Regulation of hair cell and stomatal size by a hair cell-specific peroxidase in the grass Brachypodium distachyon

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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。     

Single-cell transcriptome analysis reveals three sequential phases of gene expression during zebrafish sensory hair cell regeneration

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Modes of neuronal arbor enlargement in the ear of a postembryonic fish,Astronotus ocellatus

New hair cells are added during postembryonic life in several species of fishes and birds. The production of new hair cells appears to require enlargement of eighth nerve arbors during growth since, at least in fish, eighth nerve neurons are added more slowly than hair cells or not at all. This situation provides an intriguing opportunity to study the mechanisms of growth of the neuronal arbors. In this paper, we report the results of studies on the postembryonic growth of eighth nerve dendritic arbors in the saccular epithelium of the cichlid fish Astronotus ocellatus. Arbor sizes and shapes were compared in small and large fish using the axonal tracer cobaltouslysine. Our data suggest that postembryonic eighth nerve arbors enlarge in 2 ways. First, arbors add new terminal endings to their distal ends. Second, whole new branches appear to be added at locations up to hundreds of micrometers proximal to the terminal endings. These 2 modes of growth suggest that more than one mechanism may be operative in controlling arbor enlargement.     

Dark hair cells in the inner ear of the rainbow trout. A study of the influence of different fixation methods

The occurrence of dark staining cells in different tissues has been suggested to be artefactual and caused during the fixation process. In inner ear sensory epithelia, dark hair cells (DHC) have been suggested to be apoptotic cells. We have examined whether dark cells represent dying cells or whether they are the results of fixation artefacts. The effects of buffer osmolarity and different fixation methods on the incidence of dark hair cells in the inner ear macula sacculi of the rainbow trout (Oncorhynchus mykiss) were investigated by light and electron microscopy. Glutaraldehyde in phosphate buffer with osmolarities of 0, 135, 225, 425, and 560 mosmol were used for fixation by immersion. For comparison, fixation by vascular perfusion as well as the effects of mechanical injury and delayed fixation were studied. DHC were found in all examined saccular maculae except for the delayed fixation protocol where almost all the sensory cells were lost. The number of DHC accounted for 2.5–12.9‰ of the sensory cells. Neither the buffer osmolarity nor the fixation method had significant effects on the frequencies of DHC. Mitotic cell division events were seen exclusively in the apical cell strata of the sensory epithelium. The DHC are suggested to be associated with apoptosis rather than fixation artefacts.     

Isolation and characterization of the CD133+ precursors from the ventricular zone of human fetal brain by magnetic affinity cell sorting

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti-CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133(+) cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133(-) cells, these CD133(+) cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.     

卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响

目的:探讨卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响。方法:取20只豚鼠随机分为2组,实验组连续14d肌肉注射硫酸卡那霉素,200mg/(kg.d),对照组连续14d等量肌肉注射生理盐水,停药14d处死动物后制作耳蜗标本,处死前检测其ABR的变化,免疫组化及原位杂交法测定Bcl-2的表达。结果:豚鼠卡那霉素耳慢性中毒后,ABR阈值较对照组明显上升,Bcl-2阳性表达减低,与对照组比较差异有显著性(P<0.05)。结论:卡那霉素耳慢性损害可能与抑制Bcl-2的表达有关。     

Regeneration in zebrafish lateral line neuromasts: expression of the neural progenitor cell marker sox2 and proliferation-dependent and-independent mechanisms of hair cell renewal

Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.     

NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis

We previously identified functional N-methyl-D-aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and Rank ligand (RankL). Using RT-PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi-quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose-dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western-blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF-kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF-kappa B pathway.     

中华蜜蜂工蜂咽下腺胚后发育过程中细胞的增殖和凋亡

【目的】咽下腺(hypopharyngeal gland)是蜜蜂重要的外分泌腺,是工蜂合成和分泌蜂王浆的主要腺体。本研究的目的在于了解中华蜜蜂Apis cerana cerana工蜂咽下腺的胚后发育特点。【方法】通过组织形态学、Brd U免疫组织化学和TUNEL细胞凋亡检测等技术,对中华蜜蜂工蜂咽下腺的胚后发育过程及组织结构特点进行了比较研究。【结果】中华蜜蜂工蜂的咽下腺起源自预蛹阶段口器内壁的陷入,细胞分裂活动的高峰期集中在蛹发育的前3 d,随后分裂细胞数减少,并一直持续到蛹发育的第7天左右结束;分泌腺泡的出现大约在蛹发育的第5天。到蛹发育的末期,咽下腺已基本形成,但是没有发育完全;哺育蜂的咽下腺高度发育,分泌活动旺盛;采集蜂的咽下腺中有许多分泌细胞凋亡。【结论】本研究揭示了中蜂工蜂咽下腺胚后发育过程中细胞增殖和凋亡的模式,为昆虫咽下腺的发育和功能研究提供了一定的理论依据。     

