Retinoic acid signaling pathways.

Retinoic acid (RA), a metabolite of retinol (vitamin A), functions as a ligand for nuclear RA receptors (RARs) that regulate development of chordate animals. RA-RARs can activate or repress transcription of key developmental genes. Genetic studies in mouse and zebrafish embryos that are deficient in RA-generating enzymes or RARs have been instrumental in identifying RA functions, revealing that RA signaling regulates development of many organs and tissues, including the body axis, spinal cord, forelimbs, heart, eye and reproductive tract. An understanding of the normal functions of RA signaling during development will guide efforts for use of RA as a therapeutic agent to improve human health. Here, we provide an overview of RA signaling and highlight its key functions during development.

Zebrafish embryos exposed to deltamethrin exhibit abnormalities despite induced expression of related genes ( you, you-too, momo and u-boot).

Evaluation of the toxic effects of a widely used synthetic pyrethroid, deltamethrin (DM), was carried out in this study. This pesticide is preferred for pest control because of its low environmental persistence and toxicity. We investigated the expression pattern of four genes, namely, you ( you), yot ( you-too), momo ( mom) and ubo ( u-boot) during early development of zebrafish, that is, from 12 hpf to 48 hpf stages. These stages are selected as most of the important developmental aspects take place during this period. All four genes are known to play a vital role in development of notochord and somites. To understand the effect of DM on development, embryos of 4 hpf stage were exposed to two concentrations (100 and 200 µg/L) of DM, and observations were made at 12, 24 and 48 hpf stages. Our earlier studies have shown phenotypic abnormalities such as notochord bending, tail deformation, yolk sac and pericardial edema, lightening of body and eye pigmentation and interfered in somite patterning, during these stages of development. Understanding the relationship of phenotypic abnormalities with these four genes has been our primary objective. These four genes were analyzed by Reverse transcription (RT)-polymerase chain reaction and intensity of the bands has shown induction in their expression after exposure to the toxicant. In spite of the expression of genes, it was noticed that DM caused abnormalities. It can be said from the results that translational pathway could have been affected.

Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression.

Expression of developmental genes in brown fat cells grown in vitro is linked with lipid accumulation.

En1, Nr2f1, Gpc4, Sfrp2, Shox2, Tbx15 and Thbd are among the genes involved in development process of an organism in a number of tissues, in particular adipose tissue. Considering the involvement of isobutyl-methyl-xanthine (IBMX), indomethacin, dexamethasone (DEX), triiodothyronine (T3), and insulin in adipocyte differentiation, we propose that these differentiation-inducing agents may regulate differentiation in brown adipose tissue through a developmental process. Stromavascular cells isolated from interscapular brown fat of mice were cultured in DMEM-LG medium. Proliferating brown preadipocytes were differentiated in the presence of IBMX, indomethacin, DEX, T3 and insulin. Pref1 (marker of proliferation stage) and uncoupling protein 1 (UCP1, marker for differentiation stage) were upregulated during proliferation and differentiation, respectively. Relative expression of Pref1, UCP1 and developmental genes was determined in different stages of adipogenesis. En1, Gpc4, Nr2f1, Sfrp2 and Shox2 were upregulated during differentiation. Differentiation of preadipocytes in the absence of IBMX, indomethacin, and DEX resulted in drastic reduction in fat accumulation in differentiated adipocytes with simultaneous decrease in En1, Gpc4, Nr2f1, Sfrp2, Shox2 and Tbx15 gene expression. T3 upregulated the expression of En1, Gpc4, Sfrp2 and Tbx15 genes during differentiation and downregulated Shox2 expression as compared to proliferated state. Insulin upregulated the expression of Shox2.

Ciona intestinalis as a marine model system to study some key developmental genes targeted by the diatom-derived aldehyde decadienal.

