Development 1989 Supplement, 133-140(1989)Printed in Great Britain © T h e Company of Biologists Limited 1989

133

Steroid hormone receptor homologs in development

ANTHONY E. ORO 1 2 , KAZUHIKO UMESONO1 and RONALD M. EVANS1

' Howard Hughes Medical Institute, Salk Institute for Biological Sciences, La Jolla, California 92138-9216, USA2 Department of Biological Sciences, University of California, San Diego, La Jolla, California 92093, USA

Summary

The steroid/thyroid receptor superfamily are llgand-dependent transcription factors which consist of distinctfunctional domains required for transcriptional controlof a network of genes. Members of this superfamily arebeginning to be studied for their contribution to embryo-genesis. Two human receptors for the vertebrate mor-phogen retinoic acid have been isolated and furthercharacterized on model promoters. Moreover, the pres-ence of homologs of these receptors in Drosophila reveals

that members of this superfamily predate the divergenceof the vertebrates and invertebrates. One locus is knirps-related (knrl), whose product is closely related to that ofthe gap segmentation gene knirps (knf). knrl is one of themost diverged steroid receptor-like molecules and dis-plays a spatially restricted blastoderm pattern.

Key words: steroid hormone, thyroid receptor,transcriptional control, Drosophila, knirps.

I. Development as a problem of differential generegulation

In the 1930s T. H. Morgan theorized that the processof development occurred by the differential expression ofsmall invisible entities called genes and that expression ofdifferent sets of these genes gives rise to differences in theadult organism (Morgan, 1934). Indeed differential geneexpression does appear to play an important part innormal development as many classes of transcriptionalregulators have been recently isolated and shown to playimportant roles in embryogenesis (Dressier and Gruss,1988). One of them is the steroid/thyroid receptorsuperfamily of proteins. These proteins are receptors forsmall hydrophobic molecules which as nuclear hormone-receptor complexes mediate their effects. The biologi-cally active ligands play roles in both homeostasis in theadult and development, as illustrated in Table 1. Forexample, the adrenal steroids widely influence energymetabolism, controlling glycogen, muscle, and mineralmetabolism as well as mediating behavioral responses toperceived stress. They have widespread effects on theimmune and nervous systems and influence the determi-nation of neural crest fate. The sex steroids provoke thedevelopment and determination of body sexual dimor-phisms including the reproductive organs embryonically,the central nervous system perinatally, and reproductivebehavior in the adult. Aberrant production of thesehormones have been associated with a broad spectrum ofclinical disease. Further, both thyroid and steroid hor-mones appear to be important in metamorphosis. Thyroi-dectomy inhibits tadpole maturation to a frog, butaddition of thyroxine to its drinking water induces all ofthe changes into a terrestrial adult (Schwind, 1933).

Similarly, ecdysteroids are required for insect metamor-phosis, allowing the various molts into adulthood (Ash-burner, 1971).

An initial insight into how small, relatively simplemolecules elicit such a diversity of complex responses wasprovided by the identification of steroid and thyroidhormone receptors with radiolabeled ligands in the early1970s (Jensen and DeSombre, 1972). After the additionof hormone, the receptor apparently underwent a confor-mational change such that it associated with high affinitybinding sites in chromatin and induced or repressed alimited number of genes (Ivaric and O'Farrell, 1978).This type of experiment led to the idea that thesereceptors controlled a specific network of genes in orderto facilitate homeostasis. Purification and biochemicalcharacterization of the glucocorticoid receptor was ac-companied by the identification of a variety of glucocorti-coid responsive genes (Yamamoto, 1985; Ringold, 1985).Each gene contained a short m-acting sequence (about20 bp) in the promoter region which was required forhormone-dependent activation of transcription (called ahormone responsive element or HRE, Scheidereit et al.1983; Chandler et al. 1983; Karine/a/. 1984). Selectivityof gene expression is achieved by synthesis of the cognateligand, restricted expression of a receptor in specific cellsand tissues, the presence of an HRE in the promoter of aparticular gene, and the presence of other transcriptionfactors required for that promoter to function at normallevels.

