The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

Commentary to Gorelenkova Miller and Mieyal (2015): sulfhydryl-mediated redox signaling in inflammation: role in neurodegenerative diseases.

Gorelenkova Miller and Mieyal (Arch Toxicol 89(9): 1439-1467, 2015) recently published a review paper suggesting that reversible cysteine plays a key role in redox-linked signal transduction via alteration of protein function, resulting in an association with many diseases including neurodegenerative disorders. Following their suggestions, we considered the correlation between sulfhydryl-mediated redox signaling and neurodegenerative diseases by focusing on RET proteins, a protein tyrosine kinases (PTKs) potentially sited upstream of the signal transduction cascade. c-RET is the receptor for glial cell line-derived neurotrophic factor family ligands. c-RET has been reported to be involved in not only Hirschsprung disease via development of the enteric nervous system but also neurodegenerative diseases including Parkinson's disease and amyotrophic lateral sclerosis. We also showed that c-RET might be associated with hearing loss via neurodegeneration of spiral ganglion neurons in the inner ear after birth in mice and humans. Moreover, we have reported that three kinds of oxidative stress, ultraviolet light-induced stress, osmotic stress and arsenic-induced stress, modulate kinase activity of RET-PTC1 without an extracellular domain as well as c-RET by conformational change of RET protein (dimerization) via disulfide bond formation. The oxidative stresses also modulate kinase activity of RET-PTC1 with cysteine 365 (C365) replaced by alanine with promotion of dimer formation, but not with cysteine 376 (C376) replaced by alanine. Since C376 of Ret-PTC-1 or its equivalent is most highly conserved and crucial for activity in PTKs, the cysteine could be one of major targets for oxidative stresses.

Limited effectiveness of household sand filters for removal of arsenic from well water in North Vietnam.

Since well water utilized for domestic purposes in the Red River Delta of North Vietnam has been reported to be polluted by arsenic, barium, iron, and manganese, household sand filters consisting of various components are used. Information regarding the effectiveness of various sand filters for removal of the four toxic elements in well water is limited. In this study, arsenic levels in 13/20 of well water samples and 1/7 of tap water samples exceeded World Health Organization (WHO) health-based guideline value for drinking water. Moreover, 2/20, 6/20, and 4/20 of well water samples had levels exceeding the present and previous guideline levels for barium, iron, and manganese, respectively. Levels of iron and manganese, but not arsenic, in well water treated by sand filters were lower than those in untreated water, although previous studies showed that sand filters removed all of those elements from water. A low ratio of iron/arsenic in well water may not be sufficient for efficient removal of arsenic from household sand filters. The levels of barium in well water treated by sand filters, especially a filter composed of sand and charcoal, were significantly lower than those in untreated water. Thus, we demonstrated characteristics of sand filters in North Vietnam.

Cadherin-dependent differential cell adhesion in Xenopus causes cell sorting in vitro but not in the embryo.

Adhesion differences between cell populations are in principle a source of strong morphogenetic forces promoting cell sorting, boundary formation and tissue positioning, and cadherins are main mediators of cell adhesion. However, a direct link between cadherin expression, differential adhesion and morphogenesis has not yet been determined for a specific process in vivo. To identify such a connection, we modulated the expression of C-cadherin in the Xenopus laevis gastrula, and combined this with direct measurements of cell adhesion-related parameters. Our results show that gastrulation is surprisingly tolerant of overall changes in adhesion. Also, as expected, experimentally generated, cadherin-based adhesion differences promote cell sorting in vitro. Importantly, however, such differences do not lead to the sorting of cells in the embryo, showing that differential adhesion is not sufficient to drive morphogenesis in this system. Compensatory recruitment of cadherin protein to contacts between cadherin-deprived and -overexpressing cells could contribute to the prevention of sorting in vivo.

Large-scale mechanical properties of Xenopus embryonic epithelium.

Epithelia are planar tissues that undergo major morphogenetic movements during development. These movements must work in the context of the mechanical properties of epithelia. Surprisingly little is known about these mechanical properties at the time and length scales of morphogenetic processes. We show that at a time scale of hours, Xenopus gastrula ectodermal epithelium mimics an elastic solid when stretched isometrically; strikingly, its area increases twofold in the embryo by such pseudoelastic expansion. At the same time, the basal side of the epithelium behaves like a liquid and exhibits tissue surface tension that minimizes its exposed area. We measure epithelial stiffness (~1 mN/m), surface tension (~0.6 mJ/m(2)), and epithelium-mesenchyme interfacial tensions and relate these to the folding of isolated epithelia and to the extent of epithelial spreading on various tissues. We propose that pseudoelasticity and tissue surface tension are main determinants of epithelial behavior at the scale of morphogenetic processes.

