Although usp is transcribed throughout embyonic development, highest levels are attained in the late third instar larva. A 2.7-kb USP mRNA is detected throughout development, although its abundance varies. Among premetamorphic stages, both the 2.7-kb transcript and USP protein attain their highest levels in the late third larval instar. The 2.7-kb USP transcript is also found in adult stages and a 1.2-kb transcript is detected in the polyadenylated RNA fraction of both mature adult females and early embryos. USP is especially prevalent in wing discs, salivary gland and larval fat body cells. The fat body, like the salivary gland, responds transcriptionally to ecdysteroids (Henrich, 1994). The USP protein is required in the eye-antennal imaginal disc for normal eye morphogesis. In mutants a sunken phenotype with marked ventral-dorsal polarity is caused by a lack of usp function in cells residing between eye and antennal anlage. usp functions include cell autonomous and non-autonomous components, suggesting use of multiple pathways. usp is also required for somatic and germline tissues of adult females for fetilization, eggshell morphogenesis and embryonic development (Oro, 1992).

The propagation of the morphogenetic furrow requires coordinate action of multiple gene systems. One system involves hedgehog expressed posterior to the furrow, and leads to the induction of decapentaplegic. Hh acts by antagonizing the activity of patched and cyclic AMP-dependent protein kinase A. A second gene system involves coordinate activity of Hairy and Extramachrochaetae. hairy is transiently expressed in a line of cells anterior to the morphogenetic furrow as it traverses the eye disc. Both Hairy and Extramachrochaete negatively regulate the progression of the morphogenetic furrow in the developing eye. A third gene system is required for normal morphogenetic furrow movement. ultraspiracle, the gene encoding the Drosophila cognate of the Retinoid X Receptor, is required for normal morphogenetic furrow movement and ommatidial cluster formation. Examination of the expression of genes involved in regulating the furrow suggests that ultraspiracle defines a novel regulatory pathway in eye differentiation. patched and cyclic AMP-dependent protein kinase A activity are normal and neither hairy nor extramachrochaetae are altered in usp clones. Usp functions to repress differentiation and furrow movement. Within usp mutant clones, ommatidial clusters are misaligned with respect to one another and with respect to adjacent wild-type tissue. Disruption of patterning may be due to the premature or abnormal differentiation and recruitment of cells into ommatidial clusters. Ultraspiracle may be involved in regulating eye morphogenesis with respect to the molt cycle, or it may be a competency factor to down regulate the response to fibroblast growth factor in regions anterior to the morphogenetic furrow, in a role homologous to the role of FGF in vertebrate limb growth (Zelhof, 1997).

SMRT-related ecdysone receptor-interacting factor (SANT domain protein ) antibodies were prepared in order to examine its cytological and chromosomal localization patterns. Consistent with its action as a corepressor of EcR, Smrter was localized to nuclei of salivary glands. Whether Smrter is associated with the EcR:Usp complex on chromosomes was examined. The fact that Usp and EcR colocalize with each other on polytene chromosomes, allowed the use of the Usp staining pattern as an index for EcR's presence on chromosomes. Chromosomal spreads prepared from the salivary glands of wandering third instar larvae (prior to pupariation) were subjected to simultaneous immunological staining with antibodies against Smrter and Usp. Indirect immunofluorescence staining reveals that Smrter is a chromosome-bound protein and colocalizes with Usp at a majority of chromosomal sites. The strongest Smrter staining is primarily associated with the boundary between band and interband regions as well as within the interband regions of chromosomes counterstained with DAPI. This result confirms that, as an EcR-associating factor, Smrter is recruited by the EcR:Usp heterodimers to their specific target chromosomal loci. Interestingly, Smrter staining can still be detected in puffed regions, such as the 2B puff. Since the polytene chromosomes consist of a parallel arrangement of several hundred to two thousand copies of the euchromatic portions of the chromosomes, an individual binding protein like Smrter may be cycling on and off, resulting in a steady state of signals detected in the broader chromatin regions. Whether or not Smrter levels actually change prior to or after the peak of ecdysone pulses remains to be established (Tsai, 1999).

