Hormone receptor-like in 46
See the embryonic expression pattern of Hr46 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Hr46 mRNA is detected during midembryogenesis (9-18) hours (Koelle, 1992). Hr46 is induced following germ band retraction [Images] and can be detected until cuticle deposition late in embryogenesis. Hr46 is widely expressed during mid-embryogenesis in tissues that include the gut, salivary gland, ventral nerve cord and epidermis. The embryonic central nervous system contains little, if any DHR protein (Lam, 1997).
Hr46 mRNA is detected during the first two larval stages or instars and at the end of the third larval instar (Koelle, 1992).
The temporal developmental profile for Hr46 expression closely parallels that for the ecdysone titer and for the ecdysone-inducible E75 and E74 Drosophila early genes. The structural similarity to a Manduca early gene and the expression similarities to Drosophila early genes suggest that the Hr46 gene may also belong to the early gene class (Koelle, 1992).
Hr46 is expressed in the larval and imaginal cells of the salivary gland, as well as the fat bodies, Malpighian tubules and imaginal discs of 0-2-hour prepupae. Although expression is clearly evident in the peripodial membranes that surround imaginal discs, Hr46 protein is also present in the disc epithelial monolayer. In addition, Hr46 is found in the midgut, garland cells, trachea, ring gland, hemocytes, lymph gland and pericardial cells of early prepupae. Only low levels of Hr46 protein are present in the 2-hour prepupal central nervous system and no protein can be detected in the proventriculus (Lam, 1997).
Many nuclear receptor genes are localised in the polytene chromosome puffs of
the salivary gland previously classified as intermoult, early or early-late puff loci. Early-late puffs include Hr46/DHR3, DHR-39 and E78B. These puffs appear after those of Ecdysone receptor, E74A and E75A, but prior to betaFTZ-F1 and the late puffs. Transcripts from certain early-late puffs are induced in parallel with the early transcripts and are thus hierarchically equivalent. Hr46 is not induced in the absence of ecdysone. The induction of Hr46 does not require protein synthesis and is a primary response to hormone. Particularly striking are the mirror image of Hr46 and Ecdysone receptor in vivo. Hr46 does not become activated while EcR levels are high. This suggests that the best candidate for EcR repression is a gene whose regulation is similar to that of Hr46, and this cross-talk may continue later in the prepupal period (Huet, 1995).
Pulses of the steroid hormone 20-hydroxyecdysone (20E) trigger the larval-to-adult metamorphosis of Drosophila by reprogramming gene expression throughout the
organism. 20E directly induces a small set of early regulatory genes that repress their own expression and induce a large set of late secondary-response genes. Two members of the Drosophila nuclear hormone receptor superfamily, Hr46/DHR3 and DHR39, are rapidly induced by 20E, in parallel with the early regulatory
genes. Both genes also require protein synthesis at high 20E concentrations for their maximal induction by the hormone. DHR39 is induced in mid third instar larvae and expressed throughout most of third instar larval and prepupal development, while Hr46 is briefly expressed in late third instar larvae and early prepupae. The 20E-induction and temporal patterns of Hr46 and DHR39 transcription strongly suggest that these genes function together with the
early regulatory genes to coordinate the complex gene networks that direct the early stages of Drosophila metamorphosis (Horner, 1995).
Drosophila metamorphosis is characterized by diverse
developmental phenomena, including cellular proliferation, tissue
remodeling, cell migration, and programmed cell death. Cells undergo
one or more of these processes in response to the hormone
20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end
of the third larval instar and before puparium formation (PF) via a
transcriptional hierarchy.
Additional pulses of ecdysone further coordinate these processes during
the prepupal and pupal phases of metamorphosis. Larval tissues such as
the gut, salivary glands, and larval-specific muscles undergo
programmed cell death and subsequent histolysis. The imaginal discs
undergo physical restructuring and differentiation to form rudimentary
adult appendages such as wings, legs, eyes, and antennae. Ecdysone also
triggers neuronal remodeling in the central nervous system (White, 1999).
Wild-type patterns of gene expression in
D. melanogaster during early metamorphosis were examined by assaying whole
animals at stages that span two pulses of ecdysone. Microarrays were constructed containing
6240 elements that included more than 4500 unique cDNA expressed
sequence tag (EST) clones along with a number of
ecdysone-regulated control genes having predictable expression
patterns. These ESTs represent
approximately 30% to 40% of the total estimated number of genes in the
Drosophila genome. In order to gauge
expression levels, microarrays were hybridized with fluorescent probes
derived from polyA+ RNA isolated from developmentally
staged animals. The time points examined are relative to PF, which
last approximately 15 to 30 min, during which time the larvae cease to move
and evert their anterior spiracles. Nineteen arrays were examined representing
six time points relative to PF: one time point before the late larval
ecdysone pulse; one time
point just after the initiation of this pulse (4 hours BPF), and time
points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of
ecdysone occurs 9 to 12 hours APF (White, 1999).
