Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.     

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

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

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius'' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.     

Identifying MicroRNAs Involved in Degeneration of the Organ of Corti during Age-Related Hearing Loss

MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA targets, are important regulators of cellular senescence and aging. We questioned which miRNAs are involved in age-related degeneration of the organ of Corti (OC), the auditory sensory epithelium that transduces mechanical stimuli to electrical activity in the inner ear. Degeneration of the OC is generally accepted as the main cause of age-related hearing loss (ARHL), a progressive loss of hearing in individuals as they grow older. To determine which miRNAs are involved in the onset and progression of ARHL, miRNA gene expression in the OC of two mouse strains, C57BL/6J and CBA/J, was compared at three different ages using GeneChip miRNA microarray and was validated by real-time PCR. We showed that 111 and 71 miRNAs exhibited differential expression in the C57 and CBA mice, respectively, and that downregulated miRNAs substantially outnumbered upregulated miRNAs during aging. miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family, which are known regulators of pro-apoptotic pathways. In contrast, miRNAs that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family, which are known to be important for proliferation and differentiation, respectively. The shift of miRNA expression favoring apoptosis occurred earlier than detectable hearing threshold elevation and hair cell loss. Our study suggests that changes in miRNA expression precede morphological and functional changes, and that upregulation of pro-apoptotic miRNAs and downregulation of miRNAs promoting proliferation and differentiation are both involved in age-related degeneration of the OC.     

青年羊驼耳部和背部皮肤组织中miRNA差异表达研究

羊驼是毛用型经济动物,其耳部和背部的毛发品质和生长速度存在差异.MicroRNA(miRNA)是新发现的一类在转录后水平调控基因表达的非编码RNA分子,为比较miRNA在羊驼耳部和背部皮肤的表达差异,从而探讨miRNA在羊驼皮肤和毛囊发育过程中的调控作用,本实验提取羊驼皮肤总RNA,制备了羊驼皮肤miRNA芯片,通过与Affymetrix多物种miRNA芯片跨物种杂交对耳部和背部皮肤的miRNA进行筛选,并通过实时荧光定量PCR进行了验证,同时利用在线生物信息软件预测miRNA靶基因.结果显示,羊驼耳部和背部皮肤中高表达差异2倍以上的miRNA有39个,实时荧光定量PCR检测let-7b和miR-24在2个部位皮肤中的差异表达量与miRNA基因芯片结果一致;预测到let-7b和miR-24的靶基因中包含有与毛囊生长发育和毛发品质相关的基因,提示这些miRNA可能参与羊驼皮肤和毛囊的生长发育、更新以及毛发品质的调控.     

FGF信号通路在内耳发育调控和毛细胞再生中的作用

内耳是感受听觉和平衡觉的复杂器官。在内耳发育过程中,成纤维生长因子(fibroblast growth factor, FGF)信号通路参与了听基板的诱导、螺旋神经节(statoacoustic ganglion, SAG)的发育以及Corti器感觉上皮的分化。FGF信号开启了内耳早期发育的基因调控网络,诱导前基板区域以及听基板的形成。正常表达的FGF信号分子可促进听囊腹侧成神经细胞的特化,但成熟SAG神经元释放的过量FGF5可抑制此过程,形成负反馈环路使SAG在稳定状态下发育。FGF20在Notch信号通路的调控下参与了前感觉上皮区域向毛细胞和支持细胞的分化过程,而内毛细胞分泌的FGF8可调控局部支持细胞分化为柱细胞。人类FGF信号通路异常可导致多种耳聋相关遗传病。此外,FGF信号通路在低等脊椎动物毛细胞自发再生以及干细胞向内耳毛细胞诱导过程中都起到了关键作用。本文综述了FGF信号通路在内耳发育调控以及毛细胞再生中的作用及其相关研究进展,以期为毛细胞再生中FGF信号通路调控机制的阐明奠定理论基础。     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

PGC-Enriched miRNAs Control Germ Cell Development

Non-coding microRNAs (miRNAs) regulate the translation of target messenger RNAs (mRNAs) involved in the growth and development of a variety of cells, including primordial germ cells (PGCs) which play an essential role in germ cell development. However, the target mRNAs and the regulatory networks influenced by miRNAs in PGCs remain unclear. Here, we demonstrate a novel miRNAs control PGC development through targeting mRNAs involved in various cellular pathways. We reveal the PGC-enriched expression patterns of nine miRNAs, including miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301, using miRNA expression analysis along with mRNA microarray analysis in PGCs, embryonic gonads, and postnatal testes. These miRNAs are highly expressed in PGCs, as demonstrated by Northern blotting, miRNA in situ hybridization assay, and miRNA qPCR analysis. This integrative study utilizing mRNA microarray analysis and miRNA target prediction demonstrates the regulatory networks through which these miRNAs regulate their potential target genes during PGC development. The elucidated networks of miRNAs disclose a coordinated molecular mechanism by which these miRNAs regulate distinct cellular pathways in PGCs that determine germ cell development.     

MicroRNAs and epigenetic regulation in the mammalian inner ear: implications for deafness

Sensorineural hearing loss is the most common sensory disorder in humans and derives, in most cases, from inner-ear defects or degeneration of the cochlear sensory neuroepithelial hair cells. Genetic factors make a significant contribution to hearing impairment. While mutations in 51 genes have been associated with hereditary sensorineural nonsyndromic hearing loss (NSHL) in humans, the responsible mutations in many other chromosomal loci linked with NSHL have not been identified yet. Recently, mutations in a noncoding microRNA (miRNA) gene, MIR96, which is expressed specifically in the inner-ear hair cells, were linked with progressive hearing loss in humans and mice. Furthermore, additional miRNAs were found to have essential roles in the development and survival of inner-ear hair cells. Epigenetic mechanisms, in particular, DNA methylation and histone modifications, have also been implicated in human deafness, suggesting that several layers of noncoding genes that have never been studied systematically in the inner-ear sensory epithelia are required for normal hearing. This review aims to summarize the current knowledge about the roles of miRNAs and epigenetic regulatory mechanisms in the development, survival, and function of the inner ear, specifically in the sensory epithelia, tectorial membrane, and innervation, and their contribution to hearing.     

褪黑激素对绒山羊皮肤中毛囊周期相关miRNAs表达模式的影响

褪黑激素(Melatonin, MT)和miRNAs在毛囊的生长发育过程中发挥重要的作用, 但MT对绒山羊皮肤毛囊miRNAs表达模式的影响尚未见报道。为探索MT从miRNAs层次影响山羊绒生长的机制, 文章在内蒙古绒山羊中实施了褪黑激素埋植试验：5只青年母羊作为实验组埋植褪黑激素, 另外5只青年母羊作为对照。利用荧光定量PCR检测褪黑激素埋植前后毛囊周期相关miRNAs的表达变化。结果表明, 埋植MT明显改变了6个绒毛相关miRNAs的表达规律：在一个绒毛周期内, 除let-7a外, miR-203、miR-205、miR-96、miR-183和miR-199a的表达量均发生3次跃迁;埋植MT改变了miRNAs之间的共表达模式。对照组各miRNA之间相关系数范围为0.87~0.99(P<0.01)。与对照组相比, 埋植组中let-7a与miR-96、miR-199a、miR-205, miR-203与miR-96、miR-199a, miR-96与miR-183, miR-183与miR-199a之间的相关系数被明显消弱;MT通过下调6月份埋植组各miRNA的表达量提早诱发二次生绒。