J Biol Chem, Vol. 274, Issue 38, 26968-26977, September 17, 1999

Glypican-6, a New Member of the Glypican Family of Cell Surface Heparan Sulfate Proteoglycans^*

Mark Veugelers^§, Bart De Cat^§, Helga Ceulemans, Anne-Marie Bruystens, Christien Coomans, Joachim Dürr, Joris Vermeesch^¶, Peter Marynen^¶, and Guido David^**

From the  Laboratory for Glycobiology and Developmental Genetics, ^¶ Laboratory for Human Genome Analysis, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Leuven B-3000, Belgium

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, we identified a sixth member of the glypican family. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (identity of 63%). Expression of this protein in Namalwa cells shows a core protein of ~60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily comprising so far glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might in part be regulated by the presence of glypican-6 on the cell surface.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Heparan sulfate proteoglycans are present at the surfaces of most adherent cells and in the extracellular matrices that support or surround these cells. The heparin-like moieties of these molecules bind and modulate the activities of several matrix components, growth factors, proteinase inhibitors, cell-cell, and cell-matrix adhesion molecules (1). Substantial evidence suggests that cell surface-associated heparan sulfate proteoglycans act as "co-receptors" for FGFs¹ (2), epidermal growth factors (3), Wnts (4), and members of the transforming growth factor-BMP superfamily (5, 6), facilitating the interactions of these ligands with specific signal-transducing receptors and adding an important dimension of regulation to these signaling systems.

Two major proteoglycan families account for most of the heparan sulfate that is integrally associated with cell surfaces, the syndecans (7) and the glypicans (8). While syndecans are transmembrane proteins, glypicans are linked to the cell surface through a glycosylphosphatidylinositol anchor. Glypicans are also characterized by a unique pattern of 14 cysteine residues, conserved in all glypicans identified to date, including the glypican encoded by the dally gene in Drosophila (9). The core proteins of all glypicans are of roughly similar sizes (60 kDa) and contain Ser-Gly repeats in their C-terminal parts that compose sites for substitution with heparan sulfate. Since the structures of individual glypicans are extremely well conserved across species, these proteins might support additional specific functions, other than the display of heparan sulfate at the cell surface. Recently, it has been claimed that both insulin-like growth factor-2 and tissue factor pathway inhibitor are ligands for the core protein of glypican-3 (10, 11).

All glypicans are highly expressed during embryonic development. Glypican-1 is a major proteoglycan in the developing and adult brain, but it is also expressed in the kidneys, perichondrium, and several other tissues (12, 13). Glypican-2 expression, in contrast, is limited to the developing nervous system (14). Glypican-3 is highly expressed in the developing intestine and in mesoderm-derived tissues (15, 16), whereas glypican-4 shows a predominant expression in blood vessels, kidney, brain, and adrenal cortex (17). Glypican-5 shows a developmentally regulated expression in kidney, limb, and brain (18, 19), with brain expressions that persist into the adult. Consistent with a proposed role in development, the expression patterns of these glypicans often coincide with those of growth factors, such as FGFs and BMPs, and their receptors.

The first mutations described in glypican genes are associated with phenotypes that show a deregulation of cellular growth and morphogenesis, further implicating glypicans as controllers or effectors of developmental processes. Mutations of the dally locus in Drosophila affect cell division patterning and morphogenesis in the developing brain (9), and genetic evidence suggests that dally affects the cellular response to Dpp, the transforming growth factor--related morphogen encoded by decapentaplegic (5). Deletions and translocations affecting glypican-3, and occasionally also glypican-4, are associated with the Simpson-Golabi-Behmel syndrome (10, 20). This X-linked syndrome is characterized by pre- and postnatal overgrowth, visceral and skeletal anomalies, and an increased risk for the development of tumors (21). Evidence that overexpression of glypican-3 induces cell line-specific apoptosis might provide some link to the genesis of these dysmorphisms, but the precise mechanisms at work remain unclear (22).

