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The Drosophila gene alien is expressed in the muscle attachment sites during embryogenesis and encodes a protein highly conserved between plants, Drosophila and vertebrates
ELSEVIER
Mechanisms
of Development
57 (1996) 59-68
The Drosophila gene aLien is expressed in the muscle attachment sites during embryogenesis and encodes a protein highly conserved between plants, DrosophiZu and vertebrates Anette Goubeaud,
Stefan Knirr, Renate Renkawitz-Pohl,
Achim Paululat*
Entwicklungsbiologie, Zoologie, FB Biologic. Philipps-Universitiit, Kurl-van-Frisch-SrrcIsse, 35032 Marburg, Germany Received
12 February
1996; revision received I9 March 1996; accepted 20 March 1996
Abstract
We have found a novel gene (alien) that is expressed exclusively in the muscle attachment sites (apodemes) during embryogenesis in Drosophila. Antibodies raised against the Alien protein enable us to follow the developing attachments from stage 1 l/l 2 until stage 16/17. The coding region of the Drosophila alien gene is highly conserved to a gene of unknown function, isolated from a plant (Loo et al., 1995), and to the human TRIP15 gene (Lee et al., 1995). Searching for thyroid receptor interacting proteins, TRZPZ.5 was isolated as a negative regulator. Whether there is a functional correlation to Alien remains to be analyzed. Alien expression is independent of muscle formation, as shown in rolling stone mutant embryos. Even in twist and snail mutants, lacking mesodermal development, alien expression is fairly normal, showing a rather autonomous development of the apodemes. The conservation of alien suggests an important role in differentiation. Keywords: alien; Evolutionary
conservation;
Drosophila;
Muscle attachments;
1. Introduction The attachment of multinucleated myotubes to the epidermis at the muscle attachment sites, the apodemes, results in the final organization of the somatic muscles at the end of embryogenesis. During this process, the membrane of the epidermal cell and the membrane of the muscle cell interact and large adherens junctions are formed (Lai-Fook, 1967; Tepass and Hartenstein, 1994). It was shown that the integrins mediate this cell-cell interaction. Most of the cloned integrin genes encode adapters for the major components of the extracellular matrix, such as lamin, fibronectin or collagen. Furthermore, the integrins were found in the muscle cell membrane and the corresponding tendon cell membrane. For example, the aps2 integrin gene is expressed in the mesoderm and the aps&Jps complex accumulates at the muscle attachment sites (Bogaert et al., 1987). The aPslpPs integrin complex complements this expression pattern and accumulates on
* Corresponding author. Fax: +49 642 e-mail: [email protected].
I 28897 I ;
TRIP 15
the ectodermal side of the muscle attachments; that is, the basal surface of the tendon cell. The absence of this integrin complex results in the detachment of the embryonic muscles from the epidermis, which is clearly evident from myospheroid mutants (mys, BPS-) (Leptin et al., 1989; Brown, 1994), which are missing BPS integrin, and from inflated null mutants lacking the aPS2-integrin (Brown, 1994). At stage 17 both inflated and myospheroid mutant embryos have a similar ‘detached’ phenotype, although the timing of detachment differs (Brown, 1994). Several other genes have been shown to be specifically expressed in the apodemes, like delilah (Armand et al., t994), tiggrin (Fogerty et al., 1994), msp300 (Volk, 1992; Volk and Vijay-Raghavan, 1994), masquerade (Murugasu-Oei et al., 1995), p1 tubutin (Buttgereit, 1991), and srripe (Lee et al., 1995b; Frommer et al., 1996). Some are expressed in other tissues as well. Here we describe the novel Drosophila gene alien, that encodes a hydrophilic 42 kDa protein. During embryogenesis, Alien is localized exclusively in all apodemes of the somatic muscles in the head, thorax, and abdominal segments, as well as in the epidermal attachments of the pharynx. This protein pro-
09254773/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved P11: SO925-4773(96)00532-6
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A. Goubeaud et al. /Mechanisms
vides, therefore, an excellent marker for all embryonic muscle attachments and allows to follow the progenitor cells from stage 11 onward. The sequence comparison of the deduced Alien protein reveals a striking conservation between Drosophila and vertebrates (humans). Surprisingly, a homologous sequence was found in plants. 2. Results 2. I. Cloning and sequence analysis of alien alien was cloned and identified from a screen for genes involved in embryonic muscle development, based on a lethal P-element insertion line with a muscle-specific phenotype (Paululat et al., 1995). Using standard methods, P-element flanking sequences were cloned. Three transcription units were found within the region (Fig. 1B): try29F encodes a serin protease (Paululat, 1996), rolling stone is responsible for the muscle phenotype (Paululat et al., 1995, in preparation), and the third transcription unit, called alien, is described here. The P element is located 4 kb downstream of the alien gene and we have no evidence that this P-element influences the alien expression (Fig. 1B). Polytene chromosome in situ hybridization using a genomic clone that uncovers alien maps the gene on chromosome II at 29F/30A. Using genomic DNA clones as a probe for an embryonic cDNA library screen, several cDNA clones were obtained that encompass the coding region of the alien transcript, respectively. The sequence of the alien gene that we have determined using different overlapping cDNA clones is shown in Fig. 1A. The positions of three introns, determined by sequence comparison of the cDNA with genomic clones, are indicated. The putative ATG start codon is preceded by an in-frame stop codon, which confirms the translation start. A putative poly A signal was not found in the longest cDNA which we have cloned. However, in the genomic clone uncovering the putative alien trailer several poly A signals are observable (data not shown). The open reading frame has a coding capacity of approximately 42 kDa, which is coincident with our Western blot analysis of embryonic protein extracts (data not shown). The amino acid sequence by itself contains a putative nuclear localization signal (NLS; amino acids 190-216), but so far we have no evidence for a nuclear localization of Alien (see below). Moreover, a region strongly enriched for acidic amino acids (Asp) at the N-terminus is notable. This region reveals a significant homology to the Asp-enriched N-terminus of the Drosophila Modulo protein (Krejci et al., 1989).
of Development 57 (1996) 5948 2.2. Alien is highly conserved between insects, plants, and vertebrates The cDNA and the deduced protein sequence was used to search different data bases. We found a very significant homology to a partial sequence of the human TRIP15 protein (gb: L40388), isolated from a Hela cell-derived cDNA library (Lee et al., 1995a). TRIP15 has been proposed to interact with human nuclear receptors, like the thyroid hormone receptor TR/31 and the retinoid X receptor (RXR). The predicted Alien protein, described in this paper, fits very well to TRIP15 with an identity of at about 86% (205 of 235 amino acid residues within the Nterminus, that is available for TRIP15 from the database; Fig. 2). A further homology was found to two partial human sequences (gb: T34712; gb: T347 13), derived from genome projects. Both short partial cDNAs are identical to the TRIP15 gene mentioned above. In conclusion, Alien is the Drosophila homologue of the human TRIP15. Another striking similarity was observed to a partial cDNA, with unknown function, isolated from endosperm embryos from the plant Ricinus communis (gb: T14894; Loo et al., 1995). This cDNA encodes a putative protein, that fits very well to the C-terminal part of Alien, beginning at amino acid 220, with an identity of at about 76%. Onward from amino acid 315 the similarity between Alien and the plant homologue is lower. 2.3. Expression pattern of alien during embryogenesis The expression of alien during embryogenesis was assessed by Northern hybridization using total RNA preparations from various developmental stages. Using an alien cDNA clone of nearly full length as a probe, the result shows that a single transcript of approximately 1400 bp is detectable (Fig. 3). The signal decreases dramatically during embryogenesis. The strong early signal corresponds to a maternal component, whereas the zygotic transcript is much less intense. In situ hybridization to ovaries, using an alien cDNA probe, reveals a high level of transcripts during oogenesis (data not shown). The zygotic transcript is not detectable with the in situ hybridization method, which may be due to the low transcription level as it is visible in the Northern experiment. 2.4. Alien protein is localized in the muscle attachment sites To analyze the function
of alien, we used the deduced
Fig. 1. (A) Sequence of the alien cDNA derived from overlapping clones. The open reading frame and the translation product is shown. The positions of the three introns are indicated by arrows. The putative nuclear localization signal (NLS) is located between amino acids 190-216. (B) Genomic organization of the analyzed region with the three identified transcription units try29F (Paululat, in press), alien and ml!ing stone (Paululat et al., 1995, in preparation). The localization of the P-element upstream of roNing sfone is indicated by an arrow. alien expression is unaffected in the Pmutant fly as shown in Fig. 6A. The sequence of aKen is available in the GenBank database under accession no. U57758.
