Oridonin employs interleukin 1 receptor type 2 to treat noise-induced hearing loss by blocking inner ear inflammation.

NOD-like receptor protein 3 (NLRP3) inflammasomes trigger the inflammatory cascades and participate in various inflammatory diseases, including noise-induced hearing loss (NIHL) caused by oxidative stress. Recently, the anti-inflammatory traditional medicine oridonin (Ori) has been reported to provide hearing protection in mice after noise exposure by blocking the NLRP3-never in mitosis gene A-related kinase 7 (NEK7)-inflammasome complex assembly. Using RNA sequencing analysis, we further elucidated that interleukin 1 receptor type 2 (IL1R2) may be another crucial factor regulated by Ori to protect NIHL. We observed that IL1R2 expression was localized in spiral ganglion neurons, inner and outer hair cells, in Ori-treated mouse cochleae. Additionally, we confirmed that ectopic overexpression of IL1R2 in the inner ears of healthy mice using an adeno-associated virus delivery system significantly reduced noise-induced ribbon synapse lesions and hearing loss by blocking the "cytokine storm" in the inner ear. This study provides a novel theoretical foundation for guiding the clinical treatment of NIHL.

[Estradiol regulates the expression of plasma membrane Ca2+-ATPase isoform 2 in inner ear of rats].

Objective: To observe the effects of estradiol on expression of plasma membrane Ca2+-ATPase isoform 2 in inner ear of rats. Methods: Twenty-five Three-months-old female Sprague-Dawley rats were randomly and equally divided into five groups by random number table mathod,with five rats in each group. Animals in Sham group were sham-operated while others were bilateral ovariactmized. One month after modeling, the OVX groups were supplemented with estradiol (E2 group), progesterone (P group), estradiol and progesterone (E2+P group)and vehicle sesame oil (Veh group), while the Sham operation group (Sham group) was supplemented with vehicle sesame oil.All rats were sacrificed and otocysts were obtained immediately. Enzyme-linked immunosorbent assay was used to detect the changes in serum estradiol and progesterone levels of each group of rats before operation, before treatment and before sacrifice. Western blot and quantitative real-time polymerase chain reaction were used to detect the expression of total PMCA2 protein and mRNA in the inner ear of each group. Results: There was no significant difference in serum estradiol and progesterone levels among the five groups before operated(P>0.05). Before treatment, the serum estradiol and progesterone levels of rats in each group were significantly lower than those in Sham group (P<0.05). The serum estradiol level in E2 group and E2+P group was not significantly different from that in Sham group (P>0.05), while the serum estradiol level in P group and Veh group was significantly different from that in Sham group (P<0.05). The level of progesterone in P group and E2+P group was higher than that in Sham group (P<0.05), while the level of progesterone in Veh group and E2 group was lower than that in Sham group (P<0.05). Protein and mRNA expression of PMCA2 in P and Veh groups were significantly decreased compared with that of Sham group (P<0.05) while the expression levels underwent no significantly change in E2 and E2+P groups (P<0.05). Conclusion: The decrease of serum estradiol level can reduce the expression of otolith regulatory protein PMCA2 in rats, and then affect otolith metabolism, which may be an important link of estrogen affecting otolith metabolism.

[Effect of melatonin on expression of Prestin protein in the inner ear of mice following radiotherapy].

Objective: To investigate the effect of melatonin on the expression of prestin protein in the inner ear of mice following a single dose radiation therapy, so as to provide the basis for the mechanism study of radiation induced inner ear injury and its prevention. Methods: Sixty 4-week-old male mice were randomly divided into six groups, including the control group (A group), 50 mg/kg MLT group (B group), 5 mg/kg MLT group (C group), 50 mg/kg MLT + radiotherapy group (D group), 5 mg/kg MLT+ radiotherapy group (E group), and 16 Gy radiotherapy group (F group). Each experimental group was randomly subdivided into two subgroups, which were killed to harvest the cochlea on the 3rd and 7th days following 16 Gy radiation. The specimens were used for immunostaining and Western blot to detect the expression of prestin protein. SPSS 19.0 software was used for statistical analysis. Results: Prestin protein mainly distributed in the lateral membrane above the outer hair cell nucleus. When compared with A, B and C group, the expression of prestin protein in the inner ear was significantly up-regulated in F group (P<0.05). However, D and E group reduced the abnormal expression of prestin following radiotherapy when compared with F group, the difference was statistically significant (P<0.05), and the effect of D group was more significant than E group (P<0.05). Conclusions: The prestin protein of cochlea is mainly distributed in the lateral membrane above the outer hair cell nucleus. Following the high-dose radiotherapy, the prestin expression is upregulated, and melatonin can control the abnormal expression of prestin protein induced by radiotherapy with dose dependent.

