Human Molecular Genetics, 2001, Vol. 10, No. 24 2767-2773
© 2001 Oxford University Press
CRB1 has a cytoplasmic domain that is functionally conserved between human and Drosophila
Department of Human Genetics, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands and 1Institüt für Genetik, Heinrich-Heine Universität, Düsseldorf, Germany
Received July 10, 2001; Revised and Accepted September 20, 2001.
DDBJ/EMBL/GenBank accession nos A7043322–A7043325.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The CRB1 gene is expressed specifically in human retina and brain and encodes a protein homologous to the Drosophila Crumbs protein. In crumbs mutant embryos apico-basal polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be partially rescued by expression of just the membrane-bound cytoplasmic domain, and overexpression of this domain in a wild-type background results in a multilayered epidermis. A striking difference between CRB1 and Crumbs was that the latter contains a transmembrane region and a 37 amino acid cytoplasmic domain. Here we describe an alternative splice variant of human CRB1 that encodes a cytoplasmic domain 72% similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary for function in Drosophila are absolutely conserved. Rescuing and overexpression studies in Drosophila show that the cytoplasmic domains are functionally related between these distant species. This suggests that CRB1 organizes an intracellular protein scaffold in the human retina. Human homologues of proteins binding to Crumbs may be part of this complex and represent candidate genes for retinal dystrophies.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa (RP) to Leber congenital amaurosis (LCA) (1–3). Two specific types of RP are caused by mutations in the CRB1 gene, RP with a preserved para-arteriolar retinal pigment epithelium (PPRPE) and RP with Coats-like exudative vasculopathy (1,2). RP with PPRPE is a clinically distinct and severe form of RP, which is characterized by a morphological preservation of the RPE adjacent to and under the retinal arterioles, whereas there is a general loss of the RPE throughout the retina (4). Coats-like exudative vasculopathy is a relatively rare complication of RP, characterized by vascular abnormalities, yellow extravascular lipid depositions and retinal detachment. CRB1 mutations have been detected in 59% (17/29) of unrelated, isolated and autosomal recessive cases of RP with PPRPE (1; unpublished data), in 56% (5/9) of RP patients who had developed Coats-like exudative vasculopathy (2), and in 9–13% of patients with LCA, the most severe retinal dystrophy leading to blindness or severe visual impairment from birth (2,3). In our work, three patients had null mutations on both alleles, suggesting that LCA is the most severe phenotype that can be associated with mutations in the CRB1 gene (2).
The CRB1 gene is expressed in human retina, brain and fetal brain. The protein encoded by CRB1 (GenBank accession no. AF154671) was predicted as an extracellular protein with a signal peptide, 19 EGF-like domains and three laminin A G-like domains. It is likely that these domains interact with other extracellular or transmembrane proteins. CRB1 is homologous to the Drosophila Crumbs protein (1). However, Crumbs is larger, has 30 EGF-like domains, four laminin A G-like domains and is a transmembrane protein with a small, 37 amino acid cytoplasmic domain (5). The largest part of the Crumbs protein (98%) is localized extracellularly.
During embryonic development, Drosophila Crumbs is expressed mainly in the epithelia derived from the ectoderm (5,6). Epithelial cells normally show pronounced apico-basal polarity, demonstrated by a polarized cytoskeleton, an asymmetric distribution of organelles and proteins and the separation of the plasma membrane into distinct apical and basolateral domains. Crumbs is expressed on the apical face of ectodermally-derived epithelia and is particularly concentrated in a narrow region just apical to the zonula adherens (ZA), a belt-like structure encircling the apex of epithelial cells (5,7). The ZA connects apico-laterally localized actin belts of adjacent cells with each other, and thereby participates in cell–cell adhesion and intercellular communication.
Mutations in crumbs are characterized by a severe disruption of ectodermally derived epithelia and extensive cell death in the epidermal primordium and some other epithelia of ectodermal origin (5). Consequently, crumbs mutant embryos fail to develop a continuous cuticle, a secretion product of the epidermis and only develop ‘crumbs’ of cuticle (6). Loss of epithelial polarity in crumbs mutant embryos is associated with a misdistribution of DE-cadherin and Armadillo (8) and consequently the failure to assemble a ZA. The mutant phenotype can be caused by mutations abolishing transcription of crumbs as well as a mutation deleting only the last 23 amino acids of the cytoplasmic domain, emphasizing the importance of this domain (9).
