Transcriptional control underlying head morphogenesis during ...

J. Biosci., Vol. 21, Number 3, May 1996, pp 341-352. © Printed in India.

Transcriptional control underlying head morphogenesis during vertebrate embryonic development RUTH Τ YU* and KAZUHIKO UMESONO Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8915-6 Takayama, Ikoma, Nara 630-01, Japan MS received 5 October 1995 Abstract. Orphan nuclear receptors are divergent members of the steroid/thyroid/retinoic acid receptor superfamily for which ligands have yet to be identified. Unlike the classice hormone/ vitamin receptors which are found only in vertebrates, some orphan receptors are conserved between vertebrates and invertebrates, indicating a possible link in regulatory gene networks underlying key biological events like those in embryonic development. In this review we examine such an example focusing on our analysis of a novel class of orphan receptors, vertebrate TIx and fruit fly tailless, both of which are structurally related and expressed in a homologous pattern during development. Our studies show that in vitro Tlx and Tll recognize and bind similar DNA targets, and consequently in vivo they can control the same transcriptional cascades, indicating that their biochemical properties are well conserved. Based upon these observations, we will discuss possible mechanisms responsible for the creations of divergence and uniqueness in vertebrate and invertebrate morphogenetic processes. Keywords. Nuclear receptor; transcription factor; head morphogenesis.

1 . Introduction Steroids, thyroid hormones, and vitamins belong to a class of small lipophilic molecules which unlike peptide hormones and growth factors do not bind membrane receptors, but traverse cell membranes to bind intracellular receptor proteins. Transmission of hormonal signals is directly achieved by these receptors which can function as liganddependent transcription factors to control target gene expression. Structural comparison of these receptors has revealed a common modular structure which is now known to define a nuclear receptor superfamily. Members of this family are characterized by a distinct DNA binding domain (DBD) near the N-terminal and a hydrophobic ligand binding domain (LBD) at the carboxy terminal. This structure serves as the basis of their ability to act as receptor proteins for hormones and active forms of vitamins while simultaneously binding to specific DNA sites in the chromatin to function as transcription factors (for review, see Evans 1988; Parker 1993; Tsai and O'Malley 1994). After the initial cDNA cloning of several steroid hormone receptors, recognition of the high structural conservation existing among them greatly facilitated further isolation of new receptors by means of cDNA cross hybridization. As a result, receptors for mineralocorticoids, thyroid hormones, and retinoids were identified (Arriza et al 1987; Sap et al 1986; Weinberger et al 1986; Giguere et al 1987; Petkovitch et al 1987). In addition, a significant number of genes encoding nuclear receptor-like proteins have been isolated, for which ligands have yet to be found. These so-called "orphan * Corresponding author (Fax, + 81-7437-2-5559; Email, [email protected] jp).