The similarity of Taiman to steroid hormone receptor coactivators suggests that Tai might interact with one or more steroid hormone receptors. The only known steroid hormone in Drosophila is ecdysone, and the ovary is a major site of ecdysone synthesis, which peaks at stage 9. The functional ecdysone receptor is a heterodimer composed of Ultraspiracle (Usp), which is the fly retinoid X receptor (RXR) homolog, and the Ecdysone receptor. To determine whether the ecdysone receptor complex would be a good candidate for interaction with Tai, expression of ecdysone receptor subunits in egg chambers was examined using antibodies against Usp, EcR-A, and EcR-B. EcR-A and EcR-B are distinct isoforms of the EcR subunit, which are generated by alternative splicing. Usp, EcR-A, and EcR-B colocalize with Tai protein in migrating border cells; Usp and EcR-A are expressed generally, in both follicle cells and nurse cells (Bai, 2000).

These observations raise the possibility that the timing of border cell migration might be controlled by ecdysone. To test whether border cell migration is responsive to hormone, the effects of injecting hormone into female flies were examined. It was not expected that increasing the hormone concentration alone would be sufficient to cause precocious border cell migration because expression of the slbo gene and its targets are independently required for migration. Therefore, slbo was precociously expressed using transgenic flies carrying a heat-inducible slbo transgene, followed by injection of hormone. Border cell migration was assayed in stage 8 egg chambers dissected from flies treated with heat shock and hormone, and compared to control flies treated with heat shock and ethanol, or with hormone in the absence of heat shock. Precocious border cell migration was observed in 20% of egg chambers that were treated with both heat shock and hormone but not in controls. The observed effects are consistent with a role for ecdysone in regulating the timing of border cell migration (Bai, 2000).

If the rising ecdysone level at stage 9 is required to stimulate border cell migration, then reducing the ecdysone level should cause a delay in border cell migration. The ecdysoneless mutant ecd1 is temperature sensitive for production of ecdysone. Females homozygous for ecd1 are sterile when held at the nonpermissive temperature for 5 days, and egg chambers in these flies arrest development at stage 8 and subsequently degenerate. Border cells fail to develop in these arrested egg chambers. However, when ecd1 mutants are held at the nonpermissive temperature for 2 days, some stage 10 egg chambers develop, in which border cells differentiate and express Slbo protein. Greater than 50% of these egg chambers exhibit delayed border cell migration (Bai, 2000).

Since the effects on border cell migration of increasing or decreasing ecdysone levels could have been indirect, whether there is a cell autonomous requirement for the ecdysone receptor in border cells was tested. The EcR locus is proximal to available FRT insertion sites, preventing mosaic analysis. Therefore, the analysis was carried out using mutations in usp. Border cells that were homozygous mutant for a null allele of usp exhibit inhibition of border cell migration, but no obvious defects in other follicle cells (Bai, 2000).

To assess whether Tai and the ecdysone receptor are likely to associate in a complex in vivo, Tai expression was examined in third instar larvae. Antibodies against Tai react specifically with the salivary gland nuclei, as well as other larval tissues. Polytene chromosome spreads were stained with antibodies against Tai and Usp proteins in a double labeling experiment. Anti-Tai antibody labels specific loci on the polytene chromosomes. Moreover, Usp and Tai proteins colocalize precisely. Since previous experiments have shown that Usp and EcR colocalize as a complex on polytene chromosomes, these results indicated that Tai colocalizes with the functional Ecdysone receptor complex at specific target sites (Bai, 2000).

Whether expression of Tai can enhance ecdysone receptor-dependent transcriptional activation in EcR-293 mammalian cells was tested. These cells respond to hormone, either ecdysone or an analog known as ponasterone, with a substantial increase in transcriptional activation of genes placed under the control of a cis-acting sequence known as an E/GRE. Transcriptional activation was tested in cells expressing varying amounts of Tai in transient transfection assays. Tai expression increases transcriptional activation up to 5-fold, in a dose-dependent manner, specifically in the presence of hormone (Bai, 2000).