The crumbs mutant phenotype can be partially rescued by GAL4/upstream activator sequence (UAS)-mediated overexpression of full-length Crumbs protein. Major parts of the cuticle are restored, visualized by a contiguous cuticular shield with well-developed denticle belts, characteristic cuticular structures indicative of normal epidermal development (10). A similar degree of rescue was observed when overexpressing only the membrane-bound cytoplasmic domain of Crumbs, tagged with a Myc epitope (crbintra-myc) (10). Furthermore, embryos rescued by overexpression of crbintra-myc show continuous patches of epidermis, in which the ZA appears to be properly organized (11).
Moderate overexpression of either full-length Crumbs or strong overexpression of the membrane-bound cytoplasmic domain (crbintra-myc) in wild-type embryos leads to an abnormal, multilayered epidermis (10,11). The outermost cells still exhibit aspects of polarity, such as polarized distribution of apical membrane marker Stranded at Second (SAS) and basolateral membrane marker Fasciclin III (FASIII), whereas the cells in the inner layer show only basolateral characteristics (11). The cells lose their columnar shape, DE-cadherin localization is affected and a proper ZA fails to form (11). As a consequence, morphogenetic movements of the epidermis such as ‘dorsal closure’ fail, resulting in embryonic lethality. Interestingly, overexpression of crbextra, a construct containing the extracellular domain of Crumbs but lacking the transmembrane and cytoplasmic domain, does not have any obvious consequences for epithelial development (10).
The fundamental role of the cytoplasmic domain of Drosophila crumbs prompted us to re-examine the human CRB1 locus for additional transcripts and protein isoforms. This analysis identified an alternative 3' exon of CRB1 that predicts a transmembrane domain and a 37 amino acid cytoplasmic domain, which is 72% similar to Crumbs. Rescuing experiments in Drosophila crumbs mutant embryos and overexpression studies in wild-type Drosophila show that the cytoplasmic domains are functionally related between these distant species.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Alternative splicing of CRB1
The previously published CRB1 cDNA sequence consists of 4361 bp and encodes a predicted protein of 1376 amino acids (GenBank accession no. AF154671) (1). Screening of EST databases identified 13 human ESTs that showed overlap with the CRB1 cDNA sequence. Interestingly, comparison of the EST sequences revealed that they diverge, suggesting that the CRB1 gene may exhibit alternative splicing at its 3' end. EST sequences all diverged at the same position, which would result in different protein products that differ at their C-termini after amino acid 1335.
Four classes of mRNAs predicted to encode four different proteins can be distinguished (Fig. 1A). Class I molecules contain exons 11a and 11b and encode a 1376 amino acid protein, which was originally described as CRB1 (GenBank accession no. AF154671) (1). The cDNA sequence has a 94 bp 3'-UTR that contains a small poly(A) tail but lacks an upstream polyadenylation signal, which suggests that this sequence does not contain the 3' end of the transcript (1). 3'-RACE experiments using a forward primer in exon 11b resulted in a product with a 704 bp 3'-UTR that contains a poly(A) tail, which is not preceded by a polyadenylation signal. In the database we encountered one cDNA clone from the Soares fetal liver/spleen 1NFLS cDNA library that contains exons 11a, 11b and 12, and has a poly(A) tail that is preceded by a polyadenylation signal (GenBank accession nos R09831 and AY043322). Class II molecules (GenBank accession no. AY043325) contain exons 11a and 12 and are represented by an EST from the Soares fetal liver/spleen 1NFLS cDNA library (GenBank accession nos N59646 and N78199) and an EST from an anaplastic oligodendroglioma cDNA library (GenBank accession no. BF347919). The stop codon is present in exon 12, leading to a 1406 amino acid protein. Class III molecules contain exons 11a, 12a and 12, and are represented by two cDNA clones from the Soares fetal liver/spleen 1NFLS cDNA library (GenBank accession nos AA033939, AY043323, T87786 and T87787). Exon 12a starts 116 bp upstream of the splice acceptor site of exon 12. The stop codon is present in exon 12a, and the cDNA is predicted to encode a 1336 amino acid protein. Class IV molecules contain exons 11a and exon 13, a novel exon located 3 kb downstream of exon 12. This isoform is represented by two ESTs derived from a cDNA library made from a mixture of cDNA from fetal lung, testis and B-cells (GenBank accession nos AI805296 and AA909366), and by one EST from a testis cDNA library (GenBank accession no. AI026624). The stop codon is present in exon 13 and this isoform is predicted to encode a 1364 amino acid protein.
