ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.
Sonic hedgehog (SHH) is a vertebrate homologue of the secreted Drosophila protein hedgehog and is expressed by the notochord and floor plate in the developing spinal cord. Sonic hedgehog provides signals relevant for positional information, cell proliferation and possibly cell survival, depending on the time and location of expression. Although the role of SHH in providing positional information in the neural tube has been experimentally proven, the underlying mechanism remains unclear. In this study, in ovo electroporation was employed in the chicken spinal cord during chicken embryo development. Electroporation was conducted at stage 17 (E2.5), after electroporation the embryos were continued incubating to stage 28 (E6) for sampling, tissue fixation with 4% paraformaldehyde and frozen sectioning. Sonic hedgehog and related protein expressions were detected by in situ hybridization and fluorescence immunohistochemistry and the results were analysed after microphotography. Our results indicate that the ectopic expression of SHH leads to ventralization in the spinal cord during chicken embryonic development by inducing abnormalities in the structure of the motor column and motor neuron integration. In addition, ectopic SHH expression inhibits the expression of dorsal transcription factors and commissural axon projections. The correct location of SHH expression is vital to the formation of the motor column. Ectopic expression of SHH in the spinal cord not only affects the positioning of motor neurons, but also induces abnormalities in the structure of the motor column. It leads to ventralization in the spinal cord, resulting in the formation of more ventral neurons forming during neuronal formation.
Chicken embryos are used widely in the fields of developmental biology and neurobiology. The chicken embryo also serves as a model to analyze gene expression and function using in ovo electroporation. Plasmids may be injected into the spinal cord or tectum of the chicken central nervous system by microinjection for electroporation. Here, we developed a novel method that combines in ovo electroporation and neuronal culturing to study gene function in the chicken tectum during embryo development. Our method can be used to study in-vivo and in-vitro exogenous genes' function. In addition, live cell imaging microscopy, immunostaining, and transfection can be used with our method to study neuronal growth, development, neurite growth and retraction, and axonal pathfinding. Our result showed that axons were present in isolated neurons after culturing for 24 h, and cell debris was low after replacing the media at 48 h. Many GFP-expressing neurons were observed in the cultured cells after 48 h. We successfully cultured the neurons for 3 weeks. Together, this method combines in ovo electroporation and neuronal culturing advantages and is more convenient for the gene function analysis.
KEY MESSAGE: Three tau class MaGSTs responded to abiotic stress, MaGSTF1 and MaGSTL1 responded to signaling molecules, they may play an important role in the growth of banana plantlet. Glutathione S-transferases (GST) are multifunctional detoxification enzymes that participate in a variety of cellular processes, including stress responses. In this study, we report the molecular characteristics of five GST genes (MaGSTU1, MaGSTU2, MaGSTU3, MaGSTF1 and MaGSTL1) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these GSTs range from 23.4 to 27.7 kDa and their pIs are acidic. At the amino acid level, they share high sequence similarity with GSTs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaGSTs also have high similarity to GSTs of other plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. In addition, their expression is regulated by various stress conditions, including exposure to signaling molecules, cold, salinity, drought and Fusarium oxysporum f specialis(f. Sp) cubense Tropical Race 4 (Foc TR4) infection. The expression of the tau class MaGSTs (MaGSTU1, MaGSTU2 and MaGSTU3) mainly responded to cold, salinity and drought while MaGSTF1 and MaGSTL1 expressions were upregulated by signaling molecules. Our findings suggest that MaGSTs play a key role in both development and abiotic stress responses.
Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.
