Fine structure of extracellular matrix and basal laminae in two types of abnormal collagen production: L-proline analog-treated otocyst cultures and disproportionate micromelia (Dmm/Dmm) mutants.

L-Azetidine-2-carboxylic acid (LACA), a naturally occurring vegetable imino acid, can be incorporated into mammalian proteins in place of proline, thereby eliciting an inhibitory effect on collagen secretion. Exposure of explants of the embryonic mouse inner ear to LACA reduces the number of collagen fibrils in the otic capsule, gives rise to a dose-dependent derangement of the basal lamina, and ultimately results in dysmorphogenesis and retarded differentiation of the inner ear. Disproportionate micromelia (Dmm) is an incomplete dominant form of dwarfism characterized by a reduced quantity of type II collagen in the cartilaginous extracellular matrix (ECM). Abnormal morphogenesis in homozygotic Dmm mice resembles the abnormal morphogenesis observed in LACA-exposed otic explants, resulting in malformed inner ears with a bulky cartilaginous capsule and a lack or reduction of defined perilymphatic spaces (Van De Water and Galinovic-Schwartz, 1987). In this study, we examined by ultrastructural analysis LACA-exposed otic explants and inner ears of Dmm/Dmm mouse embryos for abnormalities in the collagenous constituents of the basal laminae and capsular ECM. We demonstrate, in comparison to normal embryonic mouse inner ears, a reduction in collagen fibrils and irregular cytodifferentiation of chondrocytes in the ECM of LACA-exposed and Dmm/Dmm inner ears as well as in the basal laminae of LACA-exposed specimens. In addition, we provide evidence of dysmorphogenesis of the otic capsule and perilymphatic spaces in LACA-exposed explants. Moreover, while previous studies demonstrated the anomalous development of sensory structures in otocyst explants following LACA exposure, in this study we provide evidence of the normal morphogenesis of otic epithelial-derived sensory structures in homozygotic Dmm/Dmm mouse embryos.

Retinoic acid-induced embryopathy of the mouse inner ear.

Retinoic acid (RA) is an active metabolite of vitamin A that is teratogenic when present in excess during mammalian embryogenesis. We have investigated the effect of embryonic exposure to nonphysiological levels of all-trans RA on the development of the mouse inner ear. Dysmorphogenesis of both vestibular and auditory portions of the inner ear, and abnormal formation of the surrounding capsule are produced by exposure to teratogenic levels of RA at an embryonic age of 9 days (E9). There was no observable teratogenic effect of RA when administered at earlier (i.e., E7 or E8) or later (i.e., E10) stages of otic morphogenesis. We hypothesize that exposure to high levels of RA during a critical period of early otic morphogenesis interferes with the inductive tissue interactions required for inner ear development.

The role of the neurotrophins in maturation and maintenance of postnatal auditory innervation.

Auditory hair cells produce trophic factors that directly affect maturation and survival of auditory neurons. These factors include two members of the neurotrophin family: brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Loss of hair cells, as a result of either noise trauma or ototoxic damage, results in the degeneration of auditory neurons. An in vitro model of early postnatal rat organ of Corti/spiral ganglion explants was used to study the effects of deprivation and supplementation of nerve growth factor (NGF), BDNF, and NT-3 on neuronal survival. Immunolocalization of receptors for these neurotrophins correlated with their effectiveness as promoters of neuronal survival. BDNF affected early neuronal survival, whereas NT-3 was the most important survival factor for maturing auditory neurons. NGF was shown to maintain axonal morphology. Our results support the hypothesis that changes in the expression of these neurotrophins and their specific receptors in the maturing cochlea may control the postnatal processes of neuronal apoptosis and maturation of the innervation of both inner and outer hair cells. The results suggest that these growth factors have potential for preventing neuronal degeneration as well as enhancing the repair of damaged neuronal processes in the traumatized auditory system.

NGF, BDNF and NT-3 play unique roles in the in vitro development and patterning of innervation of the mammalian inner ear.

