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.     

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.     

Vascularization of the endochondral layer with respect to the otic capsule. A comparative anatomical study.

The otic capsules of several species were examined morphometrically and compared with the otic capsule of man. Area, perimeter and diameter of vessels, radius of tissue cylinder, circumscribing hexagon, intervascular distance, and O2 exchange area were evaluated. A correlation between occurrence and expansion of the interglobular spaces ('Interglobularr?ume') on the one hand and vascularization of the endochondral layer on the other hand was found. This corresponds to results of other authors concerning the persistence of cartilage observed in tissue cultures and long bones.     

The enthesis of the elbow-joint capsule of the dog humerus

The enthesis of the elbow-joint capsule in the dog is described histologically in relation to the specific mechanical forces that operate in different regions along its line of attachment. Special attention is given to the collagen fibre-bone interface in those parts of the capsule that are highly affected by mechanical stress. The histological features of the enthesis are heterogeneous along the entire circumference of the attachment site. Three types of collagen fibre-bone interconnections can be distinguished: (1) periosteal insertion: attachment to the periosteum of the humerus; (2) bony insertion: attachment directly to peripheral osteons; (3) fibrocartilaginous insertion: attachment to the bone via fibrocartilage. The periosteal insertion covers the greatest part of the joint capsule attachment line, along the peripheral borders of the radial and olecranon fossae. In contrast, bony insertions and fibrocartilaginous insertions are focally arranged: bony insertions in the caudoproximal aspect of the olecranon fossa, related to nutrient foramina; fibrocartilaginous insertions in combination with the attachment of distinct ligaments. This distribution reflects a strict relation between the type of enthesis and the biomechanical stress at the attachment site. The periosteal insertion type is predominant in entheses adjacent to pouches of a loose joint capsule -- i.e., regions less dependent on the high tensile strength of collagen fibres. Fibrocartilaginous insertions characterise areas of the joint capsule which are subjected to high biomechanical traction during joint movement. Both structurally and functionally, the entheses of the fibrous layer of the joint capsule are similar to those of tendons and ligaments.     

Ornithine decarboxylase activity during development of the mouse inner ear in vivo and in vitro

Summary Ornithine decarboxylase activity was determined during the development of the peripheral auditory system in the murine otocyst with the goal of understanding the role of this enzyme in the morphological and functional maturation of the inner ear. At gestational days 11 and 12 enzyme activity was more than 10-fold higher than adult levels. A sharp decline occured between day 12 and 13 after which activity rose to a peak around day 15. Activity then dropped continuously until near-adult levels were reached at birth. A lower specific activity of ODC but a similar time-course was seen in otocysts explanted at gestational day 13 and subsequently cultured for 6 days. For two stages of development, enzyme activity and binding of ³H--difluoromethylornithine were compared. The four-fold difference in enzymatic activity on gestational days 15 and 17 was paralleled by a similar difference in binding. Ornithine decarboxylase activity during inner ear development therefore seems primarily regulated at the level of protein synthesis. Ornithine decarboxylase activity correlates with major inductive events in the morphogenesis of the cartilagenous otic capsule that serves as a template for the formation of the bony labyrinth. The pattern of activity may reflect the changes in the head mesenchyme that is recruited by the otocyst to aggregate and form its protective 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.     

Craniofacial and otic capsule abnormalities in a transgenic mouse strain with a Col2a1 mutation

Abnormal craniofacial features of a transgenic mouse model of chondrodysplasia with a type II collagen mutation (Gly574Ser) are described in this report. In addition to a shortened mandible and cleft palate, a misshapen otic capsule was observed. Interestingly, hearing impairment is often a component of the chondrodysplasia phenotype that results from mutations in COL2A1. To identify a potential mechanism in the hearing loss associated with type II collagen mutations, we examined the development of the otic capsule in the transgenic mice. It appeared to be smaller overall, relative to the skull proportions, and rather than the normal rounded dimensions, the transgenic capsule was flattened and elongated. We speculate that the cartilage of the developing otic capsule was less able to resist the mechanical forces from the developing brain and other tissues within the cranium and thus became deformed under pressure. We further speculate that the hearing loss associated with the chondrodysplasia phenotype is at least partially due to these defects in the developing cartilage matrix of the otic capsule.     

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.     

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.     

Lack of association between SNP rs3914132 of the RELN gene and otosclerosis in India

Otosclerosis (MIM 166800) is primarily a metabolic bone disorder of the otic capsule, which leads to bony fixation of the stapedial footplate in the oval window; it is among the most common causes of acquired hearing loss. The etiology of this disease is largely unknown, although epidemiological studies suggest the involvement of both genetic and environmental factors. Recently, a reelin gene, SNP rs3914132, located in intron 2, was shown to be associated with otosclerosis in a European population. When we sequenced blood DNA samples of 85 individuals with otosclerosis and 85 controls, four SNPs of this gene: rs3914131 (P = 0.6463), rs3914132 (P = 0.1822), rs9641319 (P = 0.7371), and rs10227303 (P = 0.5669) were not significantly associated with this disease. In one familial case, a novel variant (C/T) at contig position 2923488 was found to be inherited by the proband and affected family members.     