RU486 is a partial progesterone and estrogen receptor antagonist, functioning to actively silence progesterone receptor gene-associated transcription. For this reason, it has been used as both a contraceptive and an abortive agent. In the present study, cellular and gene specific effects of RU486 were investigated in a rat model of early pregnancy, including key phases of the window of receptivity and early implantation. As these stages are hormonally regulated by progesterone and estrogens, the focus here was to elucidate the mechanism of action of a single dose of RU486, used as a postcoital contraceptive, to successfully prevent implantation of a viable blastocyst. Immunofluorescent techniques were used to examine the change in protein levels of PR in RU486-treated endometria at days 4.5, 5.5 and 6.5 of pregnancy. Changes in the Pgr gene expression level as a consequence of RU486 administration was evaluated using quantitative real-time reverse transcription polymerase chain reaction. The progesterone receptor gene and protein expression was ubiquitously decreased throughout pregnancy as a direct consequence of RU486 administration. The overall effects of postcoital RU486 administration during early pregnancy indicate highly effective inhibition of progesterone and estrogen effects on the endometrium, mediated by their receptors. More specifically, the expression and localization of the progesterone receptor mirrors that described in ovariectomized animal models, suggesting a hormonally under-stimulated endometrium. Clearly from the present study, the precise priming of the endometrium by progesterone, in preparation for blastocyst implantation, is severely impaired by RU486, thus predisposing the uterus to pregnancy failure.
pbx1, a TALE (three-amino acid loop extension) homeodomain transcription factor, is involved in a diverse range of developmental processes. We examined the expression of pbx1 during zebrafish development by in situ hybridization. pbx1 transcripts could be detected in the central nervous system and pharyngeal arches from 24 hpf onwards. In the swim bladder anlage, pbx1 was detected as early as 28 hpf, making it the earliest known marker for this organ. Morpholino-mediated gene knockdown of pbx1 revealed that the swim bladder failed to inflate, with eventual lethality occurring by 8 dpf. The knockdown of pbx1 did not perturb the expression of prdc and foxA3, with both early swim bladder markers appearing normally at 36 and 48 hpf, respectively. However, the expression of anxa5 was completely abolished by pbx1 knockdown at 60 hpf suggesting that pbx1 may be required during the late stage of swim bladder development.
To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.
The front-end desaturases (Fads) are rate-limiting enzymes responsible for production of long-chain polyunsaturated fatty acids (LC-PUFA). The full spectrum of the transcriptional regulation of fads is still incomplete, as cloning of fads promoter is limited to a few species. Here, we described the cloning and characterisation of the zebrafish fads2 promoter. Using 5'-deletion and mutation analysis on this promoter, we identified a specific region containing the sterol regulatory element (SRE) which is responsible for the activation of the fads2 promoter. In tandem, two conserved CCAAT boxes were also present adjacent to the SRE and mutation of either of these binding sites attenuates the transcriptional activation of the fads2 promoter. An in vivo analysis employing GFP reporter gene in transiently transfected zebrafish embryos showed that this 1754 bp upstream region of the fads2 gene specifically directs GFP expression in the yolk syncytial layer (YSL) region. This indicates a role for LC-PUFA in the transport of yolk lipids through this tissue layer. In conclusion, besides identifying novel core elements for transcriptional activation in zebrafish fads2 promoter, we also reveal a potential role for fads2 or LC-PUFA in YSL during development.
Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.
Advanced paternal age at fertilization is a risk factor for multiple disorders in offspring and may be linked to age-related epigenetic changes in the father's sperm. An understanding of aging-related epigenetic changes in sperm and environmental factors that modify such changes is needed. Here, we characterize changes in sperm small non-coding RNA (sncRNA) between young pubertal and mature rats. We also analyze the modification of these changes by exposure to environmental xenobiotic 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). sncRNA libraries prepared from epididymal spermatozoa were sequenced and analyzed using DESeq 2. The distribution of small RNA fractions changed with age, with fractions mapping to rRNA and lncRNA decreasing and fractions mapping to tRNA and miRNA increasing. In total, 249 miRNA, 908 piRNA and 227 tRNA-derived RNA were differentially expressed (twofold change, false discovery rate (FDR) p ≤ 0.05) between age groups in control animals. Differentially expressed miRNA and piRNA were enriched for protein-coding targets involved in development and metabolism, while piRNA were enriched for long terminal repeat (LTR) targets. BDE-47 accelerated age-dependent changes in sncRNA in younger animals, decelerated these changes in older animals and increased the variance in expression of all sncRNA. Our results indicate that the natural aging process has profound effects on sperm sncRNA profiles and this effect may be modified by environmental exposure.
