Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13).

We report that mutation of COL11A2 causes deafness previously mapped to the DFNA13 locus on chromosome 6p. We found two families (one American and one Dutch) with autosomal dominant, non-syndromic hearing loss to have mutations in COL11A2 that are predicted to affect the triple-helix domain of the collagen protein. In both families, deafness is non-progressive and predominantly affects middle frequencies. Mice with a targeted disruption of Col11a2 also were shown to have hearing loss. Electron microscopy of the tectorial membrane of these mice revealed loss of organization of the collagen fibrils. Our findings revealed a unique ultrastructural malformation of inner-ear architecture associated with non-syndromic hearing loss, and suggest that tectorial membrane abnormalities may be one aetiology of sensorineural hearing loss primarily affecting the mid-frequencies.

New members of the collagen superfamily.

During the past year, several novel collagenous clones have been isolated from cDNA libraries and designated as new collagen types. Recent research has been concerned with describing these new collagens and their potential relationships to other previously described collagen types.

Monoclonal antibody against chicken type IX collagen: preparation, characterization, and recognition of the intact form of type IX collagen secreted by chondrocytes.

A series of monoclonal antibodies was prepared against the pepsin-resistant fragment of type IX collagen designated HMW. One of these antibodies (called 2C2) was selected for further analysis. Antibody 2C2 showed no cross-reactivity with other collagen types by inhibition enzyme-linked immunosorbent assays. It recognized an epitope present in native HMW, but failed to recognize any of the three chains of HMW fractionated after denaturation followed by reduction and alkylation of interchain disulfide bridges. Electron microscopic observations after rotary shadowing showed that the location of the epitope for antibody 2C2 was close to the carboxy-terminus of HMW. Immunofluorescent staining of sections of embryonic and adult cartilage with antibody 2C2 after removal of proteoglycans by testicular hyaluronidase digestion showed that type IX collagen is distributed throughout the cartilage matrix, and is not present in other connective tissues or skeletal muscle. The intact type IX collagen molecule, which was secreted by a suspension culture of freshly isolated embryonic chick chondrocytes, was recognized by rotary shadowing in the presence of antibody 2C2 after first precipitating the procollagens from the culture medium with ammonium sulfate (30%). Two different collagenous molecules were present in the precipitate: a longer molecule of type II procollagen (average length, 335 nm) with both amino- and carboxy-propeptides still remaining uncleaved, and a shorter molecule (average length, 190 nm) which was identified as type IX collagen. Antibody 2C2 consistently bound to the shorter molecules at a site located 136 nm from a distinctive knob at one end of the molecule, and did not bind to any specific site on the type II procollagen molecules. The structure of the intact type IX collagen molecule with the location of both collagenous and noncollagenous domains was as predicted after converting the nucleotide sequence of a cDNA clone encoding for one of the chains of type IX collagen to an amino acid sequence (Ninomiya, Y., and B. R. Olsen, 1984, Proc. Natl. Acad. Sci. USA, 81:3014-3018).

Developmental acquisition of basement membrane heterogeneity: type IV collagen in the avian lens capsule.

To investigate potential heterogeneity and developmental changes in basement membranes during embryogenesis, we performed immunohistochemical analyses on lens capsules in chicken embryos of different ages using domain-specific monoclonal antibodies against type IV collagen. We found that the capsule of the newly formed lens stained uniformly with antibodies against this component of basement membranes, but with increasing age and differentiation of the lens cells the anterior lens capsule remained brightly fluorescent while staining of the posterior capsule became relatively much less intense. This antero-posterior gradient of anti-type IV collagen antibody reactivity demonstrated that developmentally-regulated changes can occur within a single, continuous basement membrane.

A novel muscle-specific beta 1 integrin binding protein (MIBP) that modulates myogenic differentiation.

Myogenesis is regulated by cell adhesion receptors, including integrins of the beta1 family. We report the identification of a novel muscle-specific beta1 integrin binding protein (MIBP). MIBP binds to the membrane-proximal cytoplasmic region shared by beta1A and beta1D integrins, and the binding occurs in vivo as well as in vitro. Furthermore, we show that MIBP is abundantly expressed by C2C12 myogenic cells before fusion, and the expression of MIBP is dramatically downregulated during subsequent differentiation. Finally, we show that overexpression of MIBP in C2C12 cells resulted in a suppression of fusion and terminal differentiation, suggesting that MIBP may play a key role in controlling the progression of muscle differentiation.

