InteractiveFly: GeneBrief

Hormone receptor-like in 39: Biological Overview | References

Gene name - Hormone receptor-like in 39

Synonyms - ftz-F1 (confusing misnomer since there is another gene named ftz-f1)

Cytological map position - 39C-60

Function - Zinc finger transcription factor, Nuclear receptor

Keywords - female reproductive tract development, sperm-storing spermathecae, glandular parovaria, brain, axon pruning

Symbol - Hr39

FlyBase ID: FBgn0261239

Genetic map position - chr2L:21,237,237-21,256,186

Classification - NR_LBD: The ligand binding domain of nuclear receptors and C4-type zinc fingers

Cellular location - nuclear

NCBI link: EntrezGene
Hr39 orthologs: Biolitmine
Recent literature
Cattenoz, P. B., Delaporte, C., Bazzi, W. and Giangrande, A. (2016). An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system. Sci Rep 6: 37792. PubMed ID: 27886257
NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids.This study has identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans.
Praggastis, S. A., Nam, H. J., Lam, G., Child Vi, M. B., Castillo, D. M. and Thummel, C. S. (2021). Regulation of male fertility and accessory gland gene expression by the Drosophila HR39 nuclear receptor. Dev Biol 479: 51-60. PubMed ID: 34331899
Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. This study shows that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, these studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility.

Secretions within the adult female reproductive tract mediate sperm survival, storage, activation, and selection. Drosophila female reproductive gland secretory cells reside within the adult spermathecae and parovaria, but their development remains poorly characterized. With cell-lineage tracing, this study found that precursor cells downregulate lozenge and divide stereotypically to generate three-cell secretory units during pupal development. The NR5A-class nuclear hormone receptor Hr39 is essential for precursor cell division and secretory unit formation. Moreover, ectopic Hr39 in multiple tissues generates reproductive gland-like primordia. Rarely, in male genital discs these primordia can develop into sperm-filled testicular spermathecae. Drosophila spermathecae provide a powerful model for studying gland development. It is concluded that Hr39 functions as a master regulator of a program that may have been conserved throughout animal evolution for the production of female reproductive glands and other secretory tissues (Sun, 2012).

In species where fertilization takes place internally, including mammals and insects, a sperm's long and obstacle-filled journey through the female reproductive tract culminates in the penetration of the egg. Prior to reaching its target, both paternal and maternal reproductive tissues deploy mechanisms that strongly influence an individual sperm's chances for success. In particular, specialized glands in female reproductive tracts produce mucus-rich secretions that capacitate sperm to fertilize successfully, inhibit infection, and provide nutritional, maintenance, and storage factors. The interactions of sperm and seminal fluid with the female reproductive tract and its secretions in Drosophila offer an opportunity to genetically analyze these complex processes (Sun, 2012).

Two paired glands, spermathecae (SPs) and parovaria (POs), are the primary sources of secretions encountered by sperm within the Drosophila female reproductive tract (see Structure and origin of Drosophila female reproductive glands). Messenger RNAs (mRNAs) encoding serine proteases, serpins, antioxidants, immune proteins, and enzymes involved in mucus production are found in SPs. Whereas two SPs arise from the engrailed (en) and en+ domains of the A8 segment, both POs originate in the en+ domain of the A9 segment in the female genital disc during pupal development. Both types of mature gland contain large, polyploid secretory cells (SCs). Each SC connects with the gland lumen via a specialized cuticular canal equipped with a secretion-collecting 'end apparatus'. Anatomically related secretory units are found in SPs from other species and in insect epidermal glands that produce pheromones, venoms, and many other products. Despite their ubiquity, insect epidermal gland development has not been well characterized at the molecular genetic level (Sun, 2012).

Studies of genital disc development and patterning have identified multiple genes important for reproductive gland formation. lozenge (lz), encoding a runt-domain transcription factor, is essential for both SP and PO formation and may be directly regulated by the sex determination pathway. Homologous to mammalian AML-1, Lz also supports developing blood precursors and prepatterns ommatidial cells in the developing eye. The dachshund (dac) gene also acts in multiple imaginal discs and is specifically needed for spermathecal duct development. Mutations that disrupt sphingolipid metabolism also cause abnormalities in spermathecal number and structure (Sun, 2012).

