In Vitro and In Vivo Antimelanogenesis Effects of Leaf Essential Oil from Agathis dammara.

Agathis species are widely distributed around Southeast Asia, Australasia, South Pacific islands, and etc. Traditionally, Agathis species have been used as the folk medicines, the common ethnopharmacological uses of Agathis genus are the treatments of headache and myalgia. This study aims to investigate the chemical composition of Agathis dammara (Lamb.) Rich. leaf essential oil and to explore its antimelanogenesis effect. The chemical constituents of leaf essential oil are analyzed using gas chromatography-mass spectrometry (GC-MS), the major constituents of leaf essential oil are sesquiterpenoids. The major constituents are δ-cadinene (16.12%), followed by γ-gurjunene (15.57%), 16-kaurene (12.43%), ß-caryophyllene (8.58%), germacrene D (8.53%), and γ-cadinene (5.33%). As for the in vitro antityrosinase activity, leaf essential oil inhibit the tyrosinase activity of mushroom when the substrate is 3,4-dihydroxyphenylalanine (L-DOPA). Leaf essential oil prevents tyrosinase from acting as diphenolase and catalyzing L-DOPA to dopaquinone, and converting into dark melanin pigments. A. dammara leaf essential oil also exhibits the in vivo antimelanogenesis effect, leaf essential oil reduces 43.48% of melanin formation in zebrafish embryos at the concentration of 50 µg/mL. Results reveal A. dammara leaf essential oil has the potential for developing the skin whitening drug and depigmentation ingredient for hyperpigmentary disorders.

Outcomes in Dogs with Multiple Sites of Cervical Intervertebral Disc Disease Treated with Single Ventral Slot Decompression.

In dogs with acute myelopathy but showing multiple sites of spinal compression from intervertebral disc disease (IVDD) on imaging, one approach is surgical decompression of the single acute disc extrusion while ignoring other previously extruded or protruded discs. However, little is known regarding the outcomes of this approach. This study described the outcomes and investigated prognostic factors in 40 dogs with multiple sites of cervical disc extrusion or protrusion on MRI who underwent ventral slot decompression for the single acute disc. The overall recovery rate was 97.5%. The median recovery time was seven days. The number of affected discs (including disc extrusion and protrusion) and the presence and number of the affected discs causing severe spinal compression did not influence the 30-day outcome. Compared with 23 dogs with single disc extrusion treated surgically, the recovery time and outcomes were similar between the two groups. The total number of affected discs was not associated with recovery time or outcomes. In conclusion, if an acute disc could be identified, ventral slot decompression targeting the single acute disc is a viable management approach for dogs with an acute presentation but diagnosed with multiple sites of spinal cord compression from IVDD.

Correction: Chondroitin sulfate proteoglycan 4 regulates zebrafish body axis organization via Wnt/planar cell polarity pathway.

[This corrects the article DOI: 10.1371/journal.pone.0230943.].

Topical applications of allogeneic adipose-derived mesenchymal stem cells ameliorate the canine keratoconjunctivitis sicca.

BACKGROUND: Canine keratoconjunctivitis sicca (KCS) is predominantly an immune-mediated disease. Current therapy of canine KCS is mainly by immunosuppressant, but the effectiveness was limited in some patients. In the past few years, some studies showed the results of the use of mesenchymal stem cells in treating canine KCS via periocular injections. However, the periocular injection procedure requires sedation or general anesthesia, and may lead to iatrogenic or incidental injury during the injection process. The aim of this study was to investigate the efficacy of topical allogenic canine adipose-derived mesenchymal stem cells (cAD-MSCs) in clinical patients of canine KCS. RESULTS: The cAD-MSCs used in this study were characterized for their capability of tri-lineage differentiation and immunomodulatory properties. In addition, preparation methods for eye drops of cAD-MSCs was developed and its optimal preservation was tested. The canine KCS patients were recruited for clinical trial and divided into two groups based on their history of previous treatment. All patients received topical cAD-MSCs treatment once per week for 6 consecutive weeks and complete ophthalmic examinations were performed 1 week before treatment (week 0) and at 3rd, 6th, 9th weeks, respectively. The results showed that the quantity and quality of tears have improved significantly following topical cAD-MSCs treatment based on Schirmers tear test-1 and tear break-up time. More than half of all patients were found improved in the tear quantity. In particular, 56.5% of the patients that were unresponsive to prior immunosuppressant therapy had an effective increase in tear volume. The severity of clinical signs was also ameliorated according to the numeric rating scale score from both patient owners and the clinician. CONCLUSION: To sum up, topical cAD-MSCs may be beneficial especially in KCS patients with poor owner compliance for frequent daily use of eye drops or those who are unresponsive to immunosuppressant therapy.

