The Hr46/DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. Hr46 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Hr46 directly contributes to both of these regulatory responses. Hr46 protein binds to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory proteins, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Hr46 represses E74A, and to a lesser extent E74B, and it also represses BR-C, E75A, and E78B. Hr46 activates betaFTZ-F1. Three Hr46 binding sites are present downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of Hr46 regulation. Ectopic expression of Hr46 reveals that the polytene chromosome binding pattern is of functional significance. Hr46 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. Hr46 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae. This study also provides evidence for direct cross-regulation among orphan members of the nuclear receptor superfamily and further implicates these genes as critical transducers of the hormonal signal during the onset of Drosophila metamorphosis (Lam, 1997).
The beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses, adult head eversion, leg elongation and salivary gland death, in response to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. Both E74A and E75A, whose levels of expression are normally increased several orders of magnitude by ecdysone, are significantly affected in betaFTZ-F1 mutants. The severity of these effects correlates with the intensity of polytene chromosome staining by FTZ-F1 antibodies. The Br-C locus is only weakly stained, while E74 is strongly stained, and E75 is the most intensely stained site in the genome. It thus appears that betaFTZ-F1 exerts specificity to the degree to which it can enhance the ecdysone-induction of different promoters. The E93 early gene is also submaximally induced in betaFTZ-F1 mutants, consistent with the proposal that this stage-specific response is dependent on betaFTZ-F1 function. In contrast, the levels of Ecdysone receptor and Ultraspiracle mRNA are not significanty affected by betaFTZ-F1. EDG84A, a gene that encodes a pupal cuticle protein that is specifically expressed in the imaginal discs of mid-prepupae, contains a betaFTZ-F1 binding site upstream from the start site, and EDG84A transcription is delayed and reduced in betaFTZ-F1 mutants. Thus this study defines beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence (Broadus, 1999).
Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).
The nuclear localization of E93 in larval salivary glands provided an opportunity to determine if E93 binds to the salivary gland polytene chromosomes and, if so, to identify the sites bound by the protein. Salivary glands were dissected 12-14 hr after puparium formation, fixed, squashed, and photographed to acquire accurate cytology of the banding and puffing patterns for mapping. The chromosomes were then stained with affinity-purified E93 antibodies, and these patterns were compared with the original set of photographs to allow accurate mapping of the bound sites. E93 clearly binds to the polytene chromosomes in a reproducible and site-specific manner and is consistently detected at 65 chromosome sites, many of which contain ecdysone-regulated genes or programmed cell death genes. Among these sites are the 74EF and 75B early puffs, which contain the E74 and E75 ecdysone-inducible genes, as well as the 93F puff, which contains E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and contain the programmed cell death genes dredd, crq, dcp-1, and drICE, respectively. The 2B5 early puff, containing the BR-C ecdysone-inducible gene, and 75CD, containing βFTZ-F1 and the programmed cell death genes rpr, hid, and grim, were not bound by E93. These data indicate that E93 may directly regulate the genes in bound chromosome loci and may either encode a site-specific DNA binding protein or a chromatin-associated protein that functions as a transcriptional regulator (Lee, 2000).
The observations that E93 is essential for salivary gland cell death and that E93 protein binds to specific sites in the salivary gland polytene chromosomes suggest that E93 may regulate the transcription of target genes that function in steroid-triggered programmed cell death. If this hypothesis is true, then E93 mutations should impact the transcription of genes that reside in salivary gland chromosome loci bound by E93. Salivary glands were dissected from staged late third instar larvae, prepupae, and pupae of control and mutant animals. Total RNA extracted from these tissues was analyzed by Northern blot hybridization. E93 mutations have little or no effect on the timing and levels of BR-C, E74, and E75A transcription in the salivary glands of late third instar larvae and early prepupae. However, the level of expression of each of these regulatory genes is significantly reduced or absent in salivary glands 10-24 hr following puparium formation. Although the smaller E74B transcript is induced, the larger E74A RNA is not detected following the prepupal pulse of ecdysone. The levels of EcR expression in late third instar larval and prepupal salivary glands are not altered by E93 mutations, although its timing is delayed by 4-6 hr at the prepupal to pupal transition. Like EcR, βFTZ-F1 transcription is delayed but the level of this mRNA is not altered in E93 mutant salivary glands. A similar delay is observed in the parental flies that were used for mutagenesis, indicating that this effect is due to the genetic background. The induction of EcR and E74B in E93 mutant prepupae, as well as the successful completion of adult head eversion, indicates that the prepupal pulse of ecdysone occurs in these mutant animals, signaling the prepupal-pupal transition (Lee, 2000).
The E75B receptor, which lacks a complete DNA binding domain, inhibits the inductive function of Hr46/DHR3 on ßFTZF1 by forming a complex with DHR3 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. Thus the repressive function of Hr46 does not involve binding to DNA but instead involves physical interaction with the Ecdysone receptor. E75B fails to bind DNA in the absence of HR46. Thus E75B acts like a co-repressor with HR46, rather than as a competitor with Hr46 for DNA binding; the restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).
Pulses of the steroid hormone ecdysone trigger the major developmental
transitions in Drosophila, including molting and puparium formation.
The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer
that binds to specific response elements in the genome and directly regulates
target gene transcription. A novel nuclear receptor interacting
protein is described, encoded by rigor mortis (rig), that is required for ecdysone responses during larval development. rig mutants display
defects in molting, delayed larval development, larval lethality, duplicated
mouth parts, and defects in puparium formation -- phenotypes that
resemble those seen in EcR, usp, E75A and ßFTZ-F1
mutants. Although the expression of these nuclear receptor genes is
essentially normal in rig mutant larvae, the ecdysone-triggered
switch in E74 isoform expression is defective. rig encodes a
protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as
specific protein-protein interaction domains. Consistent with the presence of
these elements and the lethal phenotypes of rig mutants, Rig protein
interacts with several Drosophila nuclear receptors in GST pull-down
experiments, including EcR, USP, DHR3, SVP and ßFTZ-F1. The ligand
binding domain of ßFTZ-F1 is sufficient for this interaction, which can
occur in an AF-2-independent manner. Antibody stains reveal that Rig protein
is present in the brain and imaginal discs of second and third instar larvae,
where it is restricted to the cytoplasm. In larval salivary gland and midgut
cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially
and temporally regulated manner, at times that correlate with the major lethal
phase of rig mutants and major switches in ecdysone-regulated gene
expression. Taken together, these data indicate that rig exerts
essential functions during larval development through gene-specific effects on
ecdysone-regulated transcription, most likely as a cofactor for one or more
nuclear receptors. Furthermore, the dynamic intracellular redistribution of
Rig protein suggests that it may act to refine spatial and temporal responses
to ecdysone during development (Gates, 2003).
Mutations in rig result in prolonged second and third instar
larval stages, defects in molting, larval lethality and duplicated mouth parts. These phenotypes are characteristic of defects in ecdysone signaling, suggesting a critical role for rig in ecdysone responses during larval development. Two classes of genes produce mutant phenotypes that resemble those seen in rig
mutant animals: those required for ecdysone biosynthesis or release --
including ecdysoneless (ecd), dare and itpr -- and those encoding nuclear receptors that mediate the ecdysone signal
-- EcR, usp, E75A, and ßFTZ-F1. Unlike
ecdysone-deficient mutants, the lethal phenotypes of rig mutants
cannot be rescued by feeding 20E, indicating that ecdysone is not limiting in these animals and that rig acts downstream from hormone biosynthesis or release.
Rather, it is proposed that Rig is functioning as a nuclear receptor cofactor,
based on five lines of evidence. (1) The lethal phenotypes of rig
mutants are very similar to those defined for EcR, usp, E75A and
ßFTZ-F1, although all of these nuclear receptor genes are
expressed in an essentially normal manner in rig mutant larvae. (2) rig mutants display a defect in the ecdysone-triggered switch in E74
isoform expression that is characteristic of reduced ecdysone signaling,
indicating that rig is required for the appropriate expression of
specific ecdysone-inducible genes. (3) These effects on gene expression are likely to be indirect as the predicted Rig protein sequence contains multiple
protein-protein interaction domains and no known DNA-binding motifs. (4)
Rig protein can interact physically with several Drosophila nuclear
receptors, including EcR, USP and ßFTZ-F1, all of which have
mutant phenotypes in common with rig mutants. (5) Rig protein shuttles between the cytoplasm and nucleus of larval cells in a manner similar to the active subcellular redistribution that has been reported for known Drosophila and vertebrate nuclear receptor cofactors (Gates, 2003).
Five Drosophila nuclear receptor cofactors have been identified to
date: Alien, SMRTER, MBF1, Taiman and Bonus. Of these, only bonus appears to have
activities in common with rig, although relatively limited genetic
studies have been undertaken for most of these cofactors. No mutants have been
characterized for SMRTER or Alien, which act as co-repressors in tissue
culture transfection assays. MBF1 null mutants are viable and display a strong genetic
interaction with tdf/apontic mutants: this indicates a role in
tracheal and nervous system development. Somatic
clones of taiman mutants reveal a role in border cell migration
during oogenesis. In contrast, bonus mutants display first instar
larval lethality as well as defects in salivary gland cell death and cuticle
and bristle development, implicating a role for bonus in ecdysone
responses during development. Also like rig, bonus mutations result in
gene-specific defects in ecdysone-regulated transcription, and Bonus protein
can interact with a range of Drosophila nuclear receptors, including
EcR, USP, SVP, DHR3 and FTZ-F1. Bonus, however, interacts with these receptors
in an AF-2-dependent manner, unlike Rig. Moreover, the larval
lethal phenotypes of rig mutants do not resemble those reported for
bonus mutants and, unlike Rig, Bonus protein appears to be
exclusively nuclear in both larval and imaginal tissues. Further work is
required to determine whether bonus and rig might act
together to regulate ecdysone response pathways (Gates, 2003).