Antero-posterior tissue polarity links mesoderm convergent extension to axial patterning.

Remodelling its shape, or morphogenesis, is a fundamental property of living tissue. It underlies much of embryonic development and numerous pathologies. Convergent extension (CE) of the axial mesoderm of vertebrates is an intensively studied model for morphogenetic processes that rely on cell rearrangement. It involves the intercalation of polarized cells perpendicular to the antero-posterior (AP) axis, which narrows and lengthens the tissue. Several genes have been identified that regulate cell behaviour underlying CE in zebrafish and Xenopus. Many of these are homologues of genes that control epithelial planar cell polarity in Drosophila. However, elongation of axial mesoderm must be also coordinated with the pattern of AP tissue specification to generate a normal larval morphology. At present, the long-range control that orients CE with respect to embryonic axes is not understood. Here we show that the chordamesoderm of Xenopus possesses an intrinsic AP polarity that is necessary for CE, functions in parallel to Wnt/planar cell polarity signalling, and determines the direction of tissue elongation. The mechanism that establishes AP polarity involves graded activin-like signalling and directly links mesoderm AP patterning to CE.

Application of a human mesoderm tissue elongation system in vitro derived from human induced pluripotent stem cells to risk assessment for teratogenic chemicals.

Toxic compounds from the mother's diet and medication in addition to genetic factors and infection during pregnancy remain risks for various congenital disorders and misbirth. To ensure the safety of food and drugs for pregnant women, establishment of an in vitro system that morphologically resembles human tissues has been long desired. In this study, we focused on dorsal mesoderm elongation, one of the critical early development events for trunk formation, and we established in vitro autonomous elongating tissues from human induced pluripotent stem cells (hiPSCs). This artificial tissue elongation is regulated by MYOSIN II and FGF signaling, and is diminished by methylmercury or retinoic acid (RA), similar to in vivo human developmental disabilities. Moreover, our method for differentiation of hiPSCs requires only a short culture period, and the elongation is cell number-independent. Therefore, our in vitro human tissue elongation system is a potential tool for risk assessment assays for identification of teratogenic chemicals via human tissue morphogenesis.

Axisymmetric drop shape analysis for estimating the surface tension of cell aggregates by centrifugation.

Biological tissues behave in certain respects like liquids. Consequently, the surface tension concept can be used to explain aspects of the in vitro and in vivo behavior of multicellular aggregates. Unfortunately, conventional methods of surface tension measurement cannot be readily applied to small cell aggregates. This difficulty can be overcome by an experimentally straightforward method consisting of centrifugation followed by axisymmetric drop shape analysis (ADSA). Since the aggregates typically show roughness, standard ADSA cannot be applied and we introduce a novel numerical method called ADSA-IP (ADSA for imperfect profile) for this purpose. To examine the new methodology, embryonic tissues from the gastrula of the frog, Xenopus laevis, deformed in the centrifuge are used. It is confirmed that surface tension measurements are independent of centrifugal force and aggregate size. Surface tension is measured for ectodermal cells in four sample batches, and varies between 1.1 and 7.7 mJ/m2. Surface tension is also measured for aggregates of cells expressing cytoplasmically truncated EP/C-cadherin, and is approximately half as large. In parallel, such aggregates show a reduction in convergent extension-driven elongation after activin treatment, reflecting diminished intercellular cohesion.

Regulation of convergent extension by non-canonical Wnt signaling in the Xenopus embryo.

Non-canonical Wnt signaling is an important regulator of gastrulation in Xenopus. In particular, it has been implicated in the control of convergent extension movements. Convergent extension in the gastrula occurs primarily in the dorsal tissue of the marginal zone, and explants of this tissue will continue to undergo these movements in isolation. This observation has led to an assay to examine convergent extension movements that is unique to the Xenopus system, and is described herein.

A comprehensive study including monitoring, assessment of health effects and development of a remediation method for chromium pollution.