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To determine whether isoforms I, II, III or IV are transcribed, a reverse transcription–polymerase chain reaction (RT–PCR) was performed on RNA from 10 adult tissues, four fetal tissues and a RPE cell line. RT–PCR was performed with a sense primer in exon 10 and antisense primers chosen behind the stop codons of the different splice forms (Fig. 1A). Isoforms III and IV could not be amplified from any of these tissues (data not shown). Isoforms I and II were successfully amplified from adult retina, adult brain, fetal eye and fetal brain, but not from any other tissues (Fig. 1B). The primersets for isoform I amplified a weak product on RPE/choroid RNA, which is likely to be caused by a contamination with retinal tissue. By RNA in situ hybridization we detected expression of the gene in the outer and inner nuclear layers of the retina but not in the RPE (12).
The predicted protein sequences encoded by the class I and class II isoforms of CRB1 were subjected to four different transmembrane prediction programs (PRED-TMR, DAS, TMpred and TopPred2). For both isoforms putative transmembrane regions were predicted near the C-terminus. However, the prediction of the transmembrane region in isoform II is much stronger than that in isoform I, as can be exemplified by the prediction scores of the DAS program (5.8 versus 2.9), the TMpred program (2457 versus 1307) and the TopPred2 program (2.346 versus 1.086). The protein encoded by isoform II is predicted to contain a 22 amino acid transmembrane region and a 37 amino acid cytoplasmic domain (Fig. 1C). Although isoform I was previously described as an extracellular protein (1), detailed analysis with these transmembrane prediction programs suggests that it may contain a transmembrane region and a small, 20 amino acid cytoplasmic domain (Fig. 1C).
Conservation of residues in the cytoplasmic domains of Crumbs homologues supports the presence of two functional domains
The cytoplasmic domain of isoform II is highly (72%) similar to that of Drosophila Crumbs protein. Twenty-two out of 37 (59%) amino acids are identical between human CRB1 and Drosophila Crumbs cytoplasmic domains (Fig. 1D). Eight amino acids are identical between CRB1, Drosophila Crumbs, a Crumbs homologue (C.e. CRB1; GenBank accession no. U42839) and a Crumbs-like protein (C.e. CRL1; GenBank accession nos AL008869 and EAT-20; GenBank accession no. AB032748) both recently identified in Caenorhabditis elegans (Fig. 1D) (11,13,14). The conserved residues are clustered in two regions of the cytoplasmic domains, the C-terminal amino acids ERLI and an N-terminal region including residues at positions 8, 10, 12 and 16 of the cytoplasmic domains (G8, Y10, P12 and E16, respectively).
These two conserved regions coincide with two functional subdomains recently identified by site-directed mutational analysis in the cytoplasmic domain of Drosophila Crumbs (11). The C-terminal amino acids EERLI, as well as residues Y10 and E16 in the N-terminal region, are necessary to rescue the crumbs mutant phenotype. However, if mutated constructs are overexpressed in a wild-type background, only constructs including an intact C-terminus (the EERLI motif) produce a multilayered epidermis irrespective of replacement of additional conserved residues. The C-terminal EERLI motif binds to the PDZ-domain containing protein Discs Lost in vitro (15) and recruits it into an apically localized protein complex (11,15), whereas the region circumspanning the amino acids Y10 and E16 may be involved in other protein interactions (11). The three proline residues preceding the ERLI motif in human CRB1, Drosophila Crumbs and C.elegans CRB1 may serve to break any secondary structures, allowing free movement of the C-terminal ERLI motif.
