Drosophila Hormone Receptor 38 Functions in Metamorphosis: A Role in Adult Cuticle Formation

Corresponding author: T. Kozlova, Howard Hughes Medical Institute, Department of Human Genetics, 5200 Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, kozlova{at}howard.genetics.utah.edu (E-mail).

Communicating editor: V. G. FINNERTY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

METAMORPHOSIS in Drosophila melanogaster occurs over a four-day period and leads to a drastic transformation of the entire body pattern whereby most larval structures are histolyzed and replaced by new adult structures. Larval epidermal cells are totally replaced as the adult integument is formed by cells that originate in the imaginal discs (for the head and thorax), or the imaginal histoblast nests (for the abdominal-integument; reviewed in FRISTROM and FRISTROM 1993 ). Two cuticles are sequentially synthesized during metamorphosis: The pupal cuticle is deposited by both larval and adult epidermal cells during prepupal and early pupal stages, whereas the adult cuticle is deposited only by the latter epidermis 35–80 hr after puparium formation (FRISTROM and FRISTROM 1993 ). There are notable differences in the cuticle ultrastructure in pupae and adults, as well as regional differences within each stage (reviewed by FRISTROM and FRISTROM 1993 ). From the outside in, the adult cuticle consists of cuticulin, a protein epicuticle and a lamellate procuticle. The deposition of the inner cuticle layers also exhibits regional differences in timing and is associated with the synthesis of adult cuticle proteins of low molecular mass (CHINARA et al. 1982 ; ROTER et al. 1985 ). During tanning and hardening of the adult cuticle after fly eclosion, the adult cuticle proteins are thought to be cross-linked to each other and chitin using dopamine and ß-alanine as the linking agents (ANDERSEN 1979 ). Deposition, sclerotization and melanization of adult cuticle effectively conclude metamorphosis in the adult integument.

Metamorphosis is propelled by the action of ecdysteroids, mediated by members of the steroid hormone receptor superfamily. These receptors are ligand-dependent transcription factors that regulate expression of a large number of genes, which in turn effect the appropriate responses to the hormonal stimuli. Half of the known members of the steroid receptor superfamily in Drosophila appear to be regulated by ecdysone at the transcriptional level and, in turn, to participate in relaying the hormonal signal during early stages of metamorphosis (reviewed in THUMMEL 1995 ). Far less is known about late stages of metamorphosis, even though the highest peaks of ecdysone are achieved during this period (RIDDIFORD 1993 ). It is widely accepted that the action of ecdysone in Drosophila is mediated by a functional ecdysone receptor consisting of a heterodimer of Ecdysone receptor (EcR) and ultra-spiracle (USP) proteins (KOELLE 1992 ; THOMAS et al. 1993 ; YAO et al. 1993 ). It is now known that other members of the steroid receptor superfamily in Drosophila can modulate the action of this ecdysone receptor complex, either by competing for binding on certain DNA EcREs (ecdysone response elements) or by interacting at the protein level with one of the receptor subunits (reviewed in THUMMEL 1995 ; THUMMEL 1997 ). We have shown previously that DHR38 is a potential modulator, as it is able to interact with USP in vitro, in yeast and in Drosophila cell lines (SUTHERLAND et al. 1995 ). However, our results also demonstrated that DHR38 is able to bind DNA as a monomer in vitro, raising the possibility that both mechanisms of action are relevant in vivo in particular tissues or stages of Drosophila development. It is interesting that DHR38 homologues in mammals, the NGFI-B type receptors, can act as both monomers (ZETTERSTROM et al. 1996 ) and heterodimers with the RXR homologues of USP (FORMAN et al. 1995 ; PERLMANN and JANSSON 1995 ).

To gain insight into the mechanisms of DHR38 action we have initiated an in vivo analysis of Dhr38 gene functions in Drosophila development. Elucidation of the Dhr38 genomic organization revealed that Dhr38 spans at least 40 kb and encompasses alternative promoters and polyadenylation sites. Dhr38 transcripts are present throughout fly development with clear variations in quantity, being particularly enriched at the pupal stage. We report the isolation of mutations in the Dhr38 gene which appear to result in fragility of the adult cuticle, at least in some areas. One of the alleles is represented by a pre-existing P-element insertion, while three others are new EMS-induced alleles. The three weaker alleles result in adult lethality shortly after eclosion with flies displaying haemolymph loss and melanization in the leg joints, while the strongest EMS allele causes earlier lethality.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Recombinant DNA manipulations:
A D. melanogaster genomic library prepared in DASH II from a stock isogenic for the second chromosome (dp, cl, cn, bw) was a gift of W. GELBART. A cDNA library made from third instar larval organs treated with ecdysone and cycloheximide was generously provided by C. THUMMEL. Phage DNA purification, digestion with restriction endonucleases, subcloning, agarose gel-electrophoresis, and transfer of nucleic acids to a nylon membrane (GeneScreen, NEN, Boston, MA) were performed as described (SAMBROOK et al. 1989 ). Inserts from genomic clones were subcloned into a pBluescript plasmid vector and sequenced on both strands by the dideoxy chain termination technique (SANGER et al. 1977 ). The ³⁵S dATP (NEN) and reagents from a standard sequencing kit (United States Biochemicals) were used according to manufacturer's instructions. For cTK61, exon 3 was sequenced on both strands and exon 1 on one strand; this allowed the determination of the splice sites between exons 1, 2, and 3 by comparing the sequence with our and available [Berkeley Drosophila Genome Project (BDGP), unpublished data] genomic sequences in 38E. The composite cTK61 sequence was generated using exon 1 and 2 sequences of BDGP and our sequences of exons 3–5.

To clone genomic DNA sequences, flanking the site of P-element insertion, genomic DNA from heterozygous adult flies bearing the l(2)02306 insertion was digested with XbaI. After heat inactivation of the enzyme at 65° for 15 min, 2 fly equivalents of DNA were ligated overnight in 200 µl ligation buffer containing rATP (NEN) in the presence of 2 units of T4 DNA ligase at 14°. After precipitation and resuspension in 10 µl of H₂O, the samples were used to transform by electroporation XL1-Blue competent cells, which were plated on kanamycin-containing agar plates.