Rig is distinct from all known Drosophila nuclear receptor
cofactors in that it is not part of an evolutionarily conserved protein
family. Alien, SMRTER, MBF1, Taiman and Bonus all have vertebrate homologs,
and Taiman and Bonus are the fly orthologs of the well characterized
vertebrate nuclear receptor cofactors AIB1 and TIF1, respectively. In
contrast, Rig does not contain identifiable enzymatic activities nor the
conserved functional domains that define most nuclear receptor cofactors.
BLAST searches with the Rig protein sequence did not reveal any closely
related sequences in other organisms, although the top hits, which show
limited homology in the WD-40 repeats, are in factors known
to modify chromatin, including human histone acetyltransferase type B subunit
2 (RBBP-7) and chromatin assembly factor 1 (CAF-1) (Gates, 2003).
The WD-40 repeats that comprise about half of the Rig protein sequence are
likely to play an important role in its activity. Consistent with this
proposal, an N-terminal fragment of Rig, containing two WD-40 repeats but
missing the LXXLL motif (amino acids 1-300), is capable of interacting with
GST-DHR3 and GST-USP, suggesting that these repeats are sufficient for
Rig-nuclear receptor interactions. WD-40 repeats provide
multiple surfaces for protein-protein interactions and have been identified in
over 150 proteins that function in a wide range of processes, including
cytoskeleton assembly, transcriptional regulation, and pre-mRNA processing. In Drosophila, WD-40 repeats are associated with
several transcriptional regulators, including the p85 subunit of TFIID, the
Polycomb group protein encoded by extra sex combs, and
the Groucho corepressor. In addition, a WD-40 repeat protein, TBL1, has been
identified as part of a multiprotein complex with thyroid hormone receptor
that contains the SMRT nuclear receptor corepressor and HDAC-3. The
presence of these sequences in Rig may thus provide a scaffold for
protein-protein interactions that could mediate the formation of multiprotein
transcriptional complexes on ecdysone-regulated promoters. Further biochemical
studies of Rig should provide insights into the significance of its WD-40
repeats as well as a foundation for understanding how Rig exerts its effects
on transcription (Gates, 2003).
It is not clear how Rig expression in the brain, imaginal discs and
salivary glands of second and third instar larvae is related to the lethal
phenotypes of rig mutants, although neuroendocrine signaling is
clearly required for molting, a process that is defective in rig
mutant larvae. The subcellular localization of Rig protein at later
stages, however, correlates with the distinct fates of larval and imaginal
cells during metamorphosis. Rig protein appears to be restricted to the
cytoplasm of cells that are fated to form parts of the adult fly, including
neuroblasts, imaginal discs, and the imaginal islands of the larval midgut. In contrast, Rig shows dynamic changes in its subcellular distribution in larval salivary gland and midgut cells, both of which undergo steroid-triggered programmed cell death during metamorphosis. It is possible that these differences in subcellular localization could contribute to the distinct fates of these tissues in
response to ecdysone signaling (Gates, 2003).
In addition to this spatial correlation, there is also a temporal
correlation between the times at which Rig protein shuttles between the
cytoplasm and nucleus in larval tissues and the coordinated changes in
ecdysone-regulated gene expression that occur during the third instar. The
switch from cytoplasmic to nuclear localization in larval salivary glands and
midguts occurs at approximately the same time, 24-30 hours after the
second-to-third instar larval molt, suggesting that Rig may be
responding to a common temporal signal. Cell type-specific factors, however,
must also contribute to this regulation as Rig is localized to the nucleus of
only a subset of cells in the larval midgut. Interestingly,
this protein redistribution correlates with a poorly understood event that is
represented by widespread changes in ecdysone-regulated gene expression,
called the 'mid-third instar transition.' It is
possible that the cytoplasmic-to-nuclear transport of Rig in larval tissues
contributes to the regulation of this response, which prepares the animal for
metamorphosis one day later. Similarly, Rig returns to the cytoplasm of
salivary gland cells at puparium formation, in synchrony with the widespread
changes in ecdysone-regulated gene expression associated with the onset of
metamorphosis. This translocation, however, is not seen in the larval midgut,
where Rig protein remains in the nucleus of some cells. Rig
shuttling thus appears to be differentially controlled in both a temporally
and spatially restricted manner, correlating with major switches in
ecdysone-regulated transcription. The observation that the first of these
shifts in subcellular distribution occurs during the major lethal phase of
rig mutants -- the mid-third instar -- suggests that
these intracellular movements contribute to the critical functions of Rig
during development (Gates, 2003).
Interestingly, several recent reports have described the subcellular
redistribution of nuclear receptor cofactors in both vertebrate and
Drosophila cells. The p/CIP vertebrate nuclear receptor coactivator
is differentially distributed within the cells of the mouse female
reproductive organs. For example, p/CIP is detected primarily in the nuclei of
highly proliferative follicular cells while it is most abundant in the
cytoplasm of terminally differentiated cells of the corpus luteum. p/CIP
displays active nucleocytoplasmic shuttling in response to growth factors in
cell culture, and interacts directly with the microtubule network in the
cytoplasm. Similarly, MEK-1 kinase-mediated phosphorylation of the SMRT
mammalian corepressor leads to the translocation of this factor from the
nucleus to the cytoplasm in cell culture transfection assays.
The functional homolog of this protein in flies, SMRTER, also shows active
redistribution from the nucleus to the cytoplasm in response to a MAP kinase
pathway, in this case mediated by EGFR/Sno/Ebi in the Drosophila eye. In both of these systems, regulated phosphorylation of SMRT/SMRTER results in
dissociation of a repressor complex and derepression of target gene
transcription (Gates, 2003).
These observations raise the possibility that the subcellular location of
Rig could determine its regulatory function in different cell types. For
example, by analogy with SMRT/SMRTER, loss of Rig from the nucleus of larval
cells might disrupt a corepressor complex on specific promoters, leading to
coordinate target gene derepression. This is consistent with the proposal that
the ecdysone receptor exerts critical repressive functions during larval
development. Alternatively, Rig protein in the cytoplasm may tether one
or more nuclear receptors, preventing them from acting on their cognate target
genes in the nucleus. This model is not favored, however, because antibody
stains reveal an exclusively nuclear localization for EcR, USP and ßFTZ-F1 at the onset of metamorphosis. It is also interesting to note that Rig protein appears to localize to discrete regions within the nuclei of larval midgut cells that do not contain chromosomes while Rig co-localizes with the giant polytene chromosomes in larval salivary gland cells. Rig may thus exert some functions in the nucleus that are independent of chromatin binding. Further biochemical studies of Rig, including the identification of additional proteins that interact with this factor, should provide insights into the significance of the subcellular localization of Rig protein as well as a mechanistic understanding of how Rig contributes to ecdysone responses during Drosophila larval development (Gates, 2003).
Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still 'orphans'. The ligand binding pocket of the Drosophila nuclear receptor E75 is shown to contain a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed (Reinking, 2005).
Using electronic absorption and mass spectrometry, it has been shown that the Drosophila nuclear receptor E75 contains a single tightly associated heme prosthetic group. Thus, nuclear receptor LBDs can now be added to the limited repertoire of known heme binding motifs. All results here are consistent with a conventional heme-protein interaction mediated by a pair of coordinate bonds. Two highly conserved histidine residues are good candidates for the interacting residues (Reinking, 2005).
The results suggest three general ways in which E75-heme may function. (1) The necessity of heme for E75 LBD stability and the changes in expression levels brought about by supplemental heme suggest that E75 may function as a nuclear monitor of cellular heme levels. (2) The ability of E75-heme to switch between oxidized and reduced states and the effects of these states on CO, NO, and cofactor peptide binding suggest a possible role as a cellular redox sensor. (3) The ability of E75-heme to bind NO and CO, and for these gases to modulate cofactor binding and transcriptional activity, suggests a role in mediating NO and/or CO intercellular signaling. This is the first example of a nuclear receptor with a bipartite ligand binding system (Reinking, 2005).
Nuclear receptors were first characterized based on their ability to bind steroid hormones but are now known to bind a fairly diverse set of lipophilic molecules including fatty acids, phospholipids, retinoids, bile acids, farnesoids, and a range of xenobiotics. Like most other nuclear receptor ligands, heme is also a lipophilic molecule with polarized negative charges. Its molecular weight and solvent-excluded volume, although larger than most, are within the range of other known ligands. For example, one of the closest vertebrate homologs to E75, Peroxisome proliferator activator receptor gamma (PPARgamma), has a pocket that can easily accommodate a molecule of this size. Although heme is not dissimilar to other ligands in most physical attributes, an iron atom within a protoporphyrin ring brings an exciting new dimension to the potential molecular and physiological roles of nuclear receptor proteins (Reinking, 2005).
