Ecdysone receptor



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).

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

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).

GFP in living animals reveals dynamic developmental responses to ecdysone during Drosophila metamorphosis

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).

Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila

Steroid hormones fulfill important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. This study reports that TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 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 (shits1). The shibire gene is the Drosophila homolog of dynamin, which is involved in cellular membrane functions, including endocytosis, phagocytosis, and membrane growth. Because Shits1 protein acts as a dominant-negative at a restrictive temperature, targeted expression of Shits1 in glial cells would inhibit endocytosis and other membrane-related functions (Awasaki, 2004).

Larvae expressing Shits1 in glial cells by using the GAL4-repo driver (repo>shits1) 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>shits1 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>shits1 in the restrictive temperature were devoid of any glial processes even at 18 hr APF. This demonstrates that Shits1 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>shits1 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>shits1 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).

Dendritic growth gated by a steroid hormone receptor underlies increases in activity in the developing Drosophila locomotor system

As animals grow, their nervous systems also increase in size. How growth in the central nervous system is regulated and its functional consequences are incompletely understood. These questions were explored using the larval Drosophila locomotor system as a model. In the periphery, at neuromuscular junctions, motoneurons are known to enlarge their presynaptic axon terminals in size and strength, thereby compensating for reductions in muscle excitability that are associated with increases in muscle size. This work examined how motoneurons change in the central nervous system during periods of animal growth. I was found that within the central nervous system motoneurons also enlarge their postsynaptic dendritic arbors, by the net addition of branches, and that these scale with overall animal size. This dendritic growth is gated on a cell-by-cell basis by a specific isoform of the steroid hormone receptor ecdysone receptor-B2, for which functions have thus far remained elusive. The dendritic growth is accompanied by synaptic strengthening and results in increased neuronal activity. Electrical properties of these neurons, however, are independent of ecdysone receptor-B2 regulation. It is proposed that these structural dendritic changes in the central nervous system, which regulate neuronal activity, constitute an additional part of the adaptive response of the locomotor system to increases in body and muscle size as the animal grows (Zwart, 2013).

The implications of these observations are twofold: First, neurons can deploy structural changes in their dendritic trees as a central mechanism with which to regulate and adjust levels of neuronal activity; second, in terms of connectivity, the size of the postsynaptic dendritic arbor seems to be decisive in determining the number of connections that neurons form among available presynaptic terminals (Zwart, 2013).

Increases in body or organ size are normally accompanied by matching changes in innervation, required to maintain appropriate neuronal control. One of the best-studied examples is the neuromuscular junction where increases in muscle size lead to biophysical changes in muscle physiology, which are compensated for by a matching enlargement of the neuromuscular junction. The neural and cellular mechanisms that regulate these homeostatic adjustments have been studied in detail. This study has identified a potential additional, central mechanism associated with adjustment to growth in the neuromuscular system: In the central nervous system, motoneurons also enlarge their postsynaptic dendritic arbors as animals increase in body size; this leads to increased synaptic drive and thus prolonged periods of bursting activity (Zwart, 2013).

Morphometric quantifications of dendritic arbors during larval stages, show that motoneuron dendritic trees increase their overall dendritic length proportionately to body size. The growth of these arbors occurs by addition of new dendritic segments that 'fill in' existing territory, as well as at the perimeter of the tree, thus widening its reach. Such scaling growth of dendritic arbors in relation to body size is a widespread phenomenon and has previously been observed in different types of nerve cells, including Purkinje, pyramidal, olfactory mitral cells and sympathetic ganglionic neurons (Zwart, 2013).

An interesting discovery of this study is that the growth that normally occurs during larval stages is regulated cell-autonomously. Specifically, this study established that the B2 isoform of the ecdysone steroid hormone receptor is required cell-autonomously in motoneurons for normal dendritic growth. Expression of a dominant-negative form of EcRB2, UAS-EcR-B2W650A, in single cells prevents the characteristic increase in motoneuron dendritic arbor size during the second larval instar stage and seems to arrest neural arbors structurally at a young larval stage, despite being embedded in an otherwise normally developing ganglion. Electrical excitability of aCC and RP2 motoneurons, however, are not affected by expression of EcR-B2W650A. It was found EcR-B2 is the only isoform expressed in the larval nerve cord during early larval stages, in agreement with and complementary to previously published data. Although different functions have been ascribed to the other two EcR isoforms, A and B1, the role of the B2 isoform had until now remained unknown. This study has uncovered an important role for the EcR-B2 isoform in nervous system development, namely to permit growth in larval stages. The role of EcR-B2 is intrepeted as being a permissive factor for dendritic growth for two reasons. First, the pattern of dendritic growth during larval stages does not follow ecdysteroid titers but may be exponential. Second, precocious or overexpression of the wild-type form of EcR-B2 does not cause abnormal dendritic growth. Because the nervous system is one of the most metabolically expensive tissues, it is conceivable that gating the decision through EcR-B2 on whether or not neurons grow may provide a strategy to synchronize neural growth with the growth of the animal as a whole, as it progresses from one developmental stage to the next. Indeed, production of ecdysteroids in the Drosophila larva is under nutritional control (Zwart, 2013).

It is likely that other signaling pathways determine the extent to which neurons grow. For example, in rat superior cervical ganglion cells dendritic growth correlates with peripheral target size and NGF has been implicated. In Drosophila, three neurotrophic factors have been identified, expressed in subsets of body-wall muscles at embryonic stages, although none has thus far been reported to be expressed in the dorsal musculature, whose innervating aCC and RP2 motoneurons were analyzed in this study. Other muscle and associated glia-derived retrograde regulators of neuromuscular junctions include the TGF-β homologs Glass bottom boat and Maverick and the Wnt family member Wingless. It is conceivable that these could regulate the growth of motoneuron dendritic arbors in synchrony with that of presynaptic axon terminals. Indeed, it was fiynd that, in addition to its role in regulating dendritic growth, EcR-B2 is also required for normal neuromuscular junction growth, suggesting that EcR-B2 itself regulates the development of both pre- and postsynaptic compartments (Zwart, 2013).

As the Drosophila larva grows around 100-fold in surface area with matching increases in body wall muscle size, appropriate levels of muscle depolarization have to be maintained. The larval body-wall muscles are virtually isopotential, and as their input resistance goes down with increasing muscle size, presynaptic output at the neuromuscular junction increases in a compensatory fashion: By adjusting both terminal size and synaptic strength the amplitude of postsynaptic responses in the muscle is maintained. Similarly, at the growing vertebrate neuromuscular junction, motor endplates expand as muscles enlarge, enhancing neurotransmitter release. The findings of this study show that in addition to the well-characterized regulation of neuromuscular junction strength, in the central nervous system, motoneurons also adjust the size of their dendritic terminals. Previous studies have demonstrated that during the initial assembly of the locomotor network in the embryo neurons deploy their dendritic arbors as structural homeostatic devices, adjusting their extent to compensate for naturally occurring variations in the density of synaptic partner terminals. This study has shown that in subsequent larval stages, when animals grow rapidly, extension and elaboration of dendritic arbors leads to greater numbers of presynaptic inputs and thus increased synaptic drive. Specifically, with progression from the second to the third larval instar the duration of action potential bursts approximately doubles. Longer action potential burst periods might increase and potentially prolong muscle contractions; they could also enhance facilitation. Furthermore, the time course of excitatory junctional potentials (EJPs) at the Drosophila neuromuscular junction changes as muscles enlarge: Time constants describing both the rise and fall of EJPs increase, which, taking into account the bursting input the muscle receives, will result in a larger envelope of depolarization as the animals grows. It is therefore conceivable that the increased number of action potentials fired per burst that were observed in this study affects the strength of the neuromuscular synapse by enhancing both the process of facilitation and the envelope of depolarization (Zwart, 2013).