Gestational vitamin A deficiency: a novel cause of sensorineural hearing loss in the developing world?

Hearing loss is a substantial public health problem with profound social and economic consequences in the developing world. The World Health Organization (WHO) estimates that there are 360 million people living with disabling hearing loss globally, and 80% of these individuals are from low- and middle-income countries. The epidemiology of hearing impairment remains poorly defined in most impoverished societies. Middle ear infections in childhood are a key determinant; however, congenital anomalies may also comprise an important etiology and may arise from gestational malnutrition. While evidence exists that preventable vitamin A deficiency exacerbates the severity of ear infections and, consequently, hearing loss, antenatal vitamin A deficiency during sensitive periods of fetal development may represent an etiologically distinct and virtually unexplored causal pathway. Evidence from multiple animal systems clearly shows that fetal inner ear development requires adequate vitamin A nutriture to proceed normally. Inner ear malformations occur in experimentally imposed maternal vitamin A deficiency in multiple species in a dose-response manner. These anomalies are likely due to the loss of retinoic acid-dependent regulation of both hindbrain development and otic morphogenic processes. Based on in vivo evidence in experimental animals, we hypothesize that preventable gestational vitamin A deficiency, especially during early stages of fetal development, may predispose offspring to inner ear malformations and sensorineural hearing loss. As vitamin A deficiency affects an estimated 20 million pregnant women globally, we hypothesize that, in undernourished settings, routine provision of supplemental vitamin A at the recommended allowance throughout pregnancy may promote normal inner ear development and reduce risk of an as yet unknown fraction of sensorineural hearing loss. If our hypothesis proves correct, gestational vitamin A deficiency would represent a potentially preventable etiology of sensorineural hearing loss of substantial public health significance.

Usher proteins in inner ear structure and function.

Usher syndrome (USH) is a neurosensory disorder affecting both hearing and vision in humans. Linkage studies of families of USH patients, studies in animals, and characterization of purified proteins have provided insight into the molecular mechanisms of hearing. To date, 11 USH proteins have been identified, and evidence suggests that all of them are crucial for the function of the mechanosensory cells of the inner ear, the hair cells. Most USH proteins are localized to the stereocilia of the hair cells, where mechano-electrical transduction (MET) of sound-induced vibrations occurs. Therefore, elucidation of the functions of USH proteins in the stereocilia is a prerequisite to understanding the exact mechanisms of MET.

[Otosclerosis- <> or the loss of the natural variant of inertness (<> of the labyrinth capsule)].

The author suggests an original hypothesis of otosclerosis based on the analyses of the literature publications for many years and his personal clinical observations. The normal labyrinth capsule is considered to be bradytrophic, i.e. inert and showing an extremely low level of metabolic processes. The disturbance of bradytrophicity under the action of individual factors and/or especially their combination make it involved in the maintenance of calcium homeostasis in the body. The validity of this conjecture is confirmed by the results of histological investigations, viz. the appearance of diquide or xplasma-like, bone in the labyrinth of the patients suffering otosclerosis. Such bone resorption is known to occur in other parts of the bony skeletontoo and should be regarded as a normal physiological process contributing to the replenishment of blood calcium deficiency.The subsequent reorganization (remodeling) of any part of the bony skeleton is physiologically neutral. In the labyrinth capsule,with its small size and delicate structure, such reorganization induces the otosclerotic process responsible for dysfunction of the membranaceous labyrinth. The surgical treatment of the patients presenting with otosclerosis should be supplemented by conservative treatment intended to slow down the otosclerotic reorganization and to restore bradytrophicity of the labyrinth capsule.