In order to manage, analyze, and disseminate the large amount of data,
a searchable database was constructed that includes
the average expression differential at each time point. The analysis
set consists of all elements that reproducibly fluctuate in
expression threefold or more at any time point relative to PF, leaving
534 elements containing sequences represented by 465 ESTs and control
genes. More than 10% of the genes represented by the
ESTs display threefold or more differential expression during early
metamorphosis. This may be a conservative estimate of the percentage of
Drosophila genes that change in expression level during
early metamorphosis, because of the stringent criteria used for their
selection (White, 1999).
To interpret these data, genes were grouped according to similarity of
expression patterns by two methods. The first relied on pairwise
correlation statistics, and the second relied on the use
of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first
category contains genes that are expressed at >18 hours BFP (before
the late larval ecdysone pulse) but then fall to low or undetectable
levels during this pulse. These genes are potentially repressed by ecdysone and
make up 44% of the 465 ESTs identified in this set. The second
category consists of genes expressed at low or undetectable levels
before the late larval ecdysone pulse but then are induced during this pulse. These genes are
potentially induced by ecdysone and make up 31% of the 465 ESTs.
Consequently, 75% of genes that changed in expression by threefold or
more do so during the late larval ecdysone pulse that marks the
initial transition from larva to prepupa. This result is consistent
with the extreme morphological changes that are about to occur in these
animals. There are clearly discrete subdivisions of gene expression within these
categories (White, 1999).
Description of wild-type development is a first step in understanding
metamorphosis from a global perspective. However, it is of interest to
understand the composition of the genetic hierarchy that leads to
metamorphosis. To test whether new targets of
transcription factors could be identified in the ecdysone genetic hierarchy, the ecdysone-induced nuclear receptor DHR3 was
prematurely expressed at >18 hours BPF. DHR3 is responsible for the coordination of part of the
transcriptional program controlling metamorphosis and can act as either
a repressor or an activator of transcription, depending on the target
gene. DHR3 can induce betaFTZ-F1, a nuclear receptor
that is active during midprepupal development and is responsible for
the difference in the genetic response to ecdysone between the late
larval and prepupal ecdysone pulses.
betaFTZ-F1 induction is confirmed by
the microarray results. Several
other genes are induced by DHR3 when it is expressed at >18 hours
BPF. One of these is represented by a novel EST (LD24139) that is
induced from 3 to 9 hours APF during wild-type development. ESTs representing 12 other DHR3-induced genes that have less than
threefold induction at 3 to 9 hours APF are listed at DHR3-regulated genes.
Some of these additional genes may not normally be DHR3 targets or may
be induced by DHR3 at other stages during development (White, 1999).
DHR3 has been shown to inhibit the induction of
ecdysone-inducible genes, and with E75B it can act as a repressor of
the betaFTZ-F1 gene. DHR3 is expressed
before the ecdysone-inducible genes are up-regulated but is still
capable of repressing genes. Four out of seven such genes
belong to the cytochrome P450 (CYP) class of genes. Three of these CYP genes are normally
repressed during the late larval pulse, and this repression begins
before DHR3 induction occurs (approximately 4 hours BPF). Thus, DHR3
cannot be solely responsible for their repression, although it may
contribute to it. One function of cytochrome P450 molecules is
hydroxylation of steroids; the depletion of
transcripts of the CYP genes may provide a mechanism by
which production of the biologically active form of ecdysone
(20-hydroxyecdysone) is stifled at PF. Regulation of these
CYP genes within the ecdysone hierarchy further suggests
that they may have a role in controlling the ecdysone genetic cascade (White, 1999).
The simple cellular composition and array of distally pointing hairs has made
the Drosophila wing a favored system for studying planar polarity and the
coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).
The HR46 gene (also known as DHR3) encodes a nuclear receptor and is an essential gene known to be important for the ecdysone cascade. Large clones of loss of function alleles result in wing (folded and curved) and notum defects (rough short bristles and pale pigmentation). The expression of this gene increased 250 fold from 24 to 32 hr and then decreased 4.3 fold from 32 to 40 hr. Moderate sized wing clones of cells lacking HR46 were examined, but no clear cut phenotype was seen. In pupal wing clones examined a couple of hours after hair formation mutant hairs appeared somewhat thicker but this alteration was transient (Ren, 2005).
The Eip78CD gene encodes a related nuclear receptor. The expression of this non-essential gene increased 3 fold from 24 to 32 hr followed by a three fold drop from 32 to 40 hr (but the differences were not significant) suggesting it might be functionally redundant with HR46. To test this hypothesis Eip78CD mutants, which also contained HR46 mutant clones, were examined. No mutant phenotypes were seen in the clones, suggesting either that there is an alternative redundant gene or that HR46 is not essential for hair morphogenesis. Since the level of HR46 expression fell dramatically between 32 and 40 hrs it seemed possible that declining HR46 expression could be important for hair development. To test this the overexpression of HR46 from a transgene containing a hs promoter was induced. This resulted in a dramatic loss of hair formation leading to wings with extensive bald regions. The strongest phenotype was seen when the transgene was induced by heat shocking 6-8 hrs prior to the time of hair initiation. The phenotype was dose sensitive and directly related to the number of transgenes and length and temperature of transgene induction (Ren, 2005).
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).
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Hormone receptor-like in 46:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
date revised: 25 June 2007
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