One of the most interesting genes needed to form reproductive glands encodes the nuclear hormone receptor Hr39, an early ecdysone-response gene (Allen, 2008). Hr39 and Ftz-f1 are the only two NR5A class nuclear hormone receptors in Drosophila, a class that in mammals includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1). All four of these proteins share 60%-90% sequence identity within their DNA binding domains and bind in vitro to identical sequences. SF-1 is a master regulator of steroidogenesis and sex hormone production (Schimmer, 2010), whereas LRH-1 is required in the ovary for female fertility (Duggavathi, 2008), in embryonic stem cells for pluripotency (Guo, 2010; Heng, 2010) and in endodermal tissues for metabolic homeostasis (Lee, 2011; Lee, 2008). Weak Hr39 mutations alter the production of some SP gene products (Allen, 2008), whereas LRH-1 directly controls major secretory proteins of the exocrine pancreas (Holmstrom, 2011). Thus, NR5A class hormone receptors may play a conserved role controlling secretions from certain tissues, including female reproductive glands (Sun, 2012).

This study characterized the cell lineage of developing reproductive glands and clarify the roles of lz and Hr39. Hr39 is expressed sex-specifically in lz-positive female gland primordia beginning shortly after the ecdysone pulse that initiates prepupal development. When levels of Hr39 are reduced, lz-expressing precursors fail to protrude, divide, or remain viable, suggesting that Hr39 expression orchestrates reproductive gland development. Mouse LRH-1, but not SF-1, can partially replace Hr39 function in gland formation. Ectopic expression of Hr39 in male larvae can induce a pigmented SP-like structure containing sperm to develop in the male reproductive tract. It is proposed that Hr39 acts as a master regulator of reproductive gland development and that the production of sperm-interacting proteins in the female reproductive tract under the control of NR5A proteins has been conserved during evolution. These findings suggest new targets for controlling agriculture pests and human-disease vectors (Sun, 2012).

These studies reveal that lz and Hr39, despite their nearly identical loss-of-function phenotypes, have distinctive expression patterns during gland development. All gland precursors express both genes following puparium formation, but within 24 hr divide to produce lz+ epithelial precursors apically and lz SUPs basally. SUPs then differentiate according to a stereotyped program involving production of two transient accessory cells and a single polyploid secretory cell (Sun, 2012).

Reproductive secretory cells arise in a superficially similar manner to sensory bristles and multiple classes of mechanosensory and chemosensory sensilla. Both utilize short fixed-cell lineages that employ transient accessory cells to generate permanent extracellular structures (secretory canal, sensory bristle, etc.), but the three-cell secretory lineage analyzed in this study differs from the four asymmetric divisions producing five different cells typical of PNS differentiation (see Lineage Analysis of Secretory Unit Formation). Many other insect epidermal glands probably develop in a generally similar manner, but the precise cell lineages and mechanisms documented in this study for Drosophila reproductive glands (three cells, absence of ciliary involvement) differ from previous models (Sun, 2012).

Drosophila secretory units provide a powerful system for analyzing insect gland development. Studies in other insects suggested that an accessory cell utilizes a ciliary process to prevent the SCs from being sealed off by cuticle-secreting epithelial cells. This study found no morphological or genetic evidence that cilia are involved in forming Drosophila secretory units. However, the apical cell (AC) may fulfill this same role using normal microtubules, in much the same way that the anterior polar cells in egg chambers template the micropyle channel during oogenesis. Membranes from the basal cell (BC) likely surround this AC process, secrete the cuticular canal, and join it to the luminal cuticle. Concomitantly, the BC likely secretes the end apparatus around a large apical segment of the SC, which it surrounds (Sun, 2012).

The NR5A hormone receptor Hr39 plays multiple roles in reproductive gland development. Initially, Hr39 orchestrates gland protrusion and in the absence of Hr39 protrusion fails to occur. Among Drosophila imaginal discs, gland protrusion in genital discs is a unique process that leads to the differentiation of a gland capsule connected to the nascent reproductive tract by a tubular duct. When Hr39 is misexpressed, patches of cells within multiple imaginal discs that do not normally express Hr39 undergo changes reminiscent of early protrusion (Sun, 2012).

