InteractiveFly: GeneBrief

Hormone receptor-like in 38: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Hormone receptor-like in 38

Synonyms -

Cytological map position - 38D5--E6

Function - transcription factor

Keywords - molting, atypical ecdysteroid signalling pathway

Symbol - Hr38

FlyBase ID: FBgn0014859

Genetic map position - 2-

Classification - hormone receptor

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Chen, X., Rahman, R., Guo, F. and Rosbash, M. (2016). Genome-wide identification of neuronal activity-regulated genes in Drosophila. Elife 5. PubMed ID: 27936378
Summary:
Activity-regulated genes (ARGs) are important for neuronal functions like long-term memory and are well-characterized in mammals but poorly studied in other model organisms like Drosophila. This study stimulated fly neurons with different paradigms and identified ARGs using high-throughput sequencing from brains as well as from sorted neurons: they included a narrow set of circadian neurons as well as dopaminergic neurons. Surprisingly, many ARGs are specific to the stimulation paradigm and very specific to neuron type. In addition and unlike mammalian immediate early genes (IEGs), fly ARGs, including hr38, stripe, ring finger protein CG8910, and the protein kinase CG11221, do not have short gene lengths and are less enriched for transcription factor function. Chromatin assays using ATAC-sequencing show that the transcription start sites (TSS) of ARGs do not change with neural firing but are already accessible prior to stimulation. Lastly based on binding site enrichment in ARGs, transcription factor mediators of firing were identified, and neuronal activity reporters were created.
Adhikari, P., Orozco, D., Randhawa, H. and Wolf, F. W. (2018). Mef2 induction of the immediate early gene Hr38/Nr4a is terminated by Sirt1 to promote ethanol tolerance. Genes Brain Behav: e12486. PubMed ID: 29726098
Summary:
Drug naive animals given a single dose of ethanol show changed responses to subsequent doses, including the development of ethanol tolerance and ethanol preference. These simple forms of behavioral plasticity are due in part to changes in gene expression and neuronal properties. Surprisingly little is known about how ethanol initiates changes in gene expression or what the changes do. This study demonstrate a role in ethanol plasticity for Hr38, the sole Drosophila homolog of the mammalian Nr4a1/2/3 class of immediate early response transcription factors. Acute ethanol exposure induces transient expression of Hr38 and other immediate early neuronal activity genes. Ethanol activates the Mef2 transcriptional activator to induce Hr38, and the Sirt1 histone/protein deacetylase is required to terminate Hr38 induction. Loss of Hr38 decreases ethanol tolerance and causes precocious but short-lasting ethanol preference. Similarly, reduced Mef2 activity in all neurons or specifically in the mushroom body alpha/beta neurons decreases ethanol tolerance; Sirt1 promotes ethanol tolerance in these same neurons. Genetically decreasing Hr38 expression levels in Sirt1 null mutants restores ethanol tolerance, demonstrating that both induction and termination of Hr38 expression are important for behavioral plasticity to proceed. These data demonstrate that Hr38 functions as an immediate early transcription factor that promotes ethanol behavioral plasticity.
BIOLOGICAL OVERVIEW

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (Usp). Evidence is provided for a second ecdysteroid signaling pathway mediated by Hormone receptor-like in 38 (Hr38), the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. Hr38 also heterodimerizes with Usp, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either Hr38 or Usp. X-ray crystallographic analysis of Hr38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans (Baker, 2003).

Of the 18 Drosophila genes that encode canonical nuclear receptors, only EcR, in conjunction with Usp, has been shown to be ligand responsive. Similarly, although a wide spectrum of ecdysteroids with biological activity have been identified in the insect hemolymph, only a small subset of these can activate EcR/Usp at physiologic concentration. These results have suggested that other signaling pathways, perhaps mediated by one or more orphan nuclear receptors, may participate in ecdysteroid responses. To that end, the possibility was investigated that Hr38 (NR4A4) may be an ecdysteroid-responsive factor based on the following observations. (1) Hr38, like the ecdysteroid-responsive EcR, is the only other Drosophila nuclear receptor known to heterodimerize with Usp, the ortholog of the vertebrate retinoid X receptor (RXR). (2) Hr38 is the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors, that includes NGFI-B (NR4A1), Nurr1 (NR4A2), and NOR1 (NR4A3) (Philips, 1997; Wilson, 1991; Cheng, 1997; Paulsen, 1995; Zetterstrom, 1996; Perlmann, 1995; Forman, 1995). Like other orphan receptors that heterodimerize with RXR, the NGFI-B/RXR and Nurr1/RXR heterodimers are ligand responsive, suggesting that the Hr38/Usp heterodimer may also be ligand activated (Perlmann, 1995). (3) Like EcR and usp, Hr38 is broadly expressed during the third instar, prepupal, and pupal stages, suggesting that its temporal specificity may also be conferred by a hormone (Fisk, 1995; Kozlova, 1998). (4) Both Hr38 and usp mutant flies have abnormalities in cuticle formation that are not seen in EcR mutants, thereby uncoupling the action of the two receptor heterodimer complexes and suggesting they may govern distinct ecdysteroid signaling pathways (Kozlova, 1998; Hall, 1998). Taken together, these findings raise the possibility that Hr38 participates in an ecdysteroid response pathway that is different from the one transduced by the EcR/Usp heterodimer (Baker, 2003).

Evidence is provided for the existence of an ecdysteroid signaling pathway mediated by the orphan nuclear receptor Hr38. The existence of this pathway is supported by four independent experimental findings. (1) Transactivation assays in insect cells demonstrated that a distinct group of endogenous ecdysteroids, several with no previously known function, can potentiate Hr38-dependent transcription when heterodimerized with a preactivated partner (i.e., rexinoid bound RXR or VP16-Usp). (2) Organ explants from transgenic flies bearing a Hr38-specific reporter gene were shown to be similarly responsive to ecdysteroids, indicating that this pathway can function in vivo. Importantly, the specificity of the Hr38 ecdysteroid activators and the use of RNAi methodology have excluded the involvement of EcR in mediating this response. (3) Neither the ecdysteroid agonists nor any of the known nuclear receptor coactivators are capable of binding directly to Hr38. (4) X-ray crystallographic structure analysis of the Hr38 ligand binding domain shows that Hr38 lacks the classic binding sites for either a ligand or a conventional coactivator, features that are hallmarks of all other known inducible nuclear receptors. These findings provide compelling evidence for an atypical nuclear receptor transcriptional signaling pathway that mediates ecdysteroid responses in insects (Baker, 2003).

The insect hemolymph carries a wide range of endogenous ecdysteroids, some of which are only present at specific stages during development. These may be supplemented by phytoecdysteroids that can enter the animal through its diet. Until recently, it was thought that the vast majority of these compounds were unable to elicit a biological response. Mounting evidence, however, indicates that alternate transcriptional pathways exist that are driven by ecdysteroids other than 20E. Coordinate changes in ecdysteroid-regulated gene expression occur at several stages in the Drosophila life cycle at times when the 20E titer is known to be low. In addition, the let-7 and miR-125 small temporal RNAs are induced at puparium formation in precise synchrony with the E74A 20E-inducible gene, but in a manner that is independent of either 20E or EcR (Bashirullah, 2003). Of particular relevance to Hr38 functions, α-ecdysone has been shown to drive neuroblast proliferation during early pupal development in the hornworm Manduca sexta, providing in vivo evidence that this hormone is responsible for a specific response in insects. Similarly, 3-dehydro-20E has been shown to have a potency indistinguishable from 20E in Manduca and is also observed to have high activity in Drosophila larval fat body, while it has been noted that makisterone A and not 20E is the major ecdysteroid present during the last larval instar of the honeybee. Given the reported activity of these ecdysteroids, it seems reasonable to expect that at least one of the pathways governing these responses is mediated by the Hr38 pathway described here. Further support for the hypothesis that a Hr38/Usp heterodimer may play an essential role in ecdysteroid signaling comes from the observation that Hr38 and Usp are each required for ecdysteroid-induced cuticle formation during Drosophila development. A key to the future characterization of this developmental pathway will be the use of the Hr38/Usp heterodimer and ecdysteroid agonists as tools to identify downstream target genes, which at present remain unknown (Baker, 2003 and references therein).

An interesting feature of the Hr38 response is the broad specificity and increased sensitivity that a number of ecdysteroids have for Hr38 compared to the previously described signaling pathway mediated by EcR. Indeed, even the response to 20E appears to be an order of magnitude more potent for Hr38 than for EcR. Thus, discovery of the Hr38 response pathway may not only provide a mechanism of action for other ecdysteroids in insects, but may also provide a means of augmenting the ecdysteroid-mediated functions of EcR at specific stages in the life cycle (Baker, 2003).

Another striking feature of the Hr38 response is that it requires coactivation of its heterodimer partner to become competent for transcriptional activation via ecdysteroids. The finding that VP16-Usp is able to substitute for ligand-activated RXR in transfection assays is intriguing and suggests that in vivo, wild-type Usp is capable of activation by ligand or some other coactivation mechanism. The existence of a ligand for Usp is supported by X-ray crystal data on the Usp ligand binding domain showing the presence of a large hydrophobic pocket that can be occupied by lipophilic ligands (Billas, 2001; Clayton, 2001). The observation that the Hr38 ecdysteroid response can occur in larval organs that contain wild-type Usp supports this hypothesis. Identification of the Usp ligand and/or coactivator represents a critical next step toward defining the mechanism of Hr38 action (Baker, 2003).

Hr38's distinct ecdysteroid-regulated activity points to a role that is substantially different from that of EcR, both in terms of ligand specificity and mechanism of action. Although both receptors require heterodimerization with Usp to be ecdysteroid responsive, only the EcR response appears to require conventional binding of the ecdysteroid agonist. Furthermore, the role of Usp in the EcR heterodimer is that of a silent partner (i.e., the transcriptional activity of Usp is dispensable for the ecdysteroid response). In contrast, the Hr38 pathway requires transcriptional activation of both itself and its heterodimeric partner. Surprisingly, however, this response occurs in the absence of ecdysteroid binding directly to receptor, implying the existence of a nonclassical mechanism of action (Baker, 2003).

