucleotide (AS) to modulate otic capsule chondrogenesis are demonstrated in the inner ear and in culture. RESULTS: Endogenous TGFbetaRII and Smad2 are downregulated in the inner ear following in utero atRA treatment. In addition, a reduction in endogenous TGFbeta(1) and a marked suppression of chondrogenesis occur in RARalpha(1) AS-treated cultures in comparison to untreated or oligonucleotide-treated control cultures. This chondrogenic suppression can be partially overcome by supplementation of RARalpha(1) AS-treated cultures with exogenous TGFbeta(1) protein. CONCLUSIONS: Our findings support a role for TGFbeta in the physiological and pathological effects of RA on inner ear development.

Bone morphogenetic protein 4 (BMP4): a regulator of capsule chondrogenesis in the developing mouse inner ear.

Formation of the cartilaginous otic capsule is directed by otic epithelial-periotic mesenchymal interactions. In response to induction by otic epithelium, condensations of mesenchyme appear in the periotic region and form a chondrified otic capsule that serves as the template for the subsequent formation of the endochondral bony labyrinth. Previous studies indicate that members of the transforming growth factor beta superfamily, including transforming growth factor beta(1), participate in guiding these tissue interactions. In this study, we report the localization of bone morphogenetic protein 4 (BMP4) to the mesenchymal and epithelial-derived tissues of the mouse inner ear between 10.5 and 14 days of embryonic development. We demonstrate modulation of chondrogenesis in cultured mouse periotic mesenchyme by exogenous BMP4 protein and investigate the function of endogenous BMP4 in otic capsule chondrogenesis. We show that in the presence of the BMP antagonist, Noggin, otic capsule chondrogenesis is suppressed in culture in a dose-dependent manner. Consistent with this finding, addition of BMP4-specific antisense oligonucleotide to cultures of mouse periotic mesenchyme containing otic epithelium decreases levels of endogenous BMP4 protein and suppresses the chondrogenic response of the cultured periotic mesenchyme, providing evidence of the necessity for BMP4 in mediating otic capsule chondrogenesis. Supplementation of either Noggin- or BMP4 antisense oligonucleotide-treated cultures with BMP4 protein can restore the extent of chondrogenesis to normal levels. Our findings support BMP4 as an essential mediator of chondrogenesis in the developing otic capsule in situ.