Antimelanogenesis Effects of Leaf Extract and Phytochemicals from Ceylon Olive (Elaeocarpus serratus) in Zebrafish Model.

The melanogenesis inhibition effect in zebrafish (Danio rerio) and antityrosinase activity of the ethanolic extract and its phytochemicals from Ceylon olive (Elaeocarpus serratus Linn.) leaves were investigated in this study. Among the leaf extract and four soluble fractions, the ethyl acetate soluble fraction exhibits the best antityrosinase and antimelanogenesis activities. One phenolic acid, gallic acid, and two flavonoids, myricetin and mearnsetin, are isolated from the active subfractions through the bioassay-guided isolation; their structures are elucidated based on the 1D and 2D NMR, FTIR, UV, and MS spectroscopic analyses. These compounds have significant antityrosinase activity whether using l-tyrosine or l-DOPA as the substrate; mearnsetin shows the optimal activity. In the enzyme kinetic investigation, both gallic acid and mearnsetin are the competitive-type inhibitors against mushroom tyrosinase, and myricetin acts as a mixed-type tyrosinase inhibitor. Leaf extract and an ethyl acetate soluble fraction show effective performance in the inhibition of melanin formation in zebrafish embryos. Mearnsetin also possesses a promising antimelanogenesis effect, which is superior to the positive control, arbutin. Results reveal that the Ceylon olive leaf extract and its phytochemicals, especially mearnsetin, have the potential to be used as antimelanogenesis and skin-whitening ingredients.

Transcriptome Analysis of Dnmt3l Knock-Out Mice Derived Multipotent Mesenchymal Stem/Stromal Cells During Osteogenic Differentiation.

Multipotent mesenchymal stem/stromal cells (MSCs) exhibit great potential for cell-based therapy. Proper epigenomic signatures in MSCs are important for the maintenance and the subsequent differentiation potential. The DNA methyltransferase 3-like (DNMT3L) that was mainly expressed in the embryonic stem (ES) cells and the developing germ cells plays an important role in shaping the epigenetic landscape. Here, we report the reduced colony forming ability and impaired in vitro osteogenesis in Dnmt3l-knockout-mice-derived MSCs (Dnmt3l KO MSCs). By comparing the transcriptome between undifferentiated Dnmt3l KO MSCs and the MSCs from the wild-type littermates, some of the differentially regulated genes (DEGs) were found to be associated with bone-morphology-related phenotypes. On the third day of osteogenic induction, differentiating Dnmt3l KO MSCs were enriched for genes associated with nucleosome structure, peptide binding and extracellular matrix modulation. Differentially expressed transposable elements in many subfamilies reflected the change of corresponding regional epigenomic signatures. Interestingly, DNMT3L protein is not expressed in cultured MSCs. Therefore, the observed defects in Dnmt3l KO MSCs are unlikely a direct effect from missing DNMT3L in this cell type; instead, we hypothesized them as an outcome of the pre-deposited epigenetic signatures from the DNMT3L-expressing progenitors. We observed that 24 out of the 107 upregulated DEGs in Dnmt3l KO MSCs were hypermethylated in their gene bodies of DNMT3L knock-down ES cells. Among these 24 genes, some were associated with skeletal development or homeostasis. However, we did not observe reduced bone development, or reduced bone density through aging in vivo. The stronger phenotype in vitro suggested the involvement of potential spreading and amplification of the pre-deposited epigenetic defects over passages, and the contribution of oxidative stress during in vitro culture. We demonstrated that transient deficiency of epigenetic co-factor in ES cells or progenitor cells caused compromised property in differentiating cells much later. In order to facilitate safer practice in cell-based therapy, we suggest more in-depth examination shall be implemented for cells before transplantation, even on the epigenetic level, to avoid long-term risk afterward.

