Targeted Disruption of the Perlecan Gene
The perlecan gene was inactivated in ES cells by homologous recombination using the cre/loxP technique. A neomycin/thymidine kinase (neo/tk) cassette flanked by single loxP sites was inserted into intron 5 and a single loxP site was inserted into intron 6 ( A). Eight independently targeted ES cell lines were obtained. Two of them (clones 243 and 310) were transiently transfected with a cre recombinase expression plasmid, selected in FIAU, and analyzed for cre-mediated deletion. In three clones derived from 243 and 1 clone derived from 310, respectively, the sequence between the outermost loxP sites (including the neo/tk cassette, part of intron 5, exon 6, and part of intron 6) was deleted ( A). One clone carrying the constitutive null allele from 243 and from 310, respectively, was injected into C57BL/6 blastocysts and transferred to pseudopregnant females. Chimeric males from both clones gave germline transmission of the mutated perlecan gene.
ES Cells Carrying a Homozygous Mutation in the Perlecan Gene Do Not Secrete Perlecan
To verify that the deletion of exon 6 in the perlecan gene leads to a constitutive null mutation, ES cells carrying a homozygous mutation were generated. Heterozygous ES cells were retransfected with the original targeting construct and again selected for neo expression. Afterwards, one of the clones with a recombination event on the wild-type allele was transiently transfected with the cre expression plasmid to obtain ES clones with a homozygous deletion of exon 6 ( B).
To test whether the mutant allele was transcribed, RT-PCR was performed with total RNA extracted from normal, heterozygous, and homozygous ES cells. The amplified fragment encompasses sequences in exons 5–8. The size of the amplified DNA fragment was 640 bp for the wild-type allele and 479 bp for the mutant allele, indicating that splicing occurs between exon 5 and 7 ( C). Splicing of exon 5–7 leads to a reading frame shift and to the formation of a truncated perlecan protein consisting of the 116–NH2-terminal amino acids that make approximately half of domain I.
To determine whether the mutant mRNA was translated, normal and homozygous ES cells were grown in serum-free culture medium, and the supernatant as well as the cell extract were subjected to a highly sensitive RIA using polyclonal antibodies against laminin-1, nidogen-1, or the NH2-terminal domain I of perlecan. Whereas normal and mutant ES cells produce similar amounts of laminin and nidogen-1, only normal but not mutant ES cells produce perlecan ( D).
These data show that although the mutant perlecan allele is transcribed, no truncated protein could be detected with a domain I–specific polyclonal antibody. Most likely, the truncated form of domain I is not properly folded and is consequently degraded intracellularly as soon as it is translated. This was confirmed by episomal transfection of human EBNA-293 cells with domain I lacking 38 COOH-terminal residues that did not produce any recombinant fragment (data not shown). Therefore, the mutation in the perlecan gene is designated as a loss of function mutation.
Mice Homozygous for the Perlecan-Null Mutation Die at Two Developmental Stages: between E10 and E12 and Perinatally
Mice heterozygous for the mutation appeared normal and did not display any overt anatomical or behavioral abnormalities. No mice homozygous for the mutation were detected among 728 weaned progeny from heterozygous intercrosses (). Analysis of newborn offspring detected three homozygotes and several cannibalized mice among 98 neonates (). The three homozygotes showed exencephaly, chondrodysplasia, hemorrhage in several organs (see below), severe cleft palates, and died around birth.
Progeny of Perlecan+/− × Perlecan+/− Crosses
To determine when the remaining homozygotes die, embryos were examined from E9.5 to birth (). At E9.5, wild-type, heterozygous, and homozygous mutant embryos were represented in a normal Mendelian ratio (). Perlecan-null embryos at E9.5 were indistinguishable from wild-type and heterozygous littermates. E9.5 perlecan-null embryos formed brain vesicles, optic vesicles, branchial arches, otic pits, limb buds, a beating heart, and 20–25 somites (not shown). Therefore, development to E9.5 proceeds normally in the absence of perlecan.
By E10.5, defects began to appear in perlecan-null embryos. Although they were still present in the expected percentage () and were of normal size, ~70–80% were dead as demonstrated by the absence of the heart beating and the presence of severe hemopericardium ( B, arrow). 20–30% of homozygotes were alive and of normal appearance (not shown). By E11.5 and E12.5 the percentage of dead embryos with hemopericardium increased further, whereas some of the living embryos developed abnormally in the head region and survived to the perinatal period. At all stages analyzed, the placental development was unaffected by the loss of perlecan. The placental size, architecture, and the blood content were similar between normal and perlecan-null embryos (not shown). In addition, PECAM whole mount stainings of E9.5 and E10.5 embryos revealed that homozygotes had no defects in angiogenesis, sprouting, and remodeling to generate vessels of different sizes (not shown). At later stages of development (E13–E17), we could observe the formation of microaneurysms associated with bleedings in several tissues including lung, skin, and brain (not shown).
