Monitoring of compound resting membrane potentials of cell cultures with ratiometric genetically encoded voltage indicators.

The cellular resting membrane potential (Vm) not only determines electrical responsiveness of excitable cells but also plays pivotal roles in non-excitable cells, mediating membrane transport, cell-cycle progression, and tumorigenesis. Studying these processes requires estimation of Vm, ideally over long periods of time. Here, we introduce two ratiometric genetically encoded Vm indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting Vm between cell groups. We investigated the influence of ectopic expression of K+ channels and their disease-causing mutations involved in Andersen-Tawil (Kir2.1) and Temple-Baraitser (KV10.1) syndrome on median resting Vm of HEK293T cells. Real-time long-term monitoring of Vm changes allowed to estimate a 40-50 min latency from induction of transcription to functional Kir2.1 channels in HEK293T cells. The presented methodology is readily implemented with standard fluorescence microscopes and offers deeper insights into the role of the resting Vm in health and disease.

longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth.

Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.

Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains.

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from Polish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.

Ectopic expression of MELK in oral squamous cell carcinoma and its correlation with epithelial mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is closely correlated to metastasis formation generation and maintenance of cancer stem cells, nevertheless, the underlying mechanisms are unclear. The aim of this study is to investigate the role of maternal embryonic leucine-zipper kinase (MELK) in EMT regulation in oral squamous cell carcinoma (OSCC). We found that there was overexpression of MELK in human OSCC tissues, and high MELK expression was correlated with lymphatic metastasis and led to poor prognosis in patients with OSCC. We also confirmed that MELK is closely correlated to the EMT process using a human OSCC tissue microarray. Additionally, MELK expression was observed to be regulated in several OSCC cell lines, and knockdown of MELK genes inhibited cell proliferation, migration, invasion and EMT of OSCC cells in vitro. Furthermore, silencing of MELK suppressed tumour growth in vivo, and experimental research verified that MELK may augment OSCC development via mediating the Wnt/Notch signalling pathway. Our findings suggest that MELK serves as an oncogene to improve malignant development of OSCC via enhancing EMT, and MELK might be a potential target for anticancer therapeutic.

Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

Ectopic expression of GmHP08 enhances resistance of transgenic Arabidopsis toward drought stress.

KEY MESSAGE: Ectopic expression of Glycine max two-component system member GmHP08 in Arabidopsis enhanced drought tolerance of transgenic plants, possibly via ABA-dependent pathways. Phosphorelay by two-component system (TCS) is a signal transduction mechanism which has been evolutionarily conserved in both prokaryotic and eukaryotic organisms. Previous studies have provided lines of evidence on the involvement of TCS genes in plant perception and responses to environmental stimuli. In this research, drought-associated functions of GmHP08, a TCS member from soybean (Glycine max L.), were investigated via its ectopic expression in Arabidopsis system. Results from the drought survival assay showed that GmHP08-transgenic plants exhibited higher survival rates compared with their wild-type (WT) counterparts, indicating better drought resistance of the former group. Analyses revealed that the transgenic plants outperformed the WT in various regards, i.e. capability of water retention, prevention of hydrogen peroxide accumulation and enhancement of antioxidant enzymatic activities under water-deficit conditions. Additionally, the expression of stress-marker genes, especially antioxidant enzyme-encoding genes, in the transgenic plants were found greater than that of the WT plants. In contrary, the expression of SAG13 gene, one of the senescence-associated genes, and of several abscisic acid (ABA)-related genes was repressed. Data from this study also revealed that the ectopic expression lines at germination and early seedling development stages were hypersensitive to exogenous ABA treatment. Taken together, our results demonstrated that GmHP08 could play an important role in mediating plant response to drought, possibly via an ABA-dependent manner.

Ectopic expression of the transcription factor CUC2 restricts growth by cell cycle inhibition in Arabidopsis leaves.

Plant leaf margins produce small outgrowths or teeth causing serration in a regular arrangement, which is specified by auxin maxima. In Arabidopsis, the spatiotemporal pattern of auxin dependents on both, the transcription factor CUC2 and the signal peptide EPFL2, a ligand of the growth-promoting receptor kinase ERECTA (ER). Ectopic expression of CUC2 can have contrary effects on leaf growth. Ubiquitous expressed CUC2 suppresses growth in the whole leaf, whereas cuc2-1D mutants have enlarged leaves, through ER-dependent cell proliferation in the teeth. Here we investigated the growth dynamics of cuc2-1D leaves and the growth restricting the function of CUC2 using the ubiquitous inducible CUC2-GR transgene. In time courses, we dissected the serration promoting the function of CUC2 in the leaf margin and ectopic growth inhibition by CUC2 in the leaf plate. We found that CUC2 limits growth rather by cell cycle inhibition than by cell size control. Furthermore, endogenous CUC2 was rapidly induced by CUC2-GR indicating a possible auto-inducible feedback. In contrast, EPFL2 was quickly decreased by transient CUC2 induction but increased in cuc2-3 mutant leaves suggesting that CUC2 can also counteract the EPFL2-ER pathway. Therefore, tooth growth promotion and growth inhibition by CUC2 involve partially the same mechanism but in contrary ways.

