Glypican-3 Controls Cellular Responses to Bmp4 in Limb Patterning and Skeletal Development

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Stephenie Paine-Saunders,* Beth L. Viviano,* Joel Zupicich,† William C. Skarnes,† and Scott Saunders*,‡,1 *Division of Newborn Medicine, Department of Pediatrics, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri 63110; †Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720; and ‡Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson–Golabi–Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS. © 2000 Academic Press Key Words: heparan sulfate; glypican; proteoglycans; bone morphogenetic protein; limb; skeleton; polydactyly; Simpson– Golabi–Behmel syndrome.

INTRODUCTION tions. Although heparan sulfate is found ubiquitously on cell surfaces, these carbohydrates are carried predominantly The essential requirement of heparan sulfate glycosami- by the protein products of only two gene families, synde- noglycans for normal development has been emphasized cans (Saunders et al., 1989) and glypicans (David et al., only recently by the discovery that mutations in heparan 1990). sulfate biosynthesis cause significant developmental de- All members of the glypican family share a common fects in Drosophila (Bellaiche et al., 1998; Binari et al., mechanism of attachment to the cell membrane through 1997; Hacker et al., 1997; Haerry et al., 1997), in mice glycosylphosphatidylinositol linkages, and the character- (Bullock et al., 1998), and in humans (Lind et al., 1998; ization of amino acid sequence homologies has suggested McCormick et al., 1998). This has stimulated interest in the existence of subfamilies of structurally related verte- understanding the potential functions of specific protein brate glypicans (Paine-Saunders et al., 1999). Whether these gene products that bear these posttranslational modifica- individual glypicans have distinct or a related function is unknown; however, human mutations in a single glypican 1 To whom correspondence should be addressed at the Washing- gene, glypican-3 (Gpc3), result in an overgrowth phenotype ton University School of Medicine, 660 South Euclid Avenue, known as Simpson–Golabi–Behmel syndrome (SGBS). This Campus Box 8208, St. Louis, MO 63110. Fax: (314) 286-2893. X-linked condition is characterized by both pre- and post- E-mail: [email protected]. natal overgrowth, a distinct facial appearance, macroglos-

0012-1606/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 179 180 Paine-Saunders et al. sia, and multiple anomalies, including cleft palate, congeni- Gpc3 expression in the progress zone, in the anterior and tal heart and renal defects, imperforate vagina, umbilical posterior necrotic zones, and in the interdigital web where hernias, supernumerary nipples, vertebral and rib defects, it plays an active role in mediating critical apoptotic events and postaxial polydactyly (Behmel et al., 1984; Garganta in the development of these structures. Furthermore, Bmp4 and Bodurtha, 1992; Hughes-Benzie et al., 1992; Pilia et al., haploinsufficient mice show a phenotype implicating a role 1996). Nothing is known about the mechanism by which for BMPs in anterior–posterior patterning (Dunn et al., these in vivo mutations of Gpc3 lead to the described 1997). Therefore we decided to test genetically whether phenotypic features in humans. Nonetheless, as described GPC3 might be important in BMP4 signaling in the devel- in this study and elsewhere (Cano-Gauci et al., 1999), oping limb and have found a strong genetic interaction GPC3-deficient mice recapitulate the SGBS phenotype and between mutants of these two genes that result in a high therefore provide an excellent in vivo model system for penetrance of both postaxial polydactyly and rib malforma- evaluating the functions of vertebrate glypicans. tions. This study provides the first in vivo evidence of a role Although the mechanism of action of vertebrate glypi- for glypicans in vertebrate development and establishes a cans is unknown, the function of the Drosophila glypican previously unknown link between GPC3 and BMP4 func- dally has been evaluated genetically. These studies have tion, which suggests a mechanism for the skeletal defects revealed a role for dally in both wingless (wg; the Drosoph- seen in SGBS. ila ortholog of vertebrate Wnt) signaling during early embryonic patterning (Lin and Perrimon, 1999; Tsuda et al., 1999) and decapentaplegic (dpp; the Drosophila ortholog of MATERIALS AND METHODS vertebrate bone morphogenetic proteins) signaling during postembryonic morphogenesis (Jackson et al., 1997). The Generation of mutant mice. A 7.5-kb genomic fragment containing the first exon of Gpc3, which includes the signal peptide, mechanism of Dally function has not been experimentally was isolated from a strain 129SV genomic library in the Lambda established; however, these findings are consistent with the FIX II vector (Stratagene) using a 5Ј-specific probe from human proposed function for heparan sulfate proteoglycans as Gpc3 cDNA. The DNA targeting vector was constructed by replac- coreceptors for a number of growth factors in vitro. More- ing the first exon of Gpc3 (from approximately Ϫ50 bp to just over, it is clear from the above studies that glypican downstream of the first splice site) with the AscI fragment contain- functions are highly context and tissue dependent in Dro- ing the PGK-neo gene from pKO SelectNeo V800 (Lexicon). This sophila and are