More insight in the regulatory roles or functions of the glypicans might be gained from the identification of additional members of this gene family. By extensive analysis of EST data bases and cDNA cloning, we were able to identify a sixth member of the glypican gene family. Glypican-6 appears to be a close homologue of glypican-4. Recombinant glypican-6 is substituted with heparan sulfate, and Northern blotting and in situ RNA analyses show that glypican-6 is differentially expressed in the tissues of the developing embryo and the adult.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

cDNA Cloning-- The EST data base dbEST was screened for sequences related to known vertebrate glypicans. Two potentially novel glypican-like ESTs (GenBank^TM accession numbers AA001322 and N87558) were identified. The EST clone zh83a06, from the Soares fetal liver/spleen library (23), was obtained from the I.M.A.G.E. Consortium through Research Genetics, Inc. (Huntsville, AL). This clone (code 427858) was completely sequenced, yielding residues 1835-2748 of the composite GPC6 cDNA sequence shown in Fig. 1. Missing coding sequences were isolated by 5'-RACE, performed on a human fetal brain library (CLONTECH, Palo Alto, CA), using gene-specific primers (Table I, part A). BLAST searches revealed near identity between the 5'-UTR of human GPC6 and a human CpG island (GenBank^TM accession numbers Z54669 and Z54670). The corresponding mouse cDNA was cloned by 5'- and 3'-RACEs, performed on a cDNA library derived from a 15.5-dpc mouse embryo, using primers specific for human and mouse GPC6 (Table I, part A). Further BLAST searches identified one EST clone (GenBank^TM accession number AA123401) that extended the 3'-UTR of mGPC6. This EST was completely sequenced and corresponds to residues 2706-3515 of the composite mouse sequence.

Northern Blotting-- Membranes containing lanes with poly(A)⁺ RNA from multiple human and mouse tissues were obtained from CLONTECH. Hybridization was performed for 2 h at 68 °C, using Expresshyb solution (CLONTECH) according to the manufacturer's specifications. The probe for the analysis of human RNAs was either a ³²P-oligolabeled BamHI-XbaI fragment (corresponding to residues 2147-2483) or a HindIII-BamHI fragment (corresponding to residues 1724-2147) of the human cDNA sequence. A probe from the 3'-UTR (corresponding to residues 2706-3515 of the murine sequence) was used for the Northern analysis of the murine embryo poly(A)⁺ RNAs. Dehybridization included two washes with 2.0% SSC, 0.05% SDS (5 min at room temperature, 30 min at 60 °C) and a high stringency wash with 0.1% SSC, 0.1% SDS (30 min at 65 °C).

In Situ Hybridization-- E11.5- and E13.5-day embryos were collected, embedded in Tissue-Tek, and frozen at 80 °C, until use. 12-µm cryostat sections were recovered on silanized slides (Sigma) and fixed for 10 min in 4% paraformaldehyde. Tissues were permeabilized in 1% Triton X-100 for 5 min, acetylated for 15 min with acetic acid anhydride and triethanolamine, and prehybridized with hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's, 250 µg/ml bakers' yeast RNA, 500 µg/ml herring sperm DNA) for 30 min. Overnight hybridization was performed at 72 °C with digoxigenin-labeled RNA probes. Sense and antisense probes were synthesized using the Roche Molecular Biochemicals DIG RNA labeling kit. Probes were made from either the 3'-noncoding region (residues 2706-3515) or the 5' end region (residues 1-766) of murine glypican-6. After hybridization, the sections were washed at 72 °C in 0.2% SSC for 1 h. After washing, the sections were rinsed with MAB buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5) for 5 min. Blocking of nonspecific antibody binding was performed in MABR (MAB buffer, 1% Boehringer Blocking Reagent) for 1 h. Slides were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (dilution 1/5000) for 1 h in MABR. Color reactions were developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate in color buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂, 2 mM levamisole, pH 9.5).

Construction of Expression Plasmids, Cell Transfection, and Protein Analysis-- The complete coding sequence of human glypican-6 was introduced into pREP4 (Invitrogen, Groningen, The Netherlands), yielding the plasmid glyp6-pREP4. Namalwa cells (ATCC CRL 1432) were routinely grown in Dulbecco's modified Eagle's F12 medium supplemented with 10% fetal calf serum and L-glutamine. For transfection, the cells were prewashed with Ca²⁺- and Mg²⁺-free phosphate-buffered saline and incubated for 10 min at 4 °C (10⁷ cells in 1 ml of Ca²⁺- and Mg²⁺-free phosphate-buffered saline) with 30 µg of pREP4 or glyp6-pREP4 plasmid before electroporation at 240 V and 960 microfarads (Gene Pulser, Bio-Rad). Selection was started 48 h later with 250 µg/ml hygromycin B. Stable transfectants were obtained after 14 days. Expression of heparan sulfate in the transfectants was analyzed by Western blotting, using the anti-HS 3G10 antibody, as described before (19, 24).

Genomic Structure of GPC6-- A human genomic BAC library (Research Genetics, Huntsville, AL) was screened with the complete glypican-6 cDNA which resulted in the identification of BACs 114A17 and 182F5. In addition, a human genomic PAC library, RPCI5 (25), was screened, which yielded 24 PACs containing various parts of the GPC6 gene. BACs/PACs were characterized by Southern blotting and exon-specific PCR (Table I, part B), assuming a gene structure similar to that of GPC4 (20). Subclones were made from these BACs/PACs, and the exon/intron boundaries were sequenced using gene-specific primers derived from the GPC6 cDNA sequence.

