Histol Histopathol (2009) 24: 859-868 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology

Tissue distribution of domains III and V during embryonic and fetal human development

Matthias Roediger, Jenny Kruegel, Nicolai Miosge and Nikolaus Gersdorff Department of Prosthodontics, Tissue Regeneration Work Group, Georg-August-University Goettingen, Germany

Summary. A major component of basement membranes functions during development and in adulthood. BMs (BMs) is perlecan, a five-domain heparan sulphate divide tissues into compartments, separating the . During murine embryogenesis, nearly all epithelium and stroma, and regulate many cellular BMs of mesenchymal origin express perlecan, and it is activities, including growth, differentiation and cell believed to participate in the supramolecular assembly of migration (Timpl and Aumailley, 1989). BMs. However, the distribution of perlecan in human Laminin-111 (Timpl et al., 1979), nidogen-1 (Carlin embryonic and fetal tissues is widely unknown, except et al., 1981), nidogen-2 (Kohfeld et al., 1998), collagen for cartilage anlagen of developing extremities and the type IV (Kühn, 1995) and perlecan (Noonan and Hassel, fetal spine. Clinical syndromes, caused by perlecan- 1993) have all been identified as major components of associated mutations or -defects, suggest its BMs. multifunctional involvement during human Perlecan is a heparan sulphate proteoglycan found in development. Here we reveal the immunohistochemistry many adult connective tissues, and was first isolated of perlecan domains III and V during human from a mouse tumor (Engelbreth-Holm-Swarm, EHS) development from gestational weeks (gw) 6 to 12 in (Hassel et al., 1980). Perlecan is expressed in cartilage zones (BMZs) of the developing and several other mesenchymal tissues (French et al., brain, nervous system, blood vessels, skin, lung, heart, 1999; Hassell et al., 2002; Melrose et al., 2006), and is kidney, liver, intestine and skeletal system. Interestingly, present in nearly all BMs, as well as in all mesenchymal a difference in the distribution of the two perlecan organs, such as kidney, lung, liver and the domains was found in the endoneurium of ganglia. gastrointestinal tract. Furthermore, it is found in Domain III is strongly present from gw 6 onwards, while numerous connective tissues during vasculogenesis, as domain V shows attenuated expression at this stage and well as in heart, pericardium, blood vessels and cartilage has been detected abundantly only from gw 8 onwards, during murine embryogenesis (E11-17.5), suggesting a possibly indicating vascularization of the endoneurium crucial role in maturation and maintenance of during this early stage. We found perlecan to be present differentiated tissues (Handler et al., 1997). In addition, particularly at those stages of human development where it has been demonstrated for early-stage murine epithelial-mesenchymal interactions occur. embryogenesis (E12-17.5) that perlecan controls the response of the neuroepithelium to growth factors, and Key words: Perlecan, Basement membrane, Human that perlecan-null brains show atrophy and decreased embryogenesis, Immunohistochemistry numbers of cells in mitosis (Girós et al., 2007). In chick embryos perlecan was detected very early from stage X (morula) to Hamburger-Hamilton stage (HH) 17 (29 Introduction somites) with varying intensity in neuroepithelium, lung rudiments, aortic arches, myocardium, tubule rudiments Basement membranes (BMs) are specialized and gut. The authors suggest a crucial role for perlecan structures of the closely apposed to in maintaining BM integrity, mediation of epithelization, epithelia, endothelia, peripheral nerve axons, fat cells adhesive separation and maintenance of neuroepithelium and muscle cells. They are known to fulfil various in brain and tissue architecture in heart, aorta and gut (Soulintzi and Zagris, 2007). Offprint requests to: Nikolaus Gersdorff, Department of Prosthodontics, Many studies suggest functions for perlecan during Tissue Regeneration Work Group, Georg-August-University Goettingen, cell growth, differentiation and tissue organization, as it Robert-Koch-Str. 40, 37075 Goettingen, Germany. e-mail: has been shown to exhibit binding sites for various BM [email protected] components. Perlecan may also play a structural role in 860 Perlecan in human organogenesis

