Developmental Stage-specific Manner Research Articles

The sugar transporter inventory of tomato: genome-wide identification and expression analysis.

The mobility of sugars between source and sink tissues in plants depends on sugar transport proteins. Studying the corresponding genes allows the manipulation of the sink strength of developing fruits, thereby improving fruit quality for human consumption. Tomato (Solanum lycopersicum) is both a major horticultural crop and a model for the development of fleshy fruits. In this article we provide a comprehensive inventory of tomato sugar transporters, including the SUCROSE TRANSPORTER family, the SUGAR TRANSPORTER PROTEIN family, the SUGAR FACILITATOR PROTEIN family, the POLYOL/MONOSACCHARIDE TRANSPORTER family, the INOSITOL TRANSPORTER family, the PLASTIDIC GLUCOSE TRANSLOCATOR family, the TONOPLAST MONOSACCHARIDE TRANSPORTER family and the VACUOLAR GLUCOSE TRANSPORTER family. Expressed sequence tag (EST) sequencing and phylogenetic analyses established a nomenclature for all analyzed tomato sugar transporters. In total we identified 52 genes in tomato putatively encoding sugar transporters. The expression of 29 sugar transporter genes in vegetative tissues and during fruit development was analyzed. Several sugar transporter genes were expressed in a tissue- or developmental stage-specific manner. This information will be helpful to better understand source to sink movement of photoassimilates in tomato. Identification of fruit-specific sugar transporters might be a first step to find novel genes contributing to tomato fruit sugar accumulation.

Ceramide galactosyltransferase expression is regulated positively by Nkx2.2 and negatively by OLIG2.

Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.

Different responses to soy and estradiol in the reproductive system of prepubertal male rats and neonatal male pigs (373.5)

Concerns have been raised regarding the safety of soy infant formula based on phytochemical components such as genistein, structurally similar to estradiol (E2). To examine potential estrogenic actions on male development, we fed weanling male rats casein‐based or soy protein isolate (SPI)‐based diets (PND21‐PND33) and neonatal male piglets cow’s milk or soy infant formulas (PND2‐PND21) or treated the animals with E2. E2 reduced testis and prostate weight and serum testosterone (P<0.05) in both species. Soy diet had no effects. In the piglet, E2 increased testis tubule area, Sertoli cell and germ cell number (P<0.05) and decreased expression of CYP19 and StAR mRNA. Soy increased germ cell number (P<0.05). The major effect of E2 is to change gene expression in reproductive tissues. E2 changed expression of 2645 genes in rat testis vs. 312 genes changed by SPI >1.5‐fold. 74% of the SPI genes were common to E2. In contrast, 353 genes were changed by E2 and only 116 by feeding soy formula in piglet testis with only 34 genes (29%) in common. These data suggest that soy diets are not estrogenic in the male reproductive system but may interact with a small subset of estrogen‐regulated genes in a species and developmental stage‐specific manner.Grant Funding Source: USDA‐ARS CRIS 6251‐51999‐007‐04S

Identification of Chemicals Inducing Cardiomyocyte Proliferation in Developmental Stage–Specific Manner With Pluripotent Stem Cells

The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provides therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells. The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse pluripotent stem cell-derived cardiomyocytes and identified 4 chemical compound groups: inhibitors of glycogen synthase kinase-3, p38 mitogen-activated protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II, and activators of extracellular signal-regulated kinase. Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human pluripotent stem cells, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas extracellular signal-regulated kinase activators and Ca(2+)/calmodulin-dependent protein kinase II inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, glycogen synthase kinase-3 and p38 mitogen-activated protein kinase inhibitors substantially and synergistically induced re-entry and progression of cell cycle in neonatal but also as well as adult cardiomyocytes. Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered pluripotent stem cell-derived cardiomyocytes as a potent tool to explore chemical-based cardiac regenerative strategies.

DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation.

BackgroundGenomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes.ResultsUsing this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse.ConclusionsOur study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development.