likely to be similarly context dependent in resulted in a vector flanked with 5.5 kb of homologous sequence vertebrates. Therefore it is not obvious which signaling upstream and 2.0 kb of homologous sequence downstream. Addi- pathways might be impacted by the loss of function of a tionally, an RsrII fragment containing the RNA Pol II diphtheria specific vertebrate glypican in any particular tissue during toxin gene from pKO SelectDT V840 (Lexicon) was inserted at the 3Ј end to facilitate negative selection. The targeting vector was development. We have therefore chosen to focus on under- linearized with NotI before electroporation into RW4 ES cells. standing the function of GPC3 in the pathogenesis of a G418-resistant ES cell clones were screened for targeting by South- single phenotypic trait in vertebrates, polydactyly. ern blot analysis. Although commonly seen in human patients with SGBS, One targeted ES cell line, No. 108, was microinjected into this phenotype is never seen in GPC3-deficient mice. We C57Bl/6J blastocysts, resulting in multiple high-percentage chi- therefore speculated that these animals may represent a meric mice. Chimeric males were mated to C57Bl/6J females (The sensitized strain for glypican function in limb development Jackson Laboratory). Agouti offspring that inherited the targeted and that genetic interaction studies might point toward the allele (Gpc3tm108Sau) were backcrossed to either C57Bl/6J or C57Bl/ tm1blh TgN(␤geo)ex136Ska signaling pathway(s) mediated by GPC3 in this tissue. 6-Bmp4 (The Jackson Laboratory). The Gpc3 allele Indeed, as has been suggested by others, the identification was recovered in a screen, as described previously (Skarnes et al., 1995), whereby the pGT1.8TM vector inserted within the second of genetic modifiers provides an important pathway to intron of Gpc3 in 129 Ola ES cells. understanding the cellular and genetic interactions under- The Gpc3tm108Sau allele was identified by PCR using a Gpc3-specific lying embryonic development and the pathogenesis of hu- forward primer, Dx22099, 5Ј-ATCTCCCAGCTAAGCTCCTT-3Ј man malformations (Dunn et al., 1997; Erickson, 1996; (Ϫ187 to Ϫ168), and reverse primer derived from the neo gene, Winter, 1996). 5Ј-ATGCTCCAGACTGCCTTG-3Ј, while the wild-type allele was We have found that the distribution of Gpc3 expression identified using a reverse primer designed from the deleted exon, overlaps with the patterns of a number of gene products Dx22141, 5Ј-CCCAAGCCTAGCAGCATC-3Ј (184–201). Details are ␤ implicated in limb patterning and whose biological activi- available on request. The Gpc3TgN( geo)ex136Ska allele was followed by ties are known to be dependent in part on heparan sulfate detection of the lacZ reporter gene contained in the gene trapping proteoglycans. These include fibroblast growth factors vector (Skarnes et al., 1995), while Bmp4 knockout genotyping (Winnier et al., 1995) and sex determination (Hogan et al., 1994) were (FGFs) (Rapraeger et al., 1991; Yayon et al., 1991), Wnts (Lin both done by PCR as described elsewhere. et al., et al., and Perrimon, 1999; Reichsman 1996; Tsuda Timed pregnancies were determined by visualization of a vaginal 1999), bone morphogenetic proteins (BMPs) (Jackson et al., plug which was defined as day 0.5. 1997; Ruppert et al., 1996), and members of the vertebrate Southern analysis. ES cell clones were harvested and genomic hedgehog family (The et al., 1999). In particular, Bmp4 DNA was prepared, restriction digested with HindIII and XbaI, and expression in the developing limb is highly coincident with fractionated by agarose gel electrophoresis (George and Hynes,

1994). DNA was transferred to Zeta Probe (Bio-Rad) using alkaline transfer and hybridized with Rapid-Hyb (Amersham) at 65°C for 1 h using a random-labeled probe generated from a 0.5-kb EcoRV–XbaI genomic fragment immediately 3Ј of the targeted sequence. The hybridized targeted allele gives a restriction fragment of 2.1 kb compared with the endogenous allele of 5.2 kb. Western analysis. Heparan sulfate proteoglycans were ex- tracted from embryonic day 16.5 mouse liver in 50 mM Tris–HCl (pH 8.0), 0.15 M NaCl, 6 M urea, 1% Triton X-100, 1 mM EDTA, 1 ␮g/ml pepstatin, 0.25 mg/ml N-ethylmaleimide, 0.5 mM phenyl- methylsulfonyl fluoride and purified by DEAE chromatography as described elsewhere (Herndon and Lander, 1990). Heparitinase (Sigma) digests (0.002 U/ml) were carried out in 20 mM Tris–HCl, pH 7.5, 5 mM CaCl2 at 37°C. Following electrophoresis in 4–15% SDS–PAGE gel (Bio-Rad) and electroblotting to Immobilon-P (Mil- lipore), GPC3 was detected using a rabbit polyclonal antisera generated against a synthetic peptide corresponding to murine GPC3 amino acids 524–541. X-gal staining of tissues. Whole embryos at day 11.5, or dis- sected limbs also at day 11.5 and later, were fixed 30 min in 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA, 0.02% NP-40 in PBS. Tissues were washed 3ϫ 30 min in 0.2% NP-40 in PBS and stained overnight at 37°C in 1 mg/ml X-gal, 5 mM K3Fe(CN)6,5mMK4(CN)6, 2 mM MgCl2, 0.01% deoxycholate, 0.02% NP-40 in PBS. After staining, tissues were postfixed in 1% formaldehyde, 0.2% glutaraldehyde in PBS for 1 h before dehydra- tion to 30% saline/70% EtOH, sectioned in paraffin, and lightly counterstained with eosin. Skeleton preparations. Embryonic day 16.5 animals were fixed and their skeletons were stained with alcian blue and alizarin red and cleared with KOH/glycerine as described by McLeod (1980).