虎皮鹦鹉耳蜗感觉细胞的发育与听觉的关系

选用18d、38d、成鸟(3月以上)三种年龄的雄性虎皮鹦鹉作为实验材料,采用测量脑干听觉诱发电位和制作内耳的石蜡切片两种方法,研究了鸟类在发育时期耳蜗感觉上皮细胞的变化以及听觉功能的发育状况。鸟的听觉能力在出生后不断得到提高,38d基本达到成鸟的水平;耳蜗毛细胞的形态、内部结构逐渐趋于成熟,其灵敏度不断提高,感受听觉的能力也在增强,38d基本与成鸟的发育程度相同。耳蜗感觉上皮细胞的发育对听觉行为产生的时间和发展具有极为重要的影响。     

Appearance of hair follicle-inducible mesenchymal cells in the rat embryo

Rat vibrissa follicle morphogenesis starts around 13 days of gestation. By day 14 mesenchymal cells have already aggregated as 'condensations' beneath the initial hair bud. Some of the mesenchymal cells will form a dermal papilla, having profound effects on hair follicle formation. The appearance of follicle-inducing mesenchymal cells in the process of vibrissa follicle development was examined. Mesenchymal cells were isolated from the developing site of vibrissa follicles at 13 days or at later stages and amplified in mass culture, harvested and transplanted in association with the epithelium. It was demonstrated that 13-day mesenchymal cells did not induce any hair bulbs but those from 14 days or later stages could induce hair-producing new bulbs or new follicles depending on the association with the follicle epithelium or with the glabrous sole epidermis of the adult rats, respectively. Further, clones having hair bulb-inducing ability were obtained from 14- and 15-day mass-cultured mesenchymal cells. Based on these and other results, it was concluded that mesenchymal cells having follicle-inducing ability are present at least by 14 days in the future whisker pad region. This suggests that the differentiation of the dermal papilla cells must start before the initial hair bud stage.     

The influence of glycogen synthase kinase 3 in limiting cell addition in the mammalian ear

In the vestibular organs of the inner ear, an early postnatal decline in the capacity for cell proliferation appears to be responsible for limits to hair cell regeneration that are unique to mammals. We have investigated the time course of that decline in cell proliferation and its potential regulation by glycogen synthase kinase-3 (GSK3). Our immunoblots have revealed that inactive GSK3 beta decreases postnatally in the murine utricular epithelium, as E-cadherin and the active forms of GSK3 alpha and GSK3 beta each increase. In cultured utricular epithelia, pharmacological inhibition of GSK3 by LiCl and SB-216763 increased cell proliferation across a range of postnatal ages. LiCl treatments also led to increased levels of beta-catenin and Snail and decreased expression of E-cadherin. Transfection with a dominant-negative GSK3 beta enhanced proliferation in these epithelia in a cell-autonomous manner, while overexpression of wild-type GSK3 beta markedly reduced it. The evidence from these measurements and experimental manipulations indicates that the balance of active and inactive forms of GSK3 helps to determine whether mammalian vestibular supporting cells will proliferate; permitting proliferation during early development when inactive GSK3 predominates and progressively inhibiting proliferation, and thereby limiting the capacity for hair cell regeneration as more GSK3 becomes active during the first week of postnatal maturation.     

Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of Steel factor transgenic mice

Steel factor (SLF, also called KIT-ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro . In the present study, Kit-independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function-blocking antibody. We successfully purified these precursor or stem cells as Kit⁺CD45⁻ cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit-independent precursors. The Kit-independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit-independent nature for their survival.     

Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells

Summary. The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.Correspondence and reprints: Department of Biology, 221 Morrill Science Center II, University of Massachusetts, Amherst, MA 01003, U.S.A.Received September 30, 2002; accepted February 12, 2003 Published online September 23, 2003     

Recovery of mechano-electrical transduction in rat cochlear hair bundles after postnatal destruction of the stereociliar cross-links

Mechano-electrical transduction (MET) in the stereocilia of outer hair cells (OHCs) was studied in newborn Wistar rats using scanning electron microscopy to investigate the stereociliar cross-links, Nomarski laser differential interferometry to investigate stereociliar stiffness and by testing the functionality of the MET channels by recording the entry of fluorescent dye, FM1-43, into stereocilia. Preparations were taken from rats on their day of birth (P0) or 1–4 days later (P1–P4). Hair bundles developed from the base to the apex and from the inner to outer OHC rows. MET channel responses were detected in apical coil OHCs on P1. To study the possible recovery of MET after disrupting the cross-links, the same investigations were performed after the application of Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and allowing the treated samples to recover in culture medium for 0–20 h. We found that the structure and function were abolished by BAPTA. In P0–P1 samples, structural recovery was complete and the open probability of MET channels reached control values. In P3–P4 samples, complete recovery only occurred in OHCs of the outermost row. Although our results demonstrate an enormous recovery potential of OHCs in the postnatal period, the structural component restricts the potential for therapy in patients.     

Homogenization of freshwater fish faunas after the elimination of a natural barrier by a dam in Neotropics

Aim We quantify biotic homogenization of fish fauna caused by the elimination of a natural barrier between two freshwater ecoregions. We also evaluated fish introductions by different mechanisms such as aquaculture, angling and the aquarium trade in the homogenization of fish assemblages. The relative importance of native extinctions in the homogenization process was assessed by simulating the exclusion of threatened species in the data set. Location Paraná River, south‐eastern South America. Methods A fish species list of the Parana River Basin was organized in a subset of species distributions, according to pre‐ and post‐introductions caused by the elimination of the natural barrier and by other mechanisms. Biotic homogenization was verified by the use of Jaccard’s and Bray–Curtis’s coefficients, Whittaker’s beta diversity index, non‐metric multidimensional scaling analysis (NMDS) and nonparametric tests. Results For all subsets of species distributions, we observed an increase in the number of non‐native species in common related to the introductions. Between 40 and 52% of the species currently present in the Upper Paraná Basin dispersed upstream from the Lower Paraná after the construction of Itaipu Dam, including at least 1 class, 2 orders, 4 families and 16 genera of fish. Jaccard’s coefficient between the Upper and Lower Parana River increased by 6–7.5% only considering the Itaipu Dam influence and 10.5% considering all mechanisms of fish introductions. More than 50% of the increase in similarity was caused by the elimination of the barrier. Our results indicated functional homogenization related to large‐bodied Siluriformes (catfish). Main conclusions Itaipu Lake flooded a natural barrier and allowed hydrologic connectivity between the Upper and Lower Paraná River, and many fishes of the lower part of the river were able to colonize the upper stretches. The homogenization of the two assemblages between these adjacent aquatic regions was an unpredicted result of hydropower implementation. Introductions by dam can also shift longitudinal and latitudinal body size patterns (i.e. Bergmann's rule).     

Topical RT1640 treatment effectively reverses gray hair and stem cell loss in a mouse model of radiation‐induced canities

Gray hair is a visible sign of tissue degeneration during aging. Graying is attributed to dysfunction of melanocyte stem cells (McSCs) that results in depletion of their melanin‐producing progeny. This non‐lethal phenotype makes the hair follicle and its pigment system an attractive model for investigating mechanisms that contribute to tissue aging and therapeutic strategies to combat this process. One potential combination therapeutic is RT1640, which is comprised of two drugs that are known to stimulate hair growth (cyclosporine A [CsA] and minoxidil), along with RT175, a non‐immunosuppressive immunophilin ligand that is implicated in tissue regeneration. Using the ionizing radiation‐induced acute mouse model of hair graying, we demonstrate that RT1640, over CsA alone, promotes regeneration of the hair pigment system during and following treatment. In non‐irradiated mice, RT1640 is also physiologically active and successfully speeds hair growth and expands the McSC pool. It appears that this effect relies on the combined activities of the three drugs within RT1640 to simultaneously activate hair growth and McSCs as RT175 alone was insufficient to induce hair cycling in vivo, yet sufficient to drive the upregulation of the melanogenic program in vitro. This study sets the stage for further investigation into RT1640 and its components in McSC biology and, ultimately, melanocyte hypopigmentary disorders associated with disease and aging.