Ecdysone receptor

DEVELOPMENTAL BIOLOGY (part 1/2)

Embryonic

Except for the first three hours, EcR is present throughout embryonic development in a widely distributed pattern (Koelle, 1991).

Larval and Pupal

In the third instar larva, the EcR nuclear antigen is found in imaginal discs, fat body, trachea and salivary glands, as well as the central nervous system, gut, ring gland and cells associated with cuticular structures (Koelle, 1991).

Imaginal tissues exhibit two different patterns of EcR expression at pupariation: EcR-A is the major isoform in the imaginal discs and imaginal rings, while the abdominal histoblast nests and midgut imaginal islands express only EcR-B1. This difference is correleted with the observation that the former were entering their differentiative phase whereas the latter were preparing to begin extensive proliferation. Earlier in larval life, when imaginal disc cells are rapidly proliferating, they too express mainly EcR-B1 (Talbot, 1993 and Truman, 1994).

Most larval neurons show high levels of EcR-B1 at the start of metamorphosis, a time when they lose larval features in response to ecdysteroids. Earlier, during larval molts, the same cells have no detectable receptors and show no response to ecdysteroids. The stem cells for imaginal neurons show prominent EcR-B1 expression during the last larval stage, peaking at pupariation. This expression is correlated with the main proliferative periods for neurons. The level subsequently declines, but reappears toward the end of metamorphosis and persists into the adult stage (Truman, 1994).

Four mushroom body neuroblasts in each brain hemisphere are unique in that they generate neurons throughout metamorphosis until about 10 hours before adult emergence. Throughout this period, the younger neurons can be readily identified because they form a compact column that extends centripetally from each neuroblast down towards the mushroom body neuropil. EcR-B1 is first detected in these cells about 12 hours prior to pupariation, and the level peaks at pupariation, but declines thereafter (Truman, 1994).

Mushroom bodies (MBs) are symmetrically paired neuropils in the insect brain that are of critical importance for associative olfactory learning and memory. In Drosophila melanogaster, the MB intrinsic neurons (Kenyon cells) undergo extensive reorganization at the onset of metamorphosis. A phase of rapid axonal degeneration without cell death is followed by axonal regeneration. This re-elaboration occurs as levels of the steroid hormone 20-hydroxyecdysone (20E) are rising during the pupal stage. Based on the known role of 20E in directing many features of CNS remodeling during insect metamorphosis, it was hypothesized that the outgrowth of MB axonal processes is promoted by 20E. Using a GAL4 enhancer trap line (201Y) that drives MB-restricted reporter gene expression, Kenyon cells were identified in primary cultures dissociated from early pupal CNS. Paired cultures derived from single brains isolated before the 20E pupal peak were incubated in medium with or without 20E for 2-4 days. Morphometric analysis demonstrates that MB neurons exposed to 20E have significantly greater total neurite length and branch number when compared with neurite length and branch number for MB neurons grown without hormone. The relationship between branch number and total neurite length remains constant regardless of hormone treatment in vitro, suggesting that 20E enhances the rate of outgrowth from pupal MB neurons in a proportionate manner and does not selectively increase neuritic branching. These results implicate 20E in enhancing axonal outgrowth of Kenyon cells to support MB remodeling during metamorphosis (Kraft, 1998).

For MB neurons in culture to exhibit a cell-autonomous response to 20E, they must express ecdysone receptors. The concentrations of the various Drosophila Ecdysone receptor (EcR) isoforms within the MB neurons during metamorphosis fluctuate in a characteristic pattern. The EcR-B1 isoform is maximally expressed in the Kenyon cells at pupariation and then decreases but remains detectable for 40 hr after puparium formation. Examination of EcR-B1 expression in brain whole mounts from lacZ reported animals 5 hr after pupation reveals that MB neuronal cell bodies marked by betagal expression are positive for EcR-B1 expression. To determine whether EcR-B1 is also expressed in MB neurons isolated 5 hr after pupation and dissociated into culture, cells were plated for 2 hr from brain and optic lobes of lacZ reporter animals, and then the cells were fixed and stained to visualize betagal and EcR-B1. MB neurons identified by betagal expression are invariably positive for EcR-B1, whereas EcR-B1 is also expressed in many, but not all, betagal-negative neurons in the culture. This is consistent with whole-mount data demonstrating that EcR-B1 is expressed in Kenyon cells as well as in some other neurons of the CNS at this time during metamorphosis (Kraft, 1998).

Approximately 350 neurons in the brain and ventral CNS show another pattern characterized by expression of EcR-A at a high level during the pupal phase. These high levels are maintained throughout the remainder of metamorphosis. All these cells share the same fate; they undergo rapid degeneration after the adult emerges from the pupal case. One prerequisite for this death is the decline of ecdysteroids at the end of metamorphosis. Thus expression of the EcR-A isoform appears to set the stage for programmed cell death of pupal specific neurons. Animals treated with ecdysteroids at eclosion show a delay in cell death (Robinow, 1993).

During CNS metamorphosis, the ecdysteroids evoke diverse neuronal responses; these include remodeling, maturation, and programmed death. These responses are matched by the equally diverse expression of the two main ecdysone receptor isoforms, EcR-A and EcR-B1. Based on these patterns, it appears that each isoform is associated with a particular program of steroid response: EcR-A with maturation and synaptogenesis and EcR-B1 with synapse loss and process regression. No EcR is detected in the CNS at the start of larval life, except for a pair of brain neurons of unknown identity. EcR begins to appear by the second larval instar, but its appearance is confined to neuroblasts scattered throughout the medial brain and ventral CNS and in the optic lobe proliferation zones. These stem cells express only the B1 isoform that increases in amount through the second and most of the third larval instar but then declines and disappears as the larva leaves the food in preparation for metamorphosis. One of the suppressive actions of ecdysones late in metamorphosis is the inhibition of the death of the larval neurons that are fated to die after adult emergence. Late in metamorphosis, ecdysteroids suppress this degenerational response. Consequently, these steroids need to be withdrawn to allow neuronal death to occur. In the fly, neurons show elevated levels of EcR through metamorphosis then degenerate after the emergence of the adult. In the fly these elevated receptor leves are due exclusively to upregulation of the A isoform. Although all neurons that express high levels of EcR-A subsequently degenerate, the overexpression of EcR-A is not a generic signal for neuronal death. During the first period of neuronal death that occurs soon after the formation of the pupal stage, neurons die without overexpressing EcR-A. Even at the time of adult emergence, there are a few neurons that die that do not also show high EcR-A levels. Presumably, the differences between the type 2 neurons and these other doomed cells relate to the role of ecdysteroids in controlling their death (Truman, 1996).

The development of Drosophila midline glia during larval and pupal stages was characterized by localizing beta-gal expression in enhancer trap lines, as well as with BrdU incorporation and pulse-chase experiments. At hatching about 40 to 50 glial cells are present along the midline of the ventral nerve cord (2 to 3 dorsal and 1 to 2 ventral cells per neuromere). The cells proliferate during the third larval instar and spread dorsoventrally within the midline, increasing in number to about 230 or more (around 20 cells per neuromere). Cell divisions cease shortly after pupariation, and the cells persist for the first half of pupal life with no apparent changes in numbers or positions. Between 50 and 80% of the way through metamorphosis, however, virtually all of the midline glia undergo programmed cell death. Tissue culture experiments indicate that a peak of ecdysteroids, which occurs at pupariation, is required for the cessation of proliferation of midline glia and their subsequent degeneration. Midline glia in the central nervous system (CNS) cultured with low or no ecdysteroids, survive and continue to divide; with high ecdysteroids levels, the glia cease proliferating and later degenerate. The midline glial cells may play a role during CNS metamorphosis similar to that of their progenitors in the embryo: stabilizing outgrowing neurites that cross or run along the midline. Ecdysteroid is likely to act through an intracellular receptor to alter the gene expression of components of the regulatory machinery of the cell cycle, leading to mitotic arrest. The midline glia express the B1 isoform of the Ecdysone receptor during their proliferative phase (Awad, 1997).

In Drosophila, secretion of the steroid hormone ecdysone from the prothoracic ring gland coordinates and triggers events such as molting and metamorphosis. In the developing Drosophila compound eye, pattern formation and cell-type specification initiate at a moving boundary known as the morphogenetic furrow. The role of ecdysone has been investigated in eye development and the ecdysone signaling pathway has been found to be required for progression of the morphogenetic furrow in the eye imaginal disc of Drosophila. A temperature-sensitive mutation in ecdysoneless (ecd1) reduces ecdysone titer in vivo up to 20-fold, but does not eliminate it completely. Genetic mosaic analysis has shown that ecdysoneless is required in the ring gland (and ovary) and the mutation is thus likely to affect either ecdysone synthesis or release. Genetic disruption, either of the ecdysone signal in vivo with the ecd1 mutant, or of ecdysone response with a Broad-Complex mutant, results in disruption of morphogenetic furrow progression (Brennan, 1998).

