cDNA characterization and chromosomal mapping of two human homologues of the Drosophiladishevelled polarity gene
cDNA characterization and chromosomal mapping of two human homologues of the Drosophila dishevelled polarity geneAntonio Pizzuti1, Francesca Amati2,6, Giuseppe Calabrese3, Aldo Mari2,6, Alessia Colosimo2, Vincenzo Silani1, Luciana Giardino4, Antonia Ratti1, Donata Penso1, Laura Calzà5, Giandomenico Palka3, Gugliemo Scarlato1, Giuseppe Novelli2,* and Bruno Dallapiccola2,6
1Istituto di Clinica Neurologica, Centro `Dino Ferrari' Università di Milano, Milan, Italy, 2Cattedra di Genetica Medica e Umana, Dipartimento di Sanità Pubblica e Biologia Cellulare, Università Tor Vergata, Roma, Via di Tor Vergata 135, 00133 Rome, Italy, 3Istituto di Biologia e Genetica, Università di Chieti, Chieti, Italy, 4Clinica Otorino-Laringoiatra, Università di Milano, Milan, Italy, 5Istituto di Fisiologia Umana, Università di Cagliari, Cagliari, Italy and 6Ospedale CSS, San Giovanni Rotondo, Italy
Received January 4, 1996;Revised and Accepted April 3, 1996
The Drosophila dishevelled gene (dsh) encodes a secreted glycoprotein, which regulates cell proliferation, acting as a transducer molecule for developmental processes, including segmentation and neuroblast specification. We have isolated and characterized cDNA clones from two different human dsh-homologousgenes, designated as DVL-1 and DVL-3. DVL-1and DVL-3 putative protein products show 64% amino acid identity. The DVL-1 product is 50% identical to dsh and 92% to a murine dsh homologue (Dvl-1). Both human DVL genes are widely expressed in fetal and adult tissues, including brain, lung, kidney, skeletal muscle and heart. DVL-1 locus maps to chromosome 1p36 and DVL-3 to chromosome 3q27. DVL-1 locus on chromosome 1 corresponds to the murine syntenic region where Dvl-1 is located. DVL-1 and DVL-3 are members of a human dsh-like gene family, which is probably involved in human development. Although the precise role of these genes in embryogenesis is only conjectural at present, the structural and evolutionary characteristics suggest that mutations at their loci may be involved in neural and heart developmental defects.
A single Drosophila dishevelled segment polarity gene (dsh) has been isolated (1 ). Dsh mediates cell-cell interactions during embryogenesis and adult fly development, which in turn determine the ultimate cell polarity and fate (1 -3 ). Dsh is required for wingless (wg) function (4 ). wg is a segment polarity gene, homologous to the mammalian proto-oncogene Wnt-1, coding for a secreted protein (5 -7 ). In Drosophila, dsh expression loss ends in segmentation defects identical to wg mutated embryos (8 ). Many Drosophila polarity genes are conserved during evolution. If dsh homologues are also conserved, they may have a key role in vertebrate morphogenesis as well. Only a mouse (Dvl-1) and a Xenopusdsh (Xdsh) homologous genes have been reported so far (9 ,10 ). They encode proteins with 50% identity to the Drosophiladsh. Dvl-1 and Xdshprotein sequence comparison shows 60% identity. Conservation is highest at the amino-terminus and at the central portion of the protein, where Dvl-1 and Xdsh are virtually identical. In particular, a sequence known as GLGF repeat or disc-large homology region (DHR), a motif which is common to proteins supposed to be part of cell junctions, is completely conserved (9 ,10 ). Dvl-1 also contains two RGD tripeptides. RGD motives are associated to recognition systems for cell signalling in proteins which have a role in cell adhesion (9 ). Only one of these RGD sequences is present in Xdsh (10 ).