341

342

Ruth Τ Yu and Kazuhiko Umesono

receptors" suggest the presence of yet unknown regulatory gene networks, that in response to new hormones or vitamins, may operate through given receptor proteins to control gene transcription. Interestingly, in addition to vertebrates, orphan receptors are also widelyfound in arthropods (insects), echinoderms (seaurchin), and nematodes (C. elegans), but appear to be absent from yeast and plants (Amero et al 1992; Laudet et al 1992; Kostrouch et al 1995). This suggests that the emergence of the nuclear receptors may have coincided with the evolution of metazoan multicellular animals, and be an essential element which is required for intercellular signalling to control the patternofgene transcription. In this review, we will discuss our recent results on a novel vertebrate orphan receptor, Tlx, which is structurally and functionally related to the product of a fruit fly Drosophila melanogaster terminal/gap gene tailless (tll). Ourgoal is to better undestand how regulatory gene networks involved in morphogenetic processes may have been conserved or diverged between vertebrates and invertebrates through analyses of these orphan nuclear receptors. 2. Identification of a Tlx/Tll subclass of orphan receptors Retinoids have been shown to exert profound effects during embryonic morphogenesis. It is now known that at least two distinct classes of nuclear retinoid receptors exist, the all-trans retinoic acid receptors (RARs) and the retinoidXreceptors (RXRs) which bind 9-cis retinoic acid (Heyman et al 1992; Levin et al 1992). Three isoforms α, β, and γ have been identified for both classes. By virtue of its structure, the RXRs are more closely related to other orphan receptors than to the RARs, but identification of its ligand has set the RXRs apart from the rest of the "orphans". In addition to regulation through its ownhormonal pathway, the RXRs have been shown to serve as heterodimeric partners for the RARs, as well as for several other nuclear receptors such as the thyroid hormone and vitamin D3 receptors (Yu et al 1991; Bugge et al 1992; Kliewer et al 1992; Leid et al 1992; Marks et al 1992; Zhang et al 1992). Thus RXR occupies a unique niche among the members ofthe nuclear receptor family due to its multi-potential role in the control of different hormonal signal transduction pathways. These unique properties of the RXRs prompted us to further elucidate the role of RXR-like genes during embryonic development. As an initial step, we attempted to identify new related members from a chick embryonic cDNA library. Using the mouse RXR ß as a probe, we obtained numerous chick cDNA clones, among which one class was found to encode a novel nuclear receptor, which we referred to as Tlx (Yu et al 1994). Sequence comparison revealed that among vertebrate receptors, the degree of similarity was highest with COUP (Miyajima et al 1988; Ritchie et al 1990) and RXR (Leid et al 1992; Mangelsdorf et al 1992). But among all known receptors, the highest similarity could be seen between this new receptor. Tlx, and the product of D. melanogaster orphan receptor gene tll (Pignoni et al 1990) (figure 1). Comparison of the amino acid sequences deduced from the respective cDNA revealed that the DBDs of Tlx and Tll share 81 % identity, which suggested that these two receptors may regulate a similar set of target genes. It is known from structurefunction analyses of the nuclear receptors that discrete subregions of the DBDs such as the P-box, D-box, and T/A-box play crucial roles in determining the target gene specificity (Umesono and Evans 1989; Wilson et al 1992). The hexameric core DNA

Vertebrate embryonic development

343

Figure 1. Schematic comparison of the vertebrate Tlx receptors with other members of the nuclear receptor family. The amino acid sequences of the chick Tlx are compared for per cent identity against the mouse Tlx and other known nuclear receptors. The Drosophila Tll was found to share the highest identity in the DNA binding domain (DNA) with these new nuclear receptors, although the similarity in the ligand binding domain (LIGAND) was equivalent to that seen with a vertebrate orphan receptor, EAR3/COUP. The numbers in the boxes indicate the per cent amino acid identity within the enclosed region with the chick Tlx protein.

sites, or half-sites, are specified by the P-box region in the first zinc finger. The D-box which is located in the 2nd zinc finger is involved in modulating the spacing between the half-sites. Additional specificity is conferred by a T/A box which is located downstream of the zinc-finger region. Interestingly, in the Tlx/Tll DBDs these subregions are particularly well conserved; in their P-boxes a lysine which is absolutely conserved in all other members is replaced by either a serine (in Tlx) or an alanine (in Tll). The D-boxes encode seven amino acids instead of the usual five, and the T/A boxes also share a high degree of sequence identity (Yu et al 1994) (figure 2). On the other hand, the LBDs are more divergent with about 41 % similarity which is comparable to that seen with COUP and RXR. It is worthy to note that Tll from a different fruit fly, D. virilis, does share overall homology with that of D. melanogaster ( Liaw et al 1993). The apparent sequence divergence observed in the Tlx/Tll LBDs raised the question of whether the chick Tlx LBD is conserved in other vertebrate Til-like receptors. To resolve this point, we went ahead to isolate mouse Tlx cDNA clones (Yu 1994). Under low stringency conditions, an 11·5-day mouse embryonic cDNA library was screened with the chick Tlx as a probe. All resulting positives were found to encode one of two types of cDNAs, mouse Tlx or RXR ß, indicating that under the stringency employed, these two cDNAs are the most similar to the chick Tlx; unlike the RXRs, no isoforms (such as α or β) of the Tlx gene have been identified. Sequence comparison revealed that the conservation between chick and mouse Tlx proteins is extremely high, 100%

