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Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0-5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

Background: A large number of researches related to auricle development have been conducted in different countries, but there were few similar studies in China, especially in Chinese children. This study was designed to investigate the auricular development by measuring normal age- and sex-related dimensions of auricles in Chinese children., Methods: A total of 900 participants were evaluated according to their age and gender. From the landmarks, the dimensions of the and tragus including length and width, the relevant indices, inclination angles were measured. The average score of each item was calculated according to age and gender., Results: The dimensions of the and tragus including width and length showed certain developmental patterns respectively. No significantly difference was found for the inclination angles and relative indices between different age groups and different sides., Conclusions: The data indicated that there was a certain pattern of auricular development. There were also gender difference in the development of auricle. There were certain proportional relationships between different subunits of the auricle. These results may be useful for designing the plan of auricular reconstruction.

Background: The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability., Results: Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period., Conclusions: This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality., (© 2020 Wiley Periodicals, Inc.)

Supramolecular polymers self-assemble into nanofibers, micelles, and other nanostructures through weak noncovalent interactions between subunits. Such systems possess attractive properties for use in a variety of practical settings such as energy, sustainability, and healthcare. In regenerative medicine, a common approach involves implanting a supramolecular material containing cell and growth factor binding motifs directly into a diseased or traumatized tissue defect, whereupon it interacts with and/or recruits components of the biological system to induce tissue healing. Here we introduce a supramolecular therapeutic in which tissue regeneration is orchestrated by a supramolecular polymer prodrug implanted subcutaneously in a remote tissue. Our approach exploits a hydrophobic small-molecule inhibitor of prolyl hydroxylase enzyme as both a regeneration-inducing therapeutic and a structure-directing agent in a supramolecular polymer that forms shear-thinning nanofiber hydrogels. Subcutaneous injection of the supramolecular hydrogel in the back of mice wounded with a critical-sized defect in the ear led to transient upregulation of hypoxia inducible factor-1α and regeneration of ear tissue in a manner reminiscent of epimorphic regeneration. This drug-induced regeneration strategy utilizes a simple and translatable supramolecular design, eliminates the need for delivery of biologics ( e. g., growth factors, cells), and avoids implantation of a foreign material directly in a tissue defect.

Considerable diversity exists in porcine ear size, which is an important morphological feature of pig breeds. Previously, we localized four crucial candidate genes-high mobility group AT-hook 2 (HMGA2), LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3) and Wnt inhibitory factor 1 (WIF1)-on Sus Scrofa chromosome 5 affecting porcine ear size, then cloned LEMD3 and MSBR3. In this study, we performed rapid amplification of cDNA ends to obtain full-length cDNA sequences of 2338-bp WIF1 and 2998-bp HMGA2. Using quantitative real-time PCR, we revealed that WIF1 expression was highest in ear cartilage of 60-day-old pigs and that this is therefore a better candidate gene for ear size than HMGA2. We further screened coding sequence variants in both genes and identified only one missense mutation (WIF1:c.1167C>G) in a conserved epidermal growth factor-like domain from the mammalian WIF1 protein. The protein-altering mutation was significantly associated with ear size across the Large White × Minzhu hybrid and Beijing Black pig populations. When WIF1:c.1167C>G was included as fixed effect in the model to re-run a genome-wide association study in the Large White × Minzhu intercross population the P-value of the peak SNP on SSC5 from re-running the genome-wide association study dropped from 2.45E-12 to 7.33E-05. Taken together, the WIF1:c.1167C>G could be an important mutation associated with ear size. Our findings provide helpful information for further studies of the molecular mechanisms controlling porcine ear size., (© 2019 Stichting International Foundation for Animal Genetics.)

Background: The size and type of ears are important conformation characteristics that distinguish pig breeds. A significant quantitative trait locus (QTL) for ear size has been identified on SSC5 (SSC for Sus scrofa chromosome) but the underlying causative gene and mutation remain unknown. Thus, our aim was to identify the gene responsible for enlarged ears in pig., Results: First, we narrowed down the QTL region on SSC5 to a 137.85-kb interval that harbors only the methionine sulfoxide reductase B3 (MSRB3) gene. Then, we identified a 38.7-kb copy number variation (CNV) that affects the last two exons of MSRB3 and could be the candidate causative mutation for this QTL. This CNV showed complete concordance with genotype at the QTL of the founder animals in a white Duroc × Erhualian F 2 intercross and was found only in pigs from six Chinese indigenous breeds with large ears and from the Landrace breed with half-floppy ears. Moreover, it accounted for the significant association with ear size on SSC5 across the five pig populations tested. eQTL mapping revealed that this CNV was significantly associated with the expression of the microRNA (miRNA) miR-584-5p, which interacts with MSRB3, one of its target genes. In vivo and in vitro experiments confirmed that miR-584-5p inhibits the translation of MSRB3 mRNA. Taken together, these results led us to conclude that presence of the 38.7-kb CNV in the genome of some pig breeds affects ear size by altering the expression of miR-584-5p, which consequently hinders the expression of one of its target genes (e.g. MSRB3)., Conclusions: Our findings shed insight into the underlying mechanism of development of external ears in mammals and contribute to a better understanding of how the presence of CNV can regulate gene expression.

The ectodysplasinA receptor gene (EDAR) plays an important role in the development of ectoderm. The derived G allele of its key missense variant EDARV370A is prevalent in East Asians and Americans, but rare in Africans and Europeans. This leads to distinct ectodermal-derived phenotypes between different continental groups, such as the straighter and thicker hair, more eccrine sweat glands, feminine smaller breasts, shovel incisors characteristic of East Asians. At present, we know little about the association between EDARV370A and facial and ear morphology characteristics. To better understand the effect of EDARV370A on craniofacial phenotypes, we systematically examined the association between EDARV370A and 136 facial quantitative phenotypes, one chin ordinal phenotype and six ear ordinal phenotypes in 715 Uyghurs. The quantitative phenotypes were derived by applying our automated landmark annotation method to facial 3D photos and the ordinal phenotypes were manually graded from facial 2D photos. The analysis identified significant association (P<0.05 after multiple testing correction) between EDARV370A and eight facial phenotypes, one chin phenotype and three ear morphology phenotypes. Our study thus elucidated the pleotropic effect of EDARV370A on craniofacial phenotypes in a European-Asian admixed Uyghur population.