Cell-intrinsic Fgf signaling contributes to primordial germ cell homing in zebrafish.

Primordial germ cells (PGCs) are specified before gastrulation and migrate toward the developing gonads. Previous in vitro studies have demonstrated a cell-intrinsic requirement of fibroblast growth factors (FGFs) by PGCs; however, no evidence suggests FGFs signal directly to PGCs in vivo. Here, using zebrafish as the animal model, we identified the mRNA expressions of Fgf receptors (Fgfrs) and determined the roles of Fgf signaling in migrating PGCs. To clarify the functions of Fgf signaling, we manipulated Fgf signaling specifically in PGCs using dominant-negative (dn) and constitutively-active (ca) Fgfrs and revealed a requirement of a basal Fgf signaling level for the robust arrival of PGCs. Repression of Fgf signaling in PGCs swayed the marginal positioning of PGCs as early as 6 h post-fertilization (6 hpf) and disrupted their arrival at the gonadal ridge at 24 hpf. On the other hand, the ectopic PGC phenotypes caused by the dn-Fgfrs could be alleviated by constitutive activation of Fgf signaling. In addition, we carefully ruled out the somatic effects in mosaic embryos by injecting RNA materials into one blastomere of the four- or eight-cell stage embryos. Injection of dn-Fgfrs into one of eight blastomeres hampered the arrival of only the treated PGCs, while the other PGCs remained unaffected. Furthermore, mosaic treatment of ca-Fgfrs rescued the ectopic rates of dn-Fgfr treated PGCs, while the other PGCs remained more ectopic within the same embryos. Interestingly, PGC-specific repression of Fgf signaling did not compromise the PGC number. To our knowledge, this is the first in vivo evidence to show that Fgf signaling plays a cell-intrinsic role in the migration of vertebrate PGCs.

The single nucleotide variant at c.662A>G in human RRM2B is a loss-of-function mutation.

BACKGROUND: Mitochondrial DNA maintenance defects (MDMDs) is one of the critical pediatric dysfunction. One of the recent report indicated that a severe patient of MDMDs carries the NP_056528.2:p.Asn221Ser (N221S) variation in the RRM2B gene (NM_015713.5). However, there is no direct evidence demonstrating the nature of the N221S variation. MATERIALS AND METHODS: This study aimed to utilize zebrafish and morpholino oligomer (MO) knockdown technique to provide direct evidence for the nature of the N221S variation in the RRM2B. RESULTS: The results showed that two distinct MOs were both able to perturb the expression of rrm2b in zebrafish and dose-dependently induced morphological defects. Furthermore, co-injection of human wild-type RRM2B mRNA with MO-e4i4 successfully rescued the developmental defects, whereas co-injection of RRM2B/N221S mRNA with MO-e4i4 did not rescue the developmental defects. CONCLUSION: In conclusion, the functional assay in this study provided the direct evidence proving that the N221S variation is a loss-of-function mutation and plausibly related to the pathogenic developmental defects found in the infants of previous clinical reports.

Chondroitin sulfate proteoglycan 4 regulates zebrafish body axis organization via Wnt/planar cell polarity pathway.

Pericellular and extracellular proteoglycans play an important role in modulating morphogen gradients and signal transductions. Chondroitin sulfate proteoglycan 4 (Cspg4) is a membrane spanning proteoglycan expressed in immature progenitor cells and cancer cells. Cspg4 participates in cellular events such as proliferation, migration and signal transduction, and these events are generally important for embryo development. In this study, we characterized Cspg4 for its roles in zebrafish embryonic development. Our results demonstrated that cspg4 was maternally expressed from 0 to 3 hours post fertilization (hpf) and expressed in the anterior and posterior embryo end after 9 hpf. Knocking-down cspg4 resulted in a shorter anterior-posterior axis than control embryo, which could be rescued by co-injecting wnt11 mRNA suggesting that Cspg4 regulates body axis organization through modulating the Wnt/planar cell polarity signaling pathway. In addition, overexpressing cspg4 caused cyclopia. The Cspg4 transmembrane domain mutant embryo phenocopied the global over-expression of cspg4 mRNA and led to cyclopia with a very low penetrance. Our results demonstrated that the quantitatively and spatially accurate distribution of Cspg4 is critical for body axis and midline development during gastrulation.