Intratympanic dexamethasone as initial therapy for idiopathic sudden sensorineural hearing loss: Clinical evaluation and laboratory investigation.

OBJECTIVE: To evaluate the effect of intratympanic dexamethasone (ITD) as initial therapy for idiopathic sudden sensorineural hearing loss (ISSHL) as well as to determine the concentration-dependent time course distribution of dexamethasone in the inner ear. METHODS: Sixty-six patients with profound ISSHL were included. Twenty-two were treated with ITD and the rest as control. Audiograms were performed before the treatment and one month afterwards. In the animal study, dexamethasone of different concentrations (5, 10 and 20mg/ml) was injected into the tympanums of three groups of SD rats (Groups A, B and C), their inner ears dissected free at various postinjection survival intervals. Immunofluorescence was applied to detect the locations of dexamethasone. RESULTS: The overall rate of good prognosis was 77.27% in ITD group, which was not significantly different from 81.82% in the control group. In the animal study, the higher local concentration and longer lasting period was found in Groups B and C. CONCLUSIONS: ITD at 5mg/ml did not add effect to systemic steroids in improving hearing outcomes in patients with ISSHL. An increase in dexamethasone concentration led to large variations in pharmacokinetics in animal study, showing potential value in optimizing the drug delivery protocols and improving the therapeutic results.

Distribution of androgen receptor mRNA expression in vocal, auditory, and neuroendocrine circuits in a teleost fish.

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive-related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context-dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain-spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase-containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general.

Middle ear application of a sodium hyaluronate gel loaded with neomycin in a Guinea pig model.

OBJECTIVE: Establishing methods for topical administration of drugs to the inner ear have great clinical relevance and potential even in a relatively short perspective. To evaluate the efficacy of sodium hyaluronate (HYA) as a vehicle for drugs that could be used for treatment of inner ear disorders. METHODS: The cochlear hair cell loss and round window membrane (RWM) morphology were investigated after topical application of neomycin and HYA into the middle ear. Sixty-five albino guinea pigs were used and divided into groups depending on the type of the treatment. Neomycin was chosen as tracer for drug release and pharmacodynamic effect. HYA loaded with 3 different concentrations of neomycin was injected to the middle ear cavity of guinea pigs. Phalloidin stained surface preparations of the organ of Corti were used to estimate hair cell loss induced by neomycin. The thickness of the midportion of the RWM was measured and compared with that of controls using light and electron microscopy. All animal procedures were pe rformed in accordance with the ethical standards of Karolinska Institutet. RESULT: Neomycin induced a considerable hair cell loss in guinea pigs receiving a middle ear injection of HYA loaded with the drug, demonstrating that neomycin was released from the gel and delivered to the inner ear. The resulting hair cell loss showed a clear dose-dependence. Only small differences in hair cell loss were noted between animals receiving neomycin solution and animals exposed to neomycin in HYA suggesting that the vehicle neither facilitated nor hindered drug transport between the middle ear cavity and the inner ear. One week after topical application, the thickness of the RWM had increased and was dependent upon the concentration of neomycin administered to the middle ear. At 4 weeks the thickness of the RWM had returned to normal. CONCLUSION: HYA is a safe vehicle for drugs aimed to pass into the inner ear through the RWM. Neomycin was released from HYA and transported into the inner ear as evidenced by hair cell loss.

Ascorbic acid reduces noise-induced nitric oxide production in the guinea pig ear.

OBJECTIVES: Noise-induced hearing loss can be caused, among other causes, by increased nitric oxide (NO) production in the inner ear leading to nitroactive stress and cell destruction. Some studies in the literature suggest that the degree of hearing loss (HL) could be reduced in an animal model through ascorbic acid supplementation. To identify the effect of ascorbic acid on tissue-dependent NO content in the inner ear of the guinea pig, we determined the local NO production in the organ of Corti and the lateral wall separately 6 hours after noise exposure. STUDY DESIGN: Prospective animal study in guinea pigs. METHODS: Over a period of 7 days, male guinea pigs were supplied with minimum (25 mg/kg body weight/day) and maximum (525 mg/kg body weight/day) ascorbic acid doses, and afterwards exposed to noise (90 dB sound pressure level for 1 hour). The acoustic-evoked potentials were recorded before and after noise exposure. The organ of Corti and the lateral wall were incubated differently for 6 hours in culture medium, and the degree of NO production was determined by chemiluminescence. RESULTS: Ascorbic acid treatment reduced the hearing threshold shift after noise exposure depending on concentration. When the maximum ascorbic acid dose was substituted, NO production was significantly reduced in the lateral wall after noise exposure and slightly reduced in the organ of Corti. CONCLUSIONS: Oral supplementation of the natural radical scavenger ascorbic acid reduces the NO-production rate in the inner ear in noisy conditions. This finding supports the concept of inner ear protection by ascorbic acid supplementation.