344


Figure 2. Comparison of vertebrate and fly Tlx/Tll DNA binding domains. The DBDs of the chick/mouse Tlx are compared with that of Drosophila Tll. Three functionally important subregions implicated in modulating target gene specificity, the Ρ box, D box, and T/A box are shaded and labelled. The diamonds indicate amino acids in the fly protein which are identical to those in the vertebrate proteins.

identical in the DBDs and only 1 amino acid substitution out of the 200 amino acids in the LBDs. All the other amino acid substitutions (11 positions) are clustered in the linker region connecting the DBD and LBD (figure 1). Based upon these results, we propose that the vertebrate Tlx and invertebrate Tll comprise, with these distinct structural features, a unique subclass among the nuclear receptor superfamily. 3. Restricted expression of Tlx in developing embryos The expression of tll in fly embryos appears to be biphasic. As a terminal/gap gene, tll is initially expressed at both embryonic termini during the syncytial stage, but after cellularization becomes restricted to procephalic neuroblasts in the anterior (future brain region) (Pignoni et al 1990). The initial expression is under control of the terminal system which can be triggered by localized activation of the torso transmembrane receptor tyrosine kinase (Lu et al 1993) (figure 3). The torso signal is subsequently transmitted to the ras-raf- MAP kinase intracellular signalling cascade which culminates in the activation of a hypothetical transcription factor gene Y. It is assumed that this transcription factor Υ induces tll and another terminal gene huckbein (hkb) to establish nonmetameric domains at the anterior and posterior ends of the embryo. The


345

Figure 3. Model of a signaling pathway specifying terminal cell fates during fly embryogenesis. The figure depicts a model of the intracellular signaling pathway which can be seen in two separable steps, a cytoplasmic kinase cascade and a nuclear transcription cascade. The former initiates with the activation of torso, a transmembrane receptor tyrosine kinase, by a presumed ligand, and proceeds through Ras 1( GTPase), D-raf (serine/threonine kinase), and Dsor 1 (MAPKK) to reach MAPK (mitogen activated protein kinase). In the latter steps, upon phosphorylation a hypothetical transcription factor "Y" induces expression of TLL and HKB (huckbein) which in turn regulate the expression of downstream target genes whose products are also transcription factors, to control the body plan,

activated Tll and hkb proteins then participate in modulating the expression of downstream gap genes; they repress (Krüppel and knirps, but activate hunchback (Lu et al 1993). After cellularization, tll expression disappears from the posterior region and becomes anterior-specific. In this latter phase, the expression is localized to neuroblasts in the acron, or developing brain (although weak expression can be transiently detected in peripheral neuroblasts) and later can be seen in the optic lobe (Pignoni et al 1990). Some of the defective phenotypes seen in tll mutants are lack of brain structure in the anterior, as well as loss of hindgut structures in the posterior. The importance of tll localization during fly embryogenesis with respect to the establishment of the embryonic termini and development of the central nervous system suggests that the localization of Tlx expression in vertebrate embryos may also be crucial. In flies, the terminal pattern is found during the early syncytial stages, and the localization of the head region coincides with the late cellular blastoderm stages. If one were to propose that Tlx may participate in homologous genetic programmes during vertebrate development, it is more likely to predict that Tlx expression would follow the latter tll pattern, namely, localization to the anterior head region, since in vertebrates the syncytial stage does not exist. We examined the spatial pattern of Tlx expression in chick embryos by means of hole mount in situ hybridization (Yu et al 1994). Signals were first detectable at the

