Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the alpha1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices.

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of alpha1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express alpha1(XI), as well as 293 cells which do not express alpha1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of alpha1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.

Developmental distribution of collagen type XII in cartilage: association with articular cartilage and the growth plate.

Collagen type XII is a member of the fibril-associated collagens and is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains. Previous studies suggested that collagen XII is a component of cartilage but little is known about its spatial-temporal distribution. This study uses a polyclonal antibody to the purified NC3 domain to investigate its developmental distribution in rat forelimb. Collagen XII was present at the joint interzone on embryonic day 16 (E16d) and restricted to the presumptive articular cartilage by E18d. Labeling of the articular surface intensified as development progressed postnatally (day 1 [1d] to 28d) and extended approximately six cell diameters deep. In juvenile rats, collagen XII antibodies also labeled the longitudinal and transverse septa of stacked chondrocytes in the growth plate. However, collagen XII was not associated at any developmental stage with the cartilaginous secondary ossification center and was only weakly expressed in epiphyseal cartilage. Ultrastructural localization of the NC3 domain epitope showed labeling of the surface of collagen II fibrils both in tissue and in isolated fibrils. The results presented provide further evidence that articular cartilage differs substantially from the underlying epiphyseal cartilage and that different chondrocytic developmental fates are reflected in the composition of their extracellular matrix starting early in development. In addition, collagen XII was distributed in areas of cartilage with more organized fibril orientation and may have a role in promoting alignment or stabilizing such an organization, thereby creating a matrix capable of withstanding load-bearing forces.

Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of approximately 220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

The Cav2.1/alpha1A (P/Q-type) voltage-dependent calcium channel mediates inhibitory neurotransmission onto mouse cerebellar Purkinje cells.

The effects of voltage-dependent calcium channel (VDCC) antagonists on spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse Purkinje cells were examined using in vitro cerebellar slices. The inorganic ion Cd2+ reduced sIPSC amplitude and frequency. No additional block was seen with the Na+ channel antagonist tetrodotoxin (TTX) suggesting that all action potential-evoked inhibitory GABA release was mediated by high-voltage-activated VDCCs. No evidence was found for involvement of Cav1/alpha1C and alpha1D (L-type), Cav2.2/alpha1B (N-type) or Cav2.3/alpha1E (R-type) high-voltage-activated VDCCs or low-voltage-activated Cav3/alpha1G, alpha1H and alpha1I (T-type) VDCCs in mediating presynaptic GABA release. Blockade of sIPSCs by 200 nM omega-agatoxin IVA implicated the Cav2.1/alpha1A (P/Q-type) subtype of high-voltage-activated VDCCs in mediating inhibitory transmission. Inhibition by omega-agatoxin IVA was similar to that seen with Cd2+ and TTX. Selective antibodies directed against the Cav2.1 subunit revealed staining in regions closely opposed to Purkinje cell somata. Cav2.1 staining was colocalized with staining for antibodies against glutamic acid decarboxylase and corresponded well with the pericellular network formed by GABAergic basket cell interneurons. Antibody labelling of Cav2.3 revealed a region-specific expression. In the cerebellar cortex anterior lobe, Cav2.3 staining was predominantly somatodendritic; whilst in the posterior lobe, perisomatic staining was seen primarily. However, electrophysiological data was not consistent with a role for the Cav2.3 subunit in mediating presynaptic GABA release. No consistent staining was seen for other Cav (alpha1) subunits. Electrophysiological and immunostaining data support a predominant role for Cav2.1 subunits in mediating action potential-evoked inhibitory GABA release onto mouse Purkinje cells.

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg( downward arrow)) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for approximately 20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg(159 downward arrow)- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor alpha1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.

Axial structure of the heterotypic collagen fibrils of vitreous humour and cartilage.