Leucine-rich repeat containing 8A contributes to the expansion of brain ventricles in zebrafish embryos.

The sodium osmotic gradient is necessary for the initiation of brain ventricle inflation, but a previous study predicted that organic and inorganic osmolytes play equivalently important roles in osmotic homeostasis in astrocytes. To test whether organic osmoregulation also plays a role in brain ventricle inflation, the core component for volume-regulated anion and organic osmolyte channel, lrrc8a, was investigated in the zebrafish model. RT-PCR and whole-mount in situ hybridization indicated that both genes were ubiquitously expressed through to 12 hpf, and around the ventricular layer of neural tubes and the cardiogenic region at 24 hpf. Knocking down either one lrrc8a paralog with morpholino oligos resulted in abnormalities in circulation at 32 hpf. Morpholino oligos or CRISPR interference against either paralog led to smaller brain ventricles at 24 hpf. Either lrrc8aa or lrrc8ab mRNA rescued the phenotypic penetrance in both lrrc8aa and lrrc8ab morphants. Supplementation of taurine in the E3 medium and overexpression csad mRNA also rescued lrrc8aa and lrrc8ab morphants. Our results indicate that the two zebrafish lrrc8a paralogs are maternal message genes and are ubiquitously expressed in early embryos. The two genes play redundant roles in the expansion of brain ventricles and the circulatory system and taurine contributes to brain ventricle expansion via the volume-regulated anion and organic osmolyte channels.

Topical application of Heparanase-1 facilitates bone remodeling during the healing of bone defects in a mouse model.

BACKGROUND: Although previous studies have suggested a stimulatory role of heparanase in physiological bone turnover, the potential therapeutic role of heparanase in bone healing has not been elucidated. The purpose of this study was to assess the effect of topical application of heparanase-1 on bone healing. METHODS: Two different dosages of recombinant mouse heparanase-1 and vehicle control were prepared and delivered via an osmotic pump to provide continuous topical infusion of the therapeutic reagent in a mouse bone defect model at the distal femoral metaphysis. The bone healing progress was evaluated by micro-computed tomography and histological examination at 7, 14, and 21 days after the bone defect was created. RESULTS: The peak of trabecular bone generation was achieved earlier than anticipated with the use of heparanase as measured by medullary bone volume fraction and trabecular number observed in micro-computed tomography, while the remodeling of trabecular bone to cortical bone was also achieved earlier than anticipated with the use of heparanase as measured by connectivity density. Histopathological observation revealed a higher frequency of the presence of cartilaginous tissue in the heparanase-treated groups. Both bone mineral density and cortical bone volume fraction showed the best healing outcome with low-dose heparanase, implying a biphasic effect of its mode of action. CONCLUSION: These results indicated that with the appropriate dose of topical heparanase-1, the progress of bone healing could be accelerated in vivo.

Temperature-induced embryonic diapause in blue-breasted quail (Coturnix chinensis) correlates with decreased mitochondrial-respiratory network and increased stress-response network.

Blue-breasted quail has been recognized as a potential model animal. The aim of this study is to investigate the low-temperature-induced embryonic diapause in blue-breasted quail. To this end, the early embryonic staging in blue-breasted quail was briefly described and various incubation temperatures were tested. While the embryonic diapause in early blue-breasted quail embryos can be induced when the eggs were stored at 21°C, a lower temperature such as 16°C yielded a significantly better hatchability (P = 0.0231). Additionally, prolonged storage duration from 3, 7 to 14 d significantly reduced the hatchability (P < 0.0001). Visual examination on the unhatched eggs revealed that reduced hatchability in prolonged storage was significantly correlated with embryonic mortality during the first half of incubation period (R2 = 0.9999, P = 0.0055). High-throughput RNA sequencing with de novo assembly showed that a gene network cluster consisted of ND4, ND5, ND6, and COX3, which are components of mitochondrial respiratory complexes, was down-regulated in the cold-stored embryos, while a stress-responsive gene network cluster consisted of JUN, ATF3, and DUSP1 was up-regulated. Accordingly, cell death in the blastoderm was significantly increased as the storage duration prolonged from 3 to 10 d. Taken together, our study provided basic information on the temperature-induced embryonic diapause in blue-breasted quail. Furthermore, transcriptomic analysis sheds light for the molecular basis on how blastoderm cells respond to the prolonged cold-stress and stay diapause.