Having identified EcR-B2 as a regulator of dendritic growth of motoneurons, this study has investigated how dendritic growth relates to synaptic drive in this system. During normal development, motoneurons increase their dendritic arbor proportionally to animal body size. A biophysical consequence of increased neuronal size is increased capacitance and decreased input resistance, both of which reduce the cell’s intrinsic excitability. Indeed, this study found that motoneurons in third instar larvae (48 h ALH) have larger dendritic arbors and are less excitable than smaller cells of younger, second instar (24 h ALH) animals. It was also found that increases in dendritic arbor size are accompanied by increases in the frequency of spontaneous mEPSPs, suggesting that the dendritic growth during larval development facilitates the addition of synapses. It was asked how these changes in synaptic input, from the second to the third larval instar, might lead to extended bursting periods. Most likely, this requires the addition of synapses from new premotor partner neurons. Interestingly, at 48 h ALH EcRB2W650A– expressing motoneurons, despite being located within an otherwise unmanipulated nervous system with its third instar complexity and density of presynaptic release sites, are indistinguishable from younger neurons in a younger ganglion, in terms of dendritic arbor size, distribution of dendritic branches in the neuropil, number of synaptic sites on these, and activity patterns. This suggests that the size and geography of these dendritic arbors is decisive in determining their connectivity. Because increases were found in the duration of action potential bursts over developmental time, it is likely that as they grow aCC and RP2 motoneuron arbors establish new presynaptic contacts from additional interneurons, some of which may be in adjacent segments. These could include segmental homologs of those with whom they already form connections more proximally at earlier stages. Such a scenario could extend the duration of the synaptic drive, as was observe: As each wave of activity passes through the nerve cord during locomotion cycles, synapses in adjacent segments would have different timings that when combined on one dendritic arbor could lead to prolonged periods of action potential bursts. Indeed, tentative evidence has shown that larval Drosophila motoneurons change their connectivity qualitatively, in that neurons begin to show inhibitory responses during larval development (Zwart, 2013).

Comparable strategies have been documented in other systems. For instance, the substantia nigra compacta dopaminergic neurons change their dendritic architecture to alter the number and identity of synapses they receive and thereby also their functional properties within the network. In the case of the aCC and RP2 motoneurons in Drosophila that are studied here in this work, the identity of their excitatory presynaptic partners has not yet been characterized beyond being cholinergic and so at this point cannot be resolved conclusively (Zwart, 2013).

The electrical excitability of aCC/RP2 is not affected by expression of EcRB2W650A. The excitability of a cell is determined by the input resistance, the sum of all leak currents at rest, and the voltage-sensitive conductances that generate the action potential. The input resistance of a cell is normally inversely proportional to its size. Consistent with this notion, this study found that during normal development the electrical excitability of these neurons decreases during the second larval instar stage, as cells increase in size. Moreover, no changes were recorded in the relationship between membrane potential and action potential firing, suggesting there is no net change in the voltage-sensitive conductances that generate the action potential, and this aspect is not affected by EcR-B2W650A expression. However, the inverse relationship between cell size and excitability no longer holds for cells expressing EcR-B2W650A: At 48 h ALH, the intrinsic excitability of these comparatively small cells, which are reduced both in dendritic arbor and soma size, is similar to that of their age-matched, larger, control counterparts. These findings are compatible with at least two scenarios. First, it is possible that the location of the action potential initiation zone, a key determinant of excitability of these neurons, changes from the second to the third larval instar stage. For example, the positioning of the action potential initiation zone relative to the proximal primary neurite, which integrates dendritic currents, may change as nerve cords enlarge, and this could be independent of EcR-B2 signaling in individual motoneurons. However, in agreement with an earlier study, this study found that the intrinsic excitability of aCC and RP2 motoneurons strongly correlates with the amplitude of synaptic input (78). In this model, EcR-B2W650A–expressing neurons would undergo homeostatic adjustment to remain within the normal range of neuronal activity. Given that no changes were detected in the voltage-sensitive conductances that generate the action potential, but measured a reduction in membrane resistance over developmental time, leak channels could be involved (Zwart, 2013).

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 Sb63B 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 (PI3KDN) 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>PI3KDN 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>PI3KDN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3KDN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3KDN 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>PI3KDN 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>PI3KDN 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>PI3KDN 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>PI3KDN 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>PI3KDN 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 PI3KDN 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, Mmp1Q273 and Mmp2W307, 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 Mmp1Q273 and Mmp2W307 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. Mmp1Q112Mmp2W307 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).

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis

The fusion of epithelial sheets is an essential morphogenetic event. The development of the abdomen of Drosophila was studied as a model of bounded epithelia expansion; a complex multistep process was uncovered for the generation of the adult epidermis from histoblasts, founder cells that replace the larval cells during metamorphosis. Histoblasts experience a biphasic cell cycle and emit apical projections that direct their invasive planar intercalation in between larval cells. Coordinately, the larval cells extrude from the epithelia by apical constriction of an actomyosin ring and as a consequence die by apoptosis and are removed by circulating haemocytes. The proliferation of histoblasts and the death of larval cells are triggered by two independent extrinsic Ecdysone hormonal pulses. Histoblast spreading and the death of larval cells depend on a mutual exchange of signals and are non-autonomous processes (Ninov, 2007).

Ecdysone acts as a significant temporal signal in Drosophila, triggering each of the major developmental transitions. Although most of the genetic elements involved in Ecdysone signal transmission are known, the difficulty in visualizing morphogenetic changes in vivo and interfering with signal reception in individual cells has become a major impediment in understanding of Ecdysone actions during metamorphosis (Ninov, 2007).

In vitro culture studies have shown that Ecdysone pulses are crucial for the morphogenesis of adult appendages. Other studies have uncovered the ecdysteroid dependence of multiple differentiative and maturational responses. Nonetheless, little is known about ecdysteroid control of cell proliferation. A revealing analysis in Manduca showed that proliferating cells of the optic lobe reversibly arrest in G2 whenever the concentration of ecdysteroid drops below a critical threshold. Furthermore, earlier studies had shown that the number of histoblasts is reduced in hypomorph mutants for EcR isoforms. Whether this was the result of lack of proliferation or cell death had not been defined. This study shows in vivo a direct role for Ecdysone in cell proliferation. The rapid cell cycles experienced by abdominal histoblasts at the end of the third larval instar halt if Ecdysone-signalling reception is cell-autonomously compromised. Histoblasts remain quiescent in G2 and competent to resume proliferation in response to late Ecdysone pulses. These observations suggest that Ecdysone signalling controls the cell cycle by regulating the expression of genes involved in the G2-to-M transition (Ninov, 2007).

The destruction of larval tissues in Drosophila also results from a major transcriptional switch triggered by Ecdysone. The anterior larval muscles and larval midgut and the head and thoracic LECs degenerate during the first half of prepupal development (prepupal Ecdysone peak), while the larval salivary glands, abdominal muscles and abdominal polyploid larval epidermal cells (LECs) histolyze after the second Ecdysone pulse (pupation). Given that the exposure to Ecdysone is systemic, the stage-specific cell death responses of different cell types to Ecdysone must be differentially regulated (Ninov, 2007).

The death of abdominal LECs shows apoptotic characters and proceeds in two steps: the basal extrusion of cells initiated by the contraction of an apical actomyosin ring, and their removal by haemocytes. The cell-autonomous inhibition of EcR activity in LECs led to abortive extrusion and cell survival. Thus, the death of LECs share with other obsolete larval cells a common priming hormonal (Ecdysone) input (Ninov, 2007).

It is still not clear how cell proliferation and cell death are differentially controlled by Ecdysone. The trigger of histoblast proliferation seems to be directly dependent on Ecdysone signalling. However, it is still not known how the onset of LEC death is set. In other words, how do LECs distinguish between the late larval and the pupal Ecdysone pulses? In a plausible scenario, to avoid detrimental epithelial gaps at the surface, signalling clues from 'matured' histoblasts (after their rapid proliferation in response to the initial prepupal Ecdysone pulse), could assist Ecdysone signalling to instruct LECs to die. Indeed, LECs do not die in response to the pupal Ecdysone pulse if histoblast proliferation (and hence, 'maturation') has been experimentally delayed. The identification and characterization of this putative signal awaits further genetic and molecular analysis. Thus, Ecdysone signalling is necessary, but not sufficient, for LEC death (Ninov, 2007). The developmental control of cell cycle dynamics and diversity represents a key regulatory mechanism that directs cell size, cell number and ultimately the organ size of adult individuals. Despite numerous elegant experiments, the details of how cell division is regulated and coupled to cell growth remain poorly understood (Ninov, 2007).