II. Functional domains

The cloning of the human glucocorticoid receptor

134 A. E. Oro, K. Umesono and R. M. Evans

Table 1. Major biologically active small hydrophobic molecules

Hormone Cellular source Principal adult action Known developmental action

Glucocorticoid

Mineralocorticoid*Estrogens'AndrogensProgesteroneThyroxine"c

1,25(OH)2CholecaliferoPRetinoidsc

Ecdysoned

Juvenile hormoned

Adrenal

AdrenalGonads, Adrenal

Thyroid gland

Kidney

Liver, IntestineProthoradc glandOvaryCorpus Allatum

Carbohydrate synthesisStress behaviour

Salt and water balanceSexual reproduction

Behaviour and physiology

Basal metabolic rate

Calcium, phosphate balance

Structural elements of visionOogenesis

Oogenesis

Influence neural crest fate

9

Reproductive organ developmentand neural development

Neural developmentAmphibian metamorphosisBone development

Positional informationInsect molting and metamorphosis

Inhibition of insect maturation

Known biological actions of some hydrophobic, small molecules in different species. Selected references: a: Felig et al. 1981; b: Andersonand Axel, 1986; c: Ham and Veomott, 1980; d: Steel and Davey, 1985.

(hGR) provided the first completed structure of asteroid receptor and permitted the molecular dissectionof the receptor as a transcription factor (Hollenberg etal. 1985). An understanding of the action of steroidsrequires a mechanistic explanation for how a singletranscription factor, when bound to its ligand, acts as asequence-specific, positive and negative regulatory fac-tor and a delineation of the unique domains in thereceptor dedicated to each phenomenon. Throughcotransfection studies using the 'cis-trans vector assay',the hGR can be studied as a model hormone-hormonereceptor system. Activation of transcription can beanalyzed through a c«-vector employing the mammarytumor virus (MTV) promoter (Giguere et al. 1986),while repression of transcription can be studied on the(r-glycoprotein hormone promoter (Oro et al. 1988£>).Effects of mutations can be rapidly assayed for theirtranscriptional effects in the presence or absence ofhormone (Figure 1).

Both activation and repression by hGR share somecommon features. First, both processes demonstrate arequirement for the DNA-binding domain and reflectthe fact that positive and negative regulation are DNAsequence-specific. Deletions in this cysteine-rich, zincfinger region destroy all function. Second, the carboxylterminal deletions show that activation and repressionrequire an intact ligand binding domain and the pres-ence of hormone. Consistent with previous results foractivation (Godowski et al. 1987), removal or replace-ment of this region by heterologous sequences leads tohormone independence for both processes.

In contrast, several experiments provide criteria thatdistinguish positive and negative regulatory effects ofthe hGR. First, the amino terminal domain that con-tains a potent activator sequence (Tau 1) is not necess-ary for frarw-repression. The Tau domain can be placedon heterologous DNA-binding domains such as Gal4 ormoved to other parts of the receptor and still maintainfunction (Hollenberg and Evans, 1988). These factssubstantiate the duality of receptor function and high-

light the observation that deletion of Tau 1 engenders amore potent repressor. Second, heterologous proteinssuch as /2-galactosidase can functionally replace thehGR carboxyl terminus only in repression. Removal ofthe carboxyl terminus results in a receptor variant withgreatly reduced repression and activation activity (Fig-ure 1). The addition of a /3-galactosidase moiety selec-tively increases repression and not activation activity,while addition of a mineralocorticoid receptor carboxylterminus increases both activities (Oro et al. 1988fo).Given the lack of amino acid identity or similar chargedistribution between the hGR, hMR and y3-gal, oneplausible model is that hGR represses through itscarboxyl terminus simply by steric hindrance but re-quires specific sequences for activation activity.