The anti-proliferative effects of diatoms, described for the first time in copepods, have also been demonstrated in benthic invertebrates such as polychaetes, sea urchins and tunicates. In these organisms PUAs (polyunsaturated aldehydes) induce the disruption of gametogenesis, gamete functionality, fertilization, embryonic mitosis, and larval fitness and competence. These inhibitory effects are due to the PUAs, produced by diatoms in response to physical damage as occurs during copepod grazing. The cell targets of these compounds remain largely unknown. Here we identify some of the genes targeted by the diatom PUA 2-trans-4-trans-decadienal (DD) using the tunicate Ciona intestinalis. The tools, techniques and genomic resources available for Ciona, as well as the suitability of Ciona embryos for medium-to high-throughput strategies, are key to their employment as model organisms in different fields, including the investigation of toxic agents that could interfere with developmental processes. We demonstrate that DD can induce developmental aberrations in Ciona larvae in a dose-dependent manner. Moreover, through a preliminary analysis, DD is shown to affect the expression level of genes involved in stress response and developmental processes.

Chronic PFOS exposure alters the expression of neuronal development-related human homologues in Eisenia fetida.

PFOS is a toxic, persistent environmental pollutant which is widespread worldwide. PFOS contamination has entered the food chain and is interfering with normal development in man and is neurotoxic, hepatotoxic and tumorigenic. The earthworm, Eisenia fetida is one of the organisms which can help to diagnose soil health and contamination at lower levels in the food chain. Studying the chronic effects of sub-lethal PFOS exposure in such an organism is therefore appropriate. As PFOS bioaccumulates and is not easily biodegraded, it is biomagnified up the food chain. Gene expression studies will give us information to develop biomarkers for early diagnosis of soil contamination, well before this contaminant passes up the food chain. We have carried out mRNA sequencing of control and chronically PFOS exposed E. fetida and reconstructed the transcripts in silico and identified the differentially expressed genes. Our findings suggest that PFOS up/down regulates neurodegenerative-related human homologues and can cause neuronal damage in E. fetida. This information will help to understand the links between neurodegenerative disorders and environmental pollutants such as PFOS. Furthermore, these up/down regulated genes can be used as biomarkers to detect a sub-lethal presence of PFOS in soil. Neuronal calcium sensor-2, nucleoside diphosphate kinase, polyadenylate-binding protein-1 and mitochondrial Pyruvate dehydrogenase protein-X component, could be potential biomarkers for sub lethal concentrations of PFOS.

Transcriptome analysis of the dihydrotestosterone-exposed fetal rat gubernaculum identifies common androgen and insulin-like 3 targets.

Androgens and insulin-like 3 (INSL3) are required for development of the fetal gubernaculum and testicular descent. Previous studies suggested that the INSL3-exposed fetal gubernacular transcriptome is enriched for genes involved in neural pathways. In the present study, we profiled the transcriptome of fetal gubernaculum explants exposed to dihydrotestosterone (DHT) and compared this response to that with INSL3. We exposed fetal (Embryonic Day 17) rat gubernacula to DHT for 24 h (10 and 30 nM) or 6 h (1 and 10 nM) in organ culture and analyzed gene expression relative to that of vehicle-treated controls using Affymetrix arrays. Results were annotated using functional, pathway, and promoter analyses and independently validated for selected transcripts using quantitative RT-PCR (qRT-PCR). Transcripts were differentially expressed after 24 h but not 6 h. Most highly overrepresented functional categories included those related to gene expression, skeletal and muscular development and function, and Wnt signaling. Promoter response elements enriched in the DHT-specific transcriptome included consensus sequences for c-ETS1, ELK1, CREB, CRE-BP1/c-June, NRF2, and USF. We observed that 55% of DHT probe sets were also differentially expressed after INSL3 exposure and that the direction of change was the same in 96%. The qRT-PCR results confirmed that DHT increased expression of the INSL3-responsive genes Crlf1 and Chrdl2 but reduced expression of Wnt4. We also validated reduced Tgfb2 and Cxcl12 and increased Slit3 expression following DHT exposure. These data suggest a robust overlap in the DHT- and INSL3-regulated transcriptome that may be mediated in part by CREB signaling and a common Wnt pathway response for both hormones in the fetal gubernaculum.

