Temporal Patterns Of Gene Expression Research Articles

Dissecting the contributions of time and microbe density to variation in immune gene expression.

Widespread differential expression of immunological genes is a hallmark of the response to infection in almost all surveyed taxa. However, several challenges remain in the attempt to connect differences in gene expression with functional outcomes like parasite killing and host survival. For example, temporal gene expression patterns are not always monotonic (unidirectional slope), yielding results that qualitatively depend on the time point selected for analysis. They may also be correlated to microbe density, confounding the strength of an immune response and resistance to parasites. In this study, we analyse these relationships in an mRNA-seq time series of Tribolium castaneum infected with Bacillus thuringiensis Our results suggest that many extracellular immunological components with known roles in immunity, like antimicrobial peptides and recognition proteins, are highly correlated to microbe load. On the other hand, intracellular components of immunological signalling pathways overwhelmingly show non-monotonic temporal patterns of gene expression, despite the underlying assumption of monotonicity in most ecological and comparative transcriptomics studies that rely on cross-sectional analyses. Our results raise a host of new questions, including to what extent variation in host resistance, infection tolerance and immunopathology can be explained by variation in the slope or sensitivity of these newly characterized patterns.

IWP2 impairs the development of porcine somatic cell nuclear transfer embryos via Wnt signaling pathway inactivation.

Wnt signaling is critical in embryonic development and post-embryonic tissue homeostasis. The aim of the present study was to evaluate the expression levels of canonical Wnt signaling genes in porcine somatic cell nuclear transfer (SCNT) embryos. Quantitative polymerase chain reaction analysis was performed in porcine SCNT embryos, and the results indicated that the temporal expression patterns of canonical signaling genes were similar between in vivo and SCNT embryos from the 2-cell to the blastocyst stage. In addition, aberrant expression in a small number of Wnt signaling genes in SCNT embryos was identified. IWP2, an inhibitor of Wnt processing, was applied to the culture of SCNT embryos. The Wnt signaling pathway in the SCNT blastocysts may be inactivated via IWP2 treatment, reflecting the low expression levels of c-Myc and peroxisome proliferator-activated receptor δ. Furthermore, blastocyst hatching was damaged by IWP2 treatment. These findings indicate that the canonical Wnt signaling pathway is important for SCNT embryo development.

Spatial and temporal expression patterns of genes around nine neuroticism-associated loci

Neuroticism is a high-order personality trait. Individuals with higher neuroticism have increased risks of various psychiatric disorders and physical health outcomes. Neuroticism is related to physiological differences in the brain. A recent genome-wide association study identified nine distinct genomic loci that contribute to neuroticism. Brain development and function depend on the precise regulation of gene expression, which is differentially regulated across brain regions and developmental stages. Using multiple publicly available human post-mortem databases, we investigated, in brain and non-brain tissues and across several developmental life stages, the spatial and temporal expression patterns of genes arising from nine neuroticism-associated loci. Functional gene-network analysis for neuroticism-associated genes was performed. The spatial expression analysis revealed that the nearest genes (GRIK3, SRP9, KLHL2, PTPRD, ELAVL2, CRHR1 and CELF4) from index single-nucleotide polymorphisms (SNPs) at the nine loci were intensively enriched in the brain compared with their representation in non-brain tissues (p<1.56×10−3). The nearest genes associated with the glutamate receptor activity network consisted mainly of GRIK3 (FDR q=4.25×10−2). The temporal expression analysis revealed that the neuroticism-associated genes were divided into three expression patterns: KLHL2, CELF4 and CRHR1 were preferentially expressed during postnatal stages; PTPRD, ELAVL2 and MFHAS1 were expressed during prenatal stages; and the other three genes were not expressed during specific life stages. These findings suggest that the glutamate network might be a target for investigating the neurobiological mechanisms underlying susceptibilities to higher neuroticism and several psychiatric disorders and that neuroticism is mediated by genes specifically expressed in the brain during several developmental stages.

