View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Cell, Vol. 83, 871-877, December 15, 1995, Copyright 0 1995 by Cell Press

From Embryogenesis to Metamorphosis: Review The Regulation and Function of Drosophila Nuclear Receptor Superfamily Members

Carl S. Thummel (Pignoni et al. 1990). TLL, and its murine homolog TLX, Howard Hughes Medical Institute share a unique P box and can thus bind to a sequence Eccles Institute of Human Genetics that is not recognized by other superfamily members University of Utah (AAGTCA) (Vu et al., 1994). Interestingly, overexpression Salt Lake City, Utah 84112 of TLX in Drosophila embryos yields developmental de- fects resembling those caused by ectopic TLL expression (Vu et al., 1994). In addition, TLX is expressed in the em- The discovery of the nuclear receptor superfamily and de- bryonic brain of the mouse, paralleling the expression pat- tailed studies of receptor function have revolutionized our tern of its homolog. Taken together, these observations understanding of action. Studies of nuclear re- suggest that both the regulation and function of the TLLl ceptor superfamily members in the fruit fly, Drosophila TLX class of orphan receptors have been conserved in melanogaster, have contributed to these breakthroughs these divergent organisms. by providing an ideal model system for defining receptor The HNF4 gene presents a similar example of - function in the context of a developing animal. To date, ary conservation. The fly and vertebrate HNF4 homologs 16 genes of the nuclear receptor superfamily have been have similar sequences and selectively recognize an isolated in Drosophila, all encoding members of the heter- HNF4-binding site (Zhong et al., 1993). The murine HNF4 odimeric and orphan classes of receptors (reviewed by gene is expressed in the intestine, kidney, and liver while Mangelsdorf and Evans, 1995 [this issue of Ce//]). Some dHNF4 is expressed in some of the fly counterparts of superfamily members appear to function specifically dur- these tissues: the midgut and Malpighian tubules. Em- ing embryonic stages while others are required during mul- bryos with a chromosomal deficiency that removes dHNF4 tiple stages of development. Remarkably, half of the show defects in the development of these tissues, sug- known Drosophila superfamily members are regulated by gesting that this gene may be required during organogene- the steroid hormone and appear to contribute sis (Zhong et al., 1993; Hoshizaki et al., 1994). In contrast, to the complex developmental pathways associated with disruption of the murine HNF4 gene results in lethality with metamorphosis. This review provides a brief survey of our defects in gastrulation and mesoderm formation, indicat- current understanding of how nuclear receptor superfam- ing that the vertebrate gene performs earlier functions ily members function during Drosophila development than its fly homolog (Chen et al., 1994). and how their activities relate to those of their vertebrate Two other orphan receptors, FTZ-Fl and DHR39, are homologs. highly related to one another within the DBD (63%), and both are related to the murine SF1 orphan receptor (Table Drosophila Receptors That Function 1). The FTZ-FI gene encodes two protein isoforms with during Embryogenesis: Examples distinct amino-terminal sequences joined to identical DBD of Evolutionary Conservation and LBD sequences (Lavorgna et al., 1993). aFTZ-F7 is Embryogenesis in Drosophila occurs during the first day expressed during early embryogenesis while PFTZ-F7 is of the life cycle and involves the initial segmentation of expressed in late embryos and during metamorphosis. the animal followed by organogenesis. Of the Drosophila FTZ-Fl , DHR39, and SF1 share a unique P box sequence nuclear receptor superfamily members, eight appear to as well as the so-called FTZ-Fl box, located adjacent to perform specific functions during embryonic develop- the carboxyl terminus of the DBD. These domains direct ment, although some of these genes also act at later binding to a PyCAAGGPyCPu consensus sequence, with stages of development: knirps (kr10 (Nauber et al., 1988), the first three nucleotides recognized by the FTZ-Fl box knirps-related (knrl) (Oro et al., 1988; Rothe et al., 1989), (Ueda et al., 1992). FTZ-Fl, DHR39, and SF1 can bind embryonic gonad (egon) (Rothe et al., 1989), tailless (tll) this element as monomers, consistent with the presence (Pignoni et al., 1990), dHNF4 (Zhong et al. 1993), seven-up of only a single half-site in the binding sequence (Ueda (svp)(Mlodziket al., 199O),FTZ-F7 (Lavorgnaet al., 1991), et al., 1992; Wilson et al., 1993; Ohno et al., 1994). The and DHR39 (also called FTZ-F7/3) (Ohno and Petkovich, ability of FTZ-Fl and DHR39 to bind an identical se- 1992; Ayer et al., 1993). Three of these genes, kni, km/, quence, combined with the widespread expression of and egon, are highly divergent members of the superfamily DHR39, suggests that these two proteins may compete that have retained only the sequences encoding the DNA- for occupancy of a common set of target sequences (Ayer binding domain (DBD) (see Figure 1A in Mangelsdorf and et al., 1993; Ohno et al., 1994). Analysis of FTZ-F7 and Evans, 1995). The absence of a ligand-binding domain DHR39 mutants should provide a test of this model, as (LBD) in these proteins suggests that they function in a well as insights into their roles in both gene regulation ligand-independent manner. Of these three genes, kni has and development. It will also be interesting to determine been studied in the most detail and is required for proper whether there are any similarities to the phenotypes of Sf7 abdominal segmentation (Nauber et al., 1988). mutant mice, which are deficient in steroidogenesis and The Wgene is also required for segmentation, as well as sexual differentiation (Luo et al., 1994). the development of the brain, gut, and Malpighian tubules The svp gene is expressed in the embryonic central Cell 072