addition to its involvement in cell-matrix interactions development. (Hopf et al., 1999; Miosge et al., 2003). In this study, we systematically examined the Perlecan is involved in angiogenesis, regulation of localization of perlecan in human embryonic and fetal wound healing and bone formation (Knox and tissues with the aid of immunohistochemistry. We found Whitelock, 2006). Additionally, perlecan is postulated to perlecan to be present particularly at those mediate the binding of to the core and/or developmental stages where epithelial-mesenchymal its glycosaminoglycan (GAG) chains, and may play a interactions occur. role in the pathogenesis of human late-stage osteoarthritis (Tesche and Miosge, 2004). Materials and methods Mutations and gene defects, associated with perlecan, cause various syndromes, suggesting Tissue sources perlecan’s functional involvement in human development, for example, Schwartz-Jampel Syndrome, Aborted human embryos and fetuses were obtained a myotonic myopathy associated with chondrodysplasia according to the regulations of the Ethics Committee of (Nicole et al., 2000; Arikawa-Hirasawa et al., 2002), as the Medical Faculty of the University of Goettingen. The well as dyssegmental dysplasia of Silverman- cut-off point for the differentiation between embryos and Handmaker type, a human disorder identified to be a fetuses has been defined to range from gestational week functional null mutation of perlecan (Arikawa-Hirasawa (gw) 8 to 9 (Moore, 1982). We investigated three et al., 2001). embryos of gw 6 to 7, two of gw 8, two fetuses of gw 9, The human perlecan gene (HSPG2) consists of 97 two of gw 10 and two fetuses of gw 12. The stages were exons and is located in the telomeric region of determined according to O’Rahilly and Mueller 1. HSPG2 encodes a large 467 kDa protein (O’Rahilly and Mueller, 1987). No malformations or consisting of five distinct domains, I-V (Noonan et al., abnormalities were observed in these specimens. The 1991), with homologies to laminin, low-density abortion procedure leads to destruction of much of the lipoprotein (LDL) receptor, epithelial growth factor embryonic tissues, limiting the tissues we could (EGF) and neural cell adhesion molecule (N-CAM). investigate. The main region of GAG substitution in human perlecan is domain I, which contains a cluster of three Tissue processing potential attachment sites (Konyesi and Silbert, 1995) and promotes binding to laminin-111 and collagen type The specimens were transported in histidine- IV when substituted with (HS). There tryptophan-ketoglutarate solution at 4°C to ensure tissue are a variety of additional interactions, including preservation (Bretschneider, 1980) and were fixed with nidogen-2, fibulin-2, fibronectin, PRELP (proline/ 3.7% paraformaldehyde in phosphate buffer, pH 7.2, at arginine-rich and leucine-rich repeat protein)/prolargin, 4°C for 24 h, and then serially dehydrated in ethanol and types XIII and XVIII collagen (Hopf et al., 2001; from 30% to 100% and embedded in paraffin according Bengtsson et al., 2002; Miosge et al., 2003). to standard protocols (Gersdorff et al., 2005). Serial The N-terminal domain III of perlecan has been sections of 5 µm thickness were cut with a Reichert’s detected in BMs of several mouse organs by microtome. Every fifth section was stained with immunohistochemistry (Couchman et al., 1995) and in hematoxylin and eosin (HE) for topological orientation human adult tissues, including the pituitary gland, skin, within the anatomical regions examined. breast, thymus, prostate, colon, liver, pancreas, spleen, heart, lung, vascular BMs and in the subendothelial Antibodies regions of sinusoidal vessels of liver, spleen, lymph node and pituitary gland (Murdoch et al., 1994). Rabbit polyclonal antibodies against perlecan The C-terminal domain V of perlecan promotes beta domain III-3 (1030, Schulze et al., 1996) and perlecan 1 integrin-mediated cell adhesion and binds heparin, domain V (1056, Brown et al., 1997) were kindly nidogens and fibulin-2 (Brown et al., 1997) and is also provided by Dr. Takako Sasaki (Shriners Hospital, involved in GAG attachment, substitution and the Portland, Oregon). binding of several carbohydrate and protein ligands Secondary antibodies used for immunostaining were (Friedrich et al., 1999). The localization of perlecan purchased from Dako (Hamburg, Germany). domain V is not as well documented as that of domain III. Immunohistochemistry BM components like perlecan are well known to participate in numerous cell biological processes during Immunoperoxidase staining was performed on embryogenesis. Other than its localization in hyaline paraffin-embedded tissue sections as follows: Tissues cartilage anlagen in extremities (Melrose et al., 2006) were deparaffinised, rehydrated and rinsed for 10 min in and spine of the fetal human (Melrose et al., 2003), little phosphate buffered saline (PBS), pH 7.2. Endogenous is known about perlecan localization during early human peroxidase was inhibited with a solution of 3% H2O2 in

861 Perlecan in human organogenesis

methanol for 45 min in the dark. The sections were which are described in the text below. pretreated for 5 min with 10 µg/mL protease XXIV (Sigma Deisenhofen, Germany). The antibodies 1030 Neuronal system (perlecan domain III) and 1056 (perlecan domain V) were used at a 1:100 dilution in PBS/BSA and incubated The chorda dorsalis induces the morphogenesis of for 1 h at room temperature. Each step was followed by the neuroectoderm to form the neural tube at the a 10-min rinse in PBS. The secondary antibody was beginning of gw 4 (Sadler, 2005). Staining for perlecan applied at a 1:50 dilution in PBS/BSA, followed by was found in the endoneurium of peripheral ganglia and incubation with the PAP complex (diluted in 1:150 in the basement membrane zones (BMZs) of capillaries. PBS/BSA) for 30 min at room temperature. The sections Perlecan domain III was localized in all embryonic and were counterstained with hematoxylin. Negative controls fetal specimens from gw 6 to 12 (Fig. 1A), whereas omitted the primary antibody. perlecan domain V showed only weak staining at gw 6 and was abundantly found from gw 8 onwards (Fig. Results 1B,E). Neuronal cells were negative for staining of perlecan domain III at all stages examined and showed A complete list of our results is given in Table 1 only attenuated distribution of domain V (Fig. with the exception of those from the skeletal system, 1A,B).