Hypoxic changes in the central nervous system of noise-exposed mice.

CONCLUSION: After a noise-induced transient threshold shift, hypoxia occurred in the central nervous system, especially in the auditory cortex, the hippocampus, and the inferior colliculus. OBJECTIVES: Noise-induced inner ear hypoxia was shown by measurement of an increase in hypoxia-inducible factor-1 alpha, which is expressed? in the nucleus under hypoxic conditions. This study uses pimonidazole to localize site-specific hypoxic changes occurring in the mouse central auditory pathway during noise-induced auditory threshold shift. METHOD: BALB/c hybrid mice with normal hearing were exposed to 122 dB SPL white noise for 3 h. Immediately after exposure to the noise, and 7 d after noise exposure, the brains of mice were collected. Brains were cryosectioned into slices 15 microm thick and examined by immunofluorescence after staining with pimonidazole HCl. RESULTS: After 3 h of exposure to 120 dB SPL noise, the hearing thresholds of mice decreased to 51.1+/-8.6 dB SPL (n =14), but hearing recovered in 7 d. After noise exposure, pimonidazole signal increased in the auditory cortex, the hippocampus, and the inferior colliculus. The pimonidazole signal remained elevated after 7 d. In control mice, pimonidazole did not stain any brain region.

Early regionalization of the otic placode and its regulation by the Notch signaling pathway.

Otic neuronal precursors are the first cells to be specified and do so in the anterior domain of the otic placode, the proneural domain. In the present study, we have explored the early events of otic proneural regionalization in relation to the activity of the Notch signaling pathway. The proneural domain was characterized by the expression of Sox3, Fgf10 and members of the Notch pathway such as Delta1, Hes5 and Lunatic Fringe. The complementary non-neural domain expressed two patterning genes, Lmx1b and Iroquois1, and the members of the Notch pathway, Serrate1 and Hairy1. Fate map studies and double injections with DiI/DiO showed that labeled cells remained confined to anterior or posterior territories with limited cell intermingling. To explore whether Notch signaling pathway plays a role in the initial regionalization of the otic placode, Notch activity was blocked by a gamma-secretase inhibitor (DAPT). Notch blockade induced the expansion of non-neural genes, Lmx1 and Iroquois1, into the proneural domain. Combined gene expression and DiI experiments showed that these effects were not due to migration of non-neural cells into the proneural domain, suggesting that Notch activity regulates the expression of non-neural genes. This was further confirmed by the electroporation of a dominant-negative form of the Mastermind-like1 gene that caused the up-regulation of Lmx1 within the proneural domain. In addition, Notch pathway was involved in neuronal precursor selection, probably by a classical mechanism of lateral inhibition. We propose that the regionalization of the otic domain into a proneural and a non-neural territory is a very early event in otic development, and that Notch signaling activity is required to exclude the expression of non-neural genes from the proneural territory.

[Preliminary study on brain-targeted drug delivery via inner ear].

The article investigates the feasibility of delivering drugs to brain via inner ear, and provides a novel route for delivering drugs to the brain tissues. Dexamethasone acetate (DA)-loaded solid lipid nanoparticles (SLN) was prepared by using Compritol 888 ATO as material. HPLC assays for the determination of DA, dexamethasone sodium phosphate (DSP) and dexamethasone (Dex) were developed, separately. DA-loaded SLN and DSP solution were administered after intratympanic injection (IT) or intravenous injection (IV). Perilymph ( PL) and cerebrospinal fluid (CSF) were collected periodically. The concentrations in PL and CSF were measured by HPLC, and used to estimate pharmacokinetic parameters of Dex in CSF. The AUC of Dex in CSF following IT DA-loaded SLN or DSP solution were respectively 2.5 and 4.3-fold higher than those following IV. After IT, DA-loaded SLN increased the AUC by 13 times and extended the MRT by 19 times, compared with the solution. Moreover, the AUC of Dex in PL following IT the SLN was 76% lower than that following IT the solution. Intra-cochlear administration shows great potential and offers a promising alternative to brain-targeted drug delivery.