RESULTS AND DISCUSSION Null Alleles of Glypican-3 Model Human SGBS To evaluate the functions of GPC3 in vivo, we created and characterized two mutant Gpc3 alleles generated by independent methods. The first allele, designated tm108, was created by homologous recombination, resulting in targeted deletion of both the transcriptional and the trans- lational initiation sites, as well as nucleotides encoding the signal peptide and first 33 amino acids of the putative FIG. 1. Gene targeting, trapping, and molecular characteriza- mature protein (Fig. 1A). The second allele, designated tion. (A) Structure of the murine Gpc3 locus. In the targeting vector the neo gene was flanked on the 5Ј side with 5.5 kb of Ex136, was identified in a gene trap experiment designed to genomic DNA and on the 3Ј side with 2 kb of genomic DNA, identify mutations in secreted or cell surface proteins introducing a deletion of the first exon of Gpc3. The identified (Skarnes et al., 1995). Insertion of the gene trap vector into probe hybridizes to a 2.2-kb HindIII/XbaI restriction fragment at the second intron of Gpc3 was confirmed by sequence a correctly targeted locus (tm108) compared with a 5-kb HindIII analysis of 5Ј RACE products derived from a fusion tran- restriction fragment from the endogenous locus. (B) The trapped script (Townley et al., 1997) (Fig. 1B). Both ES cell clones allele of Gpc3 (Ex136) was the result of a random integration were successfully transmitted through the germ line of event of the gene trapping vector pGT1.8TM within the second chimeric mice following blastocyst injection. To determine intron of Gpc3 as indicated. Identification of the site of integra- Ј the functional consequence of these mutations we evalu- tion within the Gpc3 locus was determined by 5 RACE of the ated the presence of GPC3 protein in DEAE-purified pro- resulting fusion transcript. (C) A rabbit polyclonal antiserum, specific to a GPC3-derived peptide, detects the GPC3 proteogly- teoglycan extracts from hemizygous mutant and wild-type can and its core protein (as well as a previously described lower animals by Western blotting using an antipeptide antibody molecular weight fragment of GPC3) following heparitinase recognizing the core protein of murine GPC3. While this digestion in extracts from wild type, but not in extracts from antiserum readily identified the GPC3 heparan sulfate either the targeted (tm108) or the trapped (Ex136) allele of proteoglycan in wild-type extracts, as well as the core glypicans, thus indicating that both alleles are null mutations protein following enzymatic removal of the heparan sulfate for GPC3.

27/53), 23% heterozygote (Gpc3/ϩ, 12/53), and 26% hemizygote (Gpc3/Y, 14/53). By the time of weaning, however, there was a significant deviation from the expected fre- quency of affected animals with 58% wild type (ϩ/ϩ, 39/67), 25% heterozygote (Gpc3/ϩ, 17/67), and only 16% hemizygote (Gpc3/Y, 11/67). The majority of these deaths appear to occur within the first 2 days of life, but the reasons for this lethality have not been addressed. Some of these phenotypic features have recently been reported by another group who has independently targeted the Gpc3 allele (Cano-Gauci et al., 1999). However, a number of additional phenotypic traits seen in human SGBS were also apparent in our mutant alleles of Gpc3. Specifically, we found that ventral wall closure defects occur with incomplete penetrance (33%, n ϭ 12). In most FIG. 2. Mice bearing mutant alleles of Gpc3 recapitulate many of cases these consisted of small- to moderate-sized umbilical the phenotypic features of human patients with Simpson–Golabi– herniations but in one stillborn animal this was manifested Behmel syndrome. (A) Male embryonic day 16.5 embryos, bearing as an omphalocele. In addition, stained skeletal prepara- either hemizygous Ex136 (column 4) or tm108 (column 5) mutant alleles weigh approximately 30% larger (P Ͻ 0.0001) than wild- type littermate controls (column 1), while heterozygous females for either the Ex136 (column 2) or the tm108 (column 3) allele show intermediate growth properties (P Ͻ 0.0001). (B) The kidneys of Gpc3 mutants show disregulated development of the collecting system resulting in cystic (arrowhead) medullar changes of the renal parenchyma. (C and D) Subtle skeletal abnormalities are apparent in Gpc3-deficient animals, including bifurcations of the xyphoid process as seen in D compared with a wild-type littermate control in C.

chains, no detectable GPC3 was found in extracts from animals hemizygous for either mutant allele (Fig. 1C). In contrast, immunostaining of the same blot with a mono- clonal antibody recognizing the core protein of syndecan-1 detected similar levels of that heparan sulfate proteoglycan in all extracts (data not shown). Therefore we have con- cluded that both the tm108 and Ex136 alleles of Gpc3 are functionally null alleles. Consistent with the findings of Gpc3 mutations in humans, we found that mice bearing either the tm108 or the Ex136 mutation displayed a range of phenotypic features similar to human patients with SGBS, and no obvious differences between the phenotypes of mice bearing these FIG. 3. Expression of the Gpc3 promoter is dynamically regulated two different alleles were observed (Fig. 2 and data not in the developing limb as reflected by the distribution of shown). In particular, hemizygous male mouse embryos, of ␤-galactosidase expression from the trapped Ex136 allele. Whole- either allele, were 30% larger than wild-type littermate mount ␤-galactosidase staining of embryonic day 11.5 (A–E), day controls (Fig. 2A), while heterozygous female animals (pre- 12.5 (F), and day 13.5 (G) limbs, followed by paraffin infiltration and sumably mosaic due to X inactivation) showed intermedi- sectioning (C–G). Gpc3 is not expressed in the apical epidermal ate growth properties. ridge (AER) as seen in whole mount (A and B) or longitudinal Animals deficient for GPC3 also displayed medullary section (arrowheads). At embryonic day 11.5, Gpc3 is intensely expressed in the distal mesenchyme and in the proximal anterior cystic dysplasia of their kidneys (Fig. 2B) and a high fre- and posterior limb bud as seen in longitudinal section (D) and is quency of perinatal lethality, phenotypes reported in hu- asymmetrically expressed preferentially in the dorsal third of the man patients with SGBS. On embryonic day 16.5, embryos limb bud as seen in cross section at a level just below the AER (E). derived from heterozygous female (129SVJ/C57B16) and At later stages (F and G), Gpc3 expression becomes restricted to wild-type male (C57B16) matings were found at the ex- regions of cartilage differentiation in developing bone and within pected Mendelian ratios including 51% wild type (ϩ/ϩ, the interdigital webs. a, anterior; p, posterior; d, dorsal; v, ventral.