The ecd1 mutation is a hypomorphic temperature-sensitive allele thought to affect ecdysone synthesis and/or release in the ring gland. Homozygous ecd1 flies show some eye defects when shifted to 30°C for 24 hours during their final larval instar. However, consistent with the partial loss of function associated with ecd1, these phenotypes are more pronounced when this mutation is placed in trans to a deletion for the region (Df[3L]R-G7), and all data described here are from such heterozygotes. When larvae are returned to the permissive temperature (18°C) after a 24 hour exposure to 30°C during the third instar, and allowed to mature, their adult compound eyes are disrupted, with anterior nicks in the retinal tissue. Larvae that are exposed to 30°C for 24 hours during the second instar have more severe defects in adult eye morphology, showing significant intrusions of cuticle tissue into the eye field. In a study of 3rd instar effects of ecdysone signaling, discs were dissected from late 3rd instar ecd1/(Df[3L]R-G7) larvae immediately following a 24 hour exposure to 30°C (referred to as ecd-ts discs). Two neural antigens, Elav and 22C10, were visualized in wild-type. The ecd-ts discs and the mutant phenotype observed are consistent with the furrow moving much more slowly than normal, or with total arrest: relatively mature clusters at the furrow and an absence of earlier stages. This is similar to the phenotype seen with hh loss of function and other genotypes that arrest the furrow. Clusters behind the furrow continue to mature, but new clusters are not yet recruited, with the result that mature clusters with as many as five cells, and with well-grown axons, are seen right at the furrow. A wild-type furrow progresses at about the rate of one column every 1.5 hours at 25°C and somewhat faster at 30°C. Other disruptions include clusters with single cells posterior to larger clusters and altered cluster morphology. In addition, the discs are occasionally smaller than in wild-type discs (up to 25% smaller) (Brennan, 1998).

To examine early events in the furrow, a study was carried out of the expression pattern of atonal (ato), the proneural gene for the R8 photoreceptor cell, the founder of the ommatidium. Like the products of other proneural genes, Ato is first expressed broadly in an equivalence group of cells and subsequently narrows to single cells before disappearing. Due to the reiterative nature of retinal development, both phases of expression can be seen at once, with a wide zone of staining in the furrow and single cell staining in R8 founder cells posterior to the furrow. In all ecd-ts discs, the wide band of Ato expression in the furrow disappears. In some of the discs, the later R8-specific expression has also gone. It is suggested that once ecdysone signaling is disrupted, no new cells begin to express ato, but that cells already expressing this gene complete the normal expression sequence. These results confirm a failure of the recruitment of cells into the photoreceptor differentiation pathway, further supporting the interpretation of this phenotype as a failure of furrow progression (Brennan, 1998).

While the prefurrow general proliferation is not visibly altered, cell-cycle synchrony in the furrow is lost in ecd-ts discs. Observed is a general loss of BrdU incorporation in ecd-ts eye discs, in particular in the zone just posterior to the furrow. In wild-type, CyclinB is expressed generally anterior to the furrow and in a tight band just posterior to it. In ecd-ts, the anterior CycB expression remains, but the tight, postfurrow band of CycB is lost. Thus there is a loss of cell cycle regulation in the furrow of ecd-ts discs. This supports the hypothesis that the eye disc phenotypes seen after loss of ecdysone are not due to general failure of disc cell proliferation, but rather to specific effects on the furrow. By staining early 3rd instar ecd-ts eye discs with 22C10, it has been found that even the earliest phases of furrow progression are sensitive to ecdysone. Overly mature photoreceptor clusters with well-extended axonal projections can be found at the anterior edge of differentiation, just as in older discs (Brennan, 1998).

Hedgehog expression is also affected posterior to the ecd-ts furrow. Progression of the furrow beyond the first ten columns is driven by Hedgehog, expressed in the differentiating clusters and diffusing forward to induce anterior cells to enter the furrow. Two eye-specific hedgehog alleles (hh 1 and hhfse) cause the furrow to arrest about a third of the way across the eye field; when Hedgehog is removed with a temperature-sensitive allele, only the first several rows of photoreceptor clusters are formed, yielding a 'Bar' shaped eye. In all larvae where Ato expression is impaired in the typical ecd-ts manner, Hh protein in the contralateral disc is greatly reduced, far below normal levels. Hh protein appears to be lost uniformly across the disc, contrasting with Ato whose domain of expression is progressively narrowed from the anterior (Brennan, 1998).

A transgenic construct was used to reveal the domain of gene expression that is directly regulated by Ecdysone receptor in the developing eye. This construct contains a heptamer of Ecdysone Receptor-binding sites (EcREs or ecdysone response elements) from the hsp27 promoter that drives expression of beta-galactosidase and provides the most direct assay currently available of the EcR in situ in the living disc. In mid third instar discs, beta-gal is restricted to a zone in and anterior to the furrow. This domain then travels with the furrow until the end of the third larval instar, when it becomes ubiquitous (as hormone titer rises at the end of larval life). In younger discs, no beta-galactosidase expression until the furrow has produced 10 to 12 columns of ommatidia. The EcRE:lacZ may not represent the complete domain of transcriptional activation by the Ecdysone Receptor in the eye disc. Nevertheless, these results do show clear spatially restricted ecdysone-responsive transcriptional activation in the eye disc (Brennan, 1998).

To investigate how the ecdysone signal might be transduced in the eye disc, a study was carried out of the expression and role of Broad complex, an early ecdysone response gene complex known to play an important role in metamorphic responses to ecdysone. With an antibody specific to the Z1 finger-containing forms of this protein (the isoform expressed more strongly in differentiating imaginal tissues), this protein is found localized near the furrow. Z1-containing isoforms of this protein begin to be expressed just anterior to the furrow and reach maximal levels posterior to the furrow, following in time the activation of EcRE:lacZ, as visualized by beta-galactosidase staining just anterior to the furrow. This suggests that although BR-C is immediately downstream of the Ecdysone Receptor, there is a delay in its maximal expression as compared with the expression of EcRE:lacZ, which may reflect autoregulation or post-transcriptional control of expression. Both cross-sections and whole mount stainings show that, in addition to its furrow domain of expression, Br-C Z1 is also expressed ubiquitously in the peripodial membrane. Br-C Z1 expression both near the furrow and in the peripodial membrane is greatly reduced when ecdysone titer is reduced in ecd-ts flies. Males hemizygous for npr-1 (an allele null for all Broad-Complex functions), exhibit failures of furrow progression and photoreceptor recruitment. The correlation between effects of removal of ecdysone on furrow progression and Br-C expression and the effects of direct removal of Br-C function suggest that Br-C may mediate a subset of the ecdysone effect in the eye, but doesn't preclude the possibility that Br-C may also have independent functions, as does Usp (Brennan, 1998).

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo, transgenic animals were established that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of ß-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. The patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, no GAL4-LBD activation is observed in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. It is concluded that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development (Kozlova, 2002).

Spatially restricted and largely distinct patterns of GAL4-EcR and GAL4-USP activation were observed in the CNS at the onset of metamorphosis. Understanding the significance of these patterns will require more detailed studies that extend beyond the limits of this initial report. Nonetheless, there are several aspects of these activation patterns that are consistent with current understanding of the roles of EcR and USP in CNS development. (1) The cells where GAL4-EcR is most active at this stage correlate with the location of the optic proliferation zones, consistent with the known role for ecdysteroids in neuronal proliferation during metamorphosis. (2) It is also interesting to note that the pattern of GAL4-USP activation in the CNS reflects a subset of the EcR-B1 expression pattern at the onset of metamorphosis. EcR-B1 is most abundantly expressed in the mushroom body neurons and surrounding cells of the optic lobes as well as the abdominal neuromeres of the ventral nerve cord. GAL4-USP activation is strongest in a cluster of cells at the anterior end of the optic lobes that could correspond to the mushroom body neurons, and is clearly elevated in the abdominal neuromeres. (3) In addition, GAL4-USP activation in the CNS is significantly reduced in an EcR mutant background, supporting the conclusion that it is acting as a heterodimer with endogenous EcR. Interestingly, low levels of GAL4-EcR activation can also be seen in the cluster of anterior neurons in the optic lobes that show high levels of GAL4-USP activation. Unambiguous identification of these cells, however, will require more detailed studies of the patterns of GAL4-EcR and GAL4-USP activation in the CNS as well as the use of cell-type specific markers (Kozlova, 2002).