The structure of the Hr38 ligand binding domain offers an intriguing framework from which several clues about the mechanism of Hr38 action can begin to be elucidated. Although the possibility that a ligand could bind to Hr38 by an induced-fit mechanism or to an allosteric site cannot be formally ruled out, both possibilities are considered unlikely. The tight spatial constraints forced upon the protein by the four phenylalanines within the conventional ligand binding pocket almost completely exclude the induced-fit possibility. Likewise, the inability to demonstrate any type of specific ligand binding to the protein under a variety of conditions (e.g., in the presence or absence of activated heterodimer partner) using a number of assays argues against the existence of a second binding site on the protein. An equally important finding is the loss of the charge clamp, which fundamentally excludes the Hr38 ligand binding domain from interacting with the p160 family of coactivators in a conventional fashion. This finding is consistent with the inability to observe any interactions with these coactivators in either cell-based or biochemical assays. Taken together, these results provide strong evidence that the ecdysteroid response by the Hr38/Usp heterodimer occurs through a mechanism that is different from the well-documented, direct binding paradigm that has been exhibited for numerous other RXR heterodimers. Therefore, the signaling pathway between ecdysteroid and Hr38-mediated transcription must be transduced in an atypical fashion. This mechanism, however, still appears to require the AF-2 domain of Hr38. Although it is not clear how the AF-2 contributes to receptor transactivation, the data support a model in which ecdysteroids may indirectly activate Hr38, perhaps by recruiting a specific cofactor to the Hr38/Usp heterodimer. In this model, it is tempting to speculate that ecdysteroids may activate the cofactor through a direct interaction or through a second message pathway. Regardless, the requirement for a Hr38 cofactor is implicit in these findings and its future characterization will be important to fully understand the mechanism of this new signal transduction pathway (Baker, 2003).

The principles of Hr38 action may be of help in characterizing its mammalian orthologs, the NGFI-B family of receptors. Like Hr38, these orphan receptors can function as monomers or RXR heterodimers and be activated by RXR ligands (Giguere, 1999). However, little is known about the agonist or cofactor specificity of these proteins or the mechanistic details of how they promote transactivation. The analysis carried out in this study shows that the overall conservation between Hr38 and the three mammalian NGFI-B family members is well conserved in the putative ligand binding pocket. Indeed, as shown for Hr38, modeling of the 3D structure of the NGFI-B receptors predicts the absence of both a ligand binding pocket and a coactivator binding site, suggesting that a common mechanism of action may exist for governing these receptors in mammals. Given these similarities between Hr38 and its vertebrate counterparts, it should not be surprising that, like many other insect signaling pathways, there is a lot to learn from the fly (Baker, 2003).

Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles

One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of the understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. This study analyzed the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24h of metamorphosis. Ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis (Zirin, 2013).

There are three different isoforms of the EcR gene, EcR A, EcR B1, and EcR B2, each sharing the same DNA binding and ligand binding domains, but with a unique amino terminus. The different temporal and spatial expression patterns of EcR A and EcR B isoforms are thought to reflect their distinct functions during development. EcR B1 is expressed primarily in larval cells that are destined for histolysis, while EcR A is expressed primarily in imaginal tissues destined for differentiation into adult structures. Thus the response of salivary glands and midgut to ecdysone during metamorphosis is dependent on EcR B1. Nonetheless, EcR A mutants also have a defect in salivary gland histolysis, suggesting that the isoform might also contribute to this process. Furthermore, some neurons in the ventral nerve cord and brain that strongly express EcR A undergo apoptosis in response to ecdysone soon after eclosion, suggesting that ecdysone induced PCD is not strictly a function of EcR B1 signaling (Zirin, 2013).

This study examined expression of both EcR A and EcR B1 isoforms and found that only EcR B1 was detectable in the dorsal internal oblique muscles (DIOMs) and DEOMs during pupariation. Consistent with its expression pattern, knockdown of EcR B1 specifically in the muscle inhibited DEOM histolysis. The inhibition achieved with the EcR B1 isoform RNAi was not as strong as with RNAi targeting all isoforms, or with overexpression of the dominant negative EcR B1. This could be due to either differences in the efficiency of knockdown or to a role for EcR B2 in DEOM degradation. Taken together these data strongly supports the view that, like in salivary glands and midgut, ecdysone signals through EcR B1 to induce cell death in abdominal muscles. However, given that both DIOMs and DEOMs express EcR B1 at the same time, the presence of the receptor is not sufficient to explain why only the latter muscles are degraded. Another important player in the timing of salivary glands cell death is the orphan nuclear hormone receptor gene ftz fl, which is transcribed midway through prepupal development, when the ecdysone titer is relatively low. FTZ F1 has been hypothesized as a competence factor, directing the subsequent genetic responses to the ecdysone pulse at the prepupal/pupal transition. Thus FTZ F1 is required for the induction of salivary gland histolysis. In contrast, the midgut does not express ftz fl prior to its earlier ecdysone induced cell death, indicating that FTZ F1 is not required for all histolysis during Drosophila metamorphosis. Despite the fact that the timing of muscle histolysis is similar to that of the midgut, the function of FTZ F1 was more like in the salivary gland, as FTZ F1 was observed in the DEOMs starting at ~ 5 h APF, prior to caspase activation at 8 h APF. Furthermore, ftz fl was required for proper muscle histolysis, as knockdown cell autonomously delayed caspase activation and death in the DEOMs (Zirin, 2013).

These results raised the possibility that the presence of FTZ F1 in the DEOMs, but not in the DIOMs, determined the different response of these muscles to ecdysone. However, overexpression of FTZ F1 in the OJOMs, while causing severe muscle degeneration, was unable to induce caspase activity or cell death. Nor could the presence of HR39 in the DIOMs account for the different response of the muscles to ecdysone. Although a reciprocity of HR39 and FTZ F1 expression was observed in the DIOMs and DEOMs, Hr39 mutant DJOMs still persist through metamorphosis. It is concluded that FTZ F1 and HR39 expression determine the timing of the abdominal muscle response to ecdysone but that these factors do not change the nature of the response (Zirin, 2013).

It was recently shown that EcR B1 expression is regulated by FTZ F1 and HR39 in mushroom body neurons and abdominal motor neurons during metamorphosis. The current observation that EcR B1 promotes muscle degeneration is consistent with the finding that EcR B1 promotes post synaptic dismantling in the motor neuron, and supports the notion that muscle degeneration is instructive on motor neuron retraction. However, it was show that even though both ftz f1 and EcR B1 are essential for the proper histolysis of DEOMs, there was no change in EcR B1 staining upon ftz fl knockdown as was observed in the mushroom body system. This suggests that changes observed in the muscle synapse due to ftz fl knockdown are not the result of a downstream effect on EcR B1 expression in the muscle cell. Thus, the regulatory relationship between FTZ F1, HR39 and EcR B1 in early pupal abdominal muscles is distinct from the relationship reported in neurons (Zirin, 2013).

This suggests that there is an additional unknown factor whose expression dictates the fate of the OJOMs or DEOMs. This factor is unlikely to be either of the nuclear proteins EAST or Chromator (Chro ), which were previously identified as having opposing effects on the destruction of the abdominal DEOMs during metamorphosis. Breakdown of DEOMs was incomplete in Chro mutants, and promoted in east mutants, leading to the proposal that Chro activates and EAST inhibits tissue destruction and remodeling. However, neither east nor chro alleles cause histolysis of the DIOMs, nor do they alter caspase activation in either DEOMs or DIOMs. Rather these genes may affect the timing of muscle histolysis through a function downstream of PCD induction. It is proposed that there must be an additional factor present in the DIOMs which inhibits EcRB1 signaling from inducing PCD, or alternatively, a factor present in the DEOMs, which permits EcRB1 to activate a death program. The identification of this factor will be a focus of future studies (Zirin, 2013).

The previously reported cleaved caspase 3 staining in the DEOMs is the only data addressing the nature of abdominal muscle PCD prior to this study. This study addressed whether muscle histolysis is apoptotic, autophagic or some combination of both. During salivary gland histolysis, several autophagy related genes (ATGs) are upregulated, and mutations or knockdown of these ATGs specifically in the salivary gland inhibit the destruction of the tissue. Caspase activation also occurs in the histolyzing salivary glands, but overexpression of the caspase inhibitor p35 only partially blocks salivary gland degradation. Simultaneous inhibition of both autophagy and apoptosis in the salivary gland produces the strongest inhibition of death, suggesting that both pathways contribute to histolysis of this tissue). In contrast to salivary gland histolysis, midgut histolysis requires autophagy but not caspase activity. Mutations or knockdown of ATGs inhibit midgut death, but p35 expression has no effect. Based on these two model systems, it appears that there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis (Zirin, 2013).

These data serves to further highlight how distinctive PCD for each of the tissues undergoing histolysis. DEOMs stain positive for cleaved caspase 3, consistent with previous reports. TUNEL positive staining and chromatin condensation was also observed in the DEOMs at 8 h APF, both markers of apoptosis. Importantly, it was possible to suppress DEOM degradation by overexpression of the pan caspase inhibitor p35, indicating that unlike the midgut, muscle histolysis is apoptotic. To determine whether the muscle PCD was autophagic in nature, the DEOMs were examined by EM. Although some autophagic vesicles were observed in the dying muscles, they were not abundant, nor were GFP Atg8 localization to autophagosomes examined by confocal microscopy. Several essential components of the autophagic machinery were knocked down, and no effect was observed on the timing or extent of DEOM histolysis. Although knockdown efficiency is always a concern with RNAi experiments. each of the transgenes was able to strongly inhibit autophagosome formation in larval muscles. It can be said therefore with confidence that autophagy is not required for DEOM PCD, putting the abdominal muscle in the unique category of non autophagic histolysis. In future studies it will be interesting to compare muscles, salivary gland, and midgut to determine why each tissue has a distinctive type of PCD (Zirin, 2013).


REGULATION

Hr38 functions as an ecdysteroid sensor

To investigate the possibility that Hr38 may function in an ecdysteroid-mediated transcriptional pathway, a screening assay was developed in which Hr38 was heterodimerized with ligand-activated RXR. The feasibility of this approach was based on the finding that RXR can substitute for Usp as a productive heterodimeric partner for EcR. A distinct advantage of substituting RXR for Usp is that although ligands for Usp are not known, several potent RXR ligands (i.e., rexinoids) have been characterized. It was reasoned that assaying Hr38 activity in the presence of rexinoid-activated RXR may be important because previous work has shown that some RXR heterodimers require sensitization with ligand for one receptor before the partner receptor can become ligand responsive (Baker, 2003 and references therein).