Quantitative Immunocytometry-- Namalwa cells transfected with pREP4 or glyp6-pREP4 were analyzed for cell surface expression of heparan sulfate by flow immunocytometry, using the mouse monoclonal antibodies 10E4 and 3G10 as described before (24). The analyses were performed on a FACSort (Becton Dickinson, Mountain View, CA), and data were analyzed with the program Lysis II.

FISH Analysis-- BAC DNA was labeled with bio-16-dUTP (Sigma) by nick translation, using a commercial kit (Life Technologies, Inc.). Metaphase spreads were prepared from PHA-stimulated normal human peripheral blood lymphocytes cultured for 72 h. Prior to FISH, slides were treated with RNase A and pepsin as described (19). Human Cot1 DNA (Life Technologies, Inc.) was used as a competitor. Denaturation of the slides and probes, hybridization, and subsequent cytochemical detection of the hybridization signals were performed as described previously (19). Chromosomes were counterstained with 4,6-diamidino-2-phenylindole, and the slides were mounted in Vectashield mounting medium (Vector Laboratories Inc, Burlingame, CA). The signal was visualized by digital imaging microscopy using a cooled charge-coupled device camera (Photometrics Ltd, Tucson, AZ). Merging and pseudocoloring were performed using the Smart Capture software (Vysis, Stuttgart, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of a New Member of the Glypican Gene Family-- By screening the EST data base for glypican-related cDNA sequences, we identified two human ESTs (one from a fetal liver/spleen library and the other from a heart library) encoding a protein that was related to but potentially significantly different from human glypican-4 and glypican-1 (~70% amino acid identity). One of the corresponding clones contained a 913-bp insert. Sequence analysis confirmed that the insert encoded the C terminus and the 3'-UTR of a novel glypican. 5'-RACE with gene-specific primers, performed on a human fetal brain cDNA library, yielded multiple independent clones that extended the partial EST sequence. Alignment of all the clones yielded a consensus sequence of 2747 bp (Genbank^TM accession number AF105267), containing an open reading frame of 1665 bp (Fig. 1). The merged sequence contained an ATG start codon in a Kozak consensus sequence at position 586 and a TAA stop codon at position 2251. Two potential polyadenylation signals (AATAAA) were present at positions 2599 and 2691. The open reading frame encoded a protein of 555 amino acids that showed significant homology to all known glypicans (Table II). Since this protein was also clearly different from all vertebrate glypicans characterized to date, we named this protein glypican-6 (and the corresponding mRNA/cDNA GPC6).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Human GPC6 cDNA and predicted glypican-6 protein sequence. The nucleotide sequences corresponding to the primers used in the 5'-RACE experiments are indicated by single underlines. The shaded boxes highlight two AATAAA sequences. The terminal poly(A) run may not represent a genuine polyadenylation (since it is separated from the AATAAA box by 34 residues). The N-terminal and C-terminal hydrophobic signal peptide-like sequences are indicated by double underlines. The serine residues that correspond to potential heparan sulfate attachment sites are indicated by carets. The DSSAA sequence that may harbor the site (most likely Ser529) for the transamidation reaction that transfers the protein to glycosylphosphatidylinositol is indicated in bold.

Like all glypicans, glypican-6 starts and terminates with signal peptide-like sequences. By analogy, the N-terminal signal sequence is predicted to be required for the membrane translocation of the nascent polypeptide, whereas the C-terminal sequence supports the temporary membrane anchoring and the subsequent glypiation of the protein. Four Ser-Gly dipeptide repeats are present near the C-terminal end of the protein, within a short distance from a cluster of acidic amino acids, reproducing a motif that promotes the assembly of heparan sulfate in proteoglycans (26).

The complete coding region for murine glypican-6 was isolated by 5'- and 3'-RACE experiments, performed on a mouse 15-dpc embryo cDNA library, using mouse- and human-specific primers. Multiple independent clones were obtained for both the 5'- and 3'-RACEs. BLAST searches with 3'-RACE mouse GPC6 (mGPC6) sequences revealed the presence of an EST almost identical to mGPC6 (~98% identity). This EST was ordered and completely sequenced (insert size 809 bp). Alignment of all the clones yielded a combined murine sequence of 3515 bp (Genbank^TM accession number AF105268). The encoded protein, mouse glypican-6, consisted of 555 amino acids and was highly homologous to human glypican-6 (96% identity).

