ABSTRACT: Genome wide mapping of Ftz and Eve transcription factor binding sites in early Drosophila embryos Overall design: Ftz has one replicate and dyes swapping Eve has two replicates with one dye swap Mock IPs were used as controls Slides were hybridized with two color samples, but individual channels were uploaded seperately to GEO
INSTRUMENT(S): ChIP chip data for Ftz and Eve proteins during early embryogenesis
Project description:Genome wide mapping of Ftz and Eve transcription factor binding sites in early Drosophila embryos Ftz has one replicate and dyes swapping Eve has two replicates with one dye swap Mock IPs were used as controls Slides were hybridized with two color samples, but individual channels were uploaded seperately to GEO
Project description:The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern.
Project description:During development, cascades of regulatory genes act in a hierarchical fashion to subdivide the embryo into increasingly specified body regions. This has been best characterized in Drosophila, where genes encoding regulatory transcription factors form a network to direct the development of the basic segmented body plan. The pair-rule genes are pivotal in this process as they are responsible for the first subdivision of the embryo into repeated metameric units. The Drosophila pair-rule gene fushi tarazu (ftz) is a derived Hox gene expressed in and required for the development of alternate parasegments. Previous studies suggested that Ftz achieves its distinct regulatory specificity as a segmentation protein by interacting with a ubiquitously expressed cofactor, the nuclear receptor Ftz-F1. However, the downstream target genes regulated by Ftz and other pair-rule genes to direct segment formation are not known. In this study, we selected candidate Ftz targets by virtue of their early expression in Ftz-like stripes. This identified two new Ftz target genes, drumstick (drm) and no ocelli (noc), and confirmed that Ftz regulates a serotonin receptor (5-HT2). These are the earliest Ftz targets identified to date and all are coordinately regulated by Ftz-F1. Engrailed (En), the best-characterized Ftz/Ftz-F1 downstream target, is not an intermediate in regulation. The drm genomic region harbors two separate seven-stripe enhancers, identified by virtue of predicted Ftz-F1 binding sites, and these sites are necessary for stripe expression in vivo. We propose that pair-rule genes, exemplified by Ftz/Ftz-F1, promote segmentation by acting at different hierarchical levels, regulating first, other segmentation genes; second, other regulatory genes that in turn control specific cellular processes such as tissue differentiation; and, third, 'segmentation realizator genes' that are directly involved in morphogenesis.
Project description:The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos but mutations result in a pair-rule phenotype. Previously characterized Ftz-F1 target genes were co-regulated by Ftz, which is expressed in stripes, consistent with the pair-rule phenotype observed for ftz-f1 and ftz mutants. However, attempts to identify new target genes on the basis of Ftz-F1 and Ftz binding sites alone has met with only limited success. To discern the rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Overall design: Drosophila embryos from a cross between w males and FRT germ-line clone ftz-f1 females or FRT germ-line clone w females were hand selected at 3 stages of development (5, 6 and 8) for RNA extraction and hybridization on Affymetrix microarrays. --------------------------------- The authors state that "Affymetrix CEL files have been lost."
Project description:Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.
Project description:Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1) are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1) produces two splicing variants, ?Ftz-F1 and ?Ftz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30-48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna.
Project description:The NR5A-family nuclear receptors are highly conserved and function within the somatic follicle cells of the ovary to regulate folliculogenesis and ovulation in mammals; however, their roles in Drosophila ovaries are largely unknown. Here, we discover that Ftz-f1, one of the NR5A nuclear receptors in Drosophila, is transiently induced in follicle cells in late stages of oogenesis via ecdysteroid signaling. Genetic disruption of Ftz-f1 expression prevents follicle cell differentiation into the final maturation stage, which leads to anovulation. In addition, we demonstrate that the bHLH/PAS transcription factor Single-minded (Sim) acts as a direct target of Ftz-f1 to promote follicle cell differentiation/maturation and that Ftz-f1's role in regulating Sim expression and follicle cell differentiation can be replaced by its mouse homolog steroidogenic factor 1 (mSF-1). Our work provides new insight into the regulation of follicle maturation in Drosophila and the conserved role of NR5A nuclear receptors in regulating folliculogenesis and ovulation.