Overexpression of ginseng UGT72AL1 causes organ fusion in the axillary leaf branch of Arabidopsis

BackgroundGlycosylation of natural compounds increases the diversity of secondary metabolites. Glycosylation steps are implicated not only in plant growth and development, but also in plant defense responses. Although the activities of uridine-dependent glycosyltransferases (UGTs) have long been recognized, and genes encoding them in several higher plants have been identified, the specific functions of UGTs in planta remain largely unknown. MethodsSpatial and temporal patterns of gene expression were analyzed by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and GUS histochemical assay. In planta transformation in heterologous Arabidopsis was generated by floral dipping using Agrobacterium tumefaciens (C58C1). Protein localization was analyzed by confocal microscopy via fluorescent protein tagging. ResultsPgUGT72AL1 was highly expressed in the rhizome, upper root, and youngest leaf compared with the other organs. GUS staining of the promoter: GUS fusion revealed high expression in different organs, including axillary leaf branch. Overexpression of PgUGT72AL1 resulted in a fused organ in the axillary leaf branch. ConclusionPgUGT72AL1, which is phylogenetically close to PgUGT71A27, is involved in the production of ginsenoside compound K. Considering that compound K is not reported in raw ginseng material, further characterization of this gene may shed light on the biological function of ginsenosides in ginseng plant growth and development. The organ fusion phenotype could be caused by the defective growth of cells in the boundary region, commonly regulated by phytohormones such as auxins or brassinosteroids, and requires further analysis.

Genome-Wide Analysis of the Sucrose Synthase Gene Family in Grape (Vitis vinifera): Structure, Evolution, and Expression Profiles.

Sucrose synthase (SS) is widely considered as the key enzyme involved in the plant sugar metabolism that is critical to plant growth and development, especially quality of the fruit. The members of SS gene family have been identified and characterized in multiple plant genomes. However, detailed information about this gene family is lacking in grapevine (Vitis vinifera L.). In this study, we performed a systematic analysis of the grape (V. vinifera) genome and reported that there are five SS genes (VvSS1–5) in the grape genome. Comparison of the structures of grape SS genes showed high structural conservation of grape SS genes, resulting from the selection pressures during the evolutionary process. The segmental duplication of grape SS genes contributed to this gene family expansion. The syntenic analyses between grape and soybean (Glycine max) demonstrated that these genes located in corresponding syntenic blocks arose before the divergence of grape and soybean. Phylogenetic analysis revealed distinct evolutionary paths for the grape SS genes. VvSS1/VvSS5, VvSS2/VvSS3 and VvSS4 originated from three ancient SS genes, which were generated by duplication events before the split of monocots and eudicots. Bioinformatics analysis of publicly available microarray data, which was validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct temporal and spatial expression patterns of VvSS genes in various tissues, organs and developmental stages, as well as in response to biotic and abiotic stresses. Taken together, our results will be beneficial for further investigations into the functions of SS gene in the processes of grape resistance to environmental stresses.

Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.

Comparative transcriptome analyses of flower development in four species of Achimenes (Gesneriaceae)