Postnatal treatment with selective serotonin reuptake inhibitors (SSRIs) has been found to affect brain development and the regulation of reproduction in rodent models. The normal masculinization process in the brain requires a transient decrease in serotonin (5-HT) levels in the brain during the second postnatal week. Strict regulation of androgen receptor (AR) and gonadotropin-releasing hormone (GnRH) expression is important to control male reproductive activity. Therefore, this study was designed to examine the effects of a potent SSRI (citalopram) on male sexual behavior and expression levels of AR and GnRH in adult male mice receiving either vehicle or citalopram (10mg/kg) daily during postnatal days 8-21. The citalopram-treated male mice showed altered sexual behavior, specifically a significant reduction in the number of intromissions preceding ejaculation compared with the vehicle-treated mice. The citalopram-treated male mice displayed elevated anxiety-like behavior in an open field test and lower locomotor activity in their home cage during the subjective night. Although there was no change in GnRH and AR mRNA levels in the preoptic area (POA), quantified by real-time polymerase chain reaction, immunostained AR cell numbers in the medial POA were decreased in the citalopram-treated male mice. These results suggest that the early-life inhibition of 5-HT transporters alters the regulation of AR expression in the medial POA, likely causing decreased sexual behavior and altered home cage activity in the subjective night.
Oil palm, a plantation crop of major economic importance in Southeast Asia, is the predominant source of edible oil worldwide. We report the identification of the virescens (VIR) gene, which controls fruit exocarp colour and is an indicator of ripeness. VIR is a R2R3-MYB transcription factor with homology to Lilium LhMYB12 and similarity to Arabidopsis production of anthocyanin pigment1 (PAP1). We identify five independent mutant alleles of VIR in over 400 accessions from sub-Saharan Africa that account for the dominant-negative virescens phenotype. Each mutation results in premature termination of the carboxy-terminal domain of VIR, resembling McClintock's C1-I allele in maize. The abundance of alleles likely reflects cultural practices, by which fruits were venerated for magical and medicinal properties. The identification of VIR will allow selection of the trait at the seed or early-nursery stage, 3-6 years before fruits are produced, greatly advancing introgression into elite breeding material.
Pieris rapae is a serious pest of brassicas worldwide. We performed de novo assembly of P. rapae transcriptome by next-generation sequencing and assembled approximately 65,727,422 clean paired-end reads into 32,118 unigenes, of which 13,585 were mapped to 255 pathways in the KEGG database. A total of 6173 novel transcripts were identified from reads directly mapped to P. rapae genome. Additionally, 1490 SSRs, 301,377 SNPs, and 29,284 InDels were identified as potential molecular markers to explore polymorphism within P. rapae populations. We screened and mapped 36 transcripts related to OBP, CSP, SNMP, PBAN, and OR. We analyzed the expression profiles of 7 selected genes involved in pheromone transport and degradation by quantitative real-time PCR; these genes are sex-specific and differentially expressed in the developmental stages. Overall, the comprehensive transcriptome resources described in this study could help understand and identify molecular targets particularly reproduction-related genes for developing effective P. rapae management tools.