Table 1. Drosophila Nuclear Hormone Receptor Superfamily Members and Their Vertebrate Counterparts

Fly Cytogenetic DBDILBD Receptor Location Vertebrate Homolog’ Percent ldentityb Ecdysone Regulationc KNI 77E KNRL 77E EGON 798 SVP 870 COUP-TF* 94/93 TII 1 OOA TLX’ 81141 dHNF4 29E HNF4’ 90/67 FTZ-Fl 75D SF1 * 00133 USP 2c RXR’ 66149 ECR 42A Farnesoid X receptor 81134 E75 758 Rev-Erb 79135 E78 7% Rev-Erb 72135 DHR3 46F RORa 75120 DHR38 38E NGFI-B* 88154 DHR39 39c SF1 62l17 + DHR78 78D TR2 74126 + DHR96 96B Human vitamin D receptor 64128 + a Vertebrate homologs of Drosophila receptors are marked by an asterisk, indicating that the high degree of sequence similarity extends outside of the DBD. Other vertebrate receptors are among the best matches in the DBD to the corresponding Drosophila protein. b The percent amino acid identity between the DBD and LBD of the Drosophila and vertebrate receptors. c A plus sign indicates transcriptional regulation by ecdysone in cultured larval organs (either induction or repression), while a minus sign indicates no apparent response.

nervous system (CNS) and fat body as well as in photore- imaginal progenitor cells. Thus, metamorphosis results in ceptor cell precursors of the eye imaginal disc (Mlodzik a complete transformation in form and function-from a et al., 1990; Hoshizaki et al., 1994). Clonal analysis has crawling to the highly motile, reproductively active revealed that svp is required for the proper specification adult fly. Remarkably, these divergent developmental of the R7 photoreceptor cell fate (Mlodzik et al., 1990). pathways are manifested simultaneously in apparent re- Interestingly, a zebrafish homolog of svp, svp[44], is also sponse to a single steroid hormone. A focus of current expressed in the embryonic CNS and developing eye study is to understand how the systemic hormonal signal (Fjose et al., 1993). The high degree of sequence conser- is refined into the appropriate stage- and tissue-specific vation between the SVP and Svp[44] proteins (94% in developmental pathways that direct this transformation. DBD; 91% in LBD), combined with their similar spatial patterns of expression, suggests that these genes may have conserved their regulation and function. Both pro- The Drosophila Ecdysone Receptor Is a teins are highly related to the vertebrate chicken oval- Heterodimer of ECR and USP bumin upstream promoter (COUP) orphan receptor, with Ecdysone manifests its effects on development via its in- extensive sequence similarity that extends outside of the teraction with the ecdysone receptor-the only ligand- DBD and LBD. Below we consider evidence that, like dependent nuclear receptor identified to date in Drosoph- COUP, SVP can interact with retinoid X receptor (RXR) ila. Curiously, the ecdysone receptor consists of a signaling pathways. heterodimer that more closely resembles avertebrate reti- noic acid receptor than a steroid receptor. Drosophila Postembryonic Development Is One half of the ecdysone receptor is encoded by the Controlled by Pulses of Ecdysone EcR gene (Koelle et al., 1991). EcR is induced directly by Pulses of the steroid hormone 20-hydroxyecdysone (here ecdysone, providing an autoregulatory loop that increases referred to as ecdysone) direct Drosophila through its life the level of receptor protein in response to its ligand. Three cycle, with peak hormone titers signaling the major post- protein isoforms are encoded by EcR, designated ECR-A, embryonic developmental transitions (reviewed by Riddi- ECR-81, and ECR-B2 (Talbot et al., 1993). These proteins ford, 1993). Larval development progresses through three differ in their amino-terminal sequences but contain identi- , punctuated by ecdysone-triggered molting of the cal DBD and LBD sequences. Interestingly, two of these cuticle (Figure 1). A high titer pulse of ecdysone at the end isoforms are expressed in patterns that correlate with the of the third triggers puparium formation, marking divergent developmental fates of the larval and adult tis- the onset of the prepupal stage, Prepupal development sues, suggesting that they may contribute to the tissue proceeds for 12 hr, after which an ecdysone pulse initiates specificity of ecdysone responses. The ECR-Bl isoform the 3.5 days of pupal development, followed by eclosion is expressed primarily in larval cells that are fated to die, of the adult fly (Figure 1). The larval tissues are destroyed while ECR-A is expressed in developing adult structures by histolysis during the early stages of metamorphosis and and tissues (Talbot et al., 1993). ECR-A is also expressed are replaced by adult tissues that develop from clusters of in a set of neurons in the CNS that die shortly after adult Review: Regulation and Function of Drosophila Nuclear Receptors a73

Figure 1. The Ecdysteroid Titer Profile during Drosophila Development The composite ecdysteroid titer is depicted, in 20-hydroxyecdysone equivalents from whole- body homogenates (figure adapted from Riddi- ford, 1993). Each of the developmental stages of the Drosophila life cycle are also shown, be- low a timescale in days.

