Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in Drosophila provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless¹ (ecd¹) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. ecd is shown to encode an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd¹ conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd^- clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein (Gaziova, 2004).

The insect steroid ecdysone (E), and primarily its active derivative 20-hydroxyecdysone (20E), is responsible for coordination of embryogenesis, larval molting and metamorphosis, the latter involving differentiation of adult structures from precursor imaginal discs. The generic name ecdysone is used to refer to the Drosophila steroid hormone. Blood-circulating ecdysone induces tissue-specific and temporally restricted proliferation, differentiation and programmed cell death. Numerous studies, directed towards understanding how the ubiquitous hormone governs these diverse cellular responses, culminated in detailed dissection of the regulatory cascade downstream of the ecdysone signal (Gaziova, 2004).

The major and best-studied source of ecdysone in insect larvae is the prothoracic gland, which in Drosophila consists of the lateral lobes of the ring gland. After this part of the ring gland degenerates during metamorphosis, adult ovaries contribute to the whole body steroid titer in Drosophila. The main role of ecdysone in adult females is to regulate vitellogenesis. In addition, ecdysone has been implicated in egg chamber maturation during mid-oogenesis. Inactive ecdysone conjugates are maternally deposited to eggs and are mobilized during mid-embryogenesis by the amnioserosa (Gaziova, 2004).

Recently, several Drosophila genes involved in ecdysone biosynthesis have been cloned. One is dare, a homolog of the human adrenodoxin reductase that is necessary for the reduction of mitochondrial cytochrome P450 (Cyp) enzymes (Freeman, 1999). Two other genes, disembodied (dib) and shadow (sad), encode Cyp C₂₂-and C₂-hydroxylases, respectively: these two enzymes are responsible for the final two hydroxylation steps of ecdysone synthesis (Chavez, 2000; Warren, 2002). Ecdysone is the final product of the ring gland, which is secreted to the hemolymph and converted to 20E in peripheral tissues. The Cyp C₂₀-hydroxylase responsible for this conversion is encoded by shade (shd) (Petryk, 2003). The dare, dib and sad genes are all expressed in the larval lateral ring gland and in adult ovaries, and their loss-of-function phenotypes can be fully explained as a consequence of ecdysone deficiency. Thus far, only one steroidogenic factor that is not itself an enzyme, without children (woc), has been identified (Wismar, 2000; Warren, 2001). This gene encodes a zinc finger transcription factor that probably activates expression of the cholesterol 7,8-dehydrogenase that executes the first step of ecdysone biosynthesis. Mutations of woc affect a wide range of tissues, suggesting that its transcriptional function is not restricted to regulating expression of the steroidogenic enzyme. No other regulators of the steroidogenic pathway have been identified thus far (Gaziova, 2004 and references therein).

Among steroid-deficient Drosophila mutations, ecdysoneless¹ (ecd¹) is used to study ecdysone roles in development. The ecd¹ mutation is a recessive, temperature-sensitive allele that reduces whole-body ecdysone titers and causes larval arrest at a restrictive temperature, 29°C (Garen, 1977). The effect of ecd¹ on ecdysone production is autonomous, because cultured ecd¹ mutant ring glands fail to produce ecdysone when upshifted to 29°C (Henrich, 1987; Dai, 1991; Warren, 1996). Ecdysone production is also interrupted in adult ovaries upshifted to the restrictive temperature (Garen, 1977; ; Warren, 1996). After several days at 29°C, oogenesis pauses at the onset of vitellogenesis; this phenotype can be reversed by lowering the temperature (Audit-Lamour, 1981). Transplantation experiments show that this effect of ecd¹ is autonomous to the ovary (Garen, 1977; Gaziova, 2004 and references therein).

Developmental events disrupted in ecd¹ mutants include fat body protein synthesis (Lepesant, 1978), progression of the eye-forming morphogenetic furrow (Brennan, 1998), salivary gland glue secretion (Biyasheva, 2001) and motor neuron outgrowth (Li, 2001). These defects have been interpreted as consequences of the mutationally induced ecdysone deficiency. However, Redfern and Bownes (1993) caution that a range of anomalies in ecd¹ adults result from an autonomous ecd requirement for cell viability and therefore may not be attributable to ecdysone deficiency (Gaziova, 2004).

