InteractiveFly: GeneBrief

Hormone receptor 51: Biological Overview | References


Gene name - Hormone receptor 51

Synonyms - Unfulfilled

Cytological map position - 51F7-51F7

Function - Transcription factor

Keywords - CNS, brain, mushroom body, axon guidance, wing expansion

Symbol - Hr51

FlyBase ID: FBgn0034012

Genetic map position - chr2R:11,219,106-11,226,825

Classification - nuclear receptor

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Jaumouille, E., Machado Almeida, P., Stahli, P., Koch, R. and Nagoshi, E. (2015). Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr Biol 25: 1502-1508. PubMed ID: 26004759
Summary:
Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Marmor-Kollet, N. and Schuldiner, O. (2015). Contrasting developmental axon regrowth and neurite sprouting of Drosophila mushroom body neurons reveals shared and unique molecular mechanisms. Dev Neurobiol. PubMed ID: 26037037
Summary:
The molecular mechanisms regulating intrinsic axon growth potential during development or following injury remain largely unknown despite their vast importance. This study has established a neurite sprouting assay of primary cultured mushroom body (MB) neurons. This study used the MARCM technique to both mark and manipulate MB neurons, enabling quantification of the sprouting abilities of single WT and mutant neurons originating from flies at different developmental stages. Sprouting of dissociated MB neurons was dependent on wnd, the DLK ortholog, a conserved gene that is required for axon regeneration. Next, and as expected, the sprouting ability of adult MB neurons was found to be significantly decreased. In contrast, and surprisingly, it was found that pupal-derived neurons exhibit increased sprouting compared with neurons derived from larvae, suggesting the existence of an elevated growth potential state. The molecular requirements of neurite sprouting was then contrasted to developmental axon regrowth of MB neurons, a process that requires the nuclear receptor UNF acting via the target of rapamycin (TOR) pathway. Strikingly, it was found that while TOR was required for neurite sprouting, UNF was not. In contrast, PTEN was found to inhibit sprouting in adult neurons, suggesting that TOR is regulated by the PI3K/PTEN pathway during sprouting and by UNF during developmental regrowth. Interestingly, the PI3K pathway as well as Wnd were not required for developmental regrowth nor for initial axon outgrowth suggesting that axon growth during circuit formation, remodeling, and regeneration share some molecular components but differ in others.
Kozlov, A., Jaumouille, E., Machado Almeida, P., Koch, R., Rodriguez, J., Abruzzi, K. C. and Nagoshi, E. (2017). A screening of UNF targets identifies Rnb, a novel regulator of Drosophila circadian rhythms. J Neurosci [Epub ahead of print]. PubMed ID: 28592698
Summary:
Behavioral circadian rhythms are controlled by multi-oscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions. The nuclear receptor unfulfilled (unf) represents a regulatory node that provides the small ventral Lateral Neurons (s-LNvs) unique characteristics as the master pacemaker. Previous work has shown that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period (per). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, this study identified UNF target genes using chromatin immunoprecipitation. The data demonstrate that a previously uncharacterized gene CG7837, which this study has termed R and B (Rnb), acts downstream of UNF to regulate the function of s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit.

BIOLOGICAL OVERVIEW

The mushroom bodies (MBs) of Drosophila are required for complex behaviors and consist of three types of neurons, γ, α'/β' and α/β. Previously, roles for transcription factors in MB neuronal differentiation have only been described for a subset of MB neurons. This study investigated the roles of unfulfilled (unf; HR51, CG16801) in MB development. unf encodes a nuclear receptor that is orthologous to the nuclear receptors fasciculation of axons defective 1 (FAX-1) of the nematode and photoreceptor specific nuclear receptor (PNR) of mammals. Based on previous observations that unf transcripts accumulate in MB neurons at all developmental stages and the presence of axon pathfinding defects in fax-1 mutants, it is hypothesized that unf regulates MB axon growth and pathfinding. unf mutants exhibit a range of highly penetrant axon stalling phenotypes affecting all neurons of the larval and adult MBs. Phenotypic analysis of unfX1 mutants revealed that α'/β' and α/β neurons initially project axons but stall prior to the formation of medial or dorsal MB lobes. unfZ0001 mutants form medial lobes, although these axons fail to branch, which results in a failure to form the α or α' dorsal lobes. In either mutant background, γ neurons fail to develop larval-specific dorsal projections. These mutant γ neurons undergo normal pruning, but fail to re-extend axons medially during pupal development. unfRNAi animals displayed phenotypes similar to those seen in unfZ0001 mutants. Unique asymmetrical phenotypes were observed in unfX1/unfZ0001 compound heterozygotes. Expression of UAS-unf transgenes in MB neurons rescues the larval and adult unf mutant phenotypes. These data support the hypothesis that unf plays a common role in the development of all types of MB neurons. The data indicate that unf is necessary for MB axon extension and branching and that the formation of dorsal collaterals is more sensitive to the loss of unf function than medial projections. The asymmetrical phenotypes observed in compound heterozygotes support the hypothesis that the earliest MB axons may serve as pioneers for the later-born MB neurons, providing evidence for pioneer MB axon guidance in post-embryonic development (Bates, 2010).