RNA manipulations:
Total RNA was prepared from staged whole organisms using Trisolv^TM (Biotecx Laboratories Inc., Houston, TX). For preparation of poly(A)⁺RNA the Poly(A)Tract mRNA Isolation System (Promega, Madison, WI) was used according to the manufacturer's protocol. Approximately 2 µg of poly(A)⁺RNA were loaded per lane. Formaldehyde-containing agarose gel electrophoresis and nucleic acid transfer to Hybond-N membrane (Amersham) were performed as described (SAMBROOK et al. 1989 ). Antisense riboprobe corresponding to the DNA-binding and part of the ligand-binding domain of Dhr38 was stringently hybridized and washed at 68° as described by CHURCH and GILBERT 1984 . No cross-hybridization was observed to a plasmid bearing an insertion of the CF1 cDNA clone of usp, a close relative of Dhr38 (SHEA et al. 1990 ). The blot was reprobed with an rp49 control (O'CONNELL and ROSBASH 1984 ) to monitor mRNA integrity and loading.

For developmental reverse transcription PCR (RT-PCR) analysis poly(A)⁺RNA purification and cDNA synthesis was carried out as in BARRIO et al. 1996 . PCRs were done in 50 µl with standard buffer conditions (1.5 mM MgCl₂) and AmpliTaq polymerase (Perkin Elmer, Norwalk, CT), using 20 pmoles of Dhr38 specific primers. Three sets of such primers were used: (A) Flanking an intron in LBD and amplifying ca. 150 bp presumably from all the Dhr38 mRNA species (T31 5'-AACGACATCATGGAGTTCAGC-3' and T71 5'-CATCTGGAGCTGCTCCACCTT-3'); after 9 cycles (1 min steps at 94°, 60°, and 72°) the rp49A and rp49B primers (BARRIO et al. 1996 ) corresponding to D. melanogaster ribosomal protein 49 (O'CONNELL and ROSBASH 1984 ) were added at 10 pm each, and reactions were allowed to continue for 23 additional cycles. (B) Primers specifically amplifying a fragment of ca. 160 bp from the cTK61 isoform (61KS4 5'-GATCGGCTTGCTCGCTGATT-3' and 61-VV10 5'-GCATTGAGGTTCTGTCTGTA-3'). The conditions were 1 min steps at 94°, 54°, and 72° for 8 cycles without rp49 primers and 23 cycles after rp49 primers were added. (C) Primers specifically amplifying a fragment of ca. 330 bp from the cTK11 isoform (TD31T32 5'-AGCTGGAGGACCTGGTA C-3' and 11T36 5'-ATGCATGTCCGTAATTATCG-3'); the conditions were 1 min steps at 94°, 54°, and 72° for 8 cycles without rp49 primers and 24 cycles after rp49 primers were added. Aliquots of the reactions were normalized with respect to rp49 amplified fragments and analyzed in a 2% agarose gel.

Histochemical staining for ß-galactosidase:
Dissected larval and pupal tissues were stained with X-gal by a standard procedure (MLODZIK and HIROMI 1992 ) with some modifications. Briefly, the tissues were dissected in PBX (PBS + 0.3% Triton X-100) and fixed in PBX/4% formaldehyde for 10–15 minutes. Staining reactions were allowed to develop overnight at 37°.

Fly stocks and EMS mutagenesis:
Complete insertions, containing 5' and 3' UTR sequences, were subcloned from both cTK11 and cTK61, into the EcoRI site of the pCaSpeR-hs transformation vector under control of heat shock hsp70 promoter (THUMMEL and PIRROTTA 1992 ). Transgenic fly stocks were established and genetically mapped according to standard procedures.

Fly stocks bearing chromosome rearrangements with breakpoints in polytene division 38 and known mutations residing in this interval were obtained from S. PAINE-SAUNDERS (Df(2)DS9 cn, pr, b and Df(2)DS6 cn, pr, b), H. JÄCKLE (caudal alleles), M. ERDELYI (Ketel alleles and Df(2)Ketel^RX32) and the Bloomington Stock Center. The P-element insertion l(2)02306, P[lacZ,ry⁺], cn/CyO was kindly provided by A. SPRADLING. In the course of genetic mappings we realized that both Df(2)DS6 and Df(2)DS9 extend distally much further than previously reported (MOORE et al. 1983 ), uncovering caudal, Ketel, and l(2)02306 located in 38E. To isolate recessive lethal mutations in the 38D-E region, we performed an F₂ standard lethal screen using 0.025 M EMS essentially as described by GRIGLIATTI 1986 . The mutagenized second chromosome was marked with the recessive visible mutations dp, bw. In the first experiment Df(2)DS9 was used as a selective system and the EMS 2, 8, 9, 15, 18, 27, 31, 36 and 38 mutations were recovered from 2,500 chromosomes analyzed. In a second experiment the remaining EMS mutations were recovered from 2600 chromosomes analyzed against Df(2)Ketel^RX32. The mutations were subjected to complementation testing and mapped with the deficiencies. For lethal phase determination the mutations were marked with yellow (y), the stocks being established in yw background and balanced against a CyO, y⁺ chromosome. Collections of 200–400 eggs were done on apple juice-agar plates and allowed to develop for 24 hr at 25°. Live first instar y^- larvae were transferred to vials with yeast paste and allowed to develop for 10 more days. The sample size for each genotype was 50–100 first instar larvae. The pupal cases and adult animals were scored. For the rescue experiments, heat shock was administered for the indicated periods of time to cultures in plastic vials in a water bath at 37°.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genomic organization of Dhr38:
We have performed a genomic walk of approximately 50 kb in the chromosomal 38E region where Dhr38 resides. We partially sequenced a second Dhr38 cDNA clone, cTK61, which overlaps with cTK11 (SUTHERLAND et al. 1995 ), and mapped both clones with respect to genomic DNA from the region using hybridization and PCR scanning of the genomic DNA with cDNA sequencing primers to identify potential introns. Genomic sequences in 38E region corresponding to part of intron 2 of Dhr38 and further upstream are available (BDGP, unpublished results) and were supplemented with our partial sequencing of genomic subclones (data not shown; see below). Genomic and cDNA sequences were compared. Taken together these experiments showed that the Dhr38 gene extends over at least 40 kb and includes at least 5 exons (Figure 1A). The first intron is more than 20 kb in length, whereas the second through fourth introns are short (approximately 600 bp, 117 bp, and 184 bp, respectively). The first and second exons are unique to cTK61, and exon four is shared in its entirety by the two cDNAs; exon three is complete in cTK61 and incomplete in cTK11, and conversely exon five is shorter in cTK61 relative to cTK11. As previously reported (SUTHERLAND et al. 1995 ) the cTK11 clone contains an open reading frame capable of encoding the DHR38 protein with a calculated molecular mass of 61 KDa, and more than 3 kb of 3' untranslated region (3' UTR). Using BDGP genomic and our cDNA sequences, a composite sequence of cTK61 was generated (see MATERIALS AND METHODS). Computer analysis of this sequence showed a long open reading frame of 1071 amino acids beginning at nucleotide position 1121 with double methionine codons; the third methionine of this open reading frame is also unique to cTK61, whereas the fourth corresponds to the initiation codon of cTK11 (Figure 1B). Translation beginning with the first methionine codon of cTK61 would produce a polypeptide of 1071 residues and a calculated molecular mass of 117 KDa, which includes a transcription activation domain 522 amino acids longer than in the polypeptide encoded by cTK11 (Figure 1B); the DNA and ligand binding domains of the two polypeptides appear identical. A polypeptide of ca. 120 KDa is indeed detected immunochemically upon overexpression of the hsTK61 transgene (data not shown). As discussed below, expression studies using RNA blots and RT-PCR, together with the fact that both cDNA clones are terminated with a poly(A) stretch, suggest that these clones correspond to distinct RNA isoforms and that the gene contains alternative promoters and polyadenylation sites. This conclusion is reinforced by the findings of FISK and THUMMEL 1995 who described another, shorter cDNA clone of Dhr38, pLF16 (Figure 1A; see below).