Overexpression of CRB1intra-myc rescues the crumbs mutant phenotype
The crumbs mutant phenotype can be partially rescued by GAL4/UAS-mediated overexpression of either the full-length Crumbs protein or of only the membrane-bound cytoplasmic domain of Crumbs, tagged with a Myc epitope (crbintra-myc) (10). To determine whether the cytoplasmic domain of human CRB1 can substitute the cytoplasmic domain of Drosophila Crumbs in rescuing the crumbs phenotype, GAL4/UAS-mediated expression of the membrane-bound cytoplasmic domain of CRB1 tagged with a Myc epitope (CRB1intra-myc) was induced in crumbs mutant embryos. Major parts of the cuticle were restored to a contiguous cuticular shield with belts of denticles, which is a sign of normal patterning and differentiation (compare Fig. 2A and B, for comparison to a wild-type cuticle see Fig. 3A). The phenotypic rescue reached by the strongest expression line (CRB1intra-myc 9.1) is similar compared to embryos rescued by full-length Crumbs or the membrane-bound cytoplasmic domain of Crumbs (10).
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The basis for the generation of contiguous cuticle is normal epithelial polarity (not shown) and restoration of the ZA, analysed by the expression pattern of phosphotyrosine (pY)-containing epitopes that mark the ZA (compare Fig. 2C–E). Crumbs mutant embryos do not develop a ZA, whereas crumbs mutant embryos overexpressing CRB1intra-myc show a partial restoration of the ZA and a more columnar shape of the epithelial cells.
Crumbs mutant embryos show extensive, ectopic cell death in the epidermis in addition to the endogenously occurring apoptosis. Crumbs mutant embryos overexpressing CRB1intra-myc in the epidermis show much reduced epidermal cell death whereas outside the expression domain of the GAL4 activator, e.g. the central nervous system (CNS), endogenous apoptosis proceeds as normal (compare Fig. 2F and G).
Overexpression of CRB1intra-myc transforms the single-layered epidermis into a multilayered tissue
Moderate overexpression of either full-length Crumbs or strong overexpression of the membrane-bound cytoplasmic domain (crbintra-myc) in wild-type embryos leads to an abnormal, multilayered epidermis (10,11). To determine whether the cytoplasmic domain of CRB1 has a similar effect on the development of epithelia as the cytoplasmic domain of Drosophila Crumbs, GAL4/UAS-mediated expression of CRB1intra-myc was induced in a wild-type background. Overexpression of CRB1intra-myc in wild-type embryos leads to defects comparable to those caused by overexpression of crbintra-myc (10,11).
Cuticle preparations of embryos overexpressing CRB1intra-myc show normal patterning, although the embryo appears smaller, not extending the complete length of the inner eggshell. Embryonic lethality is in 98% accompanied by the incomplete morphogenetic movement of dorsal closure, resulting in large dorsal holes (compare Fig. 3A and B, arrowheads). In embryos overexpressing CRB1intra-myc, the single-layered epidermis has lost its columnar shape and has become multilayered. The outer layer of cells still exhibit aspects of polarity, such as polarized distribution of apical membrane marker SAS and basolateral membrane marker FASIII, whereas the cells in the inner layer show only basolateral characteristics (compare Fig. 3C and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previously, we described a human homologue of Drosophila Crumbs, CRB1, which exhibits striking structural and sequence similarity to Crumbs (1). The most striking difference between CRB1 and Crumbs was that the latter contains a transmembrane and 37 amino acid cytoplasmic domain, whereas CRB1 was predicted to encode an extracellular protein (1). Analysis of splice variants of CRB1 presented here revealed that the gene exhibits alternative splicing at its 3' end, and transcription of two splice forms was confirmed by RT–PCR. The transcripts are predicted to encode a 1376 amino acid extracellular or transmembrane protein, and a 1406 amino acid membrane-bound protein, respectively. It is not clear which 3'-UTR is used by the first isoform (Fig. 1A). The cDNA sequence that was described by den Hollander et al. (1) contains a 94 bp 3'-UTR with a small poly(A) tail but no polyadenylation signal. By 3'-RACE the 3'-UTR was extended to 704 bp, but the poly(A) tail of this product was not preceded by a polyadenylation signal either. One EST was identified in the database that contains exon 12 and has a poly(A) tail preceded by a polyadenylation signal. However, it is not clear whether this is the 3'-UTR used in vivo, since in that case an RT–PCR with primers in exon 10 and exon 12 would amplify two products; 557 bp for isoform II and 799 bp for isoform I (Fig. 1A and B). Extensive 3'-RACE studies using a forward primer in exon 11b only revealed the 704 bp 3'-UTR indicating that the isoform I EST containing exon 12 represents a rare transcript or is an artefact.