Effects of antioxidants on the aging inner ear.

Age-related cochlear structural changes include the degeneration of sensory, neural cells and the stria vascularis. The hypothesis that cellular degeneration results from exposure to oxidative products of respiration was tested by supplementing aged dogs with a diet high in antioxidants and mitochondrial metabolites and by genetically modifying the expression level of the antioxidant, manganese superoxide dismutase (SOD2) in mice. Aged dogs received either a high antioxidant diet or a normal, control diet for the last 3 years of their life. Cellular measures were compared among the two aged groups (10-15 years) and young dogs. Both aged groups had cellular degeneration relative to young dogs, but the animals fed the antioxidant diet showed less degeneration at the base and apex than the control-diet group. Transgenic mice, heterozygous null for SOD2, produce only half as much enzyme as a normal mouse. These mice showed no increase in the amount of hearing loss relative to the background strain. A diet containing antioxidants reduced the magnitude of cochlear degeneration. Genetic reduction of one antioxidant, however, did not increase the magnitude of hearing loss in aging mice. A reduction in one enzyme seems to be compensated while the addition of a complex of factors is effective.

Optimization of ribonucleic acid detection from archival Guinea pig temporal bone specimens.

HYPOTHESIS: The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. BACKGROUND: A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. METHODS: Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. RESULTS: Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. CONCLUSION: Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Complementary expression patterns of retinoid acid-synthesizing and -metabolizing enzymes in pre-natal mouse inner ear structures.

Retinoic acid (RA) plays a pivotal role in patterning and differentiation of the embryonic inner ear. Despite its documented effects during embryonic development, the cellular sites that synthesize or metabolize RA in the inner ear have yet to be determined. Here we describe the distribution of three synthesizing enzymes, retinaldehyde dehydrogenases 1, 2 and 3 (RALDH1, RALDH2 and RALDH3) and two catabolizing enzymes (CYP26A1 and CYP26B1) in the mouse inner ear at embryonic day 18.5 when active cell differentiation is underway. Two detection methods, radioactive and non-radioactive in situ hybridization, were employed to elucidate the tissue distribution and cellular localization of these enzymes, respectively. All of the five enzymes examined, with the exception of CYP26A1, were expressed in both vestibular and cochlear end organs. While expression of the three RALDHs was observed in various cell types, CYP26B1 expression was found only in supporting cells of the vestibular and cochlear end organs. In the cochlea, expression domains of RALDH1-3 and CYP26B1 were complementary to one another. These results reveal specific tissue- and cellular expression patterns of RA synthesizing and catabolizing enzymes in the pre-natal inner ear, and suggest that a precise control of RA concentrations in various cell types of the inner ear is achieved by the balance between RALDHs and CYP26B1 activities.

Identification and characterization of choline transporter-like protein 2, an inner ear glycoprotein of 68 and 72 kDa that is the target of antibody-induced hearing loss.

The Kresge Hearing Research Institute-3 (KHRI-3) antibody binds to a guinea pig inner ear supporting cell antigen (IESCA) and causes hearing loss. To gain insight into the mechanism of antibody-induced hearing loss, we used antibody immunoaffinity purification to isolate the IESCA, which was then sequenced by mass spectroscopy, revealing 10 guinea pig peptides identical to sequences in human choline transporter-like protein 2 (CTL2). Full-length CTL2 cDNA sequenced from guinea pig inner ear has 85.9% identity with the human cDNA. Consistent with its expression on the surface of supporting cells in the inner ear, CTL2 contains 10 predicted membrane-spanning regions with multiple N-glycosylation sites. The 68 and 72 kDa molecular forms of inner ear CTL2 are distinguished by sialic acid modification of the carbohydrate. The KHRI-3 antibody binds to an N-linked carbohydrate on CTL2 and presumably damages the organ of Corti by blocking the transporter function of this molecule. CTL2 mRNA and protein are abundantly expressed in human inner ear. Sera from patients with autoimmune hearing loss bind to guinea pig inner ear with the same pattern as CTL2 antibodies. Thus, CTL2 is a possible target of autoimmune hearing loss in humans.

