Normal Organ Development Research Articles

Caspase Activity Is Required for Engulfment of Apoptotic Cells

Clearance of apoptotic cells by phagocytic neighbors is crucial for normal development of multicellular organisms. However, how phagocytes discriminate between healthy and dying cells remains poorly understood. We focus on glial phagocytosis of apoptotic neurons during development of the Drosophila central nervous system. We identified phosphatidylserine (PS) as a ligand on apoptotic cells for the phagocytic receptor Six Microns Under (SIMU) and report that PS alone is not sufficient for engulfment. Our data reveal that, additionally to PS exposure, caspase activity is required for clearance of apoptotic cells by phagocytes. Here we demonstrate that SIMU recognizes and binds PS on apoptotic cells through its N-terminal EMILIN (EMI), Nimrod 1 (NIM1), and NIM2 repeats, whereas the C-terminal NIM3 and NIM4 repeats control SIMU affinity to PS. Based on the structure-function analysis of SIMU, we discovered a novel mechanism of internal inhibition responsible for differential affinities of SIMU to its ligand which might prevent elimination of living cells exposing PS on their surfaces.

Identification of candidate genes associated with male sterility in CMS7311 of heading Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Polima (pol) cytoplasmic male sterility (CMS) is reported to be regulated by orf224, but the molecular mechanisms of CMS are less known. In previous research, CMS7311 of heading Chinese cabbage (Brassica campestris L.ssp. pekinensis) was successfully generated from a pol-like CMS, named 77A. To further understand the molecular mechanisms of this male sterility, cDNA-AFLP and qRT-PCR were used to identify genes differentially expressed during flower bud development between cytoplasmic male-sterile plant (Ms) and male-fertile plant (Mf) derived from the hybrid of CMS7311 and a restorer (Rf). Thirty-two transcripts of different fragments (TDFs) of over 80 bp in length were identified using cDNA-AFLP. Among them, ten TDFs were found expressing in Ms bud, and the others expressing in Mf bud. Gene ontology analysis revealed that these genes were involved in stress response, synthesis and metabolism of fatty acid, cell reconstruction, etc. Further, five genes closely related to male sterile were selected and their expression during the flower bud development of Ms and Mf line were investigated by qRT-PCR. Three genes, BcGRP17, BcMS2, and BcPME31, which were necessary for normal male organ development, showed significantly lower expressions in Ms bud; two genes, BcROPGEF8 and BcTNL3, were induced with significantly higher expression in Ms bud by unknown stress and involved in the formation of male sterility. Further work will be focused on cloning these genes and identifying their functions in the male-sterile process of heading Chinese cabbage CMS7311.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis <em>In vivo</em>

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Regulation of TGFβ superfamily signaling by two separable domains of glypican LON-2 inC. elegans

Regulated intercellular signaling is critical for the normal development and maintenance of multicellular organisms. Glypicans have been shown to regulate signaling by TGFβs, hedgehogs and Wnts, in several cellular contexts. Glypicans comprise a conserved family of heparan sulfated, glycosylphosphatidylinositol (GPI)-linked extracellular proteins. The structural complexity of glypicans may underlie their functional complexity. In a recent study31, we built on previous findings that one of the two C. elegans glypicans, LON-2, specifically inhibits signaling by the TGFβ superfamily member DBL-1. We tested the functional requirements of LON-2 protein core components and post-translational modifications for LON-2 activity. We provide the first evidence that two parts of a glypican can independently regulate TGFβ superfamily signaling in vivo: the N-terminal furin protease product and a C-terminal region containing heparan sulfate attachment sites. Furthermore, we show a protein-protein interaction motif is crucial for LON-2 activity in the N-terminal protein core, suggesting that LON-2 acts by serving as a scaffold for DBL-1 and an RGD-binding protein. In addition, we demonstrate specificity of glypican function by showing C. elegans GPN-1 does not functionally substitute for LON-2. This work reveals a molecular foundation for understanding the complexity and specificity of glypican function.