The restricted activation of GAL4-EcR cannot be attributed to the distribution of endogenous USP in the CNS, which is widely expressed in this tissue at the onset of metamorphosis. Similarly, many neurons that express EcR-B1 in the optic lobes do not show high levels of GAL4-USP activation. One possible explanation for these limited patterns of activation is that EcR might function independently of USP in certain cells of the CNS. Alternatively, any of several possible mechanisms for the reduced levels of transactivation seen in late larval imaginal discs could account for these complex cell-type specific patterns of GAL4-LBD activation in the CNS (Kozlova, 2002).

Studies of Drosophila metamorphosis have been hampered by an inability to visualize many of the remarkable changes that occur within the puparium. To circumvent this problem, GFP was expressed in specific tissues of living prepupae and pupae and images of these animals were compiled into time-lapse movies. These studies reveal the dynamics and coordination of morphogenetic movements. Responses that have not been described previously include an unexpected variation in some wild-type animals, where one of the first pairs of legs elongates in the wrong position relative to the second pair of legs and then relocates to its appropriate location. At later stages, the antennal imaginal discs migrate from a lateral position in the head to their final location at the anterior end, as leg and mouth structures are refined and the wings begin to fold. The larval salivary glands translocate toward the dorsal aspect of the animal and undergo massive cell death following head eversion, in synchrony with death of the abdominal muscles. These death responses fail to occur in rbp⁵ mutants of the Broad-Complex, and imaginal disc elongation and eversion is abolished in br⁵ mutants of the BR-C. Leg malformations associated with the crol³ mutation can be seen to arise from defects in imaginal disc morphogenesis during prepupal stages. This approach provides a new tool for characterizing the dynamic morphological changes that occur during metamorphosis in both wild-type and mutant animals (Ward, 2003).

Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development

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).

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis

Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppresses not only axon pruning but also the infiltration and engulfing action of the surrounding glia. These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. It is therefore proposed that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis (Awasaki, 2004).

The dorsal and medial axon branches of the larval γ neurons have many varicosities along its trajectory that are clearly visualized when a single γ neuron is labeled with cytoplasmic-GFP (cGFP) reporter combined with the mosaic analysis with a repressible cell marker (MARCM) system and the enhancer-trap strain GAL4-201Y. These axon branches are also labeled strongly with the anti-Synaptotagmin antibody, which detects synaptic vesicle protein accumulated in the presynaptic sites. Thus, the larval γ neurons seem to be extensively interconnected with other neurons through synapses. Because synaptic terminals are condensed in the varicosities, the fate of these varicosity structures were first examined during the time course of axon pruning (Awasaki, 2004).

The number of varicosities on a dorsal axon branch was counted at various developmental stages. A single branch contained 20.7 and 21.4 varicosities on average in the brains of late third-instar larvae (L3) and in pupae just after puparium formation (0 hr APF), respectively. The number of varicosities was reduced by approximately 35% at 6 hr APF (an average of 13.6 varicosities per axon branch). To compare the timing of local axon degeneration and reduction of varicosity numbers, the frequency of disconnected axons was simultaneously examined. Only a very few axon branches were disconnected at 6 hr APF (2 of 60 observed axons) (Awasaki, 2004).

At 12 hr APF, approximately 90% of varicosities disappeared (an average of 2.0 varicosities per branch) and 75% of axons were disconnected (30 of 40). At 18 hr APF, more than 95% of varicosities disappeared (an average of 0.9 varicosities per branch) and 90% of axons were disconnected (48 of 53). In many cases, distal portions of disconnected axons remained at 12 hr APF, and only distal tips of axons frequently remained at 18 hr APF, suggesting that the shaft region of the axon branch tends to degenerate first. At 12 hr and 18 hr APF, the number of varicosities was drastically reduced even on the axons that were not yet disconnected (an average 2.8 [n = 10] and 1.4 [n = 5] varicosities per branch, respectively). These data suggest that disappearance of varicosities precedes local axon degeneration (Awasaki, 2004).

Pruning of axons is triggered by ecdysone through the complex of ecdysone receptor type B1 (EcR-B1) and Ultraspiracle (USP). If the disappearance of varicosities is associated with programmed axon pruning, the disappearance should be suppressed by inhibition of ecdysone signaling. To address this, axon branches of the clones of usp mutant γ neurons were examined. Both disappearance of varicosities and axon disconnection were significantly suppressed. Thus, these phenomena were regulated cell autonomously by the ecdysone-induced stimulation of γ neurons (Awasaki, 2004).

There are more than 2000 neurons that form the larval MB, and these results have indicated that disappearance of the varicosities proceeds gradually during the first 18 hr of the pupal stage. How do the varicosities disappear in these masses of axon branches? Do they disappear randomly, one by one, or in a coordinated process? To examine this, the detailed structure of the dorsal MB lobe was observed by using the enhancer-trap strain GAL4-201Y and the anti-Fasciclin II (FasII) antibody, both of which label axons of γ neurons from the larva to adult stage (Awasaki, 2004).

In the dorsal lobe of late larvae (L3), a mass of γ neuron axons was labeled uniformly with cGFP driven by GAL4-201Y and with anti-FasII antibody. At 6 hr APF, however, many hole-like structures of unlabeled regions appeared in cross-sections. Considering that the number of varicosities was significantly reduced at this stage whereas most axons had not been disconnected, it is highly likely that the hole-like structures correspond to the vestiges of disappeared varicosities (Awasaki, 2004).

To confirm this, lobes labeled with the targeted expression of the presynapse marker n-Synaptobrevin::GFP (nSyb::GFP) and the membrane bound reporter mCD8::GFP (mGFP) were compared. Like cGFP, nSyb::GFP strongly labels varicosities along the axon branches. Images of the sections at 6 hr APF clearly reveal the hole-like structures. In contrast, mGFP labels voluminous varicosities much less intensively than cGFP, because these subcellular regions contain less membrane surface per volume. mGFP labels the hole-like structures much less clearly than does cGFP. This further supports the hypothesis that the unlabeled regions are associated with the disappearance of varicosities (Awasaki, 2004).

Each hole-like structure was considerably larger than the size of a single varicosity. The diameter of many holes was more than 5 μm, whereas the size of the largest varicosity was 1.8 μm. This indicates that each unlabeled hole corresponds to the vestige of multiple varicosities. Because the varicosities of a single neuron were scattered along its axon branch, it is unlikely that a single hole corresponds to the varicosities that belong to a single neuron. Rather, neighboring varicosities from different neurons must simultaneously disappear in a cluster (Awasaki, 2004).

The larval neuropil of the central nervous system of Drosophila is covered by sheaths of neuropil-associating glial cells. Three-dimensional reconstruction of the confocal serial sections revealed a smooth surface of the MB dorsal lobe in the larval brain. At 6 hr APF, however, the surface appeared eroded from the outside. This suggests that glial cells surrounding the MB lobe might erode this neuropil structure. The morphologic changes of glial cells were therefore traced by using the pan-glial driver GAL4-repo and the cGFP reporter (repo>cGFP). In larvae, glial cells surrounded the lobe, but the axon branches inside the lobe were devoid of glial processes. At 6 hr APF, glial processes had infiltrated into the lobes and formed many lumps. The positions of these lumps correspond to the hole-like structures that were unlabeled with anti-FasII antibody (Awasaki, 2004).

Close observation of the glial processes revealed various patterns of infiltration at 6 hr APF. In some sections, thin glial processes surround parts of axon branches labeled with anti-FasII antibody. Glial processes also spread into the surrounded mass, forming glial lumps. Vesicle-like structures in these glial lumps contained small dots that were FasII-positive. In the specimens with massive glial infiltration, glial processes formed large lumps that occupied whole regions unlabeled with anti-FasII antibody (Awasaki, 2004).