BackgroundFlowers have an amazingly diverse display of colors and shapes, and these characteristics often vary significantly among closely related species. The evolution of diverse floral form can be thought of as an adaptive response to pollination and reproduction, but it can also be seen through the lens of morphological and developmental constraints. To explore these interactions, we use RNA-seq across species and development to investigate gene expression and sequence evolution as they relate to the evolution of the diverse flowers in a group of Neotropical plants native to Mexico—magic flowers (Achimenes, Gesneriaceae).ResultsThe assembled transcriptomes contain between 29,000 and 42,000 genes expressed during development. We combine sequence orthology and coexpression clustering with analyses of protein evolution to identify candidate genes for roles in floral form evolution. Over 25% of transcripts captured were distinctive to Achimenes and overrepresented by genes involved in transcription factor activity. Using a model-based clustering approach we find dynamic, temporal patterns of gene expression among species. Selection tests provide evidence of positive selection in several genes with roles in pigment production, flowering time, and morphology. Combining these approaches to explore genes related to flower color and flower shape, we find distinct patterns that correspond to transitions of floral form among Achimenes species.ConclusionsThe floral transcriptomes developed from four species of Achimenes provide insight into the mechanisms involved in the evolution of diverse floral form among closely related species with different pollinators. We identified several candidate genes that will serve as an important and useful resource for future research. High conservation of sequence structure, patterns of gene coexpression, and detection of positive selection acting on few genes suggests that large phenotypic differences in floral form may be caused by genetic differences in a small set of genes. Our characterized floral transcriptomes provided here should facilitate further analyses into the genomics of flower development and the mechanisms underlying the evolution of diverse flowers in Achimenes and other Neotropical Gesneriaceae.

Exploratory bioinformatics investigation reveals importance of \u201cjunk\u201d DNA in early embryo development

BackgroundInstead of testing predefined hypotheses, the goal of exploratory data analysis (EDA) is to find what data can tell us. Following this strategy, we re-analyzed a large body of genomic data to study the complex gene regulation in mouse pre-implantation development (PD).ResultsStarting with a single-cell RNA-seq dataset consisting of 259 mouse embryonic cells derived from zygote to blastocyst stages, we reconstructed the temporal and spatial gene expression pattern during PD. The dynamics of gene expression can be partially explained by the enrichment of transposable elements in gene promoters and the similarity of expression profiles with those of corresponding transposons. Long Terminal Repeats (LTRs) are associated with transient, strong induction of many nearby genes at the 2-4 cell stages, probably by providing binding sites for Obox and other homeobox factors. B1 and B2 SINEs (Short Interspersed Nuclear Elements) are correlated with the upregulation of thousands of nearby genes during zygotic genome activation. Such enhancer-like effects are also found for human Alu and bovine tRNA SINEs. SINEs also seem to be predictive of gene expression in embryonic stem cells (ESCs), raising the possibility that they may also be involved in regulating pluripotency. We also identified many potential transcription factors underlying PD and discussed the evolutionary necessity of transposons in enhancing genetic diversity, especially for species with longer generation time. ConclusionsTogether with other recent studies, our results provide further evidence that many transposable elements may play a role in establishing the expression landscape in early embryos. It also demonstrates that exploratory bioinformatics investigation can pinpoint developmental pathways for further study, and serve as a strategy to generate novel insights from big genomic data.

Mapping Gene Expression in Excitatory Neurons during Hippocampal Late-Phase Long-Term Potentiation.

The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation.

Posterior tail development in the salamander Eurycea cirrigera: exploring cellular dynamics across life stages.

During embryogenesis, the body axis elongates and specializes. In vertebrate groups such as salamanders and lizards, elongation of the posterior body axis (tail) continues throughout life. This phenomenon of post-embryonic tail elongation via addition of vertebrae has remained largely unexplored, and little is known about the underlying developmental mechanisms that promote vertebral addition. Our research investigated tail elongation across life stages in a non-model salamander species, Eurycea cirrigera (Plethodontidae). Post-embryonic addition of segments suggests that the tail tip retains some aspects of embryonic cell/tissue organization and gene expression throughout the life cycle. We describe cell and tissue differentiation and segmentation of the posterior tail using serial histology and expression of the axial tissue markers, MF-20 and Pax6. Embryonic expression patterns of HoxA13 and C13 are shown with in situ hybridization. Tissue sections reveal that the posterior spinal cord forms via cavitation and precedes development of the underlying cartilaginous rod after embryogenesis. Post-embryonic tail elongation occurs in the absence of somites and mesenchymal cells lateral to the midline express MF-20. Pax6 expression was observed only in the spinal cord and some mesenchymal cells of adult Eurycea tails. Distinct temporal and spatial patterns of posterior Hox13 gene expression were observed throughout embryogenesis. Overall, important insights to cell organization, differentiation, and posterior Hox gene expression may be gained from this work. We suggest that further work on gene expression in the elongating adult tail could shed light on mechanisms that link continual axial elongation with regeneration.