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A. Goubeaud ei (11./ Mechunisms
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57 (1996) 5948
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A. Goubeaud et al. /Mechanisms of Development 57 (1996) 59-68
62
We used both antibodies to look for the distribution of the Alien protein during embryogenesis. There are no differences between the staining pattern using the antibody against the synthetic peptide or against the bacterial fusion protein. Alien, which seems to be localized predominantly within the cytoplasm, is first detectable in segmentally arranged cells at stage 11 (Fig. 4A). During germband retraction the number of alien-expressing cells increases dramatically (Fig. 4B-D; stage 13-14) and remains restricted to the epidermis. These cells form the apodemes in which individual muscles insert. During this developmental period the intersegmental apodemes (iapo) show a strong staining, while the cells of the two intrasegmental apodemes (ina 1 and ina 2), and the cells of
amino acid sequence to synthesize a 15 amino acid peptide to generate an antibody against Alien. This peptide corresponds to residues 241-256 of the Alien protein sequence and was coupled to KLH before injection (see Section 4). The sera of the rabbits were tested against an Alien fusion protein (amino acids Sl-360), expressed in E. coli, which was also used to generate an antibody (see Section 4). Both Alien antibodies detect one major band at approximately 42 kDa in embryonic protein extracts, probed on a Western blot (data not shown). This is in agreement with the molecular weight of the predicted Alien protein and confirms the results of the Northern analysis, that only one major gene product exists during embryogenesis. DOFMCOD
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Fig. 2. Sequence comparison of the putative Alien (A), the human TRIP15 (B) (Lee et al., 1995a. partial sequence) T14894 from Ricinus communis (C) (Loo et al., 1995).
360 234 152 and the translated
partial cDNA
A. Goubeaud et al. /Mechanisms
of Development 57 (1996) 59-68 embryo to demonstrate demes.
63
that Alien is detectable
in all apo-
2.5. ah’en is expressed in the apodemes of the pharynx and the cephalic muscles
b
Fig. 3. Northern blot analysis of the alien gene. Fifty ,ug of total RNA was used in each lane and hybridized with 32P~labelled cDNA alien insert (a). To visualize the amount of RNA in each lane we rehybridized the filter with Rp49 (b) (see Section 4). The &en transcript is at about 1.4 kb in length. The gel was loaded with embryonic RNA from O-4 h (lane 1). 4-8 h (lane 2). 8-12 h (lane 3). 12-16 h (lane 4). 1620 h (lane 5).
the apodemes for the lateral transversal muscles exhibit weaker staining (Fig. 4E). We indicate the apodemes for the lateral transversal muscles, using the number of the attached muscle, according to the nomenclature developed by Bate (1990). Onward from stage 16, when the complete muscle pattern is established, all apodemes show a high level of Alien protein (Fig. 4E-H). The iapo, ina 1, ina 2, and the attachments of muscle 21-23 are indicated. In a dorsal view, the intersegmental apodemes (ina), which stop in front of the dorsal midline, are visible (Fig. 4F,G, higher magnification). A small cluster of cells at the dorsal midline anterior to the lack of the intersegmental apodemes and to the segmental border was strongly stained (Fig. 4G). The position of this apodemes, called lp according to the terminology of Armand et al. (1994), corresponds to a second attachment of muscle 1, reflecting the morphology of muscle 1 (according to the muscle numbering system of Bate, 1990) which runs parallel to the dorsal midline and splits at the posterior end into two parts. The major part attaches to the intersegmental apodemes and the minor part attaches anterior to the segmental border, just at the position were the lp staining of Alien was found. The lp apodemes are not visible in the thoracic segments (Fig. 4F,G). To elucidate this situation, we performed a double staining to visualize the apodemes with anti-Alien, and the underlying muscles with anti-B3 tubulin (Fig. 4G). The morphology of the most dorsal external muscle (muscle 1; Bate, 1990) is different in the thoracic and abdominal segments (arrowheads in Fig. 4G). A splitting into two attachments is only obvious in the abdominal segments which is coincident with the occurrence of lp apodemes (arrows in Fig. 4G). Fig. 4H shows a ventral view of a late stage 16
Beside the expression of Alien in all apodemes of the thoracic and abdominal segments, we found a specific staining in the pharyngeal apodemes, and in the epidermis of the head region (Fig. 5A-C). A double staining with an antibody against /33 tubulin, to visualize the pharyngeal and cephalic muscles, and anti-Alien (Fig. 5A,B), demonstrates that cells located most dorsally to the pharyngeal muscles cells express alien. Fig. 5C shows that these cells are organized in two rows (arrow in Fig. 5B,C). These are the apodemes for the pharynx that is attached to the dorsal ectoderm of the embryo. Another alien expression was observed in cell rows most ventral of the pharynx (not shown), which are the attachment sites of the pharynx to the roof of the mouth. Moreover, cells in the epidermis of the head reveal an Alien staining. These are the apodemes for the dorsal prothoracic pharyngeal muscle (dppm) and the ventral prothoracic pharyngeal muscle (vppm) (arrowheads in Fig. 5B,C). 2.6. alien expression is not induced by muscle attachment It was known that cells embedded in a cell-cell contact scenario regulate the transcriptional activity of some of their genes through an induction, given by the cell-cell contact. Therefore, we address the question whether alien expression in the apodemes is depending on the attachment of the underlying muscles which arise from the mesodermal germlayer. For this purpose, we used the advantages of mutant strains. The previously described rolling stone mutant shows a dramatically reduced set of muscles. This phenotype is caused by the inability of the myoblasts to fuse with muscle precursors into myotubes (Paululat et al., 1995). Therefore, we address the question whether the alien expression within the apodemes is affected in the mutant background. Fig. 6A shows a homozygous rolling stone embryo. Alien is properly expressed in apodemes without underlying muscles. Furthermore, we analyzed the Alien distribution in mutants, where the mesoderm is affected more generally. In twist and snail mutant embryos the development of the epidermis is only slightly disarranged, but the mesoderm formation reveals strong defects. Muscle fibers are more or less absent in twist and snail mutants. Fig. 6B,C demonstrates that alien is expressed in twist and snail homozygous mutant embryos. Despite the fact that the morphology of the epidermis is slightly disordered in snail (Fig. 6B) and twist mutant (Fig. 6C) embryos, it is clearly visible that the Alien protein is located in apodemes as seen in the wild type. Therefore, it is clear that alien expression in the
64
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et al. /Mechanisms
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57 (1996)
5948
Fig. 4 Developmental distribution of the Alien protein in the Drosophila embryo. Whole-mount staining was carried out using anti-Alien antibodies in embryos at stage 11-12 (A); stage 13 (B); stage 14 (C); stage 15 (D); stage 16 (E-H); stages according to Campos-Ortega and Hartenstein (1985). At stage 1 l-12 (A) segmentally arranged cells are labelled (arrows). A weak background staining is visible in the salivary gland anlagen (see arrowhead in (D)). During germband retraction (B-D) the number of alien-expressing cells increase (arrows). First, the intersegmental apodemes were stained (iapo), than, in a lateral view, the apodemes of the lateral transversal muscles are stained. Onward of stage 15-16 all apodemes arc visible. The apodemes ina 1, ina 2, iapo and the apodemes of muscles 21-23 are labelled (E). In a dorsal view (F,G) of a stage 16 embryo the intrasegmental apodemes reveal an interruption at the dorsal midline. The anterior cluster of apodem cells (labelled Ip) in the abdominal segments are shown clearly in (G) at higher magnification (arrows). The embryo was stained with a#l3 tubulin-specific antibody to detect the muscles (black, arrowheads) and the anti-alien antibody to detect the apodemes (brown). The missing of lp apodemes (arrow) in the thoracic segments corresponds to the different morphology of the most dorsal muscles in the thorax and the abdomen.