Isolation of siRNA target by biotinylated siRNA reveals that human CCDC12 promotes early erythroid differentiation.

Erythroid differentiation is a tightly regulated multi-step process that has not been fully elucidated. We previously reported that a siRNA screened from random siRNA library, siRNA clone-67, induced erythroid differentiation in human erythroleukemia K-562cell line. Here we identified that human CCDC12 (coiled-coil domain containing 12) is a target of siRNA clone-67, by target capture with biotinylated siRNA. Over-expression of CCDC12 in K-562cell up-regulated the expression of CD235, ε-globin and γ-globin, accelerated cell growth, and slightly down-regulated the expression of GATA-2. Knockdown of CCDC12 slowed down the cell growth. These data indicate that CCDC12 is a new participant that promotes early erythroid differentiation.

Oral toxicity of Photorhabdus culture media on gene expression of the adult sweetpotato whitefly, Bemisia tabaci.

The oral toxicity of culture media of the symbiotic bacteria, Photorhabdus temperata, mutually associated with entomopathogenic nematode Heterorhabditis megidis and Photorhabdus luminescens ssp. laumondii (TT01) mutually associated with Heterorhabditis bacteriophora, were investigated in the adults of Bemisia tabaci. The oral ingestion of sucrose diet solutions (20%) containing bacteria-free supernatant of the culture media from symbiotic bacteria gradually increased mortalities and was completely lethal at 60 h after the treatments, whereas the mortalities of the controls, sucrose solutions with or without media that uncultured with bacteria, were less than 17% up to 84 h of incubation. The effects of oral ingestion of symbiont culture media were demonstrated on the expression rates of several genes of B. tabaci using quantitative real-time RT-PCR analysis. Genes associated with immunity (knottin) and nervous system (acetylcholine receptor, acetylcholine esterase and sodium channel) were up-regulated while genes involved in metabolism (cytochromep450 and carboxylesterase) were down-regulated, but genes involved in development (ecdysone receptor), reproduction (vitellogenin) and stress (hsp70, hsp90 and shsp) did not change transcription rates. Our results provide information for the understanding of the mechanism of symbiont pathogenic factors for the manipulation of host physiology at the transcription level.

Yeast cells can access distinct quiescent states.

We conducted a phenotypic, transcriptional, metabolic, and genetic analysis of quiescence in yeast induced by starvation of prototrophic cells for one of three essential nutrients (glucose, nitrogen, or phosphate) and compared those results with those obtained with cells growing slowly due to nutrient limitation. These studies address two related questions: (1) Is quiescence a state distinct from any attained during mitotic growth, and (2) does the nature of quiescence differ depending on the means by which it is induced? We found that either limitation or starvation for any of the three nutrients elicits all of the physiological properties associated with quiescence, such as enhanced cell wall integrity and resistance to heat shock and oxidative stress. Moreover, the starvations result in a common transcriptional program, which is in large part a direct extrapolation of the changes that occur during slow growth. In contrast, the metabolic changes that occur upon starvation and the genetic requirements for surviving starvation differ significantly depending on the nutrient for which the cell is starved. The genes needed by cells to survive starvation do not overlap the genes that are induced upon starvation. We conclude that cells do not access a unique and discrete G(0) state, but rather are programmed, when nutrients are scarce, to prepare for a range of possible future stressors. Moreover, these survival strategies are not unique to quiescence, but are engaged by the cell in proportion to nutrient scarcity.

Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures.

The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 µM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

Gene expression profile of androgen modulated genes in the murine fetal developing lung.

BACKGROUND: Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood. METHODS: To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17) and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens. RESULTS: Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment. CONCLUSION: Our results show clearly that there is a real delay in lung maturation between male and female in this period, the latter pursuing already lung maturation while the proper is not yet fully engaged in the differentiation processes at GD17. In addition, this study provides a list of genes which are under the control of androgens within the lung at the moment of surge of surfactant production in murine fetal lung.

Transient estrogen exposure from birth affects uterine expression of developmental markers in neonatal gilts with lasting consequences in pregnant adults.