Early Intra-Amniotic Gene Transfer using Lentiviral Vectors in Murine Model

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this presentation, we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8-10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

Somitogenesis and Axial Development in Reptiles.

Among amniote vertebrates, reptiles display the greatest variation in axial skeleton morphology. Only recently have they been used in gene expression studies of somitogenesis , challenging previous assumptions about the segmentation clock and axial patterning. An increasing number of reptile genomes and transcriptomes are becoming available as next-generation sequencing becomes more affordable. Information regarding gene sequence and structure can be used to design and synthesize labeled riboprobes by in vitro transcription for gene expression analysis by in situ hybridization, thus, enabling the characterization of spatial and temporal expression patterns of genes involved in somitogenesis, a topic of great interest within evolutionary developmental studies of vertebrates.

A complex network of molecular events triggered upon environmental cues which decide the fate of gene expression: a review

Gene expression in eukaryotes depends on a web of events which are inter-related and tightly regulated. These key events can be broadly classified into post-transcriptional and post-translational processes. In general, the post-transcriptional events include pre-mRNA processing (capping, splicing, polyadenylation), RNA stability, and translation as well as chromatin modifications through regulatory RNAs (miRNA, siRNAs and long non-coding RNAs). Protein phosphorylation, ubiquitination and sumoylation are a few post-translational events. These events are constitutive as well as provoked by specific exogenous and/or endogenous stimuli. In case of the plant system, the molecular mechanisms responsible for regulating the gene expression is diverse and yet to be fully revealed. The network of post-transcriptional and post-translational events do ensure temporal and spatial suitable patterns of downstream stress-related gene expression. The current review mainly focus on a variety of molecular events which play a pivotal role in fine tuning the gene expression in eukaryotes.

The developmental transcriptome atlas of the biofuel crop Camelina sativa.

Camelina sativa is currently being embraced as a viable industrial bio-platform crop due to a number of desirable agronomic attributes and the unique fatty acid profile of the seed oil that has applications for food, feed and biofuel. The recent completion of the reference genome sequence of C.sativa identified a young hexaploid genome. To complement this work, we have generated a genome-wide developmental transcriptome map by RNA sequencing of 12 different tissues covering major developmental stages during the life cycle of C.sativa. We have generated a digital atlas of this comprehensive transcriptome resource that enables interactive visualization of expression data through a searchable database of electronic fluorescent pictographs (eFP browser). An analysis of this dataset supported expression of 88% of the annotated genes in C.sativa and provided a global overview of the complex architecture of temporal and spatial gene expression patterns active during development. Conventional differential gene expression analysis combined with weighted gene expression network analysis uncovered similarities as well as differences in gene expression patterns between different tissues and identified tissue-specific genes and network modules. A high-quality census of transcription factors, analysis of alternative splicing and tissue-specific genome dominance provided insight into the transcriptional dynamics and sub-genome interplay among the well-preserved triplicated repertoire of homeologous loci. The comprehensive transcriptome atlas in combination with the reference genome sequence provides a powerful resource for genomics research which can be leveraged to identify functional associations between genes and understand the regulatory networks underlying developmental processes.

Unravelling molecular mechanisms from floral initiation to lipid biosynthesis in a promising biofuel tree species, Pongamia pinnata using transcriptome analysis.