Filamin and Phospholipase C-ε are required for calcium signaling in the Caenorhabditis elegans Spermatheca.

Mechanical properties of the microenvironment are fundamental in orchestrating normal tissue function, disease progression, and organismal development. Studies of mechanotransduction in cultured cells on artificial substrates have revealed underlying principles, but the in vivo roles of mechanotransduction remain unclear. We recently reported that the Caenorhabditis elegans spermatheca—a myoepithelial tube composed of a cell monolayer—may be mechanosensitive. Live imaging with the genetically encoded calcium indicator GCaMP revealed that oocyte-induced stretching of the spermatheca resulted in calcium oscillations and constriction of the tube. FLN-1/filamin, a mechanosensitive cytoskeletal scaffolding protein, is required to correctly trigger the calcium transients. PLC-1/phospholipase C-epsilon and ITR-1/IP3 receptor are required to produce the calcium transients, and may function downstream of filamin. In addition to providing important insights into the biology of C. elegans, our studies offer a novel and genetically tractable model for studying mechanotransduction in a myoepithelial tissue.

Genome-Wide Epigenetics

WhaT is epiGeneTics? The term “epigenetics” was coined by Conrad Waddington to describe “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (Goldberg et al., 2007). Very broadly, the word has come to refer to the study of the regulation of genes, their expression, and how that translates into particular phenotypes, independent of any change to the underlying DNA sequence. More simply stated, epigenetics is the study of functionally relevant changes in gene expression (with subsequent changes in cellular phenotype) that result from mechanisms other than from changes in the underlying DNA nucleotide sequence. Despite the fact that there is no change in the nucleo tide sequence, epigenetic modifications may be heritable and can be passed down to subsequent generations through cell replication and division of alternative chromatin states. This “turning on or off” of genes explains why, despite having the same underlying DNA sequence, a keratinocyte looks and behaves so differently than a hepatocyte and why the epigenetic state is carried over to maintain celland tissue-type specification. Although a given cell’s (or individual’s) genome remains relatively stable over time, the epigenome can and does vary depending on a number of factors, including environmental conditions. These processes allow for many “good” functions, including normal organism development; however, aberrant epigenetic mechanisms are implicated in different disease processes, including malignancies. This article provides a brief overview of the field of epigenetics and offers a glimpse into some of the major techniques used to study it, with a particular focus on chromatin immunoprecipitation followed by sequencing (ChIP-seq), the current standard method for studying proteins and other epigenetic factors that bind to DNA. At the heart of epigenetic control is the organization of DNA into chromatin. This begins with 147 base pairs of DNA wrapped around eight histone proteins, which include the core histones H2A, H2B, H3, and H4 (Figure 1). Each of these histone octamers is referred to as a nucleosome. The nucleosomes are packaged tightly into even more compact fibers known as chromatin. Through this complex structure, epigenetic regulation occurs primarily through four mechanisms. First, DNA can undergo direct chemical modification by cytosine methylation, which is a general marker of gene silencing. Second, posttranslational modifications of the core histones can occur, primarily through methylation, acetylation, ubiquitylation, and phosphorylation, making up the primary chromatin structure (Figure 1). Acting in concert with these two aspects of the epigenetic machinery, noncoding RNAs contribute to the regulation of these processes (Greer and Shi, 2012). Finally, the chromatin is then packaged, via long-range interactions, into a higher-order structure within the cell nucleus. All of these organizational steps serve to modulate DNA accessibility and thus control gene expression. More open regions of chromatin, or euchromatin, are poised for activation by the transcriptional machinery, whereas more ADVANTAGES OF CHIP-SEQ

Mutations in epidermis‐specific HD‐ZIP IV genes affect floral organ identity in Arabidopsis thaliana

Development of the epidermis involves members of the class-IV homeodomain-leucine zipper (HD-ZIPIV) transcription factors. The Arabidopsis HD-ZIPIV family consists of 16 members, among which PROTODERMAL FACTOR2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells; however, the functions of other HD-ZIPIV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIPIV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIPIV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIPIV family in the epidermis are crucial for normal development of floral organs in Arabidopsis.