The presence of two other 3' end splice variants, represented by ESTs in the database, could not be confirmed by RT–PCR. Possibly, these splice variants are expressed at a very low level not detectable by RT–PCR. They also could be expressed during particular developmental stages or just in a small subset of cells of the retina or brain, which were not tested sufficiently in our experiments. Another possibility is that the ESTs representing these transcripts are artefacts caused by aberrant splicing. However, one of these splice forms was found on two independent EST clones from two different cDNA libraries, suggesting that this explanation is less likely. Alternative splicing has not been detected for Drosophila crumbs, although northern blot analysis shows two different mRNA transcripts (5). In embryos and pupae only a 7.5 kb transcript was detected, whereas in larvae and adult flies an additional 7.7 kb transcript was present.
The predicted 1406 amino acid protein encoded by CRB1 isoform II contains a transmembrane domain and a 37 amino acid cytoplasmic domain 72% similar to that of Crumbs. The identification of this cytoplasmic domain of CRB1 suggests that it is a true homologue of Drosophila Crumbs. At first sight, mutations in human CRB1 and Drosophila Crumbs seem to cause different phenotypes. However, diseases associated with mutations in CRB1 and phenotypes resulting from crumbs loss of function both lead to progressive degeneration of particular polarized cell types. The different phenotypes are a reflection of the expression domains of CRB1 and crumbs. CRB1, which is expressed in the retina and the brain, causes severe retinal dystrophies ranging from RP to LCA. These diseases are characterized by a progressive degeneration of the retina, a highly organized and polarized tissue. Crumbs is expressed in ectodermally derived epithelia of the embryo. Crumbs mutant embryos fail to maintain cell polarity in epithelia that are derived from the blastoderm, namely the amnioserosa, the epidermis, the fore- and hindgut, the trachea, the Malpighian tubules and the salivary glands. Depending on the epithelium considered, the defects range from slight disturbances in epithelial organization, disruption of epithelial integrity to widespread cell death. In late larval stages Crumbs is expressed in all imaginal disc cells but enriched in the developing photoreceptor clusters and the inner and outer optic anlagen of the brain (6; K.Johnson and E.Knust, unpublished data). So far, no phenotype has been described for crumbs mutant clones in adult eyes but preliminary results suggest morphological abnormalities and inducible degeneration of the photoreceptor cells (K.Johnson and E.Knust, manuscript in preparation).
The function of Drosophila Crumbs has been studied extensively, and in particular it was shown that the cytoplasmic domain is of crucial importance for the function of Crumbs. Deletion of the 23 C-terminal amino acids of the cytoplasmic domain completely abolishes crumbs function (9). Furthermore, the membrane-bound cytoplasmic domain can partially rescue the crumbs phenotype and overexpression in a wild-type background induces a multi-layered epidermis (10). In this work we show that the cytoplasmic domain of CRB1 can substitute the function of Drosophila Crumbs in rescuing and overexpression studies. Complete rescue could not be obtained, neither using the Drosophila nor the human cytoplasmic domain, because the GAL4 expression of the activator line was not sufficiently similar to the expression of wild-type Crumbs (10).