Pongamia pinnata (L.) (Fabaceae) is a promising biofuel tree species which is underexploited in the areas of both fundamental and applied research, due to the lack of information either on transcriptome or genomic data. To investigate the possible metabolic pathways, we performed whole transcriptome analysis of Pongamia through Illumina NextSeq platform and generated 2.8 GB of paired end sequence reads. The de novo assembly of raw reads generated 40,000 contigs and 35,000 transcripts, representing leaf, flower and seed unigenes. Spatial and temporal expression profiles of photoperiod and floral homeotic genes in Pongamia, identified GIGANTEA (GI) - CONSTANS (CO) - FLOWERING LOCUS T (FT) as active signal cascade for floral initiation. Four prominent stages of seed development were selected in a high yielding Pongamia accession (TOIL 1) to follow the temporal expression patterns of key fatty acid biosynthetic genes involved in lipid biosynthesis and accumulation. Our results provide insights into an array of molecular events from flowering to seed maturity in Pongamia which will provide substantial basis for modulation of fatty acid composition and enhancing oil yields which should serve as a potential feedstock for biofuel production.

Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

Temporal dynamics of the developing lung transcriptome in three common inbred strains of laboratory mice reveals multiple stages of postnatal alveolar development.

To characterize temporal patterns of transcriptional activity during normal lung development, we generated genome wide gene expression data for 26 pre- and post-natal time points in three common inbred strains of laboratory mice (C57BL/6J, A/J, and C3H/HeJ). Using Principal Component Analysis and least squares regression modeling, we identified both strain-independent and strain-dependent patterns of gene expression. The 4,683 genes contributing to the strain-independent expression patterns were used to define a murine Developing Lung Characteristic Subtranscriptome (mDLCS). Regression modeling of the Principal Components supported the four canonical stages of mammalian embryonic lung development (embryonic, pseudoglandular, canalicular, saccular) defined previously by morphology and histology. For postnatal alveolar development, the regression model was consistent with four stages of alveolarization characterized by episodic transcriptional activity of genes related to pulmonary vascularization. Genes expressed in a strain-dependent manner were enriched for annotations related to neurogenesis, extracellular matrix organization, and Wnt signaling. Finally, a comparison of mouse and human transcriptomics from pre-natal stages of lung development revealed conservation of pathways associated with cell cycle, axon guidance, immune function, and metabolism as well as organism-specific expression of genes associated with extracellular matrix organization and protein modification. The mouse lung development transcriptome data generated for this study serves as a unique reference set to identify genes and pathways essential for normal mammalian lung development and for investigations into the developmental origins of respiratory disease and cancer. The gene expression data are available from the Gene Expression Omnibus (GEO) archive (GSE74243). Temporal expression patterns of mouse genes can be investigated using a study specific web resource (http://lungdevelopment.jax.org).

Genome-Wide Discovery of Tissue-Specific Genes in Maize

Analysis of spatial and temporal gene expression pattern is instrumental to elucidation of gene networks and molecular mechanisms of tissue development. It also holds great value to applied research by providing tissue-specific (TS) promoter candidates for transgenic studies. Here, we present a large-scale systematic discovery of tissue-specific genes in maize. Profiles of TS genes were generated from a maize transcriptome atlas, with 71, 539, 23, 352, 51, and 287 genes showing expression specificity in the root, leaf, cob, endosperm, silk, and anther. Functional annotations and enrichment analysis of these TS genes identified pathways overrepresented for each tissue. Tissue specificity was experimentally confirmed by RT-PCR, mRNA in situ hybridization, and transgenic expression of promoter-fluorescent proteins. Two significantly enriched binding motifs, CATTGYCG and KGGTATCA, were identified from the promoter analysis of the anther- and endosperm-specifically expressed genes. Further, co-expression analysis on a broader set of maize germplasms identified molecular networks of TS genes and revealed a number of novel transcripts including non-coding RNA expressing the same pattern with the TS genes from B73. Our global analysis of maize TS transcriptomes could shed light on the molecular mechanisms of tissue specificity and facilitate transgenic studies by providing a valuable resource of TS promoter candidates.