During abdominal morphogenesis, the trigger of cell proliferation occurs simultaneously in all histoblast nests within each segment. Cell counting reveals that up to eight cell divisions are required to build the complete adult hemitergite. The same proportions apply to the ventral and spiracular nest. The first three histoblast divisions during pupariation are synchronous and extremely fast, skip the G1 phase and resemble the early embryonic blastoderm divisions. In this early stage, histoblast cleave and progressively reduce their size. Ecdysone signalling was found to be involved in the initiation of the proliferation programme. But, how is the histoblast cell cycle regulated to achieve fast proliferation in the absence of cell growth? Does it rely on the storage of preexistent control molecules, as in early embryos, or is it linked to signals impeding their growth? While this issue remains to be unravelled, the extreme growth of histoblasts during previous larval stages makes plausible the accumulation of G1 regulators, which, upon Ecdysone signalling, could allow a fast transition through G1 phase. Indeed, it was found that Cyclin E concentration (which regulates entry into S phase) in histoblasts builds up during the larval period. The observed deceleration of histoblast proliferation could then be the consequence of the exhaustion of the entire stock of Cyclin E. Still, the implication of growth control mechanisms in the regulation of histoblast proliferation cannot be ruled out. Multiple cell types, such as the animal-cap blastomeres from Xenopus embryos, change their cell cycles from size-independent to size-dependent after they become smaller than a critical cell size. Histoblasts might sense size in an analogous way. Thus, pathways that regulate growth, such as insulin-mediated signalling, Myc and Ras oncoproteins and the products of the Tuberous sclerosis complex 1 and 2 genes (reviewed by 22-122">Jorgensen and Tyers, 2004), should be explored to evaluate their potential roles in the coupling mechanism linking growth and cell cycle progression (Ninov, 2007).

Histoblast nest spreading initiates with the projection of leading comet-like protrusions, followed by apical cytoskeletal activity and active crawling over the underlying basal membrane, and terminates with the implementation of an apparent purse string, reminiscent of those described during dorsal closure, C. elegans ventral enclosure or wound healing (Ninov, 2007).

The comet-like protrusions of guiding histoblasts break through the LEC epithelial barrier, leading to planar intercalation of histoblast cell bodies. They account for the capacity of histoblasts to achieve migration within the bounded epithelial layer. Indeed, electron micrographs reveal that the advancing histoblasts form junctions with non-adjacent LECs before the adjacent LECs histolyze, thus insuring the continuity of the epidermis. Time-lapse observations suggest that these protrusions grow by sequential addition of actin molecules at their forward end. In this sense, they resemble, although being considerably slower, the actin tails employed by Listeria to propel through the cytoplasm of infected cells, or the actin-rich pseudopodia extended by neutrophils in response to chemoattractants. Proper actin cytoskeleton dynamics appear to be essential to build up these protrusions and the full repertoire of activities leading to the expansion of histoblast nests. The equilibrium between actin polymerization and depolymerization activities should be exquisitely regulated, and the forced polymerization of actin by Profilin overexpression not only blocks the cytoskeletal dynamics of single cells, but impedes the spreading of the whole histoblast nest. Potential roles for further actin dynamics regulators, the Arp2-Arp3 (Arp14D-Arp66B - FlyBase) complex, Dynamin (Shibire), membrane polyphosphoinositides, Cdc42, WASp-family proteins and other molecules in building up these projections remain to be explored. Further, although these protrusions appear to have a mechanical role, they also seem to be involved in the recognition of guidance cues, as they follow stereotyped paths. Indeed, gradients of cell affinity have been described for the patterning of the Drosophila abdomen, and it would be of major interest to understand how these cells interpret the larval landscape (Ninov, 2007).

The mechanisms involved in the death of LECs have been a matter of debate. While ultrastructural analysis suggests that LECs are phagocytosed, other studies suggested that LECs are histolyzed and die by autophagy. The current findings are conclusive in this respect. The death of LECs involves a caspase-mediated apoptotic process that implicates cytoskeletal remodelling and apical cellular constriction leading to delamination. The actomyosin mediated contractile force of dying LECs contributes in bringing together neighbouring histoblasts. Once the LECs initiate extrusion, they become immediate targets for circulating haemocytes, which extend membrane projections and engulf them. Finally, LECs are degraded inside haemocytes (Ninov, 2007).

Apical constriction is a process shared by multiple morphogenetic events, e.g., Drosophila mesodermal cells accumulate myosin and apically constrict during gastrulation under the control of the small GTPase Rho. Myosin activity is also sufficient to promote the apical constriction and elimination of photoreceptor cells in the Drosophila eye in response to the overexpression of an activated form of the Rok kinase. Indeed, this study found that the apical contractility of LECs depends on the level of phosphorylation of the MRLC and could be enhanced or abolished by modulating the counteracting kinase and phosphatase activities of Rok and MLCP. As a consequence, LEC delamination is either accelerated or delayed. How these regulatory activities are themselves regulated remains to be established. Yet, the LEC extrusion defects observed in weakened caspase cascade conditions after P35 overexpression strongly suggest that apoptotic signals could be involved in the trigger of actomyosin contractility in LECs. Apical contraction would thus be an early event in the LEC apoptotic process. Being particularly important to analyse the differences that modulate the activity of myosin during apical constriction of living cells and during extrusion of apoptotic cells, the replacement of LECs could become an exceptionally suitable model to unravel how myosin activity is regulated in apoptotic cells in vivo (Ninov, 2007).

The recruitment of haemocytes to dying LECs during abdominal cell replacement is extremely fast. The apical constriction of LECs takes about 2 hours, but the time that a haemocyte needs to fully engulf a LEC is less than 10 minutes. This entails a very reliable chemoattracting mechanism. In mammals, caspase 3-dependent lipid attraction signals, released by dying cells, induce the migration of phagocytes. Furthermore, several receptors are implicated in corpse recognition, including lectins, integrins, tyrosine kinases, the phosphatidylserine receptor (PSR) and scavenger receptors. In Drosophila, the elements involved in cell recognition by macrophages are mostly unknown. Haemocytes express Croquemort, a scavenger receptor homologue, which is required for the uptake of dead cells, and Pvr, a homologue of the vertebrate PDGF/VEGF receptor that seems to affect their motility. Still, the signals that haemocytes recognize in dying cells and the links between those signals and the apoptotic cascade are essentially unknown (Ninov, 2007).

As macrophages are responsible for much of the engulfment of dead cells in developing animals, an important role for macrophages in tissue morphogenesis has been suggested. However, this is not the case during abdominal morphogenesis, as the inhibition of haemocyte motility, which abrogates the removal of LECs, does not affect their replacement by histoblasts. These results are consistent with studies showing that macrophage removal of cell debris is not required for the regeneration of laser-induced wounds in Drosophila (Ninov, 2007).

Histoblast nest expansion is tightly coordinated with LEC removal. A naive view of the process of LEC extrusion suggests that their death is altruistic - it would promote the expansion of histoblasts. However, several results suggest that LECs do not execute this process autonomously. First, histoblast nests initiate their expansion in the absence of LEC death. Second, histoblast nests, during their spreading, grow, with no obvious planar orientation, by stochastic cell divisions not restricted to their edges. Finally, and most importantly, the inhibition of histoblast proliferation exerts non-autonomous effects on both extrusion and removal of LECs. A working model in which histoblast proliferation and LEC death are synchronized by a spatially and temporally controlled exchange of signals (secreted ligands or cell-to-cell communication modules) is thus strongly appealing. This potential mechanism for replacement of LECs by histoblasts somewhat resembles the elimination and death by anoikis of amnioserosa cells upon dorsal closure completion during Drosophila embryogenesis. Through this process, physical contacts and intracellular signalling among epithelial leading cells, the amnioserosa and the yolk sac coordinate the different behaviour of these cell types, which is essential for the accurate progress of both germ band retraction and dorsal closure. In this scenario, coordinated extrinsic and intrinsic events, hormonal inputs, cell contacts and cell signalling events will be responsible for the ordered proliferation and expansion of histoblasts and the extrusion and death of LECs (Ninov, 2007).