To screen for a Hr38 ecdysteroid response, transient cotransfections were performed in the Drosophila SL2 cell line using chimeric GAL4-receptor proteins (hormone receptor proteins with an inserted GAL4 transcriptional activation domain) and a GAL4-responsive luciferase reporter gene (Baker, 2000). In this assay, GAL4-EcR and GAL4-Hr38 were screened in the presence of RXR, the synthetic rexinoid LG268, and the potent plant ecdysteroid muristerone A. Similar to previous work (Baker, 2000), the GAL4-EcR/RXR heterodimer responds to 100 nM muristerone A as expected but is not activated significantly by the RXR-specific ligand LG268 alone. Addition of both ligands results in only a modest increase in EcR/RXR activity. In contrast, GAL4-Hr38/RXR, which is known to have a potent basal activity (Baker, 2000), is not induced by the addition of ecdysteroid alone but, instead, exhibits a strong, dose-dependent response to LG268. Rexinoid activation of the Hr38/RXR heterodimer is consistent with the rexinoid response seen with other NGFI-B family members when paired with RXR (Perlmann, 1995). Surprisingly, however, there is a significant, 3- to 4-fold response to muristerone A when it is added together with LG268. A similar response is obtained with the endogenous insect ecdysteroid 20E. Both rexinoid and ecdysteroid responses are abolished when a GAL4-Hr38 construct was utilized that lacks the ligand-dependent activation function-2 (AF-2) domain. Identical results are obtained (i.e., loss of rexinoid and ecdysteroid response) when the AF-2 domain of RXR is also deleted. These data suggest that the Hr38 heterodimeric complex is responsive to ecdysteroid but, like other RXR heterodimers, it requires transactivation of both receptor partners for full agonist activity (Baker, 2003).

The results above reveal the possible existence of two ecdysteroid signaling pathways, one mediated by EcR and the other by Hr38. To begin to delineate the specificity of the Hr38 pathway and show that it functions independently of the EcR pathway, double-stranded RNA (dsRNA) directed against the coding region of the EcR ligand binding domain was used to reduce the expression of endogenous EcR in the SL2 cell assay. Treatment of SL2 cells with increasing amounts of EcR dsRNA completely eliminates muristerone-A-dependent transcription when tested using endogenous EcR/Usp heterodimers on an hsp27-EcRE reporter gene. This RNAi-mediated repression of the EcR-dependent response also completely blocks the activity of exogenously transfected EcR and GAL4-EcR. In contrast, under the same experimental conditions, where the EcR response is abolished, the GAL4-Hr38/RXR heterodimer is fully responsive to ecdysteroid and LG268. These results demonstrate that the Hr38 response to ecdysteroids is independent of EcR (Baker, 2003).

To define the spectrum of potential Hr38 agonists and further delineate the differential ecdysteroidal response of Hr38 and EcR, a panel of naturally occurring Drosophila ecdysteroids, phytoecdysteroids, synthetic ecdysteroids, and a synthetic juvenile hormone (methoprene acid) were tested for activity using the reporter gene assay described above. SL2 cells were transfected with either GAL4-EcR or GAL4-Hr38 plus RXR and tested for agonist activity in the presence of 10 nM LG268. Consistent with previous results (Baker, 2000), GAL4-EcR responds selectively to the endogenous ecdysteroids 20E and makisterone A and the plant ecdysteroids muristerone A, ponasterone A, and cyasterone. In marked contrast, the GAL4-Hr38/RXR response is promiscuous for several different ecdysteroids when LG268 is included as a coagonist. In addition to the compounds that activated EcR, at least six other ecdysteroids (α-ecdysone, 3-epi-20E, 2-deoxy-20E, 3-dehydromakisterone A, 3-epimakisterone A, and 3-dehydro-20-deoxyponsterone) also exhibit significant Hr38-dependent activity. Dose-response profiles demonstrated that all of these compounds are more potent agonists for Hr38 than for EcR. In fact, 20E, which is believed to be the endogenous hormone agonist for EcR, exhibits a 100-fold greater potency for Hr38-dependent transcription. These data suggest that the Hr38/RXR heterodimer is a potent sensor of a distinct class of physiologically relevant ecdysteroids (Baker, 2003).

Activation of the Hr38/Usp heterodimer by ecdysteroids requires transactivated Usp

An unusual characteristic of the Hr38/RXR heterodimer is that it required transactivation of both receptors to elicit an ecdysteroid response. In particular, the Hr38/RXR heterodimer fails to respond to ecdysteroid in the absence of ligand-activated RXR. Interestingly, Hr38 also fails to respond to ecdysteroid when Usp, the physiologic partner of Hr38, is used instead of RXR. These results raise the intriguing possibility that Usp, like RXR, must also be transactivated (e.g., by ligand) in order to enable the ecdysteroid response. Attempts were made to address this question by using VP16-Usp, a constitutively active form of Usp that circumvents the requirement for Usp ligand by fusing the strong transcriptional activation domain of the herpes simplex viral protein-16 (VP16) to Usp. As expected, in the absence of agonist, the GAL4-Hr38/VP16-Usp heterodimer shows a high constitutive level of basal activity that effectively mimics that of ligand-activated USP. Importantly, the addition of muristerone A to the GAL4-Hr38/VP16-Usp heterodimer elicits a significant increase in reporter gene activity, analogous to the effect seen with LG268-activated GAL4-Hr38/RXR. Similar to the results obtained above with ligand-activated RXR, the GAL4-Hr38/VP16-Usp heterodimer responds to a wide variety of ecdysteroids at comparably low concentrations. These results support the idea that Hr38 mediates a distinct heterodimer-dependent ecdysteroid signaling pathway (Baker, 2003).

To test the prediction that Hr38 is activated by ecdysteroids in Drosophila, transgenic flies were created that carry a heat-inducible hs-GAL4-Hr38 transgene in combination with a GAL4-dependent UAS-nlacZ reporter gene. Since target genes for Hr38 in the fly are unknown, this model permits the assaying of Hr38 transactivation directly in fly tissues. This transgenic fly model has been used to follow the ecdysteroid-dependent activation patterns of the EcR and Usp in Drosophila and has provided data consistent with the known biochemical and genetic activities of the full-length receptors in vivo (Kozlova, 2002). This strategy has also been employed to track ligand-dependent activation of the RAR and RXR ligand binding domains in the mouse central nervous system. To determine if GAL4-Hr38 is activated by ecdysteroids, third instar larval organs from this transgenic line were dissected at ~8 hr before puparium formation and cultured in the presence of either α-ecdysone or 3-epi-20E, two ecdysteroids that activate Hr38, but not EcR in SL2 cells. In the presence of 1 μM α-ecdysone, significant activation above background is seen for GAL4-Hr38. In contrast, these ecdysteroids have no effect on GAL4-EcR, although this same transgenic line shows robust activation by 20E (Kozlova, 2002). GAL4-Hr38 is also activated by 3-epi-20E in both the epidermis and fat body, consistent with the ability of this agonist to selectively activate Hr38 in SL2 cells. These organs contain significant amounts of endogenous Usp, consistent with the interpretation that GAL4-Hr38 ecdysteroid activation is dependent on heterodimerization with a Usp partner. Although similar results were seen in several independent experiments, not all hs-GAL4-Hr38; UAS-nlacZ animals display robust activation, indicating that a specific stage might be competent to respond to the hormone. In agreement with this idea, a complex and dynamic pattern of GAL4-Hr38 activation can be seen in untreated animals. This observation is consistent with the notion that the endogenous Hr38 response may be spatially and temporally regulated by the presence of a number of factors, including ecdysteroids, Hr38/Usp-specific coactivators, and potentially a Usp ligand (Baker, 2003).

Protein Interactions and Interaction with DNA

A rapid PCR based cloning and screening strategy was used to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, three Drosophila genes, designated Hr38, DHR78, and DHR96, have been isolated. All three genes are expressed throughout third-instar larval and prepupal development. Hr38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. The two adenosines upstream from the AGGTCA half-site are critical for Hr38 binding, as has been shown for NGF1-B. Furthermore, the ability of Hr38 to bind to a single AGGTCA half-site suggests that, like NGF1-B, this protein can bind DNA as a monomer (Fisk, 1995)

In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (Usp) and ecdysone receptor (EcR), both of which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Drosophila hormone receptor 38 (Hr38) and Bombyx hormone receptor 38 (BHR38) are two insect homologs of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, Hr38 and BHR38 have been shown, in fact, to interact strongly with Usp and this interaction is evolutionarily conserved. Hr38 can compete in vitro against EcR for dimerization with Usp and consequently disrupt EcR-Usp binding to an EcRE. Moreover, transfection experiments in Schneider cells show that Hr38 can affect ecdysone-dependent transcription. This suggests that Hr38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription (Sutherland, 1995).

Dynamic regulation of Drosophila nuclear receptor activity in vivo

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, a `ligand sensor' system was used to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. These responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation were seen that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control (Palanker, 2006).

Nine GAL4-LBD ligand sensor lines described in this study show tissue-specific patterns of activity during development: EcR, USP, ERR, FTZ-F1, HNF4, E78, DHR3, DHR38 and DHR96. These transgenic lines will serve as valuable tools for the genetic and molecular dissection of the receptors they represent, the pathways they regulate and the upstream factors and co-factors that modulate their activity. Specifically, the data reported here show that these lines can be used to: (1) indicate tissues and stages in which the corresponding NRs are likely to function; (2) indicate where endogenous ligands and co-factors are likely to be found; (3) suggest NR biological functions; (4) suggest possible NR-NR interactions, cascades and target genes; (5) evaluate putative co-factors and ligands; (6) screen chemical compound libraries for new agonists and antagonists; and (7) screen genetically for new pathway components. The results of these studies will also provide important insights into the ligands, co-factors and functions of their vertebrate NR homologues (Palanker, 2006).

Hormonal regulation of GAL4-LBD activation in the amnioserosa and yolk

Examination of the nine active ligand sensor lines provided a number of insights into possible relationships between their corresponding NRs. For example, although each of these ligand sensors displays unique temporal and spatial patterns of activity, activation in specific tissues and stages is common to many. These common sites of LBD activity may indicate shared functions, hierarchical or physical interactions, or related ligands. Examples of tissues that represent hotspots for GAL4-LBD activation include the amnioserosa, yolk, midgut and fat body (Palanker, 2006).