Alignment of the glypican-6 protein sequence with all other vertebrate glypicans clearly indicates that glypican-6 is most homologous to glypican-4 (Fig. 2 and Table II). Comparison of amino acid identities between all vertebrate glypicans suggests the existence of two glypican subfamilies and possibly also subgroups within these subfamilies. In this framework, the first subfamily comprises glypican-1, -2, -4, and -6, which are highly homologous to each other (minimally 40% amino acid identity), with glypican-4 and -6 (60% identity) as a potential subgroup. The other subfamily consists of glypican-3 and -5, which are also highly homologous to each other (40% identity). Members belonging to two different subfamilies, however, show only about 20% identity at the amino acid level. Additional evidence for the existence of two subfamilies is provided by the differing genomic structures of the glypican genes (see below).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Alignment of the predicted amino acid sequences of the members of the glypican family. GPC1 is human glypican (12); GPC2 is rat cerebroglycan (14); GPC3 is human glypican-3 (10); GPC4 is human glypican-4 (20); and GPC5 is human glypican-5 (19). The residues that are conserved in all six proteins are indicated by an asterisk and in GPC1, GPC2, GPC4, and GPC6 by a period. The set of 14 conserved cysteines and the putative glycosaminoglycan attachment sites are outlined by shaded boxes. Serines occurring in SGXG sequence contexts are indicated in bold.

Expression of Glypican-6-- In Northern blotting experiments (Fig. 3), probes derived from the 3'-UTR of GPC6 detected an mRNA of ~7 kb in all the fetal and almost all adult human tissues analyzed here. This indicates the GPC6 cDNA described here is incomplete. Since all GPCs identified so far have relatively short 5'-UTRs, it most likely lacks part of the 3'-UTR. High expression of GPC6 was noted in fetal kidney and fetal lung and lower expressions in fetal liver and fetal brain. In adult human tissues the message was very abundant in ovary. High expression levels were also observed in liver, kidney, small intestine, and colon. Low expression levels were found in all other tissues examined, except peripheral blood leukocytes, which were completely negative. Adult kidney contained also an mRNA species of 5.8 kb, which indicates the transcript might be subject to alternative splicing or polyadenylation. Adult heart and skeletal message also yielded a signal corresponding to an mRNA of 3.9 kb. Possibly this was due to cross-hybridization with mRNA encoding glypican-1, which is very highly expressed in these tissues (results not shown). Signals obtained for GPC4 on the same Northern blots (20) indicated the gross tissue expression of glypican-6 was almost identical to that of glypican-4. The expression of mGPC6 was examined with Northern blots of RNA preparations from stage-specified embryos. Expression could be detected as a message of ~7 kb from 7 to 17 dpc. However, after organogenesis, which proceeds until 13 dpc, the expression of GPC6 was clearly diminished, suggesting glypican-6 may mostly operate in morphogenetic processes.

View larger version (61K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of GPC6 expression in adult and fetal tissues. A, Northern blot of human fetal brain (lane 1), lung (lane 2), liver (lane 3), and kidney (lane 4) RNA. B, Northern blot of poly(A)+ RNA from 7-day (lane 1), 11-day (lane 2), 15-day (lane 3), and 17-day (lane 4) mouse embryos. C, Northern blot of human adult heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), and pancreas (lane 8) RNA. D, Northern blot of human adult spleen (lane 1), thymus (lane 2), prostate (lane 3), testis (lane 4), ovary (lane 5), small intestine (lane 6), colon mucosal lining (lane 7), and peripheral blood leukocyte (lane 8) RNA. The upper part of each frame represents the hybridization with the GPC6 probe; the lower part the hybridization with a -actin control probe (not shown for the mouse Northern blot). The positions of RNA size markers (kb) are indicated on the abscissa.