III. A superfamily of proteins

Analysis of the amino acid sequence of the hGRrevealed a segment with striking relatedness to the viraloncogene erbA (Weinberger et al. 1985). Two groupsinitiated the characterization of the erbA proto-onco-gene product which led to its identification as thethyroid hormone receptor (Weinberger et al. 1986; Sapet al. 1986). Although steroid and thyroid hormones arenot structurally or biosynthetically related, the exist-ence of a common structure for their receptors supportsthe proposal that there is a large superfamily of geneswhose products are ligand-responsive transcription fac-tors. Apparently it is the analogous action of thehormones that is reflected in the homologous structureof their receptors. An extension of this proposal pre-dicts that other small, hydrophobic molecules mayinteract with structurally related intracellular receptorsin order to modulate the expression of specific networksof genes. Molecular cloning studies support this pro-posal as the receptors for many of the molecules listedin Table 1 including estrogen, progesterone, aldoster-one, and vitamin D have been isolated (For review seeEvans, 1988).

Steroid hormone receptor homologs in development 135

900

wt

I860*A77-262

515*

Three generalizations can be made from thesestudies. First, the core DNA-binding domain sequenceis highly conserved with nine cysteines forming the twometal-binding finger structures. The sequence conser-vation ranges from 42 to 94 % and reflects the diversityof HREs that the receptors recognize. Second, thehomology in the ligand-binding domain is more gradedand generally parallels the structural relatedness of thehormones themselves. Although the overall homologyof the carboxyl terminal region is less than 15%, eachreceptor still maintains two clusters of amino acididentities between receptors as distinct as the humanreceptors and the Drosophila receptor-like moleculeE75 (Segraves, 1988). The exact functional contri-butions these conserved regions make is unclear, withfunctions conserved between receptors as possible can-didates: hormone binding, dimerization, transactiva-tion or transrepression. Lastly, although the aminoterminus is not conserved, it may contribute to import-ant functional differences between receptors. Deletionsin this region of the glucocorticoid receptor reduceactivity by 10- to 20-fold (Hollenberg et al. 1987;Danielsen et al. 1987), whereas the A and B forms ofthe progesterone receptor, which differ by 128 aminoacids at the amino terminus, have strikingly differentcapacities to regulate gene expression (Toraef al. 1988).

IV. Steroid hormone receptor homologs indevelopment

Although it is widely believed that differential regu-

aiea CATREPRESSION-DEX

*

*

•

*

*

*

*

*

*

*

*

2218

11±2

3i±e

24 ±6

+DEX

100

140110

195±96

160118

•

*

74±11

*

*

*

•

22±8

11±2

3116

2416

MTVACTIVATION

-DEX

* *

* *

* *

* *

* *

* *

* *

* *

* *

• *

• *

10

10

40

1

+ DEX

100

10

86

10

* *

* *

* *

96

* *

* *

* *

* *

10

10

40

1

Fig. 1. Structure-and-function analysisreveals functional domains required foractivation and repression. Deletionmutants previously characterized foractivation, DNA binding and steroidbinding were assayed on the alphapromoter. The wild-type receptor consistsof 1MM, immunogenic region whichcoincides with the Tau 1 region: DNA,DNA-binding domain, Steroid, ligand-binding domain. Scale above refers toamino acid number. Numbers on the leftindicate deleted amino acids whileasterisks next to the number indicate theamino acid linker insertion mutant atwhich the receptor is truncated. % ofwild-type activity was determined byassigning RSV control plasmid as zeroactivity and the wild-type hGR as 100%in each experiment plus or minus thestandard error of the mean. The mutantswere previously assayed for activation in(Hollenberg et al. 1987). * indicatesactivity is less than 10 % of wild-typerepression activity on the alphal68promoter, ** indicates less than 1 % ofactivation activity on the MTV promoter.