Furthermore, a GST-fusion protein containing the region of Tai protein containing the LXXLL motifs predicted to interact with EcR (residues 1028 to 1235 of Tai) associates with in vitro translated EcR in a ligand-dependent manner. The same fusion protein does not associate detectably with Usp alone. However, in the presence of EcR and ligand, the Tai-GST fusion protein is able to coprecipitate Usp. Taken together, these results suggest that Tai is a bona fide ecdysone receptor coactivator (Bai, 2000).

Thus, Tai appears to be a coactivator of the p160 class based not only on amino acid sequence similarity and overall domain structure, but based also on its in vivo colocalization with EcR, its direct, ligand-dependent binding to EcR, and its ability to potentiate hormone-dependent transcription in cultured cells. The homology of Tai to SRC proteins suggests that Tai might interact with a steroid hormone receptor. Although there are more than 20 genes in Drosophila that code for proteins related to nuclear hormone receptors, ecdysone is the only known steroid hormone. Since SRC proteins require the presence of a ligand in order to interact with receptors, the ecdysone receptor seems like the best candidate partner for Tai. The colocalization of Tai protein with the ecdysone receptor complex at specific chromosomal loci in third instar larva, the direct and ligand-dependent binding of Tai to EcR in vitro, and the ability of Tai to potentiate the ecdysone response in cell culture lend substantial support to this proposal (Bai, 2000).

The ligand-dependent interaction of Tai with the ecdysone receptor suggests that ecdysone regulates border cell migration. The strongest evidence in support of this is that border cells lacking Usp are unable to migrate. Consistent with this observation, numerous unfertilized eggs were produced from females lacking usp function. Moreover usp is required specifically in somatic cells for production of a fertilizable egg. Defects in border cell migration are known to lead to the production of unfertilized eggs. Whether EcR loss of function mutations affect border cell migration could not be examined. This is because the EcR locus, at 42A, is proximal to available FRT insertions, making it impossible to make FLP-mediated mosaic clones. The frequency of X-ray induced mitotic clones is too low to be useful, and marking such clones is problematic. A temperature-sensitive allele of EcR exists and flies at the nonpermissive temperature exhibit a variety of defects in oogenesis, including arrest prior to border cell migration. Even though it was not possible to assess the effect of EcR mutations specifically in the border cells, the observations that hormone injections can lead to precocious border cell migration and that reduced ecdysone levels can lead to delayed migration provide additional support for the hormonal control of migration (Bai, 2000).

The rise in ecdysone after eclosion, specifically in females, occurs in response to adequate nutrition. In the absence of a rich diet, yolk protein synthesis is inhibited and oogenesis does not progress. Yolk protein synthesis can be restored in the absence of a rich diet by applying ecdysone or juvenile hormone (JH) to cultured ovaries. Recent studies indicate that functional ecdysone receptors are required in the germline for progression of oogenesis through vitellogenesis, the stages during which yolk is taken up by the oocyte. In summary, then, adequate nutrition appears to lead to elevated hormone levels, which in turn stimulate yolk protein synthesis and uptake, and progression of oogenesis beyond stage 8. Together with the results reported here, these findings suggest that a rising ecdysone titer coordinates a variety of events that occur in early vitellogenic egg chambers, including border cell migration (Bai, 2000).

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).

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).

Effects of Mutation or Deletion

Heterozygotes for an altered DNA binding domain of USP display thoracic defects ranging from mild to severe with a cleft extending through both scutellum and notum. The cleft may arise either from the improper fusion of the discs along the dorsal midline of the notum, or a mutant effect on cells lying along the midline of the mesothoracic disc. Flies also have bent and misshapen sensory bristles and develop severly gnarled legs (Henrich, 1994).

Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the Ecdysone receptor (EcR) and its partner Ultraspiracle (Usp). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of Usp and the EcR-A isoform in all but one of eight oocytic mesoderm-derived somatic cell types. The eight oocytic mesoderm-derived somatic cell types consist of apical cells, terminal filament cells, cap cells, the epithelial sheath, the inner germarial sheath cells, the follicle cells, the basal stalk and the oviduct. The first recognizable event of ovarian differentiation is the formation of the terminal filaments (TFs), a process of convergent extension that begins at around 12 hours after ecdysis to the third instar, and continues throughout the remainder of the final larval stage. At pupariation (the onset of the larval-pupal transition), all of the approximately 21 TF stacks have formed, and the location of these stacks prefigures the positions of the mature ovarioles (the functional units of the ovary). During TF differentiation, three additional cell types are present: germ cells (in the central region of the ovary); apical cells (anterior to the germ cells), and basal somatic cells (posterior to the germcells). At pupariation, a subset of the apical cells, the epithelial sheath cells, have begun to surround each terminal filament, and will ultimately separate the ovarioles. The epithelial sheath and the other apical cells are collectively referred to as anterior somatic cells. At pupariation, an additional somatic cell type is distinguishable: the cap cells occupy a position between the TF cells and the germ cells (Hodin, 1998).

By 12 hours after pupariation (12 h AP), the germ cells have begun to form cysts and the epithelial sheaths are continuing to advance in an apical to basal progression. Adjacent to the germ cells reside inner germarial sheath cells, which line the interior lateral edges of the germarium (the birth place of the egg chambers). At 12 h AP, the basal somatic cells remain in a predifferentiative state. By 18 h AP, follicle cells have begun to surround the first egg chambers. At 24 h AP, the epithelial sheaths have separated the ovaries into ovarioles and the basal somatic cells begin to differentiate into the basal stalks and the anterior oviduct (cells at the extreme posterior of the ovary that will eventually fuse with the genital disc-derived oviduct). By 42 h AP, the ovaries and oviducts have fused, and the basal stalks are present (Hodin, 1998).

The exceptional cell type, that is, the one in which EcR cannot be detected, is the larval TF cell, in which only Usp is detectable during cell differentiation. The onset of TF formation is accompanied by the onset of expression in the presumptive TF cells of the Bric-a-brac protein, which is required for proper TF formation. In cells destined to form the basal stalks and anterior oviduct, Usp colocalizes with what appears to be the EcR-B2 isoform. BrdU incorporation in pupal ovaries correlates with ecdysteroid levels, suggesting that ecysteroids regulate proliferation in the ovary, presumably via the EcR-Usp heterodimeric receptor. EcR-A is first detected at approximately 12 h after ecdysis into the third instar, in the somatic cells of the ovary but not in the forming TF cells. The EcR-A isoform is abundant only in the anterior cells (Hodin, 1998).

Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. In such mutants there is a significant increase in the number of TF cells formed (identified by Bric-a-brac expression), but there are fewer TF stacks. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Thus usp3 heterozygotes exhibit accelerated TF formation; the Df EcR heterozygotes show delayed TF formation, and the EcR-usp double heterozygotes are delayed both in the onset and in the completion of TF formation. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. These results demonstrate that proper expression of the Ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the Ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects. It is concluded that heterochronic shifts in ovarian differentiation have apparently been accomplished by uncoupling the process of ovarian differentiation from tissue differentiation in the rest of the animals and that alterations in timing of expression of the EcR complex may reside in a more general mechanism by which heterochronic changes in the differentiation of individual tissues have been accomplished in insect evolution (Hodin, 1998).

Pulses of the steroid hormone ecdysone function as key temporal signals during insect development, coordinating the major postembryonic developmental transitions, including molting and metamorphosis. In vitro studies have demonstrated that the Ecdysone receptor (EcR) requires an RXR heterodimer partner for its activity, encoded by the ultraspiracle locus. usp exerts no apparent function in mid-third instar larvae, when a regulatory hierarchy prepares the animal for the onset of metamorphosis. Rather, usp is required in late third instar larvae for appropriate developmental and transcriptional responses to the ecdysone pulse that triggers puparium formation. The imaginal discs in usp mutants begin to evert but do not elongate or differentiate; the larval midgut and salivary glands fail to undergo programmed cell death, and the adult midgut fails to form. Consistent with these developmental phenotypes, usp mutants show pleiotropic defects in ecdysone-regulated gene expression at the larval-prepupal transition. usp mutants also recapitulate aspects of a larval molt at puparium formation, forming a supernumerary cuticle. These observations indicate that usp is required for ecdysone receptor activity in vivo. They demonstrate that the EcR/USP heterodimer functions in a stage-specific manner during the onset of metamorphosis and implicate a role for usp in the decision to molt or pupariate in response to ecdysone pulses during larval development (Hall, 1998).

