Rewiring of the epigenome and chromatin architecture by exogenously induced retinoic acid signaling during zebrafish embryonic development.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFß) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFß signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFß inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFß inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro.

Somite morphogenesis is required for axial blood vessel formation during zebrafish embryogenesis.

Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate toward the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid-induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system.

Developmental disorders caused by cefixime in the otic vesicles of zebrafish embryos or larvae.

To explore the developmental toxicity of cefixime (CE) in the developmental disorder and toxicity mechanism of CE on otic vesicles, zebrafish embryos were used as an animal model. The results showed that CE increased mortality in a dose-dependent manner and decreased the hatching rate of zebrafish larva at 96 hpf. Interestingly, CE significantly reduced the area of the saccule and utricle, as well as the area of otic vesicles in zebrafish larvae (p < 0.001). Fibroblast growth factor 8a (Fgf8a) inhibitors and bone morphogenetic protein (BMP) inhibitors caused similar morphological changes. CE decreased the lateral hair cells of zebrafish larvae in a dose-dependent manner. Furthermore, CE caused the downregulation of cartilage and bone-related genes and Na+/K+-ATPase-related genes of zebrafish larvae at 72 hpf and 120 hpf according to RT-qPCR. A comparison with the control group revealed that 100 µg/mL CE also caused a decrease in Na+/K+-ATPase activity (p < 0.01). In addition, antibody staining verified that CE inhibited the expression of Na+/K+-ATPase in the otic vesicles and the nephridium of zebrafish larvae. The data obtained in this study suggested that CE has significant ototoxicity during embryonic development of zebrafish, which is closely related to Na+/K+-ATPase and the regulation of the Fgf8a/BMP signaling pathways. The effects and toxicity of CE on ear development in other animal models need to be further explored.

Adult pancreatic islet endocrine cells emerge as fetal hormone-expressing cells.

The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth ("neogenesis"). Here, using four conditional cell lineage tracing ("pulse-and-chase") murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies.

RNA-Seq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation.

Skeletal muscle satellite cells cultured on soft surfaces (12 kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100 kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of É£-interferon, 37 °C). The largest change in overall gene expression occurred at day 1, as cells switched from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of mRNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNA-Seq dataset will be a useful resource for understanding Pax3 expressing cells.

Amino Acids and IGF1 Regulation of Fish Muscle Growth Revealed by Transcriptome and microRNAome Integrative Analyses of Pacu (Piaractus mesopotamicus) Myotubes.

Amino acids (AA) and IGF1 have been demonstrated to play essential roles in protein synthesis and fish muscle growth. The myoblast cell culture is useful for studying muscle regulation, and omics data have contributed enormously to understanding its molecular biology. However, to our knowledge, no study has performed the large-scale sequencing of fish-cultured muscle cells stimulated with pro-growth signals. In this work, we obtained the transcriptome and microRNAome of pacu (Piaractus mesopotamicus)-cultured myotubes treated with AA or IGF1. We identified 1228 and 534 genes differentially expressed by AA and IGF1. An enrichment analysis showed that AA treatment induced chromosomal changes, mitosis, and muscle differentiation, while IGF1 modulated IGF/PI3K signaling, metabolic alteration, and matrix structure. In addition, potential molecular markers were similarly modulated by both treatments. Muscle-miRNAs (miR-1, -133, -206 and -499) were up-regulated, especially in AA samples, and we identified molecular networks with omics integration. Two pairs of genes and miRNAs demonstrated a high-level relationship, and involvement in myogenesis and muscle growth: marcksb and miR-29b in AA, and mmp14b and miR-338-5p in IGF1. Our work helps to elucidate fish muscle physiology and metabolism, highlights potential molecular markers, and creates a perspective for improvements in aquaculture and in in vitro meat production.

Role of Groucho and Groucho1-like in Regulating Metamorphosis and Ovary Development in Nilaparvata lugens (Stål).