346


late head fold stages (stage 8) in the rostral end of the neural tube. By stage 11, the anterior region of the neural tube is expanding to form the three primary brain vesicles, forebrain, midbrain, and hindbrain. At this stage Tlx expression is localized in the primary optic vesicles which will form the future eyes and forebrain. At stage 16, Tlx expression is seen throughout the forebrain with a sharp boundary at the forebrainmidbrain border. The level of Tlx expression appears graded and is absent from the midline. At later stages in chick development, sectioned in situs of embryos revealed that Tlx expression is restricted exclusively to the neuroepithelium of the forebrain and the dorsal part of the midbrain. There is strong expression in the optic stalk as well as the neuroretina. The midline and associated structures such as the pineal gland are completely lacking in Tlx expression. Tlx expression is also absent in the zona limitans intrathalamica (ZLI), which will form part of the future thalamus. Sections through the brains of day 8 chick embryos reveal that Tlx signals are localized to the ventricular zones of the neuroepithelium, which are also known as the proliferating zones since all cell division for neuronal precursors occurs here (figure 4). This observation seems to correlate Tlx expression with the pre-differentiated state of neuronal cells in the forebrain. At these later stages, weaker expression can also be seen in dorsal midbrain.

Figure 4. Expression of Tlx in E.13·5 mouse brain section. This figure presents the brightfield (left) and darkfield photographs of a parasaggital section through an embryonic day 13·5 mouse brain. The section has been processed for in situ hybridization staining with a 35-S labeled Tlx antisense probe. Note that mTlx expression is predominantly restricted to the ventricular and subventricular regions of the neuroepithelial layer. NCX, neocortex; ACX, archicortex; Th, thalamus; PT, posterior thalamus; MGE and LGE, medial and lateral ganglionic eminences; OB, olfactory bulb; OS, optic stalk; OE, olfactory epithelium; MB, midbrain; tong, tongue.

Vertebrate embryonic developmen

347

To extend our knowledge of Tlx expression to mammalian embryonic development, similar in situ hybridization experiments were carried out with mouse embryos. The expression pattern of mouse Tlx is strikingly similar to that seen in the chick. Strong signals could be detected in the head ectoderm of day 7·5 embryos. Inspection of day 13·5 embryonic head sections revealed that Tlx is expressed strongly in the ventricular side of archicortex, neocortex, olfactory bulb, and ganglionic eminences (figure 4c), all of which are derived from telencephalon (Yu et al 1994; Monaghan et al 1995). In diencephalic region, cells in ventral and dorsal thalamus express Tlx. As has been observed in chick embryos, weak expression was detected in dorsal midbrain. Interestingly, Tlx-positive cells can be found also in olfactory epithelium (figure 3c) (Monaghan et al 1995). These results demonstrate that during vertebrate embryonic development, the pattern of Tlx expression is remarkably conserved between species and furthermore appears also reminiscent of the second phase of tll expression in the developing fly brain.

4.

Tlx/Tll as DNA-binding proteins

To examine if the evolutionary conservation between Tlx and Tll extends beyond structure and pattern of expression, we addressed the issue of whether they might share functional similarity. Members of the nuclear receptor family are transcription factors and as such, their purported function is to regulate the expression of specific target genes. One major question in nuclear receptor-mediated signal transduction is how receptors recognize their DNA binding sites. Mutational and physicochemical analyses have defined the mode of receptor DBD interaction with DNA, up to the level of atomic resolution (Danielson et al 1989; Mader et al 1989; Umesono and Evans 1989; Luisi et al 1991; Schwabe et al 1993; Rastinejad et al 1995). These studies demonstrated the importance of the amino acids in the P-box in making specific contact with the nucleotide bases. In general two types of P-boxes have been identified, type I is found in the steroid receptors (glucocorticoid, mineralocorticoid, progesterone, and androgen) and type II in estrogen, nonsteroidal, and orphan receptors (figure 5b). Three amino acid substitutions exist between the type I (GSckV) and type II (EGckG) P-boxes, resulting in a corresponding change in recognition of the target DNA site from AGAACA to AGGTCA. Furthermore, crystallographic data has demonstrated that the conserved lysine residue (underlined) makes direct contact with the second guanine in the target DNA sites. As mentioned above, the DBDs of the vertebrate Tlx and fly Tll are similar in that they differ from all other nuclear receptors in the P-box such that the conserved lysine is substituted by either serine (DGcsG) or alanine (DGcaG). Otherwise they appear closely related to the type II P-box. What does this mean in terms of target gene specificity? From previous studies in D. melanogaster, it has been shown that tll is able toregulate downstream genes such as Krüppel and knirps, and several Tll binding sites have been identified in the regulatory regions of these two genes (Pankratz et al 1992; Hoch et al 1992). These reported sites can all be found to contain a common hexameric sequence motif of AAGTCA (Liaw et al 1993) (figure 5a). This motif is quite similar to the type II DNA site; the only difference is a change from G to A at the second position. This realization prompted the speculation that it was due to the absence of the lysine in the Tll P-box, that it could not specify "G" as the scond nucleotide in its target half-site. The same could be interpolated for Tlx, which also lacks the conserved lysine.