Hr39, a known member of the ecdysone response pathway, is likely to time reproductive gland cell divisions during pupal development. The initial Hr39 expression observed in the genital disc was detected shortly after the prepupal ecdysone pulse. Several additional peaks of ecdysone titer during pupal development (Urs, 2007) correspond closely with the timing this study measured of the secretory cell divisions. These observations suggest that external hormonal signals rather than internal autonomous mechanisms sometimes drive precise cell lineages. In addition to its requirement within cellular precursors, Hr39 mutations alter SP secretory gene mRNA levels (Allen, 2008), suggesting that Hr39 also regulates secretory gene expression within SCs (Sun, 2012).

Finally, Hr39 acts as a high level 'master regulator' by integrating individual pathways to elicit the production of an entire gland. Most cells expressing ectopic Hr39 could not progress past the initial stage of eversion, but in male genital discs Hr39-positive clones sometimes generated integrated structures that strongly resembled small spermathecae. They contained round heads with lumens, a pigmented layer, and rarely were connected to the male reproductive tract by ducts through which sperm were taken up. Thus, Hr39 (but not lz) can reprogram male genital cells to generate ectopic spermathecae that likely synthesize and secrete products attractive to sperm (Sun, 2012).

Drosophila reproductive gland development is unusually susceptible to perturbation. Rare adults in some wild strains contain an extra spermatheca, and females bearing weak alleles of either lz or Hr39 lose parovaria (POs) entirely and produce fewer spermathecae (SPs), which vary dramatically in size and cellular content (Allen, 2008). These effects probably result from the disparate sizes of the precursor pools for individual organs. PO pools are very small, whereas the exceptionally large posterior SP primordium may easily split in two under conditions where precursor proliferation is perturbed. The effects of dac mutations on duct structure (Keisman, 2001) are probably also due to altered precursor pools. Sphingolipids may affect gland development (Schimmer, 2010) by serving as endogenous Hr39 ligands, consistent with reports that SF-1 can bind sphingolipids (Urs, 2006; Urs, 2007; Sun, 2012 and references therein).

In mammals, sperm interact with female secretory products at multiple locations. Glands within the uterine endometrium are hypothesized to govern selective passage through the cervix, uterus, and subsequently, the uterotubal junction. Following entry into the oviduct, sperm induce and interact with the products of specialized tubal secretory cells that likely mediate capacitation. In some species, these products also allow sperm to be stored in the oviduct while retaining their ability to fertilize an egg. Mammalian female reproductive glands continue to nurture preimplantation embryos and are likely essential for successful pregnancy (Sun, 2012).

Drosophila is emerging as a valuable model with which to study multiple aspects of reproductive physiology, some of which may have been conserved during evolution. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle (Simeone, 1995), but its role in fertility is unknown. The murine Hr39 homolog LRH-1 is required for female fertility (Duggavathi, 2008), but whether it plays a role in reproductive gland secretion has yet to be tested. However, LRH-1 is required for the development of several exocrine tissues (Fayard, 2004) and in the pancreas is directly involved in the transcription of major secretory products (Crew, 1997). Thus, LRH-1 and Hr39 may both govern the formation and secretory function of exocrine tissue (Sun, 2012).

These study studies provide further support for the idea that an NR5a-dependent program of secretory cell development has been conserved in evolution. Murine LRH-1 can partially replace Hr39 function in Drosophila reproductive gland formation. Similar rescue with two other NR5A members (mammalian SF-1 or Drosophila Ftz-F1) failed and instead suppressed all gland formation. This is consistent with previous findings that Hr39 and Ftz-F1 have opposing roles in alcohol dehydrogenase and EcR expression (Ayer, 1993; Harrison, 1993). Antagonistic roles in gene regulation by the two NR5A family members may be evolutionarily conserved. Further study of the roles of Hr39 and LRH-1 should help define a fundamental program of secretory cell development that may be widely used (Sun, 2012).

The Sf1-related nuclear hormone receptor Hr39 regulates Drosophila female reproductive tract development and function

The vertebrate nuclear hormone receptor steroidogenic factor 1 (SF1; NR5A1) controls reproductive development and regulates the transcription of steroid-modifying cytochrome P450 genes. The SF1-related Drosophila nuclear hormone receptor HR39 is also essential for sexual development. In Hr39 mutant females, the sperm-storing spermathecae and glandular parovaria are absent or defective, causing sterility. These results indicate that spermathecae and parovaria secrete reproductive tract proteins required for sperm maturation and function, like the mammalian epididymis and female reproductive tract. Hr39 controls the expression of specific cytochrome P450 genes and is required in females both to activate spermathecal secretion and repress male-specific courtship genes such as takeout. Thus, a pathway that, in vertebrates, controls sex-specific steroid hormone production, also mediates reproductive functions in an invertebrate. These findings suggest that Drosophila can be used to model more aspects of mammalian reproductive biology than previously believed (Allen, 2008).