An alternative mechanism for the ordered cell substitution taking place during abdominal morphogenesis involving cell competition could also be proposed. Competition can be defined as an interaction between individuals brought about by a shared requirement leading to a reduction in the survivorship, growth and/or reproduction rates. Classical experiments in Drosophila imaginal discs have shown that cells heterozygous mutant for ribosomal protein genes (Minutes) placed beside wild-type cells are outcompeted and eliminated from the epithelium. More recent work has shown that imaginal wild-type cells are outcompeted by cells with growth advantage overexpressing the proto-oncogene Myc. Cell competition does not just apply to the fight for survival of cells with their 'fitness' experimentally altered, but also applies to the homeostasis of self-renewing cell pools such as lymphocytes or stem cells. The substitution of LECs by histoblasts closely resembles cell competition. Rapidly dividing and expanding histoblasts may become competent to displace the surrounding less-metabolically-active LECs. During normal development, having 'weaker' neighbours, histoblasts do not compete against each other, and cells from Minute clones in the abdomen are not eliminated in heterozygous animals. However, when confronted with death-resistant LECs, 'winner' histoblasts may become 'losers'. Histoblasts in an increasingly crowded environment will compete against each other, and the less fit individuals (less competent in signalling reception and transduction, or with slower proliferation rates) would eventually become more sensitive to 'killing' signals and would die (Ninov, 2007).

These findings demonstrate that the replacement of LECs by histoblasts, independently of being driven by cooperative mechanisms, cell competition or both, represents an extremely amenable morphogenetic model for the study of the dynamic control of the cell cycle and cell death, of the coordination of cytoskeleton activities and cell adhesion, and for the study of cell invasiveness (Ninov, 2007).

Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. This study implicates Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. These data thus identify Atf3 as a new functional partner of Drosophila Jun during development (Sekyrova, 2010).

Metamorphosis of Drosophila larvae into pupae and adult flies provides remarkable examples of morphogenetic changes that involve replacement of entire cell populations. Epithelia that had served larval function undergo programmed cell death while imaginal cells proliferate and differentiate to take their position. The Drosophila abdomen is an attractive system for studying the developmental replacement of one epithelial cell population with another. Unlike the adult head and thorax with appendages, all forming from pre-patterned imaginal discs, the adult abdomen derives from histoblasts that reside in each abdominal segment. Soon after the onset of metamorphosis, the diploid histoblasts undergo an initial phase of synchronized cell divisions; later the histoblasts expand while proliferating and replace the old polyploid larval epidermal cells (LECs) that cover the surface of the abdomen. To free space for the histoblasts, LECs are extruded from the epithelial monolayer. In order to maintain integrity of the epithelia, changes in cell adhesion and cell migration must be precisely orchestrated during this tissue remodeling (Sekyrova, 2010).

Rho kinase signaling, which stimulates constriction of the apical actomyosin cytoskeleton through myosin phosphorylation, is necessary for the extrusion and the ensuing apoptosis of LECs. Perturbed myosin phosphorylation leaves the process of the epithelial exchange incomplete, with residual LECs obstructing closure of the adult abdominal epidermis at the dorsal midline. A similar defect results from compromised function of the ecdysone receptor (EcR), which is required for both the initial phase of histoblast proliferation and for the removal of LECs. Other factors besides Rho signaling and EcR that regulate the epithelial cell replacement are unknown (Sekyrova, 2010).

This study implicates Atf3 (A3-3 -- FlyBase), the single Drosophila ortholog of the vertebrate Activating transcription factor 3 (ATF3) and Jun dimerization protein 2 (JDP2) in abdominal development. ATF3 and JDP2 belong among basic region-leucine zipper (bZIP) proteins, some of which play important roles in epithelial morphogenesis. Particularly the functions of Jun and Fos bZIP proteins in epithelial closure events during development are well understood owing to genetic studies in Drosophila. By contrast, no morphogenetic function has yet been reported for Atf3 in Drosophila (Sekyrova, 2010).

Mammalian ATF3 and JDP2 form homodimers but preferentially dimerize with members of the Jun subfamily (Aronheim, 1997; Hai, 1989; Hsu, 1991), functioning either as transcriptional activators (ATF3-Jun) or repressors (JDP2-Jun). Based mainly on cell-culture studies, multiple roles in cell proliferation, differentiation and apoptosis have been ascribed to ATF3 and JDP2. Atf3-/- mice are viable but suffer from altered glucose and immune homeostasis. Also Jdp2-/- mice survive but produce extra fat in their brown adipose tissue. In vivo significance of the interaction between the ATF3 or JDP2 proteins and Jun remains unclear (Sekyrova, 2010).

This study shows that Atf3 interacts biochemically and genetically with Jun in Drosophila. Temporal downregulation of atf3 transcription during metamorphosis is crucial, since sustained atf3 expression alters adhesive properties of LECs, thus preventing their extrusion and replacement by the adult epidermis. This effect of Atf3 requires the presence of Jun (Sekyrova, 2010).

Among Drosophila bZIP proteins, the predicted product of the CG11405 gene (also referred to as a3-3), located on the X chromosome, shows the closest similarity to the mammalian ATF3 and JDP2 proteins. The DNA-binding/dimerization bZIP domains of the human ATF3 and Drosophila Atf3 proteins are identical in 60% of their amino acids; there is 58% identity between Atf3 and JDP2 in this region (Sekyrova, 2010).

Dimerization between Atf3 and Jun in Drosophila has been theoretically predicted and confirmed by a yeast two-hybrid screen. To demonstrate direct binding, co-immunoprecipitation experiments were conducted. The endogenous Jun protein from Drosophila S2 cells co-precipitates with a transiently expressed Atf3 whereas Fos did not. A DNA mobility-shift assay with recombinant bZIP domains of Atf3, Jun and Fos was conducted to test for their DNA-binding properties. Atf3 specifically bound an ATF/CRE consensus element but not the AP-1 site, which was recognized by the Jun-Fos (AP-1) complex. Although Atf3 bound DNA by itself, presumably as a homodimer, the binding was enhanced in the presence of Jun. Fos did not synergize with Atf3 in DNA binding. Excess unlabeled DNA bearing the ATF/CRE binding site competed for the Atf3 bandshift activity whereas the AP-1 binding element did not. These results have shown that, like ATF3 or JDP2 in mammals, Atf3 in Drosophila selectively dimerizes with Jun, with which it cooperatively and specifically binds the ATF/CRE DNA element (Sekyrova, 2010).

To test whether Atf3 and Jun interact in vivo, experiments were conducted in the Drosophila compound eye, the precise structure of which sensitively reflects genetic interactions. Overexpression of atf3 under the GMR-Gal4 driver disrupted the ommatidial arrangement, resulting in smaller eyes with a glossy appearance. This atf3 misexpression phenotype could be completely suppressed by simultaneous RNAi-mediated knockdown of jun but not of fos. Conversely, the phenotype was exacerbated when jun was overexpressed in the eye together with atf3, suggesting that it is Atf3 in a complex with Jun that derails the normal eye development. Neither RNAi nor overexpression of jun alone had any effect on eye morphology. Interestingly, like depletion of Jun, co-expression of fos under the GMR-Gal4 driver completely averted the atf3 misexpression phenotype, restoring the normal appearance of the eye. Expression of fos or its mutant versions alone had no effect. These data can be explained by the ability of the surplus Fos to bind Jun and thus reduce its availability for interaction with the Atf3 protein. This interpretation is further supported by experiments showing that expression of the truncated bZIP domain of Fos is sufficient to suppress the Atf3 gain-of-function phenotype, whereas its transcription activation domain or phosphorylation sites are dispensable (Sekyrova, 2010).

Taken together, these results show that Atf3 cooperates with Jun, as Jun is specifically required for an effect caused by overexpression of Atf3 in the developing eye. Given the capacity of both Atf3 and Fos to bind Jun, and based on the ability of Jun to enhance and of Fos to suppress the Atf3 gain-of-function phenotype, it is suggested that Atf3 and Fos compete for their common partner Jun in vivo (Sekyrova, 2010).