348


Figure 5. Target gene specificity of Tll. (A) In flies, Tll has been shown to directly bind to the regulatory regions of downstream target genes such as Krüppel and knirps, resulting in transcriptional repression. In these Tll binding sites, a hexameric sequence motif of AAGTCA can be commonly found. (B) Relationship between the DBD Ρ box sequences and target half-site sequences. Three amino acid substitutions found between type I and type II Ρ boxes have been shown to be responsible to direct the respective half-site specificity shown to the right. However, a conserved lysine in position five is missing in the Tll/Tlx proteins, which is likely to be responsible for the resulting change in target site recognition by Tll. The reported Tll binding site differs from that of both type I and type II binding sites by a change from G to A at position 2 in the half site.

This hypothesis was directly tested by means of gel-mobility shift DNA-binding assays with full-length in vitro translated Tll and Tlx proteins (Yu et al 1994). Using oligonucleotides encoding a Tll binding site identified in the Krüppel gene enhancer region (Kr site) (Hoch et al 1992), we detected specific binding of both Tll and Tlx proteins, where RAR/RXR heterodimers or COUP failed to show any detectable binding. In order to further evaluate the sequence specificity and the importance of the second "G" in the hexameric core DNA site, we prepared oligonucleotide probes encoding a palindrome of either AGGTCA (TREp) or AAGTCA (Tllp). TREp has been shown to be a promiscuous binding site for many nuclear receptors including RAR/RXR heterodimers and COUP homodimers. The results clearly demonstrated


349

that while RAR/RXR heterodimers and COUP homodimers can bind to TREp, only Tll and Tlx are able to bind to the palindromic AAGTCA site. Judging from the mobilities on gel-shift assays, it appears that both Tll and Tlx proteins can bind DNA efficiently as a monomer, which is supported by the fact that although the Kr site encodes only one AAGTCA sequence, yet it is able to generate Tll-or Tlx-DNA complexes which comigrate with those seen with the Tllp probe. Mutational analyses of the Kr site revealed that the hexamer is the only core target site required and that the second A of the hexamer cannot be substituted by other nucleotides to retain high affinity binding of Tlx (unpublished observations). These observations indicate that as DNA binding proteins, Tll and Tlx can recognize the same target sequences, and further confirm the unique features that are displayed by this subclass of nuclear receptors. 5. Tlx/TIl function in vivo