Sequence-specific promoter elements regulate temporal-specific changes in chromatin required for testis-specific activation of the Pgk2 gene

The phosphoglycerate kinase-2 (Pgk2) gene is regulated in a tissue-, cell type-, and developmental stage-specific manner during spermatogenesis and is required for normal sperm motility and fertility in mammals. Activation of Pgk2 transcription is regulated by testis-specific demethylation of DNA and binding of testis-specific transcription factors to enhancer and core promoter elements. Here, we show that chromatin remodeling including reconfiguration of nucleosomes and changes in histone modifications is also associated with transcriptional activation of the Pgk2 gene during spermatogenesis. Developmental studies indicate that the order of events involved in transcriptional activation of the Pgk2 gene includes demethylation of DNA in T₁- and T₂-prospermatogonia, binding of a factor to the CAAT box in type A and B spermatogonia, followed by recruitment of chromatin remodeling factors, displacement of a nucleosome from the Pgk2 promoter region, binding of factors to the Pgk2 core promoter and enhancer regions, and, finally, initiation of transcription in primary spermatocytes. Transgene studies show that Pgk2 core promoter elements are required to direct demethylation of DNA and reconfiguration of nucleosomes, whereas both enhancer and core promoter elements are required to direct changes in histone modifications and initiation of transcription. These results provide novel insight into the developmental order of molecular events required to activate tissue-specific transcription of the Pgk2 gene, the distinct elements in the 5'-regulatory region of the Pgk2 gene that regulate each of these events, and the relationship among these events in that each step in this process appears to be a necessary prerequisite for the subsequent step.

Relative expression of apocarotenoid biosynthetic genes in developing stigmas of Crocus sativus L.

Saffron, the desiccated stigmas of Crocus sativus, is recognized for its attractive color, flavor, and aroma which are due to the accumulation of crocin, picrocrocin, and safranal, respectively. HPLC analysis demonstrated maximum apocarotenoid accumulation during the fully developed scarlet stage of stigma development followed by the orange and yellow stages of stigma development. Reverse Transcription-PCR analysis revealed a concurrent expression pattern of CsZCD and CsLYC genes in a developmental stagespecific manner. However, CsBCH and CsGT2 genes were specifically expressed during the mature, scarlet stage of stigma development. Real-Time PCR analysis showed a sharp increase in gene expression of CsLYC gene during stigma development indicative of its possible regulatory role in apocarotenoid biosynthesis or stigma development. Results suggest that genetic manipulation of this gene can help to improve the quality of stigma in saffron; besides highlighting its potential to monitor stigma development during in vitro experimentation.

Correction: Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

Correction: Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

Reduced Juvenile Long-Term Depression in Tuberous Sclerosis Complex Is Mitigated in Adults by Compensatory Recruitment of mGluR5 and Erk Signaling

Author SummaryTuberous sclerosis complex (TSC) is a genetic disorder that afflicts around 1 in 6,000 people and results from a mutation in one of two genes, TSC1 or TSC2. TSC patients suffer a number of neuronal symptoms including various degrees of autism, mental retardation, and epilepsy, the latter found in more than 80% of cases within the first year of life. In the TSC mutant mice that are used to model the disease, a region of the brain called the hippocampus fails to undergo long-term depression (LTD), a neuronal process that is important for learning and memory. We find that while this is the case in juvenile mutant mice, adult mice appear to have fixed this deficit. The “fix” involves the ramping up of signaling pathways involving mGluR5 and Erk. Although increased mGluR5 and Erk signaling outwardly fixes the problem of diminished LTD in adulthood, it renders the brain insensitive to the cues and inputs that normally work to control LTD. Moreover, the hippocampus in adult TSC mice is prone to seizures and impaired in learning and memory tasks. We find that drugs that target mGluR5 or Erk signaling repair the problems with excitability and learning deficits.

Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in NBN or ATM result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show in vivo consequences of conditional inactivation of both genes in neural stem/progenitor cells using Nestin-Cre mice. Genetic inactivation of Atm in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with Nbn deletion was prevented by simultaneous inactivation of Atm in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.