The mushroom bodies (MBs) of Drosophila melanogaster, which are required for olfactory learning and other complex behaviors, are ideal for studying the transcriptional regulation of interneuronal development because they form discrete axonal projections that are well-characterized and easily visualized. Four neuroblasts in each brain hemisphere sequentially generate three types of Kenyon cells, the MB neurons that begin dividing during embryogenesis and continue to divide through development. Each neuron projects dendrites that contribute to a large dendritic field in the calyx, and an axon that travels anteroventrally, forming a tightly bundled peduncle before branching medially to form the lobes, and dorsally to form the α ' and α lobes. The earliest born γ neurons initially extend axons both medially and dorsally during late embryonic and early larval stages. These larval-specific γ axons are then pruned back to the peduncle by 18 hours after puparium formation (APF) and re-extend medially during pupal remodeling; the late larval-born α'/β ' and pupal-born α/β neurons do not remodel their axonal projection patterns during metamorphosis (Bates, 2010).

Since the three different classes of MB neurons are born sequentially, generate a single dendritic field, project axons that fasciculate prior to branching medially and/or dorsally to form type-specific lobes, it is interesting to consider whether any differentiative events of the γ. α'/β', and α/β neurons are regulated by a common set of genes or whether they utilize independent transcriptional networks. Existing data on the role of transcription factors in MB differentiation provide little insight into this question. The genes eyeless, tramtrak, mushroom body miniature, chinmo, polyhomeotic, and tailless regulate proliferation, specification, and viability of MB neurons, events that precede differentiation. dachshund (dac), ecdysone receptor B1 (EcR-B1), ultraspiracle (usp), and dSmad2 act in subtype-specific pathways, consistent with the hypothesis that the differentiation of the γ, α'/β', and α/β neurons utilize independent transcriptional pathways. dac mutants display axonal branching and pathfinding defects in subsets of α'/β' and α/β MB neurons. EcR-B1 and its heterodimeric partner, Ultraspiracle (USP), both members of the nuclear receptor superfamily, form an ecdysone-regulated transcription factor that is required for the pruning of MB γ neurons at the outset of metamorphosis dSmad2 regulates the transcription of EcR-B1 in MB γ neurons during neuronal remodeling. Thus, whether any differentiative events of the γ, α'/β', and α/β neurons are regulated by a common set of genes has not been previously reported (Bates, 2010).

This study shows that the gene unfulfilled is required for the development of all three types of MB neurons, supporting the hypothesis that some differentiative events of the three types of MB neurons are regulated by a common set of genes. The unfulfilled gene (unf; HR51, CG16801) encodes the Drosophila NR2E3 member of the nuclear receptor superfamily (Sung, 2009). UNF, like all classical nuclear receptors, contains an amino-terminal transactivational domain, a DNA-binding domain, a hinge region, and a carboxy-terminal ligand-binding domain. unf is an ortholog of the Caenorhabditis elegans gene fasciculation of axons defective (fax-1) and the human gene photoreceptor specific nuclear receptor (PNR) (DeMeo, 2008). Both fax-1 and PNR mutations disrupt developmental events in a limited number of neurons and result in behavioral or sensory deficits. fax-1 mutants are uncoordinated and display axon pathfinding and neurotransmitter defects (Wightman, 1997; Much, 2000; Wightman, 2005). The observed axon pathfinding defects are inferred to be due to the misregulation of fax-1 target genes. PNR impacts neuronal identity of vertebrate photoreceptors, functions as a dimer, and acts as a dual function transcriptional regulator, able to act as a transcriptional activator and a transcriptional repressor (Haider, 2000; Haider, 2009; Peng, 2005; Chen, 1999; Chen, 2005; Cheng, 2004; Kobayashi, 1999; Bates, 2010 and reference below).

Based on previous observations that robust levels of unf transcripts accumulate in MB neurons at all developmental stages (Wong, 2009) and the axon pathfinding defects of fax-1 mutants, it was hypothesized that unf regulates MB axon growth and pathfinding. Phenotypic analysis of unf mutants revealed that MB axons stall prior to the formation of the lobes with the exception of the larval-specific γ neurons, which project axons medially, but fail to project dorsally. These axons are pruned appropriately but fail to re-extend during pupal stages. Expression of an unf transgene in the MBs in a mutant background rescued the unf mutant phenotypes, demonstrating that MB defects of unf mutants are due to loss of unf function in the MB neurons. These data demonstrate that unf is required for the proper formation of γ, α'/β', and α/β lobes, consistent with the hypothesis that at least some differentiative events of the γ, α'/β', and α/β neurons are regulated by a common set of genes (Bates, 2010).