Disruption of estrogen-sensitive, estrogen receptor (ER)-dependent events during porcine uterine development between birth (postnatal day=PND 0) and PND 14 affects patterns of uterine morphoregulatory gene expression in the neonate with lasting consequences for reproductive success. Uterine capacity for conceptus support is reduced in pregnant adult gilts exposed to estradiol valerate (EV) for 14 days from birth. Objectives here were to determine effects of EV exposure from birth through PND 13 on neonatal uterine and adult endometrial markers of growth, patterning, and remodeling. Targets included the relaxin receptor (RXFP1), estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), morphoregulatory markers HOXA10 and WNT7A, and the matrix metalloproteinases (MMP)2 and MMP9. Gilts were treated daily with EV (50 microg/kg body weight per day, i.m.) or corn oil vehicle from birth through PND 13. Uteri were obtained from neonates on PND 14 and from adults on pregnancy day 12 (PxD 12). In neonates, EV exposure from birth increased uterine RXFP1 gene expression, and both ESR1 and VEGFA proteins. At PxD 12, endometrial RXFP1 mRNA remained elevated, while ESR1 protein was reduced. Early EV treatment decreased neonatal uterine WNT7A, but increased HOXA10 expression. WNT7A expression was reduced in EV-treated adults. Transient EV exposure increased MMP9 transcripts at PND 14, whereas both latent and active MMP9 activity was increased due to early EV treatment in adults on PxD 12. Results support the hypothesis that transient, estrogen-induced disruption of porcine uterine development from birth alters early programming events that lead to functional consequences in the adult.

Expression of the genes siamois, engrailed-2, bmp4 and myf5 during Xenopus development in presence of the marine toxins okadaic acid and palytoxin.

The present investigation examines the effects of the marine toxins, okadaic acid (OA) and palytoxin (PTX), on some genes involved in the neural and muscular specification and patterning of Xenopus laevis. The RT-PCR analyses performed at different stages of embryonic and larval development (stages 11-47) demonstrated that both toxins induce an over-expression of the genes siamois and engrailed-2 and a different behaviour in bmp4 and myf5. Indeed, OA provoked a significant increase in bmp4 in the earliest stage (11) examined, a down-regulation from stages 12 to 17, and a renewed increase from the beginning of hatching onwards (stages 35-47). In contrast, myf5 was up-regulated in all stages up to 35. PTX induced an over-expression of both bmp4 and myf5 during the embryonic and early larval development stages. The results show that PTX induces an increase in expression levels in all tested genes, while the response to OA seems to be more stage-dependent, with the embryonic development stage more sensitive to the toxin than the larval stages.

The ontogeny of genes related to ovine fetal cardiac growth.

The objective of this study was to determine the ontogenetic profiles in left and right ventricle of genes implicated in cardiac growth, including mineralocorticoid (MR) and glucocorticoid (GR) receptor, 11 beta-hydroxysteroid dehydrogenase (11beta-HSD) 1 and 2 and genes of the angiotensin system and insulin-like growth factor (IGF) family. Samples from left and right ventricles (LV, RV) were collected from hearts of sheep fetuses at 80, 100, 120, 130, and 145 days of gestation and from newborn lambs. Quantitative real-time PCR was performed to determine the MR, GR, 11beta-HSD 1 and 2, angiotensin converting enzyme (ACE) 1 and 2, IGF1, IGF2, IGF receptors IGF-1R and IGF-2R, and IGF-binding proteins (IGFBP) 2 and 3. In the LV, MR and GR both decreased toward term. In the RV, MR and GR expression did not decrease, but both 11beta-HSD 1 and 2 mRNA levels increased after birth. ACE1 expression in LV and RV sharply increases just before parturition, whereas ACE2 decreased in the LV and RV in late gestation. IGF2, IGF2R, and IGFBP2 expression levels substantially decreased in late gestation in LV and RV; IGF2R also decreased with age in LV. These patterns suggest that reduced expression of genes related to IGF and angiotensin II action occur as proliferative activity declines and terminal differentiation occurs in the late gestation fetal heart.