The tight interaction generally formed between heme molecules and their protein partners, also observed with E75, and the inability to detect apo-E75 suggest that E75 and heme are unlikely to associate and dissociate readily. For this to occur, dedicated cofactors would be required to shuttle the heme in and out of the LBD and to stabilize the LBD in the absence of heme. Although no evidence was found for such cofactors, it is possible that endogenous E75 is expressed in certain tissues or stages in which such cofactors exist. If, however, these cofactors are not present, or do not exist, heme would be required as a dedicated structural component. In the latter case, the levels of free heme in the cell would determine the levels of E75 that can accumulate. Either way, heme availability would determine the levels of active E75 in the cell. If E75 target genes include regulators of heme metabolism, these properties would make E75 an ideal regulator of heme homeostasis (Reinking, 2005).
Heme is required by proteins that control an enormous range of cellular and biological processes. Examples of these processes (and proteins) include energy transfer (mitochondrial cytochromes), lipid and drug metabolism (cytochrome P450s), heme metabolism (heme oxygenase [HO]), oxygen radical detection and removal (superoxide dismutases, catalase, NADPH hydroxylase), gas transport (hemoglobin, myoglobin), iron transport (HO-FE-ATP pump), neuronal differentiation, and behavior (guanylate cyclase, nitric-oxide synthase [NOS]) and circadian rhythm (HO, NPAS2). Interestingly, NOS and HO, the enzymes that produce NO and CO in the cell, are also heme-containing proteins (Reinking, 2005 and references therein).
Of the physiological processes listed above, particularly intriguing and relevant ones are lipid and xenobiotic metabolism. In vertebrates, most of the enzymes that regulate these reactions are cytochrome P450s, which typically use heme to transfer oxygen or hydroxyl groups to or from their substrates. Homeostasis between lipid absorption/production and secretion/breakdown is controlled by nuclear receptors, largely through transcriptional regulation of cytochrome P450 (cyp) genes. In turn, the substrates or products of the P450-regulated reactions are often ligands for the nuclear receptors that regulate expression of the respective P450 genes. This triangular relationship between transcriptional regulators, enzymes, and reaction products provides the necessary feedback for homeostasis. Given that a close link between heme and lipid metabolism is also well documented, the ability of a nuclear receptor to monitor levels of heme could provide a general mechanism for coordinating these processes (Reinking, 2005).
The iron center of E75 can reversibly switch between Fe(III) and Fe(II) oxidation states. Furthermore, the interaction with an HR3-derived peptide is selective for the Fe(II) oxidation state of E75-heme. This set of characteristics sets up the intriguing possibility of E75 acting as a direct redox sensor. An interesting example of this behavior is the reported heme-based redox sensor activity of Ec-DOS, a phosphodiesterase in E. coli. Enzymatic activity is modulated by an allosteric change in the heme-containing PAS domain. The heterodimerization and transcriptional activities of the NPAS2 and Bmal1 transcription factors, which regulate circadian rhythm, have also been shown to depend on redox potential in vitro. NPAS2 has two heme-containing PAS domains, which could provide the mechanism for redox detection. Ec-DOS and NPAS2 also function as gas sensors (Reinking, 2005).
In many heme bound proteins, the heme molecule serves as a cofactor or prosthetic group for the binding of diatomic gases. This is also the case for E75-heme, and NO and CO may serve as E75 ligand(s). Although small, these diatomic gases have been shown to bring about significant changes in protein structure. In heme-containing proteins such as hemoglobin, myoglobin, guanylate cyclase, CooA, and FixL, for example, the binding of O2, CO, or NO causes allosteric rearrangements that modulate protein multimerization, enzymatic activity, and/or the ability to interact with cofactors or target molecules (Reinking, 2005).
In the case of E75, peptide binding studies suggest that CO interferes with the ability of E75 to interact with the DHR3 AF2 motif. Although unable to confirm this by direct binding assays, the results with NO on E75 transcriptional activity suggest that NO may act similarly. This may be analogous to the case of soluble guanylate cyclase, where NO and CO binding both lead to functional activation, but to varying degrees. As with other nuclear receptors, proper placement of the DHR3 AF2 helix within its ligand binding domain is most likely required for DHR3 to bind transcriptional coactivators. If E75 were to sequester the DHR3 AF2 helix, then DHR3 activity would be compromised. By reversing the AF2-E75 interaction, NO and/or CO could then restore normal DHR3-coactivator interactions (Reinking, 2005).
NO and CO are being implicated in a rapidly growing list of signalling processes in vertebrates, and insects. Their ability to diffuse readily between and within cells, and their short half-lives, make them ideal intercellular signaling molecules. In many cases, they act together or in opposition. Examples of processes regulated by NO and CO include blood pressure, cell division, cell death, inflammation, metabolism, hypoxia, diurnal cycles, behavior, and memory. The primary source of NO in tissues is the enzyme NOS and for CO, heme oxygenase. In flies, mutation of the dnos gene results in embryonic lethality. Flies also contain a single heme oxygenase gene, but genetic and functional analyses of dHO are yet to be carried out (Reinking, 2005).
The fact that CO production is dependent upon heme as a substrate, that NOS and heme oxygenase have heme centers, and that they have related physiological functions poses an interesting set of coincidences. This and the convergence of heme with lipid synthesis and related processes, NO and CO gas binding and now E75 function, suggests that E75 may provide a unifying role in the regulation of these processes (Reinking, 2005).
Numerous studies have placed E75 genetically, transcriptionally, and functionally in the ecdysone response pathway, both upstream and downstream of the ecdysone receptor. Thus, E75 appears to play roles in both ecdysone production and response. Ecdysone-regulated processes during insect development include cuticle formation, molting, programmed cell death, neurogenesis, imaginal disk development, and oogenesis. The binding of E75 to heme suggests possible connections between these processes and heme metabolism or function. This could occur at a variety of different levels. Some intriguing possibilities include the regulation of hormone-synthesis pathways, oogenesis arrest, metabolism, and the control of circadian rhythm (Reinking, 2005).
An intimate functional triangle exists between nuclear receptor function, cytochrome P450 expression, and cytochrome P450 substrates. This relationship, taken together with the apparent need for E75 in ecdysone production and response, suggests the possibility that E75 could control ecdysteroid metabolism by regulating the transcription of key cytochrome P450 genes, or more globally by sensing the levels of available heme. The possible ability of E75 to regulate hormone synthesis, and its known role in controlling the progression of molts, metamorphosis, and oogenesis, also brings up other intriguing possible functions. One example might be the ability to monitor energy resources such as lipids and to coordinate these levels with developmental progression. More specifically, the close link between heme, NO, and lipid metabolism could be used to block or postpone molting, pupariation, or oogenesis when energy resources are low, and vice versa (Reinking, 2005).
Interestingly, in mosquitoes, oogenesis is halted prior to chorion deposition until the insect obtains a blood meal. Although the blood-meal components that release the oogenesis arrest have yet to be identified, one of the major components of the blood meal is heme removed from the metabolized hemoglobin. Taken together with the observations that one of the responses of the blood meal is a pulse of ecdysone synthesis and action and that in Drosophila, E75 functions in both a feed forward and downstream role in the ecdysone signaling pathway, E75 may play a key role in this response by responding to the ingested heme and inducing ecdysone synthesis (Reinking, 2005).
A number of possible links also exist between E75 function and a role in regulating circadian rhythms. (1) NO, CO, and heme appear to be important regulators of circadian oscillators. In vertebrates, 'light' and 'dark' inputs are thought to be converted to signals of NO and CO, which in turn modulate the phase and period of the circadian cycle. Heme biosynthesis is also reciprocally regulated by the circadian clock. (2) The closest homolog to E75 in vertebrates, Rev-Erbalpha, is a well-established regulator of circadian rhythm in mammals, also acting as a transcriptional repressor and competing with the nuclear receptor RORalpha, which is a transcriptional activator of the circadian regulator Bmal1. The interaction between Rev-Erbalpha and RORalpha is analogous to that of E75 and HR3, their closest insect homologs. (3) Each larval instar molt, as well as the onset of metamorphosis and adult eclosure, are coupled to the circadian clock. Molts between first and third instars occur every 24 hr, and metamorphosis and eclosure generally begin on the mornings of the fifth and ninth days. These events are regulated by rhythmic pulses of juvenile hormone and ecdysone, which are under E75 control. In insects that take longer to develop, such as Rodnius (21 days), ecdysone levels have been shown to oscillate daily, with levels highest at night. E75 may time these oscillating events indirectly by monitoring feeding activity (lipid intake) or through a more direct role in circadian rhythm (Reinking, 2005 and references therein).
An interesting implication of this study is that the orphan receptor, Rev-Erbalpha, may also bind heme and respond to diatomic gases. If it does not, compensating evolutionary steps may have been adopted to maintain the circuitry of E75-heme-regulated processes. It is also possible that other nuclear receptors may have adopted or conserved the capacity to bind heme and diatomic gases and to regulate corresponding developmental and physiological functions (Reinking, 2005).