The high dsh-like gene interspecies conservation suggests they act during common and precocious embryogenic events. In fact, Xdsh mRNA, injected into normal or ventralized Xenopus embryos, induces a complete body axis formation, and causes neuralization when overexpressed in the committed ectodermal cells (10 ). In situ hybridization studies indicate that the mouse Dvl-1 gene is uniformly expressed at high levels in the fetal central nervous system (CNS), also suggesting a general key role in CNS development (9 ). It is therefore likely that the dsh homologue(s), if expressed in humans, play important roles in embryonic patterning. Imbalance of such protein expression might be especially involved in neural developmental defects.
We report on the isolation of cDNA clones belonging to two different human dsh-like genes, and their respective chromosome locations. Our data indicate that other DVL sequences are present in the human genome and that they altogether may represent a more complex new gene family in humans.
In order to isolate human homologous cDNAs to the mouse Dvl-1 gene, we synthesized an oligonucleotide pair on the mouse cDNA 5' translated sequence (DVL-1 5' ATG GAC GAG GAG GAG ACG CCG TAC 3' and DVL-3 5' GAA ACC ACC CGG CCA TTG AAG CAG 3'). A 200 bp fragment (DVL-1,3), with high homology to the mouse Dvl-1 sequence (97%), was obtained by RT-PCR of human fetal brain total RNA, and used to screen a human adult caudate cDNA library.
Five independent cDNA clones were isolated. When the clones were initially sequenced at both ends, only one of them (4B) revealed a complete identity to the DVL1,3 probe, suggesting 4B represented the true human homologue of the mouse Dvl-1 (we designated it as DVL-1). 4B was 1600 bp long, and comprised a 150 bp 5'UTR followed by a 1450 bp open reading frame (ORF), with 85% nucleotide homology to the mouse Dvl-1 cDNA. In order to get the complete human DVL-1 cDNA sequence, the same cDNA library was screened with a 200 bp probe representing the mouse cDNA 3' UTR. Sequencing of a 1800 bp clone (B8), partially overlapping with clone 4B, gave a full length ~3000 bp DVL-1 cDNA sequence, with 85% nucleotide homology to the mouse Dvl-1 in the longest ORF. Human DVL-1 and mouse Dvl-1 cDNA sequences quite differ at both the 5' and the 3' UTR, except at the very 3' end, where a highly conserved 80 bp sequence is present, just preceding the poly(A) tail (Fig. 1 ). The putative human DVL-1 gene product has a 92% overall identity to the mouse Dvl-1 protein, with a maximum 99% identity in the amino-terminal half, which includes both the DHR region and the two RGDs sequences. The carboxy-terminal half is slightly less conserved, with a 84% identity.
Northern blot analysis of the DVL-1 expression using poly(A)+ RNA samples from various fetal and adult human tissues, revealed a single band of ~3 kb (Fig. 3 A). Signal quantification was obtained by densitometric analysis, normalized to [beta]-actin mRNA, and single tissue relative concentration values were determined as compared with the liver mRNA levels, where the weakest DVL-1 signals were detected. The highest expression levels were found in adult skeletal muscle and pancreas (15 and 10 times more than the liver, respectively) followed by heart muscle (3.5 times). Detectable levels were also found in adult brain, placenta, lung, liver and kidney (less than two times the liver) (Fig. 3 A). The same expression pattern was observed in fetal tissues. Appreciable quantitative differences between the adult and the fetal tissues present in the blot (brain, lung, liver and kidney) were not evident (Fig. 3 B).
DVL-1 specific primers were designed on the 3'UTR and used to amplify by PCR a rodent-human somatic cell hybrids panel. Control rodent genomic DNA showed a PCR product which differed in size from the expected human product (data not shown). The latter was detected in human chromosome 1 containing hybrids. Similarly, using DVL-3 specific primers, DVL-3 was assigned to chromosome 3 (data not shown).