d 1 2 3 4 5 6 7 8 9 days 1st I 2nd I 3rd instar 1 instar ’ hstar IEmbryo I Larva & PUp3 Adult eclosion. The death of these cells depends upon a de- Many Drosophila Orphan Receptors Are Regulated crease in ecdysone titer, suggesting that the specific ex- by Ecdysone and Appear to Function pression of the ECR-A isoform is required for the appro- during Metamorphosis priate developmental fate of these cells (Robinow et al., Perhaps the most surprising recent discovery in the Dro- 1993). sophila nuclear receptor field is the large number of these All three ECR isoforms acquire the ability to bind DNA genes that are regulated by ecdysone and appear to func- upon heterodimerization with Ultraspiracle (USP) (Koelle, tion during metamorphosis. Half of the 16 known Drosoph- 1992; Yao et al., 1992; Thomas et al., 1993) (see Figure ila nuclear receptor superfamily members are either in- 3A). USP is the only known Drosophila homolog of the duced or repressed by the hormone at the level of vertebrate RXRs (Table 1) (Henrich et al., 1990; Oro et transcription-EC/?, /3FTZ-Fl, E75, E78, DHR3, DHR39, al., 1990; Shea et al., 1990). Consistent with this sequence DHR78, and DHR96 (Table 1). The discovery of most of conservation, USP can heterodimerize with a variety of these genes arose from efforts to understand how ecdy- vertebrate RXR partners, including retinoic acid receptor, sone directs the early stages of metamorphosis. Studies hormone receptor, and vitamin D receptor (Yao et of the puffing patterns in larval salivary gland polytene al., 1992). Interestingly, although ECR can bind ligand chromosomes revealed that the ecdysone signal is trans- weakly on its own, this binding is greatly stimulated by the duced and amplified via a two-step regulatory hierarchy addition of USP (Koelle, 1992; Yao et al., 1993). Ligand (Ashburner et al., 1974). Pulses of ecdysone at the ends binding both stabilizes the ECR-USP heterodimer and of larval and prepupal development directly induce asmall also increases its affinity for binding to ecdysone respon- set of widely expressed “early” genes, at least three of sive elements (EcREs). Two naturally occurring EcREs, which encode transcription factors. These early genes from the ecdysoneinducible hsp27and fip28/29 promot- both repress their own expression and induce more than ers, are bound by ECR-USP (Yaoet al., 1992). In addition, 100 “late” secondary response genes (Figure 2). The late ECR-USP can bind to direct repeats of AGGTCA se- genes, in turn, are thought to direct each ecdysone target quences separated by 3, 4, or 5 bp, much like vertebrate tissue along its appropriate developmental pathway dur- RXR heterodimers (Horner et al., 1995). Although it re- ing metamorphosis. mains possible that ECR and USP may exert distinct func- E75 is a complex early ecdysoneinducible gene that tions as homodimers, all available in vivo evidence indi- directs the synthesis of three protein isoforms, E75A, cates that these proteins function as a heterodimer. E756, and E75C, by the use of nested promoters (Feigl Antibody stains of larval salivary gland polytene chromo- et al., 1989; Segraves and Hogness, 1990). E75A and somes support this proposal, as all sitesoccupied by ECR E75C are orphan receptors that have distinct amino- also contain bound USP protein (Talbot, 1993; Yao et al., terminal sequences joined to an identical DBD and LBD. 1993). In contrast, E75B retains only one zinc finger, suggesting The uspgene was originally identified based on its lethal that it is incapable of binding DNA. Consistent with its mutant phenotypes, which suggest a role in mediating ec- ecdysone regulation, several ECR-USP-binding sites dysone responses during development (Perrimon et al., have been identified upstream of the E75A promoter (Tal- 1985; Oro et al., 1992). USP functions in abdominal cuticle bot, 1993). Antibody staining of polytene chromosomes synthesis during midembryogenesis and larval cuticle has revealed that E75A protein is bound to both early and molting. Clonal analysis has also revealed a function for late gene loci, suggesting that it may function at both levels usp in the proper selection of the R7 photoreceptor cell of the hierarchy (Hill et al., 1993). In addition, recent evi- fate in the adult eye, suggesting that it may interact with dence indicates a role for E75 in embryonic gut morpho- svp in this developmental pathway. Curiously, however, genesis (Bilder and Scott, 1995). This observation not only adult thoracic and abdominal metamorphosis can occur provides further evidence that Drosophila nuclear recep- in the absence of USP, indicating that these responses tors can perform multiple functions during development, are either not regulated by ecdysone or are dependent on but also suggests a role for the midembryonic pulse of the activity of another nuclear receptor. ecdysone (see Figure 1). Cell 874

I Figure 2. Schematic Representation of Orphan Ecdysone , Ecdysone Gene Expression during the Onset of Metamor- phosis Ecdysone pulses at the ends of third instar lar- val and prepupal development directly induce a small set of early genes, including the E75A orphan receptor. The early genes repress their own expression and induce a large set of late genes. E78B and DHR3 induction is delayed relative to that of the early genes, apparently owing to an additional requirement for early ec- dysone-induced protein synthesis. BFTZ-F7 is repressed by both ecdysone and itself, thus restricting its expression to the brief interval of low ecdysone titer in midprepupae. flFTZ-F7 appears to provide the competence for the Third lnstar I Prepupa early genes and E93 to be induced by the pre- Larva , pupal ecdysone pulse. Pupariumformation