Fig. 1. Localization of perlecan in the neuronal system during human embryogenesis. A. Immunostaining for perlecan domain III was positive in the endoneurium of a nerve (N) and ganglion (G) at gw 6 (arrows). B. Endoneurium (arrows) of a nerve (N) and neuronal cells (asterisk) at gw 6 showed weak staining for perlecan domain V. C. Localization of perlecan domain V in BMZs of neuroectoderm (open arrows) at gw 6. D. Immunostaining for perlecan domain V in BMZs of neuroectoderm (open arrows) and capillaries (arrowheads) at gw 6. E. Localization of perlecan domain V in the endoneurium of spinal ganglia (arrows), condensed mesenchyme (triangle) and cartilage anlagen (asterisk) at gw 9. Bars: A-D, 15 µm; E, 60 µm. 862 Perlecan in human organogenesis

BMZs of the neuroectoderm and of capillaries stained Blood vessels positive for both perlecan domains III and V between gw 6 to 12 (Fig. 1C,D), while no staining was detected for Already at gw 3, in the visceral mesoderm, either perlecan domain in neuroectodermal cells. angioblasts differentiate into endothelial cells (O’Rahilly

Table 1. Light microscopic immunostaining for perlecan domain III and V in human specimens of gw 6 to 12.

Developing organ gestational week 6 / 7 8 / 9 10 12 perlecan domain perlecan domain perlecan domain perlecan domain III V III V III V III V

Brain BMZ of neuroectoderm ++ ++ ++ ++ Ø Ø Ø Ø Neuroectodermal cells - - - - Ø Ø Ø Ø BMZ of capillaries ++ ++ ++ ++ Ø Ø Ø Ø Ganglia Neuronal cells - + - + Ø + - Ø Endoneurium ++ + ++ ++ Ø ++ ++ Ø BMZ of capillaries ++ - ++ ++ Ø ++ ++ Ø Blood vessels BMZ of endothelium ++ ++ ++ ++ ++ ++ ++ ++ BMZ of smooth muscle cells ++ ++ ++ ++ ++ ++ ++ ++ Endothelial cells ------Liver Hepatocytes ------BMZ of sinusoids ++ ++ ++ ++ ++ ++ ++ ++ BMZ of mesothelium ++ ++ ++ ++ ++ ++ ++ ++ Early Mesenchyme ++ ++ Ø Ø Ø Ø Ø Ø Lung Epithelium of bronchioli - - - - Ø --Ø BMZ of bronchioli ++ ++ ++ ++ Ø ++ ++ Ø Mesenchyme + + + + Ø ++Ø BMZ of capillaries ++ ++ ++ ++ Ø ++ Ø Ø Kidney BMZ of glomeruli ++ ++ ++ ++ ++ ++ ++ Ø BMZ of tubules ++ ++ ++ ++ ++ ++ ++ Ø Mesenchyme ------Ø Ø Skin Epidermis ------Dermal-epidermal BMZ ++ ++ ++ ++ ++ ++ ++ ++ Dermis ------BMZ of capillaries ++ ++ ++ ++ ++ ++ ++ ++ Mesenchyme ------Pancreas Glands - ----ØØØ BMZ of glands ++ ++ ++ ++ ++ Ø Ø Ø BMZ of capillaries ++ ++ ++ ++ ++ Ø Ø Ø Intestine Epithelium ------BMZ of epithelium ++ ++ ++ ++ ++ ++ ++ ++ Mesenchyme - - ++ ++ ++ ++ ++ ++ BMZ of capillaries ++ ++ ++ ++ ++ ++ ++ ++ Heart Cardiomyocytes - - ++ ++ ++ ++ ++ ++ BMZ of cardiomyocytes - - ++ ++ ++ ++ ++ ++ BMZ of endo-/pericardium ++ ++ ++ ++ ++ ++ ++ ++ BMZ of capillaries ++ ++ ++ ++ ++ ++ ++ ++