It is difficult to discern which of the phenotypes result from the ecd¹ mutation directly, and which are the consequence of low ecdysone titer, without knowing the primary defect in the ecdysoneless gene, whose molecular identity remained elusive for over 25 years. The ecd locus is shown in this study to encode a protein whose orthologs in several other species, including humans, have not yet been functionally described. The original ecd¹ mutation and three non-conditional lethal alleles have been mapped and assessed for their effects. The Ecd protein has been localized to both the steroidogenic and non-steroidogenic tissues, and its cell-autonomous roles in imaginal discs and ovaries have been demonstrated (Gaziova, 2004).

Although the non-conditional ecd^- mutants often die during the ecdysis to the second instar, displaying phenotypes that might imply defective ecdysone production, their lethality cannot be a direct consequence of low blood ecdysone for the following reasons. (1) ecd^- animals cannot be advanced to the second molt by 20E feeding, despite the fact that similar doses of 20E are sufficient (i) to avert second instar lethality in mutants for the steroidogenic enzyme Dare (Freeman, 1999) and (ii) to induce pupariation in ecd¹ larvae at 29°C. (2) Since some of the ecd^- animals die during the transition to the second instar, one would expect that their ecdysone titer would be lower from as early as the first instar. However, no reduction of ecdysone content was found in first instar homozygous ecd² larvae. (3) Although Ecd is abundant in the lateral ring gland during the third instar, no such expression is seen at earlier stages. By contrast, some other steroidogenic genes, such as dib and sad, are strongly expressed in the ring gland beginning at embryogenesis. Finally, development of ecd² homozygotes can be completely rescued with ubiquitous Ecd expression but not with Ecd targeted by the Feb36-Gal4 driver to the ring gland and to some other organs (Andrews, 2002). Since Ecd presence in the ring gland cannot postpone the death of ecd-null mutants, Ecd must be required prior to the initiation of the second molt in some other tissues. One could be the nervous system, because patched-driven Ecd promotes further development of the mutants (Gaziova, 2004).

A cell-autonomous effect has been demonstrated for the ecd¹ allele during differentiation of the thorax sensory bristles (Sliter, 1989). Unexpectedly, induction of ecd-null mitotic clones in the primordia of the adult thorax, the wing imaginal discs, did not produce any defective bristles. This was probably because no ecd^- clones occurred in the adult epidermis. Based on the presence of twin ecd^+/+ clones in all imaginal discs and in the adult compound eye, it is concluded that the lost ecd^- clones were replaced by proliferation of the surrounding ecd⁺ cells. Many of the defects seen in temperature-upshifted ecd¹ mutants have been ascribed to autonomous cell lethality in the imaginal discs (Redfern, 1983). However, small clones of ecd^- cells have been detected in imaginal discs upon induction of recombination during early third larval instar, and ecd^- clones also survived in the adult ovary. Thus, the loss of ecd is not generally cell lethal although it reduces the ability of the mutant cells to proliferate at the normal rate. Mosaic analyses provide direct evidence for a cell-autonomous, ecdysone-independent function of ecd, which may underlie the previously described defects in adult morphogenesis (Gaziova, 2004).

Clones of ecd^- somatic follicle cells cause profound defects, manifest as fusions of adjacent egg chambers and leading to duplications of the nurse cell set, in some cases with two vitellogenic oocytes present at the opposite poles. Similar polarity defects are caused by perturbing the Delta/Notch signaling that specifies the polar follicle cells (PFC), and by perturbing the JAK/STAT pathway through which these cells establish proper separation between egg chambers. It remains to be tested whether the egg chamber fusions in ecd mosaic ovaries might result from a compromised signaling by the PFC. Follicle cells are thought to be the major site of ecdysone production in the ovary. However, it is difficult to imagine that the relatively small ecd^- clones could significantly reduce the ecdysone titer in the female. Therefore it is concluded that, as in the case of imaginal discs, the effects of ecd² on oogenesis are independent of free-circulating ecdysone (Gaziova, 2004).