Normally, near the end of the third instar, a low titer pulse of ecdysone causes larvae to wander from the food in search of a place to pupariate. This is followed several hours later by a high titer pulse of ecdysone that triggers puparium formation: shortening the larval body, everting the anterior spiracles, and tanning and hardening the larval cuticle to form a protective puparial case. By ~6 hours after puparium formation, apolysis from the larval cuticle is complete and a thin pupal cuticle has been deposited. usp mutants fail to undergo most of these developmental transitions. usp2 mutant larvae fail to wander, while many usp4 mutants wander only a short distance from the food. Rather than forming a puparium, usp mutants maintain their larval shape, become motionless at the surface of the food, fail to respond to a touch stimulus and fail to evert their anterior spiracles. This aberrant attempt at puparium formation has been termed the stationary stage. After several hours, usp mutants begin to apolyze from their third instar cuticle, as evidenced by retraction of the animals from both the anterior and posterior cuticle ends. By 24 hours after becoming stationary, apolysis is complete and the animals easily slip free from the external third instar cuticle. Surprisingly, a supernumerary cuticle is found to cover the posterior two-thirds of the animal. This cuticle is thick, well-infiltrated with tracheae and segmentally ridged along the body, hallmarks of a larval rather than a pupal cuticle. Most usp mutants die by 72 hours after the stationary phase, as evidenced by the onset of necrosis. All of these phenotypes are fully penetrant. Larval cuticle consists of two distinct layers, a relatively thick endocuticle surrounded by a thin external epicuticle. Endocuticle is normally deposited continuously throughout the third larval instar, raising the possibility that the epidermis of usp mutants continues to synthesize a third instar endocuticle during the stationary stage rather than depositing a new cuticle. To distinguish between these possibilities, histological sections from usp mutants were examined 24 hours after the stationary stage. Two cuticles can be clearly distinguished in these mutants, each with its own epicuticle and endocuticle, although the most internal endocuticle appears disorganized. usp mutants thus appear to initiate aspects of both a larval molt and puparium formation in response to the high titer late larval pulse of ecdysone (Hall, 1998).

Several tissues were examined in order to more accurately assess the developmental status of the stationary animals. Imaginal discs look normal in usp mutant third instar larvae and begin to evert following the stationary stage, but arrest their development at a point normally seen 1 hour after puparium formation. The gastric caeca also retract in usp mutants, although this response occurs gradually over a 24 hour period. A slight compaction of the larval midgut can be observed, but the larval cells do not die and the adult midgut does not form. The number of imaginal cells in the midguts of usp mutants does not appear to change significantly in the 24 hour period following the stationary stage, indicating that imaginal cell proliferation does not occur. Larval salivary gland development is also normal until the end of the third instar, even swelling with glue proteins in preparation for puparium formation. Destruction of the larval salivary gland, however, fails to occur and the gland persists until the death of the animal. These pleiotropic defects suggest that usp mutants are unable to transduce the ecdysone signal that triggers the onset of metamorphosis (Hall, 1998).

If USP is an essential component of the Ecdysone receptor, then usp mutants should display pleiotropic defects in ecdysone-regulated gene expression at the onset of metamorphosis. To test this hypothesis, RNA was isolated from staged control and usp mutant animals and the patterns of ecdysone-regulated transcription were analyzed by Northern blot hybridization. Unexpectedly, activation of the mid-third instar regulatory hierarchy is unaffected by the usp2 mutation. EcR, E74B and the BR-C are expressed normally in usp2 mutant mid-third instar larvae, and the ng to glue gene switch occurs on time. In contrast, the response to the high titer late larval pulse of ecdysone is blocked. E74A, E75A and the BR-C are not induced in usp2 late third instar larvae. Furthermore, the Sgs-4 glue gene is not repressed at the stationary stage, and the L71-6 late gene is not induced. These observations indicate that usp mutations selectively block the late larval response to ecdysone, consistent with the observed developmental phenotypes. These results are confirmed by analysis of the puffing patterns in the salivary gland polytene chromosomes of usp2 mutants, where the glue gene puffs fail to regress and the early and late puffs do not form (Hall, 1998).