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. GPC3 in Limb Patterning and Skeletal Development 183 tions revealed subtle malformations in GPC3-deficient limb patterning. One of these is insulin-like growth pups, including bifurcations of the xyphoid process (Figs. factor-II (IGF-II). Because mutant mice with elevated serum 2C and 2D). The identification of these additional traits and tissue levels of IGF-II share several phenotypic features further supports the validity of murine GPC3 deficiency as with SGBS, including postaxial polydactyly (Eggenschwiler a model for human SGBS and the potential value of these et al., 1997), there has been considerable speculation about animals in understanding the mechanism of GPC3 func- the potential role of GPC3 as a negative regulator of IGF-II tion. Given the complex nature of the SGBS phenotype, and function (Pellegrini et al., 1998; Pilia et al., 1996). However, the evidence that the Drosophila glypican dally may have there has been no experimental evidence in support of this multiple mechanisms of action which are both tissue and hypothesis. Indeed, on the contrary it has been impossible developmental stage dependent (Jackson et al., 1997; Lin to demonstrate a direct physical interaction between the rat and Perrimon, 1999; Tsuda et al., 1999), we chose to focus GPC3 core protein and IGF-II (Song et al., 1997), as was on understanding the function of GPC3 in the development initially reported for human GPC3 (Pilia et al., 1996). of a single phenotypic trait, polydactyly. Furthermore, recent experiments have found no alteration in tissue or serum levels of IGF-II in embryonic GPC3- deficient animals (Cano-Gauci et al., 1999), a result that we Gpc3 Expression Is Dynamically Regulated during have independently confirmed by quantitative radioimmu- Murine Limb Patterning noassay (data not shown). As a first step in evaluating the possible roles of GPC3 in In contrast, there is considerable in vitro evidence for a vertebrate limb patterning, we examined the normal distri- general role of heparan sulfate proteoglycans in the signal- bution of expression of Gpc3 during limb morphogenesis ing of a number of developmentally important growth using the ␤-galactosidase reporter allele Ex136. Preliminary factors with spatial patterns of action that overlap with the studies (Paine-Saunders and Saunders, unpublished) have distribution of Gpc3 expression. These include FGFs in the established that expression of ␤-galactosidase from this progress zone, Wnts in the dorsal limb bud, and BMPs trapped allele faithfully recapitulates the normal expression within the ANZ, PNZ, and interdigital web. Furthermore, pattern of Gpc3 mRNA as detected by in situ hybridization. as discussed above, there is evidence clearly supporting a Gpc3 expression in the developing limb was seen to be role for the invertebrate glypican dally in either wg or dpp dynamically regulated in association with limb patterning signaling in the context of distinct developmental events in events. At early embryonic stages (embryonic day 11.5), Drosophila (Jackson et al., 1997; Lin and Perrimon, 1999; Gpc3 expression was clearly absent from the specialized Tsuda et al., 1999). Because of the overlapping expression signaling structure the apical epidermal ridge (AER) (Figs. pattern of GPC3 and BMP4, and the evidence of a role for 3A, 3B, and 3C), yet was intensely expressed in the under- BMP4 in anterior–posterior patterning (Dunn et al., 1997), lying mesenchyme which responds to inductive cues from we tested genetically whether GPC3 might be important in the AER. However, expression in the mesenchyme at this BMP4 signaling in the developing limb. stage was asymmetric. As seen in longitudinal sections, Bmp4 homozygous deficient animals die during early Gpc3 expression was most intense in the regions of the embryonic development before limb morphogenesis (Win- distal mesenchyme within the progress zone and within the nier et al., 1995). Animals heterozygous for a targeted proximal anterior and posterior limb bud, regions which disruption of the Bmp4 gene (Bmp4tm1blh), however, are ultimately define the anterior and posterior necrotic zones viable and display a haploinsufficient phenotype that in- (ANZ and PNZ, respectively) (Fig. 3D). On cross section of cludes defects in anterior–posterior limb patterning (Dunn the limb bud at a level just proximal to the AER, it was et al., 1997). In particular, it has been previously reported apparent that Gpc3 was asymmetrically expressed more that on the C57Bl/6 genetic background Bmp4 heterozy- abundantly in the dorsum of the developing limb bud as gotes display preaxial polydactyly of the right hindlimb well. Whether this pattern reflects positive regulation of with 12% penetrance (Dunn et al., 1997). We have observed Gpc3 gene expression in these peripheral regions of the similar results with these animals on a mixed 129SV/ limb mesenchyme or alternatively a suppression of Gpc3 C57Bl/6 background (Fig. 4F). But on closer inspection of expression in the central condensing mesenchyme region embryonic day 16.5 Bmp4 heterozygotes, we also observed cannot be currently distinguished. a small vestigial nubbin on the postaxial side of the right At later developmental ages such as embryonic days 12.5 forelimb with approximately 50% penetrance (Fig. 4B). This (Fig. 3F) and 13.5 (Fig. 3G), glypican-3 expression was nubbin typically showed little or no ossification (Fig. 4I, restricted to the interdigital webs and the regions of chon- arrowhead) and frequently was no longer visible on new- drocytic differentiation of the developing bones. born animals. When present at birth it typically formed an ectopic nail at this location. When animals were mated to produce animals heterozy- GPC3 and BMP4 Mutations Interact to Result in gous for a disrupted Bmp4 allele and hemizygous for either Postaxial Polydactyly and Other Skeletal Defects mutant Ex136 or tm108 Gpc3 allele, the nature and severity A number of factors that regulate cell proliferation, of the limb phenotype changed dramatically. Now, 100% of differentiation, or death have been implicated in vertebrate the animals showed some degree of polydactyly with vari-