lation of gene expression is the critical level at whichdevelopment is controlled, this does not provide aconceptual framework for how specific processes likespatial organization or pattern formation are achieved.Many mechanisms have been proposed to explainpatterning in different organisms, but one long-standingtheory is that certain patterns are formed through theestablishment of a gradient of a diffusible substance ormorphogen (Crick, 1975). One example of a morpho-gen is the Drosophila bicoid protein, which acts in adiffusible concentration gradient to establish anteriorpolarity of the embryo (Driever and Nusslein-Volhard,1988). Another example is the vitamin A-relatedmetabolite, retinoic acid (RA). Work by numerouslaboratories over the last several years has indicatedthat the RA manifests morphogenic properties in ver-tebrates (Maden, 1982). Evidence from work on thedeveloping chick limb bud suggested that RA wasproduced in a gradient with its highest concentrationposteriorly at the zone of polarizing activity. Recently,RA was directly shown to be present in the chick limbbud in a 2-5-fold concentration gradient across the limb(Thaller and Eichele, 1987), supporting its morpho-genic role. Moreover, reversal of the gradient by theaddition of exongenous RA resulted in duplication oflimb structures such as the digits (Tickle et al. 1975).One important question to be answered is how ashallow RA gradient can be transmitted into differentcell fates.

Two retinoic acid receptors have been identified thatare members of the steroid hormone receptor super-family. The identification of the retinoic acid receptor a-

136 A. E. Oro, K. Umesono and R. M. Evans

A.1 420 487 531

Not I Xho I

5

Cortisol

Domain Switch

hGRr

1 87 154 198 431 462

DNA1

Retinoic Acid hRAFLNot I Xho I

Chlmaeric Receptor

420 4B7 531

DNA Cortisol hGRG

B.

AC

- DEX RA

hGRNX

- DEX RA,

hRARN X

- DEX RA,

hGRG '

Fig. 2. The human retinoic acid receptor activates througha thyroid hormone response element. (A) Construction ofthe chimaeric receptor hGRG. The hGRnx and hRARnxare mutated hGR and hRARa, respectively, with commonNotl and Xhol sites in the cDNAs. The amino acidnumbers represent the possible domain boundaries in thereceptor proteins. The ligand binding domains are indicatedby their cognate hormones, DNA-binding domains by'DNA'. The chimaeric receptors were created byexchanging the DNA-binding domains at the Notl/Xholsites. (B) Transactivation of a T3 responsive reporter by thehybrid receptors. Expression plasmids encoding mutantreceptors were cotransfected into CV-1 cells together withthe AMTV-TREp linked to the chloramphenicolacetyltransferase gene reporter (CAT) in the presence orabsence of 100 nm inducer and assayed 36 h later for CATactivity. No effect on CAT activity was observed using theparent vector pRShGRnx. When pRShRARnx wascotransfected, retinoic acid mediated a 10-fold induction.AC and C are the acetylated (AC) and unacetylated (C)forms of [14C]chloramphenicol.

(Giguere et al. 1987; Petkovich et al. 1987) was facili-tated by the modular nature of these proteins. Exchang-ing the DNA-binding domain of the retinoic acidreceptor for the homologous region from the hGR, ahybrid molecule was generated that activates GREresponsive promoters (such as the MTV-LTR) in re-sponse to retinoic acid (See Figure 2A, Giguere et al.1987). RAR B had been discovered by examination ofintegration sites of the hepatitis B virus into hepato-cellular carcinomas (Dejean et al. 1986; deThe et al.1987). Subsequently, using a similar domain-swap ap-

proach, the receptor was shown to bind retinoic acidwith high affinity (Brand et al. 1988; Benbrook et al.1988). The structure of the two receptors are verysimilar (90% in the ligand-binding domain), but the Bform of the receptor transactivates at a slightly lowerconcentration of RA (Brand et al. 1988). Moreover,RNA from the two receptor genes have different tissuedistributions. RNA from the a form is expressed inhematopoietic cell lines while RNA from the B receptorhas a more complex distribution, being highest in thebrain, kidney and prostate (deThe et al. 1989).