unf mutants exhibit a range of highly penetrant axon stalling phenotypes affecting all neurons (γ, α'/β' and α/β) of the larval and adult MBs. Similar phenotypes have been observed in unf microRNA knockdown animals (Lin, 2009). unfX1/Df2426 hemizygotes and unfX1/unfX1 homozygotes fail to project larval-specific γ dorsal collaterals, fail to re-extend γ axons medially during metamorphosis, and fail to project any medial and dorsal axons of α'/β' and α/β neurons. The γ, α'/β' and α/β axons of unfZ0001/Df2426 hemizygotes only project medially, whereas MBs were normal in some unfZ0001/unfZ0001 homozygotes. These data together with previous observations (Wong, 2009)] would seem to support the hypothesis that the unfX1 allele is an amorph, a null allele, and that the unfZ0001 allele is a hypomorph, a partial loss of function allele. However, while the unfZ0001 allele behaves as a hypomorph with respect to sterility, it displays dominant properties with respect to wing expansion (Wong, 2009)]. Interestingly, the G56R allele of PNR, which displays dominant properties, is structurally equivalent to the unfZ0001 allele (G120R) (Wong, 2009). The observation that the unfX1/unfZ0001 compound heterozygotes display unique phenotypes was unexpected and demonstrates that these alleles interact, leading to the conclusion that the unfX1 allele is not a null allele. These data strongly suggest that the unfX1 allele encodes a unique isoform of the UNF protein, UNFX1, which is predicted to contain the 110 residue amino-terminal domain and the complete first zinc finger of the DNA-binding domain (Wong, 2009). These data do not allow inference the functional nature of the unfX1 allele or the mechanism of this genetic interaction (Bates, 2010).

The phenotypic variation and asymmetry observed in the MBs of unfX1/unfZ0001 compound heterozygotes supports the hypothesis that pioneer axons are established early during MB development and that the pathfinding of these pioneers is unf-dependent. The observation that the β lobe axons in one hemisphere project medially while the β lobe axons in the other hemisphere project ventromedially demonstrates that the projection of the β lobes in these unfX1/unfZ0001 compound heterozygotes is independent of their genotype. In an independent, yet genetically identical animal, unfX1/unfZ0001 α/β MB neurons assume a different fate and fail to project any medially projecting β axons. Similar observations can be made for all unfX1/unfZ0001 MB lobe axons when these and other samples are examined. The fact that the axons of the later-born α/β neurons consistently stall or misproject whenever the axons of the earlier-born α'/β' neurons stall or misproject suggests that the α'/β' axons may be acting as pioneers for the α/β axons. These data support a model of MB lobe formation in which unf is required for MB pioneer axons to navigate to their targets, and that later-born MB neurons project axons that fasciculate along these established axons. It is proposed that the variable and asymmetric phenotypes observed in unfX1/unfZ0001 compound heterozygotes are due to inappropriate targeting of pioneer axons of the MB or the stalling of pioneer axons prematurely as a result of insufficient unf function in α'/β' pioneer axons. Thus, the asymmetric β lobe projection that was observed may be due to asymmetric projections of pioneer axons, while the lack of β lobes in another instance may be due to the stalling of these pioneer axons in the peduncle. These data are supported by an analysis of non-autonomous effects of Dscam mutant clones, which suggests that the α'/β' axons may be acting as pioneers for the α/β axons at least some of the time. The observations that larval γ dorsal axons, and the α' and α dorsal lobes all fail to develop in unfZ0001/Df2426 mutants not only supports the hypothesis that the α'/β' axons act as pioneers for the α/β axons, but suggests that the larval γ axons act as pioneers for the α'/β' axons. Discerning the roles and mechanisms of unf in MB pioneers during post-embryonic MB development requires further investigation (Bates, 2010).

The data demonstrate that unf plays a common role in the early development of all three subtypes of MB neurons by regulating axon extension and branching. While it cannot be ruled out that single axons, which normally project dorsally, may be misguided and project medially, the analysis is consistent with the hypothesis that unf mutant γ, α'β', and α/β neurons fail to project dorsal axon branches. The observations that unf mutant MB neurons express subtype-specific epitopes such as Fas II and Trio suggest that unf does not impact MB neuronal subtype identity. Interestingly, Lin (2009) disagrees and concludes that unf does regulate MB neuronal subtype identity based on a series of unf RNAi knockdown experiments. It is argued that until the transcriptional codes that distinguish MB neuronal subtypes are defined, one cannot conclusively determine whether the identity of these neurons has been impacted. While unf cannot yet be placed at any specific position in a transcriptional hierarchy that regulates MB development, the data suggest that unf acts earlier than dac, EcR-B1, and usp, since these genes regulate differentiation in specific subsets of MB neurons while unf regulates the differentiation of all three MB subtypes (Bates, 2010).

Based on the homology of UNF with photoreceptor specific nuclear receptor (PNR) of mammals, it is anticipated that UNF is likely to act as a dual function transcription factor with the ability to activate the transcription of some target genes, while repressing others. Palanker (2006) has proposed that UNF may function as a transcriptional repressor. The current study proposes that the axon stalling phenotype observed in unf mutants is due to the misregulation of target genes. Two potential target genes are Tab2 and Hr39 based on the observation that the expression of the 201Y-GAL4, an enhancer trap insertion in Tab2, and c747-GAL4, an enhancer trap insertion in HR39, is unf-dependent. If the transcriptional regulation of these two enhancer trap transgenes reflects the transcriptional regulation of the genes in which they are inserted, then it follows that Tab2 and HR39 are expressed in the MBs and that this expression is unf-dependent, a hypothesis that remains to be tested (Bates, 2010).