The effect of maternal thyroid disorders (hypothyroidism and hyperthyroidism) during pregnancy and lactation on skin development in wistar rat newborns.

Previous studies have shown that thyroid hormones are necessary for normal development of many organs and because of the importance of skin as the largest and the most important organ in human body protection in spite of external environment, the study of thyroid hormones effects on skin development is considerable. In this survey we have tried to study the effects of maternal hypothyroidism on skin development in fetus during pregnancy and lactation by immunohistochemistry technique. Rats were divided into 4 groups, hypothyroids, hyperthyroids, hypothyroids are treated with levothyroxin and a control group. The rat mothers were exposed to PTU with 50 mg/lit dosage and levothyroxin with 1 mg/lit dosage and PTU and levothyroxin simultaneously and with the same dosage respectively in hypothyroid, hyperthyroid and treated hypothyroids with levothyroxin groups. After 14 days, blood sample was taken from mothers, and if thyroid hormones level had change well, mating was allowed. After pregnancy and delivery, 1th day dorsal skin (as the sample for pregnancy assay) and 10th day skin (as for lactation assay) was used for immunohystochemical and morphometric studies. In this study it was observed that maternal hypothyroidism during pregnancy and lactation causes significant increase in laminin expression, in most areas of skin, and maternal hyperthyroidism during pregnancy and lactation causes significant decrease in laminin expression. Also significant decrease was observed in hair follicles number and epidermis thickness in hypothyroidism groups. This study showed maternal hypothyroidism causes significant decrease in epidermis thickness and hair follicles number and it causes less hair in fetus. Also maternal hypothyroidism causes large changes in laminin expression in different parts of skin. At the same time,maternal hyperthyroidism causes opposite results. In fact, thyroid hormones regulate laminin expression negatively which means increase in thyroid hormone level, decreases laminin expression. So changes in thyroid hormones level can influence skin development significantly.

Abstract 3328: Caspase activation as an approach to treat multiple myeloma.

Abstract Apoptosis is a cell death program that is essential for normal organ development. Defects in apoptosis lead to uncontrolled cell accumulation resulting in cancer such as multiple myeloma (MM), an incurable plasma cell disorder. MM patients have defective apoptosis machinery largely due to the overexpression of anti-apoptotic proteins such as BCL-2, MCL-1, cIAP1, cIAP2 and XIAP. We are investigating a novel class of procaspase-3 activators as a strategy to activate apoptosis in myeloma cells. The Procaspase Activating Compounds (PAC), PAC-1 and its analog L14R8, were synthesized by Dr. Hergenrother's lab (U. of Illinois, Urbana, IL) and are being developed for clinical application by Vanquish Oncology. PACs convert the inactive zymogen procaspase-3 to caspase-3 which activates apoptosis. Mechanistically, PACs activate procaspase-3 by chelating inhibitory zinc ions thus allowing procaspase-3 to auto-activate to caspase-3. Our hypothesis is that targeting procaspase-3 with PAC agents will bypass inhibitory activity of the anti-apoptotic proteins and induce cell death in myeloma cells. In initial experiments, we examined procaspase-3 expression in a panel of 9 myeloma cell lines and observed that it was expressed in all the cell lines suggesting that PAC agents will be active in myeloma cells. Our investigations with PAC-1 and L14R8 demonstrated that L14R8, but not PAC-1, induced cell death in myeloma cells in a dose- and time-dependent manner. Treatment with 10 μM L14R8 for 24 hours induced >80% apoptosis in U266 and MM.1S cells. Furthermore, L14R8 activity was inhibited by exogenous addition of zinc confirming that the mechanism of L14R8 action is through zinc chelation. The activation of procaspase-3 resulted in the cleavage of its target PARP and decreased the levels of the anti-apoptotic proteins MCL-1 and XIAP. 10 μM L14R8 was also effective in inducing >80% cell death in myeloma cells grown in the presence of protective microenvironment as demonstrated by co-culture experiments with NKtert stromal cells. In addition, when myeloma cells were grown in the presence of exogenous growth factors such as hepatocyte growth factor (50 ng/ml) or interleukin-6 (10 or 50 ng/ml), L14R8 was still able to induce apoptosis (> 60% with 10 μM L14R8 treatment in the presence of either growth factor). Finally, L14R8 was effective in inducing apoptosis in cells resistant to lenalidomide, a standard treatment for myeloma patients. Our future plan is to examine the effectiveness of this drug in primary CD138+ plasma cells (representing malignant myeloma cells). These studies demonstrate that targeting procaspase-3 with L14R8 is an effective strategy to induce myeloma cell death. Citation Format: Shadia Zaman, Rui Wang, Varsha Gandhi. Caspase activation as an approach to treat multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3328. doi:10.1158/1538-7445.AM2013-3328