By analogy with steroid receptors, a potential modelfor transmission of positional information via themorphogen retinoic acid is through the two retinoicacid receptors. Upon ligand binding, the receptorswould trigger the activation or repression of specificnetworks of genes. Key to this model is that theRAR is a sequence-specific transcriptional activator.Interestingly, the RARawas found to activate at highlevels through a previously isolated HRE for thethyroid hormone receptor (Umesono et al. 1988). Thisresult was predicted based upon the structural related-ness of the DNA-binding domains between the RARand the thyroid hormone receptor. Although the bio-logical significance has not yet been established, the twodistinct receptor systems may indeed regulate an over-lapping set of genes. While an authentic RAR responseelement has not been characterized, another apparenttarget of regulation for the retinoic acid receptors is theB receptor gene itself. From RNA analysis of hepatomacells, the expression of the B receptor appears toincrease in a cycloheximide-independent manner whilethe expression of the a receptor remains constant inresponse to RA (deThe et al. 1989). One interpretationof these data is that the 8 receptor is autoregulated,perhaps to amplify the expression of the set of genes itregulates. How this amplification plays a role in theestablishment of positional information and to whichgenes the receptor transmits the information are ques-tions being actively pursued.

Retinoic acid has a clear role within the developingorganism in the establishment of positional infor-mation. However, other small molecule ligands mightexist that act in distinct developmental paradigms toestablish positional information or determine cell fate,and which might act via a steroid hormone receptor-likemolecule. Moreover, the need for a well-characterizedembryological and genetic system with which to analyzethe function of these molecules pointed to the Dros-ophila melanogaster system for study.

To identify homologs of the vertebrate steroid recep-tors, a Southern blot of Drosophila genomic DNA wasprobed with a cDNA fragment encoding the hRARa'DNA-binding domain (Giguere et al. 1987; Petkovich etal. 1987) Under conditions of reduced hybridizationstringency, six distinct EcoRI bands ranging in size from2 kb to greater than 12 kb were detected. Screening of aDrosophila genomic library using the same probe andhybridization conditions resulted in the isolation ofthree distinct single copy gene loci (Oro et al. 1988a).

One class of inserts mapped on the third chromosome

Steroid hormone receptor homologs in development 137

••••v

Fig. 3. Spatial localization of knrl transcripts in early embryos. Visualization of knrl transcripts by in situ hybridization tosections of wild-type Drosophila melanogaster embryos reveals both maternal and zygotic expression. Embryos are orientedwith anterior to the left and dorsal at the top. Staging followed Campos-Ortega and Hartenstein, 1985 (A (brightfield) andB (darkfield)). Cleavage-stage embryo showing the spatially uniform distribution of apparent maternal knrl transcripts.(C and D) Embryo at syncytial blastoderm showing apparent early zygotic knrl expression in an anteroventral domainextending from 80-100% of egg length along the ventral side of the embryo. Also apparent is the first appearance of themost posterior of the three cellular blastoderm expression domains (see below). (E and F) Cellular blastoderm pattern ofknrl expression. Slightly oblique section showing intense anteroventral transcript accumulation (domain I), as well as the twomore posterior transcript stripes at approximately 70 % (domain II) and 25 % (domain III) of egg length ventrally.Transcript accumulation in domain II is always observed to be at a significantly higher level ventrally than dorsally.

at cytologic position 77E, the same location as thepreviously identified gap segmentation gene kni (Nuss-lein-Volhard and Wieschaus, 1980). kni mutants hadbeen previously isolated in a genetic screen for zygoticmutants affecting embryonic pattern formation and fallsinto a small set of genes required for abdominalsegmentation (Nusslein-Volhard et al. 1987). Mutatio-nal analysis indicates that kni+ activity apparentlyinteracts with maternally derived information (Leh-mann and Nusslein-Volhard, 1986).