Considering that dorsal lobes are initially filled with FasII-positive axon mass and hole-like structures unlabeled with anti-FasII antibody are eventually filled with glial lumps labeled with repo>cGFP, images like the ones described are best interpreted as being intermediate steps. Hypothetically it would seem that at first, infiltrating glial processes surround clusters of varicosities, which are still labeled with anti-FasII antibody. Next, glial processes further penetrate between varicosities to form lumps. FasII-positive signals observed in the glial lumps might be parts of varicosities engulfed by glia. Intensity of the FasII signal within glia is remarkably reduced compared to the signals observed outside of glia. This might be because engulfed FasII protein is subject to intracellular degradation. Finally, the FasII signal totally disappears, and glial lumps replace the vestiges of varicosities (Awasaki, 2004).

If this hypothesis has validity, infiltrated parts of glia must exhibit evidence of high activity in the intracellular degradation process. To examine whether this is the case, vital staining was performed with LysoTracker, which labels acidic organelles such as late endosomes and lysosomes. In the larval lobes, no detectable signal was found with the LysoTracker staining. In the dorsal lobe at 6 hr APF, however, the LysoTracker-positive signals were constantly found. In most cases, the signals were localized in the lumps of the infiltrated glia. This finding strongly suggests that varicosities are engulfed by glia and degraded in the endosome-lysosome pathway (Awasaki, 2004).

Though most of the varicosities disappear by 12 hr APF, glial infiltration was still observed at 12 hr and 18 hr APF. Glial processes at these stages frequently form columnar structures lying along the axon branches. These columnar structures are often observed in the shaft region of the dorsal lobes. The disconnection of axons also occurs frequently in the shaft region rather than in the tip region at 12 hr APF. Thus, the formation of the columnar structures was spatially and temporally correlated with the occurrence of axon degeneration. This suggests that infiltrated glia are involved in the disconnection and degeneration of axon branches as well as the disappearance of varicosities (Awasaki, 2004).

These results so far suggested that varicosities and axon branches are engulfed and degraded in the glial lumps. There are two possibilities for the role of glia during this process: (1) axon branches of γ neurons might be degenerated by intrinsic mechanisms and infiltrating glia might just scavenge the already degraded debris; (2) degradation of the axon branches of the γ neurons may not be due to intrinsic mechanisms alone and may require infiltrating glia to actively break down the axon branches. To determine the most likely possibility, the pruning process was observed while glial cell function was conditionally inhibited by the targeted expression of the temperature-sensitive allele of shibire (shi^ts1). The shibire gene is the Drosophila homolog of dynamin, which is involved in cellular membrane functions, including endocytosis, phagocytosis, and membrane growth. Because Shi^ts1 protein acts as a dominant-negative at a restrictive temperature, targeted expression of Shi^ts1 in glial cells would inhibit endocytosis and other membrane-related functions (Awasaki, 2004).

Larvae expressing Shi^ts1 in glial cells by using the GAL4-repo driver (repo>shi^ts1) were raised at a permissive temperature (19°C) and then placed in the restrictive temperature (29°C) from 0 hr APF to either 6 hr or 18 hr APF. Raising repo>shi^ts1 pupae at the restrictive temperature results in an abnormal morphology of the glial cells. The most striking abnormality of these glia is that they did not extend their processes into the lobe at all. Whereas glial processes in a normal condition have infiltrated into lobes intensely already at 6 hr APF, dorsal lobes of repo>shi^ts1 in the restrictive temperature were devoid of any glial processes even at 18 hr APF. This demonstrates that Shi^ts1 almost completely suppresses the infiltration of glial processes (Awasaki, 2004).

Anti-FasII labeling of dorsal lobes revealed hole-like structures of unlabeled regions at 6 hr APF, and severe loss of axon branches at 18 hr APF. Neither holes of unlabeled regions nor loss of axon branches were observed in the lobes of the repo>shi^ts1 animal in the restrictive temperature at 6 hr and 18 hr APF. The phenotypes were qualitatively indistinguishable among different individuals. These indicate that axon pruning including both disappearance of clustered varicosities and local axon degeneration were severely suppressed in these pupae (Awasaki, 2004).

Because inhibition of glial infiltration caused severe suppression of axon pruning, it is highly likely that infiltrating glia actively break down and remove axon branches rather than just scavenging already degraded debris. This result, together with the engulfment and degradation of clustered varicosities by glial lumps, suggests that infiltrating glia actively eliminate clustered varicosities at the early phase of pruning (Awasaki, 2004).

If the pruning of γ neurons is suppressed by the targeted disruption of glial function, do the unpruned axon branches remain in the adult MB? The repo>shi^ts1 pupae incubated at the restrictive temperature did not survive until adult stage; therefore, glial function could not be disrupted throughout pupal development. When the temperature was shifted from the restrictive temperature to 25°C at 24 hr APF, however, a small number of survivors eclosed. In these animals (n = 10), abnormal axon branches expressing abnormal FasII-positive signals were observed shortly after hatching beside the α and β lobes, which are adult-specific branches labeled with anti-FasII antibody. Such abnormal FasII-positive branches were observed only in the distal tip of the lobes, because continuous fibers could not be traced from them to the shaft region of the lobe. Moreover, the abnormal branches were no longer visible in the brains more than 7 days after eclosion. It is therefore likely that these FasII-positive branches were the remnants of the γ neuron axons that were not pruned at 18 hr APF but gradually degenerated thereafter. Thus, transient inhibition of glial function in early pupae does not completely block axon pruning but severely delays the time course of axon pruning (Awasaki, 2004).

Axon pruning is triggered by a surge of ecdysone, which occurs just before puparium formation. The γ neurons express the ecdysone receptor EcR-B1 strongly in late third-instar larvae. The surrounding glial cells express EcR-B1 faintly at this stage. The expression of EcR-B1 in the glial cells, however, is unlikely to be associated with axon pruning. Although EcR-B mutation causes complete suppression of axon pruning, this suppression is rescued by expression of EcR-B1 only in the γ neurons. How, then, is the glial activation controlled? Because expression of EcR-B1 in γ neurons is sufficient for axon pruning, the glial cells must be activated via ecdysone stimulation of γ neurons. To test this hypothesis, expression of a dominant-negative form of the ecdysone receptor was induced in γ neurons by using the 201Y driver (201Y>EcR-DN), which drives expression in γ neurons, but not in the surrounding glia. If the activation of glia is dependent on the ecdysone-induced stimulation of γ neurons, glial infiltration would be suppressed in these pupae. Conversely, if the glial activation is independent of γ neurons, infiltration would not be disturbed. The results strongly suggest that ecdysone-induced stimulation of γ neurons is indispensable for the infiltrating and engulfing action of glial cells. Once γ neurons receive ecdysone by EcR-B1, these neurons would induce glial infiltration and engulfment. Thus, the neuron-glia interaction is essential for coordinated axon pruning (Awasaki, 2004).

The neuron-glia interactions for selective infiltration and engulfment resemble the interactions between phagocytes and apoptotic cells. It is not likely, however, that axon pruning and apoptosis use identical molecular mechanisms to activate glial cells and phagocytes. Mutation of Drosophila apoptosis activator genes (grim, hid, and rpr) and overexpression of apoptosis inhibitor genes (p35 and DIAP1) in γ neurons have no effect on axon pruning. However, Draper (Drpr), the homolog of the C. elegans cell corpse engulfment molecule CED-1, accumulates on the cell membrane of infiltrating glial processes. Engulfment of apoptotic neurons by glial cells is suppressed in the central nervous system (CNS) of drpr mutant embryos. This raises the possibility that sensing of apoptotic cells by phagocytes and sensing of unwanted axon branches by glial cells share a common molecular mechanism. Although the drpr mutant is isolated in Drosophila, it is embryonic lethal. Furthermore, clonal analysis with a flippase-mediated MARCM system does not work reliably in the larval and pupal glial cells. Analysis of the function of drpr in the engulfing glial cells with more sophisticated techniques to overcome the above problem, together with the analyses of the involvement of phosphatidylserine and lysophosphatidylcholine in the axon pruning of γ neurons, might reveal correlated mechanisms between removal of the cell corpse and unwanted axons (Awasaki, 2004).

Inhibition of glial function almost completely suppresses axon pruning until 18 hr APF, but the remaining axons still undergo degeneration in the adult. There is therefore a possibility that cell-autonomous mechanisms can prune axon branches without glial involvement. The time course of degeneration, however, is severely delayed (Awasaki, 2004).

Rapid degeneration of γ neuron axons seems vital for proper remodeling of the MB neural circuit. In the late larval brain, the α'/β' neurons extend their axons into the core of the axon bundle of γ neurons. As γ neuron axons are pruned during the early pupal stage, α'/β' neurons claim the region previously occupied by γ neurons. When pruning of γ neurons is suppressed by ectopic expression of yeast ubiquitin protease, UBP2, the unpruned axons remain in the vicinity of axon branches of α'/β' neurons in the adult. The morphology of α'/β' axon branches was distorted in this case. This suggests that proper elimination of larval axon branches might be important for the correct formation of adult axon branches. Rapid pruning with the help of the engulfing action of glia would be advantageous in such a situation (Awasaki, 2004).