Essential role of grim-led programmed cell death for the establishment of corazonin-producing peptidergic nervous system during embryogenesis and metamorphosis in Drosophila melanogaster

SummaryIn Drosophila melanogaster, combinatorial activities of four death genes, head involution defective (hid), reaper (rpr), grim, and sickle (skl), have been known to play crucial roles in the developmentally regulated programmed cell death (PCD) of various tissues. However, different expression patterns of the death genes also suggest distinct functions played by each. During early metamorphosis, a great number of larval neurons unfit for adult life style are removed by PCD. Among them are eight pairs of corazonin-expressing larval peptidergic neurons in the ventral nerve cord (vCrz). To reveal death genes responsible for the PCD of vCrz neurons, we examined extant and recently available mutations as well as RNA interference that disrupt functions of single or multiple death genes. We found grim as a chief proapoptotic gene and skl and rpr as minor ones. The function of grim is also required for PCD of the mitotic sibling cells of the vCrz neuronal precursors (EW3-sib) during embryonic neurogenesis. An intergenic region between grim and rpr, which, it has been suggested, may enhance expression of three death genes in embryonic neuroblasts, appears to play a role for the vCrz PCD, but not for the EW3-sib cell death. The death of vCrz neurons and EW3-sib is triggered by ecdysone and the Notch signaling pathway, respectively, suggesting distinct regulatory mechanisms of grim expression in a cell- and developmental stage-specific manner.

Dynamic Postnatal Developmental and Sex-Specific Neuroendocrine Effects of Prenatal Polychlorinated Biphenyls in rats

Gestational exposures to estrogenic compounds, both endogenous hormones and exogenous endocrine-disrupting chemicals (EDCs), have long-term effects on reproductive physiology and behavior. We tested the hypothesis that prenatal treatment of rats with low doses of Aroclor 1221 (A1221), a weakly estrogenic polychlorinated biphenyl mix previously used in industry, or estradiol benzoate (EB), alters development of the hypothalamus in a sexually dimorphic manner and subsequently perturbs reproductive function. Pregnant Sprague-Dawley rats were injected on embryonic days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or EB (50 μg/kg). Developmental milestones were monitored, and on postnatal days 15, 30, 45, and 90, 1 male and 1 female per litter were euthanized. Because of their key roles in the mediation of steroid actions on reproductive function, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) were punched for a low-density quantitative PCR array of 48 neuroendocrine genes and analysis of DNA methylation of a subset of genes. Gestational exposure to A1221 or EB delayed the timing of puberty in males and disrupted estrous cyclicity in females. In the AVPV, 28 genes were affected by treatment in a developmental stage-specific manner, mostly in females, which exhibited a masculinized expression profile. This included 2 clock genes, Per2 and Arntl, implicating circadian circuits as being vulnerable to endocrine disruption. DNA methylation analysis of 2 genes, Per2 and Ar, showed no effect of EDCs and suggested alternative mechanisms for the altered mRNA levels. In the ARC, 12 genes were affected by treatment, mostly in males, again with dynamic developmental changes. Bionetwork analysis of relationships among genes, hormones, and physiological markers showed sexually dimorphic effects of estrogenic EDC exposures, with the female AVPV and the male ARC being most vulnerable, and provided novel relationships among hypothalamic genes and postnatal reproductive maturation.

Immune Response and Anti-Microbial Peptides Expression in Malpighian Tubules of Drosophila melanogaster Is under Developmental Regulation

Malpighian tubules (MT) of Drosophila melanogaster are osmoregulatory organs that maintain the ionic balance and remove toxic substances from the body. Additionally they act as autonomous immune sensing organs, which secrete antimicrobial peptides in response to invading microbial pathogens. We show that the antimicrobial peptides (AMP) diptericin, cecropinA, drosocin and attacinA are constitutively expressed and are regulated in developmental stage specific manner. Their developmental expression begins from 3rd instar larval stage and an immune challenge increases the expression several folds. Spatial variatons in the level of expression along the MT tissue are observed. The mortality of 3rd instar larvae fed on bacterial food is much less than that of the earlier larval stages, coinciding with the onset of innate immunity response in MT. Ectopic expression of AMP imparts better resistance to infection while, loss of function of one of the AMP through directed RNAi reduces host survival after immune challenge. The AMP secreted from the MT exhibit bactericidal activity. Expression of the NF-κB transcription factor, Relish, also coincides with activation of immune responsive genes in MT, demonstrating that immune regulation in MT is under developmental control and is governed by the Imd pathway.