Each of these tissues, and the stages at which they score positively, correlates well with the presence of putative ligands. The yolk, for example, is believed to act as a storage site for maternally provided ecdysteroids during embryogenesis. Work with other insects has shown that these ecdysteroids are conjugated in an inactive form to vitellin proteins via phosphate bridges. Around mid-embryogenesis, these yolk proteins and phosphate bonds are cleaved, thereby releasing what are presumed to be the earliest biologically active ecdysteroids in the embryo. Interestingly though, GAL4-EcR activation in the amnioserosa depends on the disembodied (dib) gene, which encodes a cytochrome P450 enzyme required in the penultimate step of Ecdysone (E) biosynthesis, suggesting that the final steps in the linear E biosynthetic pathway are required for EcR function in this tissue and contradicting the prediction that this activity would be dependent on maternal ecdysteroids and independent of the zygotic biosynthetic machinery. The mechanisms by which dib exerts this essential role in providing an EcR ligand, however, remain to be determined (Palanker, 2006).

The response of the EcR and USP ligand sensors in the adjacent amnioserosa tissue shows that active ecdysteroids are not present until the hormone reaches the amnioserosa. A recent study of yolk-amnioserosa interactions has revealed dynamic transient projections that emanate from one tissue and contact the other, suggesting that there may be functional interactions between these two cell types. It is possible that these projections mediate the transfer of lipophilic ligand precursors from the yolk to the amnioserosa. This transfer, in turn, could determine the proper timing of EcR activation in the amnioserosa, thus triggering the major morphogenetic movements that establish the body plan of the first instar larva (Palanker, 2006).

Studies of the DHR38 receptor have demonstrated that it can be activated by a distinct set of ecdysteroids from those that activate EcR, through a novel mechanism that does not involve direct ligand binding. The activation of GAL4-DHR38 that was observed in the embryonic amnioserosa is consistent with this model of DHR38 regulation. First, exogenous 20E can only weakly activate GAL4-DHR38, relative to the strong ectopic activation seen with 20E on the EcR ligand sensor. This correlates with the weak ability of 20E to activate DHR38 in cell culture transfection assays relative to the strong 20E activation of EcR. Second, the DHR38 ligand sensor is activated in the amnioserosa earlier than the EcR construct, suggesting that it is responding to a different signal. It is possible that this signal is an ecdysteroid precursor that can act on DHR38 but not EcR - paralleling the ability of DHR38 to be activated by E, the precursor to 20E, which activates EcR. This putative ecdysteroid must be produced in a manner independent of the conventional ecdysteroid biosynthetic pathway, however, since a zygotic dib mutation has no effect on GAL4-DHR38 activation in the amnioserosa. Rather, this early activation may be due to maternal ecdysteroids that are conjugated and inactive in the yolk and transferred to the amnioserosa. These studies highlight the value of combining mutations in hormone biosynthesis with ligand sensor activation as a powerful means of dissecting hormone signaling pathways. Further studies of DHR38 function and regulation in embryos could help clarify the potential significance of this distinct activation response (Palanker, 2006).

DHR3, DHR38 and HNF4 ligand sensors appear to respond to metabolic signals Interestingly, the midgut continues to be a hotspot for ligand sensor activity long after it has engulfed the yolk during embryogenesis. This seems logical, as the midgut is responsible for most lipid absorption and release, and many vertebrate NRs are involved in fatty acid, cholesterol and sterol metabolism and homeostasis. The observed restriction of ligand sensor activity to a narrow group of cells located at the base of the gastric caeca is of particular interest. This is the site where nutrients in a feeding larva are absorbed into the circulatory system. The activation of DHR3, DHR38 and HNF4 ligand sensors in this region of the gastric caeca suggests that these receptors are activated by one or more small nutrient ligands. Moreover, this suggests that the corresponding receptors may exert crucial metabolic functions by acting as nutrient sensors (Palanker, 2006).

Further evidence of metabolic functions for DHR3, DHR38 and HNF4 arises from their ligand sensor activation patterns in the embryonic yolk and larval fat body. The yolk is the main nutrient source for the developing embryo and represents an abundant source of lipids, correlating with specific activation of DHR3, DHR38 and HNF4 ligand sensors in this cell type during embryogenesis. Upon hatching into a larva, the fat body acts as the main metabolic organ of the animal, functionally equivalent to the mammalian liver. Upon absorption by the gastric caeca, nutrients travel through the circulatory system and are absorbed by the fat body, where they are broken down and stored as triglycerides, glycogen and trehalose. Once again, the efficient activation of the DHR3, DHR38 and HNF4 ligand sensors in the fat body of metabolically active third instar larvae, and lack of sensor activity in non-feeding prepupae, supports the model that the corresponding NRs operate as metabolic sensors. This proposed function is consistent with the roles of their vertebrate orthologs. Mammalian ROR, the ortholog of DHR3, binds cholesterol and plays a crucial role in lipid homeostasis. Similarly, mammalian HNF4 can bind C14-18 fatty acids, is required for proper hepatic lipid metabolic gene regulation and lipid homeostasis, and is associated with human Maturity-Onset Diabetes of the Young (MODY1). The studies described here suggest that DHR3 and HNF4 may perform similar metabolic functions in flies, defining a new genetic model system for characterizing these key NRs (Palanker, 2006).

New insights into the regulation of Drosophila xenobiotic responses

Several vertebrate NRs play a central role in xenobiotic responses by directly binding toxic compounds and inducing the expression of key detoxification enzymes such as cytochrome P450s and glutathione transferases. Ligand sensor activation observed in the gut, epidermis, tracheae or fat body could represent xenobiotic responses insofar as toxic compounds could enter the organism through any of these tissues. Directed screens that test xenobiotic compounds for their ability to activate Drosophila NR ligand sensors will provide a means of identifying potential xenobiotic receptors. Understanding these response systems, in turn, could facilitate the production of insect resistant crops and the development of more effective pesticides (Palanker, 2006).

Like its vertebrate orthologs SXR/PXR and CAR, DHR96 has been recently shown to act in insect xenobiotic responses, providing resistance to the sedative effect of phenobarbital and lethality caused by chronic exposure to DDT (King-Jones et al., 2006). DHR96 is also required for the proper transcriptional response of a subset of phenobarbital-regulated genes. DHR96 can be activated by the CAR-selective agonist CITCO, suggesting that it may be regulated in a manner similar to that of the vertebrate xenobiotic receptors. It is also interesting to note that angelicin was found to activate the USP ligand sensor fusion. Angelicin is an angular furanocoumarin that has the furan ring attached at the 7,8 position of the benz-2-pyrone nucleus. Detailed studies have shown that insects have adapted to the presence of furanocoumarins in their host plants by expressing specific cytochrome P450 enzymes that detoxify these compounds. In the black swallowtail butterfly (Papilio polyxenes), furanocoumarins induce the transcription of P450 genes through an unknown regulatory pathway, thereby aiding in xenobiotic detoxification. The observation that angelicin, and not the linear furanocoumarins 8-methoxypsoralen (xanthotoxin) or 5-methoxypsoralen (bergapten), can activate GAL4-USP suggests that NRs may mediate this detoxification response and may be capable of distinguishing between the linear and angular chemical forms. It is possible that USP may mediate this effect on its own or, more likely, as a heterodimer partner with another NR. Similarly, the activation of GAL4-USP by fenoxycarb may represent a xenobiotic response. This activation, however, is weaker and more variable than the activation observed with angelicin. Identifying other factors that mediate xenobiotic responses in Drosophila would provide a new basis for dissecting the control of detoxification pathways in higher organisms (Palanker, 2006).

ERR activity appears to be regulated by a temporally restricted and widespread signal

GAL4-ERR displays a remarkable switch in activity during mid-embryogenesis, from strong activation in the myoblasts to specific and strong activation in the CNS. The ERR ligand sensor also shows widespread transient activation in the mid-third instar, a time when larval ERR gene expression begins, together with a global switch in gene expression that prepares the animal for entry into metamorphosis 1 day later. This so-called mid-third instar transition includes upregulation of EcR, providing sufficient receptor to transduce the high titer late larval 20E hormone pulse, upregulation of the Broad-Complex, which is required for entry into metamorphosis, and induction of the genes that encode a polypeptide glue used to immobilize the puparium for metamorphosis. The signal and receptor that mediate this global reprogramming of gene expression remain undefined. The widespread activation of GAL4-ERR at this stage raises the interesting possibility that it may play a role in this transition. Moreover, given that the only ligand sensors to display widespread transient activation are EcR and USP, in response to 20E, it is possible that this response reflects a systemic mid-third instar pulse of a ERR hormone. Vertebrate members of the ERR family can bind the synthetic estrogen diethylstilbestrol and the selective ER modulator tamoxifen, as well as its metabolite, 4-hydroxytamoxifen, suppressing their otherwise constitutive activity in cell culture. This is notably different from the highly restricted patterns of ERR ligand sensor activity that was detected in Drosophila, which suggests that it does not function as a constitutive activator in vivo. Rather, it is envisioned that the patterns of ERR activation are precisely modulated by protein co-factors and/or one or more ligands to direct the dynamic shifts in activation that are detect during embryogenesis and third instar larval development. Functional studies of the Drosophila homolog of the ERR receptor family may provide a basis for understanding these dynamic shifts in LBD activation, as well as revealing a natural ligand for this NR (Palanker, 2006).


DEVELOPMENTAL BIOLOGY

Hr38 is expressed throughout third-instar larval and prepupal development (Fisk, 1995).

Expression of the Hr38 gene in Drosophila embryogenesis was analyzed using RNA (Northern) blots, and multiple mRNA species were observed even in high stringency hybridization experiments. A prominent 2-kb band (sometimes resolved as a doublet of ~1.8 and 2.0 kb) is present in all embryonic mRNA preparations. A ~4.0-kb species is very abundant in the late embryos (19-23 hr post-egg laying) but is also detectable at lower levels earlier, especially in 15-19-hr embryos. A ~5.0 species is the least abundant in embryogenesis but is clearly present in 15-19-hr embryos. Of the multiple developmentally regulated transcripts of Hr38, the ~4.0- and 5.0-kb species correspond in size and might be represented by the cDNA clones described above (cTK61 and cTK11, respectively). The pLF16 cDNA clone described by Fisk (1995) might be represented by the 1.8- or 2.0-kb transcript, depending on the length of the poly(A)+ tail. The 4.0-kb species and the 1.8-2.0-kb doublet were detected in Schneider's S2 cell line as well. All mRNAs of the Hr38 gene are of low abundance; blotting of purified poly(A)+ RNA and probing by antisense riboprobes was required to detect them. Moreover, it appears that at least the 4.0-kb species is unstable because it is enriched in S2 cells treated with cycloheximide; the ~2.0-kb bands are unaffected by cycloheximide (Kozlova, 1998).