Although there are many examples of axon pruning, the underlying mechanisms have not been identified except for Drosophila γ neurons and the mossy fiber pathway of the mouse hippocampal neurons. While axon pruning in Drosophila γ neurons is mediated by the degeneration of axons, which occurs within approximately 18 hr, the axon pruning of the mossy fiber pathway requires the retraction of axons over 500 μm in length, which occurs over more than 30 days. Further identification of the cellular and molecular mechanisms that mediate degeneration and retraction of axons might elucidate the differences and similarities in the strategy of each type of axon pruning (Awasaki, 2004).

Ecdysteroid control of metamorphosis in the differentiating adult leg structures of Drosophila

During insect metamorphosis, the steroid hormone 20-hydroxyecdysone (20E) is responsible for coordinating the differentiation of adult structures. Several structures of the Drosophila melanogaster adult leg, the six distalmost joints, the bristles, and the pretarsal claws, were examined to investigate how 20E controls their development in vitro. Joints, bristles, and claws were dependent on 20E for differentiation between 20-22 and 24-26 h after puparium formation (APF). After 26-28 h APF, differentiation becomes hormone independent. Tissue-specific markers in 20E-free cultures show that the bristle and joint cells do not undergone any further morphogenetic progression. In contrast, the pretarsi undergo partial differentiation. The concentration of 20E required for differentiation is structure specific; tarsal joints require higher concentrations of 20E (greater than 400 ng 20 E/ml) than pretarsal claws, bristles, and other joints (greater than 40 ng 20E/ml). The 20E precursor ecdysone (E) is also able to induce differentiation at concentrations over 700 ng E/ml, but does not show any synergistic interactions with 20E. Lastly, leg structures have a finite ability to respond to 20E; tarsal joints lose competence to respond after 32-34 h APF, while the remaining structures became incompetent after 44-46 h APF (Mirth, 2005).

In the absence of 20E at 20-22 h APF, bristles and joints did not undergo any further differentiation, but they arrested at different stages along their differentiative pathway. Bristle cells had already begun their differentiative trajectory. They arrested mid-differentiation, having undergone the appropriate number of cell divisions and arranged themselves in the crooked line formation, but failing to undergo the additional cell rearrangements and shape changes to form the adult structure. In contrast, joints had yet to undergo any differentiative events at all. In both cases, however, differentiation could occur without 20E after 26 h APF. This stage corresponds to the beginning of distal joint cell constriction and elongation, and the time at which the bristle shaft is starting to elongate. What determines the stage at which morphogenesis can become hormone independent is unclear (Mirth, 2005).

In other tissues, certain phases of differentiation appear always to require 20E. Early stages of differentiation of the neurons in the optic lobe in Manduca sexta require constant hormone input to progress. Optic lobe neurons will continue to divide as long as 20E is present at suprathreshold concentrations in the medium. Reciprocally, when 20E is not present, divisions arrest until the hormone is added back into culture. Even after 24 h without hormone, neurons are able to respond and divide when presented with 20E, whereas Drosophila leg tissue became unresponsive to hormone after 24 h without 20E. Differences in the stage of differentiation may be responsible for these distinctions, with early differentiative processes such as cell division requiring continuous hormone input, and late differentiative processes like cell shape changes requiring only temporary hormone stimulus (Mirth, 2005).

In many tissues, hormone concentrations act as a temporal switch for controlling different stages of differentiation. The behaviors of cells in the optic lobe and eye discs of M. sexta are largely dependent on hormone concentration, with early maturational responses induced by mid-range concentrations and late responses by high 20E concentration. In both cases, switching between differentiative states can occur by modifying the hormone concentration in cultures from tissues of the same age (Mirth, 2005).

Adult leg structures also require 20E to be present in concentrations above a threshold to metamorphose. Furthermore, these results have shown that threshold concentrations differed between the structures of the leg, most notably differing between the joints. The joints least sensitive to 20E, the tarsal joints, are also the last structures to complete their differentiation, and it could be hypothesized that they require a higher concentration of 20E to switch into their developmental programs. However, considering all joints begin metamorphosis at the same time and become independent of 20E at the same time, it is unlikely that concentrations of hormones are acting as a switch to control different phases of leg differentiation (Mirth, 2005).

Moreover, hormone concentration may not be determining developmental time at this stage. Hormone concentrations have already reached titers sufficient to stimulate joint invagination at 18 h APF, but this process does not begin until 24 h APF. Leg structures did not appear to use threshold concentration as a measure of time for tissue differentiation (Mirth, 2005).

Why the development of some tissues might rely on continuous hormone presence, instead of transient hormone input, is likely to do with their developmental context. In M. sexta eye discs, early and late developmental responses can be induced at the same developmental stage depending on 20E concentration. Here, the early stages of differentiation, furrow progression and ommatidial recruitment, continue in a 20E-dependent manner over several days until an increase in 20E concentration induces ommatidial maturation. This requirement for continuous hormone presence for early stages of metamorphosis could reflect a mechanism that allows a tissue to undergo a developmental process for a sustained period, then to use increasing 20E levels as a temporal marker to switch to later differentiative events (Mirth, 2005).

Conversely, cells that are ready to undergo their final differentiation, like the joints in D. melanogaster and the ommatidia in M. sexta, might only need to receive the hormone signal transiently to complete differentiation. In this scenario, competence to respond to suprathreshold levels of hormone within a limited time might be a more useful mechanism. To fully understand these mechanisms, more detailed studies of how concentrations and timing of hormone pulses affect specific tissues in terms of tissue organization, cell behavior, and gene regulation are required to make more accurate predications about how 20E acts to control tissue differentiation during metamorphosis (Mirth, 2005).

This work shows that, like earlier stages of leg development, the differentiation of adult leg structures is dependent on 20E. It would be of great interest to explore whether during the pupal 20E pulse, the genes involved in imaginal disc morphogenesis are also necessary to transmit the 20E signal and effect morphogenesis in the bristles, claws, and joints. Microarray data show that E74 and βFTZ-F1, both genes in the 20E signaling cascade that are important for disc morphogenesis, exhibit peaks of expression during the pupal 20E pulse. Genes downstream of the ecdysone cascade that are involved in disc morphogenesis, such as Stubble (Sb) and crooked legs, also show peaks of expression during these stages. Furthermore, animals homozygous for the Sb^63B mutation sometimes show fusions between the tarsal segments and have obvious bristle formation defects, suggesting that Sb is involved in later differentiative events. Examining whether the genes important for imaginal disc morphogenesis are later involved in the differentiation of adult leg structures would provide an example of how the 20E cascade controls differentiation in a single organ throughout prepupal and pupal development (Mirth, 2005).

Antagonistic actions of ecdysone and of insulins, acting through Foxo and 4E-BP, determine final size in Drosophila

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).

In metazoans, the insulin/IGF signaling pathway (IIS) plays a key role in regulating growth and metabolism. In Drosophila, a family of insulin-like molecules called Dilps activates a unique insulin receptor (InR) and a conserved downstream kinase cascade that includes PI3-kinase (PI3K) and Akt/PKB. Recent genetic experiments have established that this pathway integrates extrinsic signals such as nutrition with the control of tissue growth during larval stages. The larval period is critical for the control of animal growth, since it establishes the size at which maturation occurs and, consequently, the final adult size. Maturation is itself a complex process that is controlled by the steroid 20-hydroxyecdysone (20E). Peaks of 20E determine the timing of all developmental transitions, from embryo to larva, larva to pupa, and pupa to adult. Ecdysteroids are mainly produced by the prothoracic gland (PG), a part of a composite endocrine tissue called the ring gland. Final adult size thus mainly depends on two parameters: the speed of growth (or growth rate), which is primarily controlled by IIS, and the overall duration of the growth period, which is limited by the onset of the larval-pupal transition and timed by peaks of ecdysone secretion. Very little is known concerning the mechanisms that coordinate these two parameters during larval development (Colombani, 2005).