Analysis of age-related global DNA methylation in chicken.

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.

Effect of neonatal administration of estrogen, antiestrogen, and testosterone on the histological picture and estrogen receptor pattern of the adult rat prostate

Background Normal sexual development and functioning of the male reproductive organs are primarily controlled by androgens. However, estrogen also plays a role in the normal development, although this is not well defined. Aim The aim of this study was to assess the effect of neonatal administration of estrogen (E), antiestrogen (AE), and testosterone (T) on the histological picture and estrogen receptor (ER) pattern of the adult rat prostate. Materials and methods In all, 40 male albino rats at the age of 2 days were divided into four equal groups: untreated controls, rats that received E, those that received AE, and those that received T orally for 5 days starting from the second day. All rats were euthanized at the age of 6 weeks, after which specimens from the ventral lobe of the prostate were obtained. Main outcome measures Histopathological and immunohistochemical analysis of the investigated sections. Results In the E-treated rats, the diameter of the prostatic acini was reduced with increased fibromuscular stroma and epithelial hyperplasia. AE-treated or T-treated rats showed no histological changes compared with controls. The prostate of E-treated rats exhibited strong immunoreactivity against the ER compared with that of AE-treated or T-treated rats. The mean area percentage of ER immunoreactivity showed significant increase in E-treated rats compared with the controls, AE-treated rats, and T-treated rats. Conclusion The prostate, despite being an androgen-dependant gland, on exposure to E early in life could undergo structural disturbances that might lead to the development of prostatic disorders later.

The Expression Profile of microRNAs in Wistar Rats with Lipopolysaccharide-Induced Periventricular Leukomalacia

Over the recent decades, with numbers of premature infants being cured, clinical diseases on brain damage like periventricular leukomalacia (PVL) have become much more common. Meanwhile, since the discovery of first miRNA lin-4, an increasing number of important studies about this small RNA have been performed not only in the normal organ development but also in the pathogenic mechanism of diseases. However, throughout the past several years, there have been rare miRNA researches discussing the connection between the PVL and miRNA. In view of this situation, we constructed an animal model of PVL induced by lipopolysaccharide (LPS) and performed a miRNA microarray which was repeated three times to profile the expression of microRNAs (miRNAs) between two groups (PVL group versus control group). Then, miRNAs with notable fold changes (fold change >1.5) were found; some of them were further validated by real-time PCR. As a result, 104 differentially expressed miRNAs were identified using the microarray, including 64 upregulated and 40 downregulated miRNAs. Then, five miRNAs of them were selected, characterized by consistent trend in expression in all three microarrays. Among these five miRNAs (miRNA-451, miRNA-200b, miRNA-29a, miRNA-21, and miRNA-138), we subsequently selected miRNA-451 and miRNA-200b for real-time PCR because they possess the highest fold changes. Finally, the results of PCR are basically in accord with the microarray. We guess these new identified miRNAs may play an important role in the pathogenesis of PVL and may provide certain pathophysiological basis for the future research of related diseases in preterm infants.