The resemblance of Tlx and fly tll in structure, pattern of gene expression and DNA binding specificity all support the prediction that they may regulate homologous genetic cascades during embryonic development. However, their structural similarity exists primarily in their DBDs; this degree of similarity is much lower in the LBDs where it is comparable to that seen with another orphan receptor, COUP (figure 1). The mode of in vitro DNA binding allows Tlx to be distinguished from Tll; Tlx forms homodimers far more readily than Tll (Yu et al 1994). We believe that this property likely resides in the COUP-like LBD, which has been shown to promote stable homodimerization of COUP even in absence of DNA. This difference in the in vitro properties of Tlx and Tll LBDs raises the question of whether Tlx and Tll share a common ligand or if any ligand is required for their transcriptional function. These points were tested by means of ectopic expression of Tlx in flies. It has been shown that ectopic expression of tll in fly embryos results in a repression of segmentation which is depicted by denticle fusion and complete absence of some segments (Steingrimmson et al 1991). This is accompanied by alterations in the expression domains of downstream target genes such as hunchback, Krüppel, knirps, and engrailed. Using a hsp 70 promoter, the effect of ectopic expression of Tlx in fly embryos was examined (Yu 1994). In the wild-type Canton-S background, embryos which underwent the same treatment as those carrying the heatshock constructs exhibit eight normal abdominal segments. The transgenic hs-tll and hs-Tlx embryos gave rise to similar phenotypes, which ranged from partial repression of individual segments, to more severe phenotypes such as fusion and complete repression of several segments. We examined whether this phenotypic change in hs-Tlx was accompanied by similar changes in the pattern of expression of Tll target genes by checking the expression patterns of knirps and engrailed by in situ hybridization and found that ectopic expression of Tlx represses knirps and engrailed expression in a manner similar to Tll (Yu et al 1994). These results demonstrate that Tlx can replace Tll function to control target gene transcription resulting in specific alteration of the body pattern in flies, indicating that Tlx contains sufficient information and structures necessary for Til function. It is still not clear whether a common ligand exists for both receptors or whether they are ligand

350


independent, but the results of this ectopic expression indicate that at least in flies, either Tlx does not require a ligand for its transcriptional control or that the ligand is present ubiquitously in the fly embryo.

6. Conclusions We have discussed results here which illustrate an example of remarkable conservation in the genetic programs which direct morphogenesis in vertebrates and invertebrates. Albeit the enormous gap in the structural differences between the brains of insects, birds, and mammals, parallels can be drawn between the pattern of Tlx and Tll expression. Both are present in what appear to be precursor cells of CNS neurons and lack of Tll in flies has been shown to result in mutants which are defective in brain development. But as mentioned previously, the fly Tll has a biphasic mode of action, only the latter of which seems conserved in vertebrates. Our in vitro studies have shown that as transcriptional factors, Tlx and Tll are able to recognize and bind to similar DNA sequences; that their biochemical properties are equivalent is not surprising based upon the degree of similarity in their amino acid sequences. Taking this one step further, the ability of ectopically expressed chick Tlx to alter the body segmentation in fly embryos, demonstrates that these fundamental biochemical properties are sufficient for Tlx to mimic the gap gene action of Tll. In this respect, another example is the relationship between D. melanogaster eyeless and the vertebrate Pax-6, both of which can generate ectopic eyes upon Overexpression in fly imaginal discs (Haider et al 1995). These observations bring up the quenstion of what makes Tlx different from Tll. Both are expressed in the neural precursors in brain and seem to be important in CNS development. However, the resulting brain structure is quite different, and furthermore, the area where Tlx is strongly expressed in vertebrates, the cortex, shows extremely high diversity among different animal species. To what extent does Tls contribute to generate such diversity? One of the answers to this quenstion will soon be answered with the generation of mice lacking Tlx function of means of gene knockout experiments. Another aspect is the mode of Tlx expression in comparison with that of Tll. As mentioned above, Tll protein is employed twice during the developmental strategy in flies; first as a terminal/gap gene in the syncytial stage, and later as a neuroblast- specific transcription factor. This biphasic Tll expression appears to be controlled through distinct regulatory elements in the promoter region. It has been shown that a terminal enhancer exists in the proximal portion of the promoter, which seems separable from the neuroblast enhancer (Liaw et al 1993). One can imagine that Tll and Tlx may utilize similar mechanisms to direct specific expression in neural precursor cells, but that the terminal enhancer will not be required for Tlx function in vertebrate embryos since the developmental tactics employed during early pattern formation are so divergent between invertebrates and vertebrates. We are currently analysing elements which may be responsible for directing the head-specific expression of Tlx in forebrain neuroblasts. Detailed analysis of these elements will offer more insight in better understanding the extent of similarity and divergence in the control of genetic programs upstream and downstream of the Tlx/Tll class of nuclear receptors.