A. Goubeuud
et (11./Mechanisms
apodemes is independent from the ventral furrow formation and mesoderm differentiation. 2.7. alien is expressed in the stripeI
mutant
stripe encodes a transcription factor of the zinc finger family. During embryogenesis stripe is expressed in the developing muscle attachment sites (Lee et al., 1995b; Frommer et al., 1996). Thus, Stripe may be a potential regulator of Alien. In stripe16, a small deletion removes the gene. We addressed the question whether alien is expressed in the stripe mutant background. Fig. 6D demonstrates, that Alien is obvious at the muscle attachments. Therefore, alien expression is independent of Stripe. 3. Discussion In this paper we described the cloning and the characterization of the novel gene alien, that is expressed in all muscle attachment sites during embryogenesis in Drosophila. 3.1. Alien is highly conserved between plants, Drosophila and humans The amino acid sequence of Alien is extremely conserved between different organisms. The database analysis revealed homologues sequences from a plant (Ricinus communis, gb: T14894) and humans (gb: L40388 (TRIPIS); gb: T34712; gb: T34713). In spite of the fact that these sequences derived from partial cDNAs, the conservation on protein level is extremely high (>76% for Ricinus, >86% for TRIPlS; Fig. 2). The TRIP15 sequence is the only one for which a function is postulated. To find TRIP15, the authors (Lee et al., 1995a) used the yeast two-hybrid system to isolate human proteins that interact with the rat thyroid hormone receptor. Interaction was observed in cells grown in the absence of hormone, but not in its presence. Whether this gene indeed encodes a protein, that interacts with steroid receptors remains to be clarified. So far, for the Drosophila attachment cells a relevance of steroid receptors is not known. The conservation of Alien and TRIP15 may be taken as evidence for a role of steroid receptors in these tissues. As far as we could analyze the intracellular distribution, Alien seems
Fig. 5. The Alien protein staining in the apodemes of the pharynx. (A,B) The same stage 15 embryo double stained with an anti-/33 tubulin antibody to visualize muscle cells (black) and the anti-Alien antibody (red-brown) focused on the pharynx muscles (A) or more dorsally on the Alien-positive cells (arrow) (B). (C) Dorsal view of a stage 15 embryo, comparable to (A,B), but a single staining for Alien was used to resolve the two rows of cells dorsal to the pharynx which express alien (arrow). These cells are located at positions where the pharynx is attached to the epidermis. In (B,C) a further staining in the epidermis is visible. These cells are the apodemes for external head muscles (arrowheads).
c$Development
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Fig. 6. Alien staining tachments.
pattern in homozygous
rolling stone (A), snai! (B), twist (C), and stripeI
to be located predominantly in the cytoplasm of the muscle attachment cells. Probably Alien is involved in modifying the receptor within the cytoplasm. So far, alien-specific mutations are not available. Tested deletions in that region do not remove the alien function, respectively. Nevertheless, we plan to perform a mutagenesis to isolate alien-specific mutants to analyze the alien function in a further project. Potential defects may concern the development, as well as the differentiation of the embryonic apodemes. The potential role of Alien as a thyroid receptor-interacting protein led us assume that downstream genes of this particular transcriptional cascade may be influenced in its expression. 3.2. Expression
of alien during embryogenesis
As a first step of our analysis we used the antibodies against Alien to visualize the protein distribution during embryogenesis. We could show that Alien is detectable very early at stage 11 in cells of the epidermis. During germband retraction the number of Alien-expressing cells increases dramatically and gives a complete pattern of the embryonic apodemes. The Alien distribution coincides with the timing of muscle attachment to the epidermis, that occurs between 10 and 16 h of development. Therefore, we addressed the question whether alien expression is induced by the underlying muscles. Our experiments using rolling stone, twist, and snail mutant embryos demonstrate that the transcription of alien is not induced by the attachment of the underlying muscles as it was shown for the 81 tub&in gene (Buttgereit, 1993). Delilah (Armand et al., 1994) Stripe (Lee et al.,
(D) mutant embryos. Arrows indicate the muscle at-
1995b; Frommer et al., 1996) and Extramacrochaetae (Cubas et al., 1994) are transcription factors, expressed in the apodemes, and therefore potential regulators of alien. Only Delilah and Stripe were found in the majority of the apodemes and probably at the right time. We could show that alien is expressed in the stripe mutant (Fig. 6D), therefore Stripe is not an activator of alien. Furthermore, this result indicates that the apodemes are properly developed in stripe 16, therefore Stripe seems to be involved in transcriptional control within the already formed cells, rather than in the development of the apodem cells. So far, mutants for delilah are not available, but further analysis will show whether alien and delilah are expressed in overlapping sets of apodemes and a similar time course. Interestingly, Delilah, a basic helix loop helix protein has the potential to heterodimerize with E12like proteins, and from the sequence analysis the authors predict that Delilah exhibits optimal binding to an E-boxrelated sequence (CANNTG), different from other HLH proteins. Nevertheless, the alien gene contains several Eboxes in the upstream region of the putative translational start codon (data not shown). A detailed promoter analysis will show whether these E-boxes are essential for the transcriptional regulation of the alien gene through bHLH proteins. 4. Experimental
procedures
4. I. Drosophila stocks The following fly stocks were used: the snail allele (2288 = Df(2R)sna) and the twist allele (2213 = twiSm)
A. Goubeaud et al. /Mechanisms
were obtained from the Bloomington stock-keeping centre, and furthermore obtained from Dr. R. Reuter (Tiibingen) who has balanced these line with a marked CyO chromosome. stripeJ6, a small deficiency for the stripe gene, was kindly provided from Dr. N. Brown (Cambridge, UK). The rolling stone allele (rostz3) was previously described (Paululat et al., 1995). The wild type Oregon S stock was used from our stock in Marburg. 4.2. Cloning and sequencing
of alien
All standard molecular biology procedures were performed according to Sambrook et al. (1989). We used an 11 kb genomic fragment, isolated from a lambda gt 11 genomic library (gift from Dr. D. Tautz, Miinchen, Germany) as a probe to screen an embryonic O-16 h cDNA library (gift from Dr. Bernd Hovemann, Bochum, Germany). The cDNA inserts were subcloned into Bluescript plasmid vector (Stratagene) or amplified by PCR, using primers deduced from the lac Z gene sequence, flanking the insertion sites of lambda gt 11, as the Eco RI sites used to establish the library were not always intact. The cDNA sequence was determined by sequencing both strands of overlapping clones using the dideoxy chaintermination method (sequencing kit, Pharmacia). Exonintron boundaries were analysed by sequence comparison of cDNA and genomic clones. 4.3. Northern blot analysis RNA was prepared from different developmental stages using the RNAzol method. From each developmental stage, 5Opg of total RNA were separated on formaldehyde/agarose gels, transferred to nylon membranes (Amersham’s Hybond N). Hybridization was at 42°C overnight in 50% Formamid, 5 X SSC, 5 X Denhardt’s solution. The filters were washed twice in 2 X SSC/O.l% SDS at room temperature and four times in 2 X SSC/ 0.1% SDS at 42°C before autoradiography. To visualize the amount of RNA in each lane, we rehybridized the filter with RP49 (O’Connell, 1984). The detected transcript size was determined using a RNA size ladder (BRL).