E78 and DHR3 expression comprises a delayed early set the system, allowing the reinduction of the early/late response to ecdysone. E78 encodes two protein isoforms, genetic response to ecdysone as well as providing the E78A and E78B (Stone and Thummel, 1993). E78A is ex- competence for stage-specific transcriptional responses pressed during a brief interval in midpupal development to the hormone (Figure 2). Interestingly, BFTZ-F7 re- while E78B is expressed at puparium formation and imme- presses its own transcription, ensuring that the compe- diately following E78A in pupae. DHR3 also comprises a tence it provides will be of short duration (Figure 2). The complex locus, encoding at least three size classes of identity of PFTZ-Fl as an orphan receptor raises the possi- ml?NA (Koelle et al. 1992). Both E78B and DHR3 are in- bility that it may interact with the ECR-USP heterodimer, duced directly by ecdysone, but differ from the early genes allowing the receptor to activate a set of stage-specific in thatthey require ecdysoneinduced protein synthesis for promoters in prepupae. This interaction could be mani- their maximal levels of transcription (Stone and Thummel, fested either by binding to adjacent sequences in the DNA 1993; Horner et al., 1995). This requirement for trans- or through the formation of novel heterodimer combina- acting factors may account for the delay observed in E78B tions. and DHR3 expression relative to the early genes (Figure The remaining four Drosophila orphan receptors are ex- 2). It seems likely that this delay is of significance, allowing pressed throughout the early stages of metamorphosis: these orphan receptors to perform their regulatory func- DHR38, DHR39, DHR78 (also called XR78E/F), and tions in newly formed prepupae, after the early gene prod- DHR96 (Ohno and Petkovich, 1992; Ayer et al., 1993; Fisk ucts have peaked in activity. The functions of these genes, and Thummel, 1995; Sutherland et al., 1995; Zelhof et however, remain unknown. al., 1995b). Experiments with cultured larval organs have Curiously, in spite of its similarity to the vertebrate Rev- demonstrated that DHR39, DHR78, and DHR96 are in- Erb orphan receptor, E78A does not bind to a Rev-Erb duced by ecdysone, although the latter two genes show response element (Horner et al., 1995). Further studies only a modest response to the hormone (Fisk and Thum- are required to determine whether E78A requires a partner mel, 1995; Horner et al., 1995; Zelhof et al., 1995b). protein for its DNA-binding activity or whether E78A binds DHR38 is the fly homolog of the vertebrate nerve growth to a distinct target sequence. Only one DHR3 isoform has factor-induced NGFI-B orphan receptor while DHR78 and been described, with sequence similarity to the retinoid- DHR96 are novel orphan receptors(Table 1). As expected, related orphan receptor a (RORa) family of orphan recep- DHR38 can bind as a monomer to an NGFI-B response tors (Table 1) (Koelle et al., 1992; Gig&e et al., 1994). element (Fisk and Thummel, 1995; Sutherland et al., Like RORa, DHR3 can bind DNA as a monomer and re- 1995). In contrast, DHR78 and DHR96 can interact with quires an A at position -4 relative to the AGGTCA core distinct subsets of sequences recognized by the ECR- sequence (Giguere et al., 1994; Horner et al., 1995). USP heterodimer. DHR78 binds to direct or inverted re- Experiments with cultured larval salivary glands have peats of an AGGTCA half-site, while DHR96 binds selec- demonstrated that /3FTZ-F7 transcription is repressed by tively to the hsp27 EcRE, a palindromic arrangement of ecdysone (Woodard et al. 1994). Consistent with this regu- the imperfect half-sites AGtgCA and gGtTCA (Fisk and lation, /?FTZ-F7 is expressed during the brief interval of Thummel, 1995; Zelhof et al., 1995b). These distinct bind- low ecdysone titer in midprepupae, following E78B and ing specificities are consistent with the unique DHR96 P DHR3 (Figure 2). Ectopic expression of PFTZ-f 7 can en- box sequence (ESCKA). hance theecdysone induction of the early genes, including E75A (Figure 2). In addition, BFTZ-F7 is sufficient to direct Mechanisms for Negative Regulation the stage-specific ecdysone induction of the E93 early of Ecdysone Responses gene in prepupal salivary glands (Woodard et al., 1994). Recent functional studies of Drosophila orphan receptors Thus, /?FTZ-f7 expression in midprepupae appears to re- in transfected tissue culture cells have indicated that these Review: Regulation and Function of Drosophila Nuclear Receptors 075

A Figure 3. Models for Negative Regulation of Ecdysone Signaling Pathways Based on Tis- sue Culture Cotransfection Assays (A) The ECR-USP heterodimer binds ecdy- sone (E) and an EcRE in the DNA, activating a downstream promoter (arrow). (8) A DHR78 homodimer binds to the EipPW 29 EcRE, thus preventing the ECR-USP ecdy- sone receptor from activating the target pro- moter (Zelhof et al., 1995b). (C) DHR38 forms an inactive heterodimer with USP, preventing activation of the target pro- moter (Sutherland et al., 1995). (D) SVP forms an inactive heterodimer with ECR, preventing activation of the target pro- moter (Zelhof et al., 1995a).