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 (more ...)
These results indicate that a null mutation in the perlecan gene leads to two patterns of development. Many of the perlecan-null embryos die during an early crisis (E10.5–E12.5) characterized by hemopericardium and heart arrest. The few remaining mutants survive to the perinatal period (), but develop severe brain and skeletal defects (see below).
Early Lethality in Perlecan-deficient Embryos Is Due to Myocardial Defects
At E10–11, many perlecan-null embryos manifested several signs of cardiac insufficiency characterized by intrapericardial hemorrhages ( B), weak heartbeats, or cardiac arrest. To investigate these defects, wild-type and alive perlecan-null embryos were analyzed by immunohistochemical and ultrastructural methods. The high resolution analysis of plastic-embedded hearts derived from E10.5 wild-type embryos showed a continuous wall of several layers of cardiomyocytes covered on both sides by a single cell layer of endothelial cells forming the endocardium and epicardium ( and ). In a large number of perlecan-null embryos, the compact layer of cardiomyocytes was interrupted by small intercellular clefts that were often covered by an intact layer of endo- and epicardium ( and F–H). In a few embryos, the clefts in the myocardium were filled with endocardial cells ( and ). The defects were associated with blood cell leakage into the pericardial cavity ( and ). Adjacent to the myocardial defects, the pericardial tissue was thickened because of an increase in cell number and matrix deposition ( D). No defects were observed in the blood vessels of affected hearts (not shown).
At E9.5, thin and normally appearing BMs were observed around perlecan-null cardiomyocytes (not shown). However, at E10.5 normal cardiomyocytes were covered by a continuous BM ( I), whereas perlecan-null cardiomyocytes lacked BM deposition ( J) or showed small patches of electron dense material on their cell surface ( K). The formation of sarcomeres ( J) and tight junctions were normal in perlecan-null cardiomyocytes. Ultrastructural analysis of BMs in other tissues including skin ( and ) and gut (not shown) revealed no abnormalities, suggesting that at E10.5 the BM defects are restricted to the heart where they are exposed to mechanical stress. To test whether the BM defects in the perlecan-null myocardium are associated with abnormal expression of BM proteins, immunostaining for perlecan ( and ), laminin-1 ( and ), and collagen IV (not shown) was performed on normal and perlecan-null cardiac tissue. With the exception of perlecan, all BM components were similarly expressed in mutant tissue ( and ). These data demonstrate that perlecan-deficient cardiac muscle cells lack BM or are covered by abnormal BM, which disrupts the integrity of the myocardium and leads to the formation of small clefts in myocardial tissue and finally to blood leakage into the pericardial cavity.
Development of Exencephaly and Neuronal Ectopias
About 80% of the perlecan-null embryos surviving the first crisis developed an exencephalic malformation that was first visible between E10.5 and E11.5. To test whether the absence of perlecan results in an abnormal closure of the neural tube, scanning electron microscopy was performed with normal and perlecan-null E10.5 embryos. All normal (n = 3) and perlecan-null embryos (n = 11) analyzed, displayed properly closed neuropores (, A–C). This was also confirmed by histological studies on E9.5 and E10.5 cephalic regions derived from mutant embryos (not shown). Some perlecan-null embryos had holes in the fore- and midbrain and showed collapsed brain vesicles ( C). The cephalic region of normal embryos was covered by an intact layer of ectodermal cells ( D). However, at higher magnification, the surface ectoderm of seven out of nine homozygotes showed small clefts that were 20–30 μm in width that contained round cells with small extensions ( E). In nullizygotes with severe defects, round cells with small extensions traversed the cephalic mesenchyme to reach the surface ectoderm ( F). To exclude that the clefts were caused artificially during embryo handling, heads were incubated with colloidal gold-conjugated antibodies binding to laminin-1 ( G) and analyzed by scanning electron microscopy. Preparation artefacts occurring after antibody incubation were devoid of staining ( and , and arrow in I and K), whereas clefts already present in homozygotes before handling showed colloidal gold staining of the exposed BM ( and ).
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 (more ...)
Histological analysis of brain sections from normal and perlecan-null E9.5 embryos revealed normal BM between neural tissue and mesenchyme (not shown). At E11.5, 70% of homozygotes showed areas in which the BM surrounding the telencephalic vesicles was disrupted ( and ), and the brain tissue had invaded into the cephalic mesenchyme and fused with the overlaying ectoderm ( H). Immunostaining revealed that the ectopias contained many nestin-positive cells (a marker for neuroepithelial cells; and ), but lacked βIII isotype tubulin-positive cells (a marker for committed neurons; and ). In the ectopic region, the neuroepithelium appeared thickened and the cells in the ventricular zone region were round instead of elongated as observed in the neocortex of normal embryos ( and ). At E11.5, clusters of neuroepithelial cells were exposed to the amniotic cavity and formed small disruptions of 5–10 μm ( M). Immunostaining for proliferative cells with Ki-67 antibodies and for apoptotic cells with TUNEL labeling revealed no abnormalities in E10.5 and E11.5 perlecan-null brain tissues, neither in ectopias nor in normal appearing areas of the neocortex (not shown). At later stages, several homozygotes without obvious exencephaly showed a ruffled brain surface because the marginal zone of the neocortex was studded with large ectopias associated with a severe distortion of the laminar architecture of the cortex ( and ).