In the hindlimb, the change in the polydactylous phenotype was even more dramatic. Here 66% of the animals had postaxial polydactyly, primarily of the right hindlimb, of a form similar to that which is seen in human patients with SGBS. The expressivity of this phenotype was complete, with all animals displaying well-formed branched bifurcations of the fifth digit identical to those shown in Figs. 4G and 4H and identical to those seen in human patients with SGBS. Similar to the forelimb defects, these structures contained ossified bone as well, containing an ectopic triphalangeal digit bifurcating from a common fifth metatarsal bone (Fig. 4K). We found no differences in severity or penetrance of polydactyly whether the targeted tm108 or the trapped Ex136 mutant allele of Gpc3 was combined with heterozygotes for Bmp4. We therefore conclude that these two alleles of Gpc3 are equivalent with respect to limb patterning and, because they represent independently generated mutant alleles of Gpc3, that the phenotypes seen are specific to a loss of GPC3 function in combination with a reduction in BMP4 function. On close examination of the remainder of the skeletons of the animals null for Gpc3 and heterozygous for Bmp4, we determined that there were other significant skeletal abnormalities with apparently complete penetrance. These animals, compared with littermate controls (Figs. 5A and 5B), FIG. 4. Combined genetic deficiency for Gpc3 and haploinsuffi- had markedly abnormal chests with deformed and abnor- ciency for Bmp4 results in postaxial polydactyly. Forelimbs (A–D, mally fused ribs (Figs. 5C and 5D). In addition, these I) and hindlimbs (E–H, J, K) of embryonic day 16.5 mice bearing animals were found to have dual ossification centers heterozygous mutations for Bmp4 either alone or in combination throughout the length of their sternum (Fig. 5F). These with hemizygous deficiencies for Gpc3. All animals bearing muta- skeletal abnormalities were not seen in either Gpc3 null or tions in Gpc3 alone show normal limb patterning (not shown) as do Bmp4 heterozygous animals alone and were apparent only the majority of animals haploinsufficient for Bmp4 mutations alone (A and E), while 10% of Bmp4 haploinsufficient animals as a result of genetic interaction between these two mutant display preaxial polydactyly of the right hindlimb and 50% display alleles. Interestingly, these phenotypic features are quite a small vestigial postaxial nubbin (B, arrow) of the right forelimb. reminiscent of those reported in association with a ho- This nubbin displays little to no ossification as shown by skeletal mozygous deficiency of another Bmp gene, Bmp7 (Luo et preparation (I). In contrast, 100% of animals bearing either hemi- al., 1995). However, in contrast to the high penetrance of zygous targeted (tm108) or trapped (Ex136) alleles, in combination this phenotype in animals deficient for Gpc3 and haploin- with a heterozygous mutation for Bmp4, develop postaxial poly- sufficient for Bmp4, this feature was found inconsistently dactyly. Forelimbs frequently reveal bilateral abnormalities involv- in Bmp7-deficient animals. This may be consistent with ing more severe duplications of the fifth digit, which include both the hypothesis that GPC3 is involved in a pathway well-ossified triphalangeal duplications (J, asterisk). The hindlimbs that impacts multiple BMP family members and the specu- of these animals display postaxial polydactyly with 66% penetrance, and all display well-patterned and ossified duplications of lation that some structurally related BMPs, such as BMP4 the terminal fifth digit (K, asterisk). There were no differences in and BMP7, may display certain redundancies in function severity or penetrance of these phenotypes between either allele of during development (Dudley and Robertson, 1997; Katagiri Gpc3. et al., 1998). However, we found that a genetic interaction was not evident in all tissues in which GPC3 and BMP4 (or other BMP genes) are coexpressed, suggesting that, like Dally, GPC3 may function in a tissue-dependent manner. able severity (n ϭ 36). In the forelimbs, the phenotype demonstrated a clear increase in the severity of the pheno- GPC3 Modulation of BMP4 Signaling type observed in Bmp4 heterozygous animals alone. Fre- quently both forelimbs were associated with well-formed This study represents the first genetic evidence of a role bifurcations of the fifth digit (Figs. 4C and 4D). The redun- for glypicans in the control of cellular responses to BMPs dant structures in these animals contained ossified bone in during vertebrate development. Here we report that the loss the form of an ectopic triphalangeal duplication branching of GPC3 results in both enhanced and synthetic phenotypes from the fifth metatarsal bone (Fig. 4J), a defect never seen in association with BMP4 heterozygous deficiencies. As in Bmp4 heterozygous mutants alone. outlined below, we believe that this genetic interaction