These studies show that the nuclear receptor encoded by Hr39 is not a redundant gene, but is essential for the development of spermathecae and parovaria. Previously, a genetic requirement for this gene was not detected through studies of the Hr39k13215 allele (Horner, 1997). Although, the Hr39k13215 mutation reduces Hr39 expression in adults, its effects on spermathecal and parovarial development were the weakest of any studied Hr39 allele. Differences between the alleles, which probably result from the insertions blocking promoter access to multiple enhancers located in the first two introns and from disrupting splicing, were useful in practice. Although no single allele was completely null for Hr39 function, Hr39ly92 appeared close to null for spermathecal development and Hr3907154 was close to null for adult function. Additional insight into Hr39 function will probably require analyzing double mutants with ftz-f1. The closely related FTZ-F1 protein may be expressed in tissues where loss of HR39 did not cause a detectable phenotype, such as in developing ovarian follicles (Allen, 2008).

Clearly, the most sensitive tissue requiring Hr39 function is the anterior genital disc at the time of metamorphosis. Previous studies have localized the primordial of both spermathecae and parovaria in this region and documented the rapid growth, migration, eversion and differentiation of spermathecal and parovarial cells during the first 18 hours after the prepupal molt (Keisman, 2001). Either autonomously or non-autonomously, the current studies show that these events depend in a dose-sensitive manner on Hr39 gene action. All of the phenotypic effects observed could be explained if the amount of an Hr39-dependent product influenced the number (and/or behavior) of progenitor cells in a spermathecal field that arises during early pupal development, with excess cells leading to additional spermathecae and cell deficits leading to smaller abnormal glands. The regulation, as well as the timing, of spermathecal and parovarial development appear to be closely connected, as evidenced by their common expression and requirement for the lozenge transcription factor. Among all the female genital disc derivatives, parovaria are unique in arising from the otherwise male-specific A9 segment (Keisman, 2001) and this may somehow result in the special Hr39 requirement for the development of both tissues. Fortunately, these developmental issues did not detract from the usefulness of the Hr39 alleles in studying the roles played by spermathecae, parovaria and Hr39 in female reproduction (Allen, 2008).

The data reported it this study strongly argue that spermathecae and parovaria are redundantly required for female fertility owing to their production of a secretory product that acts throughout the female reproductive tract. Fertility correlates strongly with the number of spermathecae, arguing that it is the presence of this tissue rather than some other defect in the Hr39 mutants that is responsible for their reduced fertility and fecundity. Moreover, the demonstration that the spermathecae that do form in mutant animals are frequently still defective in secretion, and that Hr3904443 mutant spermathecae lack secretion entirely and have parovaria with increased secretory activity, all support this conclusion. The observation that at least one major serine protease, CG17012, is expressed in both tissue provides one example of this redundancy (Allen, 2008).

Many steps are required before the gametes produced by the ovary and testis can undergo successful fertilization. After mating, sperm are introduced into the female reproductive tract along with dozens of proteins (Acps) that mediate sperm storage and behavior, and can even reduce female lifespan (reviewed by Bloch Qazi, 2003). Multiple Acps undergo proteolytic processing within the female reproductive tract, and seminal fluid contains serine proteases and protease inhibitors (serpins) that may interact with female-produced factors to regulate this process. At least seven Acps, including four serpins, enter the sperm storage organs after mating. For example, the male-produced Acp36DE, which is required for efficient sperm storage, can be found in the spermathecae and is proteolytically processed after transfer to the femal. The serpin encoded by Acp62F, which is required for fertility, enters the spermathecae). The many spermathecal secretory proteins identified, including at least eight serine proteases and a serpin, are candidates for the female factor in these interactions. Consistent with this idea, some spermathecal serine protease genes are induced by mating and undergo rapid selective evolution (Allen, 2008).

The current experiments show that the spermathecal and parovarial secretion acts after sperm have been transferred to the female reproductive tract and successfully stored. Hr39 mutant females lacking spermathecae still mated successfully and stored normal amounts of sperm in their seminal receptacles, yet they were sterile in the absence of a spermatheca. This implies that the secretion normally mixes with sperm in the reproductive tract and acts to make them fertilization competent regardless of their eventual storage site. It is unclear why these results differed from studies based on lozenge mutations that suggested a spermathecal requirement for efficient sperm storage. It is possible that, in the absence of spermathecae and parovaria, the processing of Acps and of sperm is altered or slowed. These defects must not prevent storage, but the resulting sperm may remain incapable of fertilization (Allen, 2008).