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 (more ...)
All perlecan-null embryos analyzed so far, including those without exencephaly, exhibited neuronal ectopias in the ventral telencephalic region of the brain, when examined at E11.5 and later stages (). The ectopias appeared as small, compact clusters of βIII isotype tubulin-positive cells (, A–F) that had invaded the mesenchyme (, , , , , and ) at areas where the basement membrane is disrupted as shown by immunostaining for laminin-1 ( and ). Immunostaining for laminin-1 and perlecan at E13.5 revealed that both molecules were expressed around brain vessels and in the leptomeninges surrounding the brain tissue ( and ). No perlecan expression was found in the brain parenchyma of normal mice ( K).
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. (more ...)
Chondrodysplasia Associated with Abnormal Endochondral Ossification
All perlecan-null embryos that did not exhibit apparent heart defects continued their intrauterine development but died perinatally. Between E15 and the newborn stage, these animals developed a severe osteochondrodysplasia characterized by dwarfism, cleft palate, short limbs, and a short and abnormally bended vertebral column ( A and not shown). Approximately 80% of homozygotes had exencephaly and lacked calvarial bones ( A, −/−b). Homozygotes without exencephaly had a domed skull ( A, −/−a). Whole mount skeletons of E17.5 embryos showed that all bones were present in perlecan-null embryos, except in exencephalic embryos, which lacked frontal and parietal bones ( B, −/−a and −/−b). Detailed inspection of the mutant skeleton showed that long bones were approximately half of the size of wild-types. In addition, the cortical bone was thickened. In the skull, the mandible and nasal bone were shortened, and the structures of the middle and inner ear were poorly developed ( B). The bones of chondrocranium (occipital, sphenoidal, and ethmoidal) were shortened and undermineralized (data not shown).
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 (more ...)
Histological analysis at various stages of development revealed that the cartilage anlage of all long bones occurred normally in the mutant mice (not shown). Between E13 and 14, the first alterations in size and shape of the perlecan-null long bones became apparent ( and ). Mutant bones had disorganized growth plates characterized by the absence of the typical columnar arrangement of hypertrophic chondrocytes. In addition, hypertrophic chondrocytes showed an atypical morphology, and growth plates were always dissociated from their epiphyses ( F). Such gaps in the tissue were never observed in normal bones and, therefore, suggest a mechanical weakness of the mutant cartilage leading to damage during tissue processing. Perlecan-null bones had small marrow cavities.
Impaired Mineralization and Reduced Collagen Content
Safranin-O staining was reduced in perlecan-null cartilage, suggesting a decreased proteoglycan content ( and ). van Kossa staining revealed that normal bones showed clear mineralization in the longitudinal septa of the late hypertrophic zone ( A), perlecan-null tissue had minimal or no mineral deposits in the matrix around hypertrophic chondrocytes, and the calcified trabecula were transversely oriented in perlecan-null bones ( B). Immunohistochemistry showed expression of perlecan in cartilage as well as the surrounding mesenchyme of normal but not mutant mice ( and ). Matrix proteins including collagen types II, IX, X, and XI, aggrecan, matrilin-1 and -3, and COMP were expressed in homozygotes ( and , and not shown).
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 (more ...)
The ultrastructure of cartilage tissue derived from an E17.5 limb showed that wild-type hypertrophic chondrocytes were electron lucent with a paucity of organelles in the cytosol ( and ). The wild-type chondrocytes also showed contacts with the surrounding matrix that was homogeneously filled with fibrillar collagen ( A). In contrast, hypertrophic chondrocytes of perlecan-null mice displayed an increased density of organelles and distended cisternae of ER ( and ). The cytosol was enriched with free ribosomes and polysomes ( D, arrowheads). The collagen fibrils in wild-type growth plate cartilage showed a random distribution, had uniform length and diameter, and formed a typical network ( E). The perlecan-null growth plate cartilage lacked such collagen network and the fibrils were shorter in length ( 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). (more ...)
To test whether an increased expression of cartilage ECM genes was responsible for the high metabolic activity in perlecan-null chondrocytes, Northern assays were performed with the total RNA derived from cartilage of normal and homozygous E18 limbs. Analysis of optical densities of mRNA signals such as shown in G revealed that Col2a1 expression was increased threefold, and matrilin-3 and COMP expression was increased fivefold in mutant cartilage.