factors can interfere with ecdysone signaling pathways by ability to interact with RXR-dependent pathways (Forman either binding to EcREs or forming inactive heterodimers et al., 1995; Perlmann and Jansson, 1995). with ECd or USP. Studies of DHR78 have led to a model Recent evidence also suggests that negative regulation for inhibition of ecdysone responses through DNA-binding of ecdysone signaling may be achieved through hetero- site competition (Zelhof et al., 1995b). DHR78 cannot het- dimerization with ECR. Studies using the yeast two-hybrid erodimerize with either ECR or USP, but it can bind as system have demonstrated that SVP can interact with ECR a homodimer to the Eip28/29 EcRE. DHR78 expression (Zelhof et al., 1995a). SVP can also reduce the ability of significantly inhibits the ability of ECR and USP to induce ECR and USP to transactivate transcription through the transcription through the Eip28/29 EcRE, suggesting that hsp27 EcRE, although SVP is unable to bind this se- its binding to this sequence can prevent the binding of the quence. Taken together, these observations suggest that ecdysone receptor complex (Figure 38). Consistent with SVP may form an inactive heterodimer with ECR, reducing its inability to bind to the hsp27 EcRE, DHR78 has no effect the levels of functional ecdysone receptor in the cell (Fig- on the ability of ECR-USP to transactivate transcription ure 3D). This regulatory function provides yet another ex- through this sequence. Thus, DHR78 can function to regu- ample of evolutionary conservation, since the SVP homo- late ecdysone responses negatively depending on the se- log, COUP, negatively regulates RXR-signaling pathways quence of the target EcRE. A similar mechanism of action in mammalian cell culture (Kliewer et al., 1992; Tran et has been proposed for the DHR96 orphan receptor (Fisk al., 1992). Thus, both direct DNA binding and the formation and Thummel, 1995). of distinct heterodimer combinations provide different lev- The identification of USP as an RXR homolog immedi- els at which ecdysone responses can be negatively con- ately raised the possibility that this protein could hetero- trolled. dimerize with Drosophila receptors other than ECR, thus increasing the spectrum of potential regulatory interac- Future Directions tions. Similarly, heterodimer combinations among orphan In addition to their possible contribution to novel hetero- receptors or between orphan receptors and ECR could dimers, the Drosophila orphan receptors provide the po- generate an array of distinct receptors with different target tential for transducing different hormonal signals during gene specificities (Richards, 1992; Segraves, 1994). In development. Although ecdysone is the only known ste- spite of intensive efforts to search for such interactions, roid with potent biological activity in Drosophila, the hemo- however, only two alternate heterodimers have been dis- lymph contains many other ecdysteroids as well as the covered. Assays for dimerization using the yeast two- sesquiterpenoid hormone (JH). JH plays a critical hybrid system revealed a strong and specific interaction role in determining the developmental response to an ec- between USP and DHR38 (Sutherland et al., 1995). In dysone pulse in many , suggesting that it may per- transfected tissue culture cells, addition of DHR38 can form a similar function during the fly life cycle (Riddiford, disrupt ECR-USP binding to an hsp27EcRE and suppress 1993). Given that JH shares structural features with the the ability of ECR-USP to induce transcription through retinoids, it seems reasonable to predict that this hormone this sequence. These observations suggest a model for will function through a member of the nuclear receptor negative regulation of ecdysone responses in which superfamily. Indeed, Harmon et al. (1995) have recently DHR38 heterodimerizes with USP, thus blocking its ability demonstrated that the JH analog methoprene is an RXR- to form a functional receptor complex with ECR (Figure specific ligand, although this ligand cannot be bound by 3C). A similar regulatory interaction has been described USP. for the vertebrate homolog of DHR38, NGFI-B, indicating It should also be noted that several Drosophila orphan that the fly and vertebrate receptors have conserved their receptors appear to act in a ligand-independent manner. Cell 876