Juvenile hormone and ecdysone are key regulators in the metamorphosis and development. Grocho (Gro) is a highly conserved protein required for metamorphosis and development. Brown planthopper (Nilaparvata lugens) is a major pest affecting rice production in China and many Asian countries. Although the molecular function of Gro has been investigated in holometabolous insects such as Aedes aegypti and Drosophila melanogaster, their role in the hemimetabolous insect, brown planthopper, and the relationship between NlGro/NlGro1-L and JH/ecdysone signaling pathway, remained unknown. In this study, NlGroucho (NlGro) and NlGroucho1-like (NlGro1-L) were cloned. An analysis of the predicted protein sequence showed that NlGro has highly conserved Q domain and WD40 domain, and NlGro1-L has a highly conserved WD40 domain. The expression profiles of both genes were studied by quantitative real-time PCR (qRT-PCR). Their relative expressions were high in egg, head, wing, ovary, and testis. NlGro and NlGro1-L were found to interact genetically with juvenile hormone and ecdysone signaling by hormone treatment and RNAi of JH/ecdysone signaling-related genes. Moreover, when NlGro or NlGro1-L was down-regulated alone, the survival rate was decreased, the ovarian development was delayed, and the oviposition was also affected. All defects were aggravated when NlGro and NlGro1-L were down-regulated together. This study will help to develop new pesticides on the basis of the function of NlGro and NlGro1-L, and provide new possibilities for the control of Nilaparvata lugens.

Icariin inhibits epithelial mesenchymal transition of renal tubular epithelial cells via regulating the miR-122-5p/FOXP2 axis in diabetic nephropathy rats.

Epithelial mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs) dominates the pathology of diabetic nephropathy (DN). microRNAs (miRNAs) can influence the fate of DN via regulation of EMT. This study aimed to analyze the role of Icariin (ICA) in EMT of RTECs, hoping to provide theoretical basis for DN management. The DN rat model was established using streptozocin, followed by ICA treatment, histopathological observation, and detection of creatinine and blood urea nitrogen. In vitro cell models were established using high glucose (HG), followed by assessment of cell proliferation, apoptosis, and migration, and E-cadherin, α-SMA, miR-122-5p, and FOXP2 expressions. Cells were transfected with miR-122-5p mimics or si-FOXP2 for joint experiments with ICA. The targeting relationship between miR-122-5p and FOXP2 was verified. ICA repaired renal dysfunctions and glomerular structure abnormities of DN rats in a dose-dependent manner. In vitro, ICA improved proliferation while suppressed migration, apoptosis, and EMT of RTECs. miR-122-5p was up-regulated in DN rats and suppressed by ICA, and miR-122-5p targeted FOXP2. miR-122-5p up-regulation or FOXP2 down-regulation reversed the protective effects of ICA on HG-induced RTECs. Overall, our finding ascertained that ICA inhibited miR-122-5p to promote FOXP2 transcription, thereby attenuating EMT of RTECs and renal injury in DN rats.

Genome-Wide Analysis in Drosophila Reveals the Genetic Basis of Variation in Age-Specific Physical Performance and Response to ACE Inhibition.

Despite impressive results in restoring physical performance in rodent models, treatment with renin-angiotensin system (RAS) inhibitors, such as Lisinopril, have highly mixed results in humans, likely, in part, due to genetic variation in human populations. To date, the genetic determinants of responses to drugs, such as RAS inhibitors, remain unknown. Given the complexity of the relationship between physical traits and genetic background, genomic studies which predict genotype- and age-specific responses to drug treatments in humans or vertebrate animals are difficult. Here, using 126 genetically distinct lines of Drosophila melanogaster, we tested the effects of Lisinopril on age-specific climbing speed and endurance. Our data show that functional response and sensitivity to Lisinopril treatment ranges from significant protection against physical decline to increased weakness depending on genotype and age. Furthermore, genome-wide analyses led to identification of evolutionarily conserved genes in the WNT signaling pathway as being significantly associated with variations in physical performance traits and sensitivity to Lisinopril treatment. Genetic knockdown of genes in the WNT signaling pathway, Axin, frizzled, nemo, and wingless, diminished or abolished the effects of Lisinopril treatment on climbing speed traits. Our results implicate these genes as contributors to the genotype- and age-specific effects of Lisinopril treatment and because they have orthologs in humans, they are potential therapeutic targets for improvement of resiliency. Our approach should be widely applicable for identifying genomic variants that predict age- and sex-dependent responses to any type of pharmaceutical treatment.

Root-specific CLE3 expression is required for WRKY33 activation in Arabidopsis shoots.