Ectopic expression of the AaFUL1 gene identified in Anthurium andraeanum affected floral organ development and seed fertility in tobacco.

Anthurium andraeanum is a high-grade potted flower that enjoys global popularity. Its floral organs have been substantially modified, and its ornamental value is based on its petaloid bracts. MADS-box gene products are important transcription factors that control plant development. In particular, the APETALA1 (AP1)/FRUITFULL (FUL) family of MADS-box genes plays a key role in flowering transitions and out-whorl floral organ identity specification. In this report, one FUL-like gene was cloned from Anthurium andraeanum and named AaFUL1 after bioinformatics identification. Subsequent subcellular localization experiments confirmed that the AaFUL1 protein was located in the nucleus, and data obtained from an expression analysis indicated that the relative expression level of AaFUL1 was the highest in bracts and inflorescences, while its expression was relatively low in stems and roots. Next, an AaFUL1 overexpression vector was constructed and ectopically expressed in tobacco. The transformants did not show any early flowering phenotype, but the average internode length of the inflorescence branch was significantly higher than that observed in the control, and its petal color had substantially faded. The morphology of the petal and pistil was clearly changed, the fruit was deformed, and the seed was largely aborted. These data indicate that even though the sequence of AaFUL1 is relatively conserved, its function differs from that of other orthologs, and the FUL subfamily of MADS-box transcription factors may have taken on new functions during the evolution processes. The results of this experiment enrich our knowledge of FUL transcription factors in monocotyledon plants.

Ectopically Expressed IL-34 Can Efficiently Induce Macrophage Migration to the Liver in Zebrafish.

Interleukin-34 (IL-34) is a known cytokine that plays an important role in the survival, proliferation, and differentiation of macrophages. In previous studies, IL-34 can induce macrophage migration through syndecan-1 or focal adhesion kinase and extracellular signal-related kinase 1 and 2 pathway. These studies mainly focused on in vitro experiments, but the effect of IL-34 on macrophage migration in vivo is less understood. In our study, we artificially induced macrophage, but not neutrophil, enrichment in the skin or liver by overexpressing IL-34 in epidermal cells or hepatocytes in zebrafish. Live imaging showed that the enrichment of macrophages in the liver is due to the direct attraction of macrophages by IL-34. Our results demonstrated that ectopically expressed IL-34 can induce macrophage migration to liver in vivo.

Ectopic expression of a pea apyrase enhances root system architecture and drought survival in Arabidopsis and soybean.

Ectoapyrases (ecto-NTPDases) function to decrease levels of extracellular ATP and ADP in animals and plants. Prior studies showed that ectopic expression of a pea ectoapyrase, psNTP9, enhanced growth in Arabidopsis seedlings and that the overexpression of the two Arabidopsis apyrases most closely related to psNTP9 enhanced auxin transport and growth in Arabidopsis. These results predicted that ectopic expression of psNTP9 could promote a more extensive root system architecture (RSA) in Arabidopsis. We confirmed that transgenic Arabidopsis seedlings had longer primary roots, more lateral roots, and more and longer root hairs than wild-type plants. Because RSA influences water uptake, we tested whether the transgenic plants could tolerate osmotic stress and water deprivation better than wild-type plants, and we confirmed these properties. Transcriptomic analyses revealed gene expression changes in the transgenic plants that helped account for their enhanced RSA and improved drought tolerance. The effects of psNTP9 were not restricted to Arabidopsis, because its expression in soybeans improved the RSA, growth, and seed yield of this crop and supported higher survival in response to drought. Our results indicate that in both Arabidopsis and soybeans, the constitutive expression of psNTP9 results in a more extensive RSA and improved survival in drought stress conditions.

Partial Rescue of Ocular Pigment Cells and Structure by Inducible Ectopic Expression of Mitf-M in MITF-Deficient Mice.