Age, but not short-term intensive swimming, affects chondrocyte turnover in zebrafish vertebral cartilage.

Both age and intensive exercise are generally considered critical risk factors for osteoarthritis. In this work, we intend to establish zebrafish models to assess the role of these two factors on cartilage homeostasis. We designed a swimming device for zebrafish intensive exercise. The body measurements, bone mineral density (BMD) and the histology of spinal cartilages of 4- and 12-month-old zebrafish, as well the 12-month-old zebrafish before and after a 2-week exercise were compared. Our results indicate that both age and exercise affect the body length and body weight, and the micro-computed tomography reveals that both age and exercise affect the spinal BMD. However, quantitative analysis of immunohistochemistry and histochemistry indicate that short-term intensive exercise does not affect the extracellular matrix (ECM) of spinal cartilage. On the other hand, the cartilage ECM significantly grew from 4 to 12 months of age with an increase in total chondrocytes. dUTP nick end labeling staining shows that the percentages of apoptotic cells significantly increase as the zebrafish grows, whereas the BrdU labeling shows that proliferative cells dramatically decrease from 4 to 12 months of age. A 30-day chase of BrdU labeling shows some retention of labeling in cells in 4-month-old spinal cartilage but not in cartilage from 12-month-old zebrafish. Taken together, our results suggest that zebrafish chondrocytes are actively turned over, and indicate that aging is a critical factor that alters cartilage homeostasis. Zebrafish vertebral cartilage may serve as a good model to study the maturation and homeostasis of articular cartilage.

Stage-dependent piRNAs in chicken implicated roles in modulating male germ cell development.

BACKGROUND: The PIWI/piRNA pathway is a conserved machinery important for germ cell development and fertility. This piRNA-guided molecular machinery is best known for repressing derepressed transposable elements (TE) during epigenomic reprogramming. The extent to which piRNAs are involved in modulating transcripts beyond TEs still need to be clarified, and it may be a stage-dependent event. We chose chicken germline as a study model because of the significantly lower TE complexity in the chicken genome compared to mammalian species. RESULTS: We generated high-confidence piRNA candidates in various stages across chicken germline development by 3'-end-methylation-enriched small RNA sequencing and in-house bioinformatics analysis. We observed a significant developmental stage-dependent loss of TE association and a shifting of the ping-pong cycle signatures. Moreover, the stage-dependent reciprocal abundance of LINE retrotransposons, CR1-C, and its associated piRNAs implicated the developmental stage-dependent role of piRNA machinery. The stage dependency of piRNA expression and its potential functions can be better addressed by analyzing the piRNA precursors/clusters. Interestingly, the new piRNA clusters identified from embryonic chicken testes revealed evolutionary conservation between chickens and mammals, which was previously thought to not exist. CONCLUSIONS: In this report, we provided an original chicken RNA resource and proposed an analytical methodology that can be used to investigate stage-dependent changes in piRNA compositions and their potential roles in TE regulation and beyond, and also revealed possible conserved functions of piRNAs in developing germ cells.

Correction to: Stage-dependent piRNAs in chicken implicated roles in modulating male germ cell development.

Following publication of the original article [1], the authors reported that one of the authors' names is spelled incorrectly.