KEY MESSAGE: This study focused on the role of CLE1-7 peptides as defense mediators, and showed that root-expressed CLE3 functions as a systemic signal to regulate defense-related gene expression in shoots. In the natural environment, plants employ diverse signaling molecules including peptides to defend themselves against various pathogen attacks. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes (CLE1-7) respond to biotic stimuli. CLE3 showed significant up-regulation upon treatment with flg22, Pep2, and salicylic acid (SA). Quantitative real-time PCR (qRT-PCR) analysis revealed that CLE3 expression is regulated by the NON-EXPRESSOR OF PR GENES1 (NPR1)-dependent SA signaling and flg22-FLAGELLIN-SENSITIVE 2 (FLS2) signaling pathways. We demonstrated that SA-induced up-regulation of CLE3 in roots was required for activation of WRKY33, a gene involved in the regulation of systemic acquired resistance (SAR), in shoots, suggesting that CLE3 functions as a root-derived signal that regulates the expression of defense-related genes in shoots. Microarray analysis of transgenic Arabidopsis lines overexpressing CLE3 under the control of a ß-estradiol-inducible promoter revealed that root-confined CLE3 overexpression affected gene expression in both roots and shoots. Comparison of CLE2- and CLE3-induced genes indicated that CLE2 and CLE3 peptides target a few common but largely distinct downstream genes. These results suggest that root-derived CLE3 is involved in the regulation of systemic rather than local immune responses. Our study also sheds light on the potential role of CLE peptides in long-distance regulation of plant immunity.

Molecular and behavioral assessment in larval zebrafish (Danio rerio) following exposure to environmentally relevant levels of the antineoplastic cyclophosphamide.

Antineoplastics treat cancers and enter aquatic ecosystems through wastewater and hospital effluent. Risks associated with antineoplastics are not well characterized in aquatic organisms. We conducted zebrafish embryo/larvae toxicity assays to evaluate responses to cyclophosphamide (0.01-50 µM). Zebrafish survival was affected by 5 µM cyclophosphamide and deformities were noted at > 1 µM. Oxidative respiration remained unchanged in embryos with exposure up to 200 µM. Reactive oxygen species were not increased by 50 µM cyclophosphamide exposure. More than 15 oxidative stress and immune-related transcripts were measured. Superoxide dismutase 2 and heat shock protein 70 and 90a were induced in larvae by cyclophosphamide. Immune-related transcripts were assessed due to immunosuppressive properties of cyclophosphamide, and mmp9 and myd88 levels were altered in expression. Hyperactivity of larvae was noted following 5 µM cyclophosphamide exposure. There was no change in anxiety-related endpoints (light-dark preference). Risks for larval fish exposed to cyclophosphamide in the environment may be low.

Methylmercury-induced hair cell loss requires hydrogen peroxide production and leukocytes in zebrafish embryos.

Hearing impairment and deafness is frequently observed as one of the neurological signs in patients with Minamata disease caused by methylmercury (MeHg) poisoning. Loss of hair cells in humans and animals is a consequence of MeHg poisoning. However, it is still not clear how MeHg causes hearing deficits. We employed the hair cells of the lateral line system of zebrafish embryos as a model to explore this question. We exposed transgenic zebrafish embryos to MeHg (30-360 µg/L) at the different stages, and scored the numbers of hair cells. We find that MeHg-induced reduction of hair cells is in a concentration dependent manner. By employing antisense morpholino against to pu.1, we confirm that loss of hair cells involves the action of leukocytes. Moreover, hair cell loss is attenuated by co-treating MeHg-exposed embryos with pharmacological inhibitors of NADPH oxidases named diphenyleneiodonium (DPI) and VAS2870. In situ gene expression analysis showed that genes encoding the SQSTM1-Keap1-Nrf2 systems involved in combating oxidative stress and immune responses are highly expressed in the lateral line organs of embryos exposed to MeHg. This suggests that induction of hydrogen peroxide (H2O2) is the primary effect of MeHg on the hair cells. Genes induced by MeHg are also involved in regeneration of the hair cells. These features are likely related to the capacity of the zebrafish to regenerate the lost hair cells.

The role of polypeptide PDTLN1 in suppression of PI3K/AKT signaling causes cardiogenetic disorders in vitro and in vivo.