DVL-1 and DVL-3 cDNAs were more finely mapped, to bands 1p36 and 3q27 respectively, using the cloned cDNAs for FISH analysis (Fig. 6 A and B).
We report the cloning of human cDNAs encoding for two homologues of the Drosophila dsh gene, and their mapping at different loci in the human genome. The only other known vertebrate dsh homologues so far described are the XenopusXdsh and the mouse Dvl-1 gene (9 ,10 ). A second mouse gene (Dvl-2) has been deposited on GenBank, but no report has been issued yet. On the basis of the extensive nucleotide sequence homologies, the putative peptide sequence resemblance and the map position (human DVL-1 and mouse Dvl-1 genes map on known regions of synteny), human DVL-1 should correspond to the mouse Dvl-1 gene. No human Dvl-2 homologous cDNA has been isolated yet.
The second dsh-like gene we characterized and mapped has no mouse counterpart yet. We therefore designated it as DVL-3. It is likely that the human dsh-like gene family is much wider than the single Drosophila dsh gene. We have mapped a DVL 3' UTR-like sequence (90% identical to the DVL-1/9B sequence in Fig. 1 ) on the DiGeorge syndrome critical region (DGS/SRO), on chromosome 22q11 (11 ). We also isolated a chromosome 22 cosmid clone, which cross-hybridizes to the DVL-1 locus on chromosome 1 in FISH experiments. This cosmid, which maps well outside the DGS/SRO, specifically hybridizes to sequences representing the 5' coding region of DVL-1 cDNA (unpublished data). Moreover, the detection of multiple hybridization bands in DVL-3 Northern blotting experiments, which are very different in size, could also indicate, rather than the presence of different alternatively spliced forms of the same transcript, the presence of other very closely related genes. However, as we could not isolate any cDNA clone representing the 5.0 and the 5.9 kb `DVL-3 forms' so far, this issue remains still open to discussion.
DVL-1 and DVL-3 expression is widespread, both in adult and in fetal tissues, with particularly high levels in heart and striated muscle, as demonstrated by Northern analysis. In situ experiments during early human embryogenesis indicate that DVL-1 is more selectively expressed in the neural tube, in particular in its dorsal portion, at those very precocious developmental stages. These data somehow reminds the abundant Dvl-1 expression observed in mouse embryo where localization is strong throughout the neural folds and in mouse fetus where it is very abundant in the spinal cord, in particular on the ventral horns (9 ).
Purification of nucleic acids, restriction analysis, gel electrophoresis, DNA ligation, cloning, subcloning and Sanger sequencing (automated), probe radiolabelling, northern and Southern analysis, library screening were performed according to established protocols (19 ). The human adult caudate cDNA library was purchased from Stratagene. Northern blot analysis was performed on commercially available filters containing about 2 [mu]g per lane of poly(A)+ enriched RNAs from adult and fetal tissues (Clontech, Bios Laboratories). Hybridizations were conducted at 65oC in 125 mM sodium phosphate (pH 7.2), 250 mM NaCl, 7% SDS, 10% PEG for Southern blots and at 42oC in 50% (v/v) formamide, 125 mM sodium phosphate (pH 7.2), 0.1% SDS, 5* Denhardt's solution, 100 g/l denatured ssDNA for Northern blots. Southern filters were washed at the same temperature of hybridization with a final stringency ranging from 0.5* SSC to 0.1* SSC and at 50oC in 0.25* SSC/0.2% SDS for Northern filters. Automatic DNA sequencing was performed on an Applied Biosystems 370A apparatus.