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.     

Développement de la partie antérieure de la mandibule de Salmo trutta fario L. (Pisces, Teleostei, Salmonidae)

A study of the origin and development of the bony complex ("dentaire" s.l.) later forming the anterior part of the mandible of Salmo fario leads to the following conclusions: (1) various modes of ossifications occur in the ontogeny of this bony complex, (2) the main component is the dermal (dento-splénial) whereas the enchondral one (mentomeckelien) is much reduced, (3) the proposed term "dento-splénio-mentomeckelien" focuses attention, at the same time, on the order of appearance and relative importance of the two components.     

Reproduction and differentiation of the cells in enchondral osteogenesis

Light and electron-microscopical 3H-thymidine autoradiography was used to study the dynamics of cell populations in the zones of enchondral osteogenesis in a tubular bone. In the early postnatal ontogenesis little differentiated perivascular cells are characterized by the highest proliferative activity in this region; they are considered as a population containing initial forms of the histogenetic sequence (differon) of the stromal fibroblast-like cells including osteoblasts. Differentiation of osteogenic cells from the initial forms to the mature osteoblasts proceeds through a number of successive divisions (1-3 divisions) and is accompanied by a decrease in the proliferative activity due to the increase in the generation time and decrease in the cell proliferative pool. The major part of osteoblasts is outside the mitotic cycle. At the later stages of ontogenesis the intensity of growth processes in the bone is provided for by changes in the proliferative pool of the committed precursor cells (preosteoblasts) which make a part of endosteum, vascular channels and bone marrow stroma.     

Fine structure of the mammalian renal capsule: The atypical smooth muscle cell and its functional meaning

Summary The present electron microscopic study on the fine structure of the renal capsule of some mammals (mouse, rat, mole, guinea pig and rabbit) shows that, although there are some variations in the structure, the general morphology is the same.The renal capsule of these animals consists of two layers, a connective tissue layer and an atypical smooth muscle cell layer, and is bound to the renal parenchyma by a thin peritubular loose connective tissue. The atypical smooth muscle cell is characterized by the existence of fine cytoplasmic filaments usually arranged along the long axis of the cell, and the cells also show a complicated interlocking among adjacent cells. The atypical smooth muscle cells gradually undergo a transition to fibroblasts of the upper connective tissue layer, losing their similarities to smooth muscle cells.When intrarenal pressure is elevated and the renal capsule is distended, the intercellular space among interdigitating or overlapping atypical smooth muscle cells is extensively dilated.Tracers such as horseradish peroxidase and ferritin injected intravenously or intraperitoneally can transverse the renal capsule.From the present study, it is concluded that the renal capsule of mammals possesses common structures, and contains atypical smooth muscle cells. These morphological characteristics suggest that the renal capsule could play a certain role related to the renal function.The author wishes to acknowledge the helpful advices of Prof. T. Yamamoto     

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.     

Ontogeny of the partial secondary wall of the otoccipital region of the endocranium in prehatching Alligator mississippiensis (Archosauria, Crocodylia)

The ontogeny of the posterior otic and anterior occipital portions of the neural endocranium of prehatching Alligator mississippiensis was investigated by reconstruction from sectioned material. In Stage 6 of this species, in which the endochondral ossification of the otoccipital region of the neural endocranium is only in its very early stage, two bony outgrowths-laminae-are present at the external wall of the posterior portion of the neural endocranium. The anterior lamina arises from the external surface of the basal plate at the level of the posterior margin of the subcapsular process; the posterior lamina arises from the external surface of that portion of the pila occipitalis that forms the posteroventral wall of the metotic fissure. During ontogeny, both laminae lying in the anteroposterior sequence ossify in membrane, fuse together, grow laterodorsally, and fuse with the lateral wall of the lateral semicircular canal and the crista parotica. This lamina forms a new, secondary wall enclosing the posterior section of the otic capsule and contains the large external jugular foramen (or foramen vagi) in its basal portion. The laminae, designated lamina juxtaotica anterior and posterior (lamina juxtaotica when fused together), have not been recorded previously in crocodylians and are absent in all other Recent reptiles. From the functional point of view, the juxtaotic lamina 1) forms the margins of the external jugular foramen, and 2) forms the floor of the posterior section of the Eustachian tube. In birds, the structure called the metotic cartilage, which arises in ontogeny as an independent element, has a similar position as the juxtaotic lamina. However, the two structures differ in their developmental origins and their relation to the Eustachian tube and the ramus hyomandibularis of the facialis nerve. Moreover, there is no external jugular foramen in birds.