View larger version (66K): In this window In a new window Download PPT slide   Figure 1. Genomic organization of Dhr38. (A) Positions of genomic clones are shown above the composite restriction map. Three cDNA clones, cTK11 (SUTHERLAND et al. 1995 ), cTK61 (this report) and pLF16 (FISK and THUMMEL 1995 ) are depicted below the enlarged map with protein coding regions represented by open boxes, untranslated regions by solid lines and introns by angled thin lines; positions of Zn fingers are denoted with two ovals. Distances are given in kb, and 0 corresponds to the start of the cTK11 clone; EcoRI, HindIII and SalI restriction sites are indicated as R, H, and S, respectively. The site of the l(2)02306 P-element insertion is shown as an open triangle. (B) Protein sequence of the putative activation domain encoded by the cTK61 isoform. The sequence shared by cTK11 and cTK61 is in bold. The locations of introns 1, 2, and 3 are indicated by arrows (following nucleotides 1553, 1867, and 3013 of cTK61), and methionines are underlined; the fourth methionine corresponds to the apparent initiation codon of cTK11, and the fifth to that of pLF16. (C) DNA sequence near the P insertion site (marked by an open triangle). The putative TATA box is underlined and the cap-site consensus (CHERBAS and CHERBAS 1993 ) is boxed; the start and direction of transcription in the cTK61 clone are shown with an arrow.

Expression of Dhr38 in Drosophila development:
We analyzed the expression of the Dhr38 gene in Drosophila embryogenesis using RNA (Northern) blots, and observed multiple mRNA species even in high stringency hybridization experiments (Figure 2A). A prominent 2-kb band (sometimes resolved as a doublet of ca. 1.8 and 2.0 kb) is present in all embryonic mRNA preparations. A ca. 4.0-kb species is very abundant in the late embryos (19–23 hr post-egg laying) but is also detectable at lower levels earlier, especially in 15–19-hr embryos. A ca. 5.0 species is the least abundant in embryogenesis but is clearly present in 15–19-hr embryos. Of the multiple developmentally regulated transcripts of Dhr38, the ca. 4.0- and 5.0-kb species correspond in size and might be represented by the cDNA clones described above (cTK61 and cTK11, respectively). The pLF16 cDNA clone described by FISK and THUMMEL 1995 might be represented by the 1.8- or 2.0-kb transcript, depending on the length of the poly(A)⁺ tail. We have detected the 4.0-kb species and the 1.8–2.0-kb doublet in Schneider's S2 cell line as well (Figure 2B). All mRNAs of the Dhr38 gene are of low abundance; blotting of purified poly(A)⁺ RNA and probing by antisense riboprobes was required to detect them. Moreover, it appears that at least the 4.0-kb species is unstable because it is enriched in S2 cells treated with cycloheximide; the ca. 2.0-kb bands are unaffected by cycloheximide (Figure 2B).

View larger version (64K): In this window In a new window Download PPT slide   Figure 2. RNA blot analysis of Dhr38 expression. (A) Dhr38 expression in Drosophila embryogenesis. Four mRNA species of ca. 5.0, 4.0, 2.0 and 1.8 kb, indicated with dots, hybridize with Dhr38 antisense probe (upper panel). The same blot was hybridized with the control rp49 probe to monitor RNA integrity and loading (lower panel). Poly(A)+RNA was prepared from yw embryos collected at the indicated hours after egg laying. (B) Dhr38 expression in the S2 cell line (upper panel). Two predominant transcripts of ca. 2.0 and 1.8 kb are detected in S2 cells incubated in Scheider's medium for 1 or 4 hr in the absence of cycloheximide (right). A low abundance transcript of ca. 4.0 kb which is detectable in untreated cells upon much longer exposure (not shown) is greatly enriched in the cells treated with 10 µg/ml cycloheximide for 1 or 4 hr (left). The same blot was hybridized with the control rp49 probe to monitor poly(A)+RNA integrity and loading (lower panel).

To analyze the expression of the Dhr38 gene during all stages of Drosophila development, we took advantage of a more sensitive technique, RT-PCR, and designed primers that would specifically amplify fragments corresponding to either the cTK61 or the cTK11 cDNA isoforms. We also used a pair of common primers, flanking the fourth intron in the ligand binding domain, which amplify a fragment present in all three cDNA clones described so far. The results of these experiments are summarized in Figure 3. The Dhr38 gene is expressed during most of Drosophila development but with some notable variations in quantity. The common fragment (Figure 3A) indicates that the combined Dhr38 mRNAs are present in 0–8-hr embryos at very low levels, which are significantly elevated in late embryogenesis and through the larval stages. They become notably enriched in pre-pupal and especially pupal stages, and are again somewhat reduced in adult flies. The mRNAs are absent from the ovaries, but relatively concentrated in third instar larval imaginal discs and brain complexes. The expression profiles for individual isoforms (Figure 3B, Figure C) are consistent with the profile of the common fragment (Figure 3A), but show some interesting variations: the pupal enrichment is most dramatic for the cTK11 isoform, and the adult has a substantial amount of cTK11 but virtually no cTK61 transcript. In overall terms, the cTK11 (ca. 5.0 kb) isoform is enriched in pupae and adults relative to the cTK61 (ca. 4.0 kb) isoform, which is more characteristic of the larvae.