The ear can be subdivided into three distinct parts, each with significantly distinct structural and functional differences, the outer, middle, and inner ear, the latter housing the specialized sensory hair cells that act as transducers. There are numerous manuscripts documenting the anatomical development of the inner, middle, and outer ear in humans, rodents, chick, and zebrafish, dating back to the early 20th Century, and these developmental processes of these components are further compared in a number of review articles (Anthwal & Thompson, ; Basch, Brown, Jen, & Groves, ; Sai & Ladher, ). This article presents a review of both pre- and postnatal development of the inner ear, discusses recent molecular genetic advances toward our understanding of hair cells responsible for the sensory functions of the inner ear. Finally, a survey of comparative ear biology is used to pull together our understanding of the species differences, similarities, and key time points of definitive organ development of the ear., (© 2017 Wiley Periodicals, Inc.)

The current paper is a continuation of our work described in Rot and Kablar, 2010. Here, we show lists of 10 up- and 87 down-regulated genes obtained by a cDNA microarray analysis that compared developing Myf5-/-:Myod-/- (and Mrf4-/-) petrous part of the temporal bone, containing middle and inner ear, to the control, at embryonic day 18.5. Myf5-/-:Myod-/- fetuses entirely lack skeletal myoblasts and muscles. They are unable to move their head, which interferes with the perception of angular acceleration. Previously, we showed that the inner ear areas most affected in Myf5-/-:Myod-/- fetuses were the vestibular cristae ampullaris, sensitive to angular acceleration. Our finding that the type I hair cells were absent in the mutants' cristae was further used here to identify a profile of genes specific to the lacking cell type. Microarrays followed by a detailed consultation of web-accessible mouse databases allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have associated inner ear abnormalities. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X-linked deafness in humans. Our current study suggests an epigenetic role that mechanical, and potentially other, stimuli originating from muscle, play in organogenesis, and offers an approach to finding novel genes responsible for altered inner ear phenotypes.

Porcine ear size is an important characteristic for distinguishing among pig breeds. In a previous genome-wide association study of porcine ear size, LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3), high mobility group AT-hook 2 (HMGA2), and Wnt inhibitory factor 1 (WIF1) were implicated as important candidate genes for ear size. This study investigated the expression levels of four candidate genes for ear size in Erhualian and Large White pigs. Ten Erhualian pigs with large ears and eight Large White pigs with small ears at 60 days of age were examined. The mRNA expression levels of the four candidate genes were quantified by real-time polymerase chain reaction. WIF1 mRNA expression was significantly higher in Large White than in Erhualian pigs (P < 0.05), whereas the expression levels of the other three genes were not significantly different between the two breeds. The protein expression levels of the four genes were analyzed using western blot. WIF1 protein expression was significantly higher in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 protein expression was significantly higher in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 is the main causal gene for ear size in pigs.

Background: With the advent of computer-assisted three-dimensional surface imaging and rapid data processing, oral and maxillofacial surgeons and orthodontists are enabled to analyze facial growth three dimensionally. Normative data, however, are still rare and inconsistent. The aim of the present study was to establish a valid reference system and to give normative data for facial growth., Methods: Three-dimensional facial surface images were obtained from 344 healthy Caucasian children (aged 0 to 7 years). The images were put in correspondence by means of six landmarks close to the skull base (exocanthion, endocanthion, otobasion inferius). Growth curves for 21 landmarks were estimated in the three dimensions., Results: Facial regions close to the skull base (orbit and ear) showed a biphasic growth pattern, with accelerated growth during the first year of life that subsided to a decreased and linear velocity thereafter. Landmarks on the nose, lips, and chin demonstrated either a curvilinear or a linear growth pattern., Conclusions: The rapid increase of the orbit and ear region in infancy is a secondary phenomenon to the rapid growth of the neurocranium during the first year of life. Thereafter, maxillary and mandibular growth prevails. The present study gives three-dimensional normative data for an expanded growth span between birth and childhood.

The ability of adult and subadult piebald shrews (Diplomesodon pulchellum) to produce 160Hz seismic waves is potentially reflected in their vocal ontogeny and ear morphology. In this study, the ontogeny of call variables and body traits was examined in 11 litters of piebald shrews, in two-day intervals from birth to 22 days (subadult), and ear structure was investigated in two specimens using micro-computed tomography (micro-CT). Across ages, the call fundamental frequency (f0) was stable in squeaks and clicks and increased steadily in screeches, representing an unusual, non-descending ontogenetic pathway of f0. The rate of the deep sinusoidal modulation (pulse rate) of screeches increased from 75Hz at 3-4 days to 138Hz at 21-22 days, probably relating to ontogenetic changes in contraction rates of the same muscles which are responsible for generating seismic vibrations. The ear reconstructions revealed that the morphologies of the middle and inner ears of the piebald shrew are very similar to those of the common shrew (Sorex araneus) and the lesser white-toothed shrew (Crocidura suaveolens), which are not known to produce seismic signals. These results suggest that piebald shrews use a mechanism other than hearing for perceiving seismic vibrations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Neurosensory hearing loss is a growing problem of super-aged societies. Cochlear implants can restore some hearing, but rebuilding a lost hearing organ would be superior. Research has discovered many cellular and molecular steps to develop a hearing organ but translating those insights into hearing organ restoration remains unclear. For example, we cannot make various hair cell types and arrange them into their specific patterns surrounded by the right type of supporting cells in the right numbers. Our overview of the topologically highly organized and functionally diversified cellular mosaic of the mammalian hearing organ highlights what is known and unknown about its development. Following this analysis, we suggest critical steps to guide future attempts toward restoration of a functional organ of Corti. We argue that generating mutant mouse lines that mimic human pathology to fine-tune attempts toward long-term functional restoration are needed to go beyond the hope generated by restoring single hair cells in postnatal sensory epithelia., (© 2015 WILEY Periodicals, Inc.)

Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Background: The otic placode comprises the progenitors of the inner ear and the neurons that convey hearing and balance information to the brain. Transplantation studies in birds and amphibians demonstrate that when the otic placode is morphologically visible as a thickened patch of ectoderm, it is first committed to an otic fate. Fibroblast growth factor (FGF) signaling initiates induction of the otic placode, and levels of FGF signaling are fine-tuned by the Sprouty family of antagonists of receptor tyrosine kinase signaling., Results: Here, we examined the size of the otic placode and cup by combinatorial inactivation of the Sprouty1 and Sprouty2 genes. Interestingly, in a Sprouty gene dosage series, early enlargement of the otic placode was progressively restored to normal. Restoration of otic size was preceded by normal levels of FGF signaling, reduced cell proliferation and reduced cell death., Conclusions: Our study demonstrates that excess otic placode cells, which form in response to increased FGF signaling, are not maintained in mammals. This suggests that growth plasticity exists in the mammalian otic placode and cup, and that FGF signaling may not be sufficient to induce the genetic program that maintains otic fate., (Copyright © 2014 Wiley Periodicals, Inc.)

Handling (H) and cross-fostering (CF) rodent pups during postnatal development triggers changes in maternal behavior which in turn trigger long-term physiological changes in the offspring. However, less is known about the short-term effects of H and CF on infant development. In this study we hypothesized that manipulations of maternal care affect the onset of hearing in Wistar rats. To test this hypothesis we obtained auditory brainstem responses (ABRs) and micro-CT x-ray scans to measure changes in the development of the auditory periphery in H and CF pups manipulated at postnatal day (P)1, P5, or P9. We found evidence of changes in hearing development in H and CF pups compared with naive pups, including changes in the percentage of animals with ABRs during development, a decrease in ABR thresholds between P13 and P15, and anatomical results consistent with an accelerated formation of the middle ear cavity and opening of the ear canal. Biochemical measurements showed elevated levels of thyroid hormone in plasma from naive and CF pups. These results provide evidence that manipulations of maternal care accelerate hearing onset in Wistar rats. Understanding the mechanisms by which maternal care affects hearing onset opens new opportunities to study experience-dependent development of mammalian hearing.

The present study investigated whether a recloning procedure would affect the reproductive performance or the germline transmission capacity of recloned transgenic pigs. This study has also laid the foundation for the development of elite transgenic swine breeds in the future. Recloned transgenic pigs were developed from ear tissue fibroblasts of primary transgenic cloned pigs using a recloning procedure, and their reproductive performance and exogenous gene transmission were analyzed. Two transgenic cell lines with different genetic backgrounds (derived from a female miniature pig and a male Landrace pig) with stable expression of green fluorescent protein (GFP) were established successfully. Furthermore, recloned transgenic embryos were developed to full term successfully. One female Chinese experimental miniature piglet (CEMP) (GFP+) and three male Landrace piglets (GFP+) were delivered naturally. Furthermore, the index values for the reproductive characteristics of the recloned transgenic pigs, such as puberty, gestation period, sperm volume and sperm concentration, were not significantly different from those of conventionally bred pigs. In addition, 53% of the F1 offspring of the recloned transgenic pigs were GFP positive. These results demonstrate that ear tissue fibroblasts from primary transgenic cloned pigs efficiently support the full-term development of recloned transgenic embryos. Furthermore, recloned transgenic pigs maintain normal reproductive performance and stable germline (genetic) transmission capacities.

Peroxisome proliferator-activated receptor beta/delta (PPARD) is a crucial and multifaceted determinant of diverse biological functions including lipid metabolism, embryonic development, inflammatory response, wound healing and cancer. Recently, we proposed a novel function of porcine PPARD (sPPARD) in external ear development. A missense mutation (G32E) in an evolutionary conservative domain of sPPARD remarkably increases external ear size in pigs. Here, we investigated the underlying molecular mechanism of the causal mutation at the cellular level. Using a luciferase reporter system, we showed that the G32E substitution reduced transcription activity of sPPARD in a ligand-dependent manner. By comparison of the subcellular localization of wild-type and mutated sPPARD in both PK-15 cells and pinna cartilage-derived primary chondrocytes, we found that the G32E substitution promoted CRM-1 mediated nuclear exportation of sPPARD. With the surface plasmon resonance technology, we further revealed that the G32E substitution had negligible effect on its ligand binding affinity. Finally, we used co-immunoprecipitation and luciferase reporter assays to show that the G32E substitution greatly reduced ubiquitination level by blocking ubiquitination of the crucial A/B domain and consequently decreased transcription activity of sPPARD. Taken together, our findings strongly support that G32E is a functional variant that plays a key role in biological activity of sPPARD, which advances our understanding of the underlying mechanism of sPPARD G32E for ear size in pigs.

Conclusions: The changes in the cochlin isoforms in the perilymph may provide important insights to the understanding of cochlin function and the pathogenesis of related inner ear diseases., Objectives: Cochlin is involved in various pathologies of the inner ear. Altered levels of cochlin isoforms in developing inner ear tissue were reported previously. The purpose of this study was to elucidate the cochlin isoform expression in the perilymph of rats during postnatal development in relation to Coch gene mRNA expression., Methods: We studied the cochlin isoforms in the rat perilymph during postnatal development by Western blot analysis. Real-time PCR was also performed to elucidate the expression level of Coch mRNA in the developing inner ear of rats., Results: Western blot analysis showed that the expression of p63s in the perilymph was highest on the 12th day after birth (DAB12), the earliest age at which we could identify the perilymphatic space microscopically, and it decreased gradually as the cochlea developed. On the other hand, the expression of Cochlin-tomoprotein (CTP)was lowest on DAB12 and increased gradually up to DAB24. COCH mRNA was detected from DAB3 and gradually increased to DAB15, and then gradually decreased to DAB70.