These studies suggest new parallels between Drosophila and mammalian reproductive biology. Following completion of their development within the testis, mammalian sperm move through the lumen of the epididymis, where they undergo a complex process of maturation. Epididymal cells secrete proteases, protease inhibitors, antioxidants, anti-bacterial proteins and other molecules into the epididymal fluid, and they also take up and modify or degrade materials shed by sperm. Drosophila sperm are exposed to similar classes of molecules after transfer to the female and storage in the spermathecae or seminal receptacle. Thus, the spermathecae and parovaria may play a similar role to that carried out by the caudal epididymis, where under the influence of products secreted by epididymal cells, sperm become motile, fertilization competent and can be stored for long periods. It is possible that the final steps of maturation can be accomplished in the reproductive tracts of either sex, but that some advantage exists in carrying them out at the storage site (Allen, 2008).

Several studies have been carried out on the genes expressed in the epididymis. These include antioxidant glutathione peroxidases, which are thought to protect against the peroxidation of polyunsaturated fatty acids within sperm plasma membranes. Drosophila spermathecae express the similar genes (Prx6005, PHGPx, GstS1 and CG1633). Two genes comprising the 'polyol' pathway are found to be associated with membranous vesicles in the epididymal fluid known as `epididymosomes' aldose reductase and sorbitol dehydrogenase. Sorbitol dehydrogenase 2 is expressed in spermathecae and its transcript level falls 19 times to undetectable levels in Hr39 mutants. Whether any of these genes carries out an important function in the spermathecae remains to be tested genetically (Allen, 2008).

Mammalian sperm are motile, but still not fully fertilization competent when they leave the epididymis. In the female they continue to interact with maternal products, such as the mucins that line the reproductive tract and retard movement, as well as other products secreted by the reproductive tract epithelia and the specialized glands it contains, such as Bartholin's gland. In addition to secreting molecules that assist in sperm maturation and preservation, these studies show that spermathecae expressed genes involved in carbohydrate and lipid metabolism, including two genes, GlcAT-P and PAPS, that are strongly associated with glycoprotein, sulfoprotein and lipoprotein secretion. Products dependent on these genes may enter the reproductive tract, especially at the anterior uterus where the spermathecae and parovaria connect to the reproductive tract. This is the region that sperm must traverse en route to the micropyle of the egg and fertilization. How this process occurs remains almost completely unknown. However, the presence of specific glycoproteins, glycolipids, sphingolipids and sulfated molecules might facilitate this final step, and ensure that sperm arriving at the micropyle are fully capacitated for fertilization, which, in Drosophila, must be very highly efficient. Thus, the fertility-essential functions of the spermathecae lie in its secretion rather than in sperm storage, a view consistent with the presence of a separate sperm-storage organ and the independent evolution of spermathecae from these structures (Allen, 2008).

The similarities between Hr39 expression and function in Drosophila and SF1 in mammals suggest that these genes play roles that at least in part have been conserved during evolution. The expression of Hr39 in reproductive and steroid-producing tissues, in gonadal duct progenitors that develop differentially between the sexes, and in regulating cytochrome P450 genes are all strikingly similar to Sf1 or Lrh1. HR39 function, however, appears to be confined to female development. Male Hr39 mutants were viable, fertile and apparently normal. Indeed, the major function of the gene is in the development of spermathecae and parovaria. Hr39 is also likely to control gene expression within spermathecae in adults, based on the specific gene expression defects observed in Hr3904443 spermathecae. This is analogous to SF1 and steroid hormone-dependent production of numerous products throughout multiple mammalian reproductive tissues (Allen, 2008).

Further evidence that Hr39 has not simply evolved a new role in controlling the spermathecae was the observation that Hr39 mutant females turn on male courtship genes. Expression of Cyp4d21, takeout and Obp99b are normally undetectable in the reproductive tracts of wild type females, but all three are expressed in the fat body of male heads. The current studies suggest that Cyp4d21 expression might control production of a male specific steroid in the fat body that is responsible for inducing the other genes. This pathway might have been retained from a time when Hr39 played a wider role in controlling reproduction in both sexes. Perhaps a wider role for a conserved regulatory pathway will be uncovered by examining the effects of removing both ftz-f1 and Hr39 at various times during development. However, even if the role of Sf1-like genes is much more limited in Drosophila than in mammals, the finding of any conservation has important implications for our understanding of the evolution of sex-determination mechanisms (Allen, 2008).