View larger version (100K): In this window In a new window Download PPT slide   Figure 3. RT-PCR analysis of Dhr38 expression during Drosophila development. (A) Expression profile corresponding to all known Dhr38 species at the indicated stages and hours of development; A = adult, ID = imaginal discs and brain complexes of third instar larvae, OV = ovary. (B) specific expression profile of the cTK61 isoform. (C) specific expression profile of the cTK11 isoform. Rp 49 represents a semiquantitative control of amplification and loading.

The nature and significance of the ca. 2.0-kb transcript(s) merit further attention. FISK and THUMMEL 1995 described a ca. 1.9-kb transcript as predominant at larval and pre-pupal stages, and this is clearly true for the ca. 2.0-kb doublet in S2 cells in the absence of cycloheximide (Figure 2B), as well as in early embryos at 0–16 hours (Figure 2A). However, the ca. 4.0-kb isoform clearly becomes dominant in the late embryos (19–23 hr; Figure 2A), and is enriched in cycloheximide-treated samples unlike the ca. 2.0-kb forms (Figure 2B). We cannot exclude the possibility that at least one or both of the ca. 2.0-kb bands correspond to alternative Dhr38 RNA isoforms that have not yet been isolated and may not be amplifiable with our common primers. It is also possible that one or both bands correspond to a closely related gene, although we have hybridized our blots at high stringency (see MATERIALS AND METHODS).

Analysis of the lethal l(2)02306 P-element insertion in the vicinity of the Dhr38 gene:
To find a mutation affecting Dhr38 we screened the available P-element insertion collections and discovered one candidate mapping to polytene division 38E1-2, within the limits of a chromosomal deletion, Df(2)Ketel^RX32. We cloned the genomic DNA sequences flanking the site of l(2)02306 P-element insertion and by hybridizing the rescued construct to cloned genomic sequences derived from the 38E chromosomal walk we mapped the site of the insertion very close (less than 1.5 kb) to the 5' end of cTK61 (data not shown; Figure 1A). Subsequent sequence comparisons actually showed that the distance is only 34 bp (Figure 1C). The insertion site is found 8 bp downstream of a potential TATA box and 17 bp upstream of a TCAGT motif which is commonly associated with transcription start sites (± 10 bp; CHERBAS and CHERBAS 1993 ). Thus, it would not be surprising if this P insertion affects at least some aspects of Dhr38 expression.

The l(2)02306 insertion line is listed as homozygous lethal. We determined the effective lethal phase to be at the adult stage, for both homozygous and hemizygous individuals in combination with Df(2)Ketel^RX32 (Table 1). Homozygous insertion mutants develop normally through embryonic, larval and pupal stages, and the majority of the flies eclosing from the pupal cases look phenotypically normal. However, the cuticle in the leg joints becomes ruptured (occasionally during eclosion but predominantly later), as evidenced by leakage of haemolymph. The flies subsequently die within a few hours, displaying heavy melanization in the leg joints and occasionally in the proboscis, antennae and wing hinge (Figure 4D). In combination with Df(2)Ketel^RX32 the flies exhibit a slightly stronger phenotype, showing rupture of the cuticle and melanization predominantly during eclosion from the pupal case. Our interpretation is that the adult cuticle in mutant flies is not formed properly and is fragile at least in some areas, where it ruptures by mechanical stress when the flies start moving; this leads to leakage of haemolymph and melanization of the damaged spots, and eventual death possibly from desiccation. This phenotype is reverted and the viability is restored after precise excision of the P element (data not shown).

View larger version (82K): In this window In a new window Download PPT slide   Figure 4. Epidermal staining and mutant phenotype of the l(2)02306 P insertion line. (A) Histochemical X-gal staining is essentially absent in second instar larvae (L2) but is clearly detectable in the epidermis of mid-third instar larvae (marked L3). (B) The staining becomes very strong in wandering third instar animals. (C) Staining persists in imaginal epidermis, becoming localized to the regions of leg joints in the late pupae. (D) Mutant phenotype caused by the P insertion in the vicinity of the Dhr38 gene. Note the heavy melanization in the leg joints. In (C) and (D), corresponding body regions of the fly are marked with an arrow (leg joint between femur and tibia) and arrowhead (the base of the coxae).

We performed histochemical staining for ß-galactosidase at different stages of development using animals bearing the P-element insertion. No staining was detected in first and second larval stages in any tissue; staining first appears in the epidermis of feeding third instar larvae (Figure 4A), becomes much stronger in wandering third instar larvae (Figure 4B) and persists in pre-pupal and pupal stages. In early pupal stages the imaginal epidermis is stained homogeneously (data not shown), but about 70 hr after puparium formation (BAINBRIDGE and BOWNES 1981 ) the staining becomes localized to the leg joints, hinge region of the wing, proboscis and antennae (Figure 4C), locations which show the melanization phenotype with variable frequency (leg joints always, the rest sporadically). The same staining pattern persists in newly eclosed flies (data not shown). At all stages the staining is predominantly epidermal, with no internal staining observed in larvae and only very weak, presumably background, staining in pupae. Therefore the l(2)02306 enchancer trap line is under the control of and may interfere with mostly epidermis-specific enchancer(s), which presumably regulate the Dhr38 gene. However, as assayed by RT-PCR, Dhr38 mRNA is still detected in homozygous mutant animals at levels comparable to controls (data not shown). This is not surprising considering the localized nature of the phenotypic defects observed and the complexity of the Dhr38 transcription unit, which apparently includes alternative promoters (Figure 1A).

Generation of EMS-induced alleles in the Dhr38 gene:
A standard F₂ EMS lethal screen was performed on 5,000 mutagenized chromosomes to isolate mutations in the 38E region that do not complement the l(2)02306 P-element insertion and potentially correspond to Dhr38. The induced EMS mutations were subjected to complementation analysis and mapped genetically with respect to deficiencies with breakpoints in 38E, as summarized in Figure 5. In situ hybridization to polytene chromosomes of the Df(2)DS9 stock and PCR on genomic DNA prepared from homozygous Df(2)Ketel^RX32 first instar larvae had shown that Dhr38 sequences are removed in both deficiences (data not shown). We identified five complementation groups, each with multiple alleles, in the region of overlap of these two deficiencies. Two of these correspond to previously known genes, Ketel (LINDSLEY and ZIMM 1992 ; ERDELYI et al. 1997 ) and diaphanous (CASTRILLON and WASSERMAN 1994 ), and two others, l(2)38Ea and l(2)38Eb, are new. Most importantly, the fifth group includes three EMS-induced alleles that do not complement l(2)02306. Two of them, 43 and 57, are weak alleles which show a delayed melanization similar to that of l(2)02306 when crossed to Df(2)Ketel^RX32, but produce some viable escapers when homozygous (Table 1). The third allele, 56, behaves as an earlier, pupal lethal in combination with Df(2)Ketel^RX32. Two thirds of the hemizygous animals develop normally until the late pupal stage (85–90 hr after pupariation [AP]; BAINBRIDGE and BOWNES 1981 ), and pharate adults look externally normal when dissected from pupal cases (data not shown). However, when the pharate flies start moving shortly before eclosion (around 90 hr AP at 25°), the adult cuticle is ruptured followed by leakage of haemolymph and melanization within the pupal case. Effectively all pupariated hemizygous mutant flies exhibit this pupal lethal phenotype at 25° (Table 1). Homozygous 56/56 mutants die at larval stages, possibly because of a second-site lethal mutation present elsewhere on the chromosome or because Dhr38⁵⁶ is a neomorphic mutation.