Background: Ear size and shape are distinct conformation characteristics of pig breeds. Previously, we identified a significant quantitative trait locus (QTL) influencing ear surface on pig chromosome 5 in a White Duroc×Erhualian F2 resource population. This QTL explained more than 17% of the phenotypic variance., Methods: Four new markers on pig chromosome 5 were genotyped across this F2 population. RT-PCR was performed to obtain expression profiles of different candidate genes in ear tissue. Standard association test, marker-assisted association test and F-drop test were applied to determine the effects of single nucleotide polymorphisms (SNP) on ear size. Three synthetic commercial lines were also used for the association test., Results: We refined the QTL to an 8.7-cM interval and identified three positional candidate genes i.e. HMGA2, SOX5 and PTHLH that are expressed in ear tissue. Seven SNP within these three candidate genes were selected and genotyped in the F2 population. Of the seven SNP, HMGA2 SNP (JF748727: g.2836 A>G) showed the strongest association with ear size in the standard association test and marker-assisted association test. With the F-drop test, F value decreased by more than 97% only when the genotypes of HMGA2 g.2836 A>G were included as a fixed effect. Furthermore, the significant association between g.2836 A>G and ear size was also demonstrated in the synthetic commercial Sutai pig line. The haplotype-based association test showed that the phenotypic variance explained by HMGA2 was similar to that explained by the QTL and at a much higher level than by SOX5. More interestingly, HMGA2 is also located within the dog orthologous chromosome region, which has been shown to be associated with ear type and size., Conclusions: HMGA2 was the closest gene with a potential functional effect to the QTL or marker for ear size on chromosome 5. This study will contribute to identify the causative gene and mutation underlying this QTL.

Objective: The aim of this study was to investigate the maturational time course of the occlusion effect in infants with normal hearing. The objectives were (i) to investigate the occlusion effect in a larger group of young infants, (ii) to determine whether the occlusion effect is seen in bone conduction auditory steady state responses (ASSRs) for older infants, and (iii) to investigate the mechanisms that underlie bone conduction hearing in unoccluded and occluded ears in infants by measuring sound pressure in the ear canal., Design: Experiments 1A and 1B: The SPL in the ear canal to 500, 1000, and 2000 Hz bone-conducted pure tones were compared in 22 young infants (0-7 mo), 10 older infants (10-22 mo), and 34 adults, all with normal hearing, for unoccluded and occluded ears. Experiment 2: Bone conduction behavioral thresholds in 17 adults were compared for unoccluded and occluded ears at 500, 1000, 2000, and 4000 Hz. Experiment 3: Bone conduction ASSR thresholds and amplitudes were compared in 22 young infants, 10 older infants, and 20 adults for an unoccluded and occluded test ear. Stimuli were bone-conducted amplitude/frequency-modulated tones presented simultaneously at 500, 1000, 2000, and 4000 Hz., Results: There were significant increases in sound pressure in the ear canal for stimuli presented at 40 dB HL when ears were occluded at 500 and 1000 Hz for all age groups. Infants showed the largest increases in SPL at 500 and 1000 Hz (5-8 dB > adults). Young infants showed no significant decreases in ASSR thresholds (2-6 dB) and amplitudes (0-10 nV) across frequency with occlusion; however, a significant number of infants had an occlusion effect at 500 Hz. Older infants showed a nonsignificant decrease in ASSR thresholds with occlusion (8 dB), a significant increase in ASSR amplitudes at 1000 Hz (6-21 nV), and a significant number of infants with an occlusion effect at 1000 Hz. Adult behavioral thresholds decreased significantly when ears were occluded at 500 and 1000 Hz; for ASSRs, thresholds also decreased (6-7 dB) and amplitudes increased (3-11 nV) at both 500 and 1000 Hz, but the mean trends and statistical findings were not in agreement in all cases. A significant number of adult subjects had an occlusion effect at 500 and 1000 Hz for both behavioral and ASSR thresholds., Conclusions: Our findings suggest that the occlusion effect for ASSR thresholds in young infants is small but emerging at 500 Hz but negligible at 1000 Hz and that the occlusion effect in older infants is emerging at both 500 and 1000 Hz. The clinical implications of these findings are that it is appropriate to conduct bone conduction testing on young infants without compensating for an occlusion effect; however, for older infants, it is prudent to remove insert earphones during bone conduction testing. For both young and older infants, occluding the ear canal increases the sound pressure near the tympanic membrane; however, this pathway appears to contribute less to bone conduction hearing when ears are occluded compared with adults as measured by ASSRs.

Ear development requires interactions of transcription factors for proliferation and differentiation. The proto-oncogene N-Myc is a member of the Myc family that regulates proliferation. To investigate the function of N-Myc, we conditionally knocked out N-Myc in the ear using Tg(Pax2-Cre) and Foxg1(KiCre). N-Myc CKOs had reduced growth of the ear, abnormal morphology including fused sensory epithelia, disrupted histology, and disorganized neuronal innervation. Using Thin-Sheet Laser Imaging Microscopy (TSLIM), 3D reconstruction and quantification of the cochlea revealed a greater than 50% size reduction. Immunochemistry and in situ hybridization showed a gravistatic organ-cochlear fusion and a "circularized" apex with no clear inner and outer hair cells. Furthermore, the abnormally developed cochlea had cross innervation from the vestibular ganglion near the basal tip. These findings are put in the context of the possible functional relationship of N-Myc with a number of other cell proliferative and fate determining genes during ear development., (Copyright © 2011 Wiley-Liss, Inc.)

