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.     

Treatment with all-trans-retinoic acid decreases levels of endogenous TGF-beta(1) in the mesenchyme of the developing mouse inner ear

Background: Previous studies have shown that in utero exposure of the mouse embryo to high doses of all-trans-retinoic acid (atRA) produces defects of the developing inner ear and its surrounding cartilaginous capsule, while exposure of cultured periotic mesenchyme plus otic epithelium to high doses of exogenous atRA results in an inhibition of otic capsule chondrogenesis. Methods: In this study, we examine the effects of atRA exposure on the endogenous expression of transforming growth factor-beta(1) (TGF-beta(1)), a signaling molecule that mediates the epithelial-mesenchymal interactions that guide the development of the capsule of the inner ear. Results: Our results demonstrate a marked reduction in immunostaining for TGF-beta(1) in the periotic mesenchyme of atRA-exposed embryos of age E10.5 and E12 days in comparison with control specimens. Consistent with these in vivo findings, high-density cultures of E10.5 periotic mesenchyme plus otic epithelium, treated with doses of atRA that suppress chondrogenesis, showed significantly decreased levels of TGF-beta(1), as compared with TGF-beta(1) levels in untreated control cultures. Furthermore, we demonstrate a rescue of cultured periotic mesenchyme plus otic epithelium from atRA-induced chondrogenic suppression by supplementation of cultures with excess TGF-beta(1). Conclusions: Our results support the hypothesis that TGF-beta(1) plays a role in mechanisms of atRA teratogenicity during inner ear development.     

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.     

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 TGFβ₁, 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. Birth Defects Res (Part B) 2008. ©2008 Wiley‐Liss, Inc.     

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.     

Tbx1 and Brn4 regulate retinoic acid metabolic genes during cochlear morphogenesis

Background  

In vertebrates, the inner ear is comprised of the cochlea and vestibular system, which develop from the otic vesicle. This process is regulated via inductive interactions from surrounding tissues. Tbx1, the gene responsible for velo-cardio-facial syndrome/DiGeorge syndrome in humans, is required for ear development in mice. Tbx1 is expressed in the otic epithelium and adjacent periotic mesenchyme (POM), and both of these domains are required for inner ear formation. To study the function of Tbx1 in the POM, we have conditionally inactivated Tbx1 in the mesoderm while keeping expression in the otic vesicle intact.     

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. We show here that, during the precartilage and cartilage stages, Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, we analyzed the inner ears of mice homozygous for Fgf9 null alleles. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. Our results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis.     

Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear

The vertebrate inner ear, a complex sensory organ with vestibular and auditory functions, is derived from a single ectoderm structure called the otic placode. Currently, the molecular mechanisms governing the differentiation and specification of the otic epithelium are poorly understood. We present here a detailed expression study of LMO1-4 in the developing mouse inner ear using a combination of in situ hybridization and immunohistochemistry. LMO1 is specifically expressed in the vestibular and cochlear hair cells as well as the vestibular ganglia of the developing inner ear. LMO2 expression is detected in the periotic mesenchyme of the developing mouse cochlea from E12.5 to E14.5. The expression of LMO3 expression is first observed in the cochlea at E13.5 and becomes confined to the lesser epithelial ridge (LER) from E14.5 to E17.5. LMO3 is also expressed in some of the vestibular ganglion cells. LMO4 is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista, and the spiral ganglia throughout embryogenesis. Thus, the regionalized expression patterns of LMO1-4 are closely associated with the morphogenesis of the inner ear.     

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.     

BMP pathways are involved in otic capsule formation and epithelial-mesenchymal signaling in the developing chicken inner ear

The vertebrate inner ear consists of a complex labyrinth of epithelial cells that is surrounded by a bony capsule. The molecular mechanisms coordinating the development of the membranous and bony labyrinths are largely unknown. Previously, using avian retrovirus encoding Noggin (RCAS-Noggin) or beads soaked with Noggin protein, we have shown that bone morphogenetic proteins (BMPs) are important for the development of the otic epithelium in the chicken inner ear. Here, using two additional recombinant avian retroviruses, dominant negative and constitutively active forms of BMP receptors IB (BMPRIB), we show that BMPs, possibly acting through BMPRIB, are important for otic capsule formation. We also show that Bmp2 is strongly expressed in the prospective semicircular canals starting from the canal outpouch stage, suggesting that BMP2 plays an important role in canal formation. In addition, by correlating expression patterns of Bmps, their receptors, and localization of phosphorylated R-Smad (phospho R-Smad) immunoreactivity, an indicator of BMP activation, we show that BMPs emanating from the otic epithelium influence chondrogenesis of the otic capsule including the cartilage surrounding the semicircular canals.     

Inner ear malformations induced by isotretinoin in hamster fetuses.