Comparison of ectopic bone formation process induced by four calcium phosphate ceramics in mice

Phase composition played a key role in the biodegradation of calcium phosphate ceramics (CaP), which in turn influences the osteoinductive ability. The in vivo biological mechanism is still poorly understood. In this study, four types of porous CaP ceramics were investigated, namely, hydroxyapatite (HA), β-tricalcium phosphate (TCP), and biphasic calcium phosphates BCP1 and BCP2, with HA to β-TCP ratios of 70/30 and 30/70, respectively. The four types of ceramics were implanted into thigh muscle of mice for 16weeks. Longitudinal ectopic bone formation process at gene, protein, and tissue level induced by the material was assessed. Histological analysis revealed that BCP2 was the only group that had promoted new bone formation after 16weeks. In micro-CT analysis of biodegradation, the BCP2 group had the least increment of porosity due to the new bone formation, resulting in a significant elevation in material density. Instead of a steady increase, multiple peaks were observed in most of the temporal gene expression patterns. The gene expression results were further confirmed by immunohistochemical staining of the corresponding proteins. Among the target genes, Osterix and type I collagen were activated successively. The osteoinductive BCP2 group showed earlier and significantly higher peaks in BMP2, BMPR1A, and OPG expressions than non-bone forming groups. These findings revealed that the occurrence time and magnitude of these osteogenetic gene expression peaks can be crucial in the osteoinduction process.

Transcriptome analysis reveals self-incompatibility in the tea plant (Camellia sinensis) might be under gametophytic control.

BackgroundSelf-incompatibility (SI) is under genetic control and prevents inbreeding depression in angiosperms. SI mechanisms are quite complicated and still poorly understood in many plants. Tea (Camellia sinensis L.) belonging to the family of Theaceae, exhibits high levels of SI and high heterozygosity. Uncovering the molecular basis of SI of the tea plant may enhance breeding and simplify genomics research for the whole family.ResultsThe growth of pollen tubes following selfing and crossing was observed using fluorescence microscopy. Self-pollen tubes grew slower than cross treatments from 24 h to 72 h after pollination. RNA-seq was employed to explore the molecular mechanisms of SI and to identify SI-related genes in C. sinensis. Self and cross-pollinated styles were collected at 24 h, 48 h and 72 h after pollination. Six RNA-seq libraries (SP24, SP48, SP72, CP24 CP48 and CP72; SP = self-pollinated, CP = cross-pollinated) were constructed and separately sequenced. In total, 299.327 million raw reads were generated. Following assembly, 63,762 unigenes were identified, and 27,264 (42.76 %) unigenes were annotated in five public databases: NR, KOG, KEGG, Swiss-Port and GO. To identify SI-related genes, the fragments per kb per million mapped reads (FPKM) values of each unigene were evaluated. Comparisons of CP24 vs. SP24, CP48 vs. SP48 and CP72 vs. SP72 revealed differential expression of 3,182, 3,575 and 3,709 genes, respectively. Consequently, several ubiquitin-mediated proteolysis, Ca2+ signaling, apoptosis and defense-associated genes were obtained. The temporal expression pattern of genes following CP and SP was analyzed; 6 peroxidase, 1 polyphenol oxidase and 7 salicylic acid biosynthetic process-related genes were identified. The RNA-seq data were validated by qRT-PCR of 15 unigenes. Finally, a unigene (CL25983Contig1) with strong homology to the S-RNase was analyzed. It was mainly expressed in styles, with dramatically higher expression in self-pollinated versus cross-pollinated tissues at 24 h post-pollination.ConclusionsThe present study reports the transcriptome of styles after cross- and self-pollination in tea and offers novel insights into the molecular mechanism behind SI in C. sinensis. We believe that this RNA-seq dataset will be useful for improvement in C. sinensis as well as other plants in the Theaceae family.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2703-5) contains supplementary material, which is available to authorized users.