We have compared the axial structures of negatively stained heterotypic, type II collagen-containing fibrils with computer-generated staining patterns. Theoretical negative-staining patterns were created based upon the "bulkiness" of the individual amino acid side-chains in the primary sequence and the D-staggered arrangement of the triple-helices. The theoretical staining pattern of type II collagen was compared and cross-correlated with the experimental staining pattern of both reconstituted type II collagen fibrils, and fibrils isolated from adult and foetal cartilage and vitreous humour. The isolated fibrils differ markedly in both diameter and composition. Correlations were significantly improved when a degree of theoretical hydroxylysine glycosylation was applied, showing for the first time that this type of glycosylation influences the negative-staining pattern of collagen fibrils. Increased correlations were obtained when contributions from types V/XI and IX collagen were included in the simulation model. The N-propeptide of collagen type V/XI and the NC2 domain of type IX collagen both contribute to prominent stain-excluding peaks in the gap region. With decreasing fibril diameter, an increase of these two peaks was observed. Simulations of the fibril-derived staining patterns with theoretical patterns composed of proportions of types II, V/XI and IX collagen confirmed that the thinnest fibrils (i.e. vitreous humour collagen fibrils) have the highest minor collagen content. Comparison of the staining patterns showed that the organisation of collagen molecules within vitreous humour and cartilage fibrils is identical. The simulation model for vitreous humour, however, did not account for all stain-excluding mass observed in the staining pattern; this additional mass may be accounted for by collagen-associated macromolecules.

Morphology of local axon collaterals of electrophysiologically characterised neurons in the rat medial septal/ diagonal band complex.

Neurons in the medial septal/diagonal band complex (MS/DB) in vivo exhibit rhythmic burst-firing activity that is phase-locked with the hippocampal theta rhythm. The aim was to assess the morphology of local axon collaterals of electrophysiologically identified MS/DB neurons using intracellular recording and biocytin injection in vitro. Cells were classified according to previous criteria into slow-firing, fast-spiking, regular-spiking, and burst-firing neurons; previous work has suggested that the slow-firing neurons are cholinergic and that the other types are GABAergic. A novel finding was the existence of two types of burst-firing neuron. Type I burst-firing neurons had significantly longer duration after hyperpolarisation potentials when held at -60 mV, and at -75 mV, type I neurons exhibited a low-threshold spike with more rapid activation and inactivation kinetics than those of type II neurons. We have, also for the first time, described the main features of the local axon collaterals of the five neuron types. All filled neurons possessed a main axon that gave forth 1-12 local primary axon collaterals. All electrophysiological types, except for the type I burst-firing neuron, had a main axon that coursed toward the fornix. Myelination of the main axon was a prominent feature of all but the slow-firing neurons. Branching of the primary axon collaterals of the fast-spiking and type I burst-firing neurons was more extensive than that of the other cell types, with those of the slow-firing neurons exhibiting the least branching. All cell types possessed axon collaterals of the en passant type, and some in addition had twiglike or basketlike axon terminals. All cell types made synapses on distal dendrites; a proportion of the fast-spiking and burst-firing cells in addition had basketlike terminals that made synaptic contacts on proximal dendrites and on somata. Two morphological types of somata were postsynaptic to the basket cells: large (20-30-microm) oval cells with dark cytoplasm, and large oval cells with paler cytoplasm, often with an apical dendrite. The presence of lamellar bodies in the large dark neurons suggests that they may be cholinergic neurons, because previous work has localised these structures in some neurons that stain for choline acetyltransferase. Our work suggests therefore that there may be GABAergic neurons in the MS/DB that form basket synaptic contacts on at least two types of target cell, possibly cholinergic and GABAergic neurons, which means that the basket cells could play a key role in the generation of rhythmic activity in the MS/DB.

Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells.

Using patch-clamp techniques, a hyperpolarization-activated current (I(h)) was recorded from synaptic terminals of mouse cerebellar basket cells. Ih was blocked quickly and reversibly by 2 mM Cs(+), and subtraction revealed a rapidly activating and deactivating I(h) current. Similar gating and block of presynaptic I(h) were also seen with the more selective inhibitor ZD 7288 (10 microM). The time constant of activation (tau (a))of presynaptic I(h) current became faster with membrane hyperpolarization, being approximately 74 ms at -130 mV, changing e-fold for a 33 mV change in membrane potential. Whole-cell recordings from basket cell somata also revealed an I(h) current, which was similarly sensitive to block by ZD 7288. Inhibition of I(h) by 10 microM ZD 7288 reduced the frequency ( approximately 34 %) and amplitude ( approximately 26 %) of spontaneous IPSCs (sIPSCs) recorded in Purkinje cells, one of the principal synaptic targets of basket neurones. This is the first report of an I(h) current in mammalian inhibitory presynaptic terminals, which may be an important target for neuromodulation in the cerebellum. Comparing the biophysical properties and distribution of cloned hyperpolarization-activated cation channels, we also suggest a molecular candidate underlying I(h) at these synapses.