Chromium (Cr) pollution caused by wastewater from tanneries is a worldwide environmental problem. To develop a countermeasure, we performed a comprehensive study using Hazaribagh, the tannery area in Dhaka City, Bangladesh, as a model. Our environmental monitoring indicated that the soluble form of Cr, but not barium or arsenic, in Buriganga River is derived from Hazaribagh. Our chemical analysis next showed that Cr, the primary pollutant in canal water at Hazaribagh, consisted of ≤0.7 µM hexavalent Cr [Cr(VI)] and ≤1705 µM trivalent Cr [Cr(III)]. Our biological study then showed that coexposure to Cr(VI) and Cr(III) at possible ratios in canal water at Hazaribagh synergistically promotes transforming activity of human non-tumorigenic HaCaT keratinocytes with activated MEK/ERK and AKT. Our environmental engineering study finally indicated that a magnesium and iron-based hydrotalcite-like compound (MF-HT), our original depurative, can maximally adsorb 9.0 mg/g Cr(VI) and 1041 mg/g Cr(III). Our results suggested the importance of removal of Cr(III) as well as Cr(VI) by showing that Cr(III), which is generally recognized as a chemical with low toxicity, synergistically promoted carcinogenicity of a low level of Cr(VI). Therefore, we propose the use of our original high-efficient and low-cost depurative as a countermeasure to address the worldwide problem of environmental Cr pollution.

Increased expression level of Hsp70 in the inner ears of mice by exposure to low frequency noise.

Previous studies showed that people in urban areas are possibly exposed to 60-110 dB of low frequency noise (LFN) defined as noise of ≤100 Hz in their daily life. Previous studies also showed increased health risks by exposure to high levels (130-140 dB) of LFN in animals. However, little is known about the health effects of exposure to an ordinary level of LFN. We biochemically and immunohistochemically assessed the effects of exposure to inaudible LFN for mice (12 h/day of 100 Hz LFN at 95 dB for 5 days), at a level to which people are possibly exposed in daily life, on a murine inner ear by targeting 9 stress-reactive molecules. There was more than a 5-fold increased transcript level of heat shock protein 70 (Hsp70) in the whole inner ear exposed to LFN. However, the transcript levels of the other 8 stress-reactive molecules including Hsp27 and Hsp90 were comparable in LFN-exposed and unexposed murine inner ears. Only the transcript level of Cebpß among the previously reported 4 transcriptional activators for Hsp70 expression was more than 3-fold increased by LFN exposure. Hsp70 transcript expression levels in the inner ears 3 days after LFN exposure were comparable to those in unexposed inner ears. The protein level of Hsp70, but not the levels of Hsp27 and Hsp90, was also increased in the vestibule by LFN exposure. However, hearing levels as well as expression levels of Hsp70 protein in the cochleae were comparable in LFN-exposed mice and unexposed mice. Our results demonstrated that the inner ear might be one of the organs that is negatively affected by stress from inaudible LFN exposure. Moreover, LFN exposure might increase Hsp70 expression level via Cebpß in the inner ear. Thus, Hsp70 and Cebpß levels could be candidates of biomarkers for response to LFN exposure.

Arsenic level in toenails is associated with hearing loss in humans.

Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12-55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz [odds ratio (OR) = 4.27; 95% confidence interval (CI): 1.51, 12.05], 8 kHz (OR = 3.91; 95% CI: 1.47, 10.38) and 12 kHz (OR = 4.15; 95% CI: 1.55, 11.09) by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails (r = 0.8113, p = 0.0014) in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans.

Impairments of Inner Ears Caused by Physical Environmental Stresses.

The inner ears contain the organ of Corti, vestibule and semicircular canal. The organ of Corti is crucial for hearing, while the vestibule and semicircular canal play important roles in maintaining balance. Exposure to noise at excessive levels is known to cause damages of the inner ears, resulting in noise-induced hearing loss. On the other hand, noise levels (dB) are used for the evaluation of health risks by exposure to noise, although noise consists of sound with broad frequencies (Hz). Thus, information about the frequency-dependent effect of noise on health is largely unknown. In this review, we first introduce noise-induced hearing loss caused by exposure to audible noise. We then describe the imbalance in mice exposed to low-frequency noise (100 Hz).

Risk Assessment of Neonatal Exposure to Low Frequency Noise Based on Balance in Mice.

General electric devices and ventilation systems are known to generate low frequency noise (LFN) with frequencies of <100 Hz. Previous studies showed that exposure to LFN caused impairments of balance in humans and mice during adulthood. On the other hand, a previous study showed that noise levels in the neonatal intensive care unit (NICU) were greater than those in general home or office environments. Therefore, it is possible that neonates have a potential risk to be exposed to LFN in the NICU. However, the risk of neonatal exposure to LFN remains unclear in humans and mice. In this study, male ICR mice were exposed to LFN at 100 Hz for 4 weeks after birth and then subjected to rotarod and beam crossing tests in order to assess LFN-mediated risk of imbalance during the neonatal period. Exposure to LFN at 70 dB, but not exposure to LFN up to 60 dB, during the neonatal period significantly decreased performance scores for rotarod and beam crossing tests compared to the scores of the control group. The number of calbindin-positive hair cells in the saccule and utricle was decreased in mice exposed to LFN at 70 dB for 4 weeks in the neonatal phase. Cessation of exposure for 10 weeks did not result in recovery of the decreased performance in rotarod and beam crossing tests. Thus, our results suggest that 70 dB is a possible threshold for exposure to LFN for 4 weeks during the neonatal period causing unrecoverable imbalance in mice.