AIMS: A new polypeptide, PDTLN1, derived from the human Talin-1 protein, which is highly expressed in both myocardial tissue and maternal peripheral blood of aborted fetuses with congenital heart disease (CHD). However, its role in cardiac developmental disorders has not been disclosed till now. In the present study, we aim to assess the functions of PDTLN1 in heart development of zebrafish and cellular viability, proliferation, and apoptosis of P19 cells. MAIN METHODS: Cellular viability was assessed by Cell Counting Kit-8, the EdU Kit was used to evaluate cellular proliferation, and apoptosic rate of P19 was examined using FITC Annexin-V staining followed by flow cytometry. The zebrafish embryos were divided into three groups: PEP group and NC group were microinjected with polypeptides, WT group without any intervention. The protein expression of PI3K/AKT were evaluated by western blotting. KEY FINDINGS: PDTLN1 could suppress the proliferation, and facilitate apoptosis. PDTLN1 caused abnormal heart development of zebrafish embryos and the PDTLN1 (50 µM)-injected group showed an aberrant expression pattern of vmhc, amhc and cmlc2. Compared to the CTL group and SC79 group of P19 cells, the PDTLN1 group had a lower phosphorylated PI3K/AKT proteins level, decreased cellular viability and lower proliferation activity. SIGNIFICANCE: PDTLN1 caused cardiac developmental defects in zebrafish, inhibited cellular viability, proliferation, and promoted apoptosis of P19 cells via suppressing the PI3K/AKT signaling pathway. Our findings provide a fresh perspective on the functional mechanism of human-derived peptides and may promote novel diagnostic biomarkers detection and therapeutic targets in CHD.

Isopimpinellin extends antiangiogenic effect through overexpression of miR-15b-5p and downregulating angiogenic stimulators.

BACKGROUND: Angiogenesis is the formation of new blood vessels from an existing vasculature through a series of processes such as activation, proliferation, and directed migration of endothelial cells. Angiogenesis is instrumental in the metastatic spread of tumors. Isopimpinellin, a furanocoumarin group of phytochemicals, is an anticarcinogenic agent. However, no studies have proven its antiangiogenic effects. The current study thus aimed to screen the antiangiogenic effect of isopimpinellin. METHODS AND RESULTS: Human Umblical Vein Endothelial Cell (HUVEC) as an in vitro model and zebrafish embryos as an in vivo model was used in this study. The experimental results showed that isopimpinellin effectively inhibited HUVEC proliferation, invasion, migration, and tube formation, which are the key steps in angiogenesis by markedly suppressing the expression of pro-angiogenic genes VEGF, AKT, and HIF-1α. In addition, isopimpinellin exerts its anti-angiogenic effect through the regulation of miR-15b-5p and miR-542-3p. Furthermore, in zebrafish embryos, isopimpinellin inhibited the development of intersegmental vessels (ISVs) through the significant downregulation of all pro-angiogenic genes vegf, vegfr2, survivin, angpt-1, angpt-2, and tie-2. CONCLUSION: Collectively, these experimental findings offer novel insights into the antiangiogenic nature of isopimpinellin and open new avenues for therapeutic approaches.

Effects of lanthanum nitrate on behavioral disorder, neuronal damage and gene expression in different developmental stages of Caenorhabditis elegans.

Rare earth elements (REEs) are widely used in the industry, agriculture, biomedicine, aerospace, etc, and have been shown to pose toxic effects on animals, as such, studies focusing on their biomedical properties are gaining wide attention. However, environmental and population health risks of REEs are still not very clear. Also, the REEs damage to the nervous system and related molecular mechanisms needs further research. In this study, the L1 and L4 stages of the model organism Caenorhabditis elegans were used to evaluate the effects and possible neurotoxic mechanism of lanthanum(III) nitrate hexahydrate (La(NO3)3·6H2O). For the L1 and L4 stage worms, the 48-h median lethal concentrations (LC50s) of La(NO3)3·6H2O were 93.163 and 648.0 mg/L respectively. Our results show that La(NO3)3·6H2O induces growth inhibition and defects in behavior such as body length, body width, body bending frequency, head thrashing frequency and pharyngeal pumping frequency at the L1 and L4 stages in C. elegans. The L1 stage is more sensitive to the toxicity of lanthanum than the L4 stage worms. Using transgenic strains (BZ555, EG1285 and NL5901), we found that La(NO3)3·6H2O caused the loss or break of soma and dendrite neurons in L1 and L4 stages; and α-synuclein aggregation in L1 stage, indicating that Lanthanum can cause toxic damage to dopaminergic and GABAergic neurons. Mechanistically, La(NO3)3·6H2O exposure inhibited or activated the neurotransmitter transporters and receptors (glutamate, serotonin and dopamine) in C. elegans, which regulate behavior and movement functions. Furthermore, significant increase in the production of reactive oxygen species (ROS) was found in the L4 stage C. elegans exposed to La(NO3)3·6H2O. Altogether, our data show that exposure to lanthanum can cause neuronal toxic damage and behavioral defects in C. elegans, and provide basic information for understanding the neurotoxic effect mechanism and environmental health risks of rare earth elements.