of Development 57 (I 996) 59-68
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we performed Western blots loaded with the synthetic peptide, an ITPG-induced fusion protein extract from E. coli containing the pQE31 plasmid (Quiagen) with a fragment of the alien cDNA corresponding to amino acids 81-360 of the deduced protein sequence. The serum of immunized rabbits recognizes specifically the peptide as well as the bacteria1 translated Alien fusion protein (data not shown). To generate an antibody against the bacteria1 fusion protein, we injected approximately 50 ng of the isolated and purified induced protein into rabbits. After boosting (3-4 times), the serum was tested. Without further purification, only after 2-3 h preincubation against fixed embryos, the serum shows a specific staining at a final dilution of I:1000 to 1:5000, when used for antibody stainings of embryos (see below). We found no differences in the staining pattern using the antibody against the bacterial fusion protein or against the synthetic peptide. 4.5. Antibody staining of embryos Eggs laid by flies of the appropriate genetic constitution were collected over a 24 h period on agar-apple juice plates. Eggs were dechorionized, permeabilized and fixed essentially as described by Leiss et al. (1988). After washing and blocking in BBT (0.15% crystalline BSA, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 40 mM MgC12, 20 mM glucose, 50 mM sucrose, 0.1% Tween 20), the eggs were incubated overnight with an appropriate dilution of the antibody. Anti-Alien serum was used after a preincubation with fixed embryos for at least 2 h at a final dilution of 1: 1000 to 1:5000. The preimmunserum shows no staining at any dilution. For staining of mesodermal derivatives, the anti-/?3 tubulin antibody was used (Leiss et al., 1988). The bound antibody was detected with a biotinylated secondary antibody and stained with the Vectastain ABC Elite-kit (Vector Labs.) using diaminobenzidine as detection agent. Double stainings were performed as described by Lawrence et al. (1987). The stained embryos were embedded in Epon and photos taken under Nomarski optics with a Zeiss Axiophot microscope (Kodak, Ektar 25). Acknowledgements
4.4. Generation
of antibodies
The peptide CGG KMH LRE GEF EKA H, which corresponds to residues 241-256 of the deduced Alien protein sequence, was synthesized, coupled to KLH and injected into rabbits using the commercial service of Eurogentec (Belgium). The serum was used directly for embryonic whole mount antibody stainings and Western blots without further purification at dilutions between 1:lOOO and 1: 10 000. Control experiments with the preimmunserum at dilutions from 1: 10 to 1:5000 show no specific staining. To verify the specificity of the antibody,
We thank R. Hyland for excellent technical assistance and D. Buttgereit for stimulating discussions and for critical reading of the manuscript. The work was supported by grants from the Deutsche Forschungsgemeinschaft (Re628/7-l/7-2) and the Fonds der Chemischen Industrie to R.R.-P. References Armand,P., Knapp, AC., Hirsch, A.J., Wieschaus, (1994) Mol. Cell. Biol. 14, 41454154.
E.F. and
Cole, M.D.
68
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Bate, M. (1990). Development 110, 791-804. Bogaert, T., Brown, N. and Wilcox, M. (1987) Cell 51,929-940. Brown, N.H. (1994) Development 120, 1221-1231. Buttgereit, D., L&s, D., Michiels, F. and Renkawitz-Pohl, R. (1991) Mech. Dev. 33, 107-l 18. Buttgereit, D. (1993) J. Cell Sci. 105, 721-727. Campos-Ortega, J.A. and Hartenstein, V. (1985) The Embryonic Development of Drosophila melanogasrer, Springer, Berlin. Cubas, P., Modolell, J. and Ruiz-Gbmez, M. (1994) Development 120, 2555-2565. Fogerty, F.J., Fessler, L.I., Bunch, T.A., Yaron, Y., Parker, C.G., Nelson, R.E., Browner, D.L., Gullberg, D. and Fessler, J.H. (1994) Development 120, 1747-1758. Frommer, E., Vorbniggen, E., Pasta, E., Jackie, H. and Volk, T. (1996) EMBO J. 15, 1642-1649. Krejci, E., Garzino, V., Mary, C., Bennani, N. and Pradel, J. (1989) Nucleic Acids Res. 17, 8101-8116. Lai-Fook, J. (1967) J. Morphol. 123.503-508. Lawrence, P.A., Johnston, P., MacDonald, P. and Struhl, G. (1987) Nature 328.440442. Lee, J.W.. Choi, H.-S.. Gyuris, J.. Brent, R. and Moore, D.D. (1995a) Mol. Endocrinol. 9, 243-254.
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Lee, J.C., Vijay-Raghavan, K., Celniker, SE. and Tanouye, M.A. (1995b) Proc. Nad. Acad. Sci. USA 92, 10344-10348. Leiss, D., Hinz, U., Gasch, A., Mertz, R. and Renkawitz-Pohl, R. (1988) Development 104.525-531. Leptin, M., Bogaert, T., Lehmann, R. and Wilcox, M. (1989) Cell 56, 401-408. Loo, van de F.J., Turner, T. and Somerville, C. (1995) Plant Physiol. 108,1141-1150. Murugasu-Oei, B., Rodrigues, V., Yang, X. and Chia, W. (1995). Genes Dev. 9, 139-l 54. O’Connell, P. and Rosbash, M. (1984) Nucleic Acids Res. 12, 54955513. Paululat, A., Burchard, S. and Renkawitz-Pohl, R. (1995) Development 121,261 l-2620. Paululat, A. (1996) Gene, in press. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Tepass, U. and Hartenstein. V. (1994) Dev. Biol. 161, 563-596. Volk, T. (1992) Development 116, 721-730. Volk. T. and Vijay-Raghavan, K. (1994) Development 120,59-70.
