Olena Jacenko

Phenotypic and biochemical consequences of collagen X mutations in mice and humans

Skeletal biology has entered an exciting period with the technological advances in murine transgenesis and human genetics. This review focuses on how these two approaches are being used to address the role of collagen X, the major extracellular matrix component of the focal zone of endochondral ossification, the hypertrophic cartilage zone. The hypothesized role of this unique collagen in skeletal morphogenesis and the phenotypic and biochemical consequences resulting from the disruption of its function are discussed. Specifically, data from three murine models, including transgenic mice with a dominant interference phenotype for collagen X, and two sets of mice with an inactivated collagen X gene through gene targeting and homologous recombination, as well as the human disorder of Schmid metaphyseal chondrodysplasia resulting from mutations in collagen X, are summarized and compared. Several inconsistencies and unresolved issues regarding the murine and human phenotypes and the function of collagen X are discussed.

Calcium deficiency induces expression of cartilage-like phenotype in chick embryonic calvaria

A detailed histological study of the chick embryonic calvarium was carried out to characterize the effect of calcium deficiency on cell differentiation during embryonic bone formation. Calcium deficiency on cell differentiation during embryonic bone formation. Calcium deficient chick embryos, produced by means of long-term shell-less (SL) culture, developed skeletal anomalies. In addition to reduced mineralization as detected by alizarin staining, significant changes were also observed in the extracellular matrix of the embryonic bones. First, the undermineralized matrix of the calvaria of SL embryos appeared to be more acidic as shown by more intense hematoxylin staining of the trabecular regions compared to controls. Secondly, the presence of sulfated proteoglycans was suggested by specific Alcian blue staining of the calvaria of Day 14 SL embryos. In addition, indirect fluorescence immunohistochemistry confirmed the developmental appearance of type II collagen in calcium-deficient calvaria, and localized it to undermineralized regions of the bone. These observations demonstrate the emergence of a chondrogenic phenotype in a typically osteogenic tissue during, and perhaps in response to, severe systemic calcium deficiency in the developing chick embryo.

Heparan Sulfate Proteoglycans: A GAGgle of Skeletal-Hematopoietic Regulators

This review summarizes our current understanding of the presence and function of heparan sulfate proteoglycans (HSPGs) in skeletal development and hematopoiesis. Although proteoglycans (PGs) comprise a large and diverse group of cell surface and matrix molecules, we chose to focus on HSPGs owing to their many proposed functions in skeletogenesis and hematopoiesis. Specifically, we discuss how HSPGs play predominant roles in establishing and regulating niches during skeleto‐hematopoietic development by participating in distinct developmental processes such as patterning, compartmentalization, growth, differentiation, and maintenance of tissues. Special emphasis is placed on our novel hypothesis that mechanistically links endochondral skeletogenesis to the establishment of the hematopoietic stem cell (HSC) niche in the marrow. HSPGs may contribute to these developmental processes through their unique abilities to establish and mediate morphogen, growth factor, and cytokine gradients; facilitate signaling; provide structural stability to tissues; and act as molecular filters and barriers. Developmental Dynamics 237:2622–2642, 2008.

Linking Hematopoiesis to Endochondral Skeletogenesis through Analysis of Mice Transgenic for Collagen X

Each skeletal element where marrow develops is first defined by a hypertrophic cartilage blueprint. Through programmed tissue substitution, the cartilaginous skeletal model is replaced by trabecular bone and marrow, with accompanying longitudinal tissue growth. During this process of endochondral ossification, hypertrophic cartilage expresses a unique matrix molecule, collagen X. Previously we reported that transgenic mice with dominant interference collagen X mutations develop variable skeleto-hematopoietic abnormalities, manifested as growth plate compressions, diminished trabecular bone, and reduced lymphatic organs (Nature 1993, 365:56). Here, histology and flow cytometry reveal marrow hypoplasia and impaired hematopoiesis in all collagen X transgenic mice. A subset of mice with perinatal lethality manifested the most severe skeletal defects and a reduction of marrow hematopoiesis, highlighted by a lymphocyte decrease. Thymic reduction is accompanied by a paucity of cortical immature T cells, consistent with the marrows inability to replenish maturing cortical lymphocytes. Diminished spleens exhibit indistinct lymphatic nodules and red pulp depletion; the latter correlates with erythrocyte-filled vascular sinusoids in marrows. All mice display reduced B cells in marrows and spleens, and elevated splenic T cells. These hematopoietic defects underscore an unforeseen link between hypertrophic cartilage, endochondral ossification, and establishment of the marrow microenvironment required for blood cell differentiation.