Endothelial-derived extracellular matrix ameliorate the stemness deprivation during ex vivo expansion of mouse bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) hold great potential in cell therapies by virtue of the regenerative effects and immunomodulatory properties, but the scarce nature of MSCs makes ex vivo expansion indispensable prior to transplantation purposes. However, potential loss of stemness ensuing culture expansion has hindered the advancements in MSCs-based treatments. In principle, stemness could be preserved by reconstructing the stem cell niche. To test whether the endothelial cells (ECs) participate in the constitution of the stem cell niche for mesenchymal stem cells (MSCs), ECs derivatives including extracellular matrix (ECM) and conditioned medium (CM) prepared from aortic endothelial cells (AECs) and Mile Sven 1 endothelial cell line (MS1) were investigated for the potential to maintain MSCs stemness. MSCs expanded on endothelial ECMs, especially on MS1-ECM, possessed a more juvenile morphology and showed delayed proliferation, when compared with untreated MSCs and MSCs on MSC-ECM and in CMs. Once induced, MS1-ECM group showed better tri-lineage differentiations indicating that MS1-ECM could better preserve MSC stemness. MSCs on MS1-ECM showed stronger immune-modulatory potential and had significantly higher H3K27me3 with lower Kdm6b expression. Taken together, MS1-ECM shapes an inhibitory chromatin signature and retains MSCs stemness. Our work provided supportive evidence that MSCs can reside in a perivascular niche, and a feasible novel approach for MSCs expansion.

Outbreak of thiamine deficiency in cats associated with the feeding of defective dry food.

Objectives The objective of this study was to determine disease progression, association between neurological signs and magnetic resonance imaging (MRI) findings, and long-term outcome in feline thiamine deficiency associated with defective dry food. Methods The clinical records of 17 cats diagnosed with thiamine deficiency related to a defective dry food were examined and data collected. The thiamine level in the food was analysed by liquid chromatography-tandem mass spectrometry. Results The thiamine level in the food was below the recommendation of the National Research Council. Fifteen cats were fed the food exclusively. Prior to the acute development of neurological signs, most cats displayed non-specific signs such as anorexia, lethargy or vomiting. Vestibular signs of varying severity were observed in 94% of the cats, and all but one of these presented with bilateral dysfunction. Other main neurological signs included altered mentation (76%), blindness (59%) and seizures (59%). Moreover, 80% of the cats with seizures presented with cluster seizures or status epilepticus. MRI abnormalities consistent with findings reported in the previous literature were detected in five cases. MRI was unremarkable in one cat with ongoing severe neurological signs even though thiamine had been administered. Most surviving cats recovered rapidly within 2 weeks of treatment and had either returned to normal or had minimal neurological signs at the 2 month follow-up. One cat recovered slowly over 6 months. Most cats with seizures in the initial stage of the disease remained seizure free at the 24 month follow-up. Conclusions and relevance This study documented the association between feline thiamine deficiency and defective dry food. MRI examination provided valuable information in the diagnosis. However, normal MRI findings do not exclude the diagnosis of feline thiamine deficiency, especially once thiamine has been supplemented. MRI findings also may not always reflect the neurological status or severity. If treated promptly, most cats will recover rapidly with a good outcome. Occasionally, recovery may be slow and take several months.

Sterol O-Acyltransferase 2 Contributes to the Yolk Cholesterol Trafficking during Zebrafish Embryogenesis.

To elucidate whether Sterol O-acyltransferase (Soat) mediates the absorption and transportation of yolk lipids to the developing embryo, zebrafish soat1 and soat2 were cloned and studied. In the adult zebrafish, soat1 was detected ubiquitously while soat2 mRNA was detected specifically in the liver, intestine, brain and testis. Whole mount in situ hybridization demonstrated that both soat1 and soat2 expressed in the yolk syncytial layer, hatching gland and developing cardiovascular as well as digestive systems, suggesting that Soats may play important roles in the lipid trafficking and utilization during embryonic development. The enzymatic activity of zebrafish Soat2 was confirmed by Oil Red O staining in the HEK293 cells overexpressing this gene, and could be quenched by Soat2 inhibitor Pyripyropene A (PPPA). The zebrafish embryos injected with PPPA or morpholino oligo against soat2 in the yolk showed significantly larger yolk when compared with wild-type embryos, especially at 72 hpf, indicating a slower rate of yolk consumption. Our result indicated that zebrafish Soat2 is catalytically active in synthesizing cholesteryl esters and contributes to the yolk cholesterol trafficking during zebrafish embryogenesis.

Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy.

Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

BACKGROUND: Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. RESULTS: We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. CONCLUSION: We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.