Molecular signatures to define spermatogenic cells in common marmoset (Callithrix jacchus)

Germ cell development is a fundamental process required to produce offspring. The developmental program of spermatogenesis has been assumed to be similar among mammals. However, recent studies have revealed differences in the molecular properties of primate germ cells compared with the well-characterized mouse germ cells. This may prevent simple application of rodent insights into higher primates. Therefore, thorough investigation of primate germ cells is necessary, as this may lead to the development of more appropriate animal models. The aim of this study is to define molecular signatures of spermatogenic cells in the common marmoset, Callithrix jacchus. Interestingly, NANOG, PRDM1, DPPA3 (STELLA), IFITM3, and ZP1 transcripts, but no POU5F1 (OCT4), were detected in adult marmoset testis. Conversely, mouse testis expressed Pou5f1 but not Nanog, Prdm1, Dppa3, Ifitm3, and Zp1. Other previously described mouse germ cell markers were conserved in marmoset and mouse testes. Intriguingly, marmoset spermatogenic cells underwent dynamic protein expression in a developmental stage-specific manner; DDX4 (VASA) protein was present in gonocytes, diminished in spermatogonial cells, and reexpressed in spermatocytes. To investigate epigenetic differences between adult marmoset and mice, DNA methylation analyses identified unique epigenetic profiles to marmoset and mice. Marmoset NANOG and POU5F1 promoters in spermatogenic cells exhibited a methylation status opposite to that in mice, while the DDX4 and LEFTY1 loci, as well as imprinted genes, displayed an evolutionarily conserved methylation pattern. Marmosets have great advantages as models for human reproductive biology and are also valuable as experimental nonhuman primates; thus, the current study provides an important platform for primate reproductive biology, including possible applications to humans.

Molecular and functional properties of three different peroxiredoxin isotypes in Chinese cabbage.

Peroxiredoxins (Prxs), which are classified into three isotypes in plants, play important roles in protection systems as peroxidases or molecular chaperones. The three Prx isotypes of Chinese cabbage, namely C1C-Prx, C2C-Prx, and C-PrxII, have recently been identified and characterized. The present study compares their molecular properties and biochemical functions to gain insights into their concerted roles in plants. The three Prx isotype genes were differentially expressed in tissue- and developmental stage-specific manners. The transcript level of the C1C-Prx gene was abundant at the seed stage, but rapidly decreased after imbibitions. In contrast, the C2C-Prx transcript was not detected in the seeds, but its expression level increased at germination and was maintained thereafter. The C-PrxII transcript level was mild at the seed stage, rapidly increased for 10 days after imbibitions, and gradually disappeared thereafter. In the localization analysis using GFP-fusion proteins, the three isotypes showed different cellular distributions. C1C-Prx was localized in the cytosol and nucleus, whereas C2C-Prx and C-Prx were found mainly in the chloroplast and cytosol, respectively. In vitro thiol-dependent antioxidant assays revealed that the relative peroxidase activities of the isotypes were CPrxII > C2C-Prx > C1C-Prx. C1C-Prx and C2C-Prx, but not C-PrxII, prevented aggregation of malate dehydrogenase as a molecular chaperone. Taken together, these results suggest that the three isotypes of Prx play specific roles in the cells in timely and spatially different manners, but they also cooperate with each other to protect the plant.

DNA Methylation Profile: A Composer-, Conductor-, and Player-Orchestrated Mammalian Genome Consisting of Genes and Transposable Genetic Elements

Epigenetic systems play crucial roles in the differentiation of a mammalian fertilized egg into hundreds of cell types exhibiting distinct phenotypes, using a set of DNA molecules comprising about 3 billion nucleotides. Genome-wide analyses of epigenetic marks have revealed the remarkably well-established and well-maintained structure of the epigenome, consisting of DNA methylation and histone modifications that vary their state in a tissue type- and developmental stage-specific manner at numerous genomic loci. DNA methylation profiles comprising numerous tissue-dependent and differentially methylated regions (T-DMRs), found at such loci, are unique to every type of cell and tissue, and illuminate molecular networks that represent their phenotypes. T-DMRs are located in not only genic but also nongenic regions-including transposable genetic elements, such as short interspersed transposable element. Epigenetic studies indicate that the molecules that perform these modifications directly, such as DNA methyltransferases and eukaryotic histone methyltransferases, or indirectly, such as CpG-binding protein and noncoding RNAs-and combinations of these-contribute to the DNA methylation profile. It remains to be addressed how these molecules precisely find their target genomic loci.