BMZ: basement membrane zone; ++: positive reaction; +: intermediate reaction; -: negative reaction; Ø: tissues not available. 863 Perlecan in human organogenesis

and Müller, 1996). Perlecan was found in endothelial Intestine BMZs of arteries and veins as well as in BMZs of capillaries between gw 6 to 12. BMZs of smooth muscle Already at gw 4, the early foregut forms. Connective cells of the developing tunica media revealed perlecan tissues and the musculature of the gastro-intestinal tract staining, while endothelial cells remained negative. derive from the surrounding mesenchyme (O’Rahilly and Müller, 1996). The developing intestine stained Liver positive for perlecan in the epithelial BMZ, as did the BMZs of the capillaries in the tunica submucosa and Development starts with the budding of entodermal adventitia. Perlecan was absent in epithelial cells, cells from the distal end of the foregut (O’Rahilly and mesenchymal cells and the developing lamina Müller, 1996). From gw 6 onwards to week 12, in the muscularis prior to gw 8. From gw 8 onward, perlecan developing liver, both perlecan domains were present in domains III and V were expressed in the lamina BMZs of sinusoids and capillaries (Fig. 2F). Perlecan muscularis and submucosa (Fig. 2K,L). was also detected in the early mesenchyme at gw 6 and 7 (Fig. 2F). However, hepatocytes and hematopoetic cells Heart were negative at all stages examined. In embryonic week 4, the heart anlage consists of the Lung endocardial tube and the developing myocardial layer separated from each other by the cell-free, homogeneous Lung buds emerge between gw 4 and 5 also as a cardiac jelly (CJ), which also surrounds individual diverticulum of the entoderm. Branching morphogenesis myocytes (Wenink, 1976). Perlecan was detected at gw 6 results in development of bronchioli and alveoli at gw 6 in the BMZs of the endo- and pericardium, as well as of to 8, while cartilage and smooth muscles originate from capillaries, and was present until gw 12. visceral mesoderm (Burri, 1984; O’Rahilly and Müller, Perlecan was not detected between gw 6 and 7 in 1996). In the embryonic lung, perlecan domains III and developing cardiomyocytes. At gw 8, the mesenchyme V were specifically detected in BMZs of the bronchioli of the endocardial cushion tissues stained positive for and of the bronchial capillaries from gw 6 to 12 (Fig. perlecan domain V (Fig. 2H). Perlecan was also detected 2A,B). Low levels of perlecan were shown for intracellularly and in BMZs of cardiomyocytes from gw mesenchymal cells (Fig. 2B). 8 to 12 (Fig. 2I,J).

Kidney Skeletal system

From cranial to caudal, three renal systems evolve The condensed mesenchyme at gw 6 and 7 (pronephros, mesonephros and metanephros). At gw 6, represents the first developmental step towards cartilage urinary secretion starts in the mesonephros lasting up to anlagen, and exhibits weak staining for perlecan gw 12 (Saxen and Sariola, 1987). At all stages of renal domains III and V. However, perlecan was localized to development examined, perlecan domains III and V the BMZs of capillaries (Fig. 3A). At gw 8, late were expressed in the BMZs of glomeruli, tubules and condensed mesenchyme and early vertebral cartilage capillaries. The surrounding mesenchymal cells, anlagen were positive for perlecan domain III (Fig. 3B), however, were negative for perlecan (Fig. 2C-E). and perlecan domain V (Fig. 3C). Both perlecan domains were expressed in capillaries. At gw 9, Skin chondrocytes and the pericellular matrix of rib cartilage anlagen were perlecan-negative, whereas the Skin development starts from two compartments. interterritorial matrix and BMZs of capillaries stained The epidermis derives from the ectoderm and the dermis positive (Fig. 3D,E). Ossification of the long bones starts from the mesoderm (Smith and Holbrook, 1986). The around gw 8 and localization of perlecan domains III BMZs of dermal-epidermal junctions and of capillaries and V was also observed in zones of chondral and stained positive for perlecan domains III and V, while desmal ossification. Hypertrophic chondrocyte staining keratinocytes of the epidermis, dermis and the loose decreased with increasing levels of perlecan domain III mesenchyme were negative (Fig. 2G). toward the metaphysis (Fig. 3F,G). Proliferating chondrocytes of the epiphyses of long bones at gw 12 Pancreas exhibited staining for perlecan domain III in the interterritorial matrix, and in contrast to cartilage BMZs of the developing exocrine gland were anlagen at gw 9, perlecan was additionally expressed in positive for perlecan at all developmental stages the pericellular matrix (Fig 3H,I). Furthermore, BMZs of examined. Furthermore, perlecan was localized to the capillaries exhibited staining for perlecan domain III, BMZs of glandular capillaries, while epithelial cells while the perichondrium was negative at all stages revealed no staining for perlecan (Table 1). examined.