The genomic and corresponding cDNA clones for theRAR homolog were sequenced and predicted aminoacid sequence was found to have a striking similarity tothe predicted amino acid sequence of the kni product(Nauber et al. 1988) and thus called knirps-related {knrl)(Oro et al. 1988a). Southern blots of kni mutant andwild-type genomic DNA revealed mutant DNAs XT1and XT106 removed both kni and knrl loci localizingboth genes to cytological positions 77E3-5. Further, knimutant FC'3 contained a two kb deletion in the knitranscription unit while leaving knrl apparently intact(Oro, unpublished). No phenotypic differences in theabdominal region are seen between the kni mutants (R.Lehmann, personal communication), indicating that

the kni function is epistatic to the putative knrl func-tion. Loss-of-function alleles for knrl are requiredbefore the developmental role of the knrl product canbe addressed.

The knrl gene is expressed early in development.Northern blot of stage-specific RNA showed a singleRNA species of approximately 3-8 kb expressed at lowlevels between 0 and 3 h after egg-laying (AEL) and atsignificantly higher levels in later embryos, larvae andadults. The spatial location of knrl transcripts wasassayed by in situ hybridization on sections of 0-2 and2-4 hour embryos (Figure 3). After egg deposition anduntil approximately the 8th nuclear division, a weak,spatially uniform distribution of apparently maternaltranscript was detected (Figures 3A and 3B). The firstapparently zygotic expression is detected at nucleardivision 12, when the knrl transcript is localized to asmall anteroventral region of the embryo (Figures 3Cand 3D), at approximately 80-100% of egg length (EL)on the ventral side (domain I). Expression in thisdomain intensifies through the cellular blastodermstage, and two additional circumferential bands oftranscript become detectable, centered at approxi-mately 70 % EL ventrally (domain II) and 25 % EL

138 A. E. Oro, K. Umesono and R. M. Evans

A Amino Acid Alignment ol DNA-bincSng Domains

kari 10 HDTcncEiPwermM'TcreacvFasrm.ss isKw«ci i incnm/wiAcnaci j iYe«s^rumiiMrKiHCUQE<W) ic*Ulrps 1 MQTCnCSfPJW£FIFWFTCEKtVFCtSnilIST ISECOCWClIIWCainCKACRUnninCHSXEeSlirtSRSWFKIHCLLOtHtQ 97hTU 9S ....CVWS0«ie™TWlICEeCl(tfFR«TII^LHRTSCICTt6<CYIDOTIB«lCqtCJIfia£ITVSM 195Mtua 94 crvctXKSScrHTtvsACEGaafnasiQiotff ricMtoocnainrntnicQrciiiaKCFEvet \nh U 417 ....CLVCSOEASSCItYtVlTCtSCICVFFDUVEGQHI nCAGmOCIIIXIHUJOAairMCLOWX 511

B.

ATG11

14

1

fin

I I

1 i

TGA\

648

J knrl

429

kni

1 102 1 8 9 1 9 5 456

1 88 1 5 3 1 7 8 463

14714 hRARa

421 486511

hGRDNA Ugand-ttndlng

Fig. 4. Comparison of the predicted knrl product to vertebrate steroid/thyroid hormone receptors. (A) Alignment of theDNA-binding domains of representative members of the superfamily, showing the conserved amino acids and the extensivestructural similarity between knrl and kni. Note that the identity of knrl and kni extends past the conserved Gly and Metresidues of the DNA-binding domain. (B) Overall structural comparison of the predicted protein sequence of knrl to othermembers of the steroid/thyroid hormone receptor superfamily. Comparisons of the region marked DNA are to the 66-68amino acid DNA-binding domains and the region marked Ligand Binding is compared with the amino acids starting at apoint 25 amino acids past the conserved Gly and Met residues of the DNA binding domain. The intervening 25 amino acidsare highly related between knrl and kni, but not significantly related to the other vertebrate receptors (indicated by anasterisk). As there is no significant similarity of knrl to the other receptors in the carboxyterminal region, no specificalignment of these regions is shown. The programs of Devereux et al. 1984 were used for comparison. Numbers indicateamino acids as detailed in Oro et al. 1988; Nauber et al. 1988 Weinberger et al. 1986; Giguere et al. 1987 and Hollenberg etal. 1985 for knrl. km, hTR£, hRAR, and hGR, respectively.

ventrally (domain III) (Figures 3E and 3F). It is note-worthy that expression in domain II appears signifi-cantly more intense ventrally than dorsally.