Identification of Glypican-6 as a Heparan Sulfate Proteoglycan-- To test if glypican-6 drives the synthesis of heparan sulfate, as one would predict from its structure, the GPC6 insert was subcloned in the pREP4 episomal expression vector and transfected in Namalwa cells. Namalwa cells provide an interesting background for such experiments, since these cells express very little endogenous heparan sulfate but do support the synthesis of large amounts of heparan sulfate when transfected with cDNAs that encode syndecans or glypican-1.² Control- and GPC6-transfected Namalwa cells were characterized for cell surface heparan sulfate expression by quantitative immunofluorescence flow cytometry, using an antibody specific for native heparan sulfate (10E4) and an antibody specific for -HS (3G10), a neo-epitope that includes the -glucuronate generated by the digestion of heparan sulfate by heparitinase. FACSort analyses on heparitinase-treated (3G10) and on non-heparitinase-treated (10E4) cells indicated these epitopes were abundant on GPC6 transfectants but near absent from control cells (Fig. 4A). Proteoglycan was then purified from detergent extracts of these cells by ion exchange chromatography on DEAE. Eluted proteoglycans were treated with glycosidase (heparitinase, chondroitinase ABC, both, or no digestion) and fractionated by SDS-PAGE. Analysis of these proteoglycan fractions by Western blotting, using the -HS-specific monoclonal antibody 3G10, confirmed that the transfectant cells expressed a heparan sulfate proteoglycan core protein of ~65 kDa, as would be predicted from the molecular mass of glypican-6. No bands were detected in the control cells (Fig. 4B).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Heparan sulfate expression in control and GPC6 transfectant Nawalwa cells. A, cell surface heparan sulfate expression in control and GPC6 transfectant Namalwa cells, as detected by fluorescence-activated cell sorter analysis of non-digested and heparitinase-digested cells, using the native HS-specific 10E4 antibody (on non-digested cells) and the -HS-specific 3G10 antibody (on heparitinase-digested cells). Control, Namalwa cells, transfected with pREP4. GPC6, Namalwa cells, transfected with the plasmid glyp6-pREP4. Solid line, no first antibody. Dashed line, heparitinase-treated cells, stained with 3G10. Stippled line, non-treated cells, stained with 10E4. B, Western blotting of non-digested, heparitinase-digested, doubly heparitinase- and chondroitinase ABC-digested, and chondroitinase ABC-digested proteoglycan fractions, using the -HS-specific monoclonal antibody 3G10. This antibody reacts with the desaturated uronates that are generated by heparitinase and that remain in association with the core protein after the enzyme treatment and during electrophoresis. After heparitinase it therefore detects all heparan sulfate proteoglycan core proteins present in the sample. The positions of protein size markers are indicated on the abscissa. Hase, heparitinase; Case, chondroitinase ABC. Controls, wild-type Namalwa cells (WT) and Namalwa cells transfected with pREP4 (). GPC6, Namalwa cells, transfected with glyp6-pREP4.

Genomic Structure of GPC6-- So far, in humans, only the genomic structures of GPC3 and GPC4 have been reported (20, 27). Both genes localize to chromosome Xq26, where they form a cluster of genes organized in tandem array (20). Whereas GPC4 contains nine exons, the GPC3 gene is composed of only eight exons. We tested whether glypican-6, most homologous to glypican-4, might be encoded by a gene with a structure similar to that of GPC4. The complete GPC6 cDNA was used as a probe to screen BAC and PAC filters for the identification of genomic clones. In total 2 BACs and 24 PACs were identified, and all contained parts of GPC6. Initial analysis of the PACs indicated that GPC6 spans more than 1 megabase.² Subcloning of these PACs and rescue of plasmids containing GPC6 exons allowed us to establish that the gene contained nine exons, with all exon/intron boundaries complying to the AG/GT rule (Table III) and that all exon/intron boundaries were at similar positions as in GPC4. Further FISH analysis (Fig. 5) localized BACs 114A17 and 182F5, which encode parts of glypican-6, to chromosome 13q32, where prior experiments had also localized GPC5 (19), the gene encoding glypican-5 (which is most homologous to glypican-3).

View larger version (53K): [in this window] [in a new window]   Fig. 5.   FISH mapping of GPC6 to 13q32. BAC 114A17 and BAC 182F5 (not shown) were used for chromosomal localization of GPC6 by FISH, using metaphase spreads prepared from PHA-stimulated normal peripheral blood leukocytes. Chromosomes were counterstained with 4,6-diamidino-2-phenylindole, and the images were taken using a cooled CCD device. Arrows indicate the positive signals.

Developmental Expression of Gpc6-- To investigate the expression of the mouse glypican-6 gene (Gpc6) at the cellular level, we localized Gpc6 transcripts in embryos of 11.5 and 13.5 dpc, by in situ hybridization. Two different antisense RNA probes were used, with similar results. High levels of GPC6 mRNA were limited to mesenchymal tissues. Although Gpc6 was mostly expressed in mesoderm-derived tissues, signals were also present in some neurectoderm-derived sites. Cross-sections of whole embryos at 11.5 and 13.5 dpc are represented in Fig. 6, A and C. Striking localizations at 13.5 dpc are rendered in more detail in Figs. 6-8.

