20-Hydroxyecdysone (ecdysterone or 20E ..
It has previously been shown that ecdysone is synthesized within the egg chambers during oogenesis, although the precise cells that are crucial for ecdysone synthesis are not known (; see for review). It is also not known whether the multi-step process of ovarian ecdysone synthesis takes place exclusively in one cell type in the egg chamber, or whether some steps of the synthesis are carried out in the germline derived cells, such as nurse cells or oocyte, while others are performed in the FCs. Expression of some enzymes in the biosynthetic pathway was detected in the germline cells, whereas others are found predominantly in FCs (; ; ; ; ; ; ; ). It is therefore possible that certain intermediates in the pathway diffuse between the cell types. Phm RNA was detected in follicle cells at stage 8/9 and may be present later, possibly at low levels, in nurse cells (; ). We showed that this step of ecdysone synthesis takes place in the follicle epithelial cells and not sufficiently in the germline cells (neither in nurse cells nor in the oocyte) to promote border cell migration. Similarly, we demonstrated that the synthesis of 20-hydroxyecdysone from ecdysone via Shd also takes place within the follicular epithelium, and that the germline cannot rescue the absence of Shd activity in the follicle cells.
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In order to discover new genes involved in the control of the cell motility and developmental timing, we identified a set of mutants affecting the migration of BCs in a mosaic screen on the X-chromosome. Two mutants in particular, FL99 and FT59, specifically affected BC migration and did not show any other defects in FC differentiation. Here we demonstrate that FL99 and FT59 carry mutations in the gene phantom (phm), which is known to be required for ecdysone synthesis. We show that Drosophila Phm acts during oogenesis to initiate BC migration, and its function is required in the FCs and not in the germline cells. Ecdysone production seems autonomous to each egg chamber, such that wild-type FCs from adjacent egg chambers cannot rescue an egg chamber surrounded by mutant FCs. We further found that about 8–15% of FCs (50–100 cells), independent of their position within the egg chamber are sufficient to produce required levels of ecdysone to initiate BC migration. A similar phenotype was observed with mutations in the gene shade (shd), which catalyzes the last step in the ecdysone pathway converting ecdysone to active 20-hydroxyecdysone. Mutations in shd also lead to defects in border cell migration, and the function of Shd is required only in the follicular epithelium. In addition, we analyzed the migration of BCs in egg chambers cultured in vitro, and demonstrated that BC migration could be rescued in phm mutants by adding synthetically synthesized 20-hydroxyecdysone, the biologically active form of ecdysone, to the culture medium.
The ecdysteroids are steroid hormones that, in combination with juvenile hormones, program stage specific gene expression during insect development. Ecdysteroid levels show specific pulses during different stages of embryogenesis to initiate molting and metamorphosis. During the adult stage of many insects, ecdysteroids play important roles in reproduction, physiology and behavior (see for review). Ecdysone is the only known steroid hormone in Drosophila, and is produced by the prothoracic glands in immature insects (see for review). Little is known about tissue-specific production of ecdysone in adult animals due to its lower levels in comparison to the earlier developmental stages, and thus difficulties in its detection. However, ecdysone was detected in all 3 body segments of adult flies, in hemolymph, and in specific tissues such as testes, gut, Malpighian tubules and in ovaries (see for review). During the adult stage, the ovary is a major site of ecdysone synthesis, which peaks at stage 9 (), when BC migration takes place. More recently, a novel function of ecdysone was demonstrated in the regulation of timing of BC migration in the developing egg chambers in the Drosophila females (; ; ). The precise source of the ecdysone that initiates border cell migration, was however not known. In the work reported here, we isolated two lethal alleles of Drosophila phm, a gene encoding for a cytochrome P450 ecdysteroidogenic enzyme, which converts ketodiol to ketotriol, the intermediate products in the ecdysone synthetic chain. We demonstrated that BC migration is severely disrupted in the phm mutants due to the lack of Phm activity in the follicle cells of the developing egg chambers, and we also found that the same egg chamber autonomous requirement applies to Shade, the last enzyme in the 20-hydroxyecdysone pathway.
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Based on data obtained in Manduca and Bombyx (, ), a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. Here we show that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval–prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. We propose that this autocrine system acts downstream of the CW checkpoint to allow the larval–prepupal transition. We speculate that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions.
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Our findings that Phm activity is specifically required in the follicle epithelium to promote normal border cell migration led us to examine whether this indicates a more general requirement for the ecdysteroid synthesis pathway in the follicle cells of each egg chamber. We therefore repeated the analysis using mutations in shade (shd). Shd, which encodes 20-hydroxylase, is the P450 enzyme responsible for converting Ecdysone into 20-hydroxyecdysone, the active steroid that functions as the morphogenetic regulator in Drosophila (; ). We generated shd2 mosaic ovaries to determine whether the activation to 20-hydroxyecdysone also occurs within the follicle epithelium of each egg chamber, and is important for migration of the border cells.