View larger version (13K): In this window In a new window Download PPT slide   Figure 5. Lethal mutations in the 38D-E region. The extent of the deficiencies in the region is shown by solid lines in the upper part of the figure (Df). The complementation groups (CG) and gene symbols (Dhr38, Ketel, dia and cad) are shown in bold italics. Complete names of the complementation groups are abbreviated [eg. l(2)38Da is represented by Da, and so on]. New EMS-induced alleles assigned to complementation groups are designated by plain numbers (Alleles). The Da-De cluster is defined by not being uncovered by Df(2)DS9 and the EFa-EFf by not being uncovered by Df(2)KetelRX32; within each of these clusters the order of complementation groups is unknown because of the absence of relevant chromosomal rearrangements. Within the Ea-dia cluster, Ea is most distal because it is uncovered by Df(2)DS9 but not Df(2)DS6; Eb and Dhr38 are uncovered by all three deficiencies. Ketel and dia, placed in 38E cytologically (LINDSLEY and ZIMM 1992 ; CASTRILLON and WASSERMAN 1994 ), were positioned proximally to Dhr38 by sequence analysis (Berkeley Drosophila Genome Project, as reported in FLYBASE).

To prove that the l(2)02306/EMS^43,56,57 complementation group corresponds to the Dhr38 gene, we used transgenic copies of Dhr38 to rescue the pupal lethality of the most extreme mutation, 56. We created transgenic fly stocks bearing the inserts of the cTK11 and cTK61 cDNA clones under the control of an hsp70 heat shock promoter. Figure 6 diagrams the final rescue cross using the hsTK11 construct in the transgenic line, P[11SA3, w⁺], and reports the numbers of eclosing adult progeny in each phenotypic marker class. One of the progeny classes (male ywCy⁺) is the 56/Df(2)Ketel^RX32 hemizygous mutant control, which shows absolutely no adult eclosion. In contrast, the hemizygous mutant class bearing the P[11SA3, w⁺] insert (female yw⁺Cy⁺ class) is represented by a substantial number of eclosed adults, documenting convincing rescue of the pupal lethality. Quantitative comparison with the number of control female heterozygotes (y⁺w⁺Cy class), in which the deficiency or Dhr38⁵⁶ chromosome is balanced with the Curly chromosome, showed that the rescue is partial but robust: after a mild heat shock, 48–69% of the expected eclosed yw⁺Cy⁺ adults were observed in two independent experiments. A weaker (12%) rescue at 25° was observed and can be explained by the known low-level leakage of the hsp70 promoter at room temperature documented in similar experiments (BAYER et al. 1997 ; VAN DE WETERING et al. 1997 ). The transgene-dependent, heat-shock-induced rescue is also partial, in that the adult yw⁺Cy⁺ flies die within a few hours after eclosion, displaying the melanotic phenotype described earlier for l(2)02306. More vigorous heat shock treatments for obtaining fuller rescue were precluded by the deleterious effect of massive DHR38 overexpression observed with the hsTK11 inserts, even in wild-type backgrounds (T. KOZLOVA and F. C. KAFATOS, unpublished results). For the same reason, we chose to test rescue of pupal lethality with the 56/Df(2)Ketel^RX32 hemizygotes, rather than larval lethality with 56/56 homozygotes; the latter would have required repeated heat shocks in both the larval and pupal stages, as well as being subject to complications from possible second site lethals on the mutagenized chromosome. Despite these limitations, the reality of the rescue is verified by similar results obtained with a second independent hsTK11 insert, 11SC1 (between 40 and 60% rescue of pupal lethality in several independent experiments; data not shown). However, even the simultaneous presence of both hsTK11 and hsTK61 constructs in the same animal is not sufficient to produce viable rescued flies in similar experiments (data not shown).

View larger version (14K): In this window In a new window Download PPT slide   Figure 6. Transgenic rescue of the Dhr3856 mutation with a P[11SA3, w+] construct. Females of a balanced stock bearing a deficiency Df(2)KetelRX32 (hereafter referred to as RX32) were crossed to males bearing the P[11SA3, w+] transgene on the X chromosome and the EMS-induced Dhr3856 mutation on the second chromosome balanced against a CyO,y+ chromosome. The resultant F1 progeny genotypes (italics), respective phenotypes (Roman type) and expected ratio for flies of particular phenotype are shown; since all the stocks are in a yw background, the w+ phenotype is caused by the presence of the transgene. The outcome of the rescue crosses with a mild heat shock treatment (four heat shocks of 10 min at 37°) at the pupal stage and at 25° is shown. The numbers represent observed eclosed adults, and those in parentheses represent the expected number of flies as calculated by comparison to the corresponding control class (a or b, respectively; note that the controls are expected at double the number of the experimentals).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dhr38 has a complex genomic organization and produces developmentally regulated isoforms:
In terms of its large size and complex structure, the Dhr38 gene is a typical member of the steroid receptor superfamily in Drosophila (SEGRAVES and HOGNESS 1990 ; KOELLE et al. 1992 ; STONE and THUMMEL 1993 ; TALBOT et al. 1993 ; RUSSELL et al. 1996 ). It spans at least 40 kb in length and includes four introns varying in size from 117 bp to more than 20 kb. At least four developmentally regulated mRNA species are detected, apparently generated by alternative promoters and polyadenylation sites. Since only three of the four putative mRNA isoforms have been characterized, each by a single cDNA clone, the transcriptional structure of the gene may be more complex than is currently known. The Dhr38 mRNA isoforms corresponding to cTK11 and cTK61 differ in the untranslated regions, suggesting a possible complex post-transcriptional regulation. In particular, cTK61 has a 5' UTR of 1.1 kb, while cTK11 has more than 3 kb of 3' UTR containing multiple ATTTA motifs which presumably affect mRNA stability (DECKER and PARKER 1994 ). The isoforms also differ in the encoded putative transcription activation domains, but not the domains corresponding to DNA binding, dimerization and possible ligand binding. In particular, the cTK61 clone encodes a putative polypeptide containing 522 N-terminal amino acids in addition to those encoded in cTK11. This difference is unlikely to affect the DNA binding or dimerization properties described for DHR38 (SUTHERLAND et al. 1995 ), but might well alter the transcriptional activation properties. Similarly, the polypeptide encoded by the pLF16 clone (FISK and THUMMEL 1995 ) appears to differ only in the putative transcriptional activation domain; this clone is missing the first initiation codon of cTK11 and, if it is complete, presumably utilizes a second in-frame methionine as a translation start site, resulting in an N-terminal domain truncated by 34 amino acids relative to cTK11. Three Dhr38 homologues exist in well-characterized vertebrate species such as mouse or human. Two of them, NGFI-B/Nur77/N10 and Nurr1, are relatively short (ca. 14 kb) and include six introns (RYSECK et al. 1989 ; WATSON and MILBRANDT 1989 ; ZETTERSTROM et al. 1997 ). The third member of the family, NOR-1, spans at least 35 kb and contains seven introns (OHKURA et al. 1996 ). There is no correlation in the sizes of introns between the fly gene and its vertebrate homologues, and the positions of the first three introns are not conserved. However, the last intron in the putative ligand binding domain of DHR38 does correspond in location to that of intron six of NGFI-B/Nur77/N10 (RYSECK et al. 1989 ; WATSON and MILBRANDT 1989 ) and intron seven of NOR-1 (OHKURA et al. 1996 ).