The cytoplasmic domain of Crumbs has been shown to contain two functional subdomains, the C-terminal EERLI motif and a N-terminal region containing residues Y10 and E16, which are both highly conserved between Crumbs homologues (Fig. 1D). The EERLI motif binds the multi-PDZ-domain protein Discs Lost in vitro (15) and is essential for its recruitment into an apical protein complex in epithelial cells (11). Crumbs, discs lost and stardust may constitute a pathway to organize the apical cortical scaffold that influences the assembly of a continuous ZA. Stardust encodes a scaffolding protein of the MAGUK family and also interacts with the C-terminal EERLI motif in the yeast two-hybrid system (16).
Similarly, the C-terminus of CRB1 may interact with a human homologue of Discs Lost and/or Stardust to organize a protein scaffold in the human retina. It has been speculated that CRB1 may play a role in localizing the phototransduction complex to the apical membrane of the photoreceptors (17). The subsequent lack of coordinated phototransduction activity in the photoreceptors may lead to their progressive decay and indirectly affect the supporting retinal pigment epithelium, resulting in LCA or RP phenotypes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Analysis of EST clones
To identify splice variants at the 3' end of the CRB1 gene, a BLAST search was performed with the CRB1 cDNA sequence against the dbEST database. Thirteen human cDNA sequences were identified that showed overlap with the CRB1 cDNA sequence. These ESTs were derived from retina (GenBank accession no. AI444814), brain (GenBank accession nos BF347919 and R35113), testis (GenBank accession no. AI026624), fetal liver/spleen (GenBank accession nos N78199, N59646, AA033939, R09831, T87786 and T87787) and mixed tissue (fetal lung, testis, B-cell; GenBank accession nos AI805296, AA909366 and AA906086) cDNA libraries. ESTs with GenBank accession nos N78199 and N59646 represent the same IMAGE clone (IMAGE clone no. yv74f07) sequenced from both sides, as well as ESTs with GenBank accession nos T87786 and T87787 (IMAGE clone no. yd93e09). The two cDNA clones represented by GenBank accession nos AA033939 (IMAGE clone no. zi06c01) and R09831 (IMAGE clone no. yf30a11), were ordered from the Resource Center/Primary Database of the German Human Genome Project (RZPD), Berlin, Germany. Plasmid DNA was prepared using a miniprep plasmid DNA isolation kit (Qiagen) and analysed by cycle sequencing. The complete sequences of the inserts of these clones were deposited in the GenBank database under accession nos AY043322 and AY043323.
RT–PCR analysis of 3' end splice variants
For RT–PCR analysis, total RNA was isolated from adult human retina, ARPE-19, RPE/choroid and testis by RNAzol B (Campro Scientific) and from fetal eye and fetal cochlea by CsCl purification. Total RNA samples from human liver, kidney, lung, skeletal muscle, placenta, heart, brain, fetal liver and fetal brain were purchased from Clontech. Randomly primed cDNA was synthesized from DNase I-treated RNA using random hexanucleotides as described by den Hollander et al. (18). RT–PCR reactions were performed for four putative CRB1 3' end splice variants, using a forward primer in exon 10 (2529; 5'-TGCAGACAGAGCAGATTACC-3') and reverse primers downstream of the predicted stop codons. Primers were located in exon 11b for isoform I (2530; 5'-CAGAGATCTAAAATGAATCAAG-3'), in exon 12 for isoform II (3123; 5'-TCAGGTATGTCAGAGATACC-3'), in exon 12a for isoform III (3850; 5'-ACAATGGAACTACTCAGG-3') and in exon 13 for isoform IV (3851; 5'-TTGCATCCAGCAGGCACGG-3'). PCR was performed as described by den Hollander et al. (18) for 35 cycles.
Transmembrane prediction
Transmembrane predictions of CRB1 protein sequences were performed with four different transmembrane prediction programs. Prediction with PRED-TMR was performed at the University of Athens, Biophysics Laboratory (http://o2.db.uoa.gr), with DAS at the Stockholm Bioinformatics Center (http://www.sbc.su.se), with TopPred2 at the Institut Pasteur (http://bioweb.pasteur.fr) and with TMpred at the European Molecular Biology Network (http://www.ch.embnet.org).