Axon stalling and branching phenotypes in unf mutants suggest that genes encoding axon guidance cues are likely to be regulated by unf. Genetic screens have already identified a number of guidance genes that are expressed in the MBs. For example, genes encoding cell adhesion molecules like volado, an α-integrin, Notch, a transmembrane receptor and transcription factor, and semaphorinla and plexinA, which encode a ligand-receptor pair that is largely involved in axonal repulsion, have been identified. Ephrin receptor tyrosine kinase and Dscam, other well-known guidance cues, are necessary for the proper guidance of MB axons. Misregulation of these or other guidance genes could disrupt the normal balance of attractive and repulsive cues resulting in inappropriate axon pathfinding and stalling (Bates, 2010).

These data support the hypothesis that unf plays a common role in the early development of all three subtypes of MB neurons, γ, α'/β', and α/β, by regulating axon extension and branching during the initial phases of larval and pupal outgrowth. Expression of a UAS-unf transgene in MB neurons of unf mutants rescues the unf mutant MB phenotypes, demonstrating that the MB defects are due to the lack of unf. The phenotypic variation and asymmetry observed in the MBs of unfX1/unfZ0001 compound heterozygotes suggests a role for unf in the targeting of pioneer axons (Bates, 2010).

The nuclear receptor unfulfilled is required for free-running clocks in Drosophila pacemaker neurons

An intricate neural circuit composed of multiple classes of clock neurons controls circadian locomotor rhythms in Drosophila. Evidence indicates that the small ventral lateral neurons (s-LNvs, M cells) are the dominant pacemaker neurons that synchronize the clocks throughout the circuit and drive free-running locomotor rhythms. Little is known, however, about the molecular underpinning of this unique function of the s-LNvs. This study shows that the nuclear receptor gene unfulfilled (unf; DHR51) is required for the function of the s-LNvs. Unfulfilled is rhythmically expressed in the s-LNvs, and unf mutant flies are behaviorally arrhythmic. Knockdown of unf in developing LNvs irreversibly destroys the ability of adult s-LNvs to generate free-running rhythms, whereas depletion of Unf from adult LNvs dampens the rhythms of the s-LNvs only in constant darkness. These temporally controlled LNv-targeted unf knockdowns desynchronize circuit-wide molecular rhythms and disrupt behavioral rhythms. Therefore, Unf is a prerequisite for free-running clocks in the s-LNvs and for the function of the entire circadian circuit (Beuchle, 2012).

Evidence is presented that Unf is required for the proper functioning of the s-LNvs as dominant pacemaker neurons. Unf accumulates rhythmically in the s-LNvs and unf mutant flies are arrhythmic. Unf depletion in developing or adult s-LNvs disables free-running molecular clocks in adult s-LNvs as well as disrupts rhythms in E cells. This is the first study demonstrating the direct involvement of a nuclear receptor in the Drosophila circadian clock, highlighting the similarity to the mammalian circadian system, where nuclear receptors play important roles (Beuchle, 2012).

Importantly, none of the LNv-targeted unf knockdowns impaired behavioral rhythms in LD, even by highly efficient knockdown at 29°C. Consistent with this notion, the cellular clocks in the s-LNvs in knockdown flies cycle with a high amplitude in LD. Therefore, the action of Unf in the LNvs is specific to the function of free-running molecular clocks in differentiated s-LNvs. In contrast, unf mutants were arrhythmic in both LD and DD. This observation suggests that there are additional mechanisms through which unf contributes to the behavior in LD. Because ubiquitous knockdown of unf by tubulin-GAL4 recapitulates the abnormal LD behavior of unf mutants, unf in cells other than the LNvs and MB neurons are involved in generating normal LD behavior (Beuchle, 2012).

The results demonstrate that Unf interacts with the molecular clock feedback loops at least in adult s-LNvs, because adult-only unf knockdown severely dampens and slows down the circadian clock in DD. Strikingly, unf knockdown in developing s-LNvs leads to an even stronger impairment of the molecular clocks and concomitant behavioral arrhythmia in the adult. Per expression during development is not required for rhythmicity in adult flies, whereas CLK/CYC activity in the developing LNvs is required for the adult rhythmicity. Published studies together with the current results suggest the possibility that Unf is required for the normal activity of CLK/CYC complex. Alternatively, unf may control genetic programs that are independent of molecular clocks but are required for the functional development of the s-LNvs. The unf ortholog in C. elegans, fax-1, controls the expression of the genes required for neuronal identity specification, such as neurotransmitters and neurotransmitter receptors. unf in MB neurons appears to share a similar function. Therefore, it is plausible that Unf is required to establish and maintain the neuronal identity and connectivity of the s-LNvs during development, which are required for adult s-LNvs to free run. In addition, Unf might be involved in the control of clock output in the s-LNvs to synchronize downstream clock neurons. Identification of the Unf target genes in developing and adult s-LNvs will clarify the role of Unf in molecular clockwork and circuit synchronization (Beuchle, 2012).