To find out whether Atf3 is required for Drosophila development and whether its absence might resemble a phenotype caused by loss of its partner Jun, atf3 mutant flies were generated. The longest deletion (line atf376) obtained by imprecise excision of a P element, removed the entire bZIP domain of Atf3, and atf376 hemizygous (male) larvae lacked detectable atf3 mRNA. Thus, atf376 probably represents a null allele. Most atf376 larvae die soon after hatching and during all three larval stages. Only a few (approximately 2%) reach the third instar but die before metamorphosis as defective pseudopuparia. Expression of atf3 cDNA under the ubiquitous armadillo (arm-Gal4) driver rescued some atf376 hemizygotes to adults, confirming that loss of atf3 was the cause of the lethal phenotype. Interestingly, the moribund atf376 larvae abnormally enlarged lipid droplets in their fat body, thus displaying a phenotype reminiscent of that in mice lacking one of the Atf3 orthologs, JDP2. However, in contrast to viable Jdp2 or Atf3 knockout mice, atf3 is an essential gene in Drosophila (Sekyrova, 2010).

Fly embryos lacking the function of Jun or Fos die because of the failed dorsal closure. However, atf376 embryos develop normally, without the dorsal open defect, even when derived from atf3-deficient germline clones induced in atf376/ovoD1 mothers. Thus, unlike its partner Jun, Atf3 is not required for dorsal closure, suggesting that dorsal closure is regulated by Jun-Fos dimers and that the Atf3-Jun complex has another function later in development (Sekyrova, 2010).

Consistent with the vital requirement for Atf3 during larval stages, atf3 mRNA was expressed in embryos and larvae. Expression then sharply declines by the late-third larval instar, and no atf3 mRNA was detected by northern blot hybridization in wandering larvae and during metamorphosis from the time of puparium formation until the second day of pupal development. Detailed RT-PCR analysis showed that atf3 downregulation coincided with the cessation of feeding and the onset of metamorphosis [0 hours after puparium formation (APF)]. A pulse of expression occurred at 6 hours APF. RT-PCR from isolated fat body and abdominal integuments, together with in situ hybridization performed on puparia at this stage, showed that atf3 mRNA was primarily present in the larval epidermis (LECs) during the expression peak at 6 hours APF. From the time of head eversion (12 hours APF) the mRNA level remained low until the second day of pupal development, and then it grew steadily during morphogenesis of the adult. Quantitative RT-PCR revealed a 4.3-fold difference in atf3 mRNA abundance between 0 and 72 hours APF. In contrast to the tight regulation of atf3, the mRNAs of fos and jun fluctuated little during the examined period. Therefore, unlike Jun or Fos, Atf3 was dynamically regulated during metamorphosis at the level of transcription (Sekyrova, 2010).

The precise temporal control of atf3 expression suggested that the rise and subsequent fall of Atf3 during metamorphosis might be critical for the complex morphogenesis occurring in fly pupae. This possibility was tested by means of sustained expression of the full-length Atf3 protein using the UAS-Gal4 system with various drivers. A striking, fully penetrant metamorphic defect was observed with the pumpless (ppl) Gal4 driver. Although ppl>atf3 animals developed normally until the pupal stage, they failed to complete fusion of the adult abdominal epidermis. A dorsal cleft in the abdomen remained that could not be covered with the adult cuticle, and consequently 86% of the flies died inside the puparium. All of the ppl>atf3 adults that did eclose showed abdominal lesions filled with the old pupal cuticle lacking adult pigmentation and bristles, often with a clot covering a bleeding wound. Adults with the same abdominal cleft (but otherwise normal) also emerged when atf3 was moderately and ubiquitously misexpressed under the arm-Gal4 driver, suggesting that abdominal morphogenesis was the process most sensitive to ectopic Atf3 (Sekyrova, 2010).

The adult fly abdomen derives from histoblasts that proliferate, replace LECs and finally differentiate, giving rise to the adult cuticle. Therefore, the observed abdominal defect suggested a compromised function of the epidermis, either LECs, histoblasts or both cell types. To distinguish between these possibilities, expression of the ppl-Gal4 driver was first examined in the epidermis. It was found that ppl-Gal4 was active in LECs but not in histoblasts. Second, another driver, Eip71CD-Gal4, which was inactive in histoblasts but strongly expressed in LECs, was examined. Eip71CD-Gal4-driven misexpression of atf3 mostly produced lethal pupae lacking adult cuticle, but it occasionally yielded adults with a dorsal abdominal cleft. In addition to being active in LECs, both ppl-Gal4 and Eip71CD-Gal4 (data not shown) were also expressed in the fat body. However, no abdominal defects occurred when atf3 was misexpressed under either of three fat-body-specific Gal4 drivers, Lsp2, Cg or C7. Third, to rule out the possibility that ectopic Atf3 affected the imaginal epidermis, its expression was directed to histoblasts by using the escargot (esg) and T155 Gal4 drivers; in neither case the fusion of the adult abdominal epidermis was affected (Sekyrova, 2010).

To finally confirm that abdominal morphogenesis was disrupted by sustained atf3 activity in LECs, atf3 was induced by using the flp-out technique. Owing to the timing of heat-shock induction to the mid-third instar, this method triggers expression in the polyploid larval cells but not in the diploid histoblasts . Misexpression of atf3 under the actin promoter following the flp-out event invariantly led to an abdominal cleft. The lesions were often more severe than those observed in ppl>atf3 animals, affecting also lateral and ventral parts of the abdomen. Together, the above data demonstrate that the sustained expression of atf3 prevents fusion of the adult abdominal epidermis by acting upon LECs, suggesting that the replacement of these obsolete larval cells by adult histoblasts requires the developmental downregulation of atf3 expression (Sekyrova, 2010).

To understand the cellular events underlying the incomplete epithelial closure in ppl>atf3 animals, cell membranes were visualized by antibody staining of the septate junction component, Discs large 1 (Dlg1), or used a transgenic DE-cadherin::GFP fusion protein (shg::gfp). In wild-type animals 24 hours APF, LECs covering the surface of the abdomen gave way to the rapidly expanding nests of histoblasts that began to fuse laterally and ventrally. In ppl>atf3 pupae the histoblast nests also spread, and at least at 16 hours APF, before their fusion, they comprised normal numbers of histoblasts. By 48 hours APF a control abdomen was fully covered with adult epidermis consisting exclusively of histoblasts, now forming sensory bristles. Histoblasts in ppl>atf3 abdomens also differentiated the adult cuticle with sensory bristles, although polarity of the bristles near the dorsal cleft was altered. However, in contrast to the control, a large population of LECs remained in the dorsal abdomen of ppl>atf3 animals at 48 hours APF. The membranes of the persisting LECs accumulated the Dlg protein, and although these cells became severely deformed they survived throughout metamorphosis to the adult stage. When visualized in live ppl>atf3 pupae, the apical junctions of the remaining LECs displayed interdigitation and accumulation of DE-cadherin::GFP. Another adherens junction component, the Drosophila β-catenin Armadillo, was also enriched in atf3-expressing LECs (Sekyrova, 2010).

Cooperation between adherens junctions and the apical ring of actomyosin cytoskeleton is required for basal extrusion of LECs. The altered pattern of DE-cadherin and β-catenin therefore suggests that excessive Atf3 might prevent LEC extrusion through stabilization of the cell-cell adhesion complex. To examine the effect of Atf3 on LECs in further detail, the flp-out technique, which allows comparisons of atf3-misexpressing and control LECs within one tissue, was employed. Membrane interdigitation occurred between atf3-positive LECs already at 18 and 24 hours APF, even in areas where the LECs had no contact with histoblasts. At 48 hours APF only LECs expressing atf3 persisted, apparently being squeezed by the expanding histoblasts. The membrane-associated DE-cadherin::GFP signal was stronger in adjacent atf3-positive LECs compared with non-induced LECs, and quantitative analysis of confocal images acquired at 18 hours APF and at 24 hours APF both revealed a statistically significant 1.4-fold increase of the DE-cadherin::GFP signal intensity upon atf3 induction. Enrichment of DE-cadherin on apical membranes of atf3-expressing LECs was further confirmed on confocal cross sections (Sekyrova, 2010).