A Dominant Interference Collagen X Mutation Disrupts Hypertrophic Chondrocyte Pericellular Matrix and Glycosaminoglycan and Proteoglycan Distribution in Transgenic Mice

Collagen X transgenic (Tg) mice displayed skeleto-hematopoietic defects in tissues derived by endochondral skeletogenesis.(1) Here we demonstrate that co-expression of the transgene product containing truncated chicken collagen X with full-length mouse collagen X in a cell-free translation system yielded chicken-mouse hybrid trimers and truncated chicken homotrimers; this indicated that the mutant could assemble with endogenous collagen X and thus had potential for dominant interference. Moreover, species-specific collagen X antibodies co-localized the transgene product with endogenous collagen X to hypertrophic cartilage in growth plates and ossification centers; proliferative chondrocytes also stained diffusely. Electron microscopy revealed a disrupted hexagonal lattice network in the hypertrophic chondrocyte pericellular matrix in Tg growth plates, as well as altered mineral deposition. Ruthenium hexamine trichloride-positive aggregates, likely glycosaminoglycans (GAGs)/proteoglycans (PGs), were also dispersed throughout the chondro-osseous junction. These defects likely resulted from transgene co-localization and dominant interference with endogenous collagen X. Moreover, altered GAG/PG distribution in growth plates of both collagen X Tg and null mice was confirmed by a paucity of staining for hyaluronan and heparan sulfate PG. A provocative hypothesis links the disruption of the collagen X pericellular network and GAG/PG decompartmentalization to the potential locus for hematopoietic failure in the collagen X mice.

Craniofacial abnormalities in mice carrying a dominant interference mutation in type X collagen

Type X collagen is a short, non‐fibril forming collagen restricted to hypertrophic cartilage, and has been hypothesized to play a role in endochondral ossification. The purpose of the study was to investigate the consequences resulting from the interference of type X collagen function on the growth and development of the craniofacial skeleton through analysis of transgenic mice with a dominant interference mutation for type X collagen. The craniofacial tissues of 21‐day‐old transgenic mice were examined by: cephalometric and radiographic densitometry analyses, conventional histology, and immunohistochemistry using antibodies specific for either endogenous mouse type X collagen or the transgene product. Genotypically positive mutant mice showed moderate but statistically significant craniofacial skeletal abnormalities, including the underdevelopment of the chondrocranium and mandible, but no cleft palate. Mean radiographic optical densities of the mutant condylar cartilage and the subchondylar areas were 32% less than the corresponding areas of normal mandibles, while mean radiographic optical density measured at the incisor tooth point remained constant. Histologically, transgene‐positive mice revealed compressed hypertrophic cartilage zones and reduced trabeculae in both the mandibular condyle and the synchondroses of the chondrocranium. In the normal condyle, mouse type X collagen was localized by the monospecific antibody against a synthetic rat type X collagen NC1 peptide throughout the hypertrophic cartilage layer; in the mutant condyle, immunoreactivity to endogenous type X collagen was only seen sporadically. The truncated type X collagen transgene product, identified with the monoclonal antibody against an epitope within the chick type X collagen NC2 domain, persisted in the lower hypertrophic cartilage layer and the primary spongiosa, rather than being removed by subsequent endochondral ossification. The data suggested that the expression of the chick type X collagen transgene product was strongly associated with the craniofacial skeletal abnormalities that were distinct from other cartilage‐related phenotypes. Dev. Dyn. 208:544–552, 1997.

Polyionic regulation of cartilage development : promotion of chondrogenesis in vitro by polylysine is associated with altered glycosaminoglycan biosynthesis and distribution

The development of cartilage nodules in cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23/24) is significantly promoted when the culture medium is supplemented with (poly-L-lysine (PL) (M(r) greater than or equal to 14K) (San Antonio and Tuan, 1986. Dev. Biol. 115: 313). Here we present findings consistent with the hypothesis that PL may promote chondrogenesis by interacting electrostatically with sulfated glycosaminoglycans (GAGs): (1) poly-L-ornithine, poly-L-histidine, poly-D,L-lysine, and lysine-containing heteropolypeptides stimulate chondrogenesis in proportion to their contents of cationic residues; (2) the effects of PL are diminished when limb mesenchyme cultures are supplemented with exogenous GAGs, including heparin, dermatan sulfate, and chondroitin sulfate; (3) in high density cultures of limb bud mesenchyme, the release of sulfated macromolecules, but not of proteins in general, into the culture medium was significantly inhibited by PL (398K M(r)) treatment, and a net increase in total GAG content of the PL-treated cultures was observed; and (4) in monolayer cultures of cells derived from other chick embryonic tissues, including liver, skeletal muscle, and calvaria, PL treatment promoted the cell layer-associated retention of sulfated GAG. These effects were not observed using the nonstimulatory, low M(r) PL (4K). Based on the above findings and those from previous studies, it is proposed that PL may promote chondrogenesis by interacting electrostatically with cartilage GAGs, thus trapping the extracellular matrix around the newly emerging cartilage nodules and thereby stabilizing their growth and differentiation.