Tissues from chromosomally normal first-trimester spontaneous abortions specimens (5.3 weeks of gestational age) were obtained according to the current Italian regulation. Tissues were washed in PBS and frozen in liquid nitrogen. Fourteen [mu]m thick sections were cut in a cryostat (Leitz Kryostat 1720) at -20oC and twad-mounted on to precleaned microscope glass slides (ProbeOn, Fisher Scientific, Pittsburgh, USA). Oligonucleotide probe with sequence complementary to mRNA encoding DVL-1 (5' CGC TCC TTG AAG CCC TCC ACG TGT GTG TAC AGC CAG TCC ACC AC 3') was synthesized and purified through Pharmacia NAP-10 column. The oligonucleotide probe was labeled at the 3'-end with 35S-dATP (New England Nuclear, Boston, MA) using terminal deoxynucleotidiltransferase (IBI, New Haven, CT, or Amersham, England) in a buffer containing 10 mM CoCl2, 1 mM dithiothreitol (DTT), 300 mM Tris base, and 1.4 M potassium cacodylate (pH 7.2). Afterwards, the labeled probe was purified through Nensorb-20 columns (New England Nuclear) and DTT was added to a final concentration of 10 mM. The specific activity obtained ranged from 1 to 4*106 d.p.m./ng oligonucleotide. Sections were brought to room temperature, air dried, covered with a hybridization buffer containing 50% formamide, 4* SSC, 1* Denhardt's solution, 1% sarcosyl, 0.02 M phosphate buffer (pH 7.0), 10% dextran sulfate, 500 g/ml heat-denatured salmon sperm DNA, 200 mM DTT and 40 ng/l of the labeled probe. The slides were placed in a humid chamber and incubated for 15-20 h at 50oC. Afterwards, the sections were rinsed in 1* SSC at 55oC for 1 h with six changes and washed in the same buffer for 1 h at room temperature. Finally, the slides were rinsed in distilled water and 60 and 95% ethanol (2 min each) and air dried. The sections were dipped in NTB2 nuclear track emulsion (Kodak). Sections were exposed for a period of time of 3 weeks before being developed with Kodak D19 for 3 min and fixed with G333 (AGFA Gevaert, Leverkusen, Germany) for 10 min. The tissue was counterstained with toluidine blue. Emulsion-dipped tissue sections were examined in a Nikon Microphot-FX microscope equipped with dark- and bright-field condensers and photographed with Tri-X 100 (Kodak) black and white film.
DVL-1 and DVL-3 were mapped respectively to chromosomes 1 and 3 using somatic cell hybrids from BIOS Laboratories and the monochromosomal somatic cell hybrid panel (UK Human Genome Mapping Project Resource Centre). The following primers were used to amplify human-specific DVL-1 and DVL-3 DNA fragments: DVL-1, sense (5' AGC CGT GAC GGG ATG GAC AAC G 3'), and antisense (5' TCA AGC TCG CTG CTG AGG GC 3'); DVL-3 sense (5' CTT CCG CAT GGC CAT GGG AAA C 3') and antisense (5' GAC GGA CGG ATG GAG AGA GGA AC 3').
Metaphase chromosomes were prepared from human peripheral blood lymphocytes obtained from normal individuals as described (20 ). Purified cDNA probes were labeled with biotinylated dATP and digoxigenated dUTP by nick translation (Life Technologies Inc., USA) and hybridized to chromosome spreads according to routine procedures. Chromosomes were counterstained with DAPI and visualized as described (20 ). At least 25 metaphases for each probe were scored. Images were CCD captured and merged through a Vidas Image Analyzer (Zeiss, Germany).
Data bank search (GenBank, GenEmbl, SwissProt and PIR) were run through the BlastX and BlastN network service. Sequence analysis was performed using the GCG package. For pairwise alignments, the gap program was used. Known protein motifs were searched with PROSITE.
We are grateful to D. Sussman and T. Wynshaw-Boris for communicating results prior to publication and for useful comments on this work. We thanks the UK Human Genome Mapping Project for reagents and computer resources, M. Gennarelli and M. Lucarelli for experimental help. This work was supported by grants from EEC, Biomed-1 Project, MURST, Italian Ministry of Health and Telethon Italia (Grant No.D27).
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*To whom correspondence should be addressed
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