E75A is transcribed during each stage in the life cycle in bursts that accompany ecdysteroid peaks in the life cycle, as well as in late embryos in synchrony with the E74A early mRNA (Segraves, 1988; Thummel, 1990). The three E75 transcripts accumulate with distinct kinetics at the onset of metamorphosis, with peaks of E75A mRNA in late third instar larvae and late prepupae in synchrony with the ecdysteroid pulses, consistent with its direct induction by ecdysteroids (Andres, 1993; Huet, 1993; Karim, 1992; Segraves, 1990). E75B mRNA accumulates to peak levels in 2–4 hr prepupae, while E75C mRNA peaks in 10–12 hr prepupae (Bialecki, 2002).
In Drosophila, pulses of the steroid hormone ecdysone function as temporal signals that trigger the major postembryonic developmental transitions. The best characterized of these pulses activates a series of puffs in the polytene chromosomes as it triggers metamorphosis. A small set of early puffs is induced as a primary response to the hormone. These puffs encode regulatory proteins that both repress their own expression and activate a large set of late secondary response genes. Northern blot analysis of RNA isolated from staged animals and cultured organs has been used to study the transcription of three primary response regulatory genes, E75, BR-C and EcR. Remarkably, their patterns of transcription in late larvae can be defined in terms of two responses to different ecdysone concentrations. The class I transcripts (E74B and EcR) are induced in mid-third instar larvae in response to the low, but increasing, titer of ecdysone. As the hormone concentration peaks in late third instar larvae, these transcripts are repressed and the class II RNAs (E74A, E75A and E75B) are induced. The BR-C RNAs appear to have both class I and class II characteristics. These data demonstrate that the relatively simple profile of a hormone pulse contains critical temporal information that is transduced into waves of primary response regulatory gene activity (Karim, 1992).
The Drosophila E75 early gene has been isolated and two of its products, E75A and E75B, have been shown to be members of the steroid receptor superfamily. Antisera directed against A- and B-specific regions of the E75 proteins has been prepared. Antisera and a monoclonal antibody raised against E75A, the major larval protein product of the E75 gene, bind to discrete sites in native salivary gland chromosomes. These sites are closely correlated with early and late ecdysone responsive loci (Hill, 1993).
The ecdysone response hierarchy mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates (Buszczak, 1999).
During stage 10, the follicle cell expression of E75 becomes enriched in the dorsal anterior cells. This suggested that inputs in addition to ecdysone are needed to refine E75 expression. Previous work has shown that follicle cell polarity is established during mid- to late-oogenesis and depends on the interaction between Gurken and the Drosophila homolog of the mammalian EGF receptor (Egfr). To determine whether E75 expression is under control of the dorsoventral signaling pathway, ovarian E75 mRNA distribution was examined in dorsalized and ventralized mutant backgrounds. In fs(1)K10 mutants, mislocalization of Grk protein results in activation of Egfr in all anterior follicle cells surrounding the oocyte. In fs(1)K10 mutant egg chambers, E75 expression expands to a ring of anterior follicle cells surrounding the oocyte. Mutations in Egfr prevent signal transduction by the receptor and lead to the ventralization of the eggshell and embryo. In situ analysis indicates that stage 10 follicle cells overlying the oocyte in Egfr mutants no longer express E75. However, E75 expression in the nurse cells is unaffected. These experiments show that the Egfr signaling pathway regulates E75 expression in the dorsal follicle cells but not in the germline (Buszczak, 1999).
Many of the 21 members of the nuclear receptor superfamily in Drosophila are transcriptionally regulated by the steroid hormone ecdysone and play a role during the onset of metamorphosis, including the EcR/USP ecdysone receptor heterodimer. The temporal patterns of expression for all detectable nuclear receptor transcripts were examined throughout major ecdysone-regulated developmental transitions in the life cycle: embryogenesis, a larval molt, puparium formation, and the prepupal-pupal transition. An unexpected close temporal relationship was found between DHR3, E75B, and betaFTZ-F1 expression after each major ecdysone pulse examined, reflecting the known cross-regulatory interactions of these genes in prepupae and suggesting that they act together at other stages in the life cycle. In addition, E75A, E78B, and DHR4 are expressed in a reproducible manner with DHR3, E75B, and betaFTZ-F1, suggesting that they intersect with this regulatory cascade. Finally, known ecdysone-inducible primary-response transcripts are coordinately induced at times when the ecdysteroid titer is low, implying the existence of novel, as yet uncharacterized, temporal signals in Drosophila (Sullivan, 2003).
Total RNA was isolated from two independent collections of embryos staged at 2-h intervals throughout the 24 h of Drosophila embryonic development. Five Northern blots were prepared using equal amounts of RNA from each time point. These blots were sequentially hybridized, stripped, and rehybridized with radioactive probes derived from each of the 21 nuclear receptor genes encoded by the Drosophila genome. This approach allowed the generation of time courses of nuclear receptor gene expression that could be directly compared between family members. The transcripts detected are consistent with reported sizes. Transcripts from eight nuclear receptor genes were not detectable during embryonic development: E75C, E78, CG16801, DHR38, DHR83, dsf, eg, and svp (Sullivan, 2003).
Transcripts from nine nuclear receptor genes can be detected at the earliest time point (0-2 h): usp, EcR-A, FTZ-F1, DHR39, DHR78, DHR96, dERR, dHNF-4, and tll. This expression is consistent with the known maternal contribution of usp, EcR, and FTZ-F1. The observation that transcripts from DHR39, DHR78, DHR96, dERR, and dHNF-4 are undetectable by the next time point examined (2-4 h) suggests that these mRNAs are maternally loaded and rapidly degraded. EcR-B and usp transcripts are induced in early embryos, up-regulated at 6-8 h after egg laying (AEL), and maintain expression through the end of embryogenesis, with down-regulation of EcR-B in late embryos. EcR-A, in contrast, is expressed for a relatively brief temporal window, at 8-14 h AEL (Sullivan, 2003).
Six nuclear receptor genes are expressed in brief intervals during midembryonic stages. DHR39 and E75A are initially induced at 4-6 and 6-8 h AEL, respectively, and peak at 8-12 h AEL. This is followed by induction of DHR3, DHR4, and E75B at 8-12 h AEL, followed by ßFTZ-F1 expression at 12-18 h AEL. DHR39 appears to exhibit an expression pattern reciprocal to that of ßFTZ-F1, with lowest levels of mRNA at 14-16 h AEL and reinduction at 16-18 h as ßFTZ-F1 is repressed. This is followed by a second peak of E75A transcription at 18-22 h AEL (Sullivan, 2003).
A second group of nuclear receptors, DHR78, DHR96, dHNF-4, and dERR, is more broadly expressed at low levels throughout embryogenesis. DHR78 accumulates above its constant low level of expression between 8 and 14 h AEL. dERR exhibits an apparent mRNA isoform switch between 14 and 18 h AEL. dHNF-4 regulation also appears complex, with two size classes of mRNA induced at approximately 8-10 h AEL. While the 4.6-kb dHNF-4 mRNA is expressed throughout embryogenesis, the 3.3-kb mRNA is down-regulated at 14-16 h AEL. This timing is consistent with observations that dHNF-4 is expressed primarily in the embryonic midgut, fat body, and Malpighian tubules. Finally, nuclear receptors known to exert essential functions in patterning the early embryo, tll, kni, and knrl, are expressed predominantly during early stages (Sullivan, 2003).
Two genes that are not members of the nuclear receptor superfamily, BR-C and E74, were also examined in this study, as transcriptional markers for ecdysone pulses during development. Unexpectedly, both of these genes are induced late in embryogenesis, several hours after the rise in ecdysone titer at 6 h AEL. An approximately 7-kb BR-C transcript is induced at 10-12 h AEL and is present through the end of embryogenesis while E74B is induced at 14-16 h AEL and repressed as E74A is expressed from 16-20 h AEL. This BR-C expression pattern is consistent with the identification of the BR-C Z3 isoform in specific neurons of the embryonic CNS (Sullivan, 2003).
First-instar larvae were synchronized as they molted to the second instar, aged and harvested at 4-h intervals throughout second-instar larval development. Two Northern blots were prepared using equal amounts of total RNA isolated from a single collection of animals. Each blot was sequentially hybridized, stripped, and rehybridized to detect nuclear receptor transcription. The following transcripts were not detectable during the second instar: E75C, dERR, CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).
EcR-B expression is induced in mid-second-instar larvae, but does not reach maximum levels until 68-72 h AEL, just before the molt. In contrast, usp is expressed throughout the instar. A sequential pattern of nuclear receptor expression is observed that resembles the pattern seen in midembryogenesis. DHR39 and E75A are expressed in the early second instar. This is followed by induction of E75B, E78B, DHR3, and DHR4, followed by expression of ßFTZ-F1 at the end of the instar. DHR39 again shows a pattern that is approximately reciprocal with ßFTZ-F1, with highest levels during the first half of the instar. Similarly, DHR78, DHR96, and dHNF-4 exhibit broad expression patterns throughout second-instar larval development. E74A, E75A, and DHR38 are coordinately up-regulated with EcR-B at the end of the instar, between 64-72 h AEL. Finally, an approximately 9-kb BR-C transcript is detected throughout the second-larval instar (Sullivan, 2003).