Leukemogenic MicroRNA-29a Causes Myeloproliferation by Inhibiting Apoptosis and Inducing Aberrant Self-Renewal in a Developmental Stage Specific Manner

Abstract 2556We recently reported that miR-29a is highly expressed in AML as well as in hematopoietic stem cells (HSC). Overexpression of miR-29a in mouse bone marrow resulted in myeloproliferative disease characterized by a granulocyte/macrophage differentiation bias and progressive disease culminating in AML. Prior to the development of AML, committed myeloid progenitors including common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP) aberrantly acquired the ability to self-renew, demonstrated by long-term engraftment of FACS-purified transplanted cells. In order to elucidate the mechanisms of miR-29a induced leukemogenesis, we characterized the immature hematopoietic compartment during the myeloproliferative phase of disease. Similar to our prior studies, miR-29a primary chimeras exhibited granulocytic hyperplasia as soon as 6–8 weeks after transplantation, with increased frequencies of GFP+Gr-1+Mac-1+ granulocytes in the peripheral blood of animals transplanted with miR-29a versus empty vector transduced cells (mean 34.7% vs. 9.2%, p<0.05). miR-29a expressing primary chimeras also showed increased frequencies of LSK (Lin-Sca1+c-kit+) cells and myeloid progenitors (Lin-Sca1-c-kit+) compared with empty vector controls (2.34% vs. 0.54%, p<0.05; and 6.85% vs. 14.3%, p<0.05, respectively). miR-29a expressing LSK cells also exhibited decreased apoptosis as assessed by annexin V staining (mean 10.8% vs. 5.9%, p<0.05), as well as a marked increase in the number of cells in G0/G1 (mean 85.3% vs. 64%; p<0.05); both of these effects were significantly attenuated or absent in more mature myeloid progenitors. Evaluation of miR-29a target mRNAs by qPCR revealed down-regulation of DNMT3a and DNMT3b transcripts in miR-29a progenitor cells. Secondary transplants were performed with FACS-sorted cells including myeloid-biased (My-bi, Lin-Sca1+c-kit+CD150hiCD34-) and lymphoid-biased (Ly-bi, Lin-Sca1+c-kit+CD150lowCD34-) HSC, multipotent progenitors (MPP, Lin-Sca1+c-kit+CD150negCD34+), and myeloid progenitors. Five months after transplant, mice transplanted with miR-29a expressing MPP (4/5) or myeloid progenitors (3/7) exhibited a myeloid expansion, consistent with acquisition of aberrant self-renewal and transfer of disease-initiating cells. In contrast, GFP+ chimerism was either low or undetectable in secondary recipients transplanted with My-bi HSC (1/4) or Ly-bi HSC (0/6). Finally, we measured miR-29a expression levels in AML blasts from several mouse models of AML including MLL-AF9, AML-ETO9a, and NUP98-HOXD13 and show that miR-29a expression is increased 6–12 fold over normal c-kit+ cells, consistent with our previously published results demonstrating high levels of miR-29a in primary AML blasts compared to their normal HSC/progenitor counterparts. Together, these results indicate that miR-29a induces myeloproliferation by inducing self-renewal in hematopoietic progenitors including MPP and committed myeloid progenitors. The enhanced self-renewal and expansion of LSK cells is likely due to the anti-apoptotic effect of miR-29a, which is developmental stage specific and associated with decreased cell cycling and decreased DNMT3a/b expression. These findings indicate that myeloproliferation may be due to expansions caused by increased self-renewal of progenitors and not necessarily through enhanced HSC function. Disclosures:No relevant conflicts of interest to declare.