To analyze the expression of the Hr38 gene during all stages of Drosophila development, advantage was taken of a more sensitive technique, RT-PCR, and primers were designed that would specifically amplify fragments corresponding to either the cTK61 or the cTK11 cDNA isoforms. A pair of common primers, flanking the fourth intron in the ligand binding domain, was used to amplify a fragment present in all three cDNA clones described so far. The Hr38 gene is expressed during most of Drosophila development but with some notable variations in quantity. The common fragment indicates that the combined Hr38 mRNAs are present in 0-8-hr embryos at very low levels, which are significantly elevated in late embryogenesis and through the larval stages. Transcript levels become notably enriched in pre-pupal and especially pupal stages, and are again somewhat reduced in adult flies. The mRNAs are absent from the ovaries, but relatively concentrated in third instar larval imaginal discs and brain complexes. The expression profiles for individual isoforms are consistent with the profile of the common fragment, but show some interesting variations: the pupal enrichment is most dramatic for the cTK11 isoform, and the adult has a substantial amount of cTK11 but virtually no cTK61 transcript. In overall terms, the cTK11 (~5.0 kb) isoform is enriched in pupae and adults relative to the cTK61 (~4.0 kb) isoform, which is more characteristic of the larvae (Kozlova, 1998).

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-beta/BMP signaling and orphan nuclear receptors

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. This study describes the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, this study shows that phagocytes have a key role in this process. Importantly, the presence of peripheral glial cells is shown close to the neuro-muscular junction that retracts before the motor neuron. In muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. This study interrupted TGF-β signaling in motor neurons using expression of dominate negative Wishful thinking. It is proposed that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation (Boulanger, 2012).

It is a general feature of maturing brains, both in vertebrates and in invertebrates, that neural circuits are remodeled as the brain acquires new functions. In holometabolous insects, the difference in lifestyle is particularly apparent between the larval and the adult stages. These insects possess two distinct nervous systems at the larval and adult stages. A class of neurons is likely to function in both the larval and the adult nervous systems. The neuronal remodeling occurring during this developmental period is expected to be necessary for the normal functioning of the new circuits (Boulanger, 2012).

The pruning of an axon can involve a retraction of the axonal process, its degeneration or both a retraction and degeneration. The MB γ axon is pruned through a local degeneration mechanism. In contrast, axons may retract their cellular processes from distal to proximal in the absence of fragmentation and this mechanism is called retraction. Interestingly, the two mechanisms can occur sequentially in the same neuron, as in the case of the dendrites of the da neurons, where branches degenerate and the remnant distal tips retract (Boulanger, 2012).

This study provides evidence that the motor neuron innervating larval muscle 4 (NMJ 4) is pruned predominantly through a retraction mechanism. The first morphological indication of motor neuron retraction is the absence of fragmentation observed with anti-HRP staining at the level of the presynapse in all the developmental stages analyzed, together with a decrease in perimeter size observed after 2 h APF. The continuity of this HRP staining is in contrast to the pronounced interruptions between blebs observed with an antibody against mCD8 in γ axons. A molecular indication of motor neuron retraction in these studies is the fact that β-Spectrin disappears at the synapse 5 h APF, before motor neuron pruning takes place. Indeed, it has been shown using an RNA interference approach that loss of presynaptic β-Spectrin leads to presynaptic retraction and synapse elimination at the NMJ during larval stages. The modifications of the microtubule morphology that were observed, such as an increase in microtubule thickness and withdrawal, provide additional evidence of axonal retraction during NMJ remodeling. Finally, a strong argument in favor of a motor neuron retraction mechanism is the fact that Tubulin is present at the NMJ throughout all stages of axonal pruning at the start of metamorphosis (0-7 h APF). This stands in clear contrast to the abolition of Tubulin expression observed before the first signs of γ axon degeneration. It is also interesting to note that the motor neuron retraction observed in this study at metamorphosis and at larval stages are morphologically different. During metamorphosis, retraction bulbs or postsynaptic footprints, which have been reported at larval stages, were never visualized. The fact that the postsynapse dismantles at metamorphosis before motor neuron retraction might explain these discrepancies. Worth noting is the mechanistic correlation between accelerated debris shedding observed here for NMJ pruning at the start of metamorphosis and axosome shedding occurring during vertebrate motor neuron retraction (Boulanger, 2012).

In vertebrates, glia play an essential role in the developmental elimination of motor neurons. In Drosophila, the role of glia in sculpting the developing nervous system is becoming more apparent. Clear examples of a role for engulfing glial cells in axon pruning are well documented during the MB γ axon degeneration at metamorphosis. Also, glia are required for clearance of severed axons of the adult brain. A distinct protective role of glia has been recently discovered during the patterning of dorsal longitudinal muscles by motor neurobranches. This study describes the presence of glia processes close to the end of the pupal NMJ. The observations suggest that the glial extensions retract at 5 h APF, just before motor neuron retraction is observed. When the glial dynamic is blocked, the NMJ dismantling might be also blocked. It is hypothesized that during development in larvae and early pupae, glial processes have a protective role and aid in the maintenance of the NMJ. Then, between 2 and 5 h APF, glial retraction would be a necessary initial step that allows NMJ dismantling. In accordance with this hypothesis, glia play a protective role in the maintenance of NMJ during pruning of second order motor neuron branches 31 h APF (Boulanger, 2012).

Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body (Awasaki, 2004). One can note that at the NMJ the role of the glia is proposed to be essentially opposite from its role in MB γ axons pruning but in both cases blocking the glia dynamics results in a similar blocking of the pruning process (Boulanger, 2012).

In vertebrates, phagocytes are recruited to the injured nerve where they clear, by engulfment, degenerating axons. In Drosophila, phagocytic blood cells engulf neuronal debris during elimination of da sensory neurons. This study shows that blocking phagocyte dynamics with shi produces a strong blockade of the NMJ dismantling process. One possibility is that phagocytes attack and phagocytose the postsynaptic material, a process blocked by compromising shi function resulting in postsynaptic protection. In accordance, it has been shown that phagocytes attack not only the da dendrites to be pruned, but also the epidermal cells that are the substrate of these dendrites (Boulanger, 2012).

During NMJ dismantling, the muscle has an instructive role for motor neuron retraction. In all the situations where postsynapse dismantling is blocked, the corresponding presynaptic motor neuron retraction is also blocked. Therefore, it is sufficient to propose that both glial cells and phagocytes affect only the postsynaptic compartment. Nevertheless, one cannot rule out that these two cell types both act directly at the pre and at the postsynapse (Boulanger, 2012).

ECR-B1 is highly expressed and/or required for pruning in remodeling neurons of the CNS. MB γ neurons and antennal lobe projection neurons remodeling require both the same TGF-β signaling to upregulate EcR-B1. In the MBs only neurons destined to remodel show an upregulation of EcR-B1. At least two independent pathways insure EcR-B1 differential expression. The TGF-β pathway and the nuclear receptor pathway are thought to provide the necessary cell specificity of EcR-B1 transcriptional activation. This study shows that in the motor neuron pruning these two pathways are also necessary to activate EcR-B1. Noteworthy, showing an analogous requirement of ftz-f1/Hr39 pathway in two different remodeling neuronal systems unravels the fundamental importance of this newly described pathway (Boulanger, 2012).

The following model is proposed for the sequential events that are occurring during NMJ dismantling at early metamorphosis. First, EcR-B1 is expressed in the muscle under the control of FTZ-F1. FTZ-F1 activates EcR-B1 and represses Hr39. This repression is compulsory for EcR-B1 activation. Importantly, TGF-β/BMP signaling does not appear to be required for EcR-B1 activation in this tissue, however, a result of EcR-B1 activation in the muscle would be the production of a secreted TGF-β family ligand. Then, this secreted TGF-β family ligand reaches the appropriate receptors and activates the TGF-β signaling in the motor neuron. Finally, TGF-β signaling in association with the nuclear receptor pathway activates EcR-B1 expression resulting in motor neuron retraction. Since glial cells and phagocytes are required for the dismantling process, it is possible that a TGF-β/BMP family ligand(s) be produced by one or both of these cell types and not by the postsynaptic compartment. Noteworthy, a recent study shows that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila MBs (Awasaki, 2011). Nevertheless, one can note that the requirement of the two pathways in the motor neuron provides a simple molecular explanation of the instructive role of postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation. It was proposed that in the MBs, the association of these two pathways provides the cell (spatial) specificity of pruning. In this paper, this association is proposed to provide the temporal specificity of the events. Future studies will be necessary to understand how EcR-B1 controls the production of a TGF-β/BMP ligand(s) in the muscle, the reception of this signal by the motor neuron and the ultimate response by the motor neuron to initiate retraction. These steps will be necessary to unravel the molecular mechanisms underlying the NMJ dismantling process and related phenomenon in vertebrate NMJ development and disease. Interestingly, it appears that TGF-β ligands on the one hand are positive regulators of synaptic growth during larval development and on the other hand, they are positive regulators of synaptic retraction, at the onset of metamorphosis. In both situations signaling provides a permissive role, sending a signal from the target tissue to the neuron. The consequence of this signal would be dependent on developmental timing thus, on a change in context (Boulanger, 2012).

Visualization of neural activity in insect brains using a conserved immediate early gene. Hr38

Many insects exhibit stereotypic instinctive behavior, but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. This study identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, comprehensive activity patterns of the silkmoth brain were mapped in response to female sex pheromones. Hr38 could also be used as a neural activity marker in the fly Drosophila melanogaster. Using Hr38, a neural activity map of the fly brain was mapped that partially overlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies (Fujita, 2013).