Developing cochleovestibular ganglion (CVG) neurons depend upon interaction with the otocyst, their peripheral target tissue, for both trophic support and tropic guidance. RT-PCR of E11 through E14 otocyst-CVG RNA extracts have shown that NGF as well as BDNF and NT-3 are expressed in the developing inner ear (in situ RT-PCR on tissue sections of E12 otocysts localized all three neurotrophins to the otocyst). To evaluate the functional significance of NGF, BDNF and NT-3 expression, E10.5 otocyst-CVG explants were treated with antisense oligonucleotides and compared to sense treated and control cultures. Confocal microscopic analysis revealed that treatment with BDNF antisense resulted in extensive neuronal cell death, downregulation of NGF caused an inhibition of neuritogenesis and a decrease in the neuronal population of the CVG, whereas treatment with NT-3 antisense resulted in a loss of target directed CVG neuritic ingrowth in this in vitro model. The effect of NGF or BDNF antisense treatment could be prevented by the simultaneous addition of the respective growth factor. These findings demonstrate that each of the three neurotrophins have important roles during the onset of neuritic ingrowth of the CVG neurons to the otocyst.

Induction of chondrogenesis: requirement for synergistic interaction of basic fibroblast growth factor and transforming growth factor-beta.

Interactions between the epithelial anlage of the developing mouse inner ear and its associated periotic mesenchyme control the differentiation of the cartilaginous otic capsule. Transforming growth factor-beta 1 (TGF-beta 1) is a naturally occurring signal peptide that is present in these tissues at times of active differentiation and morphogenesis. Previous studies have shown that TGF-beta 1 alone is not a sufficient stimulus to initiate chondrogenesis in cultured periotic mesenchyme. In this study, we provide evidence that basic fibroblast growth factor (bFGF) can elicit a specific but limited chondrogenic response in cultured periotic mesenchymal cells. We also demonstrate that simultaneous addition of bFGF and TGF-beta 1 to cultured periotic mesenchyme results in a full chondrogenic response comparable to that which occurs when periotic mesenchyme is grown in the presence of its natural inductor tissue (i.e. otic epithelium). Utilizing antibodies directed against bFGF, we show localization of endogenous bFGF in the otic epithelium in vivo and in mixed epithelial-mesenchymal cultures. Additionally, we demonstrate the presence of FGF-like activity in medium conditioned by otic epithelium. Blocking of epithelial elicited chondrogenesis by a combination of both alpha bFGF and alpha TGF-beta 1 antibodies provides further evidence of the necessity for these growth factors in the chondrogenic differentiation of periotic mesenchyme in vitro. Our results suggest a role for both bFGF and TGF-beta 1 in the regulation of chondrogenesis during otic capsule formation in situ.

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.

Nerve growth factor stimulates neurite regeneration but not survival of adult auditory neurons in vitro.

Injury to either the peripheral or central nervous system results in the accumulation of growth factors at the wound site. Some of these growth factors have been shown to participate in the neural repair process. Adult auditory neurons grown in dissociated spiral ganglion cell cultures are injured (i.e. bilateral axotomy) as a result of the initial preparation of these cultures. Therefore, cell cultures of dissociated spiral ganglia provide a model for the study of repair processes of adult auditory neurons (e.g. effects of exogenous growth factors on the process of neuritogenesis by injured neurons). Auditory neurons do not survive in these dissociated ganglion cell cultures when only exogenous NGF is added to the defined culture medium. Previous work has identified substrate bound basic fibroblast growth factor (bFGF) as a survival factor for adult auditory neurons in vitro. Auditory neurons cultured on substrate bound bFGF also do not show increased survival in response to the addition of increasing concentrations of nerve growth factor (NGF) to the defined medium. This is in sharp contrast to the pronounced neurite outgrowth-promoting effects (concentration dependent) observed when exogenous NGF is added to adult auditory neurons cultured on substrate bound bFGF. We propose that several neuronotrophic factors (e.g. TGFB1, bFGF, NGF and other neurotrophins) are active in the spiral ganglions' response to injury. Several of these growth factors (i.e. bFGF, NGF) act in cooperation to promote the regeneration or repair of severed or traumatized neuritic processes.