regard has been the identification of genetic modifiers of BMP function. For example, several mutations that modify the phenotype of weak dpp alleles in Drosophila have been identified (Ferguson and Anderson, 1992a,b; Raftery et al., 1995), and the characterization of these gene products, and their vertebrate homologs, has led rapidly to new informa- tion about the downstream signaling pathways activated by BMP and about the secreted proteins that modify BMP activities. Significantly, the Drosophila glypican Dally has been shown to modify Dpp function in at least some postembryonic developmental events in imaginal tissue (Jackson et al., 1997). In most of these tissues, mutations of dally enhance the phenotype associated with reduced dpp expression while suppressing phenotypes associated with ectopic dpp expression, and dally mutants display reduced expression of dpp target genes, suggesting that Dally directly affects cellular responses to Dpp. Similarly, we have discovered that loss of a vertebrate glypican, GPC3, modifies BMP4 function in the mouse limb. Interestingly, not only is this genetic interaction between Gpc3 and Bmp4 mutants in mice particularly strong, but the polydactylous phenotype seen in these animals is identical to the phenotype seen in human patients with Gpc3 deficiency alone. Taken together, the results of our studies and previous work in Drosophila demonstrate that the regulation of cellular responses to BMP is an evolutionarily conserved function of glypicans. FIG. 5. Combined genetic deficiency for Gpc3 and haploinsuffi- There are multiple mechanisms by which Dally and ciency for Bmp4 results in other skeletal abnormalities. Animals GPC3 could function. For example, they could affect a bearing heterozygous null mutations for Bmp4 alone (A and B), as parallel signaling pathway that alters cellular responses to well as animals bearing hemizygous null mutations for Gpc3 alone BMPs. Alternatively, they could more directly modulate (Fig. 2D and data not shown), have normally formed rib cages. In the BMP signaling pathway. By analogy with the role of contrast, all animals with combined deficiency for Gpc3 and another heparan sulfate proteoglycan, betaglycan, which haploinsufficiency for Bmp4 display markedly abnormal chest directly potentiates TGF-␤ binding to its signaling receptor, walls with multiple rib fusions (C and D) as well as dual ossifica- it has been suggested that Dally could function as a core- tion centers of the sternum (F). ceptor for Dpp (Jackson et al., 1997) and GPC3 could have a similar function with respect to BMP4 in the vertebrate limb. Consistent with this model, Drosophila Dpp (Groppe argues for a direct role for GPC3 in the control of cellular et al., 1998) and vertebrate BMP2, -3, -4, and -7 (Ruppert et responses to BMP4. al., 1996; Sampath et al., 1987, 1992; Wang et al., 1988) all Although controversy remains as to whether BMPs func- bind to heparin with high affinity in vitro and therefore tion through short-range interactions or over longer ranges likely bind to heparan sulfate in vivo. However, the conse- through the establishment of morphogenetic gradients, it is quence of this interaction to BMP function remains un- clear that the quantity of BMP protein available in the clear. For example, although heparin potentiates the activ- target tissue is critical for achieving the necessary develop- ity of BMP2 in a chick limb bud differentiation model in mental response. This is evident from several studies which vitro, as would be expected if it were serving a coreceptor have now established, in both vertebrate and invertebrate function, so does destruction of the N-terminal heparin species, patterning abnormalities associated with heterozy- binding domain of BMP2 or addition of a peptide corre- gous null mutants for BMPs, which presumably express half sponding to the N-terminal heparin binding domain of the normal levels of these gene products (Dunn et al., 1997; BMP2 in these same assays (Ruppert et al., 1996). These Green, 1968; Irish and Gelbart, 1987; Katagiri et al., 1998; latter results can be interpreted as evidence that at least McPherron et al., 1999). Therefore, understanding the mo- some heparan sulfate-containing proteins actually seques- lecular mechanisms that modify the strength of BMP sig- ter BMPs in vivo and serve to suppress rather than enhance nals or the cellular response to these signals in vivo is BMP functions. essential to deciphering the role of BMPs in the regulation Another likely mechanism by which GPC3 could directly of morphogenesis. affect BMP4 activity in the developing limb could be One strategy that has been particularly effective in this through regulation of the distribution or level of accumu-

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 186 Paine-Saunders et al. lation of a BMP antagonist, several of which are known to dylinositol-anchored membrane heparan sulfate proteoglycan bind to heparin with high affinity. In particular, the heparin from human lung fibroblasts. J. Cell Biol. 111, 3165–3176. binding BMP antagonist Gremlin has recently been shown Dudley, A. T., and Robertson, E. J. (1997). Overlapping expression to mediate the SHH/FGF4 feedback loop in vertebrate limb domains of bone morphogenetic protein family members poten- buds through direct BMP antagonism (Zuniga et al., 1999). tially account for limited tissue defects in BMP7 deficient embryos. Dev. Dyn. 208, 349–362. Loss of the heparan sulfate proteoglycan GPC3 could result Dunn, N. R., Winnier, G. E., Hargett, L. K., Schrick, J. J., Fogo, A. B., in the increased