Eosinophil‐derived cytokines in health and disease: unraveling novel mechanisms of selective secretion

Over the past two decades, our understanding of eosinophils has evolved from that of categorically destructive effector cells to include active participation in immune modulation, tissue repair processes, and normal organ development, in both health and disease. At the core of their newly appreciated functions is the capacity of eosinophils to synthesize, store within intracellular granules, and very rapidly secrete a highly diverse repertoire of cytokines. Mechanisms governing the selective secretion of preformed cytokines from eosinophils are attractive therapeutic targets and may well be more broadly applicable to other immune cells. Here, we discuss recent advances in deciphering pathways of cytokine secretion, both from intact eosinophils and from tissue-deposited cell-free eosinophil granules, extruded from eosinophils undergoing a lytic cell death.

Histone H4 Lysine 20 methylation: key player in epigenetic regulation of genomic integrity

Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. Nuclear DNA is packaged into chromatin, and thus genome maintenance can be influenced by distinct chromatin environments. In particular, post-translational modifications of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic instability, demonstrating the important functions of H4K20 methylation in genome maintenance. In this review, we explain molecular mechanisms underlying these defects and discuss novel ideas for furthering our understanding of genome maintenance in higher eukaryotes.

External and internal shell formation in the ramshorn snail Marisa cornuarietisare extremes in a continuum of gradual variation in development

BackgroundToxic substances like heavy metals can inhibit and disrupt the normal embryonic development of organisms. Exposure to platinum during embryogenesis has been shown to lead to a “one fell swoop” internalization of the shell in the ramshorn snail Marisa cornuarietis, an event which has been discussed to be possibly indicative of processes in evolution which may result in dramatic changes in body plans.ResultsWhereas at usual cultivation temperature, 26°C, platinum inhibits the growth of both shell gland and mantle edge during embryogenesis leading to an internalization of the mantle and, thus, also of the shell, higher temperatures induce a re-start of the differential growth of the mantle edge and the shell gland after a period of inactivity. Here, developing embryos exhibit a broad spectrum of shell forms: in some individuals only the ventral part of the visceral sac is covered while others develop almost “normal” shells. Histological studies and scanning electron microscopy images revealed platinum to inhibit the differential growth of the shell gland and the mantle edge, and elevated temperature (28 - 30°C) to mitigate this platinum effect with varying efficiency.ConclusionWe could show that the formation of internal, external, and intermediate shells is realized within the continuum of a developmental gradient defined by the degree of differential growth of the embryonic mantle edge and shell gland. The artificially induced internal and intermediate shells are first external and then partly internalized, similar to internal shells found in other molluscan groups.

Effect of different contents of extruded linseed in the sow diet on piglet fatty acid composition and hepatic desaturase expression during the post-natal period

n-3 polyunsaturated fatty acids (n-3 PUFA) contribute to the normal growth and development of numerous organs in the piglet. The fatty acid composition of piglet tissues is linked to the fatty acid composition of sow milk and, consequently, to the composition of sow diet during the gestation and lactation period. In this study, we investigated the impact of different contents of extruded linseed in the sow diet on the fatty acid composition and desaturase gene expression of piglets. Sows received a diet containing either sunflower oil (low 18:3n-3 with 18:3n-3 representing 3% of total fatty acids) or a mixture of extruded linseed and sunflower oil (medium 18:3n-3 with 9% of 18:3n-3) or extruded linseed (high 18:3n-3 with 27% of 18:3n-3) during gestation and lactation. Fatty acid composition was evaluated on sow milk and on different piglet tissues at days 0, 7, 14, 21 and 28. The postnatal evolution of delta5 (D5D) and delta6 (D6D) desaturase mRNA expression was also measured in the liver of low 18:3n-3 and high 18:3n-3 piglets. The milk of high 18:3n-3 sows had higher proportions of n-3PUFA than that of low 18:3n-3 and medium 18:3n-3 sows. Piglets suckling the high 18:3n-3 sows had greater proportions of 18:3n-3, 20:5n-3, 22:5n-3 and 22:6n-3 in the liver, and of 22:5n-3 and 22:6n-3 in the brain than low 18:3n-3 and medium 18:3n-3 piglets. D5D and D6D mRNA expressions in piglet liver were not affected by the maternal diet at any age. In conclusion, extruded linseed in the sow diet modifies the n-3PUFA status of piglets during the postnatal period. However, a minimal content of 18:3n-3 in the sow diet is necessary to increase the n-3PUFA level in piglet liver and brain. Moreover, modifications in the n-3PUFA fatty acid composition of piglet tissue seem linked to the availability of 18:3n-3 in maternal milk and not to desaturase enzyme expression.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