To investigate the function of ecdysone in controlling organismal growth, a genetic approach was developed that allowed modulation basal levels of ecdysteroids in Drosophila. The initial rationale was to modify the mass of the ring gland in order to change the level of ecdysteroid production. For this goal, the levels of PI3- kinase activity were manipulated in the PG by crossing P0206-Gal4 (P0206>), a line with specific Gal4 expression in the PG and corpora allata (CA), with flies carrying UAS constructs allowing expression of either wild-type (PI3K) or dominant-negative (PI3K^DN) PI3-kinase. As expected, these crosses produced dramatic autonomous growth effects in the ring gland, and particularly in the PG: tissue size was increased upon PI3K activation and decreased upon inhibition. Surprisingly, the changes in ring gland growth were accompanied by opposite effects at the organismal level. P0206>PI3K animals (with large ring glands) showed reduced growth at all stages of development and produced emerging adults with reduced size and body weight (78% of wt). Conversely, reducing PI3K activity in the ring gland of P0206>PI3K^DN animals led to increased growth and produced adults with 17% greater weight on average. Adult size increase was attributable to an increase in cell number in the wing and the eye. Adult size reduction was accompanied by a decrease in cell number in the wing and in cell size in the eye (Colombani, 2005).

Importantly, the timing of embryonic and larval development of these animals was comparable to control. Both the L2 to L3 transition as well as the cessation of feeding (wandering) occurred at identical times. Further, animals entered pupal development at the same time, except for P0206>PI3K^DN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3K^DN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3K^DN animals, albeit by less than 4 hours following 10 days of development. In contrast, the speed of larval growth was found to be increased in P0206>PI3K^DN animals and decreased in the P0206>PI3K animals background at the earliest stage that could be measured (early L2 instar). Because none of these effects were observed when PI3K levels were modified specifically in the CA using the Aug21- Gal4 driver, it was concluded that the observed phenotypes are solely due to PI3K modulation in the PG. Together, these results demonstrate that manipulating PI3K levels in the PG induces non-autonomous changes in the speed of larval growth (growth rate effects), without changing the timing of larval development (Colombani, 2005).

To investigate whether these effects could be attributed to changes in 20E levels, ecdysteroid titers were measured in third instar larvae of the different genotypes. Early after ecdysis into third instar (74hrs AED) ecdysteroids are present at basal level. They accumulate to an intermediate plateau around 90hrs AED and reach peak levels before pupariation (120hrs AED). Because early L3 levels are below the detection limit of the EIA assay, ecdysteroid titers were measured at the intermediate plateau (90hrs AED). In these conditions, a very modest increase of ecdysteroids was observed in P0206>PI3K animals larvae and a small but significant decrease in P0206>PI3K^DN animals animals. This was further confirmed by measuring the transcript levels of a direct target of 20E, E74B, which responds to low/moderate levels of 20E. However, in early L3 larvae with basal ecdysteroid levels (74hrs AED), differences in E74B transcripts were clearly visible, with a 1.9-fold increase seen for P0206>PI3K animals and a 1.7-fold decrease for P0206>PI3K^DN animals. This establishes that basal circulating levels of 20E are modified in response to manipulation of PI3K levels in the PG. It also suggests that the differences observed on basal 20E level off with the strong global increase of ecdysteroids in mid/late L3 (Colombani, 2005).

Several related lines of evidence strengthen these results: (1) the increase in growth rate and size observed in P0206>PI3K^DN animals can be efficiently reverted by adding 20E to their food; (2) feeding wild-type larvae 20E recapitulates the effects observed in P0206>PI3K animals animals; (3) ubiquitous silencing of EcR using an inducible EcR RNAi construct results in a growth increase similar to that observed in P0206>PI3K^DN larvae. Finally, the phantom (phm) and disembodied (dib) genes, which are specifically expressed in the PG and encode hydroxylases required for ecdysteroid biosynthesis, showed 1.65- and 2.2- fold increased expression, respectively, upon PI3K activation in the ring gland. This supports the notion that 20E biosynthesis is mildly induced in these experimental conditions. In line with previous results, neither 20E treatment nor EcR silencing has any effect on developmental timing. Overall, the results indicate that manipulating basal levels of 20E signaling in various ways modifies the larval growth rate without affecting the timing of the larval transitions (Colombani, 2005).

Variations in ecdysone levels in animals with different sized ring glands could be due to changes in the PG tissue mass or, alternatively, to a specific effect of PI3K signaling in the secreting tissue. To distinguish between these two possibilities, extra growth was induced in the PG using either dMyc or CyclinD/Cdk4, two potent growth inducers in Drosophila. Although the size of the larval ring gland was markedly increased under these conditions, no effect on pupal or adult size was observed, implying that the size of the ring gland is not the critical factor in the control of body size. Instead, it is likely that the InR/PI3K signaling pathway can specifically activate ecdysone production from the PG (Colombani, 2005).

The possibility was tested that ecdysone signaling opposes the growth-promoting effects of IIS in the larva. To test this, larvae were fed 20E and xPI3K activity was assessed in vivo using a GFP-PH fusion (tGPH) as a marker. Membrane tGPH localization shows a marked decrease in the fat body of 20E-fed animals, and this effect can be reverted by specifically silencing EcR in the fat body. This indicates that ecdysone-induced growth inhibition correlates with decreased IIS, and is mediated through the nuclear receptor EcR. Conversely, larvae with PI3K^DN expression in the PG show a 4.2-fold increase in the global levels of dPKB/Akt activity, as measured by the phosphorylation levels of serine 505. In Drosophila cells (as in other metazoan cells) high levels of PI3K/AKT activity cause the transcription factor dFOXO to be retained in the cytoplasm, while low PI3K/AKT activity allows dFOXO to enter the nucleus where it promotes 4E-BP transcription. In larvae with ectopic PI3K expression in the PG, a strong increase is observed in nuclear dFOXO in fat body cells. Similar results were obtained by feeding larvae with 20E. Conversely, inactivation of EcR signaling in fat body cells was carried out using the clonal over-expression of a dominant-negative form of EcR (EcRF645A). In these conditions, a reduction was observed in the accumulation of dFOXO in the nuclei of EcRF645A-expressing cells. As an expected consequence of the increased nuclear dFOXO, global accumulation of 4E-BP transcripts was consistently higher in P0206>PI3K animals as well as in 20E-fed early L3 larvae as compared to control animals, and reduced in arm>EcR-RNAi animals. Together, these results indicate that ecdysone-dependent inhibition of larval growth correlates with a general alteration of insulin/IGF signaling, and a relocalization of dFOXO into the cell nuclei. To more directly test the role of dFOXO in the growth-inhibitory function of ecdysone signaling, the effects of increasing ecdysone levels were examined in a dFOXOmutant genetic background. Although homozygous dFOXO21 animals do not display a detectable growth phenotype, introducing the dFOXO21 mutation was sufficient to totally revert the growth defects of P0206>PI3K animals animals, either when homozygous or heterozygous. This data establishes that dFOXO is required for 20E-mediated growth inhibition (Colombani, 2005).

The endocrine activities of the brain and the fat body have previously been implicated in the humoral control of larval growth. In order to test for possible roles of these two organs in mediating the systemic growth effects of ecdysone, EcR expression was silenced specifically in the brain cells that produce insulins (IPCs) or in the fat body. While specific expression of EcR RNAi in the IPCs fails to reproduce the overgrowth observed in armGal4>EcR-RNAi animals, EcR silencing in the fat body elicits an acceleration of larval growth and a remarkable increase in pupal size. Moreover, no detectable delay in the larval timing was observed in pplGal4>EcR-RNAi animals. Thus, specifically reducing 20E signaling in the fat body is sufficient to recapitulate the systemic effects of global EcR silencing. This demonstrates that the fat body is a major target for ecdysone, and that this tissue can act to relay the 20E growth-inhibitory signal to all larval tissues (Colombani, 2005).

In summary, these results establish an additional role for 20E in modulating animal growth rates. This function is mediated by an antagonistic interaction with IIS that ultimately targets dFOXO function. A similar antagonistic interaction between 20E and insulin signaling controls developmentally-regulated autophagy in Drosophila larva (Colombani, 2005).

Although a direct effect of ecdysone on the cellular growth rate of all larval tissues cannot be ruled out, the experiments reveal a key role for the fat body in relaying ecdysone-dependent growth control signals. Together with previous work, these data suggest that various inputs such as nutrition and ecdysone converge on this important regulatory organ, which then controls the general IIS to modulate organismal growth (Colombani, 2005).

How then is growth connected to developmental timing? The finding that 20E can modulate growth rates in addition to developmental transitions places this hormone in a central position for coordinating these two key processes and controlling organismal size (Colombani, 2005).