Germline clones completely lacking ecd function arrest at pre-vitellogenic stages, probably earlier than egg chambers carrying the ovo^D1 mutation, thus showing that ecd is autonomously required for oocyte maturation. This result is consistent with the phenotype of ecd¹ mutant ovaries: ecd¹ females become sterile after a few days at 29°C, with a majority of egg chambers at pre-vitellogenic stages (Audit-Lamour, 1981). Interestingly, the steroidogenic enzyme Dare, and the ecdysone response proteins EcR and E75, are similarly required in the nurse cells for egg maturation, since germline clones mutant for these genes arrest as pre-vitellogenic egg chambers as well. This has led to a proposal that ecdysone synthesis by the germline is necessary in an autocrine manner for the progression of oocytes to the vitellogenic stage. Since normal ecd function is required for autonomous ecdysone production by the ovary (Garen, 1977), the pre-vitellogenic arrest of the ecd^- germline clones is consistent with an autocrine germline function (Gaziova, 2004).

By inducing ecd² mutant clones in adult females, mosaic egg chambers were created in which some nurse cells were null for ecd, whereas others carried the ovo^D1 dominant mutation that unconditionally blocks oogenesis. Surprisingly, these mixed-genotype egg chambers continued to mature much beyond the phase of arrest caused by either the ecd² or ovo^D1 mutations acting alone. This suggests a functional rescue among the cells within the egg chamber. Since nurse cells are interconnected by ring canals, it is speculated that the ecd⁺ ovo^D1 cells and their ecd^-, ovo⁺ sisters exchange materials that complement them and consequently permit oocyte development. In the light of the autocrine germline hypothesis, an intriguing possibility is that the product of the ecd⁺ ovo^D1 clones might be ecdysone (Gaziova, 2004).

Although the ecdysoneless gene encodes a protein with highly conserved regions, no data was found that would describe the function of these regions and thus enlighten the mode of Ecd action. The only published report has implicated the human ortholog of Ecd, which compensates for the loss of an unrelated yeast protein GCR2 in transcriptional regulation (Deminoff, 2001). An antibody detects Ecd predominantly in the cytoplasm, and thus does not directly support the possibility that Ecd acts at the level of transcription. Yeast two-hybrid studies have been initiated to address the mechanism of Ecd action by identifying its protein partners. Until the exact function of Ecd is known, interpretations of results obtained with the ecdysone-deficient ecd¹ mutants should consider its non-steroidogenic effects (Gaziova, 2004).

GENE STRUCTURE

cDNA clone length - 2148

Exons - 2

Bases in 3' UTR - 85

PROTEIN STRUCTURE

Amino Acids - 684

Structural Domains

Genetic mapping placed ecd among 10 genes predicted by the Berkeley Drosophila Genome Project to be within region 62D. Four partially overlapping genomic fragments harboring subsets of these 10 genes were used for germline transformation. All three obtained transgenic lines carrying the E5 genomic fragment rescued the otherwise lethal ecd genotypes: ecd²/ecd², ecd²/ecd^l(3)23, ecd²/ecd^g24, ecd²/Df(3L)R+R2, ecd¹/ecd² (29°C) and ecd¹/ecd^g24 (29°C) to adulthood. A shorter construct S4, containing only the CG5714 gene, rescued the ecd mutants to the same extent as E5. In all cases, a single transgenic copy of the CG5714 gene was sufficient for the complete rescue. These results clearly identify CG5714 as ecdysoneless (Gaziova, 2004).

The sequence of the deduced Ecd protein reveals a broad evolutionary conservation. Putative Ecd orthologs have been found in the mosquito Anopheles gambiae (43% overall amino acid identity), humans and mouse (31%), zebrafish (30%), Arabidopsis thaliana (26%) and the fission yeast Schizosaccharomyces pombe (21% identity). The human Ecd ortholog, known as Suppressor of GCR2 (SGT1), is expressed in a wide range of human organs (Sato, 1999) and functionally rescues a mutation of GCR2, a transcriptional regulator of glycolytic enzyme genes in the fission yeast (Deminoff, 2001). However, GCR2 is not homologous to SGT1 and thus the normal role of SGT1 in humans is unknown. Interestingly, although several highly conserved motifs are evident among the aligned orthologs, none of these correspond to any known functional domain. There is a putative ATP/GTP-binding motif (P-loop) near the C terminus of the Drosophila and Anopheles orthologs, as recognized by the PROSITE database (Gaziova, 2004).

date revised: 20 September 2004

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