A comparison of the predicted knrl protein withother members of the steroid/thyroid receptor super-family is shown in Figure 4. First, sequence alignmentdemonstrates greatest similarity with the other recep-tors in the 67 amino acids of the putative knrl DNA-binding domain (Figure 4A). Between amino acids 14and 80 of knrl there is 85 % amino acid identity with thekni product, 49% with the human thyroid receptor,47 % with the human retinoic acid receptor and 43 %with the human glucocorticoid receptor. Interestingly,the knrl and kni DNA-binding domains both contain aglycine in the region linking the two zinc fingers(residues 39 and 30 in knrl and kni, respectively), at aposition which in all other receptors is either an arginineor lysine. This further suggests a common origin forthese two genes. Second, amino acid sequence analysis

reveals a highly conserved stretch of 30 amino acidsimmediately following the DNA-binding domain. Be-tween other steroid receptors little or no homologyexists (Figure 4), while this region in knrl or kni is moreconserved than the DNA-binding domain. Other tran-scription factors also contain regions of high conser-vation outside of the DNA-binding domain, such as thePOU domain in homeoproteins (Herr et al. 1988).Perhaps this region plays a novel role in receptorfunction.

Further, the homology of the predicted knrl geneproduct to vertebrate steroid receptors suggests that itsfunction is ligand-dependent. If this is the case, such aligand might constitute a previously unrecognizedsmall-molecule morphogen, and some of the genesinvolved in regulating knrl function might affect thesynthesis of the ligand or storage of a ligand precursor,rather than regulating knrl expression. However, theunrelatedness of the knrl carboxy terminus to that of

Steroid hormone receptor homologs in development 139

the other receptors makes it difficult to predict apotential ligand. As mentioned above, even distantreceptors have particular structural similarities in thecarboxyl terminus. Perhaps the knrl product is a consti-tutive transcriptional regulator, and functions entirelywithout a ligand. Finger swap experiments similar tothose used to identify the retinoic acid receptor mayilluminate these differences.

Conclusion

Development of an organism is a complex biochemicalprocess. Part of the understanding of the mechanismshas come about from the study of classes of transcrip-tion factors such as the steroid/thyroid hormone recep-tors whose ligands share common biophysical proper-ties and a common mechanism of action inenvironmentally modulating gene expression and trig-gering specific networks of genes. The identification ofthe retinoic acid receptor has allowed the proposal thatthe morphogenic properties this ligand exerts are me-diated through a hormone-hormone receptor complexwhich regulates a network of genes. Elucidation ofproteins that interact with the receptors and their ligandas well as the receptor target genes, their spatial patternof expression and function will allow new insight intofurther mechanisms of vertebrate development. Analy-sis of related receptor systems will identify new para-digms of receptor action. Characterization of one classof Drosophila retinoic acid receptor homologs ident-ified a locus highly related in amino acid sequence to thegap segmentation gene kni with a spatially restrictedexpression pattern. These two receptors are the mosthighly diverged members of the gene superfamily;functional analysis may reveal a new class of transcrip-tional activators. Finally, the characterization of Dros-ophila steroid receptor homologs may reveal receptorsystems common to both invertebrates and vertebrates.Although the gross structural features of developingembryos are distinct, common mechanisms using com-mon or related molecules may be uncovered.

The authors would like to acknowledge Mike McKeown,Jon Margolis, Jim Posakony and Charles Zuker for help withthe Drosophila work and Chris Glass, Vincent Giguere andM. G. Rosenfeld for work on retinoic acid receptor acti-vation. R.M.E. is an investigator for the Howard HughesMedical Institute and also acknowledges NIH support.A.E.O. is supported by the Medical Scientist Training Pro-gram, General Medical Grant PSH GM07198.

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