Mechanisms
of Development
57 (1996) 59-68
The Drosophila gene aLien is expressed in the muscle attachment sites during embryogenesis and encodes a protein highly conserved between plants, DrosophiZu and vertebrates Anette Goubeaud,
Stefan Knirr, Renate Renkawitz-Pohl,
Achim Paululat*
Entwicklungsbiologie, Zoologie, FB Biologic. Philipps-Universitiit, Kurl-van-Frisch-SrrcIsse, 35032 Marburg, Germany Received
12 February
1996; revision received I9 March 1996; accepted 20 March 1996
Abstract
We have found a novel gene (alien) that is expressed exclusively in the muscle attachment sites (apodemes) during embryogenesis in Drosophila. Antibodies raised against the Alien protein enable us to follow the developing attachments from stage 1 l/l 2 until stage 16/17. The coding region of the Drosophila alien gene is highly conserved to a gene of unknown function, isolated from a plant (Loo et al., 1995), and to the human TRIP15 gene (Lee et al., 1995). Searching for thyroid receptor interacting proteins, TRZPZ.5 was isolated as a negative regulator. Whether there is a functional correlation to Alien remains to be analyzed. Alien expression is independent of muscle formation, as shown in rolling stone mutant embryos. Even in twist and snail mutants, lacking mesodermal development, alien expression is fairly normal, showing a rather autonomous development of the apodemes. The conservation of alien suggests an important role in differentiation. Keywords: alien; Evolutionary
conservation;
Drosophila;
Muscle attachments;
1. Introduction The attachment of multinucleated myotubes to the epidermis at the muscle attachment sites, the apodemes, results in the final organization of the somatic muscles at the end of embryogenesis. During this process, the membrane of the epidermal cell and the membrane of the muscle cell interact and large adherens junctions are formed (Lai-Fook, 1967; Tepass and Hartenstein, 1994). It was shown that the integrins mediate this cell-cell interaction. Most of the cloned integrin genes encode adapters for the major components of the extracellular matrix, such as lamin, fibronectin or collagen. Furthermore, the integrins were found in the muscle cell membrane and the corresponding tendon cell membrane. For example, the aps2 integrin gene is expressed in the mesoderm and the aps&Jps complex accumulates at the muscle attachment sites (Bogaert et al., 1987). The aPslpPs integrin complex complements this expression pattern and accumulates on
* Corresponding author. Fax: +49 642 e-mail: [email protected].
I 28897 I ;
TRIP 15
the ectodermal side of the muscle attachments; that is, the basal surface of the tendon cell. The absence of this integrin complex results in the detachment of the embryonic muscles from the epidermis, which is clearly evident from myospheroid mutants (mys, BPS-) (Leptin et al., 1989; Brown, 1994), which are missing BPS integrin, and from inflated null mutants lacking the aPS2-integrin (Brown, 1994). At stage 17 both inflated and myospheroid mutant embryos have a similar ‘detached’ phenotype, although the timing of detachment differs (Brown, 1994). Several other genes have been shown to be specifically expressed in the apodemes, like delilah (Armand et al., t994), tiggrin (Fogerty et al., 1994), msp300 (Volk, 1992; Volk and Vijay-Raghavan, 1994), masquerade (Murugasu-Oei et al., 1995), p1 tubutin (Buttgereit, 1991), and srripe (Lee et al., 1995b; Frommer et al., 1996). Some are expressed in other tissues as well. Here we describe the novel Drosophila gene alien, that encodes a hydrophilic 42 kDa protein. During embryogenesis, Alien is localized exclusively in all apodemes of the somatic muscles in the head, thorax, and abdominal segments, as well as in the epidermal attachments of the pharynx. This protein pro-
09254773/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved P11: SO925-4773(96)00532-6
60
A. Goubeaud et al. /Mechanisms
vides, therefore, an excellent marker for all embryonic muscle attachments and allows to follow the progenitor cells from stage 11 onward. The sequence comparison of the deduced Alien protein reveals a striking conservation between Drosophila and vertebrates (humans). Surprisingly, a homologous sequence was found in plants. 2. Results 2. I. Cloning and sequence analysis of alien alien was cloned and identified from a screen for genes involved in embryonic muscle development, based on a lethal P-element insertion line with a muscle-specific phenotype (Paululat et al., 1995). Using standard methods, P-element flanking sequences were cloned. Three transcription units were found within the region (Fig. 1B): try29F encodes a serin protease (Paululat, 1996), rolling stone is responsible for the muscle phenotype (Paululat et al., 1995, in preparation), and the third transcription unit, called alien, is described here. The P element is located 4 kb downstream of the alien gene and we have no evidence that this P-element influences the alien expression (Fig. 1B). Polytene chromosome in situ hybridization using a genomic clone that uncovers alien maps the gene on chromosome II at 29F/30A. Using genomic DNA clones as a probe for an embryonic cDNA library screen, several cDNA clones were obtained that encompass the coding region of the alien transcript, respectively. The sequence of the alien gene that we have determined using different overlapping cDNA clones is shown in Fig. 1A. The positions of three introns, determined by sequence comparison of the cDNA with genomic clones, are indicated. The putative ATG start codon is preceded by an in-frame stop codon, which confirms the translation start. A putative poly A signal was not found in the longest cDNA which we have cloned. However, in the genomic clone uncovering the putative alien trailer several poly A signals are observable (data not shown). The open reading frame has a coding capacity of approximately 42 kDa, which is coincident with our Western blot analysis of embryonic protein extracts (data not shown). The amino acid sequence by itself contains a putative nuclear localization signal (NLS; amino acids 190-216), but so far we have no evidence for a nuclear localization of Alien (see below). Moreover, a region strongly enriched for acidic amino acids (Asp) at the N-terminus is notable. This region reveals a significant homology to the Asp-enriched N-terminus of the Drosophila Modulo protein (Krejci et al., 1989).
of Development 57 (1996) 5948 2.2. Alien is highly conserved between insects, plants, and vertebrates The cDNA and the deduced protein sequence was used to search different data bases. We found a very significant homology to a partial sequence of the human TRIP15 protein (gb: L40388), isolated from a Hela cell-derived cDNA library (Lee et al., 1995a). TRIP15 has been proposed to interact with human nuclear receptors, like the thyroid hormone receptor TR/31 and the retinoid X receptor (RXR). The predicted Alien protein, described in this paper, fits very well to TRIP15 with an identity of at about 86% (205 of 235 amino acid residues within the Nterminus, that is available for TRIP15 from the database; Fig. 2). A further homology was found to two partial human sequences (gb: T34712; gb: T347 13), derived from genome projects. Both short partial cDNAs are identical to the TRIP15 gene mentioned above. In conclusion, Alien is the Drosophila homologue of the human TRIP15. Another striking similarity was observed to a partial cDNA, with unknown function, isolated from endosperm embryos from the plant Ricinus communis (gb: T14894; Loo et al., 1995). This cDNA encodes a putative protein, that fits very well to the C-terminal part of Alien, beginning at amino acid 220, with an identity of at about 76%. Onward from amino acid 315 the similarity between Alien and the plant homologue is lower. 2.3. Expression pattern of alien during embryogenesis The expression of alien during embryogenesis was assessed by Northern hybridization using total RNA preparations from various developmental stages. Using an alien cDNA clone of nearly full length as a probe, the result shows that a single transcript of approximately 1400 bp is detectable (Fig. 3). The signal decreases dramatically during embryogenesis. The strong early signal corresponds to a maternal component, whereas the zygotic transcript is much less intense. In situ hybridization to ovaries, using an alien cDNA probe, reveals a high level of transcripts during oogenesis (data not shown). The zygotic transcript is not detectable with the in situ hybridization method, which may be due to the low transcription level as it is visible in the Northern experiment. 2.4. Alien protein is localized in the muscle attachment sites To analyze the function
of alien, we used the deduced
Fig. 1. (A) Sequence of the alien cDNA derived from overlapping clones. The open reading frame and the translation product is shown. The positions of the three introns are indicated by arrows. The putative nuclear localization signal (NLS) is located between amino acids 190-216. (B) Genomic organization of the analyzed region with the three identified transcription units try29F (Paululat, in press), alien and ml!ing stone (Paululat et al., 1995, in preparation). The localization of the P-element upstream of roNing sfone is indicated by an arrow. alien expression is unaffected in the Pmutant fly as shown in Fig. 6A. The sequence of aKen is available in the GenBank database under accession no. U57758.