Ectopic expression studies of svp, t/l, and/?FTZ-Fl indicate hormone receptor gene family is expressed in the 20-OH-ecdysone that these genes can function at times and places that are inducible puff 758 in . Nucl. Acids Res. 77, 7167-7178. different from their normal domains of expression, sug- Fisk, G.J., and Thummel, C.S. (1995). Isolation, regulation, and DNA- gesting that ligand availability does not restrict their activ- binding properties of three Drosophila nuclear hormone receptor su- ity (Steingrimsson et al., 1991; Hiromi et al., 1993; Wood- perfamily members. Proc. Natl. Acad. Sci. USA 92, 10604-10608. ard et al., 1994). These effects contrast with the absence Fjose, A., Nornes, S., Weber, U., and Mlodzik, M. (1993). Functional of any phenotypic consequences caused by ectopic ex- conservation of vertebrate seven-up related genes in neurogenesis pression of the ligand-dependent USP receptor (Oro et and eye development. EMBO J. 72, 1403-1414. al., 1992). The brief expression of E75A, E78B, DHR3, and Forman, B.M., Umesono, K., Chen, J., and Evans, R.M. (1995). Unique BFTZ-FI for only a few hours during the onset of metamor- response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 87, 541-550. phosis is also difficult to reconcile with an additional re- Giguere, V., Tini, M., Flock, G., Ong, E., Evans, R.M., and Otulakowski, quirement for a temporally restricted ligand. G. (1994). Isoform-specific amino-terminal domains dictate DNA- Finally, the regulation of seven Drosophila orphan re- binding properties of RORa, a novel family of orphan hormone nuclear ceptor genes by ecdysone raises the interesting possibility receptors. Genes Dev. 8, 538-553. that the expression of vertebrate orphan receptors may Harmon, M.A., Boehm, M.F., Heyman, R.A., and Mangelsdorf, D.J. also be hormonally controlled. This aspect of vertebrate (1995). Activation of mammalian retinoid X receptors by the growth regulator methoprene. Proc. Natl. Acad. Sci. USA 92, 6157- orphan receptor regulation has not yet been examined 6160. and potentially provides another point for convergence in Henrich, V.C., Sliter, T.J., Lubahn, D.B., Maclntyre, A., and Gilbert, the regulation and function of nuclear receptor superfamily L.I. (1990). A steroid/thyroid hormone receptor superfamily member members. in Drosophila melanogaster that shares extensive sequence similarity The future holds the possibility of opening up new areas with a mammalian homologue. Nucl. Acids Res. 78, 4143-4148. of receptor research. One distinct advantage offered by Hill, R.J., Segraves, W.A., Choi, D., Underwood, P.A., and MacAvoy, Drosophila is the ability to study receptor function in the E. (1993). The reaction with polytene chromosomes of antibodies raised against Drosophila E75A protein. Insect Biochem. Mol. Biol. context of defined genetic hierarchies, integrating these 23, 99-104. regulators into well-characterized developmental path- Hiromi, Y., Mlodzik, M., West, S.R., and Rubin, G.M. (1993). Ectopic ways. In addition, by performing genetic screens for genes expression of seven-up causes cell fate changes during ommatidial that interact with Drosophila nuclear receptors, it may be assembly. Development 7 78, 1123-i 135. possible to identify previously unexpected regulatory inter- Horner, M., Chen, T., and Thummel, C.S. (1995). Ecdysone regulation actions in hormone signaling pathways. These studies and DNA binding properties of Drosophila nuclear hormone receptor may both reinforce and dispel some of the prevailing para- superfamily members. Dev. Biol. 768, 490-502. digms in the hormone receptor field. We can hope that Hoshizaki, D.K., Blackburn, T., Price, C., Ghosh, M., Miles, K., Ra- gucci, M., and Sweis, R. (1994). Embryonic fat-cell lineage in Drosoph- the next 10 years of receptor research will yield further ila melanogaster. Development 720, 2489-2499. insights into the conserved regulation and function of nu- Kliewer, S.A., Umesono, K., Heyman, R.A., Mangelsdorf, D.J., Dyck, clear receptors during development as well as provide J.A., and Evans, R.M. (1992). Retionoid X receptor-COUP-TF interac- new directions in our understanding of hormone signaling tions modulate retinoic acid signalling. Proc. Natl. Acad. Sci. USA 89, pathways. 