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Inter-Organ Steroid Hormone Signaling Promotes Myoblast Fusion via Direct Transcriptional Regulation of a Single Key Effector Gene

ABSTRACT: Steroid hormones regulate tissue development and physiology by modulating the transcription of a broad spectrum of genes. In insects, the principal steroid hormones, ecdysteroids, trigger the expression of thousands of genes through a cascade of transcription factors (TFs) to coordinate developmental transitions such as larval molting and metamorphosis. However, whether ecdysteroid signaling can bypass transcriptional hierarchies to exert its function in individual developmental processes is unclear. Here, we report that a single non-TF effector gene mediates the transcriptional output of ecdysteroid signaling in Drosophila myoblast fusion, a critical step in muscle development and differentiation. Specifically, we show that the 20-hydroxyecdysone (commonly referred to as “ecdysone”) secreted from an extraembryonic tissue, amnioserosa, acts on embryonic muscle cells to directly activate the expression of antisocial (ants), which encodes an essential scaffold protein enriched at the fusogenic synapse. Not only is ants transcription directly regulated by the heterodimeric ecdysone receptor complex composed of Ecdysone receptor (EcR) and Ultraspiracle (USP) via ecdysone response elements, but more strikingly, expression of ants alone is sufficient to rescue the myoblast fusion defect in ecdysone signaling-deficient mutants. We further show that EcR/USP and a muscle-specific transcription factor Twist synergistically activate ants expression in vitro and in vivo. Taken together, our study provides the first example of a steroid hormone directly activating the expression of a single key non-TF effector gene to regulate a developmental process via inter-organ signaling and provides a new paradigm for understanding steroid hormone signaling in other developmental and physiological processes.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE236183 | GEO | 2024/03/20

REPOSITORIES: GEO

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Project description:Methoxyfenozide (RH-2485) represents the group of non-steroidal ecdysteroid agonists with a dibenzoylhydrazine structure, that are used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ~1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis is also predicted to gain insights into the mechanism of resistance and the crosstalk between ecdysteroid signaling and cell proliferation-linked processes. In this microarray experiment three biological conditions were under study: (i) untreated S2 cells (S2), (ii) a subpopulation of these untreated cells, which were grown under continuous pressure of methoxyfenozide and which were shown to become resistant to this compound (S2res) and (iii) a subpopulation of the resistant cells, which were again cultured without methoxyfenozide added to the medium (S2res-woMF). For each condition, two subpopulations (which were treated as biological replicates, although they are not exactly independent), were isolated and used for the RNA extractions. The overall experimental design was an n+2 A-optimal design. When each hybridisation is respresented by an arrow (Cy5 ? Cy3), the design can be respresented as follows: First loop = S2.1 ? S2res.1 ? S2.2 ? S2res.2 ? S2.1; Second loop = S2.1 ? S2res-woMF.1 ? S2.2 ? S2res-woMF.2 ? S2.1 (numbers following the condition name correspond to the biological replicate numbers). The two loops are thus connected by S2.1 and S2.2. Two analyses were performed: S2res compared to S2 and S2res-woMF compared to S2.

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Inter-Organ Steroid Hormone Signaling Promotes Myoblast Fusion via Direct Transcriptional Regulation of a Single Key Effector Gene

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