Inner ear malformations induced in anotic hamster fetuses following maternal treatment with 50 mg/kg isotretinoin (13-cis-retinoic acid) on gestational day 8 are described. Computer-assisted three dimensional reconstruction was used. Two general types of defective vestibulocochlear development were seen. Defects were bilateral and correlated with extent of middle ear deficiency and severity of mandibular defects. In the more severely affected fetuses the inner ear was limited to an epithelial sac with occasional small projections, no apparent innervation and a correspondingly reduced otic capsule. In most of the fetuses examined the inner ear was less severely affected and was characterized by a reduction in the number of semicircular ducts and alterations in the size and shape of the cochlear duct. These defects are similar to those seen in a child with the isotretinoin embryopathy. Pathogenesis may result from a direct effect on otic epithelium or from faulty inductive interactions with the rhombencephalon or with periotic neural crest cells.     

Canonical Wnt signaling regulates the proliferative expansion and differentiation of fibrocytes in the murine inner ear

Otic fibrocytes tether the cochlear duct to the surrounding otic capsule but are also critically involved in maintenance of ion homeostasis in the cochlea, thus, perception of sound. The molecular pathways that regulate the development of this heterogenous group of cells from mesenchymal precursors are poorly understood. Here, we identified epithelial Wnt7a and Wnt7b as possible ligands of Fzd-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated periotic mesenchyme (POM). Mice with a conditional deletion of Ctnnb1 in the POM exhibited a complete failure of fibrocyte differentiation, a severe reduction of mesenchymal cells surrounding the cochlear duct, loss of pericochlear spaces, a thickening and partial loss of the bony capsule and a secondary disturbance of cochlear duct coiling shortly before birth. Analysis at earlier stages revealed that radial patterning of the POM in two domains with highly condensed cartilaginous precursors and more loosely arranged inner mesenchymal cells occurred normally but that proliferation in the inner domain was reduced and cytodifferentiation failed. Cells with mis/overexpression of a stabilized form of Ctnnb1 in the entire POM mesenchyme sorted to the inner mesenchymal compartment and exhibited increased proliferation. Our analysis suggests that Wnt signals from the cochlear duct epithelium are crucial to induce differentiation and expansion of fibrocyte precursor cells. Our findings emphasize the importance of epithelial-mesenchymal signaling in inner ear development.     

Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

Addition of the BMP4 antagonist, noggin, disrupts avian inner ear development

Bone morphogenetic protein 4 (BMP4) is known to regulate dorsoventral patterning, limb bud formation and axis specification in many organisms, including the chicken. In the chick developing inner ear, BMP4 expression becomes localized in two cell clusters at the anterior and posterior edges of the otic epithelium beginning at stage 16/17 and is expressed in presumptive sensory tissue at later stages. This restricted spatiotemporal pattern of expression occurs just prior to the otocyst's transition to a more complex three-dimensional structure. To further analyze the role of BMP4 in avian otic morphogenesis, cells expressing BMP4 or its antagonist, noggin, were grown on agarose beads and implanted into the periotic mesenchyme surrounding the chick otocyst. Although the BMP4-producing cells had no effect on the mature inner ear structure when implanted alone, noggin-producing cells implanted adjacent to the BMP4 cell foci prevented normal semicircular canal development. Beads implanted at the anterior BMP4 focus eliminated the anterior and/or the horizontal canals. Noggin cells implanted at the posterior focus eliminated the posterior canal. Canal loss was prevented by co-implantation of BMP4 cell beads next to noggin beads. An antibody to the chick hair cell antigen (HCA) was used to examine sensory cell distribution, which was abnormal only in the affected tissues of noggin-exposed inner ears. These data suggest a role for BMP4 in the accurate and complete morphological development of the semicircular canals.     

Analysis of Netrin 1 receptors during inner ear development

Netrin 1 plays key roles in axon guidance and neuronal migration during central nervous system (CNS) development. Outside the CNS, Netrin 1 has been shown to be involved in epithelial morphogenesis of various organs. We have shown that Netrin 1 is essential for inner ear semicircular duct formation, but the involvement of Netrin 1 receptors in this process has remained unknown. Netrin 1 receptors include members of the Deleted in colorectal cancer (Dcc), Unc5-homologue and integrin families. Here we have analysed the expression of these receptor genes during inner ear development and verified the inner ear phenotypes of several receptor mutant mice. Special interest was directed to receptors that could cooperate with Netrin 1 during semicircular duct formation. We show that Neogenin (Neo1), Unc5c as well as integrin b1 (Itgb1) are expressed in periotic mesenchyme, while Dcc, Unc5b, Unc5c, Itga3, Itga6 and Itgb1 are expressed in different parts of the otic epithelium. In spite of the broad and strong expression of several receptors in ear region, none of the analysed receptor mutant embryos showed any defects in inner ear development.