Manganese-Mediated Decrease in Levels of c-RET and Tyrosine Hydroxylase Expression In Vitro.

Previous studies showed that overexposure to manganese causes parkinsonism, a disorder of dopaminergic neurons. Previous studies also showed that activity of c-RET kinase controls dopamine production through regulation of tyrosine hydroxylase (TH) expression, suggesting the involvement of c-RET in the development of parkinsonism. To our knowledge, however, there is no report showing a correlation between manganese-mediated parkinsonism and c-RET. In this study, we examined the effect of manganese on the expression and/or activation levels of c-RET and TH in human TH-expressing cells (TGW cells). We first found that treatment with 30 and 100 µM manganese resulted in reduction of c-RET transcript level and degradation of c-RET protein through promotion of ubiquitination. We then examined the biological significance of manganese-mediated decrease of c-RET protein expression. Decreased TH expression with decreased c-RET kinase activity was observed in c-RET protein-depleted TGW cells by treatment with manganese (30 µM) as well as by c-RET siRNA transfection. Since TH protein has been shown to be involved in the dopamine-producing pathway in previous studies, our results indicate the possibility that manganese-mediated reduction of TH expression and phosphorylation via decreased expression of c-RET protein in neural cells is involved in parkinsonism induced by manganese.

Improved efficiency of definitive endoderm induction from human induced pluripotent stem cells in feeder and serum-free culture system.

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies, we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition, keeping cell density low during endoderm induction is important for the efficiency. On the other hand, we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm, indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless, these results should enable better control in the production of definitive endoderm-derived cells.

Tissue surface tension measurement by rigorous axisymmetric drop shape analysis.

Certain behaviours of embryonic cell aggregates can be modelled by ascribing to them a tissue surface tension, with each cell analogous to a liquid molecule. Under normal gravity, aggregates are nearly spherical, but they can be partially flattened in a centrifuge. This allows measurement of their tissue surface tensions by a drop shape method such as axisymmetric drop shape analysis (ADSA). We study ectodermal embryonic cells from the frog Xenopus laevis subjected to centrifugation at 100 x g and 200 x g. We show that ADSA can be applied to irregular aggregate profiles and compare results with those from a previous, simpler version called ADSA-IP. With a modification in the experimental method, the two algorithms give similar results and the aggregate profiles more closely follow Laplacian curves. The ADSA fitting error allows an estimate of the relative uncertainty in the results.

Epithelial coating controls mesenchymal shape change through tissue-positioning effects and reduction of surface-minimizing tension.

Signalling between mesenchymal and epithelial cells has a profound influence on organ morphogenesis. However, less is known about the mechanical function of epithelial-mesenchymal interactions. Here, we describe two principal effects by which epithelia can regulate shape changes in mesenchymal cell aggregates. We propose that during formation of the embryonic body axis, the epithelial layer relieves surface minimizing tensions that would force cell aggregates into a spherical shape, and controls the serial arrangement of cell populations along the axis. The combined effects permit the tissue to deviate from a spherical form and to elongate.

Parallel microtubules and other conserved elements of dorsal axial specification in the direct developing frog, Eleutherodactylus coqui.

Specification of the dorsal axis in commonly studied frogs, such as Xenopus laevis and Rana pipiens, depends on a microtubule-mediated movement of cytoplasm in the fertilized egg. The Puerto Rican tree frog, Eleutherodactylus coqui, has an egg that is twenty times the volume of that of X. laevis, raising the question as to whether the mechanism of dorsal axial specification is conserved in these large eggs. Fertilized eggs of E. coqui develop a transient array of parallel microtubules, similar to other frogs, but proportionately larger. The array persists after first cleavage, longer than in other frogs, and is gone by the third cleavage. Correlated with the longer life of the parallel microtubules, both 2- and 8-cell E. coqui embryos remain sensitive to gravity-mediated axial specification, a sensitivity lost in X. laevis before the 2-cell stage. Activation of the Wnt/beta-catenin pathway by injected Xwnt8 RNA causes axial formation as in X. laevis. The results indicate that elements of dorsal axial specification are conserved in E. coqui, but they occur later compared to in X. laevis.