qfDeveiopment
A. Goubeaud ei (11./ Mechunisms
A
57 (1996) 5948
61
AAATATTCTAGATTGGCAATAATTTGCTCATAAAGCGTACAAAGCCAAACTCGTAAAATG
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CTGAAGGAGGAGGAGCCCAAAGCGGCGCTGGCCAGTTTCCAAAAGGTGCTCGACCTGGAA L
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AATGGCGAAAAAGGAGAGTGGGGCTTCAAGGCGCTGAAGCAGATGATCAAGATCAACTTT
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A. Goubeaud et al. /Mechanisms of Development 57 (1996) 59-68
62
We used both antibodies to look for the distribution of the Alien protein during embryogenesis. There are no differences between the staining pattern using the antibody against the synthetic peptide or against the bacterial fusion protein. Alien, which seems to be localized predominantly within the cytoplasm, is first detectable in segmentally arranged cells at stage 11 (Fig. 4A). During germband retraction the number of alien-expressing cells increases dramatically (Fig. 4B-D; stage 13-14) and remains restricted to the epidermis. These cells form the apodemes in which individual muscles insert. During this developmental period the intersegmental apodemes (iapo) show a strong staining, while the cells of the two intrasegmental apodemes (ina 1 and ina 2), and the cells of
amino acid sequence to synthesize a 15 amino acid peptide to generate an antibody against Alien. This peptide corresponds to residues 241-256 of the Alien protein sequence and was coupled to KLH before injection (see Section 4). The sera of the rabbits were tested against an Alien fusion protein (amino acids Sl-360), expressed in E. coli, which was also used to generate an antibody (see Section 4). Both Alien antibodies detect one major band at approximately 42 kDa in embryonic protein extracts, probed on a Western blot (data not shown). This is in agreement with the molecular weight of the predicted Alien protein and confirms the results of the Northern analysis, that only one major gene product exists during embryogenesis. DOFMCOD
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Fig. 2. Sequence comparison of the putative Alien (A), the human TRIP15 (B) (Lee et al., 1995a. partial sequence) T14894 from Ricinus communis (C) (Loo et al., 1995).
360 234 152 and the translated
partial cDNA
A. Goubeaud et al. /Mechanisms
of Development 57 (1996) 59-68 embryo to demonstrate demes.
63
that Alien is detectable
in all apo-
2.5. ah’en is expressed in the apodemes of the pharynx and the cephalic muscles
b
Fig. 3. Northern blot analysis of the alien gene. Fifty ,ug of total RNA was used in each lane and hybridized with 32P~labelled cDNA alien insert (a). To visualize the amount of RNA in each lane we rehybridized the filter with Rp49 (b) (see Section 4). The &en transcript is at about 1.4 kb in length. The gel was loaded with embryonic RNA from O-4 h (lane 1). 4-8 h (lane 2). 8-12 h (lane 3). 12-16 h (lane 4). 1620 h (lane 5).
the apodemes for the lateral transversal muscles exhibit weaker staining (Fig. 4E). We indicate the apodemes for the lateral transversal muscles, using the number of the attached muscle, according to the nomenclature developed by Bate (1990). Onward from stage 16, when the complete muscle pattern is established, all apodemes show a high level of Alien protein (Fig. 4E-H). The iapo, ina 1, ina 2, and the attachments of muscle 21-23 are indicated. In a dorsal view, the intersegmental apodemes (ina), which stop in front of the dorsal midline, are visible (Fig. 4F,G, higher magnification). A small cluster of cells at the dorsal midline anterior to the lack of the intersegmental apodemes and to the segmental border was strongly stained (Fig. 4G). The position of this apodemes, called lp according to the terminology of Armand et al. (1994), corresponds to a second attachment of muscle 1, reflecting the morphology of muscle 1 (according to the muscle numbering system of Bate, 1990) which runs parallel to the dorsal midline and splits at the posterior end into two parts. The major part attaches to the intersegmental apodemes and the minor part attaches anterior to the segmental border, just at the position were the lp staining of Alien was found. The lp apodemes are not visible in the thoracic segments (Fig. 4F,G). To elucidate this situation, we performed a double staining to visualize the apodemes with anti-Alien, and the underlying muscles with anti-B3 tubulin (Fig. 4G). The morphology of the most dorsal external muscle (muscle 1; Bate, 1990) is different in the thoracic and abdominal segments (arrowheads in Fig. 4G). A splitting into two attachments is only obvious in the abdominal segments which is coincident with the occurrence of lp apodemes (arrows in Fig. 4G). Fig. 4H shows a ventral view of a late stage 16
Beside the expression of Alien in all apodemes of the thoracic and abdominal segments, we found a specific staining in the pharyngeal apodemes, and in the epidermis of the head region (Fig. 5A-C). A double staining with an antibody against /33 tubulin, to visualize the pharyngeal and cephalic muscles, and anti-Alien (Fig. 5A,B), demonstrates that cells located most dorsally to the pharyngeal muscles cells express alien. Fig. 5C shows that these cells are organized in two rows (arrow in Fig. 5B,C). These are the apodemes for the pharynx that is attached to the dorsal ectoderm of the embryo. Another alien expression was observed in cell rows most ventral of the pharynx (not shown), which are the attachment sites of the pharynx to the roof of the mouth. Moreover, cells in the epidermis of the head reveal an Alien staining. These are the apodemes for the dorsal prothoracic pharyngeal muscle (dppm) and the ventral prothoracic pharyngeal muscle (vppm) (arrowheads in Fig. 5B,C). 2.6. alien expression is not induced by muscle attachment It was known that cells embedded in a cell-cell contact scenario regulate the transcriptional activity of some of their genes through an induction, given by the cell-cell contact. Therefore, we address the question whether alien expression in the apodemes is depending on the attachment of the underlying muscles which arise from the mesodermal germlayer. For this purpose, we used the advantages of mutant strains. The previously described rolling stone mutant shows a dramatically reduced set of muscles. This phenotype is caused by the inability of the myoblasts to fuse with muscle precursors into myotubes (Paululat et al., 1995). Therefore, we address the question whether the alien expression within the apodemes is affected in the mutant background. Fig. 6A shows a homozygous rolling stone embryo. Alien is properly expressed in apodemes without underlying muscles. Furthermore, we analyzed the Alien distribution in mutants, where the mesoderm is affected more generally. In twist and snail mutant embryos the development of the epidermis is only slightly disarranged, but the mesoderm formation reveals strong defects. Muscle fibers are more or less absent in twist and snail mutants. Fig. 6B,C demonstrates that alien is expressed in twist and snail homozygous mutant embryos. Despite the fact that the morphology of the epidermis is slightly disordered in snail (Fig. 6B) and twist mutant (Fig. 6C) embryos, it is clearly visible that the Alien protein is located in apodemes as seen in the wild type. Therefore, it is clear that alien expression in the
64
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et al. /Mechanisms
of Development
57 (1996)
5948
Fig. 4 Developmental distribution of the Alien protein in the Drosophila embryo. Whole-mount staining was carried out using anti-Alien antibodies in embryos at stage 11-12 (A); stage 13 (B); stage 14 (C); stage 15 (D); stage 16 (E-H); stages according to Campos-Ortega and Hartenstein (1985). At stage 1 l-12 (A) segmentally arranged cells are labelled (arrows). A weak background staining is visible in the salivary gland anlagen (see arrowhead in (D)). During germband retraction (B-D) the number of alien-expressing cells increase (arrows). First, the intersegmental apodemes were stained (iapo), than, in a lateral view, the apodemes of the lateral transversal muscles are stained. Onward of stage 15-16 all apodemes arc visible. The apodemes ina 1, ina 2, iapo and the apodemes of muscles 21-23 are labelled (E). In a dorsal view (F,G) of a stage 16 embryo the intrasegmental apodemes reveal an interruption at the dorsal midline. The anterior cluster of apodem cells (labelled Ip) in the abdominal segments are shown clearly in (G) at higher magnification (arrows). The embryo was stained with a#l3 tubulin-specific antibody to detect the muscles (black, arrowheads) and the anti-alien antibody to detect the apodemes (brown). The missing of lp apodemes (arrow) in the thoracic segments corresponds to the different morphology of the most dorsal muscles in the thorax and the abdomen.