Although some atf3-positive LECs began the extrusion process, they could not detach from the apical surface even when entirely surrounded by histoblasts, possibly being tethered to it by the excessive adhesion protein. By contrast, control LECs did completely separate from the epithelium. In addition, LECs overexpressing atf3 displayed apical enrichment of moesin, an actin-binding protein of the ERM (ezrin, radixin, moesin) family, which links transmembrane proteins to cortical actin filaments. Interestingly, prominent accumulation of DE-cadherin was also observed in atf3-expressing clones of epithelial cells within the hinge region of wing discs that form the adult thorax, indicating that the effect of Atf3 on cell adhesion components may not be limited to larval epithelia (Sekyrova, 2010).

In summary, these results show that deregulation of atf3 expression causes marked changes of cell membranes, including interdigitation and accumulation of cell adhesion molecules, suggesting that LEC adhesiveness might be increased. Although some of the affected LECs initiate extrusion, this process stays incomplete. Consequently, the adhering LECs present a physical barrier for the migrating histoblasts (Sekyrova, 2010).

Rho kinase (Rok)-dependent phosphorylation of myosin regulatory light chain was shown to be required for LEC extrusion. To examine a possible relationship between the Rok-dependent cytoskeletal regulation and Atf3, the function of the GTPase Rho1 (also called RhoA), which acts immediately upstream of Rok, was disrupted. RNAi silencing of Rho1 using the ppl-Gal4 driver produced a phenocopy of atf3 misexpression, causing a dorsal abdominal cleft in 100% of ppl>Rho1(RNAi) adults, of which most died in the puparium and about 12% eclosed, similar to ppl>atf3 animals. However, when Rho1 RNAi and misexpression of atf3 in LECs were combined, the abdominal defect became more severe, not allowing any pharate adults to eclose. Conversely, co-expression of a dominantly active Rho1V14 protein suppressed the otherwise fully penetrant abdominal defect in some ppl-atf3 flies. Surprisingly, it was found that the endogenous Rho1 protein was mislocalized in atf3-misexpressing LECs, showing a diffuse cytoplasmic signal, compared with membrane localization in control LECs. These results suggest a genetic interaction between Rho signaling and atf3, and support the idea that excess Atf3 prevents extrusion of LECs by altering their cell adhesion properties (Sekyrova, 2010).

Disturbed function of the ecdysone receptor (EcR) has been shown to prevent extrusion of LECs, causing a dorsal abdominal cleft that closely resembles the Atf3 gain-of-function phenotype. Therefore whether stimulating EcR-dependent signaling by addition of the natural agonist 20E might overcome the defect caused by sustained atf3 expression was examined. Indeed, supplying third-instar ppl>atf3 larvae with dietary 20E increased the number of eclosing adults, the abdominal scars of which were in 22% of the cases partially or completely sealed with normal adult cuticle (Sekyrova, 2010).

Atf3 interacts with Jun to form a DNA-binding complex and genetically when overexpressed in the developing compound eye. To see if this interaction is biologically relevant during abdominal morphogenesis, whether Atf3 relies on the presence of Jun to cause the dorsal cleft phenotype was tested. First, it was confirmed that Jun is indeed expressed in LECs during metamorphosis. RNAi-mediated depletion of Jun in animals that misexpressed atf3 under the ppl-Gal4 driver restored viability of adults from 14% (atf3 alone) to 100%. Strikingly, 87% of the ppl>atf3, jun(RNAi) adults eclosed with a completely normal abdomen. By contrast, RNAi knockdown of Fos in ppl>atf3 background did not improve the abdominal defect. RNAi silencing of either jun or fos alone under the ppl-Gal4 driver had no effect on the abdomen. These results demonstrate that Atf3 requires its partner Jun but not Fos to disrupt abdominal morphogenesis. Similar to the situation in the compound eye, the effect of misexpressed atf3 can be neutralized by simultaneously expressing Fos or its truncated bZIP domain under the ppl-Gal4 driver. Therefore, the model in which Atf3 and Fos compete for their common partner Jun may be extended to the developing abdomen (Sekyrova, 2010).

This study has identified Atf3 as a new partner of Jun in Drosophila. Previously, Jun has only been known to dimerize with itself and with the Drosophila homolog of Fos. Functional analysis of Atf3 has not yet been reported. These biochemical data show that, similar to mammalian ATF3 and JDP2, the Atf3 protein selectively binds Jun but not Fos. Also consistent with the properties of ATF3 and JDP2 is the ability of Atf3 to bind the ATF/CRE response element alone or synergistically with Jun. In contrast to its mammalian counterparts, however, neither Atf3 alone nor in complex with Jun bound to the AP-1 element under the same conditions. The selective interactions of Atf3 point to distinct biological roles for the Atf3-Jun and the Fos-Jun dimers, respectively (Sekyrova, 2010).

This study has shown a genetic interaction between Atf3 and Jun. The evidence is based on the ability of ectopic Atf3 to disturb morphogenesis of the adult abdomen and the compound eye, which strictly depends on the availability of Jun. Importantly, none of the Atf3 gain-of-function phenotypes could be induced by misexpression of the truncated bZIP domain of Atf3, suggesting that the functional Atf3 protein in complex with Jun is required. Based on the selectivity of Atf3 in a DNA-binding assay, it is predicted that the Atf3-Jun complex regulates specific target genes distinct from those targeted by Fos-Jun dimers (Sekyrova, 2010).

The data also reflect a relationship between the AP-1 and Atf3-Jun complexes. Although Fos does not dimerize or bind DNA with Atf3, its ability to suppress the Atf3 misexpression phenotype in the eye suggests that Fos and Atf3 compete in vivo for their common partner Jun. The fact that the same suppression can be achieved by overexpressing either the truncated Fos bZIP domain or Fos lacking phosphorylation sites indicates that the suppression does not rely on a transcriptional function of Fos but probably occurs through sequestering of Jun, even by a transcriptionally inactive Fos protein. Early in vitro studies have proposed a competition model for the AP-1 and Atf3 proteins to explain a temporal regulation of gene expression in the regenerating liver. However, to date such a relationship among Fos, Jun and Atf3 has not been supported with direct genetic evidence (Sekyrova, 2010).

Removal of LECs is normally complete by 36 hours APF, at which time the sheets of histoblasts reach the dorsal midline. The data strongly support the argument that the temporal downregulation of atf3 expression during abdominal morphogenesis is necessary for LECs to be replaced by the adult epidermis. When experimentally sustained, atf3 activity in LECs interfered with this exchange by blocking extrusion and death of the LECs. This was evident as the atf3-expressing LECs survived within the epithelial layer for days after their scheduled destruction (Sekyrova, 2010).

Interdigitation of cell membranes and accumulation of adherens junction proteins in LECs suggested that ectopic Atf3 caused adjacent LECs to reinforce their mutual contacts. This probably resulted from altered distribution of the proteins, as levels of the shg (DE-cadherin) mRNA remained unchanged in LECs of ppl>atf3 animals. By contrast, junctions between atf3-expressing LECs and their normal neighbors or histoblasts were smooth and presumably less rigid. DE-cadherin was similarly enriched in clones of imaginal disc cells. These observations suggested that differential adhesion of atf3-expressing cells might have led to their sorting out from the surrounding epithelium. Even modest differences in cadherin levels have been shown to cause segregation of cells within a population by altering their adhesiveness (Sekyrova, 2010).

Recent live imaging data have revealed that migrating histoblasts push the LECs ahead of themselves towards the dorsal midline, where histoblasts fuse last. The atf3-expressing LECs that adhered to each other were probably moved and pressed by the expanding histoblasts to the dorsal side, whereas non-induced LECs were eliminated. This explains why the abdominal lesions primarily occurred at the dorsal midline, although flp-out experiments showed that atf3 misexpression could affect LECs in other areas as well. Strengthened contacts among persisting LECs probably blocked invasion of histoblasts in between them and inhibited LEC extrusion, eventually causing gaps in the adult epidermis (Sekyrova, 2010).