View larger version (96K): [in this window] [in a new window]   Fig. 6.   Expression of glypican-6 RNA in murine embryos. Expression of glypican-6 RNA, in embryonic day 11.5 (A) and 13.5 (BG) mouse tissues, as detected by in situ hybridization with digoxigenin-labeled mGPC6 sense (B) and antisense (A and CG) RNA probes. The result shown for the sense probe (B) is representative for the levels of background staining that were obtained in several different experiments. In the 11.5 dpc embryo, only shown at low magnification (A), strong glypican-6 stainings occurred in the mesenchyme of the mandibular process, the mesenchyme surrounding the lung bud, the smooth muscle cells of the dorsal aorta, the submucosal cell layers of the intestine, and the intervertebral disks. Weaker expressions were noted in the ventral wall of the otic vesicle and in the forebrain. Low expression levels were seen in the neuroepithelium of the hindbrain and the telencephalic vesicle. The low magnification of the 13.5 dpc embryo (C) reveals the relative strengths of the signals, with prominent stainings in the intestine, lung, outflow tract of the heart, and tooth. The expressions in lung (D), dorsal aorta, and intervertebral disks (E), outflow tract of the heart (F), and kidney (G) are also shown at higher magnification. Expression in lung is seen in the mesenchyme immediately surrounding the epithelium. In blood vessels, staining is confined to the smooth muscle cell layer underlying the endothelium. Expression in the kidney is highest in the metanephric mesenchyme. The kidney epithelium is negative. Scale bar in A, B, and C, 1 mm. Scale bar in DG, 100 µm.

In the lungs of 11.5- and 13.5-dpc embryos, Gpc6 transcripts were mostly restricted to the peri-bronchial mesenchymal cells (Fig. 6D). Control hybridizations for Gpc1 transcripts, in contrast, showed an epithelial staining at stage 13.5 dpc (not shown), indicating the specificity of the staining patterns and non-overlapping expressions for these two glypicans at this site and developmental stage. In both 11.5- and 13.5-dpc embryos, Gpc6 was expressed in the aorta and other major blood vessels, in the subendothelial smooth muscle cell layers (Fig. 6E). It was also expressed in the outflow tract of the heart ventricle, but other parts of the heart were virtually negative (Fig. 6F). In the developing kidney, at 13.5 dpc, Gpc6 signals were detectable in the metanephric cap mesenchyme of the cortical region, in the condensing mesenchyme surrounding the ureteric branches (Fig. 6G). No staining was seen in the adrenal glands, an organ for which relatively high levels of glypican-4 expression were reported (17). The liver parenchyme was also negative, both at 11.5 and 13.5 dpc, but the liver septae were weakly stained.

Both the stomach (Fig. 7A) and intestine (Fig. 7B) were very strongly stained. Staining occurred in the submucosal layers, in the condensing splanchnic mesenchyme that will subsequently form the connective tissue and smooth muscle cells of these organs. At 13.5 dpc Gpc6 was also expressed in the mesenchymal cells of pancreas (not shown), gonad (Fig. 7A), mesonephric tissue (Fig. 7C), and genital eminence (not shown). The thymus was also positive (Fig. 7D). At 11.5 dpc strong expression was seen in the mesenchyme of the mandibular process, with highest expression in the mesenchymal cell layer just below the oral epithelium. No staining was present in the overlying epithelium. At 13.5 dpc Gpc6 was highly expressed in the dental mesenchyme surrounding the epithelial bud. A very strong staining was also present near the top of the lip furrow (Fig. 7E). The tongue was also positive. At 13.5 dpc, cartilage primordia of the ear and snout expressed Gpc6. Although the notochord and vertebrae were negative, intervertebral disks were strongly expressing Gpc6, both at 11.5 and 13.5 dpc (Fig. 7F and also see Fig. 6E). Gpc6 was also highly expressed in mesenchymal condensations of both the forelimb and hindlimb (not shown).

View larger version (160K): [in this window] [in a new window]   Fig. 7.   Expression of glypican-6 RNA in dpc 13.5 murine embryos. Expression in stomach (A) and intestine (B) occurs in the prospective smooth muscle layer. Mesenchymal stainings in mesonephros (C) and thymus (D). In the developing tooth (E) expression is limited to the dental mesenchyme. Note particularly strong mesenchymal stainings at the top of the lip furrow. Very distinctive stainings occur also at the level of the caudal intervertebral dics, but the remnants of the notochord are negative (F). Expression occurs also in the gonad (A, lower left corner, under the stomach). Scale bar, 100 µm.