Dhr38 expression was investigated in the brain of a naive male fly stimulated with a decapitated virgin female body. D. melanogaster males recognize conspecific females through visual, olfactory, and gustatory cues and show courtship behavior even to decapitated females. In response to female stimulation, Dhr38 was robustly expressed in various brain regions. In particular, strong signals were detected in the cells located dorsal to the AL (defined as area 1) and around the MBs (area 2). Signals were also reproducibly detected between the protocerebrum (PC) and OL (area 3), around the SOG (area 4), and around the lobula (area 5). The number of Dhr38-positive cells was comparable between male brains stimulated with one or three decapitated virgin females, suggesting that Dhr38 detection is sensitive enough to detect neural activity induced by a single virgin females (Fujita, 2013).

The gross expression pattern of Dhr38 was similar between the brains of males stimulated with a virgin and those stimulated with a mated female. Although mated females emit an antiaphrodisiac male pheromone, cis-vaccenyl acetate, they are still attractive to naive males and induce courtship behaviors (Fujita, 2013).

Next the contribution of female pheromones to Dhr38 expression in male brains was investigated. To examine the contribution of contact pheromone input, the Dhr38 expression pattern was examined in the brains of males with both foreleg tarsi surgically removed. In males with foreleg amputations, virgin female stimulation still induced a Dhr38 expression pattern similar to that in intact males. Then the contribution of olfactory input was evaluated using males whose antennae were surgically removed. Antennae amputation led to a significant decrease in the number of Dhr38-positive cells in all brain areas in response to female stimulation. The remaining Dhr38 expression completely disappeared when foreleg amputation was combined with antennae removal. In addition, in anosmic Orco mutants, female stimulation induced Dhr38 expression in a smaller number of cells than in wild-type, and Dh38 expression was decreased by foreleg amputation. The Dhr38 expression remaining after foreleg amputation might be derived from Orco-independent olfactory inputs. These findings indicate that Dhr38 expressed in response to female stimulation is derived from both olfactoryand contact-dependent neural pathways. Further, decapitated male stimulation induced Dhr38 expression in a moderate number of cells in areas 1, 2, and 5, indicating that these areas comprise heterologous neurons responsive to females and males. Dhr38 expression was examined in Or47b mutants, because Or47b is thought to be a female pheromone receptor (van der Goes van Naters, 2007). Double in situ hybridization of Dhr38 and Or47b confirmed that Or47b-expressing cells are responsive to virgin female stimulation. In Or47b mutants, female stimulation induced Dhr38 expression in a smaller number of cells than in wild-type, and this response was significantly decreased by foreleg amputation. These findings indicate that a large part of the neural activity induced by female stimulation is regulated by chemical inputs from contact pheromones and female odors (Fujita, 2013).

Male fly courtship behavior is regulated by neural circuits comprising fruitless (fru)-expressing neurons. Thus, it was asked whether virgin female-induced Dhr38 expression overlaps with fru-expressing neurons. To address this question, Dhr38 expression was examined in the brains of males whose fru-expressing neurons were visualized using an NP21-GAL4 strain, which covers 82% of Fru-expressing neurons. In areas 1, 4, and 5, Dhr38 was not expressed in fru-expressing cells. In contrast, in area 2, a small portion of Dhr38-positive cells was positive for GFP. Because the majority of Dhr38-positive cells were located dorsal to the MB calyces and fru-expressing cells were located ventral to the MB calyces, this area was analyzed in detail by focusing on each cell cluster. Dhr38-positive cells were detected in GFP-positive P1 and P4 cells (one or two double-positive cells per cluster) in three specimens. No Dhr38-positive cells were detected in P2 and P3 clusters. Interestingly, Dhr38-positive cells in area 3 frequently colocalized with fru-expressing neurons and are assumed to be Lv1+Ld and Lv2 cluster cells, which extend neurites to the OLs. P1 neurons are the master command neurons of male courtship behavior and are activated on contact with females through the foreleg tarsus (Kimura, 2008; Kohatsu, 2011). Consistent with this notion, no P1 neurons were positive for Dhr38 in males with both foreleg tarsi amputated. In contrast, the number of cells positive for both Dhr38 and GFP in area 3 was not affected by foreleg amputation, indicating that Dhr38-positive area 3 neurons are not involved in contact pheromone recognition. Taken together, these findings indicate that the neural circuit activated by virgin female stimulation partially overlaps with that comprising fru-expressing cells, supporting the notion that Dhr38 can be used to detect physiologically relevant neural activities (Fujita, 2013).

This study has identified Hr38 as a conserved IEG that can be used as a neural activity marker in insect brains. HR38 is the sole insect ortholog of the NR4A nuclear receptor family, which is highly conserved among metazoans and whose expression is increased by a variety of cellular signals. It is therefore reasonable to assume that neural activity-dependent Hr38 expression is widely conserved among insects. Because the DNA binding domain of NR4A family genes is highly conserved among species and in situ hybridization is feasible in nontransgenic animals, this study provides a powerful approach for neuroethologic studies in a wide variety of animalss (Fujita, 2013).

Hr38 was previously identified as an interaction partner of Ultraspiracle that binds to Ultraspiracle in competition with the ecdysone receptor, which is suggested to contribute to the fine-tuning of the ecdysone signaling pathway. Recently, ecdysone signaling was confirmed to be involved in memory formation in vinegar flies. Activity-dependent Hr38 expression suggests that ecdysone signaling may be modified in a neural activity-dependent manner, leading to the hypothesis that Hr38 has important roles in higher neural function, such as memory formation. Further studies are needed to elucidate the mechanism regulating activity-dependent Hr38 expression and its neural functions (Fujita, 2013).


EFFECTS OF MUTATION

Four alleles have been characterized of Hr38; these consist of a P-element enhancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Hr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Hr38 plays an important role in late stages of epidermal metamorphosis. The lethal phases of available EMS and P-element induced mutations indicate that Hr38 is important for late stages of metamorphosis; the haemolymph leakage and melanization phenotype suggest that all presently available alleles affect adult cuticle formation, possibly leading to incomplete sclerotization. In the three weaker alleles the defects appear to be specific to the thoracic cuticle of the leg joints, because abdominal and head structures are not visibly affected. Overall morphology of the mutant flies bearing the stronger EMS allele, including tanning of the bristles, is normal in Hr3856/Df(2)KetelRX32 hemizygotes at 80-90 hr after puparium formation. It is unlikely that these mutations represent complete loss-of-function alleles. The weak Hr3843 and Hr3857 alleles behave as hypomorphs in genetic assays, and both mRNA and Hr38 protein are still present in the Hr3856/Df(2)KetelRX32 hemizygous mutant animals. Therefore either a specific epidermal function of Hr38 is affected in these mutants, specific epidermal cells are most sensitive to altered levels of Hr38 expression, or Hr38 is dispensable in tissues other than epidermis (Kozlova, 1998).


EVOLUTIONARY HOMOLOGS

NGFI-B subfamily members: Interaction with RXR

Heterodimerization is a common paradigm among eukaryotic transcription factors. The 9-cis retinoic acid receptor (RXR) serves as a common heterodimerization partner for several nuclear receptors, including the thyroid hormone receptor (T3R) and retinoic acid receptor (RAR). This raises the question as to whether these complexes possess dual hormonal responsiveness. A strategy was devised to examine the transcriptional properties, either of each receptor individually or when tethered to a heterodimeric partner. The intrinsic binding properties of RXR are masked in T3R-RXR and RAR-RXR heterodimers. In contrast, RXR is active as a non-DNA-binding cofactor with the NGFI-B/Nurr1 orphan receptors. Heterodimerization of RXR with constitutively active NGFI-B/Nurr1 creates a novel hormone-dependent complex. These findings suggest that allosteric interactions among heterodimers create complexes with unique properties. It is suggested that allostery is a critical feature underlying the generation of diversity in hormone response networks (Forman, 1995).

In addition to its role as a 9-cis retinoic acid receptor, RXR has an important role in the regulation of multiple hormonal pathways through heterodimerization with nuclear receptors. Two orphan receptors, NGFI-B and NURR1, which have been shown to interact with DNA as monomers, also can heterodimerize with RXR. These heterodimers bind selectively to a class of retinoic acid response elements composed of direct repeats spaced by 5 nucleotides. In this respect they are similar to heterodimers formed between RXR and the receptor for all-trans retinoic acid, RAR. However, whereas RXR is inhibited in the RXR-RAR heterodimer, NGFI-B/NURR1 promote efficient activation in response to RXR ligands and therefore shift RXR from a silent to an active heterodimerization partner. These data show that NGFI-B and NURR1 can increase the potential of RXR to modulate gene expression in a ligand-dependent manner by allowing a distinct class of direct repeats to serve as specific RXR response elements. Because expression of both NGFI-B and NURR1 is rapidly induced by various growth factors, these findings also suggest a novel mechanism for convergence between vitamin A or retinoid and growth factor signaling pathways (Perlmann, 1995).

Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands. However, the specific physiological roles of Nurr1 monomers and RXR-Nurr1 heterodimers remain to be elucidated. The aim of this study was to define structural requirements for RXR-Nurr1 heterodimerization. Several amino acid substitutions were introduced in both Nurr1 and RXR in the I-box, a region known to be important for nuclear receptor dimerization. Single amino acid substitutions introduced in either Nurr1 or RXR abolish heterodimerization. Importantly, heterodimerization-deficient Nurr1 mutants exhibit normal activities as monomers. Thus, by introducing specific amino acid substitutions in Nurr1, monomeric and heterodimeric properties of Nurr1 can be distinguished. Interestingly, substitutions in the RXR I-box differentially affect heterodimerization with Nurr1, retinoic acid receptor, thyroid hormone receptor, and constitutive androstane receptor, demonstrating that the dimerization interfaces in these different heterodimers are functionally unique. Furthermore, heterodimerization between RXR and Nurr1 had a profound influence on the constitutive activity of Nurr1, which is diminished as a result of RXR interaction. In conclusion, these data show unique structural and functional properties of RXR-Nurr1 heterodimers and also demonstrate that specific mutations in Nurr1 can abolish heterodimerization without affecting other essential functions (Aarnisalo, 2002).

