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Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

ABSTRACT: Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti. Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methoprene-tolerant (Met) showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. Sequence motif analysis revealed the consensus for a 9-mer Met-binding motif, CACG(C)/TG(A)/G(T)/AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi-down-regulated (LPE) genes but not in Met RNAi-up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody supershift analyses confirmed the binding properties of the Met consensus motif variants. There was a striking temporal separation of expression profiles among major functional gene groups, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction.

SUBMITTER: Zou Z

PROVIDER: S-EPMC3683779 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

Zou Zhen Z Saha Tusar T TT Roy Sourav S Shin Sang Woon SW Backman Tyler W H TW Girke Thomas T White Kevin P KP Raikhel Alexander S AS

Proceedings of the National Academy of Sciences of the United States of America 20130430 24

Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti. Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methop ...[more]

PMID: 23633570

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Project description:Arthropod-specific juvenile hormones control numerous essential functions in development and reproduction. In the dengue-fever mosquito Aedes aegypti, in addition to its role in immature stages, juvenile hormone III (JH) governs post-eclosion (PE) development in adult females, a phase required for competence acquisition for blood feeding and subsequent egg maturation. During PE, JH through its receptor Methoprene-tolerant (Met) regulate the expression of many genes, causing either activation or repression. Met-mediated gene repression is indirect, requiring involvement of intermediate repressors. Hairy, which functions downstream of Met in the JH gene-repression hierarchy, is one such factor. Krüppel-homolog 1, a zinc-finger transcriptional factor, is directly regulated by Met and has been implicated in both activation and repression of JH-regulated genes. However, the interaction between Hairy and Kr-h1 in the JH-repression hierarchy is not well understood. Our RNAseq-based transcriptomic analysis of the Kr-h1-depleted mosquito fat body revealed that 92% of Kr-h1 repressed genes are also repressed by Met, supporting the existence of a hierarchy between Met and Kr-h1 as previously demonstrated in various insects. Notably, 130 genes are co-repressed by both Kr-h1 and Hairy, indicating regulatory complexity of the JH-mediated PE gene repression. A mosquito Kr-h1 binding site in genes co-regulated by this factor and Hairy was identified computationally. Moreover, this was validated using electrophoretic mobility shift assays. A complete phenocopy of the effect of Met RNAi depletion on target genes could only be observed after Kr-h1 and Hairy double RNAi knockdown, suggesting a synergistic action between these two factors in target gene repression. This was confirmed using a cell-culture-based luciferase reporter assay. Taken together, our results indicate that Hairy and Kr-h1 not only function as intermediate downstream factors, but also act together in a synergistic fashion in the JH/Met gene repression hierarchy.

Project description:The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

Project description:Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.

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Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

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Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

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