Nuclear receptor gene expression was also examined throughout the third larval instar and into the early stages of metamorphosis, encompassing the ecdysone-triggered larval-to-prepupal and prepupal-to-pupal transitions. Third-instar larvae were staged relative to the molt from the second instar and harvested at 4-h intervals throughout the 48 h of the instar. Prepupae were synchronized relative to puparium formation (±15 min) and harvested at 2-h intervals up to 16 h after puparium formation (APF). Total RNA was isolated from whole animals and analyzed by Northern blot hybridization. Five blots were prepared from two independent collections of animals. These blots were sequentially hybridized, stripped, and rehybridized to detect nuclear receptor gene expression. The following transcripts were not detectable during third-instar larval or prepupal stages: CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).
Most nuclear receptor genes show little or no detectable expression in early and mid-third-instar larvae, a time when the ecdysone titer is low. Similar to the pattern seen in second-instar larvae, usp is expressed at relatively low levels throughout the instar and up-regulated at puparium formation, while EcR-B is induced at approximately 100 h AEL and rapidly down-regulated at puparium formation. This is followed by a sequential pattern of nuclear receptor expression similar to that seen at earlier stages. DHR39, E75A, and E78B are induced at 116-120 h AEL, in concert with the late larval ecdysone pulse, followed by maximum accumulation of E75B, DHR3, and DHR4 at 0-4 h APF. ßFTZ-F1 is expressed from 6-10 h APF, with a pattern that is approximately reciprocal to that of DHR39. EcR-A is expressed in parallel with E75B, DHR3, and DHR4 in midprepupae, similar to their coordinate expression during embryogenesis (Sullivan, 2003).
DHR78, DHR96, and dHNF-4 continue to exhibit broad expression profiles throughout third-instar larval and prepupal development. An E75 isoform not detected in embryos or second-instar larvae, E75C, is also detectable at low levels throughout most of the third instar and up-regulated in correlation with the late-larval and prepupal pulses of ecdysone. DHR38 is detectable at very low levels in early third-instar larvae, in synchrony with the early induction of E74B and BR-C. E74B is repressed, E74A is induced, and BR-C transcripts are up-regulated in late third-instar larvae, in synchrony with the late-larval ecdysone pulse. The prepupal pulse of ecdysone occurs at 10-12 h APF, marking the prepupal-to-pupal transition. EcR-A, E75A, E78B, DHR4, dERR, E75C, dHNF-4, and E74A are all induced at 10-12 h APF, in apparent response to this hormone pulse. These results are consistent with a microarray analysis of gene expression at the onset of metamorphosis where the temporal profiles of about half of these genes have been reported (Sullivan, 2003).
Most nuclear receptors can be divided into one of four classes based on this study: (1) those that are expressed exclusively during early embryogenesis (kni, knrl, tll); (2) those that are expressed throughout development (usp, DHR78, DHR96, dHNF-4); (3) those that are expressed in a reproducible temporal cascade at each stage tested (E75A, E75B, DHR3, DHR4, FTZ-F1, DHR39), and (4) those that are undetectable in these assays (CG16801, DHR83, dsf, eg, svp) (Sullivan, 2003).
Three nuclear receptor genes appear to be expressed exclusively during early embryogenesis: kni, knrl, and tll. This restricted pattern of expression fits well with the functional characterization of these genes, which have been shown to act as key determinants of embryonic body pattern. Eight genes (usp, EcR, FTZ-F1, DHR39, DHR78, DHR96, dERR, and dHNF-4) were identified that appear to have maternally deposited transcripts and thus possible embryonic functions. Indeed, maternal functions have been defined for usp, EcR, and alphaFTZ-F1 (Sullivan, 2003).
Four nuclear receptor genes are broadly expressed through all stages examined: usp, DHR78, DHR96, and dHNF-4. dHNF-4 mRNA is first detectable at 6-10 h AEL, as the ecdysone titer begins to rise. In addition, peaks of dHNF-4 expression are seen at 0, 12, and 16 h APF, in synchrony with the E74 and E75C early ecdysone-inducible genes. These observations raise the interesting possibility that this orphan nuclear receptor is regulated by ecdysone (Sullivan, 2003).
DHR38 transcripts are difficult to detect in these assays. This is consistent with studies which used RT-PCR or riboprobes for this purpose. Nonetheless, DHR38 mRNA can be detected during third-instar larval development, consistent with the widespread expression reported in earlier studies. DHR38 expression peaks at late pupal stages, consistent with its essential role in adult cuticle formation (Sullivan, 2003).
dERR and E75C display related temporal profiles of expression that do not fit with other nuclear receptor genes described in this study. Both of these genes are specifically transcribed during prepupal development, with increases in expression at 0 and 10-12 h APF. dERR, but not E75C, is also expressed during embryogenesis, with an initial induction at approximately 6 h AEL. These increases occur in synchrony with ecdysone pulses, suggesting that these orphan nuclear receptor genes are hormone inducible, although in a stage-specific manner. Further studies of dERR regulation, as well as a genetic analysis of this locus, are currently in progress (Sullivan, 2003).
Interactions between the DHR3 and E75B orphan nuclear receptors contribute to appropriate ßFTZ-F1 regulation during the onset of metamorphosis. DHR3 is both necessary and sufficient to induce ßFTZ-F1 and appears to exert this effect directly, through two response elements in the ßFTZ-F1 promoter. E75B can heterodimerize with DHR3 and is sufficient to block the ability of DHR3 to induce ßFTZ-F1. These three factors thus define a cross-regulatory network that contributes to the timing of ßFTZ-F1 expression in midprepupae. ßFTZ-F1, in turn, acts as a competence factor that directs the appropriate genetic and biological responses to the prepupal pulse of ecdysone. The patterns of DHR3, E75B, and ßFTZ-F1 expression observed at the onset of metamorphosis are consistent with these regulatory interactions as well as the expression patterns reported in earlier studies (Sullivan, 2003).
Unexpectedly, the tight linkage of DHR3, E75B, and ßFTZ-F1 expression seen at the onset of metamorphosis is recapitulated at earlier stages, after each of the major ecdysone pulses examined, in midembryogenesis and second-instar larval development. This observation suggests that the regulatory interactions between these receptors is not restricted to metamorphosis, but rather may recur in response to each ecdysone pulse during development. It is possible that this regulatory cascade contributes to cuticle deposition, which is dependent on ecdysone signaling in embryos, larvae, and prepupae. In support of this proposal, DHR3 and ßFTZ-F1 mutants exhibit defects in larval molting, suggesting that they act together to regulate this early ecdysone response (Sullivan, 2003).
Three other orphan nuclear receptor genes, E75A, DHR4, and DHR39, are expressed in concert with DHR3, E75B, and ßFTZ-F1, after the embryonic, second-instar, and third-instar ecdysone pulses. A peak of E75A expression marks the start of each genetic cascade, correlating with the rising ecdysone titer in 6- to 8-h embryos, the first half of the second instar, and in late third-instar larvae. This is followed by DHR3, E75B, and DHR4 expression which, in turn, is followed by a burst of ßFTZ-F1 expression. E78B is expressed in synchrony with DHR4 in late second and third-instar larvae, but not in embryos. These patterns of expression raise the interesting possibility that E75A, DHR4, and E78B may intersect with the cross-regulatory network defined for DHR3, E75B, and ßFTZ-F1. E75B and E78B are related to the Rev-erb vertebrate orphan nuclear receptor and are both missing their DNA binding domain. E75B and E78B null mutants are viable and fertile, suggesting that they exert redundant regulatory functions. E75A mutants die during larval stages, with no known direct regulatory targets. DHR4 mutants have not yet been described, although recent work indicates that this gene exerts essential roles in genetic and biological responses to the late larval ecdysone pulse. Further functional studies of these nuclear receptor genes should provide insight into their possible contribution to the regulatory circuit defined by DHR3, E75B, and ßFTZ-F1 (Sullivan, 2003).
Interestingly, DHR39 displays a reproducible pattern of expression that is inversely related to that of ßFTZ-F1, defining possible repressive interactions. DHR39 and ßFTZ-F1 have a similar DNA binding domain (63% identity) and bind to identical response elements, suggesting that they may exert cross-regulatory interactions. Moreover, DHR39 can repress transcription through the same response element that is activated by ßFTZ-F1. It would be interesting to determine whether the reciprocal patterns of DHR39 and ßFTZ-F1 expression during development is of functional significance (Sullivan, 2003).
The transcription of BR-C, EcR, E74, and E75 has been extensively characterized during the onset of metamorphosis, due to their rapid and direct regulation by the steroid hormone ecdysone at this stage in development. Surprisingly, however, their expression appears to be disconnected from the high-titer ecdysteroid pulses during embryonic and second-instar larval stages. As expected, EcR is induced early in embryonic development, in coincidence with the rising ecdysone titer at 4-10 h AEL, with EcR-B transcripts appearing first followed by EcR-A. BR-C mRNA, however, is not seen until 10-12 h AEL and E74B mRNA is induced even later, at 14-16 h AEL, when the ecdysteroid titer has returned to a basal level. Both EcR-B and E74B are repressed from 16-20 h AEL as E74A and E75A are induced, a switch that has been linked to the high-titer ecdysone pulse in late third-instar larvae; however, this response occurs during late embryogenesis when the ecdysteroid titer is low. A similar observation has been made for E75A expression in the Manduca dorsal abdominal epidermis, where a brief burst of E75A mRNA is detected immediately before pupal ecdysis, after the ecdysteroid titer has returned to basal levels (Sullivan, 2003).