Purpose: Complete deficiency of microphthalmia transcription factor (MITF) in Mitfmi-vga9/mi-vga9 mice is associated with microphthalmia, retinal dysplasia, and albinism. We investigated the ability of dopachrome tautomerase (DCT) promoter-mediated inducible ectopic expression of Mitf-M to rescue these phenotypic abnormalities. Methods: A new mouse line was created with doxycycline-inducible ectopic Mitf-M expression on an Mitf-deficient Mitfmi-vga9 background (DMV mouse). Adult DMV mice were phenotypically characterized and tissues were collected for histology, immunohistochemistry, and evaluation of Mitf, pigmentary genes, and retinal pigment epithelium (RPE) gene expression. Results: Ectopic Mitf-M expression was specifically induced in the eyes, but was not detected in the skin of DMV mice. Inducible expression of Mitf-M partially rescued the microphthalmia, RPE structure, and pigmentation as well as a subset of the choroidal and iris melanocytes but not cutaneous melanocytes. RPE function and vision were not restored in the DMV mice. Conclusions: Ectopic expression of Mitf-M during development of Mitf-deficient mice is capable of partially rescuing ocular and retinal structures and uveal melanocytes. These findings provide novel information about the roles of Mitf isoforms in the development of mouse eyes.

The diversified function and potential therapy of ectopic olfactory receptors in non-olfactory tissues.

Olfactory receptors (ORs) are mainly distributed in olfactory neurons and play a key role in detecting volatile odorants, eventually resulting in the production of smell perception. Recently, it is also reported that ORs are expressed in non-olfactory tissues including heart, lung, sperm, skin, and cancerous tissues. Interestingly, ectopic ORs are associated with the development of diseases in non-olfactory tissues. For instance, ectopic ORs initiate the hypoxic ventilatory responses and maintain the oxygen homeostasis of breathing in the carotid body when oxygen levels decline. Ectopic ORs induce glucose homeostasis in diabetes. Ectopic ORs regulate systemic blood pressure by increasing renin secretion and vasodilation. Ectopic ORs participate in the process of tumor cell proliferation, apoptosis, metastasis, and invasiveness. Ectopic ORs accelerate the occurrence of obesity, angiogenesis and wound-healing processes. Ectopic ORs affect fetal hemoglobin levels in sickle cell anemia and thalassemia. Finally, we also elaborate some ligands targeting for ORs. Obviously, the diversified function and related signal pathway of ectopic ORs may play a potential therapeutic target in non-olfactory tissues. Thus, this review focuses on the latest research results about the diversified function and therapeutic potential of ectopic ORs in non-olfactory tissues.

Production of red-flowered oilseed rape via the ectopic expression of Orychophragmus violaceus OvPAP2.

Oilseed rape (Brassica napus L.), which has yellow flowers, is both an important oil crop and a traditional tourism resource in China, whereas the Orychophragmus violaceus, which has purple flowers, likely possesses a candidate gene or genes to alter the flower colour of oilseed rape. A previously established B. napus line has a particular pair of O. violaceus chromosomes (M4) and exhibits slightly red petals. In this study, the transcriptomic analysis of M4, B. napus (H3), and O. violaceus with purple petals (OvP) and with white petals (OvW) revealed that most anthocyanin biosynthesis genes were up-regulated in both M4 and OvP. Read assembly and sequence alignment identified a homolog of AtPAP2 in M4, which produced the O. violaceus transcript (OvPAP2). The overexpression of OvPAP2 via the CaMV35S promoter in Arabidopsis thaliana led to different levels of anthocyanin accumulation in most organs, including the petals. However, the B. napus overexpression plants showed anthocyanin accumulation primarily in the anthers, but not the petals. However, when OvPAP2 was driven by the petal-specific promoter XY355, the transgenic B. napus plants produced red anthers and red petals. The results of metabolomic experiments showed that specific anthocyanins accumulated to high levels in the red petals. This study illustrates the feasibility of producing red-flowered oilseed rape, thereby enhancing its ornamental value, via the ectopic expression of the OvPAP2 gene. Moreover, the practical application of this study for insect pest management in the crop is discussed.

Ectopic expression of CITED2 prior to reprogramming, promotes and homogenises the conversion of somatic cells into induced pluripotent stem cells.

Cited2 plays crucial roles in mouse embryonic stem cells self-renewal, the initiation of the somatic reprogramming process into induced pluripotent stem cells (iPSC) and the suppression of cell senescence. Here, we investigated the potential of CITED2 expression in combination with the Oct4, Sox2, Klf4 and c-Myc factors for reprogramming of primary mouse embryonic fibroblasts (MEF) at passage 2 and 4. The ectopic CITED2 expression in primary MEF prior to the onset of the reprogramming process, generated iPSC with less variability in the expression of endogenous pluripotency-related genes. In contrast, part of the MEF reprogrammed without ectopic expression of CITED2 at passage 4 originated partially reprogrammed iPSC or pre-iPSC. However, the overexpression of CITED2 in the pre-iPSC was insufficient to complete the reprogramming process into iPSC. These results indicated that ectopic CITED2 expression at the onset of the reprogramming process in combination with the reprogramming factors promotes a complete and homogeneous conversion of somatic cells into iPSC.