Electrophysiological characteristics of non-bursting, glutamate decarboxylase messenger RNA-positive neurons of the medial septum/diagonal band nuclei of guinea-pig and rat.

Nuclei of the medial septum/diagonal band region of the mammalian forebrain contain neurons that give rise to the septohippocampal pathway, which has separate cholinergic and GABAergic components. This pathway is known to influence hippocampal-dependent memory and learning processes, but the precise role of each component is unclear. In this study, we tested the hypothesis that fast-firing, non-bursting medial septum/diagonal band neurons are GABAergic. We used brain slice preparations from young adult guinea-pigs and rats, or from weanling rats, to perform current-clamp recordings from medial septum/diagonal band neurons. Recorded neurons were injected with biocytin for subsequent visualization with fluorescent avidin, and then hybridized with a 35S-labeled riboprobe for glutamate decarboxylase-67 messenger RNA. As a positive control, guinea-pig cerebellar Purkinje cells were labeled and hybridized with the riboprobe. As expected, labeled Purkinje cells were glutamate decarboxylase-67 messenger RNA positive. Slow-firing, cholinergic (choline acetyltransferase-positive) guinea-pig medial septum/diagonal band neurons were glutamate decarboxylase-67 messenger RNA negative. Contrary to our hypothesis, of the guinea-pig neurons, only three of 11 fast-firing neurons were glutamate decarboxylase-67 positive. Of the rat medial septum/diagonal band neurons, three of four were positive for glutamate decarboxylase-67 messenger RNA. These data suggest that fast-firing, non-bursting neurons of the medial septum/diagonal band, as sampled by sharp-electrode intracellular recordings in brain slices, may be a heterogeneous group of neurons, some of which are GABAergic. Together with recent data demonstrating the presence of another GABAergic marker, parvalbumin, in fast-firing septal neurons, we conclude that GABAergic septohippocampal neurons include a population of fast-firing, non-bursting neurons. The influence of these neurons on the hippocampus is likely to occur on a shorter time-scale and over a wider range of firing frequencies as compared to slowly firing cholinergic septohippocampal neurons.

Developmentally regulated alternative splicing of the alpha1(XI) collagen chain: spatial and temporal segregation of isoforms in the cartilage of fetal rat long bones.

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the alpha1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of alpha1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.

Perineuronal nets ensheath fast spiking, parvalbumin-immunoreactive neurons in the medial septum/diagonal band complex.

Perineuronal nets, composed of extracellular matrix material, have previously been associated with parvalbumin-immunoreactive neurons in the medial septum/diagonal band (MS/DB) complex of the rat. The aim of this study was to correlate the presence of perineuronal nets with electrophysiological properties and parvalbumin immunoreactivity in MS/DB neurons. Intracellular recordings were made from cells in a brain slice preparation maintained in vitro, and neurons were characterized into four populations: (i) slow-firing neurons, (ii) burst-firing neurons, (iii) fast spiking neurons with narrow action potentials and a small degree of spike frequency adaptation, and (iv) regular spiking neurons with broader action potentials and a high degree of spike frequency adaptation. Following electrophysiological characterization, neurons were filled with biocytin, processed for parvalbumin immunoreactivity and stained for perineuronal nets using Wisteria floribunda lectin. The three substances were viewed with triple fluorescence. Fast spiking, nonadapting neurons, shown previously to contain parvalbumin immunoreactivity, were nearly all ensheathed by perineuronal nets. There was a population of small parvalbumin-immunoreactive neurons which did not possess perineuronal nets, and which were not encountered with the intracellular electrodes. The other three neuron types in the MS/DB did not contain parvalbumin immunoreactivity or perineuronal nets. In keeping with this neurochemical profile for electrophysiologically identified neurons, burst-firing neurons had action potential parameters more similar to those of regular spiking than of fast spiking neurons. We conclude that fast spiking neurons, presumed to be GABAergic septohippocampal projection neurons, are surrounded by supportive structures to enable the high level of neuronal discharge required for producing disinhibition of hippocampal pyramidal neurons.

Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI.

Collagen XI is a heterotrimeric molecule found predominantly in heterotypic cartilage fibrils, where it is involved in the regulation of fibrillogenesis. This function is thought to involve the complex N-terminal domain. The goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism. The amino-propeptide (alpha1-Npp) alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography. Cys-1-Cys-4 and Cys-2-Cys-3 disulfide bonds were detected by liquid chromatography-tandem mass spectrometry. This pattern is identical to the homologous alpha2-Npp, indicating that the recombinant proteins were folded correctly. Anomalous elution on molecular sieve chromatography suggested that the variable region was extended, which was confirmed using rotary shadowing; the alpha1-Npp formed a globular "head" and the variable region an extended "tail." Circular dichroism spectra analysis determined that the alpha1-Npp comprised 33% beta-sheet, whereas the variable region largely comprised non-periodic structure. Taken together, these results imply that the alpha1-Npp cannot be accommodated within the core of the fibril and that the variable region and/or minor helix facilitates its exclusion to the fibril surface. This provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis.

Parvalbumin-immunoreactive, fast-spiking neurons in the medial septum/diagonal band complex of the rat: intracellular recordings in vitro.

The medial septum/diagonal band complex is composed predominantly of cholinergic and GABAergic neurons, and it projects to the hippocampal formation. A proportion of the GABAergic neurons contain parvalbumin, a calcium-binding protein that has previously been localized in fast-spiking, non-accommodating GABAergic neurons in the cerebral cortex and neostriatum. The aim of the present study was to determine whether parvalbumin is localized preferentially in a similar electrophysiological class of neuron in the medial septum/diagonal band complex. The study was carried out using in vitro intracellular recording, intracellular biocytin filling and parvalbumin immunocytochemistry. Three main classes of neurons were identified according to standard criteria: burst-firing, slow-firing and fast-firing neuronal populations. The fast-firing neurons were subdivided into two subpopulations based on whether or not they displayed accommodation. The fast-spiking, non-accommodating cells were furthermore found to be spontaneously active at resting potentials, and to possess action potentials of significantly (P < 0.05) shorter duration (half width: 0.61 +/- 0.12 ms) than those of the regular-spiking, accommodating neurons (1.0 +/- 0.34 ms). Of the neurons that were successfully filled with biocytin and processed for parvalbumin immunoreactivity, 82% of the fast-spiking, non-accommodating cells possessed parvalbumin immunoreactivity, while none of the regular-spiking, accommodating neurons were found to be immunoreactive for parvalbumin. The slow-firing neurons, shown previously to be cholinergic, did not stain for parvalbumin immunoreactivity, in agreement with studies showing parvalbumin to be localized solely in GABAergic neurons in the medial septum/diagonal band complex. In conclusion, these findings suggest the presence of a previously uncharacterized population of neurons in the medial septum/diagonal band complex that generate high-frequency, non-adaptive discharge. This property correlates with the localization of parvalbumin in these neurons, which suggests that parvalbumin fulfils the same role in the medial septum/diagonal band complex that it does in other parts of the brain. The fast-spiking neurons in the medial septum/diagonal band complex may play an essential role in the GABAergic influence of the septum on the hippocampal formation.

Temporal and spatial expression of alternative splice-forms of the alpha1(XI) collagen gene in fetal rat cartilage.