Transcriptional Regulation via Nuclear Receptor Crosstalk Required for the Drosophila Circadian Clock

Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

This study has identified the nuclear receptors E75 and UNF as components of the molecular clocks in the s-LNvs. E75 is the closest homolog of mammalian REV-ERB α and REV-ERB β, which play important roles in the molecular clock feedback loops. In contrast with Rev-Erb α/β, which represses transcription, the results demonstrated that E75 is neither a potent repressor nor a strong activator but potentiates the activation of per transcription by UNF. Despite these mechanistic divergences, the notion that Rev-Erb homologs are integral to the molecular oscillators in both Drosophila and mammals highlights the significance of transcriptional regulations via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Rev-Erb α and Rev-Erb α are rhythmically transcribed by the CLOCK/BMAL1 transcriptional activators, and REV-ERBs periodically repress the transcription of Bmal1, thereby forming a feedback loop to ensure robust molecular oscillations of the mammalian clock. A previous study demonstrated that E75 is a cycling target of Clk/Cyc in the fly head (Kumar, 2014). Because E75 has three isoforms, it was not possible to determine whether any of the isoforms were rhythmically expressed in the LNvs from the RNA profiles of the isolated LNvs. Nonetheless, the results indicate that E75 together with Unf (which is not a Clk/Cyc target) reinforces the main loop of the core fly clock composed of Clk/Cyc and Per/Tim through a feedforward mechanism, showcasing the mechanistic parallels between fly and mammalian clocks (Jaumouille, 2015).

E75 has been demonstrated to covalently bind to heme, and its binding appears to stabilize the E75 and facilitates the binding of nitric oxide (NO) and carbon monoxide (CO). The NO/CO binding to E75 modulates the transcriptional activity of its known heterodimeric partner DHR3. To test whether similar mechanisms are involved in the action of E75 in the s-LNvs, attempts were made to disrupt cellular heme metabolism by knocking down the enzymes in the heme biosynthesis pathway, coproporphyrinogen oxidase (Coprox) and protoporphyrinogen oxidase (Ppox), and the key enzyme in the heme degradation pathway, heme oxygenase (Ho). These experiments were inconclusive, as no effect on the behavioral rhythms were observed by any knockdown with Pdf-GAL4, and knockdown with Tim-GAL4 was lethal (Jaumouille, 2015).

S2 cell experiments showed that Unf is a transcriptional activator of per, and concurrent expression of E75 and Unf increases the turnover of Unf binding to per regulatory sequences. This high turnover is correlated with higher transcriptional activity. The finding that E75 acts through Unf on transcription is consistent with in vivo data: (1) depletion of both Unf and E75 in adult LNvs abolishes the behavioral rhythms; (2) E75 overexpression has no effect on the behavioral rhythms; and (3) E75 overexpression does not rescue Unf knockdown. Although unf mRNA levels do not oscillate, Unf protein levels cycle in the s-LNvs, peaking at zeitgeber time (ZT)2 and lowest at ZT14. Low Unf levels may reflect the degradation as a consequence of higher transcriptional activity. Indeed, per is most actively transcribed around ZT13 when Unf levels are minimum in the s-LNvs. Nonetheless, downregulation and arrhythmia of Per levels in the s-LNvs is most probably not the sole cause of the altered locomotor rhythms in the Unf knockdown, E75 knockdown, and Unf/E75 double knockdown. A recent study showed the implication of E75 in the repression of Clk transcription, although the current results are not in concordance with this observation probably due to the differences in the reagents used for E75 knockdown and the timing of knockdown. Deciphering whether E75 and Unf heterodimerize or bind to adjacent sequences, how they cooperate with Clk/Cyc, and whether any ligand is involved in their transcriptional regulation will yield new insights into the diverse mode of nuclear receptor crosstalk and their critical roles in circadian biology (Jaumouille, 2015).

Nuclear receptor Unfulfilled regulates axonal guidance and cell identity of Drosophila mushroom body neurons.

Nuclear receptors (NRs) comprise a family of ligand-regulated transcription factors that control diverse critical biological processes including various aspects of brain development. Eighteen NR genes exist in the Drosophila genome. To explore their roles in brain development, individual NRs ware nocked down through the development of the mushroom bodies (MBs) by targeted RNAi. Besides recapitulating the known MB phenotypes for three NRs, it was found that unfulfilled (unf), an ortholog of human photoreceptor specific nuclear receptor (PNR), regulates axonal morphogenesis and neuronal subtype identity. The adult MBs develop through remodeling of γ neurons plus de-novo elaboration of both α′/β′ and α/β neurons. Notably, unf is largely dispensable for the initial elaboration of γ neurons, but plays an essential role in their re-extension of axons after pruning during early metamorphosis. The subsequently derived MB neuron types also require unf for extension of axons beyond the terminus of the pruned bundle. Tracing single axons revealed misrouting rather than simple truncation. Further, silencing unf in single-cell clones elicited misguidance of axons in otherwise unperturbed MBs. Such axon guidance defects may occur as MB neurons partially lose their subtype identity, as evidenced by suppression of various MB subtype markers in unf knockdown MBs. In sum, unf governs axonal morphogenesis of multiple MB neuron types, possibly through regulating neuronal subtype identity (Lin, 2009).