NGFI-B subfamily members: Structural studies

Nurr1, a member of the nuclear hormone receptor superfamily, is of critical importance in the developing central nervous system where it is required for the generation of midbrain dopamine cells. Nuclear receptors encompass a transcriptional activation function (activation function 2; AF2) within their carboxyl-terminal domains important for ligand-induced transcriptional activation. Since a Nurr1 ligand remains to be identified, the role of the Nurr1 AF2 region in transcriptional activation is unclear. The Nurr1 AF2 has been shown to contribute to constitutive activation independent of exogenously added ligands in human embryo kidney 293 cells and in neural cell lines. Extensive mutagenesis indicates a crucial role of the AF2 core region for transactivation but also identifies unique features differing from previously characterized receptors. In addition, Nurr1 does not appear to interact with, and is not stimulated by, several previously identified coactivators such as the steroid receptor coactivator 1. In contrast, adenovirus protein E1A, stably expressed in 293 cells, was shown to contribute to AF2-dependent activation. Finally, while the AF2 core of RXR is required for ligand-induced transcriptional activation by Nurr1-RXR heterodimers, the functional integrity of Nurr1 AF2 core is not critical. These results establish that the ligand binding domain of Nurr1 has intrinsic capacity for transcriptional activation depending on cell type and mode of DNA binding. Furthermore, these results are consistent with the possibility that gene expression in the central nervous system can be modulated by an as yet unidentified ligand interacting with the ligand binding domain of Nurr1 (Castro, 1999).

Nur77/NR4A1 is an 'orphan member' of the nuclear hormone receptor superfamily. Nur77 and its close relatives Nurr1 and NOR-1 bind as monomers to a consensus binding site, the nerve growth factor induced protein I-B (NGFI-B)-binding response element (NBRE). The Nur77/NURR1/NOR1 nuclear receptors are classified as immediate early response genes that are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, and apoptosis. However, the mechanism of coactivation and ligand independent trans-activation remains unclear. The molecular basis of Nur77-mediated cofactor recruitment and activation has been examined. Nur77 trans-activates gene expression in a cell-specific manner, and operates in an activation function-1 (AF-1)-dependent manner. The AB region encodes an uncommonly potent N-terminal AF-1 domain delimited to between amino acids 50 and 160 and is essential for the ligand-independent activation of gene expression. Steroid receptor coactivator-2 (SRC-2) modulates the activity of the N-terminal AF-1 domain. Moreover, SRC-2 dramatically potentiates the retinoid induced RXR-dependent activation of the Nur77 ligand binding domain (LBD). Interestingly, the N-terminal AB region (not the LBD) facilitates coactivator recruitment and directly interacts with SRC, p300, PCAF, and DRIP-205. Consistent with this, homology modeling indicates that the Nur77 LBD coactivator binding cleft is substantially different from that of retinoic acid receptor gamma, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors is replaced with a much more hydrophilic surface with a distinct topology. This observation accounts for the inability of this nuclear receptor LBD to directly mediate cofactor recruitment. Furthermore, the AF-1 domain physically associates with the Nur77 C-terminal LBD and synergizes with the retinoid X receptor LBD. Thus, the AF-1 domain plays a major role in Nur77-mediated transcriptional activation, cofactor recruitment, and intra- and inter-molecular interactions (Wansa, 2002).

NOR-1/NR4A3 is an 'orphan member' of the nuclear hormone receptor superfamily. NOR-1 and its close relatives Nurr1 and Nur77 are members of the NR4A subgroup of nuclear receptors. Members of the NR4A subgroup are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, T-cell apoptosis, chondrosarcomas, neurological disorders, inflammation, and atherogenesis. However, the mechanism of transcriptional activation, coactivator recruitment, and agonist-mediated activation remain obscure. The molecular basis of NOR-1-mediated activation has been examined. NOR-1 was found to trans-activate gene expression in a cell- and target-specific manner; moreover, it operates in an activation function (AF)-1-dependent manner. The N-terminal AF-1 domain delimited to between amino acids 1 and 112, preferentially recruits the steroid receptor coactivator (SRC). Furthermore, SRC-2 modulates the activity of the AF-1 domain but not the C-terminal ligand binding domain (LBD). Homology modeling indicates that the NOR-1 LBD is substantially different from that of hRORbeta, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors is replaced with a very hydrophilic surface with a distinct topology. This observation may account for the inability of this nuclear receptor LBD to efficiently mediate cofactor recruitment and transcriptional activation. In contrast, the N-terminal AF-1 is necessary for cofactor recruitment and can independently conscript coactivators. Finally, the purine anti-metabolite 6-mercaptopurine, a widely used antineoplastic and anti-inflammatory drug, is shown to activate NOR-1 in an AF-1-dependent manner. Additional 6-mercaptopurine analogs all efficiently activate NOR-1, suggesting that the signaling pathways that modulate proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A activity. It is hypothesized that the NR4A subgroup mediates the genotoxic stress response and suggest that this subgroup may function as sensors that respond to genotoxicity (Wansa, 2003).

NGFI-B subfamily members: Effects of mutations

The transcription factor Nur77 (NGFI-B), a member of the steroid nuclear receptor superfamily, is induced to a high level during T-cell receptor (TCR)-mediated apoptosis. A transgenic dominant-negative Nur77 protein can inhibit the apoptotic process accompanying negative selection in thymocytes, while constitutive expression of Nur77 leads to massive cell death. Nur77-deficient mice, however, have no phenotype, suggesting the possible existence of a protein with redundant function to Nur77. To explore this possibility, the role of two Nur77 family members, Nurr1 and Nor-1, in TCR-induced apoptosis have been characterized. Nor-1 and Nurr1 can transactivate through the same DNA element as Nur77, and their transactivation activities can be blocked by a Nur77 dominant-negative protein. In thymocytes, Nor-1 protein is induced to a very high level upon TCR stimulation and has similar kinetics to Nur77. In contrast, Nurr1 is undetectable in stimulated thymocytes. Furthermore, constitutive expression of Nor-1 in thymocytes leads to massive apoptosis and up-regulation of CD25, suggesting a functional redundancy between Nur77 and Nor-1 gene products. As in the case of Nur77-FL mice, FasL is not detectable in the thymocytes of Nor-1 transgenic mice. Constitutive expression of Nur77 in gld/gld mice rescues the lymphoproliferative phenotype of the FasL mutant mice. Thus, Nor-1 and Nur77 demonstrate functional redundancy in an apparently Fas-independent apoptosis (Cheng, 1997).

NGFI-B subfamily members: Modification by phosphorylation

The immediate-early gene NGFI-B (also called nur77) encodes an orphan nuclear receptor that activates transcription through a unique response element (NBRE). NGFI-B is rapidly induced and modified via phosphorylation by a variety of stimuli that induce cells to differentiate or to proliferate. The in vitro phosphorylation of Ser350 located within the 'A-box,' a motif necessary for DNA binding by NGFI-B, results in a decrease in the binding of NGFI-B to its response element. Nerve growth factor (NGF)-induced changes in the in vivo phosphorylation of Ser350 accompany transcriptional deactivation of NGFI-B in PC12 cells: membrane depolarization and NGF treatment cause differential phosphorylation of NGFI-B, and the transcriptional activation caused by exogenous expression of NGFI-B or membrane depolarization can be inhibited by NGF treatment. In addition, the mutation of Ser350 to Ala abolishes the inhibitory effect of NGF on the transcriptional activation of NGFI-B in PC12 cells. These data could provide new insights into the regulation of transcriptional activity required for some neurons to switch from activity-dependent survival to neurotrophin-dependent survival during development (Kitagiri, 1997).

The immediate early gene NUR77 (also called NGFI-B) is required for T cell antigen receptor-mediated cell death and is induced to very high levels in immature thymocytes and T cell hybridomas undergoing apoptosis. The Akt (PKB) kinase is a key player in transduction of anti-apoptotic and proliferative signals in T cells. Because Nur77 has a putative Akt phosphorylation site at Ser-350, and phosphorylation of this residue is critical for the transactivation activity of Nur77, whether Akt regulates Nur77 was investigated. Coimmunoprecipitation experiments show the detection of Nur77 in Akt immune complexes, suggesting that Nur77 and Akt physically interact. Akt specifically phosphorylates Ser-350 of the Nur77 protein within its DNA-binding domain in vitro and in vivo in 293 and NIH 3T3 cells. Because phosphorylation of Ser-350 of Nur77 is critical for its function as a transcription factor, the effect of Akt on this function was examined. By using luciferase assay experiments, it has been shown that phosphorylation of Nur77 by Akt decreases the transcriptional activity of Nur77 by 50% to 85%. Thus, Akt interacts with Nur77 and inactivates Nur77 by phosphorylation at Ser-350 in a phosphatidylinositol 3-kinase-dependent manner, connecting the phosphatidylinositol 3-kinase-dependent Akt pathway and a nuclear receptor pathway (Pekarsky, 2001).

The NGFI-B (Nur77) subfamily of orphan nuclear receptors (NRs), which also includes Nurr1 and NOR1, bind the NurRE regulatory element as either homo- or hetero-dimers formed between subfamily members. These NRs mediate the activation of pituitary proopiomelanocortin (POMC) gene transcription by the hypothalamic hormone corticotropin-releasing hormone (CRH), an important link between neuronal and endocrine components of the hypothalamo-pituitary-adrenal axis. CRH effects on POMC transcription do not require de novo protein synthesis. CRH signals activate Nur factors through the cyclic AMP/protein kinase A (PKA) pathway. CRH and PKA rapidly increase nuclear DNA binding activity of NGFI-B dimers but not monomers. Accordingly, CRH- or PKA-activated Nur factors enhance dimer (but not monomer) target response elements. p160/SRC coactivators are recruited to Nur dimers (but not to monomers) and coactivator recruitment to the NurRE is enhanced in response to CRH. Moreover, PKA- and coactivator-induced potentiation of NGFI-B activity are primarily exerted through the N-terminal AF-1 domain of NGFI-B. The TIF2 (SRC-2) glutamine-rich domain is required for this activity. Taken together, these results indicate that Nur factors behave as endpoint effectors of the PKA signaling pathway acting through dimers and AF-1-dependent recruitment of coactivators (Maira, 2003).

NGFI-B subfamily members: Nurr1 is a molecular target of 6-amino purine

The purine anti-metabolite 6-mercaptopurine is one of the most widely used drugs for the treatment of acute childhood leukemia and chronic myelocytic leukemia. Developed in the 1950s, the drug is also being used as a treatment for inflammatory diseases such as Crohn's disease. The antiproliferative mechanism of action of this drug and other purine anti-metabolites has been demonstrated to be through inhibition of de novo purine synthesis and incorporation into nucleic acids. Despite the extensive clinical use and study of 6-mercaptopurine and other purine analogues, the cellular effects of these compounds remain relatively unknown. More recently, purine anti-metabolites have been shown to function as protein kinase inhibitors and to regulate gene expression. Interestingly, in an attempt to find small molecule regulators of the orphan nuclear receptor Nurr1, 6-mercaptopurine was identified as a specific activator of this receptor. A detailed analysis of 6-mercaptopurine regulation of Nurr1 demonstrates that 6-mercaptopurine regulates Nurr1 through a region in the amino terminus. This activity can be inhibited by components of the purine biosynthesis pathway. These findings indicate that Nurr1 may play a role in mediating some of the antiproliferative effects of 6-mercaptopurine and potentially implicate Nurr1 as a molecular target for treatment of leukemias (Ordentlich, 2003).