Objectives: Multiple auditory steady state responses (ASSRs) will likely be included in the diagnostic test battery for estimating infant auditory thresholds in the near future; however, the effects of single- versus multiple-stimulus presentation in infants has never been investigated. In adults, there are no interactions (reduced amplitudes) between responses to multiple simultaneous stimuli presented at 60 dB SPL or lower. Maturational differences, however, may lead to greater interactions in infants; thus, it is unknown whether the single-stimulus technique or the multiple-stimulus technique is more efficient for testing infants. Two studies were carried out to address this issue., Design: All infants in study A participated in three stimulus conditions, which differed in the number of stimuli presented simultaneously. The monotic single (MS) condition consisted of 500, 1000, 2000, and 4000 Hz tones, which were presented singly to one ear. The monotic multiple (MM) condition was composed of four tones (500, 1000, 2000, and 4000 Hz) presented to one ear simultaneously. The dichotic multiple (DM) condition consisted of eight tones presented simultaneously to both ears (four tones to each ear). ASSR amplitudes were obtained from 15 normal infants (mean age: 23.1 wks) in response to multiple (MM, DM) and single (MS) air conduction amplitude-modulated (AM) tones (77 to 105 Hz modulation rates; 60 dB SPL). In study B, ASSR thresholds were determined for 500-Hz stimuli in the single- and DM-stimulus conditions (14 infants; mean age: 20.2 wks)., Results: Mean single-stimulus ASSR amplitudes for 500, 1000, 2000, and 4000 Hz were 30, 39, 45 and 43 nV, respectively. Presentation of multiple AM tones (i.e., four octave-spaced frequencies) to one ear resulted in ASSR amplitudes that were 97%, 87%, 82%, and 70% (for 500, 1000, 2000, and 4000 Hz, respectively) of the single-stimulus ASSR amplitudes. Results for the dichotic presentation of eight AM tones show ASSR amplitudes that were 70%, 77%, 67%, and 67% relative to the MS condition. Although decreases in amplitude occurred using multiple stimuli in infants, the multiple ASSR remained more efficient than the single-stimulus ASSR (i.e., multiple-stimulus amplitudes were greater than single-stimulus amplitudes divided by √K, where K is the number of stimuli). Results from study B indicate that ASSR thresholds for 500 Hz presented in the DM condition were elevated 3 dB compared with that obtained in the 500-Hz single-stimulus condition. This statistically nonsignificant difference is within the range of acceptable test-retest variability and is thus not of clinical significance., Conclusions: The amplitude reductions seen in the multiple-stimulus conditions in infants, not seen in adults, could be related to maturational differences in the ear canal, middle ear, cochlea, and/or brain stem. Because greater interactions occur in the DM-stimulus condition compared with the monotic multiple-stimulus condition and baseline single-stimulus condition, brain stem origins of these interactions are likely. Study B revealed statistically nonsignificant differences between threshold for 500 Hz when presented in the single- and DM-stimulus conditions. In summary, as with adults, multiple-stimulus presentation in infants is more efficient than single AM tones, at least for 60 dB SPL stimuli.

Basaloid skin tumors, including basal cell carcinoma (BCC) and basaloid follicular hamartoma, are associated with aberrant Hedgehog (Hh) signaling and, in the case of BCC, an expanding set of genetic variants including keratin 5 (encoded by KRT5), an intermediate filament-forming protein. We here show that genetic ablation of keratin 17 (Krt17) protein, which is induced in basaloid skin tumors and co-polymerizes with Krt5 in vivo, delays basaloid follicular hamartoma tumor initiation and growth in mice with constitutive Hh signaling in epidermis. This delay is preceded by a reduced inflammation and a polarization of inflammatory cytokines from a Th1- and Th17-dominated profile to a Th2-dominated profile. Absence of Krt17 also attenuates hyperplasia and inflammation in models of acute dermatitis. Re-expression of Krt17 in Gli2(tg); Krt17(-/-) keratinocytes induces select Th1 chemokines that have established roles in BCC. Our findings establish an immunomodulatory role for Krt17 in Hh driven basaloid skin tumors that could impact additional tumor settings, psoriasis and wound repair.

We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into one cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton., ((c) 2010 Wiley-Liss, Inc.)

The long latency auditory evoked potential (LLAEP) has been used for tracking changes in latency and morphology of the P1 peak in order to evaluate the maturation of the auditory system in children with cochlear implants (CIs). Cochlear implants can induce an artefact in the recordings when sounds are presented, which makes the analysis of LLAEPs much harder. Independent component analysis (ICA) has been used to remove this artefact. In this paper we apply a procedure based on ICA to reduce the CI artefact, to detect the LLAEPs and to use the changes in the spatial projections of their independent components (ICs) for a robust evaluation of the maturation of the auditory system in children with CIs.

The impact of small RNA function has resonated throughout nearly every aspect of eukaryotic biology and captured the varied interests of researchers, whether they are endeavoring to understand the basis of development and disease or seeking novel therapeutic targets and tools. The genetic regulatory roles of microRNAs (miRNAs) are particularly interesting given that these often highly conserved factors post-transcriptionally silence many complementary target genes by inhibiting messenger RNA translation. In this regard, miRNAs can be considered as counterparts to transcription factors, the ensemble of which establishes the set of expressed genes that define the characteristics of a specific cell type. In this review, evidence supporting a resounding role for small RNAs in development and maturation of sensory epithelia in the mouse inner ear will be considered with an emphasis on the contribution of one hair cell miRNA family (miR-183, miR-96, and miR-182). Although there is much yet to be explored in this fledgling aspect of ear biology, the breadth of miRNA expression and functional requirement for ear development are already sounding off.

Cicadas (Homoptera: Auchenorrhyncha: Cicadidae) use acoustic signalling for mate attraction and perceive auditory signals by a tympanal organ in the second abdominal segment. The main structural features of the ear are the tympanum, the sensory organ consisting of numerous scolopidial cells, and the cuticular link between sensory neurones and tympanum (tympanal ridge and apodeme). Here, a first investigation of the postembryonic development of the auditory system is presented. In insects, sensory neurones usually differentiate during embryogenesis, and sound-perceiving structures form during postembryogenesis. Cicadas have an elongated and subterranian postembryogenesis which can take several years until the final moult. The neuroanatomy and functional morphology of the auditory system of the cicada Okanagana rimosa (Say) are documented for the adult and the three last larval stages. The sensory organ and the projection of sensory afferents to the CNS are present in the earliest stages investigated. The cuticular structures of the tympanum, the tympanal frame holding the tympanum, and the tympanal ridge differentiate in the later stages of postembryogenesis. Thus, despite the different life styles of larvae and adults, the neuronal components of the cicada auditory system develop already during embryogenesis or early postembryogenesis, and sound-perceiving structures like tympana are elaborated later in postembryogenesis. The life cycle allows comparison of cicada development to other hemimetabolous insects with respect to the influence of specially adapted life cycle stages on auditory maturation. The neuronal development of the auditory system conforms to the timing in other hemimetabolous insects.