It thus seems likely that the second instar ecdysone pulse occurs during the first half of the instar. This profile is consistent with the early induction of E75A. EcR-B and E74A, however, are not induced until the second half of the second instar, with a peak at the end of the instar. BR-C mRNA levels remain steady throughout the second instar. Finally, EcR-B, E74B, and BR-C are induced in early to mid-third-instar larvae, a time when one or more low-titer ecdysone pulses may occur. It is curious that E74B is poorly expressed relative to E74A during embryonic and second-instar larval stages, disconnecting its expression from that of EcR. This pattern is not seen in studies that focused on the onset of metamorphosis. Taken together, the temporal profiles of early gene expression (EcR, BR-C, E74, E75A) during late embryonic and late second-instar larval stages appear to be unlinked to the known ecdysteroid pulses at these stages. This could indicate that these promoters are activated in a hormone-independent manner at these stages in the life cycle. Alternatively, these ecdysone primary-response genes may be induced by a novel temporal signal that remains to be identified (Sullivan, 2003).
Several lines of evidence indicate that 20-hydroxyecdysone is not the only temporal signal in Drosophila. A major metabolite of this hormone, 3-dehydro-20-hydroxyecdysone, was shown to be as effective as 20-hydroxyecdysone in inducing target gene transcription in the hornworm, Manduca sexta. Similarly, 3-dehydro-20-hydroxyecdysone is more efficacious than 20-hydroxyecdysone in inducing Fbp-1 transcription in the Drosophila larval fat body. A high-titer pulse of alpha-ecdysone, the precursor to 20-hydroxyecdysone, can drive the extensive proliferation of neuroblasts during early pupal development in Manduca. This is the first evidence that alpha-ecdysone is responsible for a specific response in insects. It is unlikely, however, that this signal is transduced through the EcR/USP heterodimer, which shows only very low transcriptional activity in response to this ligand. Rather, recent evidence indicates that alpha-ecdysone may activate DHR38 through a novel mechanism that does not involve direct hormone binding (Sullivan, 2003).
Studies of ecdysteroid-regulated gene expression in Drosophila have also provided evidence for hormone signaling pathways that may act independently of 20-hydroxyecdysone. Several studies have identified a large-scale switch in gene expression midway through the third larval instar, an event that has been referred to as the mid-third-instar transition. It is not clear whether this response is triggered by a low-titer ecdysteroid pulse, another hormonal signal, or in a hormone-independent manner. Similarly, the let-7 and miR-125 micro-RNAs are induced at the onset of metamorphosis in Drosophila in tight temporal correlation with the E74A early mRNA, but not in apparent response to 20-hydroxyecdysone. These studies indicate that 20-hydroxyecdysone cannot act as the sole temporal regulator during the Drosophila life cycle (Sullivan, 2003).
The number of Drosophila egg chambers is controlled by the nutritional status of the female. There is a developmental checkpoint at stage 8, which is controlled by BR-C in the follicle cells along with ecdysteroid. During this period, developmental decision is made in each egg chamber to determine if it will develop or die. During nutritional shortage, inducing apoptosis in the nurse cells of stages 8 and 9 egg chambers reduces the number of egg chambers. Ecdysone response genes E75A and E75B are involved in inducing or suppressing apoptosis. It is thus possible that the E75 isoforms A and B are involved in the decision to develop or die in oogenesis. This study establishes part of the pathway by which ecdysone response genes control apoptosis of the nurse cells and hence select between degeneration or development of individual egg chambers at stages 8 and 9 (Terashima, 2005a).
Drosophila egg production depends upon the nutrition available to females. When food is in short supply, oogenesis is arrested and apoptosis of the nurse cells is induced at mid-oogenesis via a mechanism that is probably controlled by ecdysteroid hormone. Expression of some ecdysone-response genes is correlated with apoptosis of egg chambers. Moreover, ecdysteroid injection and application of juvenile hormone induces and suppresses the apoptosis, respectively. In this study, an investigation was carried out to see which tissues show increases in the concentration of ecdysteroids under nutritional shortage to begin to link together nutrient intake, hormone regulation and the choice between egg development or apoptosis made within egg chambers. Ecdysteroid levels in the whole body, ovaries and haemolymph samples were measured by RIA, and it was found that the concentration of ecdysteroid increased in all samples. This contributes to the idea that nutritional shortage leads to a rapid high ecdysteroid concentration within the fly and that the high concentration induces apoptosis. Low concentrations of ecdysteroid are essential for normal oogenesis. It is suggested there is threshold concentration in the egg chambers and that apoptosis at mid-oogenesis is induced when the ecdysteroid levels exceed the threshold. Starvation causes the ovary to retain the ecdysteroid it produces, thus enabling individual egg chambers to undergo apoptosis and thus control the number of eggs produced in relation to food intake (Terashima, 2005b).
The prothoracic glands, which are the principal source of ecdysone in the immature stages, are no longer present in adults. The egg chambers produce ecdysone, which, at least in some insects, accumulates in the oocyte. In the fat body, ecdysone is converted to 20E, the active hormone, and shade, which encodes 20-hydroxylase for converting ecdysone to 20E, is expressed in nurse cells and follicle cells in the ovary and fat body (Terashima, 2005b).
Ecdysteroid synthesis is affected by the nutritional status of the female, and ecdysteroids affect oogenesis in many insects. Egg production in mosquitoes is triggered by a blood meal. The digested products of the blood meal stimulate the brain to secrete egg development neurosecretory hormone (EDNH), which is also known as ovarian ecdysteroidogenic hormone (OEH). EDNH stimulates the ovary to synthesize ecdysteroids, which instruct the fat body cells to make vitellogenin for the oocytes. Vitellogenin is critical for egg production, thus without the blood meal there is no vitellogenin and no eggs, so to produce mature eggs ecdysteroids are essential. In contrast, nutritional shortage induces an increase in ecdysteroid concentration in Drosophila females, ecdysteroid concentration increases in Drosophila whole body, haemolymph and ovaries during starvation. Feeding suppresses the high ecdysteroid concentration that is induced by nutritional shortage (Terashima, 2005b).
Under starvation, apoptosis of nurse cells in stage-8 and -9 egg chambers is induced; 20E injection into the females under adequate nutrition also induces the apoptosis and JHA treatment of females under nutritional shortage suppresses this apoptosis. Presumably high ecdysteroid concentrations in the haemolymph and/or the ovary, which are induced by starvation, may induce the apoptosis of nurse cells in stage-8 and -9 egg chambers. However, ecdysteroid is indispensable to produce mature eggs in Drosophila. Oogenesis in ecd-1 mutants is arrested at mid-oogenesis, and germline clones of EcR mutations led to developmental arrest; egg chambers degenerated during mid-oogenesis in Drosophila. Presumably, there is an ecdysteroid threshold for inducing apoptosis of nurse cells at stages 8 and 9 and ecdysteroids induce normal development when below the threshold concentration and induce apoptosis of nurse cells at stages 8 and 9 when over the threshold. Starvation induces an increase in ecdysteroid concentration to above the threshold level in the haemolymph and the ovary through activation of the ecdysone synthesis pathway in the egg chamber. Ecdysteroid secretion from the ovary decreased following nutritional shortage. Thus, ecdysteroid secretion from the fat body or other ecdysteroid-synthesizing tissues must be stimulated to induce the high ecdysteroid concentration observed in haemolymph (Terashima, 2005b).
JHA suppresses the high ecdysteroid concentration that is induced by starvation. JH and JHA suppress ecdysone synthesis/secretion from the prothoracic glands in larvae of Maduca sexta. It is likely that JHA suppression decreases the high ecdysteroid concentration in the ovary that induces apoptosis of nurse cells in stage-8 and -9 egg chambers under starvation, and therefore JHA treatment retains minimal ecdysteroid levels needed for inducing normal oogenesis (Terashima, 2005b).
There is a developmental checkpoint at stage 8 of oogenesis. YP synthesis commences at stage 8 and YP is accumulated during development into mature eggs. Drosophila egg chambers normally transit through stages 8 and 9 during a 6-h period, but starvation induced an accumulation of stage-8 and -9 egg chambers in Drosophila oogenesis. The number of stage-8 egg chambers is increased during a 5-12-h period after starvation starts, but the number of stage-9 egg chambers does not increase for 0-12 h after starvation started. This means that oogenesis progresses from stage 7 to 8, but does not progress from stage 8 to 9 and then to 10 under nutritional shortage. When 20E is injected into the fed flies, the accumulation of stage-8 chambers is not seen; therefore this arrest of oogenesis at stage 8 was not caused by the increasing 20E concentration in haemolymph and ovary. Perhaps starvation signals induce the arrest of oogenesis at stages 8 and 9 directly, or they could inhibit YP uptake. Some nutrient- and stress-response genes exhibit different expression patterns in the ovaries of females under adequate nutrition and starvation. It is suggested that the genes which respond directly to stress and nutrients interact with the ecdysone-synthesis pathway, resulting in the induction of apoptosis of nurse cells in stage-8 and -9 egg chambers through activation of BR-C Z2, Z3 and E75A expression in the follicle cells. Other genes could have altered their expression levels, so as to arrest oogenesis at stages 8 and 9 and to check the developmental status of the egg chamber. As a result, the decision is made to develop into a mature egg or undergo apoptosis at stages 8 and 9. The arrest in the progression of oogenesis at stages 8 and 9 is independent of increasing ecdysteroid levels (Terashima, 2005b).