In general, low levels of expression were observed in the nervous system. At 11.5 dpc low expression levels were seen in the neuroepithelium of the hindbrain and the telencephalic vesicle. The neuro-epithelial cells lining the mesencephalic vesicle were also weakly positive. At 13.5 dpc Gpc6 was expressed in the roof of the neopallial cortex, which gives rise to the future cerebral cortex. Weak expression was also visible in the medulla oblongata, the choroid plexus, and the ventral mantle layer of the spinal cord. Stronger signals were seen in the ganglia of the glossopharyngeal nerve (not shown).

The mesenchyme surrounding the olfactory epithelium was strongly stained, both in 11.5- and 13.5-dpc embryos, but the epithelium itself was negative (Fig. 8A). The mesenchymal tissues lining the dorsal root ganglia (perineurium), but not the ganglia themselves, were clearly stained (Fig. 8B). There were, however, also a few examples of clear epithelial stainings. At 11.5 dpc Gpc6 was also expressed in the ventromedial wall of the otic vesicle. At 13.5 dpc, the cochlea of the inner ear, which is derived from the ventral wall of the otic vesicle, was clearly outlined (Fig. 8C). In the eye (Fig. 8D) not only the neural retina but also the cells that compose the wall of the lens vesicle were stained. Both the otic vesicle and lens vesicle are of ectodermal origins.

View larger version (114K): [in this window] [in a new window]   Fig. 8.   Expression of glypican-6 RNA in dpc 13.5 murine embryos. Detail images of the olfactory epithelium and surrounding mesenchyme (A), dorsal root ganglia (B), cochlea (C), and eye (D). Note the epithelial stainings in the cochlea and lens but absence of stain in the olfactory epithelium. Scale bar, 100 µm.

In general the results obtained by in situ hybridization in the mouse were consistent with the results obtained on Northern blots from human tissues (with the possible exception of liver).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Bio-informatics and RACEs have led to the identification of a sixth member of the human glypican gene family and of its mouse orthologue. As might have been expected from the characterization of the other members, the human and mouse forms of glypican-6 are highly similar in structure, with 96% identity.

Ectopic expression of this protein in Namalwa cells shows that glypican-6 can be substituted with heparan sulfate. Whereas the syndecans frequently contain both heparan sulfate and chondroitin sulfate (28), the recombinant glypican-6 that is substituted with heparan sulfate in Namalwa cells apparently only carries this type of glycosaminoglycan. This is an observation that has also been reported for other glypicans (12, 14, 17, 19, 29), implying that glypicans may be substrates that are particularly well suited for the priming of this glycosaminoglycan or that are exclusively routed to membrane compartments that are endowed with this specific biosynthetic machinery. Whether and how this might relate to the highly conserved "glypican" structural motif that is common to all these proteins is still an unresolved issue. It is clear, however, that glypican-6 extends the list of proteins that can ensure the expression of heparan sulfate at cell surfaces and of potential co-receptors for heparin-dependent growth factors and morphogens.

Based upon sequence alignments only, the glypicans might already be divided in two subfamilies. The glypicans -1, -2, -4,and -6 form a separate group with high homology (40-60%) to each other but only 20% identity to the other group comprising glypican-3 and glypican-5 (also 40% identical to each other). The hypothesis of different subfamilies is further strengthened by an analysis of the genomic structures of the corresponding genes as follows: GPC1, GPC2, GPC4, and GPC6 consist of nine exons, whereas GPC3 and GPC5 contain only eight exons (20, 27).² Interestingly, glypican-6 is most homologous to glypican-4 (both proteins are 63% identical). The GPC6 gene maps close to the GPC5 gene on human chromosome 13q32, whereas GPC4 maps to chromosome Xq26, where it flanks GPC3, the gene encoding the glypican which so far is most homologous to glypican-5. This suggests glypicans arose from a series of gene and genome duplications and may herald extension of the gene family by additional members and gene clusters. Whether members of the same subfamily share some common functions and members belonging to different subfamilies have different functions remains to be established. However, it is interesting to note that glypican-3 shows an overlapping expression with glypican-6 in the developing kidney (at least in mouse), whereas glypican-6 may not substitute for the loss of GPC3 function in Simpson-Golabi-Behmel syndrome (since that syndrome is often associated with kidney malformation).