accumulation and/or a broader distribution and Hogan, B. L. M. (1997). Haploinsufficient phenotypes in of Gremlin protein in the developing limb fields, thus BMP4 heterozygous null mice and modification by mutations in resulting in an increase in signaling through this feedback Gli3 and Alx4. Dev. Biol. 188, 235–247. loop and therefore development of redundant postaxial Eggenschwiler, J., Ludwig, T., Fisher, P., Leighton, P. A., Tilghman, structures. S. M., and Efstratiadis, A. (1997). Mouse mutant embryos over- Further experimentation will be necessary to carefully expressing IGF-II exhibit phenotypic features of the Beckwith– evaluate each of these hypotheses about the role of GPC3 in Wiedemann and Simpson–Golabi–Behmel syndromes. Genes BMP signaling and vertebrate limb patterning. This analysis Dev. 11, 3128–3142. will not be trivial because heparan sulfate proteoglycans Erickson, R. P. (1996). Mouse models of human genetic disease: have been ascribed with various other functions, both in Which mouse is more like a man? BioEssays 18, 993–998. vitro and in vivo, and GPC3 has not yet been specifically Ferguson, E. L., and Anderson, K. V. (1992a). decapentaplegic acts as a morphogen to organize dorsal–ventral pattern in the Drosophila tested for these functions. The potential promiscuity with embryo. Cell 71, 451–461. which GPC3 may carry out the regulation of cellular Ferguson, E. L., and Anderson, K. V. (1992b). Localized enhance- processes introduces unique challenges to the understand- ment and repression of the activity of the TGF-␤ family member, ing of its in vivo function. decapentaplegic, is necessary for dorsal–ventral pattern formation in the Drosophila embryo. Development 114, 583–597. Garganta, C. L., and Bodurtha, J. N. (1992). Report of another family ACKNOWLEDGMENTS with Simpson–Golabi–Behmel syndrome and a review of the literature. Am. J. Med. Genet. 44, 129–135. We thank Jonathan Gitlin, Dave Wilson, and Ralph Sanderson George, E. L., and Hynes, R. O. (1994). Gene targeting and genera- for critical review of the manuscript. These studies were supported tion of mutant mice for studies of cell–extracellular matrix by National Institutes of Health Grant DK56063 (S.S.) and March of interactions. Methods Enzymol. 245, 386–420. Dimes Birth Defects Foundation Grant 6-FY99-441 (S.S.). S.S. was Green, M. C. (1968). Mechanism of the pleiotropic effects of the also supported as both a Spoehrer Scholar in Pediatrics and a short ear mutant gene in the mouse. J. Exp. Zool. 167, 129–150. Scholar of the Child Health Research Center of Excellence in Groppe, J., Rumpel, K., Economides, A. N., Stahl, N., Sebald, W., Developmental Biology at Washington University School of Medi- and Affolter, M. (1998). Biochemical and biophysical character- cine (HD33688). W.C.S. is a 1998 Searle Scholar. ization of refolded Drosophila DPP, a homolog of bone morpho- geneic proteins 2 and 4. J. Biol. Chem. 273, 29052–29065. Hacker, U., Lin, X., and Perrimon, N. (1997). The Drosophila REFERENCES sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development Behmel, A., Plochl, E., and Rosenkranz, W. (1984). A new X-linked 124, 3565–3573. dysplasia gigantism syndrome: Identical with the Simpson dys- Haerry, T. E., Heslip, T. R., Marsh, J. L., and O’Connor, M. B. plasia syndrome? Hum. Genet. 67, 409–413. (1997). Defects in glucuronate biosynthesis disrupt wingless Bellaiche, Y., The, I., and Perrimon, N. (1998). Tout-velu is a signaling in Drosophila. Development 124, 3055–3064. Drosophila homologue of the putative tumor suppressor EXT-1 Herndon, M. E., and Lander, A. D. (1990). A diverse set of develop- and is needed for Hh diffusion. Nature 394, 85–88. mentally regulated proteoglycans is expressed in the rat central Binari, R. C., Staveley, B. E., Johnson, W. A., Godavarti, R., nervous system. Neuron 4, 949–961. Sasisekharan, R., and Manoukian, A. S. (1997). Genetic evidence Hogan, B., Beddington, R., Costantini, F., and Lacey, E. (1994). that heparin-like glycosaminoglycans are involved in wingless “Manipulating the Mouse Embryo: A Laboratory Manual,” 2nd signaling. Development 124, 2623–2632. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Bullock, S. L., Fletcher, J. M., Beddington, R. S., and Wilson, V. A. NY. (1998). Renal agenesis in mice homozygous for a gene trap Hughes-Benzie, R. M., Hunter, A. G., Allanson, J. E., and MacKen- mutation in the gene encoding heparan sulfate zie, A. E. (1992). Simpson–Golabi–Behmel syndrome associated 2-sulfotransferase. Genes Dev. 12, 1894–1906. with renal dysplasia and embryonal tumors: Localization of the Cano-Gauci, D. F., Song, H. H., Yang, H., McKerlie, C., Choo, B., gene to Xqcen–q21. Am. J. Med. Genet. 43, 428–435. Shi, W., Pullano, R., Piscione, T. D., Grisaru, S., Soon, S., Irish, V. F., and Gelbart, W. M. (1987). The decapentaplegic gene is Sedlackova, L., Tanswell, A. K., Mak, T. W., Yeger, H., Lock- required for dorsal–ventral patterning of the Drosophila embryo. wood, G. A., Rosenblum, N. D., and Filmus, J. (1999). Glypican- Genes Dev. 1, 868–879. 3-deficient mice exhibit developmental overgrowth and some of Jackson, S. M., Nakato, H., Sugiura, M., Jannuzi, A., Oakes, R., the abnormalities typical of Simpson–Golabi–Behmel syndrome. Kaluza, V., Golden, C., and Selleck, S. B. (1997). Division J. Cell Biol. 146, 255–264. abnormally delayed, a Drosophila glypican, controls cellular David, G., Lories, V., Decock, B., Marynen, P., Cassiman, J.-J., and responses to the TGF-␤ related morphogen, Dpp. Development Berghe, H. V. d. (1990). Molecular cloning of a phosphati- 124, 4113–4120.