Chicken Collagen X Regulatory Sequences Restrict Transgene Expression to Hypertrophic Cartilage in Mice

Collagen X is produced by hypertrophic cartilage undergoing endochondral ossification. Transgenic mice expressing defective collagen X under the control of 4.7- or 1.6-kb chicken collagen X regulatory sequences yielded skeleto-hematopoietic defects (Jacenko O, LuValle P, Olsen BR: Spondylometaphyseal dysplasia in mice carrying a dominant-negative mutation in a matrix protein specific for cartilage-to-bone transition. Nature 1993, 365:56-61; Jacenko O, Chan D, Franklin A, Ito S, Underhill CB, Bateman JF, Campbell MR: A dominant interference collagen X mutation disrupts hypertrophic chondrocyte pericellular matrix and glycosaminoglycan and proteoglycan distribution in transgenic mice. Am J Pathol 2001, 159:2257-2269; Jacenko O, Roberts DW, Campbell MR, McManus PM, Gress CJ, Tao Z: Linking hematopoiesis to endochondral ossification through analysis of mice transgenic for collagen X. Am J Pathol 2002, 160:2019-2034). Current data indicate that the hematopoietic abnormalities do not result from extraskeletal expression of endogenous collagen X or the transgene. Organs from mice carrying either promoter were screened by immunohistochemistry, in situ hybridization, and Northern blot; transgene and mouse collagen X proteins and messages were detected only in hypertrophic cartilage. Likewise, reverse transcriptase-polymerase chain reaction revealed both transgene and mouse collagen X amplicons only in the endochondral skeleton of mice with the 4.7-kb promoter; however, in mice with the 1.6-kb promoter, multiple organs were transgene-positive. Collagen X and transgene amplicons were also detected in marrow, but likely resulted from contaminating trabecular bone; this was supported by reverse transcriptase-polymerase chain reaction analysis of rat tibial zones free of trabeculae. Our data demonstrate that in mice, the 4.7-kb chicken collagen X promoter restricts transcription temporo-spatially to that of endogenous collagen X, and imply that murine skeleto-hematopoietic defects result from transgene co-expression with collagen X. Moreover, the 4.7-kb hypertrophic cartilage-specific promoter could be used for targeting transgenes to this tissue site in mice.

Altered endochondral ossification in collagen X mouse models leads to impaired immune responses

Disruption of collagen X function in hypertrophic cartilage undergoing endochondral ossification was previously linked to altered hematopoiesis in collagen X transgenic (Tg) and null (KO) mice (Jacenko et al., [ 2002 ] Am J Pathol 160:2019–2034). Mice displayed altered growth plates, diminished trabecular bone, and marrow hypoplasia with an aberrant lymphocyte profile throughout life. This study identifies altered B220+, CD4+, and CD8+ lymphocyte numbers, as well as CD4+/fox3P+ T regulatory cells in the collagen X mice. Additionally, diminished in vitro splenocyte responses to mitogens and an inability of mice to survive a challenge with Toxoplasma gondii, confirm impaired immune responses. In concert, ELISA and protein arrays identify aberrant levels of inflammatory, chemo‐attractant, and matrix binding cytokines in collagen X mouse sera. These data link the disruption of collagen X function in the chondro‐osseous junction to an altered hematopoietic stem cell niche in the marrow, resulting in impaired immune function. Developmental Dynamics 237:2693–2704, 2008.

Congenic mice confirm that collagen X is required for proper hematopoietic development.

The link between endochondral skeletal development and hematopoiesis in the marrow was established in the collagen X transgenic (Tg) and null (KO) mice. Disrupted function of collagen X, a major hypertrophic cartilage matrix protein, resulted in skeletal and hematopoietic defects in endochondrally derived tissues. Manifestation of the disease phenotype was variable, ranging from perinatal lethality in a subset of mice, to altered lymphopoiesis and impaired immunity in the surviving mice. To exclude contribution of strain specific modifiers to this variable manifestation of the skeleto-hematopoietic phenotype, C57Bl/6 and DBA/2J collagen X congenic lines were established. Comparable disease manifestations confirmed that the skeleto-hematopoietic alterations are an inherent outcome of disrupted collagen X function. Further, colony forming cell assays, complete blood count analysis, serum antibody ELISA, and organ outgrowth studies established altered lymphopoiesis in all collagen X Tg and KO mice and implicated opportunistic infection as a contributor to the severe disease phenotype. These data support a model where endochondral ossification-specific collagen X contributes to the establishment of a hematopoietic niche at the chondro-osseous junction.