GPC6, like GPC1 and GPC4 (13, 17), is widely expressed in fetal and adult tissues, but GPC6 mRNA is not ubiquitous. In situ hybridization studies in mouse embryos suggest highly specific and regulated expressions of glypican-6 during development, partly overlapping with and partly different from those reported for other glypicans. High and highly distinctive expressions can be inferred in the dental mesenchyme, metanephric cap mesenchyme, intestinal mesenchyme, and blood vessels, suggesting a specific function for glypican-6 in the development of these organs. In the developing tooth the expression pattern of glypican-6 overlaps with that reported for BMP4, at least during the developmental stages examined here (30). BMP4 is the closest human homologue of the product of decapentaplegic. Since dally influences the signaling processes supported by decapentaplegic, it is tempting to speculate that glypican-6 might act as a co-receptor for BMP4 or modulator of the BMP4 signaling pathway during tooth development. Glypicans in general might have a role during tooth development since loss of GPC3 in Simpson-Golabi-Behmel syndrome patients is associated with dental malocclusion (31). In the kidney (at the mRNA level), the expression of glypican-6 overlaps with the expression pattern of BMP7 (6) and also with that of the heparan sulfate-modifying enzyme, heparan sulfate-2-O-sulfotransferase (32). All three are highly expressed in the metanephric mesenchyme. The heparan sulfate present at these sites appears to be necessary for kidney development as mice deficient in heparan sulfate-2-O-sulfotransferase exhibit bilateral renal agenesis (32). By its nature, glypican-6 qualifies as a potential substrate for this enzyme. Mice deficient in BMP7 show a gradual cessation of nephrogenesis associated with apoptosis of the metanephric mesenchyme. Interestingly, BMP7 expression in the metanephric mesenchyme requires proteoglycans (6). However, in addition to glypican-6, at least glypican-3 and syndecan-2 are also expressed in the kidney mesenchyme and might equally qualify as regulators of the signaling of this growth factor (16, 33).

The expression of glypican-6 indeed partially overlaps that of several other heparan sulfate proteoglycans, including glypican-4, the proteoglycan that structurally is most closely related to glypican-6. Based on prior reports by others (17) overlapping GPC4 and GPC6 expressions occur in the prospective smooth muscle layers of the blood vessels and digestive system. Both genes also appear to be co-expressed in the nervous system, where they are mainly restricted to the ventricular zones. Yet, in most tissues the two members of this glypican subgroup appear to be differentially expressed. In the kidney, where GPC4 (along with GPC1) is highly expressed in pretubular aggregates, GPC6 expression is limited to the cortical mesenchyme, where it shows overlap with GPC3 expression (16). In general, both glypican-1 and glypican-4 show epithelial expressions, whereas glypican-6, like glypican-3, shows a predominant expression in mesenchymal cells. Intriguingly, in the otic vesicle and cochlea, one of the rare examples of epithelial stainings for GPC6, glypican-6 appears to be expressed along with glypican-5 (18), suggesting coordinated expression of two members from the same gene cluster (assuming a similar gene organization in man and mouse) at this site. Future studies will undoubtedly provide additional details on these expressions and might also reveal possible relationships and hierarchies between genes belonging to similar or different subfamilies and gene clusters. At present, we are left wondering about the functional significance of this extensive proteoglycan diversity. In summary, glypican-6 is a novel heparan sulfate proteoglycan that shows a highly distinctive and regulated developmental expression, extending the repertoire of candidate morphogen cofactors or modulators.

	ACKNOWLEDGEMENTS

We thank Jenny Peeters, Dirk de Valck, and Frank Luyten for helpful suggestions regarding in situ hybridizations; Bernadette Vanderschueren and Guy Debrock for helpful discussions and interpretation of in situ data; and Karel Rondou for assistance in preparing the illustrations.

	FOOTNOTES

^* This work was supported in part by Grant G.0234.95 from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, by the Geconcerteerde Onderzoeksacties 1996-2000, by the Interuniversity Network for Fundamental Research sponsored by the Belgian Government, and by the Flanders Interuniversity Institute for Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF105267 and AF105268.

^§ Beneficiaries of a fellowship from the Vlaams Instituut voor de Bevordering van het Wetenschappelijk- Technologisch Onderzoek in de Industrie.

Research Directors of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.

^** To whom correspondence should be addressed: Center for Human Genetics, University of Leuven, Campus Gasthuisberg, O & N6, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-345863; Fax: 32-16-3457166; E-mail: guido.david@med.kuleuven.ac.be.

² M. Veugelers, B. De Cat, H. Ceulemans, A.-M. Bruystens, C. Coomans, J. Dürr, J. Vermeesch, P. Marynen, and G. David, unpublished results.

	ABBREVIATIONS

The abbreviations used are: FGF, fibroblast growth factor; GPC, glypican; HS, heparan sulfate; BMP, bone morphogenetic protein; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; FISH, fluorescent in situ hybridization; dpc, days post-coitum; BAC/PAC, bacterial/P1-derived artificial chromosome; UTR, untranslated region; kb, kilobase; bp, base pair.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES