Perlecan maintains the integrity of cartilage and some basement membranes

Abstract

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Figure 1

Targeting strategy, Southern blots, PCR, and RIA analysis of ES cells and mice lacking perlecan. (A) Structure of the wild-type perlecan allele, targeting construct, and targeted perlecan allele before and after cre-loxP–mediated deletion. The dark boxes show exons of the perlecan gene, the open box shows the neo-tk cassette, and the loxP sites are indicated as triangles. Probe 1 was used to detect homologous recombination, and probe 2 was used to identify the null allele. Restriction sites are as follows: E, EcoRI; B, BamHI; and H, HindIII. (B) Southern blot analysis of EcoRI-digested genomic DNA derived from wild-type, heterozygous, and homozygous mutant mice hybridized with probe 1. (C) Detection of mutant mRNA lacking exon 6 by RT-PCR using the total RNA prepared from wild-type, heterozygous, and homozygous ES cells. Primers from exons 5 and 8 amplified 640 bp from the wild-type and 479 bp from the mutant allele. PCR products were probed with exon 7–specific radiolabeled oligonucleotide. (D) Radioimmunoassay measurement of laminin-1, nidogen-1, and perlecan in the serum-free medium derived from wild-type and mutant ES cells. Values are means ± SD of triplicate culture dishes and are expressed as micrograms per milliliter of supernatant.

Figure 2

Development of the hemopericard in E10.5 perlecan-null embryos. (A and B) Whole mount pictures of E10.5 wild-type (A) and perlecan-deficient embryos showing blood leakage into the pericardial cavity (B, arrow). (C–H) Light microscopy of semi-thin sections of wild-type (C and E) and perlecan-null hearts (D and F–H) stained with methylene blue. (C) Myocardial wall with well developed trabeculation separated from the thin pericardium by a cell-free cavity. (E) Higher magnification shows that cardiomyocytes are covered by an endo- and epicardial cell layer. (D) Myocardial wall of homozygotes is disrupted (arrow), whereas the endo- and epicardial cell layers are intact. The pericardial cavity contains blood cells and is surrounded by a thickened pericardium. (F–H) Three different perlecan-null hearts with small ruptures in the myocardial wall (arrows). (I–K) Electron microscopy of a normal (I) cardiac muscle cell with BM composed of a lamina rara and densa (arrow). Mutant cardiac muscle cell shows a typical myofilament organization but lacks a BM (arrow in J) or with BM-like material (arrow in K) on the cell surface. (L and M) Typical BM with a lamina rara and densa (arrow) beneath the dermal epithelium of wild-type (L) and perlecan-null (M) embryos. (N-Q) Immunostaining for perlecan (N and O) and laminin-1 (P and Q) of a wild-type (N and P) and perlecan-null (O and Q) heart. Perlecan is absent but laminin-1 is expressed in homozygous hearts. Abbreviations: v, ventricle; m, myocardium; p, pericardium; mf, muscle filament; and e, epithelial cell. Bars: (C and D) 250 μm; (E–H) 100 μm; (I–M) 250 nm; and (N–Q) 250 μm.

Figure 3

Scanning electron microscopy revealed brain defects in perlecan-null embryos. (A–C) Scanning electron microscopy shows that the neural tube is closed in wild-type (A) and perlecan-deficient (B and C) E10.5 embryos. Some perlecan-null embryos show holes (C, arrows) in the fore- and midbrain and collapsed brain vesicles. (D–F) High magnification of the surface ectoderm. In wild-type embryos (D), the cephalic region is covered with an intact layer of ectodermal cells. Perlecan-null embryos (E) show small clefts that are 20–30 μm in width and contain round cells with small extensions (arrow). In other perlecan-deficient embryos (F), round cells with extensions burst through the surface ectoderm (arrow). G shows an antibody coupled to colloidal gold reacting with purified laminin. (H–K) Backscattered electron analysis shows that small defects in the ectoderm of normal mice caused during the preparation are not labeled with the gold-conjugated antibody (secondary electron image H and backscattered electron image J), while the clefts in perlecan-null embryos are labeled (secondary electron image I, backscattered electron image K). Arrow in K indicates an ectodermal defect caused during the preparation. Bars: (D–F and H–K) 20 μm; and (G) 50 nm.

Figure 4

Exencephaly and neuronal ectopias develop in the anterior region of the forebrain. (A and B) Hematoxylin/eosin staining of sagittal brain sections from wild-type (A) and perlecan-null (B) E11.5 embryos. Note the extension and the thinning of the anterior part of the forebrain in the perlecan-null embryo (upper box in B). Ectopias are visible ventral of the medial ganglionic eminence (arrow) in the homozygous embryo. (C–F) Higher magnification of the lower boxes indicated in A and B. In a normal brain, the neuroepithelium and the underlying mesenchyme are separated by a BM (C) that contains laminin-1 (E). In the perlecan-null brain, the BM is discontinuous (D, arrow) and shows interrupted laminin-1 staining (F, arrow). (G and H) Higher magnification of the upper boxes indicated in A and B. In a normal brain, the neuroepithelium and the overlying mesenchyme are separated by a BM (G). In the perlecan-null brain, neuroepithelial cells have invaded the overlying ectoderm (H, arrow). (I–L) Immunohistochemical localization of nestin and β-tubulin type III in the neocortex of normal and perlecan-null embryos. The neocortex of the wild-type embryo contains nestin-positive cells (I) and a few β-tubulin type III–positive cells in the cortical subplate (K). Ectopic cells in the perlecan-null embryo are positive for nestin (J) but negative for β-tubulin type III (L). (M) Hematoxylin/eosin staining of the forebrain region of an E11.5 homozygote showing a defect in the ectoderm and a small hole of 5–10 μm (arrow). Note that the amniotic membrane is still intact (arrowhead). (N–Q) Nissl staining of coronal sections of wild-type (N and P) and perlecan-null (O and Q) E17.5 forebrain regions. The perlecan-null brain shows a ruffled surface, large ectopias, and abnormal lamination (O). Posterior to this area, the ruffles and defects in lamination are less severe (Q). Abbreviations: mz, marginal zone; cp, cortical plate; iz, intermediate zone; and vz, ventricular zone. Bars: (C–L) 125 μm; (M) 250 μm; and (N–Q) 500 μm.