351

References Amero S A, Kretsinger R H, Moncrief Ν D, Yamamoto Κ R and Pearson W R 1992 The origin of nuclear receptor proteins: a single precursor distinct from other transcription factors; Mol. Endocrinol. 6 3-7 Arriza J L, Weinberger C, Cerelli G, Glaser Τ Μ, Handelin Β L, Housman D Ε and Evans R Μ 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor; Science 237 268-275 Bugge Τ Η, Pohl J, Lonnoy Ο and Stunnenberg Η G 1992 RXR alpha, a promiscuous partner ofretinoic acid and thyroid hormone receptors; EM BO J. 11 1409-1418 Danielson M, Hinck L and Ringold G Μ 1989 Two amino acids within the knuckle of the first zinc finger specify response dement activation by the glucocorticoid receptor; Cell 57 1131-1138 Evans R Μ 1988 The steroid and thyroid hormone receptor superfamily; Science 240 889-895 Giguere V, Ong Ε S, Segui Ρ and Evans R Μ 1987 Identification of a receptor for the morphogen retinoic acid; Nature (London) 330 624-629 Haider G, Callaerts Ρ and Gehring W 1995 Induction of ectopiceyes by targeted expression of the eyless gene in Drosophila; Science 267 1788-1792 Heyman R A, Mangelsdorf D J, Dyck J A, Stein R B, Eichele G, Evans R Μ and Thaller C 1992 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor; Cell 68 397-406 Hoch Μ, Gerwin Ν, Taubert Η and Jackle Η 1992 Competition for overlapping sites in the regulatory region of the Drosophila gene Krüppel; Science 256 94-97 Kliewer S A, Umesono K, Mangelsdorf D J and Evans R Μ 1992 Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling; Nature (London) 355 446-449 Kostrouch Z, Kostrouchova Μ and Rall J Ε 1995 Steroid/thyroid hormone receptor genes in Caenorhabditis elegans; Proc. Natl. Acad. Sci. USA 92 156-159 Laudet V, Hanni D, Coll J, Catzeflis F and Stehelin D 1992 Evolution of the nuclear receptor gene superfamily; EM BO J. 11 1003-1013 Liaw G J, Steingrimsson E, Pignoni F, Courey A J and Lengyel J A 1993 Characterization of downstream elements in a Raf-1 pathway; Proc. Natl. Acad. Sci. USA 90 858-862 Leid Μ, Kästner Ρ, Lyons R, Nakshatri Η, Saunders Μ, Zacharewski Τ, Chen J Υ, Staub A, Garnier J Μ and Mader S 1992 Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently; Cell 68 377-395 Levin A A, Struzenbecker L J, Kazmer S, Bosakowski T, Huselton C, Allenby G, Speck J, Kratzeisen C, Rosenberger Μ and Lovey A 1992 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha; Nature (London) 355 359-361 Lu X, Perkins L A and Perrimon Ν 1993 The torso pathway in Drosophila: a model system to study receptor tyrosine kinase signal transduction; Development (Suppl.) 47-56 Luisi Β F, Xu W X, Otwinowskik Z, Freedman L P, Yamamoto Κ R and Sigler Ρ Β 1991 Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA; Nature (London) 352 497-505 Mader S, Kumar V, de Vereneuil Η and Chambon Ρ 1989 Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive receptor; Nature (London) 338 271-274 Mangelsdorf D J, Borgmeyer U, Heyman R A, Zhou J Y, Ong Ε S, Oro A E, Kakizuka A and Evans R Μ 1992 Characterization of three RXR genes that mediate the action of 9-cis retinoic acid; Genes Dev. 6 329-344 Marks Μ S, Hallenbeck Ρ L, Nagata T, Segars J H, Appella E, Nikodem V Μ and Ozato Κ 1992 H-2RIIBP (RXR-beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes; EMBO J. 11 1419-1435 Miyajima N, Kadowaki Y, Fukushige S, Shimizu S, Semba K, Yamanashi Y, Matsubara K, Toyoshima Κ and Yamamoto Τ 1988 Identification of two novel members of erb A superfamily by molecular cloning; the gene products of the two are highly related to each other; Nucleic Acids Res. 16 11057-11074 Monaghan A P, Grau E, Bock D and Schütz G 1995 The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain; Devlopment 121 839-853 Pankratz Μ J, Busch Μ, Hoch Μ, Seifert Ε and Jackie Η 1992 Spatial control of the gap gene knirps in the Drosophila embryo by posterior morphogen system; Science 255 986-989 Parker Μ G (ed.) 1993 Steroid hormone action (New York: IRL Press, Oxford University Press) Petkovitch M, Brand Ν J, Krust A and Chambon Ρ 1987 A human retinoic acid receptor which belongs to the family of nuclear receptors; Nature (London) 330 444-450