Silencing individual NRs through development of the MBs by transgenic miRNAs has allowed identification of unf as another NR (in addition to EcR, usp, and tll) that regulates MB development. Previously, unf was shown to be essential for the wing expansion and fertility of adult flies, and abundantly expressed in the developing MBs. The function of unf was further studied in detail and it was learned that unf acts in all three major types of MB neurons to promote proper neuron subtype identity and axon guidance. Comparable axon stalling defects of adult MB neurons, as well as missing larval MB dorsal axonal branches were observed in unf mutant organisms (Bates, 2010), validating this study of unf's mechanism of action by targeted RNAi (Lin, 2009).

The involvement of unf in cell fate determination is evident by the loss of subtype-specific markers in the unf knockdown MB. Similar mechanisms have been shown in C. elegans and human. In human, the unf ortholog PNR is specifically expressed in rod photoreceptor cells to promote the rod-cell identity by repressing the expression of S-cone cell specific genes (Milam, 2002; Chen, 2006). Mutations in PNR leads to enhanced S-cone syndrome (ESCS) which is an inherited disease causing hypersensitivity to short-wave light due to increased numbers of S-cone cells at the expense of rod photoreceptor cells. However, in the present study, reciprocal cell number change was not observed in unf knockdown MBs. Thus, unlike PNR in human, unf in flies is not used to repress a default cell fate. Given that unf is required for the proper gene expression in all three major types of MB neurons, it may play a general role in assisting the temporal identity factors, such as chinmo, to diversify neuronal cell fates (Lin, 2009).

Loss of proper identity might underlie the axon guidance defect observed in the unf knockdown MB. The C. elegans ortholog of unf, fax-1, has been suggested to regulate cell identities of 18 neurons, including both motor- and interneurons; in fax-1 mutant, some neurotransmitters and synaptic proteins are not properly expressed in the neurons normally expressing fax-1. Notably, like unf in fly MB neurons, fax-1 is also required for axonal pathfinding for several C. elegans neurons, indicating a functional conservation among unf orthologs in different species (Lin, 2009).

unf has been hypothesized to act as a transcriptional repressor, because its ligand-binding domain (LDB) failed to activate gene expression. The function of the human ortholog PNR to repress S-corn specific genes supports this hypothesi. Contrary to the human PNR, in both flies and worms, loss of unf or fax-1 leads to the down-regulation of many neural genes. However, since there is no evidence for gene activation by direct binding of unf or fax-1, it is possible that unf and fax-1 regulate these neural genes indirectly by repressing other repressors (Lin, 2009).

DHR51, the Drosophila melanogaster homologue of the human photoreceptor cell-specific nuclear receptor, is a thiolate heme-binding protein

Heme has been recently described as a regulating ligand for the activity of the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta and their Drosophila homologue E75. This study reports the cloning, expression in Escherichia coli, purification, and screening for the heme-binding ability of 11 NR ligand-binding domains of Drosophila melanogaster (DHR3, DHR4, DHR39, DHR51, DHR78, DHR83, HNF4, TLL, ERR, FTZ-F1, and E78), of unknown structure. One of these NRs, DHR51, homologous to the human photoreceptor cell-specific nuclear receptor (PNR), specifically binds heme and exhibits a UV-visible spectrum identical to that of heme-bound E75-LBD. EPR and UV-visible absorption spectroscopy indicates that, like in E75, the heme contains a hexa-coordinated low spin ferric iron. One of its axial ligands is a tightly bound cysteine, while the other one is a histidine. A dissociation constant of 0.5 microM for the heme was measured by isothermal titration calorimetry. DHR51 is shown to bind NO and CO and the possibility is described that DHR51 may be either a gas or a heme sensor (de Rosny, 2008).

In the MBs, unf primarily governs axonal morphogenesis during later larval and pupal development when different MB neuron types need to make distinct projections. For instance, in early pupae, α/β neurons undergo de-novo axonogenesis to form the α/β lobes while γ axons regenerate to make up the adult-specific γ lobe. Given the notion that unf promotes MB axonogenesis possibly through regulating neuron subtype identity, its selective involvement in late MB morphogenesis could simply reflect the importance of neuron subtype identity in ensuring diverse subtype-specific axonal morphogenesis. However, the larval γ neurons of unf knockdown MBs show normal cell fate as evidenced by proper expression of EcR-B1. This argues for a stage-specific function of unf in MB development. Notably, despite its stage-specific requirement, unf is enriched in the MBs through different developmental stages and into the adult, raising the possibility that its dynamic activity is patterned through temporal control of ligand availability. Recently, an in vitro study suggested that UNF is a heme binding protein (de Rosny, 2008). Given the known relationship between heme and lipid metabolism, heme levels can serve as an indicator for energy resource and developmental progress. Perhaps by detecting heme levels, unf may potentially coordinate the timing of the unf-medicated adult-specific cell fate determination and axonogenesis. However, in vivo evidence for the interaction between heme and unf remains lacking (Lin, 2009).