The Dhr38 gene is expressed broadly during fly development but at a relatively low level, requiring for detection poly(A)⁺ RNA selection (for RNA blot analysis) or more than 30 amplification cycles (for RT-PCR). The level of expression sharply increases in pre-pupal and pupal stages, suggesting participation in metamorphic events. Of the two best characterized mRNA species, the one corresponding to cTK61 is relatively more larval-specific while cTK11 shows the most dramatic enchancement in pupae and persists into the adult stage. Neither of these isoforms is significantly expressed in the ovary or early embryos. The l(2)02306 enchancer trap insertion located very close to Dhr38 exhibits epidermal-specific expression in late larval, pre-pupal and pupal stages. Undoubtedly this only corresponds to a sub-pattern of Dhr38 expression; the regulatory regions of Dhr38 must be quite complex judging from genomic structure analysis. We detected DHR38 protein by immunostaining in the epidermis of third instar larvae but also in additional tissues and stages (J. D. SUTHERLAND, T. KOZLOVA and F. C. KAFATOS, unpublished results).

Mutations in Dhr38 result in localized fragility of the adult cuticle:
The lethal phases of available EMS and P-element induced mutations indicate that Dhr38 is important for late stages of metamorphosis; the haemolymph leakage and melanization phenotype suggest that all presently available alleles affect adult cuticle formation, possibly leading to incomplete sclerotization. In the three weaker alleles the defects appear to be specific to the thoracic cuticle of the leg joints, as abdominal and head structures are not visibly affected. Overall morphology of the mutant flies bearing the stronger EMS allele, including tanning of the bristles, is normal in Dhr38⁵⁶/Df(2)Ketel^RX32 hemizygotes at 80–90 hr after puparium formation. It is unlikely that these mutations represent complete loss-of-function alleles. The weak Dhr38⁴³ and Dhr38⁵⁷ alleles behave as hypomorphs in genetic assays, and both mRNA and DHR38 protein are still present in the Dhr38⁵⁶/Df(2)Ketel^RX32 hemizygous mutant animals (T. KOZLOVA and F. C. KAFATOS, unpublished results). Therefore either a specific epidermal function of Dhr38 is affected in these mutants, specific epidermal cells are most sensitive to altered levels of Dhr38 expression, or Dhr38 is dispensable in tissues other than epidermis.

We proved by the genetic rescue experiments that the mutations generated indeed correspond to Dhr38. Transgenic copies of Dhr38 under the control of the hsp70 promoter permit a robust, albeit not complete, rescue under various heat shock regimens. At least four reasons could explain the incomplete rescue. First, we may not have generated sufficient DHR38 protein, although we know that DHR38 is produced in excess under these conditions as compared to endogenous protein (J. D. SUTHERLAND, T. KOZLOVA and F. C. KAFATOS, unpublished results). Second, we were not able to precisely adjust the timing of inductions to reproduce the complex dynamics of Dhr38 in vivo expression, which were crucial in similar experiments described for the Broad-Complex (BAYER et al. 1997 ). Third, it is formally possible that a Dhr38 isoform which we have not tested is necessary to rescue the later adult lethality; BAYER et al. 1997 showed that the rescue of particular Broad-Complex alleles requires isoform-specific transgenes which, separately, are unable to rescue a complete loss-of-function allele. Fourth, a great excess of DHR38 protein results in dominant lethality in many developmental stages, making it practically impossible to reconstitute the in vivo Dhr38 expression pattern with heat shock constructs.

What is the mechanism of DHR38 action in the cuticle?
Since Dhr38 mutations have a phenotype suggesting localized effects on the adult cuticle, they might interfere directly or indirectly with important structures or enzymatic components of the cuticle. The first adult specific cuticle protein, Dacp-1 (Drosophila adult cuticle protein 1), has been recently characterized molecularly and shown to be expressed exclusively in the epidermis underlying the head and thoracic cuticle; it is believed to participate in a specialized function such as cuticle sclerotization (QIU and HARDIN 1995 ). DOPA decarboxylase (DDC) is a key enzyme in the biochemical pathway responsible for cuticle sclerotization and tanning (reviewed in WRIGHT 1996 ). It is well documented that Ddc is regulated by ecdysone during development (KRAMINSKY et al. 1980 ; CLARK et al. 1986 ), and the phenotypes of particular Ddc alleles retaining 5–10% of the enzyme activity closely resemble the phenotype of the l(2)02306 mutation (LINDSLEY and ZIMM 1992 ; WRIGHT 1996 ). We analyzed the expression of both Dacp-1 and Ddc in Dhr38 mutants. Assays were performed on RNAs extracted from Dhr38⁵⁶/Df(2)Ketel^RX32 hemizygous or l(2)02306 homozygous pupae 80–90 hr after puparium formation, as compared to the heterozygous sibling controls bearing a balancer chromosome. However, no significant effect on the level of either Dacp-1 or Ddc transcripts was detected in these preliminary experiments. This is not altogether surprising, considering that both the late pupal expression pattern of the insertion line and the apparent cuticle defects are highly localized.