The observations that Hr39, like Sf1, controls the expression of a small set of cytochrome P450 genes, raises the issue of whether it might act by mediating the production of steroids other than ecdysone and 20-OH ecdysone. Many other steroids have been found in Drosophila and other insects, but none has been clearly implicated in sex-specific reproductive functions. By defining specific biological functions and specific target Cyp genes, it will now be easier to further investigate the mechanism of Hr39 action, and to determine whether it involves the production of new steroid derivatives. Such studies have the potential to significantly deepen understanding of how reproduction is regulated and how this regulation evolved (Allen, 2008).

ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling

Developmental axon pruning is a general mechanism that is required for maturation of neural circuits. During Drosophila metamorphosis, the larval-specific dendrites and axons of early gamma neurons of the mushroom bodies are pruned and replaced by adult-specific processes. The nuclear receptor ftz-f1 is required for this pruning, activates expression of the steroid hormone receptor EcR-B1, whose activity is essential for gamma remodeling, and represses expression of Hr39, an ftz-f1 homologous gene. If inappropriately expressed in the gamma neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. EcR-B1 was previously identified as a target of the TGFbeta signaling pathway. This study found that the ftz-f1 and Hr39 pathway apparently acts independently of TGFbeta signaling, suggesting that EcR-B1 is the target of two parallel molecular pathways that act during gamma neuron remodeling (Boulanger, 2011).

Developmental axon pruning is a fundamental process underlying nervous system maturation. The developmental axon pruning of γ neuron in mushroom bodies is a process for localized degeneration and shares some molecular and cellular features with axon degeneration after nerve injury. Seminal studies have found that a cell-autonomous TGFβ signaling pathway is involved in modulating the EcR-B1 and usp ecdysone receptor during γ axon pruning. During Drosophila metamorphosis, several primary response genes, induced directly by the ecdysone-ECR-USP complex, have been identified. Many direct targets of this complex are nuclear receptors. Particularly, three early regulatory genes, the Broad Complex, E74 and E75, are primary targets of the ecdysone cascade. Notably, these primary targets are dispensable for mushroom body neuronal remodeling, leading to the hypothesis that previously unknown downstream genes are involved in regulating developmental axon pruning. Nevertheless, a recent microarray study found that global ECR targets are also targets of ecdysone in mushroom body neurons despite the fact that most are not required for axon pruning. This study found that homologous nuclear receptors ftz-f1 and Hr39 are important for the pruning process. Neither ftz-f1 nor Hr39 were found to be ECR targets in the aforementioned global genomic analysis of neuronal remodeling. The data clearly demonstrate a requirement for ftz-f1 expression and a simultaneous silencing of Hr39 in γ neurons for appropriate pruning to occur. A role for FTZ-F1 in nervous system development has not been described previously. The results suggest that FTZ-F1 has an essential role in γ neuron remodeling and open the door for new studies of FTZ-F1 function in the control of nervous system development (Boulanger, 2011).

Because Hr39 has to be silenced (or at least kept to a low level of expression) in the γ neurons for the pruning to occur, the mechanism of its repression is of fundamental importance. The results exclude the obvious candidates that may downregulate Hr39 in the γ neurons, TGFβ and babo signaling and EcR-B1 itself. Instead, it was found that Hr39 was downregulated in the γ neurons by FTZ-F1 (Boulanger, 2011).

Mushroom bodies are involved in olfactory and courtship conditioning memory. How memory is affected in pruning-deficient adult flies that display immature larval-stage neuronal circuitry may help to unravel the functional role of neuron remodeling. Unlike EcR-B1, usp or ftz-f1 null mutants, flies overexpressing Hr39 are viable as adults and make it possible to ultimately assess the requirement for wild-type mushroom body pruning in memory by testing adults with γ pruning defects (Boulanger, 2011).