In conclusion, patterned NR activities govern various temporally regulated neural developmental processes of interest. UNF and its orthologs probably promote subtype neuronal differentiation in temporally controlled manners. Elucidating the regulation of NR activities and their control of neuron subtype identity should shed additional light on how diverse neuron types undergo differential morphogenesis and acquire different subtype-specific projections to construct the complex brain (Lin, 2009).

The unfulfilled/DHR51 gene of Drosophila modulates wing expansion and fertility

Drosophila unfulfilled (unf; DHR51) is the NR2E3 nuclear receptor superfamily ortholog of C. elegans fax-1 and human PNR. Both fax-1 and PNR mutations disrupt developmental events in a limited number of neurons, resulting in behavioral or sensory deficits. An analysis of two independent unf alleles revealed that unf mutants are characterized by one of two phenotypes. A proportion of the mutants eclosed but fail to expand their wings and are poorly coordinated. The remainder completed wing expansion but display severely compromised fertility. Consistent with the restricted neural expression of fax-1 and PNR, unf expression was detected in situ only in mushroom body neurons and a small number of other cells of the central nervous system (CNS). These data support the hypothesis that the wing expansion failure and the compromised fertility of unf mutants are the result of underlying neural defects (Sung, 2009).

Genetic analyses show that unfX1 and unfZ0001 are not complete loss-of-function alleles and suggest that the behavior of each is dependent upon the unf genotype. These data are consistent with a model in which UNF protein functions as a homodimer like the human ortholog PNR (Chen, 2005). Such a model predicts that the product of each mutant allele, UNFX1 or UNFZ0001 has the capacity to dimerize with itself or other UNF isoforms, and that each potential dimer (UNFZ0001/UNFZ0001, UNFX1/UNFX1, UNFX1/UNFZ0001, UNFX1/UNF+, UNFZ0001/UNF+) may uniquely regulate the transcription of target genes. This model suggests that the transcriptional properties of UNF dimers are the basis for the genotype-dependent behaviors of the unfX1 and unfZ0001 alleles (Sung, 2009).

The predicted product of the unfZ0001 allele, UNFZ0001 is consistent with this model because the lesion in the first zinc finger of the DNA binding domain is not expected to impact dimerization functions. However, prediction of the coding potential of the unfX1 allele was inconsistent with this model because unfX1 was engineered to generate a truncated isoform that lacked dimerization domains. This prediction may be inaccurate because the unfX1 allele generates unanticipated transcripts that may encode novel UNFX1 isoforms capable of dimerization (Sung, 2009).

The Northern and in situ hybridization data both show that unf transcripts are enriched in the nervous system although questions remain about the non-neural expression of the 3.3-kb transcript in 0–4 hr embryos and the postembryonic expression of the 0.7-kb transcript. In situ hybridization data showed that unf is expressed in a limited number of cells within the nervous system at all stages. These data are consistent with the limited expression of the unf orthologs PNR and fax-1. However, the spatial expression of unf was unlike PNR in two ways. First, PNR is expressed exclusively in the vertebrate retina, while unf expression was not detected in the fly visual system, either in the eye-imaginal disk of third instar larvae or in the developing adult visual system. Second, unf was expressed in heterogeneous neurons unlike the restricted expression of the vertebrate PNR. This broader expression of unf is similar to the expression of fax-1 in C. elegans (Much, 2000; Wightman, 2005; Sung, 2009 and references therein).

Identifying the unf-expressing cells that are responsible for the wing expansion defect and the compromised fertility of unf mutants remains a major challenge. The enriched expression of unf in the CNS (Chintapalli, 2007) is consistent with the hypothesis that the wing expansion defect and the compromised fertility of unf mutants may be due to defects in the nervous system. Chintapalli also concluded that unf was enriched 5.8-fold in testes but not enriched in ovaries suggesting an alternative, somewhat paradoxical hypothesis; that the compromised fertility of unf mutant males is due to unf-dependent processes of the testes while the compromised fertility of females is the result of unf-dependent processes in the nervous system. The observation that unf mutants of both sexes do, although rarely, produce progeny, supports the interpretation that the male and female reproductive organs of unf mutants are functional, and that the compromised fertility may be due to behavioral deficits (Sung, 2009).