Project description:SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.
Project description:The Nod-like receptor protein 3 (NLRP3) inflammasome activation not only serves as an intracellular machinery triggering inflammation but also produces uncanonical effects beyond inflammation such as changing cell metabolism and increasing cell membrane permeability. The present study was designed to test whether this NLRP3 inflammasome activation contributes to the "two-hit" injury during nonalcoholic steatohepatitis (NASH) and whether it can be a therapeutic target for the action of Fufang Zhenzhu Tiaozhi (FTZ), a widely used herbal remedy for hyperlipidemia and metabolic syndrome in China. We first demonstrated that NLRP3 inflammasome formation and activation as well as lipid deposition occurred in the liver of mice on the high-fat diet (HFD), as shown by increased NLRP3 aggregation, enhanced production of IL-1? and high mobility group box 1 (HMGB1), and remarkable lipid deposition in liver cells. FTZ extracts not only significantly reduced the NLRP3 inflammasome formation and activation but also attenuated the liver steatosis and fibrogenic phenotype changed. In in vitro studies, palmitic acid (PA) was found to increase colocalization of NLRP3 components and enhanced caspase-1 activity in hepatic stellate cells (HSCs), indicating enhanced formation and activation of NLRP3 inflammasomes by PA. PA also increased lipid deposition. Nlrp3 siRNA can reverse this effect by silencing the NLRP3 inflammasome and both with FTZ. In FTZ-treated cells, not only inflammasome formation and activation was substantially attenuated but also lipid deposition in HSCs was blocked. This inhibition of FTZ on lipid deposition was similar to the effects of glycyrrhizin, an HMGB1 inhibitor. Mechanistically, stimulated membrane raft redox signaling platform formation and increased O2•- production by PA to activate NLRP3 inflammasomes in HSCs was blocked by FTZ treatment. It is concluded that FTZ extracts inhibit NASH by its action on both inflammatory response and liver lipid metabolism associated with NLRP3 inflammasome formation and activation.
Project description:Objective:To investigate the effect of FTZ on high-glucose-induced oxidative stress and underlying mechanisms. Methods:We used a ? cell dysfunction and diabetes model that was induced in rats fed a high-fat high-sugar diet (HFHSD) for 6 weeks and injected once with 35 mg/kg streptozocin (STZ). Then, 3 and 6 g/kg of FTZ were administered by gavage for 8 weeks. In addition, an ex vivo model of oxidative stress was induced by stimulating INS-1 cells with 25 mmol/L glucose for 48 h. Result:The levels of fasting blood glucose (FBG) in diabetic model rats were obviously higher than those in the normal group; furthermore with reduced levels of ? cells, catalase (CAT), superoxide dismutase (SOD), and Bcl-2 increased lipid peroxide malondialdehyde (MDA) and caspase-3 in the pancreatic tissue of the diabetic model rats. Afterward, the cells were incubated with FTZ-containing serum and edaravone. The 25 mmol/L glucose-induced SOD reduction increased MDA and intracellular ROS. The protein expression level of Mn-SOD and CAT in the model group decreased significantly compared with that in the control group. Conclusion:FTZ treatment significantly improved the alteration in the level of SOD, CAT, Bcl-2, caspase-3, and MDA coupled with ? cell dysfunction in diabetic rats. Oxidative stress in INS-1 cells was closely associated with a higher rate of apoptosis, increased production of ROS and MDA, enhanced Bax expression, and caspase-3, -9 activities and markedly decreased protein expression of Mn-SOD and CAT. FTZ-containing serum incubation notably reversed the high-glucose-evoked increase in cell apoptosis, production of ROS and MDA, and Bax protein levels. Furthermore, FTZ stimulation upregulated the expression levels of several genes, including Mn-SOD, CAT, and Bcl-2/Bcl-xl. In addition, FTZ decreased the intracellular activity of caspase-3, -9 in INS-1 cells. FTZ protected ?-cells from oxidative stress induced by high glucose in vivo and in vitro. The beneficial effect of FTZ was closely associated with a decrease in the activity of caspase-3, -9 and intracellular production of ROS, MDA, and Bax coupled with an increase in the expression of Mn-SOD, CAT, and Bcl-2/Bcl-xl.