Of course we cannot exclude that structural or enzymatic cuticle constituents other than those tested are affected, for example phenol oxidase or tyrosine hydroxylase, as well as some of the genes clustered in the Ddc region, which act in cuticle sclerotization and melanization (WRIGHT 1996 ). Any one of the biochemically described 15–20 adult-specific cuticle proteins (CHINARA et al. 1982 ), or larval and pupal cuticle proteins reused to synthesize adult cuticle may also be targets of Dhr38. It is interesting that regulatory regions of two of the pupal cuticle genes, EDG-78 and EDG-84, contain a response element to which DHR38 can potentially bind albeit not with highest affinity (APPLE and FRISTROM 1991 ).

Deposition of larval and pupal cuticles is known to be under hormonal control in various insects including Drosophila (HIRUMA et al. 1991 ; reviewed in FRISTROM and FRISTROM 1993 ) and expression of pupal cuticle genes is regulated by ecdysone in a complex way (FECHTEL et al. 1988 ; APPLE and FRISTROM 1991 ). It is less clear if this is also true for adult cuticle deposition, although the highest ecdysone peak is achieved between 24 and 48 hr after pupariation (reviewed in RIDDIFORD 1993 ). BAINBRIDGE and BOWNES 1988 described two ecdysone peaks during late pupal stages (20–55 hr and approximately 70 hr after pupariation). Thus, considering Dhr38 mRNA enrichment in pupae and the observed mutant phenotypes, it is tempting to speculate that DHR38 might be one of the transcriptional regulators participating in relaying the ecdysone signal in epidermis and possibly in other tissues. Further tests of this hypothesis and of possible interaction with usp awaits future experimentation, including clonal analysis.

	ACKNOWLEDGMENTS

We are grateful to A. SPRADLING, H. JÄCKLE, M. ERDELYI, S. PAINE-SAUNDERS and K. MATTHEWS (Bloomington Stock Center, Indiana) for providing fly stocks; and to W. GELBART and C. THUMMEL for genomic and cDNA libraries, respectively. The technical assistance of E. KASHEVSKY and B. MIÑANA is gratefully acknowledged. We are indebted to R. BARRIO for providing cDNA samples used for Dhr38 developmental expression studies and to members of the KAFATOS laboratory for advice and discussions. The sequence of a 600-bp genomic DNA fragment encompassing the l(2)02306 P-element insertion site and the composite sequence of cTK61 are deposited with EMBL Data Bank, accession numbers AJ001775 and AJ002073, respectively. This work was supported by INTAS grants INTAS-93-789 and INTAS-93-789-Ext.

Manuscript received October 21, 1997; Accepted for publication April 2, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ANDERSEN, S. O., 1979  Biochemistry of insect cuticle. Annu. Rev. Entomol. 24:29-61.

APPLE, R. T. and J. W. FRISTROM, 1991  20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes. Dev. Biol. 146:569-582[Medline].

BAINBRIDGE, S. P. and M. BOWNES, 1981  Staging the metamorphosis of Drosophila melanogaster.. J. Embryol. Exp. Morphol. 66:57-80[Medline].

BAINBRIDGE, S. P. and M. BOWNES, 1988  Ecdysteroid titers during Drosophila metamorphosis. Insect Biochem. 18:185-197.

BARRIO, R., M. J. SHEA, J. CARULLI, K. LIPKOW, and U. GAUL et al., 1996  The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family, defined by the adjacent, region-specific homeiotic gene spalt.. Dev. Genes Evol. 206:315-325.

BAYER, C. A., L. VON KALM, and J. W. FRISTROM, 1997  Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis. Dev. Biol. 187:267-282[Medline].

CASTRILLON, D. H. and S. A. WASSERMAN, 1994  Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene. Development 120:3367-3377[Abstract].

CHERBAS, L. and P. CHERBAS, 1993  The arthropod initiator: the capsite consensus plays an important role in transcription. Insect Biochem. Mol. Biol. 23:81-90[Medline].

CHINARA, C. J., D. SILVERT, J. AND J, and W. FRISTROM, 1982  The cuticle proteins of Drosophila melanogaster: stage specificity. Dev. Biol. 89:379-388[Medline].

CHURCH, G. M. and W. GILBERT, 1984  Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995[Abstract/Free Full Text].

CLARK, W. C., J. DOCTOR, J. W. FRISTROM, and R. B. HODGETTS, 1986  Differential responses of the dopa decarboxylase gene to 20-OH-ecdysone in Drosophila melanogaster.. Dev. Biol. 114:141-150[Medline].

DECKER, C. J. and R. PARKER, 1994  Mechanisms of mRNA degradation in eukaryotes. Trends Biochem. Sci. 19:336-340[Medline].

ERDELYI, M., E. MATHI, and J. SZABAD, 1997  Genetic and developmental analysis of mutant Ketel alleles that identify the Drosophila importin-b homologue. Acta Biol. Hung. 48:323-338[Medline].

FECHTEL, K., J. E. NATZLE, E. E. BROWN, and J. W. FRISTROM, 1988  Prepupal differentiation of Drosophila imaginal discs: identification of four genes whose transcripts accumulate in response to a pulse of 20-hydroxyecdysone. Genetics 120:465-474[Abstract/Free Full Text].

FISK, G. J. and C. S. THUMMEL, 1995  Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members. Proc. Natl. Acad. Sci. USA 92:10604-10608[Abstract/Free Full Text].

FORMAN, B. M., K. UMESONO, J. CHEN, and R. M. EVANS, 1995  Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 81:541-550[Medline].

FRISTROM, D., and J. W. FRISTROM, 1993 The metamorphic development of the adult epidermis, pp. 843–897 in The Development of Drosophila melanogaster, edited by M. BATE and A. MARTINEZ-ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

GRIGLIATTI, T., 1986 Mutagenesis, pp. 39–58 in Drosophila: A Practical Approach, edited by D. B. ROBERTS. IRL Press, Oxford.

HIRUMA, K., J. HARDIE, and L. M. RIDDIFORD, 1991  Hormonal regulation of epidermal metamorphosis in vitro: control of expression of a larval-specific cuticle gene. Dev. Biol. 144:369-378[Medline].

KOELLE, M. R., 1992 Molecular analysis of the Drosophila ecdysone receptor complex. Ph.D., Thesis, Stanford University, Stanford, CA.

KOELLE, M. R., W. A. SEGRAVES, and D. S. HOGNESS, 1992  DHR3: a Drosophila steroid receptor homolog. Proc. Natl. Acad. Sci. USA 89:6167-6171[Abstract/Free Full Text].