Starvation signals are needed to activate a number of pathways to adjust the rate of egg production in Drosophila. These pathways could be classified into two groups: one to stimulate ecdysone synthesis in the follicle cells and/or nurse cells to activate the apoptosis pathway, including BR-C Z2, Z3 and E75A expression in the follicle cells, and another one to interact with and participate in the developmental checkpoint, giving rise to an arrest in oogenesis at stage 8 under nutritional shortage. A possible scheme is presented for the regulation of oogenesis related to nutrition in Drosophila. It is likely that starvation signals from the gut activate ecdysteroid synthesis in the ovary in Drosophila under starvation. Ecdysteroid is then accumulated in the egg chamber by decreasing 20E secretion from the ovary, and the fat body secretes 20E to haemolymph. It is suggested that there are two thresholds of 20E concentration in Drosophila ovary -- one is the concentration for normal oogenesis and the other is the concentration for inducing apoptosis -- and that starvation elevates the ecdysone levels in some egg chambers over the threshold that leads to apoptosis (Terashima, 2005b).
Imprecise excision of P elements was used to generate small deletions of sequences specific to either E75A or E75B, and to create a larger deletion of common region sequences shared by all three E75 isoforms. E75A81 is a 1.8 kb deletion that removes the E75A transcription start site along with the 5'-untranslated region and 143 bp of protein-coding sequence. E75D1 is an ~3 kb deletion that removes the E75B transcription start site along with most of the first E75B exon. E75D51 is an ~30 kb deletion that removes the first exon of E75B as well as the adjacent exon, shared by all three E75 isoforms, that encodes the second zinc finger of the DNA binding domain. In homozygotes and heteroallelic combinations, E75D51 and E75e213, a putative null EMS-induced point mutation in the E75 common region (Buszczak, 1999; Segraves, 1988), led to similar lethal phases and phenotypes, arguing that the E75D51 mutation inactivates all E75 functions. E75x37 is an ~60 kb g irradiation-induced deletion that removes the E75C transcription start site as well as ~10 kb of the downstream primary transcript (Segraves, 1988). E75x37 fails to complement another E75C allele, E75e273, which is an EMS-induced 63 bp deletion that removes the E75C splice donor (Segraves, 1988). E75x37 and E75e273 generate identical lethal phenotypes when maintained over a deletion for the E75 locus, providing further evidence that E75C is the only essential E75 function affected by the x37 deficiency (Bialecki, 2002).
Similar lethal phases and phenotypes were observed for E75A81 and E75x37 mutants when examined as either homozygotes or over the E75D51 common region deficiency, suggesting that they are null alleles with respect to the affected isoform. All studies described here use an isoform-specific E75 mutation over the E75D51 common region deficiency. For simplicity, E75D51 is referred to as the deficiency (Df) for the E75 locus, E75A81/Df as E75A mutants, E75D1/Df as E75B mutants, and E75x37/Df as E75C mutants. mRNA corresponding to the affected E75 isoform was undetectable by Northern blot hybridization in each of these genotypes, consistent with the molecular nature of these lesions and provides further evidence that they are null alleles (Bialecki, 2002).
Crosses were set up between control ry506/TM6B Ubi-GFP, E75D1/TM6B Ubi-GFP, or E75x37/TM6B Ubi-GFP animals and the deficiency stock E75D51/TM6B Ubi-GFP to test for embryonic lethality among the offspring. From the ry506 control cross, 26% of the first instar larvae did not express GFP, indicating no significant embryonic lethality. Similar results were obtained from the crosses with E75B mutants (23%) and E75C mutants (25%) (Bialecki, 2002).
E75B and E75C mutant first instar larvae were collected at hatching and examined at regular intervals for phenotypes and lethality during later stages of development. E75B mutants are viable and fertile, with no detectable phenotypes. In contrast, E75C mutants display lethality during both the pharate adult and adult stages. Approximately 33% of E75C mutants die as pharate adults, with normal adult pigmentation and fully developed appendages. The remaining E75C mutants eclose and are severely uncoordinated, displaying difficulty in walking and an inability to fly. These animals die within a week following eclosion. E75C mutant adults appear morphologically normal with the exception of black spots that cover about one quarter of the surface of the eye (Bialecki, 2002).
E75B and E75C are both induced by ecdysteroids at the onset of metamorphosis, suggesting that they may function during this stage in development (Huet, 1993; Karim, 1992). Gain-of-function studies have also implicated E75B in contributing to the timing of ßFTZ-F1 expression in mid-prepupae (White, 1997). The temporal profiles of ecdysteroid-regulated gene transcription were examined in E75B and E75C mutant late larvae and prepupae. Total RNA was isolated from control, E75B, and E75C mutant late third instar larvae staged at -18, -8, and -4 hr relative to puparium formation, as well as from prepupae staged at 2 hr intervals after puparium formation. These RNA samples were analyzed by Northern blot hybridization using radiolabeled probes designed to detect 16 ecdysteroid-regulated transcripts: EcR, BR-C, E74A, E74B, E75A, E75B, E75C, E78B, DHR3, Imp-E1, Fbp-1, Sgs-4, L71-1, L71-3, ßFTZ-F1, and Edg84A (Bialecki, 2002).
All of the tested transcription units, with the exception of E75B, are expressed normally in E75B mutant larvae and prepupae. Focus was placed on the temporal profile of ßFTZ-F1 expression in this mutant, and several independent Northern blots were prepared, one of which utilized prepupae staged at 30 min intervals. No reproducible effects on ßFTZ-F1 expression, however, could be detected. It is thus concluded that E75B is not required for the appropriate timing of ßFTZ-F1 transcription during prepupal development (Bialecki, 2002).
The tested transcription units are also expressed normally in E75C mutants at the onset of metamorphosis, with four exceptions. E75B mRNA normally peaks in abundance at 2 hr after puparium formation and is reinduced ~8 hr later, in response to the prepupal ecdysteroid pulse (Huet, 1993; Karim, 1992). This reinduction is submaximal in E75C mutants. Interestingly, the E75C mutation has a similar effect on Fbp-1, L71-1, and L71-3 transcription at this stage in development. Fbp-1 encodes a larval serum protein receptor that is induced by ecdysteroids in the fat body of mid-third instar larvae, and L71-1 and L71-3 are late ecdysteroid-inducible genes that are specifically expressed in prepupal salivary glands. These genes are normally downregulated in early pupae, ~14 hr after puparium formation. In E75C mutants, however, this repression occurs prematurely, ~10 hr after puparium formation. It is at this time that E75C mRNA levels peak in abundance in wild-type animals (Andres, 1993; Karim, 1992), indicating that E75C is normally required to maintain the expression of these genes through the prepupal-pupal transition (Bialecki, 2002).
To assess possible embryonic lethality, E75A81/TM6B Ubi-GFP animals were crossed to the deficiency stock E75D51/TM6B Ubi-GFP. From this cross, 20% of the offspring hatched as first instar larvae that did not express GFP. Similarly, 20% of the offspring from E75D51/TM6B Ubi-GFP adults hatched as first instar larvae that did not express GFP. These numbers are lower than the 26% observed in the ry506 control cross, indicating some embryonic lethality. Embryonic lethality in E75 common region mutants has been shown to be associated with abnormal midgut morphogenesis (Bilder, 1995) as well as head involution defects (Bialecki, 2002).
E75A and E75D51 common region mutant first instar larvae were collected at hatching and examined at regular intervals for lethality during later stages of development. E75D51 common region mutants remain as first instar larvae for over a week before dying without any detectable morphological abnormalities. Some E75A mutants also arrest development as first instar larvae, although lethality is also observed at most other stages in the life cycle including second instar larvae, third instar larvae, early pupae, and pharate adults. Developmental delays, developmental arrests, and molting defects were observed in these animals. Subsets of first and second instar mutant larvae never molt to the next instar, surviving for up to a week before dying. Those that molt do so up to 12 hr late, while some die at the molt. These animals often have malformed mouthhooks or two sets of mouthhooks, indicative of a molting defect. E75A mutant pharate adults display no detectable morphological defects, but fail to eclose (Bialecki, 2002).
Developmental delays, developmental arrests, and molting defects seen in E75A mutants are characteristic of an ecdysteroid deficiency. Approximately 20% of E75A mutant second instar larvae display a heterochronic phenotype in which they live for several days beyond the time when they should have molted to the third instar, and then pupariate (denoted as L2 prepupae). These delayed second instar larvae continue to eat and grow, exceeding the size of wild-type second instar larvae, approaching the size of a wild-type late third instar larva. They begin to pupariate ~88 hr after the first-to-second instar larval molt, forming what will be referred to hereafter as L2 prepupae (Bialecki, 2002).
The L2 prepupae appear to be derived from second instar larvae based on three criteria: (1) L2 prepupal anterior spiracles do not evert and consist of a single club-shaped spiracular opening, identical to that of second instar larvae, in contrast to the everted spiracular papillae characteristic of a wild-type prepupa derived from a third instar larva; (2) mouthhooks dissected from L2 prepupae, although malformed to varying degrees, are more similar to wild-type second instar larval mouthhooks, both in size and tooth structure, than to wild-type third instar larval mouthhooks; (3) no ejected mouthhooks or shed cuticle could be found in the media of E75A mutant L2 prepupae by the time they pupariated. Remarkably, about 20% of the L2 prepupae develop to the pupal stage as evidenced by head eversion and leg and wing extension, indicating that these animals respond in a relatively normal manner to the prepupal ecdysteroid pulse (Bialecki, 2002).