In accord with the notion that extrusion from the epithelium is a prerequisite for LECs to undergo apoptosis, it is assumed that sustained presence of Atf3 primarily enhanced adhesiveness of LECs, which only consequently prevented their death. This view is supported by the observation that membranes of atf3-expressing LECs interdigitated and accumulated DE-cadherin as early as 18-24 hours APF, even in areas of the larval epidermis that were far from histoblasts and where control LECs did not yet extrude. In addition, the Atf3 gain-of-function phenotype was stronger than abdominal closure defects caused by caspase mutation or inhibition. When the anti-apoptotic proteins p35 or DIAP1 (Thread — FlyBase) was misexpressed under the ppl-Gal4 driver, the resulting dorsal lesions were not lethal and were clearly milder than the broad, mostly fatal scars in ppl>atf3 animals. Compared with the large contiguous populations of persisting LECs in ppl>atf3 pupae, inhibiting apoptosis with p35 only allowed small islands of LECs to survive (Sekyrova, 2010).

Ecdysone signaling promotes replacement of the abdominal epithelia by stimulating both the early histoblast proliferation and the extrusion of LECs. As atf3 misexpression affected LECs but did not impair early histoblast proliferation, the latter possibility remains, that added 20E counteracted the effect of ectopic Atf3 by facilitating the extrusion process. Since normal 20E titers was detected in ppl>atf3 larvae or prepupae, the failure of LEC extrusion was not a result of steroid deficiency. Also, 20E had no effect on atf3 mRNA levels, at least in Drosophila S2 cells or third-instar larvae. Atf3 and ecdysone signaling therefore probably influence LEC extrusion by acting independently (Sekyrova, 2010).

Although the mechanism through which ecdysone contributes to LEC removal is unknown, one attractive possibility is that it might cooperate with Rho signaling, which is required for LEC extrusion as well. It has been demonstrated that genetic interaction between the 20E-response gene broad and components of the Rho pathway including RhoGEF2, Rho1 and myosin II is important for ecdysone-dependent epithelial cell elongation during Drosophila leg morphogenesis. The current data show that Rho1 becomes mislocalized in LECs upon atf3 misexpression and that Rho1 silencing enhances the abdominal gain-of-function phenotype of atf3. The exact relationship between Atf3, Rho1 and ecdysone remains to be determined. However, Atf3 clearly represents a new intrinsic regulator of epithelial cell replacement during Drosophila metamorphosis (Sekyrova, 2010).

Metamorphosis of an identified serotonergic neuron in the Drosophila olfactory system

Odors are detected by sensory neurons that carry information to the olfactory lobe where they connect to projection neurons and local interneurons in glomeruli: anatomically well-characterized structures that collect, integrate and relay information to higher centers. Recent studies have revealed that the sensitivity of such networks can be modulated by wide-field feedback neurons. The connectivity and function of such feedback neurons are themselves subject to alteration by external cues, such as hormones, stress, or experience. Very little is known about how this class of central neurons changes its anatomical properties to perform functions in altered developmental contexts. A mechanistic understanding of how central neurons change their anatomy to meet new functional requirements will benefit greatly from the establishment of a model preparation where cellular and molecular changes can be examined in an identified central neuron. This study examined a wide-field serotonergic neuron in the Drosophila olfactory pathway and mapped the dramatic changes that it undergoes from larva to adult. Expression of a dominant-negative form of the ecdysterone receptor prevents remodeling. Different transgenic constructs were used to silence neuronal activity, and defects are reported in the morphology of the adult-specific dendritic trees. The branching of the presynaptic axonal arbors is regulated by mechanisms that affect axon growth and retrograde transport. The neuron develops its normal morphology in the absence of sensory input to the antennal lobe, or of the mushroom bodies. However, ablation of its presumptive postsynaptic partners, the projection neurons and/or local interneurons, affects the growth and branching of terminal arbors. These studies establish a cellular system for studying remodeling of a central neuromodulatory feedback neuron and also identify key elements in this process. Understanding the morphogenesis of such neurons, which have been shown in other systems to modulate the sensitivity and directionality of response to odors, links anatomy to the development of olfactory behavior (Singh, 2007).

Changes in the pattern of arborization of a mature neuron can come about as a consequence of removal of its afferent inputs or targets, chronic stress or other environmental inputs, such as delivered during learning or exercise. Many of these changes are effected through the action of growth factors and developmental signals acting in concert with steroid hormones and neuronal activity to modify the cytoskeleton or synaptic properties relevant to an altered functional setting. Metamorphosis in Drosophila (a period during which mature larval neurons are often altered to take on new adult functions) provides a context where the mechanistic underpinnings of such neuronal change can be genetically dissected (Singh, 2007).

This study used a genetic method to mark the serotonin-immunoreactive deutocerebral interneurons (CSDn), recently identified on the basis of serotonin immunoreactivity. While this preparation identifies a central neuron, it also has an important feature that allows the analysis of mechanisms underlying the changes it undergoes during remodeling. This system, because of the random nature of the RN2-FLP action, results in bilateral, unilateral or no excision of the FRT element in the Tub-FRT-CD2-FRT-Gal4 construct in the CSDn. Thus, it was possible to choose and analyze preparations where the CSDn from only one hemisphere was labeled: this facility is vital as it allows the analysis of contralateral and ipsilateral projections of the CSDn, without this being obscured by projections of the neuron from the other hemisphere to the same target sites. The GFP reporter in the RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS mCD8-GFP strain is first detected very late in embryogenesis (stage 20), after the neuron has acquired its mature larval pattern. These features thus provide a preparation where an identified central neuron, whose function is known, can be followed and genetically manipulated as it changes its form in response to external and internal cues during metamorphosis (Singh, 2007).

The neuron, present during the larval stages, undergoes well-defined changes during pupation to give rise to a more complex adult architecture. What are the factors that regulate the stereotyped pruning and re-growth of arbors in the CSDn during metamorphosis? The results suggest that the interaction of external factors and autonomous properties (some of which could be identified) establish the homeostasis required during branching and establishment of the adult form (Singh, 2007).

Arbors from the larval neuron are removed by pruning over the first 20 hours of pupation before the adult pattern is elaborated. The EcR-B1 isoform, whose expression is typically seen in neurons that alter their larval form and contribute to the circuitry in the adult, is detected in CSDn. Down-regulating EcR in the CSDn during metamorphosis results in a failure of remodeling and the 'adult' neuron retains a larval morphology. The detailed mechanisms by which EcR signaling acts to bring about sculpting of cell shape are not totally understood and reports on Manduca sexta indicate that steroid-induced modifications in dendritic shape can be regulated by activity-dependent mechanisms (Singh, 2007).

Studies on the cellular and molecular mechanisms of pruning events during metamorphosis could provide valuable insights into understanding of degeneration in higher systems. These events require ubiquitin-mediated proteolysis, and it is known that local activity of caspases is involved in dendritic pruning in an identified sensory neuron. Degeneration of specific branches is followed by migration of glial cells into the site of activity. The role of these glia in bringing about pruning and in clearing debris from the vicinity requires further study (Singh, 2007).

The assembly of complex circuits is dependent on a carefully orchestrated interplay of intrinsic and extrinsic cues. Does activity play a role in determining neuronal shape? Spontaneous and evoked activity in the CSDn were silenced using different methods and changes were observed in the dendritic arbors as well as in presynaptic terminals. The effects on the terminals and dendrites are possibly due to distinct mechanisms and will be discussed separately (Singh, 2007).

The strongest effects on presynaptic terminal branching were produced by expression of TeTxLC, which blocks synaptic release, and a dominant-negative Shi protein, which affects receptor-mediated endocytosis. Apart from blocking neuronal activity by abrogating synaptic vesicle release, both treatments could potentially affect axon growth. Consistent with this is the observation that TeTxLC expression affects re-growth of CSDn terminals during metamorphosis, while pruning occurred normally. Weak anatomical defects have also been described in other, non-modulatory neurons, some of which could be explained by a role in the regulation of levels of cell adhesion molecules (Singh, 2007).