Destruction of the larval midgut during early prepupal development is accompanied by coordinate induction of the death genes reaper (rpr) and hid. These genes are not induced in usp2 mutant animals, consistent with the failure of the larval midgut and salivary glands to undergo cell death. In contrast, the Stubble protease gene is expressed normally in usp2 mutants, although at slightly lower levels, indicating that other factors must contribute to the inability of usp mutant imaginal discs to undergo normal eversion and elongation. The expression of larval and pupal cuticle genes was also studied to more accurately determine the nature of the supernumerary cuticle. Interestingly, all three genes examined are misexpressed in usp2 mutants. The Lcp65A b larval cuticle gene is expressed long after the stationary stage in usp2 mutants, and the Pcpgart and Edg78E pupal cuticle genes are widely expressed, at both earlier and later times than their normal brief peak of expression in mid-prepupae. These observations indicate that the stage-specificity of cuticle gene expression has been disrupted by the usp2 mutation. They also indicate that the Ecdysone receptor can function as both a repressor and activator of target gene transcription, supporting an earlier study that showed that Edg78E is repressed by ecdysone (Hall, 1998).

The supernumary cuticle phenotype of usp mutants suggests a possible role for Usp in juvenile hormone signaling. The epidermis of usp mutants responds to the late larval ecdysone pulse in a manner that is distinct from the responses of the internal tissues. Whereas the larval midgut and imaginal discs attempt to initiate metamorphosis in usp mutants, the epidermis synthesizes a supernumerary cuticle, recapitulating aspects of an earlier genetic program. The production of a supernumerary cuticle in Drosophila is a novel observation. Normally, the larval abdominal epidermis is reprogrammed to produce a pupal cuticle following puparium formation. The concurrent expression of larval and pupal cuticle genes in both third instar and stationary animals confirms that the epidermal cells are receiving inappropriate cuticle production signals. EcR-B mutants secrete a pupal cuticle and form a constriction between the thoracic and abdominal regions, similar to wild-type animals. The apparent absence of a cuticular phenotype in these mutants could be due to functional redundancy with EcR-A, although this isoform is not detectable in larval epidermal cells. Alternatively, usp may play a distinct role in programming the developmental switch from larval to pupal cuticle deposition. This function could be mediated by Usp homodimers or by heterodimerization with another Drosophila nuclear receptor. These observations raise the interesting possibility that usp may regulate responses to juvenile hormone (JH) during development. Physiological studies in a variety of insects have demonstrated a role for JH in maintaining larval stages of development. Pulses of ecdysone in the presence of JH lead to molting of the larval cuticle whereas a pulse of ecdysone in the absence of JH signals the onset of metamorphosis. Consistent with this model, removal of the corpora allata (which synthesizes JH) can lead to precocious metamorphosis while implantation of the corpora allata, or ectopic JH application, can result in supernumerary larval molts. Similar effects, however, have not been demonstrated in Drosophila, raising the possibility that higher insects do not depend on JH for maintaining their larval status. Nevertheless, the JH titer in Drosophila is high during larval stages and drops during the final instar, similar to the pattern seen in insects that respond to JH treatment. Furthermore, the production of a supernumerary cuticle in usp mutants is consistent with a JH effect in Drosophila and suggests that this receptor may be functioning in a JH signaling pathway. A recent study has proposed that Usp is a JH receptor, although this binding is not saturable and is of low affinity (Jones, 1997). An effect of JH on the transactivation function of Usp has also not been demonstrated. Further biochemical and genetic studies should resolve what role, if any, JH plays during preadult Drosophila development, and whether usp functions in a JH signaling pathway (Hall, 1998).

Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, a genetic mosaic screen was conducted and one ultraspiracle (usp) allele defective in larval process pruning was discovered. Consistent with the notion that Usp forms a heterodimer with the Ecdysone receptor (EcR), it was found that the EcR-B1 isoform is specifically expressed in the MBgamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/Usp targets are dispensable for MB neuronal remodeling. This study demonstrates cell-autonomous roles for EcR/Usp in controlling neuronal remodeling, potentially through novel downstream targets (Lee, 2000).

The recently established MARCM (for mosaic analysis with a repressible cell marker) genetic mosaic system has allowed the study of functions of genes in various neural developmental processes in the Drosophila brain. The MARCM system allows unique labeling of homozygous mutant cells in a mosaic tissue, which is important for phenotypic analysis of individual mutant neurons in the complex brain. Because typical neurogenesis involves the generation of ganglion mother cells (GMCs) from neuroblasts followed by the formation of two postmitotic neurons from each GMC, the MARCM system can be used to mark the entire axonal and dendritic projections of single neurons if mitotic recombination occurs during GMC division (Lee, 2000 and references therein).

MARCM-based analysis has elucidated the cellular basis for the development of the mushroom bodies (MBs), including neuronal reorganization of the MB during metamorphosis. The MBs are prominent neuropils of the central brain that are essential for several forms of learning and memory. Each MB is composed of approximately 2,500 neurons, which are derived from four neuroblasts that undergo hundreds of asymmetric divisions through embryonic, larval, and pupal stages. Unlike most larval-born neurons, which are arrested as immature neurons until the pupal stage, MB neurons elaborate axonal and dendritic projections shortly after mitosis. In the larval brain, every MB neuron extends a single process from which dendrites branch out into the calyx. The axon extends further and then bifurcates into two major branches, one projecting medially and the other projecting dorsally. Interestingly, MB neurons generated prior to the mid-third instar stage, named gamma neurons, prune the medial and dorsal branches during early metamorphosis and subsequently project axons only into the medial gamma lobe of the adult MB. In contrast, the alpha'/beta' MB neurons that are born after the mid-third instar stage retain their larval projections during metamorphosis (Lee, 2000 and references therein).

Because the MARCM system further allows one to generate clones homozygous for any mutation of interest only in the uniquely labeled gamma neurons, the MB gamma neuron was used as a genetic model system to investigate the molecular mechanisms of neuronal remodeling. Both a forward genetic screen and a candidate gene approach have indicated that Usp, is essential for MB gamma neuron remodeling. The EcR-B1 isoform is specifically expressed in the MB neurons destined for remodeling, and it mediates the axonal pruning of MB gamma neurons independent of the surrounding cells. The individual functions of several ecdysone primary response genes, including Broad-Complex (BR-C), E74, and E75 were examined, and none of them were found to be essential for the EcR/USP-mediated MB remodeling. This study demonstrates cell-autonomous roles for EcR/USP in controlling MB neuronal remodeling, potentially through novel downstream targets (Lee, 2000).

Steroid signaling promotes stem cell maintenance in the Drosophila testis

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). This study shows that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which has been shown to maintain CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance (Li, 2014).

This work shows that the steroid hormone 20E plays an important role in maintaining stem cells in theDrosophila testis: 20E, receptors of ecdysone signaling, and downstream targets are required directly in CySCs for their maintenance. When ecdysone signaling is lost in CySCs, GSCs are also lost, but it is unclear if their maintenance requires an ecdysone-dependent or independent signal from the CySCs. The requirement for EcR in the testis is isoform-specific: expression of EcR-B2 in the CySC lineage is sufficient to rescue loss of GSCs and CySCs and increased cell death in EcR mutant testes, suggesting that there might be a temporal and spatial control of ecdysone signaling in the adult testis. In addition, evidence is provided that ecdysone signaling, as in the ovary, is able to interact with an intrinsic chromatin-remodeling factor, Nurf301, to promote stem cell maintenance. Therefore, these studies have revealed a novel role for ecdysone signaling in Drosophila male reproduction (Li, 2014).