The EcR-B1 gene is targeted by FTZ-F1 and HR39 during γ neuron pruning rather than vice versa, as would be expected. Nuclear receptor genes, including ftz-f1 and Hr39, are transcriptionally regulated by ecdysone and the expression level of their mRNA changes in phase with ecdysone pulses during development. It was predicted that the ftz-f1 and Hr39 genes, if involved in mushroom body neuron remodeling, would be targets of the ecdysone- ECR-B1- USP complex. It was found that, contrary to expectations, EcR-B1 is a genetic target and likely a direct target of ftz-f1 and Hr39 protein products. The data indicate that FTZ-F1 binds the EcR locus at several consensus sites in vivo and in the expected tissue (Boulanger, 2011).

It is proposed that ftz-f1 has two roles in γ axon pruning: directly participating in EcR-B1 activation and indirectly participating in EcR-B1 activation by directly repressing Hr39 activity. The repression of Hr39 activity is crucial because the presence of HR39 protein in the γ neurons during pruning would block neuron remodeling by downregulating EcR-B1 activation, presumably by functionally competing with FTZ-F1. Nevertheless, HR39 alone, when FTZ-F1 was absent, was able to repress EcR-B1 expression (Boulanger, 2011).

Two different pathways are involved during γ axon pruning. As has previously been described, TGFβ signaling through the dActivin receptor activates EcR-B1, although it is not known how cell type- specific responses are achieved. A second, independent pathway acting in parallel with dActivin might provide such specificity. The ftz-f1 and Hr39 pathway may be such a pathway that provides EcR-B1 activation specificity. Nevertheless, the mechanism of cell-type specificity is not obvious, as ftz-f1 seems to be expressed broadly, if not ubiquitously, in the second instar brain. A putative specific ligand or cofactor for FTZ-F1 may ensure such specificity (Boulanger, 2011).

Thus, EcR-B1 is the point of convergence for at least two independent pathways that ensure its differential expression and that is required for a specific neuron remodeling process. Understanding how these different signals are integrated to regulate EcR-B1 to facilitate axon pruning will be necessary step to unravel the molecular mechanisms underlying this fundamental process of nervous system maturation (Boulanger, 2011).


Search PubMed for articles about Drosophila HR39

Allen, A. K. and Spradling, A. C. (2008). The Sf1-related nuclear hormone receptor Hr39 regulates Drosophila female reproductive tract development and function. Development 135: 311-321. PubMed ID: 18077584

Ayer, S., Walker, N., Mosammaparast, M., Nelson, J. P., Shilo, B. Z. and Benyajati, C. (1993). Activation and repression of Drosophila alcohol dehydrogenase distal transcription by two steroid hormone receptor superfamily members binding to a common response element. Nucleic Acids Res 21: 1619-1627. PubMed ID: 8479913

Bloch Qazi, M. C., Heifetz, Y. and Wolfner, M. F. (2003). The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster. Dev Biol 256: 195-211. PubMed ID: 12679097

Boulanger, A., Clouet-Redt, C., Farge, M., Flandre, A., Guignard, T., Fernando, C., Juge, F. and Dura, J. M. (2011). ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling. Nat. Neurosci. 14: 37-44. PubMed ID: 21131955

Crew, J. R., Batterham, P. and Pollock, J. A. (1997). Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group. Dev. Genes Evol. 206: 481-493.

Duggavathi, R., Volle, D. H., Mataki, C., Antal, M. C., Messaddeq, N., Auwerx, J., Murphy, B. D. and Schoonjans, K. (2008). Liver receptor homolog 1 is essential for ovulation. Genes Dev 22: 1871-1876. PubMed ID: 18628394

Fayard, E., Auwerx, J. and Schoonjans, K. (2004). LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol 14: 250-260. PubMed ID: 15130581

Guo, G. and Smith, A. (2010). A genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state pluripotency. Development 137: 3185-3192. PubMed ID: 20823062

Harrison, D. A. and Perrimon, N. (1993). Simple and efficient generation of marked clones in Drosophila. Curr Biol 3: 424-433. PubMed ID: 15335709

Heng, J. C., Feng, B., Han, J., Jiang, J., Kraus, P., Ng, J. H., Orlov, Y. L., Huss, M., Yang, L., Lufkin, T., Lim, B. and Ng, H. H. (2010). The nuclear receptor Nr5a2 can replace Oct4 in the reprogramming of murine somatic cells to pluripotent cells. Cell Stem Cell 6: 167-174. PubMed ID: 20096661

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Biological Overview

date revised: 5 January 2013

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