The majority of E75A mutants display defects during the second larval instar. In an initial effort to understand the molecular basis of these defects, total RNA was isolated from staged control and E75A mutant second instar larvae and analyzed by Northern blot hybridization using probes to detect the expression of eight genes: E74, ßFTZ-F1, EcR, usp, dare, dib, Lcp-b, and rp49. Focus was placed on three regulatory genes that are expressed during the second instar: EcR, E74, and ßFTZ-F1. These genes are coordinately induced in wild-type second instar larvae 15–18 hr after the molt. Expression of E74 and ßFTZ-F1 is significantly affected in E75A mutant second instar larvae. E74B mRNA can be detected throughout the time course, accumulating to higher levels at later times, while E74A mRNA accumulation is both significantly reduced and delayed. ßFTZ-F1 mRNA is also significantly reduced in E75A mutant second instar larvae. The effects on EcR mRNA accumulation, however, are relatively minor, with EcR failing to be repressed at later times. These blots were also hybridized to detect usp mRNA, which is unaffected by the E75A mutation. The expression of EcR and usp mRNA in E75A mutant second instar larvae suggests that the defects in this mutant cannot be attributed to a reduced level of ecdysteroid receptor at this stage in development (Bialecki, 2002).
The pattern of E74 transcription seen in E75A mutants is similar to that seen in third instar larval organs treated with a low concentration of 20E. This observation raises the possibility that E75A mutant second instar larvae might be ecdysteroid deficient. This proposal is further supported by the lethal phenotypes of E75A mutants that resemble those seen in ecdysteroid-deficient mutants. Accordingly, the expression was examined of two genes that are known to be directly involved in the ecdysteroidogenic pathway in Drosophila: dare and disembodied (dib). dare encodes the Drosophila ortholog of adrenodoxin reductase, a mammalian enzyme that plays a central role in vertebrate steroid hormone biosynthesis by transferring electrons to all known mitochondrial cytochrome P450s. Genetic studies of dare mutants suggest that this gene plays a similar role in Drosophila ecdysteroid biosynthesis. dib encodes a presumptive target for dare action, a cytochrome P450 that is essential for ecdysteroid biosynthesis in Drosophila. Both dare and dib mRNA, however, are expressed throughout the second larval instar and appear to be unaffected by the E75A mutation. Similar results were obtained with separate collections of animals and with poly(A)+ RNA from E75A mutant second instar larvae (Bialecki, 2002).
Lcp-b expression was also examined in E75A mutant second instar larvae. This gene encodes a larval cuticle protein that is induced during the latter half of the second instar. Lcp-b mRNA is upregulated at 18 hr after the molt, in synchrony with EcR, E74A, and ßFTZ-F1, and accumulates to higher levels throughout the second instar, consistent with earlier results (Charles, 1998). Lcp-b transcription is slightly delayed and reduced in E75A mutant larvae but otherwise expressed normally, indicating that these animals can faithfully express a marker for the second instar stage (Bialecki, 2002).
A subset of E75A mutant second instar larvae fails to molt to the third instar and pupariate, forming L2 prepupae. To determine whether these animals execute genetic programs specific to later stages of development, the patterns of E74, Sgs-4, and Fbp-1 transcription were examined in E75A mutant second instar larvae. These ecdysteroid-inducible genes are normally expressed during the second half of third larval instar and thus provide molecular markers for animals that are progressing toward the onset of metamorphosis (Bialecki, 2002).
Total RNA was collected from control third instar larvae staged from 24 to 44 hr after the second-to-third instar molt, and E75A mutant second instar larvae staged from 48 to 88 hr after the first-to-second instar molt. Control larvae begin to pupariate at ~48 hr after the second-to-third instar molt, while the majority of E75A mutant second instar larvae begin to pupariate after 88 hr. RNA extracted from both sets of animals was analyzed by Northern blot hybridization (Bialecki, 2002).
E74B is expressed throughout the second half of the third larval instar in wild-type animals, and begins to be repressed as E74A mRNA is induced by the high-titer late larval ecdysteroid pulse. A similar pattern is seen in E75A mutant second instar larvae, although E74A induction is significantly delayed and reduced, detectable at 84 hr after the first-to-second instar molt. Both Sgs-4 and Fbp-1 are also induced in E75A mutant second instar larvae, although their normally coordinate induction is disrupted, with Fbp-1 induced 56–64 hr after the molt, and Sgs-4 induced 76–84 hr after the molt in mutant animals. The expression of Sgs-4 and Fbp-1 in E75A mutant second instar larvae indicates that they are capable of inducing genetic programs specific to the third instar stage (Bialecki, 2002).
Approximately 20% of E75A mutant L2 prepupae undergo head eversion, forming pupae with elongated legs and wings. In an effort to determine whether these developmental changes reflect normal transcriptional responses to ecdysteroids, the expression was examined of two key ecdysteroid-regulated genes that respond to dynamic changes in ecdysteroid titer in prepupae: E74 and ßFTZ-F1. E74A is expressed in newly formed prepupae and is repressed as E74B is induced by the rising ecdysteroid titer during prepupal development. E74B is then repressed as E74A is induced by the prepupal ecdysteroid pulse, followed by rapid repression of E74A and reinduction of E74B. In contrast, ßFTZ-F1 expression is restricted to the interval of low-ecdysteroid titer in midprepupae (Bialecki, 2002).
Total RNA was isolated from control prepupae and pupae staged at 2 hr intervals from 0 to 14 hr after puparium formation, as well as from staged E75A mutant L2 prepupae, and analyzed by Northern blot hybridization to detect E74 and ßFTZ-F1 transcription. Remarkably, the L2 prepupae display relatively normal patterns of E74 and ßFTZ-F1 expression, with a delay of ~2 hr in E74A mRNA accumulation. This observation suggests that the E75A mutants that pupariate from the second instar can execute appropriate changes in ecdysteroid titer during the onset of metamorphosis (Bialecki, 2002).
The conclusion that developmental delays, developmental arrests, and molting defects seen in E75A mutants are characteristic of an ecdysteroid deficiency is further supported by the pattern of E74 transcription in E75A mutant second instar larvae. In order to test this possibility, attempts were made to rescue the second-to-third instar larval molt by feeding ecdysteroids to E75A mutant second instar larvae. Second instar larvae staged 12–18 hr after the molt were transferred to yeast paste supplemented with either no hormone, 0.33 mg/ml 20E, 10 mg/ml 20E, or 0.66 mg/ml ecdysone. The animals were transferred to regular yeast paste after 6 hr in order to simulate the hormone pulse that triggers the molt, and then scored for animals that molted to the third instar after 18 hr. Almost all E75A mutant second instar larvae that were maintained on food without ecdysteroids failed to molt, either staying as second instar larvae or forming L2 prepupae. In contrast, the majority of larvae fed either ecdysone or 20E molted properly. Interestingly, about half of the mutant animals that molted to the third instar continued to develop to pupal and pharate adult stages, consistent with a critical role for E75A during the second larval instar (Bialecki, 2002).
These studies of E75A mutant second instar larvae suggest that they could have a reduced ecdysteroid titer at this stage of development. As a direct test of this hypothesis, the ecdysteroid titer was measured in these animals using an enzyme immunoassay (EIA). Control and E75A mutant second instar larvae were collected at 0–6, 6–12, 12–18, and 18–24 hr after the first-to-second instar larval molt. Organic extracts were prepared from these animals and the ecdysteroid titer was measured by EIA using a monoclonal antibody directed against 20E. Wild-type larvae show a peak of 20E at 6–12 hr after the molt, with the titer decreasing toward the end of the instar. In contrast, this peak is eliminated in E75A mutants, which also show a reduced basal level of 20E at all stages. To confirm these results, the EIA was repeated using a second set of control and E75A mutant larvae, collected at 0–12, 12–24, and 24–36 hr after the first-to-second instar larval molt. Similar results were obtained from these animals indicating that the ecdysteroid peak in E75A mutants is not delayed until 24–36 hr after the molt (Bialecki, 2002).
The Drosophila midgut is an excellent system for studying the cell migration, cell-cell communication, and morphogenetic events that occur in organ formation. Genes representative of regulatory gene families common to all animals, including homeotic, TGF beta, and Wnt genes, play roles in midgut development. To find additional regulators of midgut morphogenesis, a set of genomic deficiencies was screened for midgut phenotypes. Fifteen genomic intervals necessary for proper midgut morphogenesis were identified, three contain genes already known to act in the midgut. Three other genomic regions are required for formation of the endoderm or visceral mesoderm components of the midgut. Nine regions are required for proper formation of the midgut constrictions. The E75 ecdysone-induced gene, which encodes a nuclear receptor superfamily member, is the relevant gene in one region and is essential fo proper formation of midgut constrictions. E75 acts downstream of the previously known constriction regulators or in parallel. Temporal hormonal control may therefore work in conjunction with spatial regulation by the homeotic genes in midgut development. Another genomic region is required to activate transcription of the homeotic genes Antp and Scr specifically in visceral mesoderm. The genomic regions identified by this screen provide a map to novel midgut development regulators (Bilder, 1995).
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date revised: 5 April 2006
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