Cricket ears are sensitive to ultrasound as well as to lower, cricket-like sound frequencies. Ultrasound stimuli evoke negative phonotaxis in flying crickets, a behavior that has been interpreted as a defensive response against predation by echolocating bats. A recent study on a wing-dimorphic species, Gryllus texensis, showed that short-winged individuals, which are incapable of flight, are less sensitive to ultrasound, but not to lower sound frequencies, than their long-winged counterparts. The developmental decision to develop as a long- or short-winged individual is made during the last two larval instars, and there is some evidence suggesting that juvenile hormone (JH) has an instructive role, such that high levels of JH result in short-winged individuals. We show that treatment of last-instar larvae of a monomorphic long-winged species, Teleogryllus oceanicus, with a JH analog causes a decrease in sensitivity to ultrasound, but not to the lower sound frequency used for intraspecific communication.

The objective of this study was to compare bone-conduction (BC) auditory steady-state responses (ASSR) for infants and adults with normal hearing to investigate the time course of maturation of BC hearing sensitivity. Bone-conduction multiple ASSRs were recorded in 0-11-month-old (n=35), and 12-24-month-old infants (n=13), and adults (n=18). Low-frequency BC ASSR thresholds increased with age, whereas, high-frequency ASSR thresholds were unaffected by age except for a slight improvement at 2000 Hz. Compared to adults, BC ASSR amplitudes for young infants were larger for low frequencies, whereas, their amplitudes were smaller or similar for high frequencies. Compared to adults, young infants are much more sensitive to low-frequency BC stimuli, and probably more sensitive to high-frequency BC stimuli; these differences between infants and adults persist until at least two years of age. Different 'normal levels' for infants of different ages must be used and are proposed in this study.

In this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2-4, 5-7 or 8-10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p>0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p>0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p<0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.

The ability to integrate information across sensory channels is critical for both within- and between-modality speech processing. The present study evaluated the hypothesis that inter- and intramodal integration abilities are related, in young and older adults. Further, the investigation asked if intramodal integration (auditory+auditory), and intermodal integration (auditory+visual) resist changes as a function of either aging or the presence of hearing loss. Three groups of adults (young with normal hearing, older with normal hearing, and older with hearing loss) were asked to identify words in sentence context. Intramodal integration ability was assessed by presenting disjoint passbands of speech (550-750 and 1650-2250 Hz) to either ear. Integration was indexed by factoring monotic from dichotic scores to control for potential hearing- or age-related influences on absolute performance. Intermodal integration ability was assessed by presenting the auditory and visual signals. Integration was indexed by a measure based on probabilistic models of auditory-visual integration, termed integration enhancement. Results suggested that both types of integration ability are largely resistant to changes with age and hearing loss. In addition, intra- and intermodal integration were shown to be not correlated. As measured here, these findings suggest that there is not a common mechanism that accounts for both inter- and intramodal integration performance.

Audiological screening by registration of delayed evoked distortion product otoacoustic emission (DPOE) was made in 148 premature neonates (296 ears) born at gestation week 24-37. All the examinees were divided into three groups depending on the gestation term and were followed up for the first year of life. The results of the screening tests with reference to postconceptional and real age of premature infants are given. Recommendations are available on wide audiological examinations of premature infants depending on their gestational and postconceptional age.

In most of its anatomical constituents, e.g. in the Helix, etc., the external human ear is homologous to that of all Primates and Scandentia (tree shrews). Thus, its genetic basis is largely older than 60 Mio yrs. Based upon the observation of lifelong growth of the ear (e.g. Montacer-Kuhssary 1959), we aimed to elucidate the growth of the human ear in a more detailed way throughout life and in both sexes. On standardized photographical material collected randomly in Berlin (Germany), we measured N = 1448 ears from neonate children to volunteers of 92 yrs in age. 10 longitudinal measurements and 5 further anatomical parameters yielded a data set of roughly 19,000 data in total. Based upon our cross-section analysis, we quantified several sexual dimorphisms. Furthermore, we deduced ontogenetic developments and, partially, corrected their proportions for secular acceleration and body height shrinking with age. At the time of birth, in proportion to the body, the external ear was even bigger than the large head and continued growing rather linearly throughout life, reaching the highest average lengths in the volunteers aged over 85 yrs. The large yearly increases during childhood began to diminish at as early an age as 8 or 10 yrs. In all parameters where post adult growth was observed, female ears showed a lesser increase than those of men. The greatest ear length in females was 52 mm (SD +/- 4.3 mm) at birth, 61 mm (SD +/- 3.9 mm) at around 20 yrs of age and 72 mm (SD +/- 4.6 mm) in women older than 70 yrs. For the male subjects, these three values were: 52 mm (SD +/- 4.1 mm), 65 mm (SD +/- 4.0 mm) and 78 mm (SD +/- 4.8 mm), respectively. In spite of extreme premature growth of the auricle and its further lifelong growth, three anatomical features of the ear did practically not grow at all after birth: the width of the Concha auriculae and of the Incisura intertragica, as well as the diameter of the helical brim of the auricle. The problems arising concerning the functions and selective values of all these very unusual proportions and growths are discussed. The ontogenetic development of one or more pretragal skin folds could be used as a contribution to age estimations in forensic anthropology.

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

Although some studies have pointed to embryo/fetal toxicity at treatment levels that were not maternally toxic, knowledge about the potential toxic effects of the herbicide sulfentrazone is still limited. Since the results of these studies have raised some concern, the present work studied the effects of sulfentrazone maternal exposure on the physical and neurobehavioral endpoints in the development of rat pups. To accomplish that, the effects of the herbicide sulfentrazone (25 and 50mg/kg) were examined at two different developmental stages in rats: during the first 6 days of gestation, or in the organogenesis period (6-15 days). After parturition, pups were tested in a developmental test battery including measures of growth, maturational milestones, and neurobehavioral development. Maternal exposure to the herbicide resulted in significant alterations of the postnatal age at which the developmental milestones of ear and eye opening and testes descent were observed. There was a reduced weight gain rate in pups and their mothers when treated during the gestational period at the highest dose tested. Also, the functional state of the rat pup nervous system at different stages of postnatal development showed some neurodevelopmental delays in righting reflex, negative geotaxis, grip response, and motor coordination-locomotion and rearing (21-90 days of life) in the treated groups. Herbicide genotoxicity was investigated in fresh leukocytes both in mothers and pups using the comet assay: the data did not show any significant genotoxic effect induced by the herbicide. The findings of this study emphasize that sulfentrazone maternal exposure may lead to some neuromuscular and behavioral deficits in nursing pups.

To advance the understanding of genetic mechanisms involved in the patterning and the differentiation of the vertebrate auditory system, we screened for mutations affecting ear development in the zebrafish larva. Fifteen recessive mutant alleles have been isolated and analyzed. The phenotypes of these mutants involve abnormalities in ear morphology, otolith formation, or both processes in parallel. Among morphological defects, we found mutations affecting early patterning of the otic vesicle, the morphogenesis of semicircular canals, and the expansion of the ear lumen. The two most severe mutant phenotypes involve the absence of anterior and posterior cristae, as well as a severely misshapen morphology of the ear. In the category of otolith mutants, we found defects in otolith formation, growth, and shape. As it proved to be the case in past screening efforts of this type, these mutant lines represent an asset in the studies of molecular mechanisms that regulate vertebrate ear development.

In this paper on the ontogenesis and evolutionary biology of odontocete cetaceans (toothed whales), we investigate the head morphology of three perinatal pantropical spotted dolphins (Stenella attenuata) with the following methods: computer-assisted tomography, magnetic resonance imaging, conventional X-ray imaging, cryo-sectioning as well as gross dissection. Comparison of these anatomical methods reveals that for a complete structural analysis, a combination of modern imaging techniques and conventional morphological methods is needed. In addition to the perinatal dolphins, we include series of microslides of fetal odontocetes (S. attenuata, common dolphin Delphinus delphis, narwhal Monodon monoceros). In contrast to other mammals, newborn cetaceans represent an extremely precocial state of development correlated to the fact that they have to swim and surface immediately after birth. Accordingly, the morphology of the perinatal dolphin head is very similar to that of the adult. Comparison with early fetal stages of dolphins shows that the ontogenetic change from the general mammalian bauplan to cetacean organization was characterized by profound morphological transformations of the relevant organ systems and roughly seems to parallel the phylogenetic transition from terrestrial ancestors to modern odontocetes., (Copyright 2006 Wiley-Liss, Inc.)

Unlabelled: TRACP, a marker of osteoclasts, is also expressed by cells of the immune system. We identified a novel function for TRACP in the dendritic cell. DCs from TRACP knockout mice have impaired maturation and trigger reduced Th1 responses in vivo. We postulate that TRACP has an important role in the presentation of antigens to T cells., Introduction: TRACP is highly expressed by osteoclasts, activated macrophages, and dendritic cells (DCs). Knockout mice lacking TRACP have an intrinsic defect in osteoclastic resorption and macrophages that display abnormal immunomodulatory responses and cytokine secretion profiles. Our aim in this study was to investigate the significance of TRACP in the inductive phase of the immune response by examining dendritic cells from TRACP(-/-) mice., Materials and Methods: Maturational state and function of leukocyte subsets in mice was assessed by flow cytometry. The ability of the immune system to respond to nonspecific activation and to specific antigen was assessed by delayed type hypersensitivity and the presence of isotype-specific serum antibody in vivo and T-cell proliferation and cytokine production in vitro., Results: The ability of lipopolysaccharide (LPS) to upregulate MHC II and CD80 in DCs from TRACP(-/-) mice was reduced compared with wildtype mice, although production of IL-10 by DCs from TRACP-deficient animals was increased. T- and B-cell responses not involving antigen presentation (anti-CD3, TNP-ficoll) were normal in TRACP(-/-) mice, but responses to T-dependent antigens were impaired. Specifically, TRACP(-/-) mice had defective delayed hypersensitivity responses to picryl chloride and reduced proliferative responses to ovalbumin compared with wildtype mice. In response to ovalbumin, but not anti-CD3, T cells from TRACP(-/-) mice produced less interferon-gamma (IFN-gamma), but there was no difference in IL-4 production: TRACP(-/-) mice also produced less ovalbumin (OVA)-specific IgG2a after immunization., Conclusions: The finding that DCs from TRACP(-/-) mice have impaired maturation and defective Th1 responses shows that TRACP is important for polarizing responses in naïve T cells to antigen-presented dendritic cells.

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.

To determine the physiological functions of the mammalian double-stranded RNA-binding protein PACT, the single-copy mouse Pact gene was disrupted and expression of the protein was completely ablated. The most notable phenotypes of the Pact(-/-) mouse were reduced size and severe microtia. As a result of the congenital abnormality of both outer and middle ears, these mice were hearing impaired. In situ hybridization revealed that PACT mRNA was expressed in specific regions of all three parts of the ear in adult and embryonic wild-type mice. Our study demonstrated an essential role of PACT in mammalian ear development and produced the first animal model for studying human microtia.

