Effect of loss of Ecdysone-induced protein 93F (E93) on the Drosophila melanogaster antennal transcriptome.
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ABSTRACT: Ecdysone-induced protein 93F (E93) regulates local chromatin structure and/or gene transcription, raising the question of whether expression of Ir75c, Ir75b and Ir75a receptors in antenna is affected at the transcriptional level in the absence of this protein. We performed bulk RNA-sequencing (RNA-seq) of control and E93RNAi antennae. In control RNA-seq samples, we predicted the existence of seven distinct transcripts encoded by these three receptor genes. Notably, many transcripts initiating from Ir75c do not terminate at the 3’ end of this gene but rather run through the Ir75b and Ir75a exons. Similarly, a majority of transcripts initiating from Ir75b incorporate exons 2-7 of Ir75a. The pattern of transcription is consistent with the absence of canonical transcription termination/polyadenylation sequences downstream of the Ir75c and Ir75b coding regions; such a sequence is only present 3’ of Ir75a. All transcripts initiating from Ir75c are highly up-regulated in the absence of E93, consistent with the ectopic expression of receptor protein. Transcription initiating from Ir75b is strongly diminished, concordant with the loss of Ir75b protein expression. By contrast, Ir75a transcripts levels were not significantly lower in E93RNAi antennae, despite the loss of detectable Ir75a protein.
Project description:In Drosophila melanogaster, successful development relies on the precise coordination of both spatial and temporal regulatory axes. The temporal axis governs stage-specific identity and developmental transitions through a number of genes, collectively forming the Metamorphic Gene Network. Among these, Ecdysone inducible protein 93F (E93) serves as the critical determinant for adult specification, but its mechanism of action remains unclear. Here, we found that, rather than acting mainly as an instructive signal, E93 promotes adult differentiation through the repression of the pupal specifier broad (br). In the absence of E93, sustained high levels of Br during the pupal stage strongly represses pupal-specific enhancers that are essential for the terminal differentiation of the wing. Notably, RNA-seq analysis confirmed that the majority of E93-dependent transcriptomic changes in pupal wings are primarily driven by br repression. In addition, we also show that Br represses the pupal-enhancers during the larval and prepupal stages preventing the premature implementation of the adult genetic program, and that it also dampens the activity of larval enhancers during the latter stages of larval development. This mechanism of action seems to be a derived feature acquired in Diptera, as in the coleopteran Tribolium castaneum, repression of br by E93 is not sufficient to allow adult differentiation. In summary, our study elucidates the crucial role of the intricate interplay between E93 and Br as the governing mechanism in the process of terminal differentiation in Drosophila. This discovery holds significant implications for advancing our understanding of the evolution of insect metamorphosis.
Project description:How temporal cues combine with spatial inputs to control gene expression during development is poorly understood. Here, we test the hypothesis that the Drosophila transcription factor E93 controls temporal gene expression by regulating chromatin accessibility. Precocious expression of E93 early in wing development reveals that it can simultaneously activate and deactivate different target enhancers. Notably, the precocious patterns of enhancer activity resemble the wild-type patterns that occur later in development, suggesting that provision of E93 alters the competence of enhancers to respond to spatial cues. Genomic profiling reveals that precocious E93 expression is sufficient to regulate chromatin accessibility at a subset of its targets. These accessibility changes mimic those that normally occur later in development, indicating that precocious E93 accelerates the wild-type developmental program. Further, we find that target enhancers that do not respond to precocious E93 in early wings become responsive after a developmental transition, suggesting that parallel temporal pathways work alongside E93. These findings support a model wherein E93 expression functions as an instructive cue that defines a broad window of developmental time through control of chromatin accessibility.
Project description: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 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 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.
Project description:Many postmitotic tissues coordinate robust cell cycle exit with the progression of terminal differentiation. While the signals involved in initiating cell cycle exit during terminal differentiation for some tissues have been described, less is known about the mechanisms that maintain a stable, non-cycling state. We previously found that chromatin accessibility changes at a subset of rate-limiting cell cycle genes occurs during maintenance of cell cycle exit, suggesting chromatin remodelers may play a key role in maintaining a robust postmitotic state during terminal differentiation. Here we show that the chromatin remodeler Mi-2 is required to ensure a stable, postmitotic state in Drosophila eyes and wings. Mi-2 alters chromatin accessibility and gene expression during cell cycle exit and terminal differentiation. Mi-2 and a transcription factor involved in tissue maturation, E93, close chromatin accessibility at an overlapping subset of potential enhancers that include the rate-limiting mitotic regulator string (cdc25c) and genes involved in the progression of terminal differentiation. E93 is also required for a stable, postmitotic state and we verified in vivo that Mi-2 and E93 cooperate to decommission an enhancer for the essential mitotic regulator string, to coordinate the transition to a postmitotic state with terminal differentiation. Enhancer decommissioning at the string locus provides a molecular explanation for the long-term, nearly irreversible postmitotic state observed in many tissues as terminal differentiation progresses.