Increases in size and branching pattern of the dendritic trees is a robust effect occurring notably when neuronal activity was silenced by Kir2.1expression. In the third instar larva, expression of TNT-G leads to an increase in dendritic arbors with no significant effect on the presynaptic terminals. Expression using the RN2-Flp, Tub-FRT-CD2-FRT-Gal4, stock initiates in the fully developed larval neuron; hence, the changes in dendritic branches are likely to be a consequence of lack of neuronal activity, rather than a developmental effect. What are the mechanisms by which neuronal activity can alter morphologies of neurons? It has been demonstrated that tetanus toxin expression in motorneurons not only affects its presynaptic release because of cleavage of synaptobrevin, but also alters synaptic input by an as yet unknown mechanism. The finding of altered dendritic morphology supports the possibility that homeostatic alterations occur to compensate for a lack of activity (Singh, 2007).

A large body of data provides evidence for retrograde signaling in the development and consolidation of synapses. The observation of expanded dendritic trees upon expression of a dominant negative form of Glued, while intriguing, is difficult to explain in this light. The changes that were seen are in the dendritic (post-synaptic) field when retrograde transport is blocked cell-autonomously. While this needs further investigation, a possible explanation is that these effects are an indirect consequence of physiological alterations at the presynaptic terminals. Local morphological changes in neurons can be effected by sequestration of proteosomes and other molecules at different regions of the cell in response to activity, which could result in sculpting of cellular architecture due to altered protein composition at different cellular regions (Singh, 2007).

Defects in branching observed by abrogation of vesicle release at the synapse in a serotonergic neuron could implicate this modulator in paracrine or autocrine signaling in regulation of neuronal outgrowth, target selection and synapse formation. Such effects have been demonstrated in the gastropod Helisoma , as well as in Drosophila, where serotonin levels regulate neuronal branching and modulate the development of neuronal varicosities in the central nervous system. In these experiments, no significant changes were detected in the branching pattern of CSDn upon strong reduction of serotonin (and dopamine) using a temperature sensitive allele of dopa decarboxylase. Furthermore, unlike in M. sexta, where afferents are necessary for the formation of glomerular tufts of the serotonergic neuron within the antennal lobe, development of the CSDn occurs normally in the absence of sensory input from the antenna (Singh, 2007).

The olfactory pathway consists of afferent sensory neurons, local integrating neurons and projection neurons. Circuitry for an additional level of integration exists in the atypical projection neurons (aPNs), the antennal posterior superior protocerebral neuron (APSP), the giant symmetric relay interneurons (GSI) and the bilateral ACT relay interneurons (bACT). The architecture as well as the serotonergic nature of the CSDn closely resembles the S1 neuron in M. sexta, which receives input from bilateral projections in the protocerebrum and terminates in the lobe contralateral to the soma to modulate the activity of interneurons. It is proposed that the ipsilateral dendrites receive input from as-yet unidentified neural elements in the antennal lobe, while some axonal arbors are postsynaptic to interneurons in the calyx of the mushroom bodies and the lateral horn. It is speculated that the targets of the terminal arbors are either the PNs or the LNs since their ablation results in a reduction in branching. This architecture, which needs to be confirmed by electron microscopic analysis, provides circuitry for 'top-down' regulation of the primary olfactory center. It seems very likely that the CSDn, like its counterpart in the moth, responds to mechanosensory stimulation, providing an important role in responses to odor stimulation coupled with airflow, as would be expected in insects during flight. The modulatory effects of this large field neuron on its partners in the antennal lobe needs to be investigated by high-resolution functional imaging (Singh, 2007).

This study describes a serotonergic neuron whose anatomy suggests feedback integration within the antennal lobe of insects. The neuron undergoes remodeling during pupal life from a simple larval to a more complex adult pattern. These studies suggest that the morphology of the dendritic arbors that terminate in the lobe ipsilateral to the soma is regulated by neuronal activity. The arborization of terminal arbors depends on vesicle recycling, endocytosis and Dynein-dependant retrograde transport. These findings demonstrate a useful identified-neuronal preparation where developmental mechanisms and remodeling can be studied in the context of olfactory behavior (Singh, 2007).

Distinct presynaptic and postsynaptic dismantling processes of Drosophila neuromuscular junctions during metamorphosis

Synapse remodeling is a widespread and fundamental process that underlies the formation of neuronal circuitry during development and in adaptation to physiological and/or environmental changes. However, the mechanisms of synapse remodeling are poorly understood. Synapses at the neuromuscular junction (NMJ) in Drosophila larvae undergo dramatic and extensive remodeling during metamorphosis to generate adult-specific synapses. To explore the molecular and cellular processes of synapse elimination, confocal microscopy, live imaging, and electron microscopy (EM) of NMJ synapses were performed during the early stages of metamorphosis in Drosophila in which the expressions of selected genes were genetically altered. The localization of the postsynaptic scaffold protein Disc large (Dlg) becomes diffuse first and then undetectable, as larval muscles undergo histolysis, whereas presynaptic vesicles aggregate and are retrogradely transported along axons in synchrony with the formation of filopodia-like structures along NMJ elaborations and retraction of the presynaptic plasma membrane. EM revealed that the postsynaptic subsynaptic reticulum vacuolizes in the early stages of synapse dismantling concomitant with diffuse localization of Dlg. Ecdysone is the major hormone that drives metamorphosis. Blockade of the ecdysone signaling specifically in presynaptic neurons by expression of a dominant-negative form of ecdysone receptors delayed presynaptic but not postsynaptic dismantling. However, inhibition of ecdysone signaling, as well as ubiquitination pathway or apoptosis specifically in postsynaptic muscles, arrested both presynaptic and postsynaptic dismantling. These results demonstrate that presynaptic and postsynaptic dismantling takes place through different mechanisms and that the postsynaptic side plays an instructive role in synapse dismantling (Liu, 2010).

Temporally tuned neuronal differentiation supports the functional remodeling of a neuronal network in Drosophila

During insect metamorphosis, neuronal networks undergo extensive remodeling by restructuring their connectivity and recruiting newborn neurons from postembryonic lineages. The neuronal network that directs the essential behavior, ecdysis, generates a distinct behavioral sequence at each developmental transition. Larval ecdysis replaces the cuticle between larval stages, and pupal ecdysis externalizes and expands the head and appendages to their adult position. However, the network changes that support these differences are unknown. Crustacean cardioactive peptide (CCAP) neurons and the peptide hormones they secrete are critical for ecdysis; their targeted ablation alters larval ecdysis progression and results in a failure of pupal ecdysis. This study demonstrates that the CCAP neuron network is remodeled immediately before pupal ecdysis by the emergence of 12 late CCAP neurons. All 12 are CCAP efferents that exit the central nervous system. Importantly, these late CCAP neurons were found to be entirely sufficient for wild-type pupal ecdysis, even after targeted ablation of all other 42 CCAP neurons. Evidence indicates that late CCAP neurons are derived from early, likely embryonic, lineages. However, they do not differentiate to express their peptide hormone battery, nor do they project an axon via lateral nerve trunks until pupariation, both of which are believed to be critical for the function of CCAP efferent neurons in ecdysis. Further analysis implicated ecdysone signaling via ecdysone receptors A/B1 and the nuclear receptor ftz-f1 as the differentiation trigger. These results demonstrate the utility of temporally tuned neuronal differentiation as a hard-wired developmental mechanism to remodel a neuronal network to generate a scheduled change in behavior (Veverytsa, 2012; full text of article).

Ecdysone regulates morphogenesis and function of malpighian tubules in Drosophila melanogaster through EcR-B2 isoform

Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. This study conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and beta-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na+/K+-ATPase pump. B2 isoform of Ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development (Gautam, 2014).

An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila

Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. This study reports a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which was identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, Clock (Clk), and may also affect circadian output. Per inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of Per on Clk transcription. The Ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila (Kumar, 2014b).

Larval and Pupal (part 2)

Continued: Ecdysone receptor Developmental Biology part 2/2

Ecdysone receptor: Biological Overview | Evolutionary homologs | Regulation | Targets of Activity | Protein interactions | Effects of mutation | References

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