1448-l 452. Koelle, M.R. (1992). Molecular analysis of the Drosophila ecdysone Acknowledgments receptor complex. Ph.D. thesis, Stanford University, Stanford, Cali- fornia. I apologize to the authors whose work is not cited in this review owing Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas, to length restrictions. I thank D. Cimbora, S. Sakonju, A. EIHaj, and P., and Hogness, D.S. (1991). The Drosophila EcR gene encodes an members of my lab for their critical comments on this manuscript and ecdysone receptor, a new member of the steroid receptor superfamily. G. Fisk for help with preparing Table 1. C. S. T. in an Associate Investi- Cell 67, 59-77. gator with the Howard Hughes Medical Institute. Koelle, M.R., Segraves, W.A., and Hogness, D.S. (1992). DHRB: a Drosophila steroid receptor homolog. Proc. Natl. Acad. Sci. USA 89, References 6167-6171. Lavorgna, G., Ueda, H., Clos, J., and Wu, C. (1991). FTZ-Fl, a steroid Ashburner, M., Chihara, C., Meltzer, P., and Richards, G. (1974). Tem- hormone receptor-like protein implicated in the activation of fushi tar- poral control of puffing activity in polytene chromosomes. Cold Spring am. Science 252, 848-851. Harbor Symp. Quant. Biol. 38, 655-662. Lavorgna, G., Karim, F.D., Thummel, C.S., and Wu, C. (1993). Poten- Ayer, S., Walker, N., Mosammaparast, M., Nelson, J.P., Shilo, B., and tial role for a FTZ-Fl steroid receptor superfamily member in the control Benyajati, C. (1993). Activation and repression of Drosophila alcohol of Drosophila metamorphosis. Proc. Natl. Acad. Sci. USA 90, 3004- dehydrogenase distal transcription by two steroid hormone receptor 3008. superfamily members binding to a common response element. Nucl. Acids Res. 21, 1619-1627. Luo, X., Ikeda, Y., and Parker, K.L. (1994). A cell-specific nuclear Bilder, D., and Scott, M.P. (1995). Genomic regions required for mor- receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77, 481-490. phogenesis of the Drosophila embryonic midgut. Genetics 747,1087- 1100. Mangelsdorf, D.J., and Evans, R.M. (1995). The RXR heterodimers Chen, W.S., Manova, K., Weinstein, DC., Duncan, S.A., Plump, A.S., and orphan receptors. Cell 83, this issue. Prezioso, V.R., Bachvarova, R.F., and Darnell, J.E. (1994). Disruption Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C.S., and Rubin, G.M. of the HNF-4 gene, expressed in the visceral mesoderm, leads to cell (1990). The Drosophilaseven-up gene, a member of the steroid recep- death in embryonic ectoderm and impaired gastrulation of mouse em- tor gene superfamily, controls photoreceptor cell fates. Cell 60, 21 I- bryos. Genes Dev. 8, 2466-2477. 224. Feigl, G., Gram, M., and Pongs, 0. (1989). A member of the steroid Nauber, U., Pankratz, M.J., Lienlin, A., Seifert, E., Klemm, U., and Review: Regulation and Function of Drosophila Nuclear Receptors a77

Jackie, H. (1988). Abdominal segmentation of the Drosophila embryo dimerization of the Drosophila ecdysone receptor with retinoid X recep- requires a hormone receptor-like protein encoded by the gap gene tor and ultraspiracle. Nature 362, 471-475. knirps. Nature 336, 489-492. Tran, P.B., Zhang, X.K., Salbert, G., Hermann, T., Lehmann, J.M., Ohno, C.K., and Petkovich, M. (1992). FTZ-F7/3, a novel member of and Pfahl, M. (1992). COUP orphan receptors are negative regulators the Drosophila nuclear receptor family. Mech. Dev. 40, 13-24. of retinoic acid response pathways. Mol. Cell. Biol. 72, 4666-4676. Ohno, C.K., Ueda, H., and Petkovich, M. (1994). The Drosophila nu- Ueda, H., Sun, G.-C., Murata, T., and Hirose, S. (1992). A novel DNA- clear receptors FTZ-FI a and FTZ-F16 compete as monomers for bind- binding motif abuts the zinc finger domain of insect nuclear hormone ing to a site in the fushi tarazu gene. Mol. Cell. Biol. 14, 31663175. receptor FTZ-Fl and mouse embryonal long terminal repeat-binding Oro, A.E., Ong, ES., Margolis, J.S., Posakony, J.W., McKeown, M., protein. Mol. Cell. Biol. 12, 5667-5672. and Evans, R.M. (1988). The Drosophila gene knirps-related is a mem- Wilson, T.E., Fahrner, T.J., and Milbrandt, J. (1993). Theorphan recep- ber of the steroid-receptor gene superfamily. Nature 336, 493-496. tors NGFI-B and steroidogenic factor 1 establish monomer binding as Oro, A.E., McKeown, M., and Evans, R.M. (1990). Relationship be- a third paradigm of nuclear receptor-DNA interaction. Mol. Cell. Biol. tween the product of