Dendrite-specific remodeling of Drosophila sensory neurons requires matrix metalloproteases, ubiquitin-proteasome, and ecdysone signaling

During neuronal maturation, dendrites develop from immature neurites into mature arbors. In response to changes in the environment, dendrites from certain mature neurons can undergo large-scale morphologic remodeling. A group of Drosophila peripheral sensory neurons, the class IV dendritic arborization (C4da) neurons completely degrade and regrow their elaborate dendrites. Larval dendrites of C4da neurons are first severed from the soma and subsequently degraded during metamorphosis. This process is controlled by both intracellular and extracellular mechanisms: The ecdysone pathway and ubiquitin-proteasome system (UPS) are cell-intrinsic signals that initiate dendrite breakage, and extracellular matrix metalloproteases are required to degrade the severed dendrites. Surprisingly, C4da neurons retain their axonal projections during concurrent dendrite degradation, despite activated ecdysone and UPS pathways. These results demonstrate that, in response to environmental changes, certain neurons have cell-intrinsic abilities to completely lose their dendrites but keep their axons and subsequently regrow their dendritic arbors (Kuo, 2005).

To visualize abdominal C4da neurons during Drosophila metamorphosis, a pickpocket (ppk)-EGFP reporter line was used. Filleted white pupae (WP), at the onset of metamorphosis, were stained with an anti-EGFP antibody to reveal three C4da neurons, vdaB (V), v'ada (V'), and ddaC (D), in each hemisegment. Because the soma and dendritic projections of these neurons remained very close to the body surface during pupariation, live imaging was used to follow these neurons throughout metamorphosis (Kuo, 2005).

Initially at the WP stage, the C4da neurons exhibited intact, complex class IV dendritic branches that covered much of the pupal surface. Shortly after the white pupal stage, 2 h after puparium formation (APF), fine terminal dendritic branches began to disappear. By 10 h APF, most major dendritic branches were severed from the soma. This severing of dendrites has also been observed in a recent study of da neuron remodeling. During the next 8 h, which coincided with head eversion during metamorphosis, these severed and blebbing dendrites are degraded. By 20 h APF, the process of larval dendrite removal is complete, leaving C4da neurons with their axonal projections but devoid of larval dendrites. Axons from all three C4da neurons project into the VNC. By this time, V' and D neurons begin to extend fine dendritic projections. The V neurons, which do not show new dendritic projections, disappear between 30 and 35 h APF, leaving V' as the surviving neuron in the ventral hemisegment (Kuo, 2005).

Compared with the rapid sequence of larval dendritic pruning, the process of pupal dendrite regrowth is slow. By 70 h APF, both V' and D neurons begin to take on the shape of their respective adult neurons. By 95 h APF, shortly before adult eclosion, the dendritic patterns of abdominal V' neurons closely resemble larval C4da neurons before pupariation. In contrast, the D neurons take on a more elongated dendritic field, perhaps reflecting a functional divergence between these two neurons in the adult fly. These results show that C4da neurons can completely degrade their elaborate larval dendrites during early metamorphosis, survive these changes, and subsequently regrow their dendritic arbors (Kuo, 2005).

During Drosophila metamorphosis, most larval organs are replaced by adult structures. To understand the cellular environment during C4da dendrite degradation, the expression of Armadillo, an adhesive junction protein that outlines the epithelial monolayer during early metamorphosis, was examined. High-level Armadillo staining at the WP stage is completely abolished by 13 h APF but subsequently returns at 20 h APF when the pupal epithelium is reformed. Thus, the pruning of C4da neuron dendrites occurs concurrently with epithelial remodeling during metamorphosis. To determine whether the degradation of larval dendrites is a result of local tissue remodeling or neuron-intrinsic signaling, focus was placed on enzymes that are important for tissue remodeling (Kuo, 2005).

Drosophila matrix metalloproteases (metalloproteinases) Mmp1 and Mmp2 regulate tissue remodeling during metamorphosis (Page-McCaw, 2003). The weaker alleles of both genes, Mmp1^Q273 and Mmp2^W307, survive past head eversion to midpupariation, making it possible to visualize dendritic pruning of ppk-EGFP-expressing C4da neurons. Remarkably, there were abundant C4da neuron larval dendrites in both Mmp1 and Mmp2 mutants after head eversion. Whereas in WT pupae at 20 h APF all larval dendrites from C4da neurons were cleared from the extracellular space, in both Mmp1 and Mmp2 mutants, larval dendrites that are severed from the soma remain. These larval dendrites persist to much later stages at 50 and 35 h APF, just before the lethal phases of Mmp1^Q273 and Mmp2^W307 mutants, respectively. The ineffective removal of larval dendrites in Mmp mutants is not caused by a generalized delay in metamorphosis because Mmp mutant pupae had completed head eversion and epidermal remodeling, thus indicating a specific defect in dendrite degradation. Because Mmp1;Mmp2 double mutants do not survive to pupariation, it was not possible to look at dendritic pruning in the double mutant background (Kuo, 2005).

To determine whether Mmps functions on the cell surface of dendrites to regulate degradation, an Mmp inhibitor was expressed in C4da neurons to see whether the survival of larval dendrites can be prolonged. Using the ppk promoter to express transcriptional activator Gal4 (ppk-Gal4), the UAS-Gal4 system was used to express the Drosophila tissue inhibitor of metalloproteases (TIMP) in C4da neurons. Fly TIMP is closely related to mammalian TIMP-3, which associates with extracellular membrane surfaces to modulate Mmp activities. In control animals expressing GFP at 20 h APF, identical pruning of larval dendrites as in ppk-EGFP flies was seen. In contrast, when TIMP is overexpressed in C4da neurons, larval dendrites remain in the extracellular space at 20 h APF (Kuo, 2005).

The fact that TIMP inhibition can successfully delay the degradation of larval dendrites confirms Mmp's involvement in this process. But these enzymes could be synthesized by either the C4da neurons or by the surrounding cells. To identify the source of this Mmp activity, MARCM studies were performed to generate C4da clones that in both Mmps. Mmp1^Q112Mmp2^W307 double mutant C4da clones not only show dendritic branching patterns similar to WT clones during early pupariation, but live time-lapse imaging revealed complete larval dendrite removal after head eversion at 20 h APF, just like WT controls. These results show that cell-intrinsic Mmps are not required for dendritic pruning and that extracellular Mmp activity is sufficient for degrading severed larval dendrites during metamorphosis. A possible source of this extracellular activity could be phagocytic blood cells, because they have been shown to engulf dendritic debris during metamorphosis (Kuo, 2005).

Whereas removal of severed dendrites requires extrinsic metalloproteases, C4da neurons in Mmp mutants still retain their ability to sever larval dendrites from the soma during metamorphosis. To look for cell-intrinsic pathways in cleaving larval dendrites, the role of ecdysone, a steroid hormone that regulates much of Drosophila metamorphosis, was examined. Binding of ecdysone to its nuclear receptor heterodimers, consisting of Ultraspiracle (Usp) and one of three EcR isoforms (EcR-A, EcR-B1, and EcR-B2), mediates a transcriptional hierarchy that regulates tissue responses during metamorphosis. To determine whether EcR signaling plays a role in initiating dendritic pruning, EcR expression patterns were examined in the ppk-EGFP transgenic line that specifically labels C4da neurons (Kuo, 2005).

Staining with the EcR-C antibody, which recognizes the common regions of EcR family members, and staining with EcR-A and EcR-B1 specific antibodies during different stages of late larval through early pupal development, revealed that all three C4da neurons exhibit similar staining patterns. In third-instar larvae, when the ecdysone level is low before the onset of pupariation, EcR expression in C4da neurons is relatively low when compared with surrounding cells that already exhibit a high level of nuclear EcR. At the WP stage, with a transient rise in ecdysone level, EcR in C4da neurons becomes concentrated in the nucleus. Over the next 7 h, EcR gradually redistributes throughout the soma of C4da neurons, which corresponds to a rapid drop-off in ecdysone levels in the pupae after initiation of metamorphosis. Strong nuclear EcR localization in C4da neurons returns at 20 h APF, correlating with the onset of midpupal ecdysone release. Antibodies specific to either EcR-A or B1 show that whereas EcR-A expression is diffuse and weak throughout metamorphosis, EcR-B1 expression in C4da neurons corresponds to the dynamic nuclear localization patterns seen with the EcR-C antibody (Kuo, 2005).