Katagiri, T., Boorla, S., Frendo, J. L., Hogan, B. L., and Karsenty, G. and Rueger, D. C. (1992). Recombinant human osteogenic (1998). Skeletal abnormalities in doubly heterozygous Bmp4 and protein-1 (hOP-1) induces new bone formation in vivo with a Bmp7 mice. Dev. Genet. 22, 340–348. specific activity comparable with natural bovine osteogenic Lin, X., and Perrimon, N. (1999). Dally cooperates with Drosophila protein and stimulates osteoblast proliferation and differentia- Frizzled 2 to transduce Wingless signalling. Nature 400, 281–284. tion in vitro. J. Biol. Chem. 267, 20352–20362. Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lidholt, K. Sampath, T. K., Muthukumaran, N., and Reddi, A. H. (1987). (1998). The putative tumor suppressors EXT1 and EXT2 are Isolation of osteogenin, an extracellular matrix-associated, bone- glycosyltransferases required for the biosynthesis of heparan inductive protein, by heparin affinity chromatography. Proc. sulfate. J. Biol. Chem. 273, 26265–26268. Natl. Acad. Sci. USA 84, 7109–7113. Luo, G., Hofmann, C., Bronckers, A. L. J. J., Sohocki, M., Bradley, Saunders, S., Jalkanen, M., O’Farrell, S., and Bernfield, M. (1989). A., and Karsenty, G. (1995). BMP-7 is an inducer of nephrogen- Molecular cloning of syndecan, an integral membrane proteogly- esis, and is also required for eye development and skeletal can. J. Cell Biol. 108, 1547–1556. patterning. Genes Dev. 9, 2808–2820. Skarnes, W. C., Moss, J. E., Hurtley, S. M., and Beddington, R. S. McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, (1995). Capturing genes encoding membrane and secreted pro- L. E., Dyer, A. P., and Tufaro, F. (1998). The putative tumor teins important for mouse development. Proc. Natl. Acad. Sci. suppressor EXT1 alters the expression of cell-surface heparan USA 92, 6592–6596. sulfate. Nat. Genet. 19, 158–161. Song, H. H., Shi, W., and Filmus, J. (1997). OCI-5/rat glypican-3 McLeod, M. J. (1980). Differential staining of cartilage and bone in binds to fibroblast growth factor-2 but not to insulin-like growth whole mouse fetuses by alcian blue and alizarin red S. Teratology factor-2. J. Biol. Chem. 272, 7574–7577. 22, 299–301. The, I., Bellaiche, Y., and Perrimon, N. (1999). Hedgehog move- McPherron, A. C., Lawler, A. M., and Lee, S. J. (1999). Regulation of ment is regulated through tout velu-dependent synthesis of a anterior/posterior patterning of the axial skeleton by growth/ heparan sulfate proteoglycan. Mol. Cell 4, 633–639. differentiation factor 11. Nat. Genet. 22, 260–264. Townley, D. J., Avery, B. J., Rosen, B., and Skarnes, W. C. (1997). Paine-Saunders, S., Viviano, B. L., and Saunders, S. (1999). GPC6, a Rapid sequence analysis of gene trap integrations to generate a novel member of the glypican gene family, encodes a product resource of insertional mutations in mice. Genome Res. 7, structurally related to GPC4 and is colocalized with GPC5 on 293–298. human chromosome 13. Genomics 57, 455–458. Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox, Pellegrini, M., Pilia, G., Pantano, S., Lucchini, F., Uda, M., Fumi, B., Humphrey, M., Olson, S., Futch, T., Kaluza, V., Siegfried, E., M., Cao, A., Schessinger, D., and Forabosco, A. (1998). Gpc3 Stam, L., and Selleck, S. B. (1999). The cell-surface proteoglycan expression correlates with the phenotype of the Simpson– Dally regulates Wingless signalling in Drosophila. Nature 400, Golabi–Behmel syndrome. Dev. Dyn. 213, 431–439. 276–280. Pilia, G., Hughes-Benzie, R. M., MacKenzie, A., Baybayan, P., Wang, E. A., Rosen, V., Cordes, P., Hewick, R. M., Kriz, M. J., Chen, E. Y., Huber, R., Neri, G., Cao, A., Foarabosco, A., and Luxenberg, D. P., Sibley, B. S., and Wozney, J. M. (1988). Schlessinger, D. (1996). Mutations in GPC3, a glypican gene, Purification and characterization of other distinct bone-inducing cause the Simpson–Golabi–Behmel overgrowth syndrome. Na- factors. Proc. Natl. Acad. Sci. USA 85, 9484–9488. ture Genet. 12, 241–247. Winnier, G., Blessing, M., Labosky, P. A., and Hogan, B. L. M. Raftery, L. A., Twombly, V., Wharton, K., and Gelbart, W. M. (1995). Bone morphogenetic protein-4 is required for mesoderm (1995). Genetic screens to identify elements of the decapentaple- formation and patterning in the mouse. Genes Dev. 9, 2105– gic signaling pathway in Drosophila. Genetics 139, 214–254. 2116. Rapraeger, A., Krufka, A., and Olwin, B. B. (1991). Requirement of Winter, R. M. (1996). Analysing human developmental abnormali- heparan sulfate for bFGF-mediated fibroblast growth and myo- ties. BioEssays 18, 965–971. blast differentiation. Science 252, 1705–1708. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. Reichsman, F., Smith, L., and Cumberledge, S. (1996). Glycosami- (1991). Cell surface, heparin-like molecules are required for noglycans can modulate extracellular localization of the wing- binding of basic fibroblast growth factor to its high affinity less protein and promote signal transduction. J. Cell Biol. 135, receptor. Cell 64, 841–848. 819–827. Zuniga, A., Haramis, A. G., McMahon, A. P., and Zeller, R. (1999). Ruppert, R., Hoffmann, E., and Sebald, W. (1996). Human bone Signal relay by BMP antagonism controls the SHH/FGF4 feed- morphogenetic protein 2 contains a heparin-binding site which back loop in vertebrate limb buds. Nature 401, 598–602. modifies its biological activity. Eur. J. Biochem. 237, 295–302. Sampath, T. K., Maliakal, J. C., Hauschka, P. V., Jones, W. K., Received for publication April 12, 2000 Sasak, H., Tucker, R. F., White, K. H., Coughlin, J. E., Tucker, Revised May 26, 2000 M. M., Pang, R. H. L., Corbett, C., Ozkaynak, E., Oppermann, H., Accepted June 16, 2000