1 kbp 5′ upstream sequence enables developmental stage-specific expressions of globin genes in the fish, medaka Oryzias latipes

Hemoglobin of bony fish and higher vertebrates is a tetrameric protein constructed by 2 α- and 2 β-globins, which are expressed in a developmental stage-specific manner. The genomic organization of genes for embryonic and adult α- and β-globin varies from species to species. In fish, it is known that there is a unique genomic organization of globin genes, that is, α- and β-globin genes are arranged in a bi-directional and head-to-head orientation with respect to transcription start sites. In medaka, we have demonstrated that 14 globin genes are located in 2 different clusters, and 5 pairs of the α- and β-globin genes were found to be organized in a head-to-head orientation. The developmental expression patterns of the 11 globin genes were classified into 4 types. To clarify how their developmental stage-specific expressions are regulated, we produced 4 types of GFP- or RFP-transgenic medaka. Such transgenic medaka revealed that each of the 1–1.7 kbp 5′ upstream sequences from respective globin genes possesses the ability to regulate the developmental stage-specific globin gene expression. In particular, the intervals between head-to-head α3 and β3, and α4 and β4 genes controlled the synchronized expression of the globin genes located at both sides of the intervals, which is significant to understand the mechanism by which equal amounts of α- and β-globins are expressed in erythroid cells. We also demonstrated that the head-to-head intervals can control the expression of the globin genes located at both sides. These findings are significant to understand the mechanism by which α- and β-globins are equally expressed in erythroid cells.

Dark purple nectar as a foraging signal in a bird‐pollinated Himalayan plant

• Some plants secrete coloured nectar to attract pollinators, but little is known about the chemical origins of nectar colouration and its ecological function. Leucosceptrum canum stands out as the only plant with coloured nectar recorded in the Himalayas. Here, we focused on the compound associated with the dark colour of the nectar, as well as its secretion dynamics during the flowering season and its relationship to pollinators. • Fresh nectar was analysed by semi-preparative reversed-phase high-performance liquid chromatography (HPLC), LC-MS and HRESIMS (high resolution electronspray ionization mass spectroscopy) to determine which compound causes the nectar colouration. Behavioural experiments were conducted with birds and honeybees to elucidate the effect of the nectar colour and volume on pollinators. • We identified a purple anthocyanidin, 5-hydroxyflavylium, as a natural nectar product for the first time. Two short-billed birds were found to pollinate this plant, which employs two nectar-based mechanisms to direct bird pollinators to reproductively active flowers, controlling nectar palatability and presenting a foraging signal for birds by altering nectar volume and colour in a developmental stage-specific manner. • 5-Hydroxyflavylium was found to be the cause of the nectar colouration, the function of which is to act as a foraging signal to increase pollination efficiency through nectar visibility and palatability.

Alternative promoter usage and differential expression of multiple transcripts of mouse Prkar1a gene

Prkar1a gene encodes regulatory type 1 alpha subunit (RIα) of cAMP-dependent protein kinase (PKA) in mouse. The role of this gene has been implicated in Carney complex and many cancer types that suggest its involvement in physiological processes like cell cycle regulation, growth and/or proliferation. We have identified and sequenced partial cDNA clones encoding four alternatively spliced transcripts of mouse Prkar1a gene. These transcripts have alternate 5' UTR structure which results from splicing of three exons (designated as E1a, E1b, and E1c) to canonical exon 2. The designated transcripts T1, T2, T3, and T4 contain 5' UTR exons as E1c, E1a + E1b, E1a, and E1b, respectively. The transcript T1 corresponded to earlier reported transcript in GenBank. In silico study of genomic DNA sequence revealed three distinct promoter regions namely, P1, P2, and P3 upstream of the exons E1a, E1b, and E1c, respectively. P1 is non-CpG-related promoter but P2 and P3 are CpG-related promoters; however, all three are TATA less. RT-PCR analysis demonstrated the expression of all four transcripts in late postnatal stages; however, these were differentially regulated in early postnatal stages of 0.5 day, 3 day, and 15 day mice in different tissue types. Variations in expression of Prkar1a gene transcripts suggest their regulation from multiple promoters that respond to a variety of signals arising in or out of the cell in tissue and developmental stage-specific manner.