Roles of the first and second round of DNA replication in the regulation of zygotic gene activation in mice.

In the mouse embryo, expression of zygotic genes starts in the S/G2 phase of the 1-cell stage and greatly increases during the 2-cell stage. Although the timing of zygotic gene activation (ZGA) is thus established, the mechanism regulating ZGA is poorly understood. Previous studies using reporter genes have suggested that a transcriptionally repressive state is established during the 2-cell stage and that the first and second rounds of DNA replication are involved in this process. To further elucidate the respective roles of the two rounds of DNA replication in ZGA, we analyzed the expression of four ZGA genes (hsp70.1, eif-1a, muerv and zscan4d) in embryos whose DNA replication was inhibited by treatment with aphidicolin, an inhibitor of DNA polymerase. Inhibiting the first round increased the expression levels of hsp70.1, eif-1a and zscan4d but decreased that of muerv, while inhibiting the second round increased the expression levels of all four genes. These results suggest that the transcriptionally repressive state seems to be established after the second round of DNA replication.

Cloning and analysis of IFRG (interferon responsive gene) in rabbit oocytes and preimplantation embryos.

Although there is more evidence that shows that IFNs (interferons) plays a very important role in the early development of the embryo, the mechanism of IFNs is still unclear. Our study showed that IFRG is expressed from oocytes- through to the preimplantation embryo in rabbits. This finding provides some clues for better understanding the role of IFNs in the development of the embryo. The full length of rabbit IFRG cDNA (Accession No. AJ584672), with a 2794bp encoding 131 amino acid sequence, was cloned IFRG expression can be detected in 8 different tissues: ovary, heart, lung, liver, kidney, spleen, cerebra, and the 18-day whole-body embryo. Whole-mount in situ hybridization showed that IFRG was highly expressed in the inner-cell mass of rabbit blastula. IFRG may play an important role in embryo development and tissue differentiation.

Cloning and analysis of IFRG (interferon responsive gene) in rabbit oocytes and preimplantation embryos

Although there is more evidence that shows that IFNs (interferons) plays a very important role in the early development of the embryo, the mechanism of IFNs is still unclear. Our study showed that IFRG is expressed from oocytes- through to the preimplantation embryo in rabbits. This finding provides some clues for better understanding the role of IFNs in the development of the embryo. The full length of rabbit IFRG cDNA (Accession No. AJ584672), with a 2794bp encoding 131 amino acid sequence, was cloned IFRG expression can be detected in 8 different tissues: ovary, heart, lung, liver, kidney, spleen, cerebra, and the 18-day whole-body embryo. Whole-mount in situ hybridization showed that IFRG was highly expressed in the inner-cell mass of rabbit blastula. IFRG may play an important role in embryo development and tissue differentiation.(AU)

Altered expression of pro-inflammatory and developmental genes in the fetal brain in a mouse model of maternal infection.

Human studies of unexplained cerebral palsy (CP) suggest an association with maternal infection. We used an established model of maternal infection, lipopolysaccharide (LPS) administration, to investigate the molecular changes in the fetal brain that may link maternal infection and CP. We compared gene expression in brains from mouse pups exposed to LPS in utero to those from saline-treated controls. Dams were injected with 50 microg LPS or saline on E18 with surgical delivery from 0.5 to 6h later. Differential gene expression was analyzed in the whole mouse brain using RT-PCR. When compared to control mice, pups exposed to LPS showed increased expression of pro-inflammatory genes monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-1beta (IL-1beta), as well as VEGF, a regulator of vascular development and permeability, the anti-apoptotic protein Y-box-binding protein-1 (YB-1), and the neuronal differentiation factor necdin. LPS-exposed mice also showed downregulation of semaphorin 5b and groucho, involved in axon guidance and neurogenesis, respectively, providing evidence that LPS may disrupt normal developmental pathways. These data suggest possible mechanisms for adverse neurological outcomes following maternal infection involving elevated cytokine levels and altered expression of developmental genes in the fetal brain.