Accumulation of hypoxia-inducible factor-1alpha in mouse inner ear by noise stimulation.

Exposure of mice to a 120 dB SPL broad band click sound for 3 h per day induced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in the organ of Corti, lateral wall tissue and spiral ganglion cells. In the organ of Corti, HIF-1alpha was expressed in inner and outer hair cells, pillar cells and Deiters' cells, but not in Hensen's cells or the tectorial, Reissner's or basement membrane. HIF-1alpha expression was definitely increased in mice with a permanent threshold shift, where the hearing level did not recover, even after 4 weeks. These results suggest that noise may induce tissue hypoxia in the inner ear and that the further study on the role of HIF-1alpha will be needed.

Cyp26C1 encodes a novel retinoic acid-metabolizing enzyme expressed in the hindbrain, inner ear, first branchial arch and tooth buds during murine development.

Retinoic acid (RA), an active metabolite of vitamin A, is a crucial signaling molecule involved in tissue morphogenesis during embryonic development. RA distribution and concentration is precisely regulated during embryogenesis by balanced complementary activities of RA synthesizing (RALDH) and metabolizing (CYP26) enzymes. Here, we describe the identification of a novel murine p450 cytochrome belonging to the CYP26 family, mCYP26C1. Sequence alignment show that mCYP26C1 is more closely related to mCYP26B1 than mCYP26A1. At early developmental stages (E8.0-E8.5), mCyp26C1 is expressed in prospective rhombomeres 2 and 4, in the first branchial arch and along the lateral surface mesenchyme adjacent to the rostral hindbrain. At E9.5, mCyp26C1 expression persists in rhombomere 2 and in the maxillary and mandibular components of the first branchial arch, and is strongly induced in the lateral cervical mesenchyme. By mid-gestation, mCyp26C1 is weakly expressed in the cervical mesenchyme and in the maxillary component of the first branchial arch. At E11.5, mCyp26C1 can only be seen in a narrow band in the lateral cervical mesenchyme. During late gestation, mCyp26C1 exhibits region-specific expression in the inner ear epithelium and a persistent expression in the inner dental epithelium of the developing teeth. This pattern of expression suggests that mCYP26C1 may play an important role in protecting the hindbrain, first branchial arch, otocyst and tooth buds against RA exposure during embryonic development.

Retinaldehyde dehydrogenase 2 (RALDH2)- independent patterns of retinoic acid synthesis in the mouse embryo.

Knockout of the murine retinoic acid (RA)-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2) gene leads to early morphogenetic defects and embryonic lethality. Using a RA-responsive reporter transgene, we have looked for RA-generating activities in Raldh2-null mouse embryos and investigated whether these activities could be ascribed to the other known RALDH enzymes (RALDH1 and RALDH3). To this end, the early defects of Raldh2(-/-) embryos were rescued through maternal dietary RA supplementation under conditions that do not interfere with the activity of the reporter transgene in WT embryos. We show that RALDH2 is responsible for most of the patterns of reporter transgene activity in the spinal cord and trunk mesodermal derivatives. However, reporter transgene activity was selectively detected in Raldh2(-/-) embryos within the mesonephric area that expresses RALDH3 and in medial-ventral cells of the spinal cord and posterior hindbrain, up to the level of the fifth rhombomere. The craniofacial patterns of RA-reporter activity were unaltered in Raldh2(-/-) mutants. Although these patterns correlated with the presence of Raldh1 andor Raldh3 transcripts in eye, nasal, and inner ear epithelia, no such correlation was found within forebrain neuroepithelium. These data suggest the existence of additional RA-generating activities in the differentiating forebrain, hindbrain, and spinal cord, which, along with RALDH1 and RALDH3, may account for the development of Raldh2(-/-) mutants once these have been rescued for early lethality.