To examine the functional significance of EcR expression, attempts were made to disrupt ecdysone signaling specifically in C4da neurons. EcR mutants either do not survive to the pupal stage or die shortly after the onset of metamorphosis; therefore, it is not possible to look at dendritic remodeling in those mutants. The cytological location of EcR genes also precludes MARCM studies; therefore, use was made of a set of dominant-negative (DN) EcR proteins to inhibit EcR activity. When ecdysone signaling is inhibited by EcR-DN proteins, C4da neurons lose their ability to initiate larval dendrite pruning at 20 h APF. To determine whether the defects are specific to the ecdysone signaling pathway, attempts were made to rescue the EcR-DN phenotype. Coexpression of both EcR-DN and WT EcR-B1 proteins in C4da neurons resulted in complete rescue of dendritic pruning defects in all three neurons. This rescue is complete with two copies of ppk-Gal4 in C4da neurons, showing that the rescue is not caused by reduced expression of DN protein in the coexpression experiments (Kuo, 2005).

Because dimerization of EcR-B1 with its obligatory hormone receptor partner Usp is essential for transcriptional regulation, the involvement of Usp in dendrite remodeling was examined. Usp mutants do not survive to metamorphosis; however, it was possible to generate Usp MARCM clones for analysis. At 20 h APF, Usp mutant C4da clones fail to prune their larval dendrites, and this genetic mutation shows an identical phenotype to the EcR-DN experiments. Given the severity and full penetrance of this phenotype, together with the timing of EcR-B1 nuclear localization, it is concluded that the ecdysone signaling pathway plays an important cell-intrinsic role in initiating dendritic pruning in C4da neurons during metamorphosis (Kuo, 2005).

What might be the cellular machineries that carry out dendrite pruning in C4da neurons? One attractive model is a caspase-mediated local digestion and degradation of dendrites. However, overexpression of p35, an effective inhibitor of fly caspases, in C4da neurons did not prevent or delay larval dendrite degradation during metamorphosis. Another protein degradation pathway, the ubiquitin protease system (UPS), has been shown to regulate both axon and dendrite pruning of mushroom body neurons during fly metamorphosis. To test the involvement of UPS in C4da neuron remodeling, use was made of ppk-Gal4 to overexpress UBP2, a yeast ubiquitin protease, in the C4da neurons. By reversing the process of substrate ubiquination, UBP2 is an effective UPS inhibitor in the fly. Some of the C4da neurons expressing UBP2 aberrantly retained their larval dendritic arbors. Note that this pruning defect is very different from that seen in Mmp mutants. Whereas Mmp mutants accumulated severed larval dendrites in the extracellular space, UBP2 inhibition prevented efficient severing of dendrites from the soma (Kuo, 2005).

To further examine the involvement of the UPS machinery in dendritic pruning, the MARCM system was used to generate C4da clones that were either deficient in ubiquitin activation enzyme 1 (Uba1) or had mutation in the 19S particle of the proteasome (Mov34). Time-lapse imaging of Uba1 and Mov34 mutant C4da clones at WP stage and 20 h APF showed that, unlike WT clones, both mutant clones failed to efficiently sever their larval dendrites during metamorphosis. These results confirmed the requirement for an activated UPS in the severing of larval dendrites from C4da neurons during metamorphosis (Kuo, 2005).

To compare the defects in larval dendritic pruning caused by different mutations, the number of large (primary and secondary) dendritic branches that remain attached to C4da neuron soma was counted. In WT pupae at the start of metamorphosis, C4da neurons extended close to 20 large dendritic branches, none of which was retained after head eversion at 20 h APF. Mutations that disrupt ecdysone signaling, such as EcR-DN expression or Usp-deficient clones, result in the retention of 85%-90% of large dendritic branches after head eversion. Mutations in the UPS pathway, such as Uba1 and Mov34, resulted in the retention of 45%-49% of large dendritic branches at 20 h APF. Mmp1 or Mmp2 mutants retained only 3%-8% of large dendritic branches after head eversion, and Mmp1;Mmp2 mutant clones did not retain larval dendrites at 20 h APF. These data suggest that dendrite remodeling in C4da neurons starts with signals from ecdysone and UPS that result in the cleavage of larval dendrites from the soma, which then allows for the degradation of severed dendrites by Mmp activity in the extracellular matrix (Kuo, 2005).

It is possible that UPS is an upstream regulator of EcR and can lead to EcR misexpression in UPS mutants; however, normal EcR expression patterns are observed in both Uba1 and Mov34 C4da MARCM clones. It is also conceivable that EcR signaling is upstream of the UPS cascade, but this idea is difficult to demonstrate experimentally. It was reasoned that in this case, inhibition of EcR signaling should result in lower levels of protein ubiquination. However, staining in EcR-DN-expressing C4da neurons showed no significant differences in the level of ubiquitin/polyubiquitin between undegraded larval dendrites and WT dendrites before degradation. This finding does not rule out an EcR function upstream of UPS during dendritic remodeling, because EcR signaling may regulate critical factors in the UPS cascade after protein ubiquination at the level of ubiquitin ligases. The identities of such ligases are currently unknown (Kuo, 2005).

To test whether dendritic pruning in C4da neurons involves concurrent axonal remodeling, axon tracks of C4da neurons were examined in the Drosophila VNC during early metamorphosis. Direct live imaging of the ppk-EGFP transgenic line at the WP stage showed axon tracks from three C4da neurons. Axon tracing of EGFP-expressing C4da neurons at 6 h APF showed continuous axon tracks between all three C4da neurons and the VNC. At 10 h APF, the VNC appeared more compact, presumably as a result of the various remodeling events that occur in the nervous system during metamorphosis. At 20 h APF, axon tracks of EGFP-expressing C4da neurons can still be clearly identified at the VNC and are continuous with the soma, despite the complete removal of dendritic arbors of these same neurons (Kuo, 2005).

Drosophila peripheral sensory neurons generally have simple axon projections into the VNC that terminate locally. To visualize C4da neuron axon terminals during metamorphosis, the UAS>CD2>CD8-GFP system was used, together with ppk-Gal4, to generate single clones of surviving V' and D neurons. The V' C4da neuron was found to project its axon ipsilaterally upon entering the VNC to the segment immediately anterior during the WP stage. At 20 h APF, after complete pruning of larval dendrites, the V' C4da neuron keeps this axonal projection intact in the VNC. The D C4da neuron axon, in addition to having an ipsilateral branch that projects to the anterior segment, sends a commissural branch that crosses the midline at the segment where the axon enters the VNC. Likewise, at 20 h APF, the D C4da neuron keeps both axonal terminal branches intact. These data show that C4da neurons do not significantly modify their larval axons during concurrent dendrite degradation, despite the activated ecdysone and UPS pathways, which are known to facilitate axon remodeling and degradation (Kuo, 2005).

What might account for the dendrite-specific remodeling in C4da neurons, as opposed to the previously reported concurrent remodeling of both axons and dendrites? It is possible that local environments may play a role. A recent study in Manduca found central versus peripheral hormonal differences affecting axon versus dendrite remodeling. However, it remains to be tested whether the ecdysone levels are different in the fly epidermis and the VNC during metamorphosis. Anatomically, C4da neurons have distinct axon-dendrite polarity in that the cell bodies send out multiple primary dendritic arbors to the surrounding environment while each extends a single axon toward the VNC. This morphology is in contrast to most insect neurons, such as femoral depressor motoneurons and mushroom body gamma-neurons, which extend a single branch from the soma that later gives rise to both dendrites and axons. As such, C4da neurons may have developed separate mechanisms at the soma to remodel just the dendrites. Just what these mechanisms might include is currently unknown (Kuo, 2005).

This study has provided evidence that certain mature neurons have the ability to selectively degrade their dendritic projections in vivo and regrow new ones. Although fly metamorphosis is a specialized developmental process, dendrite-specific remodeling may provide a paradigm for neurons to retain part of their connections in the neuronal circuitry while responding to environmental changes such as tissue degeneration near their dendrites. Certain conditions in mammalian systems, such as trauma and injury, can induce localized degeneration and remodeling and may mimic the active tissue remodeling during metamorphosis. In the human CNS, for example, significant reorganization of granule cell projections in the dentate gyrus after human temporal lobe epilepsy has been observed. Thus, it would be of great interest to examine whether dendritic-specific remodeling of C4da neurons in Drosophila represents an evolutionarily conserved mechanism for neurons to respond to drastic changes in their environment, and to determine whether mammalian neurons have similar capacities to remodel their dendrites (Kuo, 2005).

Larval and Pupal (part 2)

Continued: Ecdysone receptor Developmental Biology part 2/2

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