A. Goubeuud
et (11./Mechanisms
apodemes is independent from the ventral furrow formation and mesoderm differentiation. 2.7. alien is expressed in the stripeI
mutant
stripe encodes a transcription factor of the zinc finger family. During embryogenesis stripe is expressed in the developing muscle attachment sites (Lee et al., 1995b; Frommer et al., 1996). Thus, Stripe may be a potential regulator of Alien. In stripe16, a small deletion removes the gene. We addressed the question whether alien is expressed in the stripe mutant background. Fig. 6D demonstrates, that Alien is obvious at the muscle attachments. Therefore, alien expression is independent of Stripe. 3. Discussion In this paper we described the cloning and the characterization of the novel gene alien, that is expressed in all muscle attachment sites during embryogenesis in Drosophila. 3.1. Alien is highly conserved between plants, Drosophila and humans The amino acid sequence of Alien is extremely conserved between different organisms. The database analysis revealed homologues sequences from a plant (Ricinus communis, gb: T14894) and humans (gb: L40388 (TRIPIS); gb: T34712; gb: T34713). In spite of the fact that these sequences derived from partial cDNAs, the conservation on protein level is extremely high (>76% for Ricinus, >86% for TRIPlS; Fig. 2). The TRIP15 sequence is the only one for which a function is postulated. To find TRIP15, the authors (Lee et al., 1995a) used the yeast two-hybrid system to isolate human proteins that interact with the rat thyroid hormone receptor. Interaction was observed in cells grown in the absence of hormone, but not in its presence. Whether this gene indeed encodes a protein, that interacts with steroid receptors remains to be clarified. So far, for the Drosophila attachment cells a relevance of steroid receptors is not known. The conservation of Alien and TRIP15 may be taken as evidence for a role of steroid receptors in these tissues. As far as we could analyze the intracellular distribution, Alien seems
Fig. 5. The Alien protein staining in the apodemes of the pharynx. (A,B) The same stage 15 embryo double stained with an anti-/33 tubulin antibody to visualize muscle cells (black) and the anti-Alien antibody (red-brown) focused on the pharynx muscles (A) or more dorsally on the Alien-positive cells (arrow) (B). (C) Dorsal view of a stage 15 embryo, comparable to (A,B), but a single staining for Alien was used to resolve the two rows of cells dorsal to the pharynx which express alien (arrow). These cells are located at positions where the pharynx is attached to the epidermis. In (B,C) a further staining in the epidermis is visible. These cells are the apodemes for external head muscles (arrowheads).
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A. Goubeaud et al. /Mechanisms of Development 57 (I 996) 59-68
66
Fig. 6. Alien staining tachments.
pattern in homozygous
rolling stone (A), snai! (B), twist (C), and stripeI
to be located predominantly in the cytoplasm of the muscle attachment cells. Probably Alien is involved in modifying the receptor within the cytoplasm. So far, alien-specific mutations are not available. Tested deletions in that region do not remove the alien function, respectively. Nevertheless, we plan to perform a mutagenesis to isolate alien-specific mutants to analyze the alien function in a further project. Potential defects may concern the development, as well as the differentiation of the embryonic apodemes. The potential role of Alien as a thyroid receptor-interacting protein led us assume that downstream genes of this particular transcriptional cascade may be influenced in its expression. 3.2. Expression
of alien during embryogenesis
As a first step of our analysis we used the antibodies against Alien to visualize the protein distribution during embryogenesis. We could show that Alien is detectable very early at stage 11 in cells of the epidermis. During germband retraction the number of Alien-expressing cells increases dramatically and gives a complete pattern of the embryonic apodemes. The Alien distribution coincides with the timing of muscle attachment to the epidermis, that occurs between 10 and 16 h of development. Therefore, we addressed the question whether alien expression is induced by the underlying muscles. Our experiments using rolling stone, twist, and snail mutant embryos demonstrate that the transcription of alien is not induced by the attachment of the underlying muscles as it was shown for the 81 tub&in gene (Buttgereit, 1993). Delilah (Armand et al., 1994) Stripe (Lee et al.,
(D) mutant embryos. Arrows indicate the muscle at-
1995b; Frommer et al., 1996) and Extramacrochaetae (Cubas et al., 1994) are transcription factors, expressed in the apodemes, and therefore potential regulators of alien. Only Delilah and Stripe were found in the majority of the apodemes and probably at the right time. We could show that alien is expressed in the stripe mutant (Fig. 6D), therefore Stripe is not an activator of alien. Furthermore, this result indicates that the apodemes are properly developed in stripe 16, therefore Stripe seems to be involved in transcriptional control within the already formed cells, rather than in the development of the apodem cells. So far, mutants for delilah are not available, but further analysis will show whether alien and delilah are expressed in overlapping sets of apodemes and a similar time course. Interestingly, Delilah, a basic helix loop helix protein has the potential to heterodimerize with E12like proteins, and from the sequence analysis the authors predict that Delilah exhibits optimal binding to an E-boxrelated sequence (CANNTG), different from other HLH proteins. Nevertheless, the alien gene contains several Eboxes in the upstream region of the putative translational start codon (data not shown). A detailed promoter analysis will show whether these E-boxes are essential for the transcriptional regulation of the alien gene through bHLH proteins. 4. Experimental
procedures
4. I. Drosophila stocks The following fly stocks were used: the snail allele (2288 = Df(2R)sna) and the twist allele (2213 = twiSm)
A. Goubeaud et al. /Mechanisms
were obtained from the Bloomington stock-keeping centre, and furthermore obtained from Dr. R. Reuter (Tiibingen) who has balanced these line with a marked CyO chromosome. stripeJ6, a small deficiency for the stripe gene, was kindly provided from Dr. N. Brown (Cambridge, UK). The rolling stone allele (rostz3) was previously described (Paululat et al., 1995). The wild type Oregon S stock was used from our stock in Marburg. 4.2. Cloning and sequencing
of alien
All standard molecular biology procedures were performed according to Sambrook et al. (1989). We used an 11 kb genomic fragment, isolated from a lambda gt 11 genomic library (gift from Dr. D. Tautz, Miinchen, Germany) as a probe to screen an embryonic O-16 h cDNA library (gift from Dr. Bernd Hovemann, Bochum, Germany). The cDNA inserts were subcloned into Bluescript plasmid vector (Stratagene) or amplified by PCR, using primers deduced from the lac Z gene sequence, flanking the insertion sites of lambda gt 11, as the Eco RI sites used to establish the library were not always intact. The cDNA sequence was determined by sequencing both strands of overlapping clones using the dideoxy chaintermination method (sequencing kit, Pharmacia). Exonintron boundaries were analysed by sequence comparison of cDNA and genomic clones. 4.3. Northern blot analysis RNA was prepared from different developmental stages using the RNAzol method. From each developmental stage, 5Opg of total RNA were separated on formaldehyde/agarose gels, transferred to nylon membranes (Amersham’s Hybond N). Hybridization was at 42°C overnight in 50% Formamid, 5 X SSC, 5 X Denhardt’s solution. The filters were washed twice in 2 X SSC/O.l% SDS at room temperature and four times in 2 X SSC/ 0.1% SDS at 42°C before autoradiography. To visualize the amount of RNA in each lane, we rehybridized the filter with RP49 (O’Connell, 1984). The detected transcript size was determined using a RNA size ladder (BRL).