KRAMINSKY, G. P., W. C. CLARK, M. A. ESTELLE, R. D. GIETZ, and B. A. SAGE et al., 1980  Induction of translatable mRNA for dopa decarboxylase in Drosophila: an early response to ecdysterone. Proc. Natl. Acad. Sci. USA 77:4175-4179[Abstract/Free Full Text].

LINDSLEY, D. S., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego.

MLODZIK, M. and Y. HIROMI, 1992  Enchancer trap method in Drosophila: its application to neurobiology. Methods in Neurosci. 9:397-414.

MOORE, G. D., D. A. SINCLAIR, and T. A. GRIGLIATTI, 1983  Histone gene multiplicity and position effect variegation in Drosophila melanogaster.. Genetics 105:327-344[Abstract/Free Full Text].

O'CONNELL, P. and M. ROSBASH, 1984  Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res. 12:5495-5513[Abstract/Free Full Text].

OHKURA, N., M. ITO, T. TSUKADA, K. SASAKI, and K. YAMAGUCHI et al., 1996  Structure, mapping and expression of a human NOR-1 gene, the third member of the Nur77/NGFI-B family. Biochim. Biophys. Acta 1308:205-214[Medline].

PERLMANN, T. and L. JANSSON, 1995  A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev. 9:769-782[Abstract/Free Full Text].

QIU, J. and P. E. HARDIN, 1995  Temporal and spatial expression of an adult cuticle protein gene from Drosophila suggests that its protein product may impart some specialized cuticle function. Dev. Biol. 167:416-425[Medline].

RIDDIFORD, L. M., 1993 Hormones and Drosophila development, pp. 899–940 in The Development of Drosophila melanogaster, edited by M. BATE and A. MARTINEZ-ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

ROTER, A. H., J. B. SPOFFORD, and H. SWIFT, 1985  Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation. Dev. Biol. 107:420-431[Medline].

RUSSELL, S. R. H., G. HEIMBECK, C. M. GODDARD, A. T. C. CARPENTER, and M. ASHBURNER, 1996  The Drosophila Eip78C gene is not vital but has a role in regulating chromosomal puffs. Genetics 144:159-170[Abstract].

RYSECK, R. P., H. MACDONALD BRAVO, M. G. MATTEI, and S. RUPPERT et al., 1989  Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor. EMBO J. 8:3327-3335[Medline].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SANGER, F., S. NICKLEN, and A. R. COULSON, 1977  DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5477[Abstract/Free Full Text].

SEGRAVES, W. A. and D. S. HOGNESS, 1990  The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev. 4:204-219[Abstract/Free Full Text].

SHEA, M. J., D. L. KING, M. J. CONBOY, B. D. MARIANI, and F. C. KAFATOS, 1990  Proteins that bind to Drosophila chorion cis-regulatory elements: a new C₂H₂ zinc finger protein and a C₂C₂ steroid receptor-like component. Genes Dev. 4:1128-1140[Abstract/Free Full Text].

STONE, B. L. and C. S. THUMMEL, 1993  The Drosophila 78C early late puff contains E78, an ecdysone-inducible gene that encodes a novel member of the nuclear hormone receptor superfamily. Cell 75:307-320[Medline].

SUTHERLAND, J. D., T. KOZLOVA, G. TZERTZINIS, and F. C. KAFATOS, 1995  Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type. Proc. Natl. Acad. Sci. USA 92:7966-7970[Abstract/Free Full Text].

TALBOT, W. S., E. A. SWYRYD, and D. S. HOGNESS, 1993  Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73:1323-1337[Medline].

THOMAS, H. E., H. G. STUNNENBERG, and A. F. STEWART, 1993  Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature 362:471-475[Medline].

THUMMEL, C. S., 1995  From embryogenesis to metamorphosis: the regulation and function of Drosophila nuclear receptor superfamily members. Cell 83:871-877[Medline].

THUMMEL, C. S., 1997  Duelling orphans—interacting nuclear receptors coordinate Drosophila metamorphosis. Bioessays 19:669-672[Medline].

THUMMEL, C. S. and V. PIRROTTA, 1992  New pCaSpeR P element vectors. Dros. Inf. Serv. 71:150.

VAN DE WETERING, M., R. CAVALLO, D. DOOIJES, M. VAN BEEST, and J. VAN ES et al., 1997  Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF.. Cell 88:789-799[Medline].

WATSON, M. A. and J. MILBRANDT, 1989  The NGFI-B gene, a transcriptionally inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction. Mol. Cell. Biol. 9:4213-4219[Abstract/Free Full Text].

WRIGHT, T. R. F., 1996  Phenotypic analysis of the Dopa decarboxylase gene cluster mutants in Drosophila melanogaster.. J. Hered. 87:175-190[Abstract/Free Full Text].

YAO, T. P., B. M. FORMAN, Z. JIANG, L. CHERBAS, and J. D. CHEN et al., 1993  Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366:476-479[Medline].

ZETTERSTRÖM, R. H., L. SOLOMIN, T. MITSIADIS, L. OLSON, and T. PERLMANN, 1996  Retinoid X receptor heterodimerization and developmental expression distinguish the orphan nuclear receptors NGFI-B, Nurr1, and Nor1. Mol. Endocrinol. 10:1656-1666[Abstract].

ZETTERSTRÖM, R. H., L. SOLOMIN, L. JANSSON, B. J. HOFFER, and L. OLSON et al., 1997  Dopamine neuron agenesis in Nurr1-deficient mice. Science 276:248-250[Abstract/Free Full Text].

This article has been cited by other articles:

				R. S. Shapiro and K. V. Anderson Drosophila Ik2, a member of the I{kappa}B kinase family, is required for mRNA localization during oogenesis Development, April 15, 2006; 133(8): 1467 - 1475. [Abstract] [Full Text] [PDF]

				A. A. Sullivan and C. S. Thummel Temporal Profiles of Nuclear Receptor Gene Expression Reveal Coordinate Transcriptional Responses during Drosophila Development Mol. Endocrinol., November 1, 2003; 17(11): 2125 - 2137. [Abstract] [Full Text] [PDF]

				T. Kozlova and C. S. Thummel Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis Development, January 4, 2002; 129(7): 1739 - 1750. [Abstract] [Full Text] [PDF]

				H. Butler, S. Levine, X. Wang, S. Bonyadi, G. Fu, P. Lasko, B. Suter, and R. Doerig Map Position and Expression of the Genes in the 38 Region of Drosophila Genetics, August 1, 2001; 158(4): 1597 - 1614. [Abstract] [Full Text] [PDF]