Two classes of proteins play a leading role in the mechanisms of differentiation. The first class presents structural proteins that are fundamental for the structure and function of organs. Proteins of the second class are less understood; they exist in minor quantities and realize their action only in determining the accomplishment by structural proteins of their terminal functional state. Here we try to clarify the role of peptides synthesized by the corneal epithelium in determining the fate of differentiating cells. The acting protein fractions were obtained from isolated corneas of adult pigs, cows and carps. Their inducing activities were tested by a standard method, i. e. by checking their action on the early gastrula ectoderm of Xenopus laevis. To study a possibility of cornea conservation, we used preferentially lyophilizing drying of the obtained substances. With some differences in frequency, the protein mixtures obtained from all corneas induced the appearance of neural tissue (brain), but in the case of lyophilized cow corneas lenses, ear vesicles and somites also appeared. The results of this work clearly show the absence of homology between the sources of inducing factors and the specificity of their action. The cornea is an ectodermal derivative, but its products induce neural derivatives and, moreover, after lyophilization they change their induction specificity and induce also mesodermal cell types due, presumably, to changes in the tertiary structure of protein molecules after lyophilization.

Objectives: To determine the range and prevalence of otologic disorders in patients with ectodermal dysplasia (ED) and provide a general review of its multiple otolaryngological manifestations., Design: Case series., Setting: Ectodermal dysplasia family conference., Patients: Sixty-nine individuals with ED were evaluated. The average age was 11 years (range, 1-56 years). Most were male patients (44 [64%]), and most had Christ-Siemens-Touraine syndrome/hypohidrotic ED (42 [61%]), with the remaining having Hay-Wells, Clouston, and ectrodactyly ED clefting syndromes and unclassified types of ED., Interventions: Questionnaire including a quality-of-life assessment modeled after the Otitis Media 6 instrument, physical examination, screening audiogram, and tympanometry., Results: Of the 69 patients, 15 (22%) had a known history of hearing loss, and over half reported some level of difficulty processing verbal information (30 patients [43%] reported speech problems and 4 patients [6%] required hearing aids). A history of otitis media was common, with 15 patients (21%) presenting with tympanostomy tubes in place. The mean +/- SD quality-of-life rating was 2.0 +/- 1.1 (range 1-7), with lower scores suggesting less of a problem. On physical examination, 18 patients (26%) had pinna anomalies. One case of advanced cholesteatoma and 2 cases of external auditory canal stenosis were identified. Of the 24 patients who received 4-tone screening audiogram, 2 (8%) had a highest pure-tone average threshold of 50 to 65 dB, whereas 5 patients (21%) had a 30- to 45-dB threshold, with the remaining having a 0- to 25-dB threshold., Conclusion: In our study, which is, to our knowledge, the largest reported collection of ED patients evaluated for otologic disease, most patients were found to have 1 or more otologic abnormalities, ranging from auricular anomaly to complications of otitis media to profound hearing loss.

This study was conducted to promote in vitro-development of clone embryos by the treatment of donor somatic cells with hemoglobin (Hb) and/or beta-mercaptoethanol (ME), based on the analysis of apoptosis after somatic cell nuclear transfer (SCNT). Prospective, randomized study was conducted and, in vitro-matured bovine oocytes and fetal fibroblasts were provided for SCNT. In the first series of experiment, embryo apoptosis after SCNT was monitored by a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling assay. As results, apoptosis occurred more (P < 0.05) frequently after SCNT than after in vitro-fertilization (IVF) of control treatment. Subsequently, donor somatic cells treated with Hb (1 microg/ml) and/or ME (10 microM) were provided for SCNT. Either Hb or ME greatly reduced apoptosis (0.083 +/- 0.006 vs. 0.058-0.068 +/- 0.005), while combined treatment did not. ME was more promotive than Hb; significant increases were found in morula compaction (86%), cell numbers of blastocyst (131.3 +/- 1.3 cells/blastocyst), and inner cell mass (31.9 +/- 0.8 cells/blastocyst) cell, and the ratio of inner cell mass to trophectodermal cell numbers (0.24 +/- 0.01). In conclusion, the treatment of donor somatic cells with ME or Hb could reduce apoptosis after SCNT, resulting improved preimplantation development., (Copyright 2004 Wiley-Liss, Inc.)

The evolution of the mechanosensory cellular module and the molecular details that regulate its development has included morphological modifications of these cells as well as the formation of larger assemblies of mechanosensory cell aggregates among metazoans. This has resulted in a wide diversity of mechanosensory organs. The wide morphological diversity of organs, including the associated morphological modifications of the mechanosensory cells, suggests parallel evolution of these modules and their associated organs. This morphological diversity is in stark contrast to the molecular conservation of developmental modules across phyla. These molecular data suggest that the evolution of mechanosensory transduction might have preceded that of distinct cellular differentiation. However, once a molecular network governing development of specialized cells involved in mechanosensory transduction evolved, that molecular network was preserved across phyla. Present data suggest that at least the common ancestor of triploblastic organisms, perhaps even the common diploblastic ancestor of bilaterian metazoans, had molecular and cellular specializations for mechanosensation. It is argued that the evolution of multicellular organs dedicated to specific aspects of mechanosensation, such as gravity and sound perception, are evolutionary transformations that build on this conserved molecular network for cellular specialization, but reflect distinct morphological solutions. We propose that the sensory neurons, connecting the craniate ear with the brain, are a derived feature of craniates, and possibly chordates, that came about through diversification of the lineage forming mechanosensory cells during development. This evolutionarily late event suggests a heterochronic shift, so that sensory neurons develop in mammals prior to mechanosensory hair cells. However, sensory neuron development is connected to hair cell development, likely in a clonal relationship. The theme of cellular conservation is reiterated in two examples of chordate otic diversification: the evolution of the horizontal canal system and the evolution of the basilar papilla/cochlea. It is suggested that here again, cellular multiplication and formation of a special epithelium predates the functional transformation to an 'organ' system for horizontal angular acceleration and sound pressure reception, respectively. Overall, evolution of the vertebrate ear needs to be understood as an interplay between and utilization of two gene networks or modules. One is at the level of the molecularly and developmentally conserved mechanosensory cellular module. The other is an increased complexity in the morphology of both adult mechanosensory cells and organs by the addition of end-stage and novel features and associated gene networks to detect specific aspects of mechanosensory stimuli.

The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells., (Copyright 2003 Wiley-Liss, Inc.)