of Development 57 (I 996) 59-68
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we performed Western blots loaded with the synthetic peptide, an ITPG-induced fusion protein extract from E. coli containing the pQE31 plasmid (Quiagen) with a fragment of the alien cDNA corresponding to amino acids 81-360 of the deduced protein sequence. The serum of immunized rabbits recognizes specifically the peptide as well as the bacteria1 translated Alien fusion protein (data not shown). To generate an antibody against the bacteria1 fusion protein, we injected approximately 50 ng of the isolated and purified induced protein into rabbits. After boosting (3-4 times), the serum was tested. Without further purification, only after 2-3 h preincubation against fixed embryos, the serum shows a specific staining at a final dilution of I:1000 to 1:5000, when used for antibody stainings of embryos (see below). We found no differences in the staining pattern using the antibody against the bacterial fusion protein or against the synthetic peptide. 4.5. Antibody staining of embryos Eggs laid by flies of the appropriate genetic constitution were collected over a 24 h period on agar-apple juice plates. Eggs were dechorionized, permeabilized and fixed essentially as described by Leiss et al. (1988). After washing and blocking in BBT (0.15% crystalline BSA, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 40 mM MgC12, 20 mM glucose, 50 mM sucrose, 0.1% Tween 20), the eggs were incubated overnight with an appropriate dilution of the antibody. Anti-Alien serum was used after a preincubation with fixed embryos for at least 2 h at a final dilution of 1: 1000 to 1:5000. The preimmunserum shows no staining at any dilution. For staining of mesodermal derivatives, the anti-/?3 tubulin antibody was used (Leiss et al., 1988). The bound antibody was detected with a biotinylated secondary antibody and stained with the Vectastain ABC Elite-kit (Vector Labs.) using diaminobenzidine as detection agent. Double stainings were performed as described by Lawrence et al. (1987). The stained embryos were embedded in Epon and photos taken under Nomarski optics with a Zeiss Axiophot microscope (Kodak, Ektar 25). Acknowledgements
4.4. Generation
of antibodies
The peptide CGG KMH LRE GEF EKA H, which corresponds to residues 241-256 of the deduced Alien protein sequence, was synthesized, coupled to KLH and injected into rabbits using the commercial service of Eurogentec (Belgium). The serum was used directly for embryonic whole mount antibody stainings and Western blots without further purification at dilutions between 1:lOOO and 1: 10 000. Control experiments with the preimmunserum at dilutions from 1: 10 to 1:5000 show no specific staining. To verify the specificity of the antibody,
We thank R. Hyland for excellent technical assistance and D. Buttgereit for stimulating discussions and for critical reading of the manuscript. The work was supported by grants from the Deutsche Forschungsgemeinschaft (Re628/7-l/7-2) and the Fonds der Chemischen Industrie to R.R.-P. References Armand,P., Knapp, AC., Hirsch, A.J., Wieschaus, (1994) Mol. Cell. Biol. 14, 41454154.
E.F. and
Cole, M.D.
68
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Bate, M. (1990). Development 110, 791-804. Bogaert, T., Brown, N. and Wilcox, M. (1987) Cell 51,929-940. Brown, N.H. (1994) Development 120, 1221-1231. Buttgereit, D., L&s, D., Michiels, F. and Renkawitz-Pohl, R. (1991) Mech. Dev. 33, 107-l 18. Buttgereit, D. (1993) J. Cell Sci. 105, 721-727. Campos-Ortega, J.A. and Hartenstein, V. (1985) The Embryonic Development of Drosophila melanogasrer, Springer, Berlin. Cubas, P., Modolell, J. and Ruiz-Gbmez, M. (1994) Development 120, 2555-2565. Fogerty, F.J., Fessler, L.I., Bunch, T.A., Yaron, Y., Parker, C.G., Nelson, R.E., Browner, D.L., Gullberg, D. and Fessler, J.H. (1994) Development 120, 1747-1758. Frommer, E., Vorbniggen, E., Pasta, E., Jackie, H. and Volk, T. (1996) EMBO J. 15, 1642-1649. Krejci, E., Garzino, V., Mary, C., Bennani, N. and Pradel, J. (1989) Nucleic Acids Res. 17, 8101-8116. Lai-Fook, J. (1967) J. Morphol. 123.503-508. Lawrence, P.A., Johnston, P., MacDonald, P. and Struhl, G. (1987) Nature 328.440442. Lee, J.W.. Choi, H.-S.. Gyuris, J.. Brent, R. and Moore, D.D. (1995a) Mol. Endocrinol. 9, 243-254.
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Lee, J.C., Vijay-Raghavan, K., Celniker, SE. and Tanouye, M.A. (1995b) Proc. Nad. Acad. Sci. USA 92, 10344-10348. Leiss, D., Hinz, U., Gasch, A., Mertz, R. and Renkawitz-Pohl, R. (1988) Development 104.525-531. Leptin, M., Bogaert, T., Lehmann, R. and Wilcox, M. (1989) Cell 56, 401-408. Loo, van de F.J., Turner, T. and Somerville, C. (1995) Plant Physiol. 108,1141-1150. Murugasu-Oei, B., Rodrigues, V., Yang, X. and Chia, W. (1995). Genes Dev. 9, 139-l 54. O’Connell, P. and Rosbash, M. (1984) Nucleic Acids Res. 12, 54955513. Paululat, A., Burchard, S. and Renkawitz-Pohl, R. (1995) Development 121,261 l-2620. Paululat, A. (1996) Gene, in press. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Tepass, U. and Hartenstein. V. (1994) Dev. Biol. 161, 563-596. Volk, T. (1992) Development 116, 721-730. Volk. T. and Vijay-Raghavan, K. (1994) Development 120,59-70.