Two pathways participate in wing expansion; a neuroendocrine cascade and tissue remodeling of the wing itself. Given the neural localization of unf transcripts, the three sets of peptidergic neurons that comprise the neuroendocrine cascade that regulates wing expansion behaviors are candidates that may be responsible for the wing expansion defects of unf mutants. These peptidergic neurons are the eclosion hormone neurons that express and release eclosion hormone (EH neurons, the crustacean cardioactive peptide (CCAP) neurons that express and release CCAP but do not express bursicon (CCAP neurons), and the CCAP neurons that co-express and release CCAP and bursicon (bursicon neurons). While it has not yet been demonstrated that unf is expressed in these neurons, the possibility exists that unf may function in any or all of these peptidergic neurons (Sung, 2009).

Of interest, the EH neurons of unfZ0001/unfZ0001 mutants and the mushroom bodies of unfZ0001/unfZ0001 mutants and unfX1/Df(2R)ED2426 mutants display axonal defects. These tantalizing observations raise the possibility that the aberrant phenotypes observed in unf mutants may be the result of a failure to develop or maintain axons of unf-dependent neurons, consistent with the behavioral and morphological phenotypes observed in C. elegans fax-1 mutants. It is proposed that unf mutants will exhibit deficits in mushroom body-dependent behaviors including olfactory learning, courtship, and locomotion. Future studies of the unf gene and unf-dependent neurons will provide insight into molecular genetic pathways that control neuronal architecture and behavior (Sung, 2009).


REFERENCES

Search PubMed for articles about Drosophila Unfulfilled

Bates, K. E., Sung, C. S. and Robinow, S. (2010). The unfulfilled gene is required for the development of mushroom body neuropil in Drosophila. Neural Dev. 5: 4. PubMed ID: 20122139

Beuchle, D., Jaumouille, E. and Nagoshi, E. (2012). The nuclear receptor unfulfilled is required for free-running clocks in Drosophila pacemaker neurons. Curr Biol 22: 1221-1227. PubMed ID: 22658597

Chen, F., et al (1999). Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Muller glial cells. Proc. Natl. Acad. Sci. 96: 15149-15154. PubMed ID: 10611353

Chen, J., Rattner, A. and Nathans, J. (2005). The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes. J Neurosci 25: 118-129. PubMed ID: 15634773

Cheng, H., et al. (2004). Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors. Hum. Mol. Genet. 13: 1563-1575. PubMed ID: 15190009

Chintapalli, V. R., Wang, J. and Dow, J. A. (2007). Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat. Genet. 39: 715-720. PubMed ID: 17534367

DeMeo, S. D., et al. (2008). Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue. BMC Mol. Biol. 9:2. PubMed ID: 18179707

de Rosny, E., et al. (2008). DHR51, the Drosophila melanogaster homologue of the human photoreceptor cell-specific nuclear receptor, is a thiolate heme-binding protein. Biochemistry 47(50): 13252-60. PubMed ID: 19086271

Haider, N. B., et al. (2000). Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat. Genet. 24: 127-131. PubMed ID: 10655056

Haider, N. B., et al. (2009). Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction. Exp. Eye Res. 89: 365-372. PubMed ID: 19379737

Jaumouille, E., Machado Almeida, P., Stahli, P., Koch, R. and Nagoshi, E. (2015). Transcriptional Regulation via Nuclear Receptor Crosstalk Required for the Drosophila Circadian Clock. Curr Biol 25: 1502-1508. PubMed ID: 26004759

Kobayashi M., et al. (1999). Identification of a photoreceptor cell-specific nuclear receptor. Proc. Natl. Acad. Sci. 96: 4814-4819. PubMed ID: 10220376

Lin, S., Huang, Y. and Lee, T. (2009). Nuclear receptor unfulfilled regulates axonal guidance and cell identity of Drosophila mushroom body neurons. PLoS One 4(12): e8392. PubMed ID: 20027309

Milam, A. H., Rose, L., Cideciyan, A. V., Barakat, M. R., Tang, W.X., et al. (2002). The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration. Proc. Natl. Acad. Sci. 99: 473-478. PubMed ID: 11773633

Much, J. W., et al. (2000). The fax-1 nuclear hormone receptor regulates axon pathfinding and neurotransmitter expression. Development 127: 703-712. PubMed ID: 10648229

Palanker, L., et al. (2006). Dynamic regulation of Drosophila nuclear receptor activity in vivo. Development 133: 3549-3562. PubMed ID: 16914501

Peng, G. H., et al. (2005). The photoreceptor-specific nuclear receptor Nr2e3 interacts with Crx and exerts opposing effects on the transcription of rod versus cone genes. Hum. Mol. Genet. 14: 747-764. PubMed ID: 15689355

Sung, C., et al. (2009). The unfulfilled/DHR51 gene of Drosophila melanogaster modulates wing expansion and fertility. Dev. Dyn. 238(1): 171-82. PubMed ID: 19097190

Wightman, B,, Baran, R. and Garriga, G. (1997). Genes that guide growth cones along the C. elegans ventral nerve cord. Development 124: 2571-2580. PubMed ID: 9216999

Wightman, B., et al. (2005). The C. elegans nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron identity by regulating the expression of glutamate receptor subunits and other neuron-specific genes. Dev. Biol. 287: 74-85. PubMed ID: 16183052


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