Figure 5

Neuronal ectopias in the ventral forebrain. (A–D) Hematoxylin/eosin staining of coronal brain sections from wild-type (A and C) and perlecan-null (B and D) E12.5 embryos. Ectopias (B and D, arrows) are visible in the ventral forebrain of homozygotes. (E and F) Cells within the ectopias are β-tubulin type III–positive (F, arrow). (G–L) Hematoxylin/eosin and immunostaining of sagittal brain sections from wild-type (G, I, and K) and perlecan-null (H, J, and L) E13.5 embryos. Ectopias (H, J, and L, arrows) are visible in the ventral forebrain. Laminin-1 is expressed in the leptomeninges and around the capillaries in both the wild-type (I) and perlecan-null (J) brain. In homozygotes, laminin-1 staining is disrupted around the ectopias (arrows). Perlecan is expressed around capillaries and in the leptomeninges but not in brain of normal embryos (K). No perlecan staining is detectable in homozygotes (L). Bars: (C–F and I–L) 250 μm.

Figure 6

Skeletal abnormalities in perlecan-null embryos. (A) Lateral view of E16.5 normal, heterozygous, and perlecan-null (−/−^a and −/−^b) embryos. Loss of perlecan results in disproportionate dwarfism with short limbs, neck, and snout. Some homozygotes have domed skull (−/−^a), others lack the roof of skull and exhibit exencephaly (−/−^b). (B) Alcian blue/alizarin red stained skeletons of normal, heterozygous, and perlecan-null (−/−^a and −/−^b) E17.5 embryos. The skeleton of heterozygotes is normal. The spine of homozygous mutants is short and displays severe kyphoscoliosis, the thorax is narrow, and the ribs, vertebrae, and long bones are malformed. Embryos with exencephaly (−/−^b) lack frontal and parietal bones of the skull. (C–F) Comparative histological analysis of developing hindlimbs from normal (C and E) and homozygotes (D and F). Sagittal sections from E14 (C and D) and E16.5 (E and F) embryos stained with hematoxylin/eosin. At E14, the mutant tibia (ti) and fibula (fi) are short, thick, and curved (D). At E16.5, the mutant tibia (F) is short and thick, the metaphyseal bone is extremely reduced, and the growth plate is disorganized. Abbreviations: ec, epiphyseal cartilage; p, zone of proliferative chondrocytes; h, zone of hypertrophic chondrocytes; and bc, bone cavity. Bar: (C–F) 100 μm.

Figure 7

ECM expression in long bones. (A and B) Safranin orange (SO) and van Kossa (vK) double staining show reduced proteoglycan content in mutant (B) as compared with wild-type (A) cartilage and the absence of mineralization of longitudinal septa in the lower hypertrophic zone (1 h) of the mutant growth plate (A and B). Also, note the transversally oriented trabecular bones in the mutant (arrows). (C–H) Immunostaining of perlecan and collagen types II (Col2) and X (Col10) on consecutive sections of elbows from normal and perlecan-null E15.5 embryos. Perlecan is present in normal cartilage, in the periosteum/perichondrium, and in the surrounding connective tissues (C). In mutant embryos, perlecan staining is absent (D). The distribution of collagen types II (E and F) and X (G and H) is similar in normal (E and G) and perlecan-null (F and H) cartilage. Bar: (A–D and G and H) 100 μm; 50 μm in E and F.

Figure 8

Ultrastructure of hypertrophic chondrocytes and territorial matrix. (A and C) The hypertrophic chondrocyte in a normal mouse femur has low organelle density. The septa are homogeneously filled with fibrillar collagen (arrows) and calcified material (c). (B and D) The hypertrophic chondrocyte in a perlecan-null femur displays increased density of organelles, and the cytosol is highly enriched with free ribosomes and polysomes (arrowheads). The adjacent pericellular matrix compartment is filled with fibrillar collagen but lacks calcification. (E and F) The territorial zone in normal cartilage shows random distribution of collagen fibrils. The collagen fibrils are of uniform diameter and length (E). The territorial zone in homozygotes lacks a well organized collagen fibrillar network. The collagen fibrils are shorter in length, lower in contrast, and the density is reduced. (G) The expression of Col2a1, matrilin-3, and COMP mRNA is increased in perlecan-deficient cartilage. Abbreviations: P, plasma membrane; N, cell nucleus; and C, calcification. Bars: (A and B) 5 μm; 1 μm (C and D); and (E and F) 0.5 μm.