Project description:Many postmitotic tissues coordinate robust cell cycle exit with the progression of terminal differentiation. While the signals involved in initiating cell cycle exit during terminal differentiation for some tissues have been described, less is known about the mechanisms that maintain a stable, non-cycling state. We previously found that chromatin accessibility changes at a subset of rate-limiting cell cycle genes occurs during maintenance of cell cycle exit, suggesting chromatin remodelers may play a key role in maintaining a robust postmitotic state during terminal differentiation. Here we show that the chromatin remodeler Mi-2 is required to ensure a stable, postmitotic state in Drosophila eyes and wings. Mi-2 alters chromatin accessibility and gene expression during cell cycle exit and terminal differentiation. Mi-2 and a transcription factor involved in tissue maturation, E93, close chromatin accessibility at an overlapping subset of potential enhancers that include the rate-limiting mitotic regulator string (cdc25c) and genes involved in the progression of terminal differentiation. E93 is also required for a stable, postmitotic state and we verified in vivo that Mi-2 and E93 cooperate to decommission an enhancer for the essential mitotic regulator string, to coordinate the transition to a postmitotic state with terminal differentiation. Enhancer decommissioning at the string locus provides a molecular explanation for the long-term, nearly irreversible postmitotic state observed in many tissues as terminal differentiation progresses.
Project description:Queen discrimination behavior in the red imported fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. While monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles do not recombine, it is difficult to determine which genes within the supergene mediate this differential worker behavior. We hypothesized that the alternate worker genotypes sense queens differently because of different patterns of gene expression in their main sensory organ, the antennae. To identify such differentially expressed genes, we sequenced RNA from four biological replicates of pooled antennae from three groups of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein coding genes with 14 encoding potential odor metabolism and perception proteins. We focused on the two differentially expressed odorant perception genes: an odorant binding protein SiOBP12 and an odorant receptor SiOR463. We found that the SiOR463 was lost in the Sb-genome. In contrast, the SiOBP12 has an Sb-specific duplication SiOBP12b’, which was expressed in the SB/Sb worker antennae, while both paralogs SiOBP12 and SiOBP12b’ were expressed in the body. This result indicates that SiOBP12b’ has gained an antennal promoter or enhancer and suggests neofunctionalization, perhaps for queen discrimination behavior.
Project description:Queen discrimination behavior in the red imported fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. While monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles do not recombine, it is difficult to determine which genes within the supergene mediate this differential worker behavior. We hypothesized that the alternate worker genotypes sense queens differently because of different patterns of gene expression in their main sensory organ, the antennae. To identify such differentially expressed genes, we sequenced RNA from four biological replicates of pooled antennae from three groups of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein coding genes with 14 encoding potential odor metabolism and perception proteins. We focused on the two differentially expressed odorant perception genes: an odorant binding protein SiOBP12 and an odorant receptor SiOR463. We found that the SiOR463 was lost in the Sb-genome. In contrast, the SiOBP12 has an Sb-specific duplication SiOBP12b’, which was expressed in the SB/Sb worker antennae, while both paralogs SiOBP12 and SiOBP12b’ were expressed in the body. This result indicates that SiOBP12b’ has gained an antennal promoter or enhancer and suggests neofunctionalization, perhaps for queen discrimination behavior.
Project description:Queen discrimination behavior in the red imported fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. While monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles do not recombine, it is difficult to determine which genes within the supergene mediate this differential worker behavior. We hypothesized that the alternate worker genotypes sense queens differently because of different patterns of gene expression in their main sensory organ, the antennae. To identify such differentially expressed genes, we sequenced RNA from four biological replicates of pooled antennae from three groups of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein coding genes with 14 encoding potential odor metabolism and perception proteins. We focused on the two differentially expressed odorant perception genes: an odorant binding protein SiOBP12 and an odorant receptor SiOR463. We found that the SiOR463 was lost in the Sb-genome. In contrast, the SiOBP12 has an Sb-specific duplication SiOBP12b’, which was expressed in the SB/Sb worker antennae, while both paralogs SiOBP12 and SiOBP12b’ were expressed in the body. This result indicates that SiOBP12b’ has gained an antennal promoter or enhancer and suggests neofunctionalization, perhaps for queen discrimination behavior.