Although ecdysone signaling is required in both ovaries and testes for stem cell maintenance, the responses in each tissue are likely to be sex-specific. In the ovary, 20E controls GSCs directly, by modulating their proliferation and self-renewal, and it acts predominantly through the downstream target gene E74. In contrast, male GSCs require ecdysone signaling only indirectly: ecdysone signaling was found to be required in the CySC lineage to maintain both CySCs and GSCs. In a previous study, RNAi-mediated knockdown of EcR, usp or E75 in the CySC lineage did not result in a significant loss of GSCs; however, the number of CySCs was not determined, and the phenotype was examined after 4 or 8 days, not 14 days as in this study. It is suspected that the earlier time points used in that study may not have allowed enough time for a significant number of GSCs to be lost (Li, 2014).

During development, 20E is produced in the prothoracic gland (PG) and further metabolized to 20E in target tissues, but the PG does not persist into adulthood. In adult female Drosophila, the ovary is a source of 20E. In contrast, the identification of steroidogenic tissues in adult male Drosophila remains the subject of active investigation. The level of 20E in adult males is significantly lower than in adult females, but it can be detected in the testis. Furthermore, RNA-seq data show that shade, which encodes the enzyme that metabolizes the prohomone ecdysone to 20E, is expressed in the adult testis, suggesting that the adult testis may produce 20E. However, the sources of 20E production in adult Drosophila males remain to be determined experimentally (Li, 2014).

20E, like other systemic hormones, can have tissue-specific effects or differential effects on the same cell type as development proceeds. These differences are mediated at least in part by the particular downstream target genes that are activated in each case. For example, in female 3rd instar larval ovaries, ecdysone signaling upregulates br expression to induce niche formation and PGC differentiation, but br is not required for GSC maintenance in the adult ovary; instead, E74 plays this role. Similarly, br is required for the establishment of intestinal stem cells (ISCs) in the larval and pupal stages but not for ISC function in adults. This study shows that ecdysone signaling in the adult testis is mediated by different target genes than in the ovary: E74, but not E75 or br, regulate stem cell function in the ovary, whereas E75 and ftz-f1 are important for stem cell maintenance in the testis. Since E75 is itself a nuclear hormone receptor that responds to the second messenger nitric oxide, it will be interesting to know whether E75's partner DHR3 also plays a role in CySCs. An intriguing question for future studies will be how different ecdysone target genes interact with the various signaling pathways that maintain stem cells in the ovary or testis (Li, 2014).

Since 20E levels can actively respond to physiological changes induced by environmental cues, it is possible that the effect of 20E on testis stem cell maintenance might reflect changes in diet, stress, or other environmental cues. For example, in Aedes aegypti, ecdysteroid production in the ovary is stimulated by blood feeding and this is an insulin-dependent process. In Drosophila, ecdysone signaling is known to interact with the insulin pathway in a complex way. Ovaries from females with hypomorphic mutations in the insulin-like receptor have reduced levels of 20E. Furthermore, ecdysone signaling can directly inhibit insulin signaling and control larval growth in the fat body. Thus, ecdysone signaling may interact with insulin signaling during testis stem cell maintenance. Previously, it was shown that GSCs in the ovary and testis can respond to diet through insulin signaling, which is required to promote stem cell maintenance in both sexes. It is possible that diet can affect 20E levels and thus regulate stem cell maintenance. In addition to diet, stress can also affect 20E levels, as is the case in Drosophila virilis, where 20E levels increase significantly under high temperature stress. A similar effect has been found in mammals, where the steroid hormone cortisol is released in response to psychological stressor. Finally, 20E levels are also influenced by mating. In Anopheles gambiae, males transfer 20E to blood-fed females during copulation, which is important for egg production. In female Drosophila, whole body ecdysteroid levels also increase after mating. Studying the roles of hormonal signaling in mediating stem cell responses to stress and other environmental cues will be an exciting topic for future studies. From this work it is now clear that, as in mammals, steroid signaling plays critical roles in adult stem cell function during both male and female gametogenesis (Li, 2014).

ultraspiracle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

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