AbstractAbstract 2332MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis.EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9.It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures:No relevant conflicts of interest to declare.

Short‐ and long‐term effects of exposure to natural and synthetic glucocorticoids during development

1.Glucocorticoids (GCs) are necessary for fetal development, but clinical and experimental studies suggest that excess exposure may be detrimental to health in both the short and longer term. 2.Exposure of the fetus to synthetic GCs can occur if the mother has a medical condition requiring GC therapy (e.g. asthma) or if she threatens to deliver her baby prematurely. Synthetic GCs can readily cross the placenta and treatment is beneficial, at least in the short term, for maternal health and fetal survival. 3.Maternal stress during pregnancy can raise endogenous levels of the natural GC cortisol. A significant proportion of the cortisol is inactivated by the placental 'GC barrier'. However, exposure to severe stress during pregnancy can result in increased risk of miscarriage, low birth weight and behavioural deficits in children. 4.Animal studies have shown that excess exposure to both synthetic and natural GCs can alter normal organ development, including that of the heart, brain and kidney. The nature and severity of the organ impairment is dependent upon the timing of exposure and, in some cases, the type of GC used and the sex of the fetus. 5.In animal models, exposure to elevated GCs during pregnancy has been associated with adult-onset diseases, including elevated blood pressure, impaired cardiac and vascular function and altered metabolic function.

The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato

The plant protein ARGONAUTE1 (AGO1) functions in multiple RNA-silencing pathways, including those of microRNAs, key regulators of growth and development. Genetic analysis of ago1 mutants with informative defects has provided valuable insights into AGO1's biological functions. Tomato encodes two AGO1 homologs (SlAGO1s), but mutants have not been described to date. To analyze SlAGO1s' involvement in development, we confirmed that both undergo decay in the presence of the Polerovirus silencing suppressor P0 and produce a transgenic responder line (OP:P0HA) that, upon transactivation, expresses P0 C-terminally fused to a hemagglutinin (HA) tag (P0HA) and destabilizes SlAGO1s at the site of expression. By crossing OP:P0HA with a battery of driver lines, constitutive as well as organ- and stage-specific SlAGO1 downregulation was induced in the F1 progeny. Activated plants exhibited various developmental phenotypes that partially overlapped with those of Arabidopsis ago1 mutants. Plants that constitutively expressed P0HA had reduced SlAGO1 levels and increased accumulation of miRNA targets, indicating compromised SlAGO1-mediated silencing. Consistent with this, they exhibited pleiotropic morphological defects and their growth was arrested post-germination. Transactivation of P0HA in young leaf and floral organ primordia dramatically modified corresponding organ morphology, including the radialization of leaflets, petals and anthers, suggesting that SlAGO1s' activities are required for normal lateral organ development and polarity. Overall, our results suggest that the OP:P0HA responder line can serve as a valuable tool to suppress SlAGO1 silencing pathways in tomato. The suppression of additional SlAGOs by P0HA and its contribution to the observed phenotypes awaits investigation.