NGFI-B subfamily members: Transcriptional regulation

The program of gene expression regulated by vascular endothelial growth factor (VEGF) remains poorly understood. The aim of this study was to identify VEGF-regulated genes in human umbilical vein endothelial cells. VEGF-regulated gene expression was analyzed by screening Affymetrix oligonucleotide arrays and quantitative, real-time, reverse transcription-polymerase chain reaction. The most strongly induced genes are the NR4A nuclear receptor family members Nur77, Nurr1, and Nor1 and the zinc-finger transcription factor Egr3. VEGF also induces rapid expression of DSCR1, cyclooxygenase-2, tissue factor, stanniocalcin-1, the serine/threonine kinase Cot, and EHD3. VEGF-induced NR4A family and Egr3 expression is blocked by a KDR inhibitor, and prostaglandin F1 and basic fibroblast growth factor weakly increases expression of these genes. Induction of NR4A genes is mediated via intracellular Ca(2+), protein kinase C- and calcineurin-dependent pathways. VEGF increases protein expression of Nurr1 and Nur77 and decreases Nur77 phosphorylation at the negative regulatory site serine 351. It is concluded that VEGF induces expression of NR4A nuclear receptors and Egr3 via KDR and KDR-mediated signaling mechanisms. The genes identified here are novel candidates as key early mediators of VEGF-induced endothelial functions (Liu, 2003).

NGFI-B subfamily members: Transcriptional targets

Three related orphan nuclear receptors that are expressed in the brain, NGFI-B, Nurr1, and NOR-1, were studied to compare their function as transcriptional activators. NGFI-B is able to activate (in the absence of added hormone) in CV1 cells both an NGFI-B-responsive luciferase reporter gene [containing eight copies of a response element for NGFI-B upstream of a basal prolactin promoter driving the luciferase gene, [NBRE(8)-LUC], a similar thyroid hormone-receptor-responsive reporter gene [TRE(3)-LUC], and a reporter gene with an authentic promoter from a Xenopus vitellogenin gene containing two binding sites for the estrogen receptor (vit-LUC). NGFI-B activates NBRE(8)-LUC and TRE(3)-LUC (but not the vitLUC) with an amino-terminal activation domain. Nurr1 is less promiscuous as a transcriptional activator, activating.the NBRE(8)-LUC better than NGFI-B, but less than NGFI-B at the other reporter genes. NOR-1 activates only the NBRE(8)-LUC reporter gene. These results indicate that closely related nuclear receptors may differentiate between response elements or promoters and that different activation mechanisms exist depending on the promoter. This may contribute to regulation of specificity of target gene expression in the brain (Paulsen, 1995).

Within the nuclear receptor family, Nur77 (also known as NGFI-B) distinguishes itself by its ability to bind a target sequence (the NBRE) as a monomer and by its role in T-cell receptor (TCR)-induced apoptosis in T cells. A novel mechanism of Nur77 action is mediated by homodimers. These dimers bind a Nur77 response element (NurRE), which has been identified as a target of CRH-induced Nur77 in the pro-opiomelanocortin (POMC) gene promoter. Both halves of the palindromic NurRE are required for responsiveness to physiological signals, like CRH in pituitary-derived AtT-20 cells. Similarly, in T-cell hybridomas, TCR activation induces NurRE but not NBRE reporters. The in vivo signaling function of Nur77 thus appears to be mediated by dimers acting on a palindromic response element of unusual spacing between its half-sites. This mechanism may represent the biologically relevant paradigm of action for this subfamily of orphan nuclear receptors (Philips, 1997).

Plasminogen activator inhibitor 1 (PAI-1) is the main fibrinolysis inhibitor, and high plasma levels are associated with an increased risk for vascular diseases. Inflammatory cytokines regulate PAI-1 through a hitherto unclear mechanism. Using reporter gene analysis, a region in the PAI-1 promoter was identified that contributes to basal expression as well as to tumor necrosis factor alpha (TNFalpha) induction of PAI-1 in endothelial cells. Using this region as bait in a genetic screen, Nur77 (NAK-1, TR3, NR4A1) was identified as an inducible DNA-binding protein that binds specifically to the PAI-1 promoter. Nur77 drives transcription of PAI-1 through direct binding to an NGFI-B responsive element (NBRE), indicating monomeric binding and a ligand-independent mechanism. Nur77, itself, is transcriptionally up-regulated by TNFalpha. High expression levels of Nur77 and its colocalization with PAI-1 in atherosclerotic tissues indicate that the described mechanism for PAI-1 regulation may also be operative in vivo (Gruber, 2003).

NGFI-B subfamily members: Developmental expression

NGFI-B, Nurr1, and Nor1 are three closely related orphan members of the steroid/thyroid hormone receptor superfamily. These receptors can bind to DNA as monomers and exhibit constitutive transcriptional activity. Moreover, two of the receptors, NGFI-B and Nurr1, form heterodimers with the retinoid X receptor (RXR). Such heterodimers as well as complexes formed between RXR and the all-trans retinoic acid receptor bind to DNA response elements composed of direct repeats spaced by five nucleotides (DR5). However, whereas retinoic acid receptor can inhibit ligand-dependent RXR activation, NGFI-B and Nurr1 allow efficient RXR activation through DR5 elements and thus define a distinct pathway for vitamin A signaling. The most recently identified member of the subfamily, Nor1, shows similar monomer DNA-binding and constitutive transactivation properties as NGFI-B and Nurr1. In contrast, however, Nor1 is unable to promote RXR signaling due to its inability to form heterodimers with RXR. To begin to understand the physiological implications of these functional differences, in situ hybridization was used to compare the distribution of Nor1, NGFI-B, and Nurr1 messenger RNAs during different developmental stages. The receptors are expressed in both distinct and overlapping patterns, predominantly in the central nervous system. Notably, Nurr1 is expressed in the prenatal ventral midbrain in a region that gives rise to dopaminergic neurons. Nor1 is also expressed during embryonic development, and all three receptors show a complex distribution in the postnatal brain. Furthermore, Nor1 colocalizes with NGFI-B in the adrenal glands and thymus, two tissues in which NGFI-B has been suggested to be functionally important. These data may indicate redundancy between members of the NGFI-B/Nurr1/Nor1 subfamily and could explain why no phenotypic disturbances have yet been found in mice in which the NGFI-B gene has been inactivated (Zetterstrom, 1996).

NGFI-B subfamily members: Function in induction of apoptosis

The immediate-early gene Nur77, which encodes an orphan nuclear receptor, is rapidly induced by various stress stimuli, including tumor necrosis factor (TNF). Nur77 has been implicated in mediating apoptosis, particularly in T cells and tumor cells. Nur77 can play a role in antagonizing apoptosis in TNF signaling. Nur77 expression is strongly induced by TNF. Interestingly, unlike most antiapoptotic molecules, this induced expression of Nur77 is largely independent of NF-kappa B. Ectopic expression of Nur77 can protect wild-type, TRAF2-/-, and RelA-/- cells from apoptosis induced by TNF, whereas expression of a dominant-negative form of Nur77 (DN-Nur77) accelerates TNF-mediated cell death in the mutant cells. In mouse embryonic fibroblasts, Nur77 remains in the nucleus in response to TNF and is not translocated to the mitochondria, where it was reported to mediate apoptosis. These results suggest that Nur77 is a survival effector protein in the context of TNF-mediated signaling (Suzuki, 1995).

Akt is a common mediator of cell survival in a variety of circumstances. Although some candidate Akt targets have been described, the function of Akt is not fully understood, particularly because of the cell type- and context-dependent apoptosis regulation. One of the mechanisms by which Akt antagonizes apoptosis involves the inhibition of Nur77, a transcription factor implicated in T-cell receptor-mediated apoptosis. It has been suggested that Akt phosphorylates Nur77 directly, but whether Akt suppresses biological functions of Nur77 remains unknown. Akt was found to inhibit the DNA binding activity of Nur77 and stimulate its association with 14-3-3 in a phosphorylation site-dependent manner. Moreover, expression of Akt suppresses Nur77-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas. The inhibition of Nur77 by Akt suggests a mechanism that explains how T-cell receptor activation can promote survival in some instances even when Nur77 is induced. Collectively, these results may suggest that Akt is a negative regulator of Nur77 in T-cell apoptosis (Masuyama, 2001).

Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). This type of macrophage death can occur in the septic mouse model, and toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal-regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role for Nur77 in caspase-independent cell death (Kim, 2003).

The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells

Successful haematopoiesis requires long-term retention of haematopoietic stem cells (HSCs) in a quiescent state. The transcriptional regulation of stem cell quiescence, especially by factors with specific functions in HSCs, is only beginning to be understood. This study demonstrates that Nurr1, a nuclear receptor transcription factor, has such a regulatory role. Overexpression of Nurr1 drives early haematopoietic progenitors into quiescence. When stem cells overexpressing Nurr1 are transplanted into lethally irradiated mice, they localize to the bone marrow, but do not contribute to regeneration of the blood system. Furthermore, the loss of only one allele of Nurr1 is sufficient to induce HSCs to enter the cell cycle and proliferate. Molecular analysis revealed an association between Nurr1 overexpression and upregulation of the cell-cycle inhibitor p18 (also known as INK4C), suggesting a mechanism by which Nurr1 could regulate HSC quiescence. These findings provide critical insight into the transcriptional control mechanisms that determine whether HSCs remain dormant or enter the cell cycle and begin to proliferate (Sirin, 2010).

Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation

Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. This study demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 and Foxa2. Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST, and then recruiting histone deacetylase 1 (Hdac1; Drosophila homolog, Rpd3), an enzyme catalyzing histone deacetylation, to DA gene promoters. Co-expression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoREST-Hdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development (Yi 2014).


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Biological Overview

date revised: 15 December 2013

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