Ectopic expression of a tomato DREB gene affects several ABA processes and influences plant growth and root architecture in an age-dependent manner.

Regulation of whole plant growth and adaptive responses by abscisic acid is complex, requires multiple regulators and largely unknown in plants other than Arabidopsis. We show that over-expression of the tomato SlDREB3/SlERF.H12 (DEHYDRATION RESPONSE ELEMENT BINDING PROTEIN3/ETHYLENE RESPONSE FACTOR. H12) gene can negatively affect many ABA-governed processes across tissues. Its expression leads to early germination in presence of ABA and in response to mannitol, NaCl and glucose. Its expression delays ABA-mediated leaf senescence and natural senescence leading to an increase in plant life by about 20days. Transgenic SlDREB3 lines show reduced ABA-mediated inhibition of conductance and transpiration and a greater sensitivity to water stress. Reduction in sensitivity to ABA-mediated stomatal closure leads to higher photosynthetic rates in transgenic plants than controls. Consequently, transgenic SlDREB3 plants produce a larger number of capsules and greater number of seeds with the increase in yield ranging from 18 to 35% in different seasons under well-watered conditions. Root growth, but not shoot growth, also undergoes a profound increase of about 50% in transgenic SlDREB3 lines. The increase occurs in an age-dependent manner with the most prominent changes being observed between 1.5 and 2.5 months in several independent experiments in different years. SlDREB3 thus seems to govern several ABA-regulated processes across tissues, partly through control over ABA levels. It may encode a factor that is most likely a component of the central ABA response machinery.

Ectopic expression of TaOEP16-2-5B, a wheat plastid outer envelope protein gene, enhances heat and drought stress tolerance in transgenic Arabidopsis plants.

Abiotic stresses, such as heat and drought, are major environmental factors restricting crop productivity and quality worldwide. A plastid outer envelope protein gene, TaOEP16-2, was identified from our previous transcriptome analysis [1,2]. In this study, the isolation and functional characterization of the TaOEP16-2 gene was reported. Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat, which were localized on the chromosomes 5A, 5B and 5D, respectively. These three homoeologues exhibited different expression patterns under heat stress conditions, TaOEP16-2-5B was the dominant one, and TaOEP16-2-5B was selected for further analysis. Compared with wild type (WT) plants, transgenic Arabidopsis plants overexpressing the TaOEP16-2-5B gene exhibited enhanced tolerance to heat stress, which was supported by improved survival rate, strengthened cell membrane stability and increased sucrose content. It was also found that TaOEP16-2 was induced by drought stress and involved in drought stress tolerance. TaOEP16-2-5B has the same function in ABA-controlled seed germination as AtOEP16-2. Our results suggest that TaOEP16-2-5B plays an important role in heat and drought stress tolerance, and could be utilized in transgenic breeding of wheat and other crop plants.

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice.

A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2-oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10-30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops.

Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3.

Application of dendritic cells (DCs) to prime responses to tumor Ags provides a promising approach to immunotherapy. However, only a limited number of DCs can be manufactured from adult precursors. In contrast, pluripotent embryonic stem (ES) cells represent an inexhaustible source for DC production, although it remains a major challenge to steer directional differentiation because ES cell-derived cells are typically immature with impaired functional capacity. Consistent with this notion, we found that mouse ES cell-derived DCs (ES-DCs) represented less mature cells compared with bone marrow-derived DCs. This finding prompted us to compare the gene expression profile of the ES cell- and adult progenitor-derived, GM-CSF-instructed, nonconventional DC subsets. We quantified the mRNA level of 17 DC-specific transcription factors and observed that 3 transcriptional regulators (Irf4, Spi-B, and Runx3) showed lower expression in ES-DCs than in bone marrow-derived DCs. In light of this altered gene expression, we probed the effects of these transcription factors in developing mouse ES-DCs with an isogenic expression screen. Our analysis revealed that forced expression of Irf4 repressed ES-DC development, whereas, in contrast, Runx3 improved the ES-DC maturation capacity. Moreover, LPS-treated and Runx3-activated ES-DCs exhibited enhanced T cell activation and migratory potential. In summary, we found that ex vivo-generated ES-DCs had a compromised maturation ability and immunogenicity. However, ectopic expression of Runx3 enhances cytokine-driven ES-DC development and acts as an instructive tool for the generation of mature DCs with enhanced immunogenicity from pluripotent stem cells.

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells.

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings.