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Genomic analysis of the ecdysone steroid signal at metamorphosis onset using ecdysoneless and EcR Drosophila melanogaster mutants.

Davis MB, Li T - Genes Genomics (2013)

Bottom Line: Around 12 % of the genome responds to the ecdysone hormone signal at the onset of metamorphosis and over half of these are independent of the receptor.In addition, a significant portion of receptor regulated genes are differentially regulated by the receptor, depending on its ligand state.Gene ontology enrichment analyses confirm known ecdysone regulated biological functions and also validate implicated pathways that have been indirectly associated with ecdysone signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Coverdell Biomedical Research Center, University of Georgia, 500 DW Brooks Dr S 270C, Athens, GA 30602 USA.

ABSTRACT
Steroid hormone gene regulation is often depicted as a linear transduction of the signal, from molecule release to the gene level, by activation of a receptor protein after being bound by its steroid ligand. Such an action would require that the hormone be present and bound to the receptor in order to have target gene response. Here, we present data that presents a novel perspective of hormone gene regulation, where the hormone molecule and its receptor have exclusive target gene regulation function, in addition to the traditional direct target genes. Our study is the first genome-wide analysis of conditional mutants simultaneously modeling the steroid and steroid receptor gene expression regulation. We have integrated classical genetic mutant experiments with functional genomics techniques in the Drosophila melanogaster model organism, where we interrogate the 20-hydroxyecdysone signaling response at the onset of metamorphosis. Our novel catalog of ecdysone target genes illustrates the separable transcriptional responses among the hormone, the pre-hormone receptor and the post-hormone receptor. We successfully detected traditional ecdysone target genes as common targets and also identified novel sets of target genes which where exclusive to each mutant condition. Around 12 % of the genome responds to the ecdysone hormone signal at the onset of metamorphosis and over half of these are independent of the receptor. In addition, a significant portion of receptor regulated genes are differentially regulated by the receptor, depending on its ligand state. Gene ontology enrichment analyses confirm known ecdysone regulated biological functions and also validate implicated pathways that have been indirectly associated with ecdysone signaling.

No MeSH data available.


Related in: MedlinePlus

Overview of experimental design for ecdysone and Ecdysone Receptor (EcR) transcriptome responses. a Normal levels of 20-hydroxyecdysone pulses measured across larval and pupal stages. Dashed arrow indicates time point of temperature shift of ecd1 mutants to restrictive temperature, removing all subsequent pulses of ecdysone. b Normal EcR expression during larval and pupal stages assessed by microarray analysis. Microarray probe isoform specificity is color-coded and indicated in legend. Dashed arrows indicate the time point where temperature rescue was ceased in EcR- mutants, effectively removing the expression of EcR. c Schematic of the ecdysoneless (ecd1) hybridization experimental design, a modified round robin comparison. The samples include two wild type and two mutant (red) conditions. Each hybridization comparison is numbered for clarity of discussion in the text. The measured variables from each comparison is indicated and were used in our mixed model ANOVA to resolve the ecdysone-specific response, confounded by heat from the temperature shift and genomic background from the control CS samples. d Schematic of the EcR- hybridization experimental design, a global reference comparison. The reference (green) includes the pooled wildtype CS samples from Blue Gut (BG), Clear Gut (CG) and White Prepupae (WPP) stages. The experimental samples include three wildtype samples and two mutant samples (red), rescued up to BG and WPP stages. (Color figure online)
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Fig1: Overview of experimental design for ecdysone and Ecdysone Receptor (EcR) transcriptome responses. a Normal levels of 20-hydroxyecdysone pulses measured across larval and pupal stages. Dashed arrow indicates time point of temperature shift of ecd1 mutants to restrictive temperature, removing all subsequent pulses of ecdysone. b Normal EcR expression during larval and pupal stages assessed by microarray analysis. Microarray probe isoform specificity is color-coded and indicated in legend. Dashed arrows indicate the time point where temperature rescue was ceased in EcR- mutants, effectively removing the expression of EcR. c Schematic of the ecdysoneless (ecd1) hybridization experimental design, a modified round robin comparison. The samples include two wild type and two mutant (red) conditions. Each hybridization comparison is numbered for clarity of discussion in the text. The measured variables from each comparison is indicated and were used in our mixed model ANOVA to resolve the ecdysone-specific response, confounded by heat from the temperature shift and genomic background from the control CS samples. d Schematic of the EcR- hybridization experimental design, a global reference comparison. The reference (green) includes the pooled wildtype CS samples from Blue Gut (BG), Clear Gut (CG) and White Prepupae (WPP) stages. The experimental samples include three wildtype samples and two mutant samples (red), rescued up to BG and WPP stages. (Color figure online)

Mentions: We have integrated classical genetics experiments with functional genomics techniques in the Drosophila model organism to elucidate the genes influenced by the 20-Hydroxyecdysone (ecdysone) steroid hormone signal. Specifically, we have resolved the target genes responding to the ecdysone signal at the specific lifecycle stage of metamorphosis onset, or pupariation (Warren et al. 2006). While this hormone is responsible for the onset of all lifecycle stages with pulses of the hormone punctuating each transition between phases of insect development (Henrich et al. 1999; Warren et al. 2006), we have chosen the pupariation pulse in order to identify both known and unknown targets during a transition of greatest diversity in morphological responses (D’Avino and Thummel 2000; Jiang et al. 2000; Kozlova and Thummel 2000; Riddiford et al. 2000). Metamorphosis onset, or the transition of Drosophila larvae into the pupal stage, is triggered by a large pulse of the ecdysone hormone (Warren et al. 2006); (Fig. 1) which is also coupled with upregulation of its receptor EcR (Fig. 1).Fig. 1


Genomic analysis of the ecdysone steroid signal at metamorphosis onset using ecdysoneless and EcR Drosophila melanogaster mutants.

Davis MB, Li T - Genes Genomics (2013)

Overview of experimental design for ecdysone and Ecdysone Receptor (EcR) transcriptome responses. a Normal levels of 20-hydroxyecdysone pulses measured across larval and pupal stages. Dashed arrow indicates time point of temperature shift of ecd1 mutants to restrictive temperature, removing all subsequent pulses of ecdysone. b Normal EcR expression during larval and pupal stages assessed by microarray analysis. Microarray probe isoform specificity is color-coded and indicated in legend. Dashed arrows indicate the time point where temperature rescue was ceased in EcR- mutants, effectively removing the expression of EcR. c Schematic of the ecdysoneless (ecd1) hybridization experimental design, a modified round robin comparison. The samples include two wild type and two mutant (red) conditions. Each hybridization comparison is numbered for clarity of discussion in the text. The measured variables from each comparison is indicated and were used in our mixed model ANOVA to resolve the ecdysone-specific response, confounded by heat from the temperature shift and genomic background from the control CS samples. d Schematic of the EcR- hybridization experimental design, a global reference comparison. The reference (green) includes the pooled wildtype CS samples from Blue Gut (BG), Clear Gut (CG) and White Prepupae (WPP) stages. The experimental samples include three wildtype samples and two mutant samples (red), rescued up to BG and WPP stages. (Color figure online)
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Fig1: Overview of experimental design for ecdysone and Ecdysone Receptor (EcR) transcriptome responses. a Normal levels of 20-hydroxyecdysone pulses measured across larval and pupal stages. Dashed arrow indicates time point of temperature shift of ecd1 mutants to restrictive temperature, removing all subsequent pulses of ecdysone. b Normal EcR expression during larval and pupal stages assessed by microarray analysis. Microarray probe isoform specificity is color-coded and indicated in legend. Dashed arrows indicate the time point where temperature rescue was ceased in EcR- mutants, effectively removing the expression of EcR. c Schematic of the ecdysoneless (ecd1) hybridization experimental design, a modified round robin comparison. The samples include two wild type and two mutant (red) conditions. Each hybridization comparison is numbered for clarity of discussion in the text. The measured variables from each comparison is indicated and were used in our mixed model ANOVA to resolve the ecdysone-specific response, confounded by heat from the temperature shift and genomic background from the control CS samples. d Schematic of the EcR- hybridization experimental design, a global reference comparison. The reference (green) includes the pooled wildtype CS samples from Blue Gut (BG), Clear Gut (CG) and White Prepupae (WPP) stages. The experimental samples include three wildtype samples and two mutant samples (red), rescued up to BG and WPP stages. (Color figure online)
Mentions: We have integrated classical genetics experiments with functional genomics techniques in the Drosophila model organism to elucidate the genes influenced by the 20-Hydroxyecdysone (ecdysone) steroid hormone signal. Specifically, we have resolved the target genes responding to the ecdysone signal at the specific lifecycle stage of metamorphosis onset, or pupariation (Warren et al. 2006). While this hormone is responsible for the onset of all lifecycle stages with pulses of the hormone punctuating each transition between phases of insect development (Henrich et al. 1999; Warren et al. 2006), we have chosen the pupariation pulse in order to identify both known and unknown targets during a transition of greatest diversity in morphological responses (D’Avino and Thummel 2000; Jiang et al. 2000; Kozlova and Thummel 2000; Riddiford et al. 2000). Metamorphosis onset, or the transition of Drosophila larvae into the pupal stage, is triggered by a large pulse of the ecdysone hormone (Warren et al. 2006); (Fig. 1) which is also coupled with upregulation of its receptor EcR (Fig. 1).Fig. 1

Bottom Line: Around 12 % of the genome responds to the ecdysone hormone signal at the onset of metamorphosis and over half of these are independent of the receptor.In addition, a significant portion of receptor regulated genes are differentially regulated by the receptor, depending on its ligand state.Gene ontology enrichment analyses confirm known ecdysone regulated biological functions and also validate implicated pathways that have been indirectly associated with ecdysone signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Coverdell Biomedical Research Center, University of Georgia, 500 DW Brooks Dr S 270C, Athens, GA 30602 USA.

ABSTRACT
Steroid hormone gene regulation is often depicted as a linear transduction of the signal, from molecule release to the gene level, by activation of a receptor protein after being bound by its steroid ligand. Such an action would require that the hormone be present and bound to the receptor in order to have target gene response. Here, we present data that presents a novel perspective of hormone gene regulation, where the hormone molecule and its receptor have exclusive target gene regulation function, in addition to the traditional direct target genes. Our study is the first genome-wide analysis of conditional mutants simultaneously modeling the steroid and steroid receptor gene expression regulation. We have integrated classical genetic mutant experiments with functional genomics techniques in the Drosophila melanogaster model organism, where we interrogate the 20-hydroxyecdysone signaling response at the onset of metamorphosis. Our novel catalog of ecdysone target genes illustrates the separable transcriptional responses among the hormone, the pre-hormone receptor and the post-hormone receptor. We successfully detected traditional ecdysone target genes as common targets and also identified novel sets of target genes which where exclusive to each mutant condition. Around 12 % of the genome responds to the ecdysone hormone signal at the onset of metamorphosis and over half of these are independent of the receptor. In addition, a significant portion of receptor regulated genes are differentially regulated by the receptor, depending on its ligand state. Gene ontology enrichment analyses confirm known ecdysone regulated biological functions and also validate implicated pathways that have been indirectly associated with ecdysone signaling.

No MeSH data available.


Related in: MedlinePlus