Type XI collagen, a member of the group of fibrillar collagens, plays a regulatory role in the formation of the collagen fibril network in cartilage and consequently plays a pivotal role in the formation of the endochondral skeleton. The mechanism by which type XI collagen limits fibril growth appears to involve the large noncollagenous amino terminal domain. Complex alternative splicing occurs within this domain in two of the three constituent subunits, alpha1(XI) and alpha2(XI). In the alpha1(XI) chain, three alternatively spliced exons encoding one very basic and two very acidic peptides generate six spliceforms and protein isoforms. In order to better understand the significance of this alternative splicing, we have examined fetal rat cartilage to determine: (a) the relationship between alternative splicing and chondrogenesis in limb bud micromass culture; (b) the relative levels of expression of each of the splice-forms by ribonuclease protection; and (c) the distribution of splice-forms and protein isoforms by in situ hybridization and immunohistochemistry. The results indicate that the pattern of alternative splicing of the alpha1(XI) chain is tightly linked to chondrogenesis. The two most abundant spliceforms in fetal rib cartilage are v(o), lacking all three exons, and v1b, containing the exon encoding the basic peptide. While most of the spliceforms show a general distribution in nasal, Meckel's, and rib cartilage, v1b was restricted to the dorsal portion of the fetal rib. This distribution appears to correlate with the portion of the rib which will ultimately ossify, rather than with any of the differentiative states of chondrocytes. Together these results suggest that alternative splicing within the amino terminal domain of the alpha1(XI) chain may contribute to the function of type XI collagen and that expression of the basic v1b peptide may play a role in endochondral ossification.

The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.

We have identified a novel missense mutation in a pseudoachondroplasia (PSACH) patient in one of the type III repeats of cartilage oligomeric matrix protein (COMP). Enlarged lamellar rough endoplasmic reticulum vesicles were shown to contain accumulated COMP along with type IX collagen, a cartilage-specific component. COMP was secreted and assembled normally into the extracellular matrix of tendon, demonstrating that the accumulation of COMP in chondrocytes was a cell-specific phenomenon. We believe that the intracellular storage of COMP causes a nonspecific aggregation of cartilage-specific molecules and results in a cartilage matrix deficient in required structural components leading to impaired cartilage growth and maintenance. These data support a common pathogenetic mechanism behind two clinically related chondrodysplasias, PSACH and multiple epiphyseal dysplasia.

Type IX collagen NC1 domain peptides can trimerize in vitro without forming a triple helix.

Synthetic peptides of the three chains of type IX collagen consisting of the carboxyl-terminal end of the COL1 domain and the complete NC1 domain were characterized by circular dichroism spectroscopy and analyzed for their ability to assemble into trimers. In vitro association and oxidation result in disulfide-linked oligomers as shown by molecular sieve chromatography and SDS-polyacrylamide electrophoresis. Whereas the individual peptides show a tendency to self-associate, when an equimolar amount of the three peptides was oxidized, a heterotrimer of the three chains was observed. This heterotrimer is recognized by a monoclonal antibody against the disulfide-linked NC1 domain of chicken type IX collagen, indicating the correct formation of the disulfide bonds. Circular dichroism measurements show that under the association conditions used, a triple helix does not form between the chains. These results indicate that these peptides contain all the necessary information for chain selection and assembly.

Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: comparison of two distinct matrix compartments.

We have characterized immunohistochemically and biochemically the collagens accumulating in two compartments of the matrix formed by mature bovine articular chondrocytes in alginate beads. At all times of the 28-day culture period, more than 90% of the collagen molecules were recovered from the rim of cell-associated matrix (CM) which encapsulates individual chondrocytes and chondrocyte clusters. Both the total amount and concentration of collagens in this matrix compartment rose progressively with time. The ratio of collagen/proteoglycan remained relatively constant with time and was always five to seven times higher in the CM than in the interterritorial matrix compartment further removed from the cells. In the CM, collagen types II, IX and XI were present on Day 28 in relative proportions (95/l/3) similar to those in adult cartilage. A higher proportion of newly synthesized collagen type XI than types II or IX molecules did not become incorporated into the pericellular rim of matrix but accumulated in the further removed matrix. Although collagen type I was synthesized in small amounts by flattened cells at the surface of the beads, it did not become incorporated as heterotrimers or homotrimers in the matrix. Mature pyridinium crosslinks, principally pyridinoline, were detected as early as Day 7 of culture but became much more abundant between Days 15 and 28, especially in the CM which contained at all times more than 90% of the crosslinks formed. The codistribution of collagen types II, IX and XI and mature collagen-specific crosslinks support the contention that mature chondrocytes cultured in alginate matrix surround themselves with a protective shell whose composition is very similar to that which encapsulated the cells in vivo.