352


Pignoni F, Baldarelli R M, Steingrimsson E, Diaz R J, Patapoutian A, Merriam J R and Lengyel J Α 1990 The Drosophila gene tailless is expressed at the embryonic termini and is a member of the steroid receptor superfamily; Cell 62 151-163 Rastinejad F, Perlmann Τ, Evans R Μ and Sigler Ρ Β 1995 Structural determinants of nuclear receptor assembly on DNA direct repeats; Nature (London) 375 203-211 Ritchie Η Η, Wang L H, Tsai S, O'Malley Β W and Tsai Μ J 1990 COUP-TF gene: a structure unique for the steroid/thyroid receptor superfamily; Nucleic Acids Res. 18 6857-6862 Sap J, Munoz A, Damm, Κ, Goldberg Υ, Ghysdael J, Leutz A, Beug Η and Vennström Β 1986 The e-erb-A protein is a high-affinity receptor for thyroid hormone; Nature (London) 324 635-640 Schwabe J W, Chapman L, Finch J Τ and Rhodes D 1993 The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements; Cell 75 567-578 Steingrimsson Ε, Pignoni F, Liaw G J and Lengyel J A 1991 Dual role of the Drosophila pattern gene tailless in embryonic termini; Science 254 418-421 Tsai Μ -J and O'Malley Β W 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members; Annu. Rev. Biochem. 63 451-486 Umesono Κ and Evans R Μ 1989 Determinants of target gene specificity for steroid/thyroid hormone receptors; Cell 57 1139-1146 Weinberger C W, Thompson C C, Ong Ε S, Lebo R, Gruol D J and Evans R Μ 1986 The c-erb-A gene encodes a thyroid hormone receptor; Nature (London) 324 641-646 Wilson Τ Ε, Paulsen R E, Padgett Κ A and Milbrandt J 1992 Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors; Science 256 107-110 Yu R Τ 1994 Molecular and genetic analysis of a brain-specific vertebrate nuclear receptor, Tlx, Doctoral thesis, UCSD Yu R T, McKeown M, Evans R Μ and Umesono Κ 1994 Relationship between Drosophila gap gene tailless and a vertebrate nuclear receptor Tlx; Nature (London) 370 375-379 Yu V C, Delsert C, Andersen B, Holloway J M, Devary Ο V, Naar A M, Kim S Y, Boutin J M, Glass C Κ and Rosenfeld Μ G 1991 RXR beta: A coregulator that enhances binding of retinoic acid, thyroid hormone and vitamin D receptors to their cognate response elements; Cell 657 1251-1266 Zhang X K, Hofmann B, Tran Ρ Β, Graupner G and Pfahl Μ 1992 Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors; Nature (London) 355 441-446