864

Fig. 2. Localization of perlecan during human embryogenesis. A. Perlecan domain V in the BMZs of bronchioli (open arrows) and of blood vessels (arrow head) at gw 8. B. Perlecan domain III in the BMZs of bronchioli (open arrows), of blood vessels (arrow head) and in the mesenchyme (M) at gw 12. C, D. Perlecan domain III localization in the BMZs of tubuli (open arrow), glomeruli (arrows) and capillaries (arrowhead) in human embryonic kidney at gw 7. E. Perlecan domain III in the BMZs of kidney tubules at gw 9 (open arrows). F. Sinusoid (S) of developing liver at gw 7. Perlecan domain III was detected in BMZs of sinusoids (open arrows) and in early mesenchyme (M). G. Perlecan domain III in dermal-epidermal BMZ (open arrows) of the skin at gw 12. Epidermis (arrows) and loose mesenchyme (LM) were negative. H. Perlecan domain V (open arrow) in endocardial cushion tissue (ECT) at gw 8. I, J. Perlecan domain III in cardiomyocytes (CM) and in BMZs of cardiomyocytes (open arrow) at gw 9. K, L. Perlecan domain III in intestinal mesenchyme (M) and in BMZs of capillaries (arrowhead) and epithelium (open arrows) at gw 10. Bars: A, D, F-H, J, L, 15 µm; B, C, E, I, K, 60 µm. 865

Fig. 3. Localization of perlecan in the skeletal system during human embryogenesis. A. At gw 6, condensed mesenchyme (triangle), the first developmental step toward cartilage anlagen revealed weak staining for perlecan domain III (asterisk), while BMZs of blood vessels (arrowhead) exhibited distinct staining. B. At gw 8, perlecan domain III was detected in BMZs of capillaries (arrowhead), in late condensed mesenchyme (triangle), as well as in vertebral cartilage anlagen (asterisk). C. At gw 8, positive staining for perlecan domain V was seen in vertebral cartilage anlagen (asterisk) and in BMZs of capillaries (arrowhead). D, E. Staining for perlecan domain III in the interterritorial matrix (open arrows) of rib cartilage anlagen (asterisk) and BMZs of blood vessels (arrowheads) at gw 9. Zones of the pericellular matrix were negative. F, G. Zones of chondral ossification (CO), desmal ossification (DO) and BMZs of capillaries (arrowhead) in developing long bones displayed positive staining for perlecan domain III at gw 9. Hypertrophic chondrocyte (asterisk) staining is reduced with increasing levels of perlecan domain III toward the metaphysis. H, I. Perlecan domain III in BMZs of capillaries (arrowhead) and in cartilage anlagen of the epiphysis of long bones (asterisk), where proliferating chondrocytes (open arrows) and pericellular matrix (arrows) displayed positive staining (gw 12). Bars: A-D, F, H, 60 µm; E, G, I, 15 µm. 866 Perlecan in human organogenesis

Discussion III was localized to BMZs of the endocardium, epicard and subendothelial BMZs of capillaries, but was absent Epithelial-mesenchymal interactions are necessary in cardial myocytes (Murdoch et al., 1994). We found for the development of several organs (Grobstein, 1956). perlecan domains III and V in the developing human The early presence of perlecan in human embryonic heart in BMZs of endo- and pericardium at gw 6. kidney in BMZs of glomeruli and tubules may indicate Perlecan was not detected in the cytoplasm and BMZs of its involvement in tubulo- and glomerulogenesis. Also, cardiomyocytes prior to gw 8. A lack of perlecan during human gut development, beginning at gw 8, expression leads to the absence of normal BM structures intestinal connective tissues showed staining for around myocardial cells, indicating the importance of perlecan, which is consistent with results for murine perlecan in cardiac development (Olsen, 1999). embryonic development (Handler et al., 1997). Perlecan Knockdown of perlecan in zebrafish resulted in was also detected in murine lung development (Handler abnormal actin filament orientation and disorganized et al., 1997). We found only low levels of perlecan sarcomeres, suggesting the involvement of perlecan in between gw 6 to 12 in mesenchymal cells of the lung of myopathies (Zoeller et al., 2008). This phenotype was human embryos. Both domains were detected partially rescued by injection of human perlecan and distinctively in the BMZs of bronchioli of the endorepellin, extending the survival time up to the larval developing lung from gw 6 onwards. Taken together, stage. BM structures may need additional support these results indicate the involvement of perlecan in starting from gw 8, while other heparansulfate epithelial-mesenchymal interactions during early human , such as agrin, may fulfill this function in embryogenesis. earlier stages. Perlecan-null mouse embryos displayed Another aspect to substantiate perlecan’s many abnormalities, including discontinuities in the significance for development, inflammation and wound myocardial wall, which result in hemorrhage or repair might be the high affinity to growth factors. For malpositioning of the larger arteries, associated with example, the interaction between fibroblast growth hyperplasia of mesenchymal cells in endocardial factor 2 (FGF-2) and perlecan enhances affinity to cushions (Costell et al., 1999, 2002). We also found the tyrosine kinase receptors and is essential for its endocardial cushion tissues positive for perlecan domain phosphorylation. Furthermore, for FGF binding and V at gw 8. Perlecan domain V may have different activation of the tyrosine kinase receptors the formation functions in mice than in humans, for example, there are of FGF-2-heparin complexes are essential (Segev et al., structural distinctions such as alternative GAG 2004). attachment sites in murine domain V compared to the During human neuronal development, perlecan human (Tapanadechopone et al., 1999). domain III is expressed in the endoneurium of ganglia Perlecan has been localized in cartilaginous fetal and in BMZs of capillaries in human embryos from gw human tissues after gw 12 and strongly associated with 6. However, only weak staining for perlecan domain V venules and arterioles in the synovial lining (Melrose et was detected in the endoneurium prior to gw 9. The low al., 2006). We found perlecan domains III and V level of domain V may indicate vascularization of the strongly expressed in cartilage anlagen beginning at gw endoneurium during this early stage. Perlecan domain V 8. Condensed mesenchyme showed low levels of (endorepellin) inhibits angiogenesis (Gonzalez et al., perlecan at earlier stages of development. Developing 2005). Processed perlecan lacking domain V might have bones stained positive for perlecan domains III and V, been detected by the anti-domain III antibody. Starting at which appeared in zones of chondral and desmal gw 9, the endoneurium and BMZs of capillaries revealed ossification at gw 8 and 9. Perlecan is involved in the distinct staining for perlecan domain V, possibly pathogenesis in late stages of osteoarthritis, as protein indicating that vascularization has been completed and mRNA levels are upregulated adjacent to the main already at this developmental stage. cartilage defect as an attempt to stabilize the Furthermore, our results lead us to hypothesize that extracellular matrix (Tesche and Miosge, 2004). perlecan might also be involved in the regulation of Perlecan-deficient mouse embryos develop severe angiogenesis in embryonic, fetal and adult human skeletal dysplasias (Arikawa-Hirasawa et al. 1999; tissues. Perlecan domains III and V were detected in the Costell et al., 1999). Growth plates of long bones BMZs of capillaries and blood vessels in all organs and deficient in perlecan are severely disrupted in the at all stages examined. Also, high levels of perlecan proliferative and hypertrophic zones (Knox and were detected in human primary liver tumors around Whitelock, 2006), consistent with our results of perlecan newly formed blood vessels (Roskams et al., 1998). localization during chondral ossification in long bones. Perlecan is postulated to play a prominent role in both Schwartz-Jampel-Syndrome results from missense and the activation and inhibition of angiogenesis, depending splicing mutations of the gene encoding perlecan on its cellular context, consistent with the expression of generating truncated versions of perlecan that lacked perlecan in both, vascularized and hypovascularized domain V or significantly reduced levels of wild-type tissues (Knox and Whitelock, 2006; Bix and Iozzo, perlecan (Nicole et al., 2000; Arikawa-Hirasawa et al., 2008). 2002). This could imply a strong association of perlecan In the heart, in adult human tissues, perlecan domain domains III and especially V with chondrogenesis. 867 Perlecan in human organogenesis