the Drosophila ultraspiracle locus and vertebrate 73,5794-5604. retinoid X receptor. Nature 347, 298-301. Woodard, C.T., Baehrecke, E.H., and Thummel, C.S. (1994). A molec- Oro, A.E., McKeown, M., and Evans, R.M. (1992). The Drosophila ular mechanism for the stage specificity of the Drosophila prepupal retanoid X receptor homolog ultraspiricle functions in both female re- genetic response to ecdysone. Cell 79, 607-615. production and eye . Development 175, 449-462. Yao, T., Segraves, W.A., Oro, A.E., McKeown, M., and Evans, R.M. Perlmann, T., and Jansson, L. (1995). A novel pathway for vitamin (1992). Drosophila ultraspiracle modulatesecdysone receptorfunction A signaling mediated by RXR heterodimerization with NGFI-B and via heterodimer formation. Cell 77, 63-72. NURRI Genes Dev. 9, 769-782. Yao, T., Forman, B.M., Jiang, Z., Cherbas, L., Chen, J.D., McKeown, Perrimon, N., Engstrom, L., and Mahowald, A.P. (1985). Develop- M., Cherbas, P., and Evans, R.M. (1993). Functional ecdysone recep- mental genetics of the 2C-D region of the Drosophila X chromosome. tor is the product of EcR and ultraspiracle genes. Nature 366, 476- Genetics 7 7 7, 23-41. 479. Pignoni, F., Baldarelli, R.M., Steingrimsson, E., Diaz, R.J., Patapou- Yu, R.T., McKeown, M., Evans, R.M., and Umesono, K. (1994). Rela- tian, A., Merriam, J.R., and Lengyel, J.A. (1990). The Drosophila gene tionship between Drosophila gap gene tailless and a vertebrate nuclear failless is expressed at the embryonic termini and is a member of the receptor Tlx. Nature 370, 375-379. steroid receptor superfamily. Cell 62, 151-163. Zelhof, AC., Yao, T., Chen, J.D., Evans, R.M., and McKeown, M. Richards, G. (1992). Switching partners? Curr. Biol. 2, 657-659. (1995a). Seven-up inhibits ultraspiracle-based signaling pathways in Riddiford, L.M. (1993). and Drosophila development. In The vitro and in viva. Mol. Cell. Biol. 15, 6736-6745. Development of Drosophila melanogaster, Volume 2, M. Bate and A. Zelhof, A.C., Yao, T., Evans, R.M., and McKeown, M. (1995b). Identifi- Martinez Arias, eds. (Cold Spring Harbor, New York: Cold Spring Har- cation and characterization of a Drosophila nuclear receptor with the bor Laboratory Press), pp. 699-939. ability to inhibit the ecdysone response. Proc. Natl. Acad. Sci. USA 92, io477-10481. Robinow, S., Talbot, W.S., Hogness, D.S., and Truman, J.W. (1993). in the Drosophila CNS is ecdysone-regulated Zhong, W., Sladek, F.M., and Darnell, J.E., Jr. (1993). The expression and coupled with a specific ecdysone receptor isoform. Development pattern of a Drosophila homolog to the mouse transcription factor 779,1251-1259. HNF-4 suggests a determinative role in gut formation. EMBO J. 72, Rothe, M., Nauber, U., and Jackie, H. (1989). Three hormone receptor- 537-544. like Drosophila genes encode an identical DNA-binding finger. EMBO J 8,3087-3094. Segraves, W.A. (1994). Steroid receptors and orphan receptors in Dro- sophila development. Semin. Cell Biol. 5, 105-113. Segraves, W.A., and Hogness, D.S. (1990). The E75 ecdysone- inducible gene responsible for the 758 early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev. 4, 204-219. Shea, M.J., King, D.L., Conboy, M.J., Mariani, B.D., and Kafatos, F.C. (1990). Proteins that bind to Drosophila chorion cis-regulatory ele- ments: a new C2H2zincfinger protein and a C2C2steroid receptor-like component. Genes Dev. 4, 1128-l 140. Steingrimsson, E., Pignoni, F., Liaw, G., and Lengyel, J. (1991). Dual role of the Drosophila pattern gene railless in embryonic termini. Sci- ence 254, 41 a-421 Stone, B.L., and Thummel, C.S. (1993). The Drosophila 78C early late puff contains E78, an ecdysoneinducible gene that encodes a novel member of the nuclear hormone receptor superfamily. Cell 75, 307- 320. Sutherland, J.D., Kozlova, T., Tzertzinis, G., and Kafatos, F.C. (1995). Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear hormone receptors of the nerve growth factor-induced protein B type. Proc. Natl. Acad. Sci. USA 92, 7966-7970. Talbot, W.S. (1993). Structure, expression, and function of ecdysone receptor isoforms in Drosophila. Ph.D. thesis, Stanford University, Stanford, California. Talbot, W.S., Swyryd, E.A., and Hogness, D.S. (1993). Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73, 1323-1337. Thomas, H.E., Stunnenberg, H.G., and Stewart, A.F. (1993). Hetero-