Hypothyroidism has been linked to infertility, but the mechanisms underlying infertility-related hypothyroidism have yet to be fully elucidated. Therefore, in this study, effects of hypothyroidism on expression of the proteins related to thyroid hormone function in the uterus, which were thought to play a role implantation, including thyroid hormone receptor (TR), thyroid stimulating hormone receptor (TSHR), retinoic acid receptor (RAR) and extracellular kinase (ERK) were identified. Pregnant female rats were rendered hypothyroid by giving methimazole (MMI), orally. Following hypothyroid induction, rats were grouped into control (non-treated) and received subcutaneous thyroxine at 20, 40, and 80 μg/kg/day for five consecutive days. At Day 6, which is the day of implantation (GD 6), rats were sacrificed and the number of embryo implantation site in the uterus was calculated. Then, uterine horns were harvested and expression of the above proteins and their mRNAs were identified by Western blotting and real-time PCR, respectively. In non-treated hypothyroid pregnant rats, the number of embryo implantation sites decreased as compared to euthyroid and hypothyroid rats receiving thyroxine treatment. Similarly, expression of TRα-1, TRβ-1, TSHR, ERK1/2 and RAR proteins and mRNA in the uterus of non-treated hypothyroid rats also decreased (P
Advancement in materials synthesis largely depends up on their diverse applications and commercialisation. Antifungal effects of phytogenic silver nanoparticles (AgNPs) were evident, but the reports on the effects of the same on agricultural crops are scant. Herein, we report for the first time, size dependent effects of phytogenic AgNPs (synthesised using Stevia rebaudiana leaf extract) on the germination, growth and biochemical parameters of three important agricultural crops viz., rice (Oryza sativa L), maize (Zea mays L) and peanut (Arachis hypogaea L). AgNPs with varied sizes were prepared by changing the concentration and quantity of the Stevia rebaudiana leaf extract. As prepared AgNPs were characterized using the techniques, such as high-resolution transmission electron microscopy, particle size and zeta potential analyser. The measured (dynamic light scattering technique) average sizes of particles are ranging from 68.5 to 116 nm. Fourier transform infrared studies confirmed the participation of alcohols, aldehydes and amides in the reduction and stabilisation of the AgNPs. Application of these AgNPs to three agricultural crop seeds (rice, maize and peanut) resulted in size dependent effects on their germination, growth and biochemical parameters such as, chlorophyll content, carotenoid and protein content. Further, antifungal activity of AgNPs also evaluated against fungi, Aspergillus niger.
The striatum is considered to be the central processing unit of the basal ganglia in locomotor activity and cognitive function of the brain. IGF-1 could act as a control switch for the long-term proliferation and survival of EGF+bFGF-responsive cultured embryonic striatal stem cell (ESSC), while LIF imposes a negative impact on cell proliferation. The IGF-1-treated ESSCs also showed elevated hTERT expression with demonstration of self-renewal and trilineage commitment (astrocytes, oligodendrocytes, and neurons). In order to decipher the underlying regulatory microRNA (miRNA)s in IGF-1/LIF-treated ESSC-derived neurogenesis, we performed in-depth miRNA profiling at 12 days in vitro and analyzed the candidates using the Partek Genome Suite software. The annotated miRNA fingerprints delineated the differential expressions of miR-143, miR-433, and miR-503 specific to IGF-1 treatment. Similarly, the LIF-treated ESSCs demonstrated specific expression of miR-326, miR-181, and miR-22, as they were nonsignificant in IGF-treated ESSCs. To elucidate the possible downstream pathways, we performed in silico mapping of the said miRNAs into ingenuity pathway analysis. Our findings revealed the important mRNA targets of the miRNAs and suggested specific interactomes. The above studies introduced a new genre of miRNAs for ESSC-based neuroregenerative therapeutic applications.
Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species.
Cell-based therapy using stem cells has emerged as one of the pro-angiogenic methods to enhance blood vessel growth and sprouting in ischaemic conditions. This study investigated the endogenous and induced angiogenic characteristics of hCDSC (human chorion-derived stem cell) using QPCR (quantitative PCR) method, immunocytochemistry and fibrin-matrigel migration assay. The results showed that cultured hCDSC endogenously expressed angiogenic-endogenic-associated genes (VEGF, bFGF, PGF, HGF, Ang-1, PECAM-1, eNOS, Ve-cad, CD34, VEGFR-2 and vWF), with significant increase in mRNA levels of PGF, HGF, Ang-1, eNOS, VEGFR-2 and vWF following induction by bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial growth factor). These enhanced angiogenic properties suggest that induced hCDSC provides a stronger angiogenic effect for the treatment of ischaemia. After angiogenic induction, hCDSC showed no reduction in the expression of the stemness genes, but had significantly higher levels of mRNA of Oct-4, Nanog (3), FZD9, ABCG-2 and BST-1. The induced cells were positive for PECAM-1 (platelet/endothelial cell adhesion molecule 1) and vWF (von Willebrand factor) with immunocytochemistry staining. hCDSC also showed endothelial migration behaviour when cultured in fibrin-matrigel construct and were capable of forming vessels in vivo after implanting into nude mice. These data suggest that hCDSC could be the cells of choice in the cell-based therapy for pro-angiogenic purpose.