Ultrastructural localization of collagen types II, IX, and XI in the growth plate of human rib and fetal bovine epiphyseal cartilage: type XI collagen is restricted to thin fibrils.

The collagen fibrils of hyaline cartilage vary in diameter depending on developmental stage and location within the tissue. In general, growth plates and fetal epiphyseal cartilages contain fibrils with diameters of less than approximately 25 nm, whereas the permanent cartilage of adult tissues contains fibrils of approximately 30-200 nm. The interstitial collagen fibrils of fetal cartilage are complex, having at least three collagen types as integral components. Type XI, a member of the fibrillar collagen class, has been proposed to limit fibril diameter. To test this proposition we sought to determine if Type XI collagen was preferentially associated with fibrils of smaller diameter. We focused our study on human juvenile rib growth plate, which has thin fibrils in the hypertrophic zone, thick fibrils in the resting zone or permanent cartilage, and a mixture of thin and thick fibrils in the proliferative zone. Tissues were examined by immunoelectron microscopy with antipeptide antibodies to the carboxyl telopeptide and to the amino terminal non-triple-helical domains of alpha 1 (XI). These studies showed that (a) both epitopes of Type XI collagen were readily accessible to antibodies at the fibrillar surface, (b) Type XI collagen was associated predominantly with fibrils < 25 nm in diameter, (c) Type XI collagen was not found in thick fibrils even after disruption with chaotropic agents, and (d) collagen Types II and IX were associated with fibrils of all sizes. These studies were extended to human newborn epiphyseal cartilage and to fetal calf cartilage, with the same result.

Alternative exon splicing within the amino-terminal nontriple-helical domain of the rat pro-alpha 1(XI) collagen chain generates multiple forms of the mRNA transcript which exhibit tissue-dependent variation.

Type XI collagen is an integral, although minor component of cartilage collagen fibrils. We have established that alternative exon usage is a mechanism for increasing structural diversity within the amino-terminal nontriple helical domain of the pro-alpha 1(XI) collagen gene. cDNA clones spanning the amino-terminal domain were selected from a rat chondrosarcoma library, and were shown to contain two major sequence differences from the previously reported human sequence. The first difference was the replacement of sequence encoding an acidic domain of 39 amino acids in length by a sequence encoding a 51-amino acid basic domain with a predicted pI of 11.9. The second difference was the absence of a sequence that would translate into a highly acidic 85-amino acid sequence downstream from the first variation. These two changes, expressed together, result in the replacement of most of the acidic domain with one that is smaller and basic. These two sequence differences serve to identify subdomains of a variable region, designated V1 and V2, respectively. V1a is defined as the acidic 39-amino acid sequence element and V1b is defined as the 51-amino acid basic sequence. Analysis of genomic DNA revealed that both V1a and V1b are encoded by separate adjacent exons in the rat genome and V2 is also encoded in a single exon downstream. Analysis of mRNA from cartilage-derived sources revealed a complex pattern of alpha 1(XI) transcript expression due to differential exon usage. In non-cartilage sources, the pattern is less complex; the most prevalent form is the one containing the two acidic sequences, V1a and V2.

A fibrillar collagen gene, Col11a1, is essential for skeletal morphogenesis.

Mice that are homozygous for the autosomal recessive chondrodysplasia (cho) mutation die at birth with abnormalities in cartilage of limbs, ribs, mandible, and trachea. Limb bones of newborn cho/cho mice are wider at the metaphyses than normal bones and only about half the normal length. By linkage analysis, the cho gene and the gene encoding the alpha 1 (XI) chain of cartilage collagen XI were mapped to the same region of chromosome 3. Deletion of a cytidine residue about 570 nt downstream of the translation initiation codon in cho alpha 1 (XI) mRNA causes a reading frame shift and introduces a premature stop codon. The data demonstrate that collagen XI is essential for normal formation of cartilage collagen fibrils and the cohesive properties of cartilage. The results also suggest that the normal differentiation and spatial organization of growth plate chondrocytes is critially dependent on the presence of type XI collagen in cartilage extracellular matrix.