Perlecan knockdown in zebrafish revealed abnormal J.R. (1995). Antibody mapping and tissue localization of globular phenotypes with a pronounced curvature of the tail and and cysteine-rich regions of perlecan domain III. J. Histochem. trunk caused by severe defects in the musculoskeletal Cytochem. 43, 955-963. system (Zoeller et al., 2008). French M.M., Smith S.E., Akanbi K., Sanford T., Hecht J., Farach- In summary, our results indicate an important role of Carson M.C. and Carson D.D. (1999). Expression of the heparan perlecan during human embryogenesis. We found sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan to be present particularly at those stages of perlecan chondrogenic activity in vitro. J. Cell Biol. 145, 1103- development where epithelial-mesenchymal interactions 1115. occur. Perlecan is a component of the BMZs that is Friedrich M.V., Gohring W., Morgelin M., Brancaccio A., David G. and ubiquitously expressed between gw 6 to gw 12 in all Timpl R. (1999). Structural basis of glycosaminoglycan modification tissues investigated. and of heterotypic interactions of perlecan domain V. J. Mol. Biol. 294, 259-270. Acknowledgements. We would like to thank Dr. Takako Sasaki (Shriners Gersdorff N., Kohfeldt E., Sasaki T., Timpl R. and Miosge N. (2005). Hospital in Portland, Oregon) for the antibodies. Laminin gamma3 chain binds to nidogen and is located in murine basement membranes. J. Biol. Chem. 280, 22146-22153. Girós A., Morante J., Gil-Sanz C., Fairén A. and Costell M. (2007). References Perlecan controls neurogenesis in the developing telencephalon. BMC Dev. Biol. 7, 29. Arikawa-Hirasawa E., Watanabe H., Takami H., Hassell J.R. and Gonzalez E.M., Reed C.C., Bix G., Fu J., Zhang Y., Gopalakrishnan B., Yamada Y. (1999). Perlecan is essential for cartilage and cephalic Greenspan D.S. and Iozzo R.V. (2005). BMP-1/Tolloid-like development. Nat. Genet. 23, 354-358. metalloproteases process endorepellin, the angiostatic C-terminal Arikawa-Hirasawa E., Wilcox W.R., Le A.H., Silverman N., Govindraj P., fragment of perlecan. J. Biol. Chem. 280, 7080-7087. Hassell J.R. and Yamada Y. (2001). Dyssegmental dysplasia, Grobstein C. (1956). Inductive tissue interaction in development. Adv. Silverman-Handmaker type, is caused by functional null mutations of Cancer. Res. 4, 187-236. the perlecan gene. Nat. Genet. 27, 431-434. Handler M., Yurchenco P.D. and Iozzo R.V. (1997). Developmental Arikawa-Hirasawa E., Le A.H., Nishino I., Nonaka I., Ho N.C., expression of perlecan during murine embryogenesis. Dev. Dyn. Francomano C.A., Govindraj P., Hassell J.R., Devaney J.M., 210, 130-145. Spranger J., Stevenson R.E., Iannaccone S., Dalakas M.C. and Hassell J., Yamada Y. and Arikawa-Hirasawa E. (2002). Role of Yamada Y. (2002). Structural and functional mutations of the perlecan in skeletal development and diseases. Glycoconj. J. 19, perlecan gene cause Schwartz-Jampel syndrome, with myotonic 263-267. myopathy and chondrodysplasia. Am. J. Hum. Genet. 70, 1368- Hassell J.R., Robey P.G., Barrach H.J., Wilczek J., Rennard S.I. and 1375. Martin G.R. (1980). Isolation of a heparan sulfate-containing Bengtsson E., Morgelin M., Sasaki T., Timpl R., Heinegard D. and proteoglycan from basement membrane. Proc. Natl. Acad. Sci. USA Aspberg A. (2002). The leucine-rich repeat protein PRELP binds 77, 4494-4498. perlecan and collagens and may function as a basement membrane Hopf M., Gohring W., Kohfeldt E., Yamada Y. and Timpl R. (1999). anchor. J. Biol. Chem. 277, 15061-15068. Recombinant domain IV of perlecan binds to nidogens, laminin- Bix G. and Iozzo R.V. (2008). Novel interactions of perlecan: Unraveling nidogen complex, fibronectin, fibulin-2 and heparin. Eur. J. perlecan's role in angiogenesis. Microsc. Res. Tech. 71, 339-348. Biocem./FEBS 259, 917-925. Bretschneider H.J. (1980). Myocardial protection. Thorac. Cardiovasc. Hopf M., Gohring W., Mann K. and Timpl R. (2001). Mapping of binding Surg. 28, 295-302. sites for nidogens, fibulin-2, fibronectin and heparin to different IG Brown J.C., Sasaki T., Gohring W., Yamada Y. and Timpl R. (1997). modules of perlecan. J. Mol. Biol. 311, 529-541. The C-terminal domain V of perlecan promotes beta1 integrin- Knox S.M. and Whitelock J.M. (2006). Perlecan: how does one mediated cell adhesion, binds heparin, nidogen and fibulin-2 and molecule do so many things? Cell. Mol. Life Sci. 63, 2435-2445. can be modified by glycosaminoglycans. Eur. J. Biochem./FEBS Kohfeldt E., Sasaki T., Gohring W. and Timpl R. (1998). Nidogen-2: a 250, 39-46. new basement membrane protein with diverse binding properties. J. Burri P.H. (1984). Fetal and postnatal development of the lung. Annu. Mol. Biol. 282, 99-109. Rev. Physiol. 46, 617-628. Kokenyesi R. and Silbert J.E. (1995). Formation of heparan sulfate or Carlin B., Jaffe R., Bender B. and Chung A.E. (1981). Entactin, a novel chondroitin/dermatan sulfate on recombinant domain I of mouse basal lamina-associated sulfated glycoprotein. J. Biol. Chem. 256, perlecan expressed in Chinese hamster ovary cells. Biochem. 5209-5214. Biophys. Res. Commun. 211, 262-267. Costell M., Carmona R., Gustafsson E., Gonzalez-Iriarte M., Fassler R. Kühn K. (1995). Basement membrane (type IV) collagen. Matrix Biol. and Munoz-Chapuli R. (2002). Hyperplastic conotruncal endocardial 14, 439-445. cushions and transposition of great arteries in perlecan-null mice. Melrose J., Smith S., Ghosh P. and Whitelock J. (2003). Perlecan, the Circ. Res. 91, 158-164. multidomain heparan sulfate proteoglycan of basement membranes, Costell M., Gustafsson E., Aszodi A., Morgelin M., Bloch W., Hunziker is also a prominent component of the cartilaginous primordia in the E., Addicks K., Timpl R. and Fassler R. (1999). Perlecan maintains developing human fetal spine. J. Histochem. Cytochem. 51, 1331- the integrity of cartilage and some basement membranes. J. Cell 1341. Biol. 147, 1109-1122. Melrose J., Roughley P., Knox S., Smith S., Lord M. and Whitelock J. Couchman J.R., Ljubimov A.V., Sthanam M., Horchar T. and Hassell (2006). The structure, location, and function of perlecan, a 868 Perlecan in human organogenesis