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro.

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen.

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.

Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

D-periodic distribution of collagen type IX along cartilage fibrils.

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs.

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific "unmasking" treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman's membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the "unmasking" did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.

Identification and characterization of a mouse protein kinase that is highly homologous to human integrin-linked kinase.

Integrin-linked kinase (ILK) is a recently identified human protein kinase that has been implicated in integrin-mediated signal transduction and tumorigenesis. We have identified a mouse molecule that is highly homologous to human ILK. The mouse ILK homologue protein is readily recognized by antibodies raised against the human ILK protein, and the gene encoding the mouse ILK homologue is widely expressed in mouse tissues. The mouse ILK homologue gene has been mapped to chromosome 7E1 band. A second locus in the mouse chromosome 9E1-3 region has also been detected with a mouse ILK homologue cDNA probe by fluorescence in situ hybridization, suggesting the possible existence of an ILK pseudo-gene or a family of ILK genes in the mouse.

Localization of collagen types I, III and V during tendon development. Changes in collagen types I and III are correlated with changes in fibril diameter.

Collagen types I, III and V were localized at different stages of tendon development: a stage when tendon architecture is established, but immature (14-day), a mature tendon (hatchling) and an intermediate point where there is a rapid growth of tendon fibrils (17-day). The tendon fascicles and their connective tissue investments (endotendenium) were studied. The data show a changing pattern of type III collagen expression in the developing metatarsal tendon. In the immature tendon at 14 days of development, collagen types I and III are codistributed throughout the fascicles and their connective tissue investments. At this stage all of the fibrils regardless of the site are small. With development the regions segregate and become easily recognizable. As this occurs, the fibril diameter distributions diverge; those in the fascicle become larger while those in the endotendenium remain small. During this period, the fascicle loses type III collagen expression, while the endotendenium becomes type III collagen rich. At the same time, the connective tissue investments develop, and the fibrils of the endotendenium remain small during this period, but then become larger in the mature tendon. The increases in diameter are associated with a decrease in type III collagen reactivity. At hatching, both significant collagen type III reactivity and small diameter fibrils are restricted to the outer sheaths. During all stages of tendon development there is a constant small, but detectable amount of type V collagen. However, no correlation between type V reactivity and fibril diameter was observed at any stage of development. These results indicate an inverse relationship between type III collagen reactivity and fibril diameter in the developing tendon.

The complete cDNA coding sequence and tissue-specific expression of the mouse laminin alpha 4 chain.

The full-length cDNA coding sequence is presented for the mouse laminin alpha 4 chain. Comparison of the encoded amino acid sequence with human laminin alpha 4 shows 88.3% identity. Northern blot analysis shows that mouse laminin alpha 4 is strongly expressed in heart, lung and skeletal muscle, with only limited expression in brain, spleen, liver, kidney and testes.

Primary structure and expression of a chicken laminin beta chain: evidence for four beta chains in birds.

Characterization of a full length cDNA sequence for a chicken laminin beta chain is described which is most closely related to the mammalian beta 2 chain. Comparison with published sequences shows that the chicken beta 2-like chain corresponds to a fragment of a previously described laminin beta chain called B1-2 (O'Rear, 1992). The sequence of the chicken beta 2-like chain differed from fragments of two other chicken laminin beta chains that were previously described and designated B1-1 (now called beta 1; O'Rear, 1992) and beta x (Ybot-Gonzalez et a1.,1995). In addition, the beta 2- like chain does not appear to be the chicken equivalent of the mammalian laminin beta 3 chain, since it differs markedly in cDNA sequence, possesses domain IV and has a transcript size of 6 kb. We therefore propose that there are at least four laminin beta chains in the chicken. Sequence comparison of the beta 2-like laminin chain with previously cloned beta 1 and beta 2 chains shows a somewhat closer relationship to rat and human beta 2 than to mouse and human beta 1, especially in domains I, II and alpha. In addition, two expressed fragments of the chicken beta 2-like chain were recognized by a monoclonal antibody (C4) regarded as specific for the rat beta 2 chain (Hunter et al., 1989a). The results therefore suggest that the laminin chain previously described as a potentially novel chain called B1-2 (O'Rear, 1992) is likely to be the chicken equivalent of the mammalian beta 2 chain.