Generation of a CRB1/UAS construct and germline transformation
Directed gene expression in Drosophila was performed as described by Brand and Perrimon (19). A GAL4-dependent target gene is constructed by subcloning it behind a tandem repeat of GAL4-binding sites, known as the UAS. To activate the target gene, flies carrying the target are crossed to flies expressing GAL4. Depending on the genomic enhancer driving GAL4 expression, GAL4 and subsequently the target gene can be expressed in a cell- or tissue-specific pattern. The UAS vector (pUAST) (19) was previously used to construct crbintra-myc, which expresses a transgene containing the N-terminal region of Crumbs including the signal peptide and the C-terminal region including the transmembrane and cytoplasmic domains, separated by a Myc epitope (10). An UAS construct expressing the cytoplasmic domain of human CRB1 (CRB1intra-myc) was generated by replacing the Drosophila cytoplasmic domain in crbintra-myc with the cytoplasmic domain of CRB1. The cytoplasmic domain of CRB1 was amplified in a RT–PCR reaction on human fetal brain cDNA using the primer combination 5'-GATCTGGCCAGGAACAAAAGGGCAACTCAGGG-3' and 5'-GATCTCTAGACTAAATCAGTCTCTCCATTGCA-3'. The primers introduced MscI and XbaI sites (underlined), respectively. The EcoRI–XbaI insert of crbintra-myc was cloned in pBluescript. The cytoplasmic domain of crumbs was excised by MscI–XbaI digestion and the PCR product containing the cytoplasmic domain of CRB1 was cloned into the MscI–XbaI sites. The EcoRI–XbaI fragment containing the signal peptide of crumbs, the Myc epitope, the transmembrane domain of crumbs and the cytoplasmic domain of CRB1 was excised from pBluescript and ligated into EcoRI–XbaI-digested pUAST. The pUAST P-element vector construct carrying CRB1intra-myc was stably transformed into the germline of flies according to Spradling (20). More than 20 lines were established, 10 of which were compared in more detail. They showed different degrees of the same phenotype depending on their genomic localization and expressivity.
Fly stocks and overexpression
Effector lines shown are CRB1intra-myc 9.1 (first chromosome) and CRB1intra-myc 18.1 (second chromosome). In a wild-type background, overexpression was mediated by GAL4daG32 in a uniform, daughterless expression pattern. Rescue experiments were carried out with GAL4385.3, crb11A22/TM3 which results in a broad, segmentally reiterated pattern of expression in the epidermis (11).
Histology, cuticle preparations and immunocytochemistry
Cuticle preparations were prepared according to Wieschaus and Nüsslein-Volhard (21). Standard fixation protocols (4% formaldehyde) were applied. Antibody dilutions were as follows: mouse anti-FASIII [7G10, 1:3 (22)], rabbit anti-SAS (1:500; E.Organ and D.Cavener, unpublished data), mouse anti-phosphotyrosine (PY20, 1:300; Tranduction Labs). Jackson Immunoresearch supplied Cy2- and Cy3-conjugated secondary antibodies. The DNA dye Yoyo-1 (Molecular Probes) was used at 1:8000. For TUNEL stainings (LaRoche), 4% paraformaldehyde-fixed embryos were devitellinized, washed in PBS, 0.3% Triton X-100, incubated in 100 mM Na-citrate, 0.1% Triton X-100 at 65°C for 30 min, washed again in PBT, then TUNEL dilution buffer, and subsequently incubated in 40 µl TUNEL reaction mix at 37°C for 3 h according to the manufacturer’s recommendations.
Stainings were analysed with a Leica TCS NT confocal microscope and images were processed and arranged using Photoshop 5.5 (Adobe) and Canvas 6 (Deneba) on an Apple Macintosh.
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ACKNOWLEDGEMENTS |
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We thank M.Luijendijk for providing fetal eye and cochlea RNA samples. A.I.d.H. and Y.J.M.d.K. are supported by the Foundation Fighting Blindness USA, Inc. K.J. and E.K. are grateful for the support of the German Research Foundation, DFG grant Kn250/15-2 and the German ‘Fonds der Chemischen Industrie’.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +31 24 3617431; Fax: +31 24 3540488; Email: a.denhollander@antrg.azn.nl Present address: A. Klebes, Department of Biochemistry, University of California in San Fransisco, San Francisco, CA, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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