Temporal pattern of innervation in the developing mouse inner ear: an immunocytochemical study of a 66-kD subunit of mammalian neurofilaments.

We have examined the expression of a 66-kD neurofilament protein (NF-66) in the developing inner ear. Mouse embryos, fetuses, and neonates were fixed in Methacarn, embedded in paraffin, and sectioned. A polyclonal antiserum raised specifically to NF-66 and unreactive to NF-L, -M, -H, and peripherin was used for immunocytochemical staining. NF-66 immunostaining was first detectable in the rhombencephalon at embryonic day (E) 9.5. Immunoreactivity was first detected in the statoacoustic ganglion (SAG) early on E10.5. By late E10.5, the first SAG axons were detectable within the intraepithelial spaces of the otocyst. At E12, NF-66 positivity was detectable in neurites that projected into areas of presumptive vestibular sensory epithelium. Neurites projecting into the presumptive acoustic sensory epithelium were negative. However, at E13, the projections from both the vestibular and the acoustic ganglion (i.e, cochlear duct) were both NF-66 positive. In the cell bodies, NF-66 expression appeared earlier in the vestibular than in the auditory neurons. By E16, neuronal somas in both ganglia were NF-66 positive.

Transforming growth factor beta: does it direct otic capsule formation?

Interactions between the epithelium of the otocyst and surrounding periotic mesenchyme direct the formation of the capsule of the mammalian inner ear. In the present study, we have characterized the temporal-spatial distribution of transforming growth factor beta 1 (TGF-beta 1) in the epithelial and mesenchymal tissues that compose the inner ear between 10 and 14 days of embryonic development. In addition, using high-density cultures of periotic mesenchyme to model otic capsule formation, we have demonstrated that exogenous TGF-beta 1 can modulate otic chondrogenesis by acting as either an enhancer or a suppressor of this process. Our immunohistochemical and in vitro results suggest a paracrine action for this growth factor in modulation of epithelial-mesenchymal tissue interactions and otic morphogenesis.

Determinants of ganglion-receptor cell interaction during development of the inner ear. A heterochronic ganglia study.

It has been suggested that inner ear sensory receptors produce attractant fields that guide neurite outgrowth from statoacoustic ganglion (SAG) neurons to appropriate target sites within the developing labyrinth. This experiment tested the temporal limitations of SAG neurons in their ability to respond to these attractant fields. Statoacoustic ganglia were excised from 12, 13, 14 and 15 gestation day (GD) mouse embryos. This temporal series of SAG was implanted into aganglionic 12 GD otocysts. All cultures were grown for 7 days in vitro, then fixed and processed for nerve fiber staining. Specimens were evaluated for the presence of neurites associated with the inner ear sensory receptors that developed within the otic explants. All of the implanted heterochronic ganglia (i.e. 13, 14 or 15 GD) as well as the homochronic (i.e. 12 GD) ganglion controls extended neurites to sensory epithelium of both vestibular and auditory character. Neurites made contact with the base of hair cells in all of the sensory structures. These findings demonstrate that SAG neurons are capable of extending processes in response to otic attractant fields for an extended period during the embryonic development of this ganglion. This observation supports the hypothesis that the onset and duration of receptor generated attractant fields may act as a controlling factor in establishing patterns of innervation within the developing inner ear.

Collagen type II in the otic extracellular matrix. Effect on inner ear development.

Immunocytochemistry was used to demonstrate type II collagen distribution during normal development of the mouse inner ear and in two malformed inner ears. Patterns of inner ear abnormalities and type II collagen distribution were compared between the malformed labyrinth of a mouse mutation (disproportionate micromelia, Dmm) and otic explants exposed to the teratogenic action of an L-proline analog, L-azetidine-2-carboxylic acid (LACA). The results suggest that type II collagen is an important constituent of the developing inner ear's extracellular matrix. Disruptions of the spatial and temporal pattern of collagen type II can adversely affect morphogenesis of the inner ear. A common mechanism of action is postulated for the causation of both the genetic and teratogen-induced inner ear malformations (i.e. disruption of the secretion of collagens to the otic extracellular matrix).

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.