Immunization with undenatured bovine zonular fibrils results in monoclonal antibodies to fibrillin.

Microfibrils were dissected from the zonular apparatus of the bovine eye, homogenized and used as an immunogen to prepare monoclonal antibodies. Initial screening of hybridomas was performed by immunoblotting to a sonicate of zonular fibrils and by immunolocalization to frozen sections of the zonular apparatus. Subsequently, monoclonal antibodies with strong immunoreactivity to zonular fibrils were shown to recognize microfibrils in a wide range of connective tissues both by immunofluorescent staining and by electron microscopic immunolocalization. All antibodies were found to recognize a single protein of 350 kDa on Western blotting of the proteins secreted by bovine aortic smooth muscle cells. A protein of the same molecular weight and properties was recognized by an antibody previously prepared by another group against fibrillin. A member of the fibrillin family therefore represents the major immunogen of intact zonular fibrils, and the results support previous evidence for a close relationship between zonular fibrils and other connective tissue microfibrils. The zonular apparatus is a suitable system to obtain purified preparations of microfibrils in order to investigate their composition and structural organization.

Molecular cloning of PARP (proline/arginine-rich protein) from human cartilage and subsequent demonstration that PARP is a fragment of the NH2-terminal domain of the collagen alpha 2(XI) chain.

We report the molecular cloning of a proline/arginine-rich protein (called PARP) from human cartilage using the polymerase chain reaction (PCR) and degenerate oligonucleotides based on the previously published amino acid sequence of bovine PARP [1]. Subsequently, a reverse transcription-polymerase chain reaction (RT-PCR) was performed with poly(A)-rich RNA from human cartilage using a sense oligonucleotide derived from PARP and an anti-sense oligonucleotide derived from the known sequence of the human collagen alpha 2(XI) chain [2]. Nucleotide sequencing of the PCR product demonstrated that PARP is a fragment of the NH2-terminal non-collagenous (NC3) domain of the collagen alpha 2(XI) chain.

Fibrillin and elastin networks in extrafusal tissue and muscle spindles of bovine extraocular muscles.

PURPOSE: Bovine extraocular rectus muscles were examined to map the distribution of elastin and fibrillin in extrafusal tissue and muscle spindles. METHODS: Immunohistochemical techniques and immunolocalization were employed to pin-point the placement of molecules relative to muscle fibers. RESULTS: Strands containing elastin and fibrillin surrounded all extrafusal fibers. They also covered the external surface of intrafusal fibers, more extensively at the equator than at the pole. Within strands elastin was placed in the center, whereas fibrillin was located in microfibrils on the periphery. CONCLUSIONS: The wide distribution in extrafusal tissue of elastin and fibrillin suggests that they are factors in determining the mechanical properties of extraocular muscles. Their placement in proximity to individual intrafusal fibers should affect the viscoelastic properties of these fibers and, thus, influence the dimensions of the afferent discharge.

Extracellular matrices in the developing avian eye: type V collagen in corneal and noncorneal tissues.

We have used immunofluorescence histochemistry to examine the temporal and spatial deposition of type V collagen in the extracellular matrices of the developing chick cornea and other selected ocular structures. Tissue sections from animals ranging in age from 4-day-old embryos to 1-day post-hatching were examined by indirect immunofluorescence employing monoclonal antibodies specific for conformational dependent sites in this molecule. In eyes from embryos younger than 6 days of development, no type V staining could be detected. Thus, the epithelially derived primary corneal stroma, which is already well formed at this time, contains little if any of this molecule. Its appearance was concomitant with the physical swelling of the primary stroma and invasion of pericorneal mesenchymal cells. Staining was initially localized in the anterior cornea; subsequently, all corneal matrices showed intense reactions, including the stroma proper and Bowman's and Descemet's membranes. In adjacent noncorneal tissues, the appearance of type V collagen occurred later in development. In some of these, such as in the ciliary body, the pattern of acquisition involved initial deposition at an epithelial-mesenchymal interface with subsequent progression of fibrous strands out into the surrounding mesenchymal tissue. Eventually, all ocular structures with a dense connective tissue component showed staining, but the intensity was appreciably less than that within the cornea. We have previously reported that in all mature tissues except Bowman's membrane, type V collagen is present in an "antigenically masked" form, and that unmasking can be achieved by pretreatment of the tissue sections with dilute acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)