Effect of benzotriazole on oxidative stress response and transcriptional gene expression in Oryzias latipes and Danio rerio embryo.

Emerging contaminants (EC) such as benzotriazole are being released into the environment in various ways, therefore it is necessary to understand how organisms are affected by EC. In this study, we exposed medaka (Oryzias latipes) and zebrafish (Danio rerio) during their embryonic period (1 day after hatching) to benzotriazole to investigate its effects on oxidative stress (ROS, GSH, GST, SOD, CAT and MDA) and changes in gene expression patterns. In both medaka and zebrafish, the influence of oxidative stress was confirmed through an increased MDA level and changes in the ROS and GSH levels. Antioxidant enzymes such as GST, CAT, and SOD were affected by benzotriazole; however, medaka and zebrafish showed different patterns in the effects by benzotriazole. Results of oxidative stress genes expression showed that medaka had either no influence or had a decrease in the gene expression profile, whereas zebrafish had a statistically significant increase in the expression of some genes. The cyp1a gene expression was increased in both species. However, vtg gene expression was increased only in zebrafish but decreased in medaka, indicating no estrogenic effects in medaka. Apoptosis genes showed changes in expression in both the species but was these changes were not dose-dependent. However, zebrafish caspase-9 gene expression was increased in all of the exposed groups, suggesting the effects on the intrinsic pathway associated with caspase-9. In conclusion, the results indicate that the toxic effects of benzotriazole differ at various levels in the two small fish medaka and zebrafish embryos.

Salmo trutta is more sensitive than Oncorhynchus mykiss to early-life stage exposure to retene.

Salmonids are known to be among the most sensitive fish to dioxin-like compounds (DLCs), but very little is known about the sensitivity of the brown trout (Salmo trutta), which has declined and is endangered in several countries of Europe and Western Asia. We investigated the sensitivity of brown trout larvae to a widespread dioxin-like PAH, retene (3.2 to 320 µg.L-1), compared to the larvae of a salmonid commonly used in toxicology studies, the rainbow trout (Oncorhynchus mykiss). Mortality, growth, cyp1a induction and the occurrence of deformities were measured after 15 days of exposure. Brown trout larvae showed a significantly higher mortality at 320 µg.L-1 compared to rainbow trout larvae. While the occurrence of deformities was only significantly increased at 320 µg.L-1 for the rainbow trout, brown trout larvae displayed pericardial edemas and hemorrhages already at 10 or 100 µg.L-1. cyp1a induction was increased significantly already at ≥3.2 µg.L-1 for the brown trout, versus ≥32 µg.L-1 for the rainbow trout. Least square regression analysis of the concentration-response relationships suggested that S. trutta larvae were at least 2 times more sensitive than O. mykiss larvae for cyp1a induction. The present study suggests that S. trutta larvae are more sensitive than O. mykiss larvae to a potent DLC, retene. As it is possible that S. trutta populations have declined partly because of pollution by DLCs, we recommend generating more data regarding the sensitivity of threatened fish populations, in order to ensure better risk assessment.

Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis.

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 â€‹hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.

Multifaceted epigenetic regulation of porcine testicular aromatase.

Testicular aromatase catalyzes the synthesis of estradiol, which contributes to regulation of porcine Sertoli cell proliferation and postpubertal maintenance of Sertoli cell numbers. Although aromatase enzymatic activity decreases with age and is persistently reprogrammed by prepubertal treatment with the aromatase inhibitor letrozole, the molecular bases for regulation have not been identified. DNA methylation was examined as a potential regulatory mechanism using DNA from Leydig cells isolated from 16-, 40-, and 68-week-old boars and from 68- week-old littermates treated with the aromatase inhibitor, letrozole. Methylation levels of individual CpG dinucleotides located in the distal untranslated exon 1 of the relevant aromatase encoding gene, CYP19A3, were quite high in Leydig cell DNA, and increased further with maturity of boar (P < 0.05), while aromatase activity and transcript abundance decreased more than two-fold. However, reduced aromatase activity following letrozole treatment was not accompanied by altered DNA methylation. Testicular expression of miR378 was altered by prepubertal treatment with letrozole. The data provide evidence for two different epigenetic mechanisms that regulate aromatase expression and enzymatic activity in the boar testis.