prominent pericellular proteoglycan of fetal, postnatal, and mature (1998). Heparan sulphate proteoglycan expression in human hyaline cartilages. J. Biol. Chem. 281, 36905-36914. primary liver tumours. J. Pathol. 185, 290-297. Miosge N., Simniok T., Sprysch P. and Herken R. (2003). The collagen Sadler T.W. (2005). Embryology of neural tube development. Am. J. type XVIII endostatin domain is co-localized with perlecan in Genet. C. Semin. Med. Genet. 135C, 2-8. basement membranes in vivo. J. Histochem. Cytochem. 51, 285- Saxen L. and Sariola H. (1987). Early organogenesis of the kidney. 296. Pediatr. Nephrol. (Berlin, Germany) 1, 385-392. Moore K.L. (1982). The developing human. Saunders. Philadelphia. Schulze B., Sasaki T., Costell M., Mann K. and Timpl R. (1996). Murdoch A.D., Liu B., Schwarting R., Tuan R.S. and Iozzo R.V. (1994). Structural and cell-adhesive properties of three recombinant Widespread expression of perlecan proteoglycan in basement fragments derived from perlecan domain III. Matrix Biol. 15, 349- membranes and extracellular matrices of human tissues as detected 357. by a novel monoclonal antibody against domain III and by in situ Segev A., Nili N. and Strauss B.H. (2004). The role of perlecan in hybridization. J. Histochem. Cytochem. 42, 239-249. arterial injury and angiogenesis. Cardiovasc. Res. 4, 603-610. Nicole S., Davoine C.S., Topaloglu H., Cattolico L., Barral D., Beighton Smith L.T. and Holbrook K.A. (1986). Embryogenesis of the dermis in P., Hamida C.B., Hammouda H., Cruaud C., White P.S., Samson D., human skin. Pediatr. Dermatol. 3, 271-280. Urtizberea J.A., Lehmann-Horn F., Weissenbach J., Hentati F. and Soulintzi N. and Zagris N. (2007). Spatial and temporal expression of Fontaine B. (2000). Perlecan, the major proteoglycan of basement perlecan in the early chick embryo. Cells Tissues Organs 4, 243- membranes, is altered in patients with Schwartz-Jampel syndrome 256. (chondrodystrophic myotonia). Nat. Genet. 26, 480-483. Tapanadechopone P., Hassell J.R., Rigatti B. and Couchman J.R. Noonan D.M. and Hassell J.R. (1993). Perlecan, the large low-density (1999). Localization of glycosaminoglycan substitution sites on proteoglycan of basement membranes: structure and variant forms. domain V of mouse perlecan. Biochem. Biophys. Res. Commun. Kidney Int. 43, 53-60. 265, 680-690. Noonan D.M., Fulle A., Valente P., Cai S., Horigan E., Sasaki M., Tesche F. and Miosge N. (2004). Perlecan in late stages of Yamada Y. and Hassell J.R. (1991). The complete sequence of osteoarthritis of the human knee joint. Osteoarthritis Cartilage 12, perlecan, a basement membrane heparan sulfate proteoglycan, 852-862. reveals extensive similarity with laminin A chain, low density Timpl R. and Aumailley M. (1989). Biochemistry of basement lipoprotein-receptor, and the neural cell adhesion molecule. J. Biol. membranes. Adv. Nephrol. Necker Hosp. 18, 59-76. Chem. 266, 22939-22947. Timpl R., Rohde H., Robey P.G., Rennard S.I., Foidart J.M. and Martin Olsen B.R. (1999). Life without perlecan has its problems. J. Cell Biol. G.R. (1979). Laminin--a glycoprotein from basement membranes. J. 147, 909-912. Biol. Chem. 254, 9933-9937. O’Rahilly R. and Muller F. (1987). Developmental stages in human Wenink A.C. (1976). Development of the human cardiac conducting embryos. Including a revision of streeter’s “horizon” and a survey of system. J. Anat. 121, 617-631. the Carnegie collection. Carnegie Institution of Washington Zoeller J.J., McQuillan A., Whitelock J., Ho S.Y. and Iozzo R.V. (2008). Publication 637. Washington. A central function for perlecan in skeletal muscle and cardiovascular O’Rahilly R. and Muller F. (1996). Human embryology and teratology. development. J. Cell Biol. 181, 381-394. 2nd ed. Wiley-Liss. New York. pp 149-157. Roskams T., De Vos R., David G., Van Damme B. and Desmet V. Accepted January 30, 2009