Analysing the Role of Growth Plate Specific Genes in Endochondral Ossification by Specific Overexpression or Deletion under the BAC Col10a1 Promoter
Final Report Abstract
Most of the bony elements of the vertebrate skeleton, in particular long bones, ribs, and vertebrae, are laid down on a cartilaginous scaffold during fetal and juvenile stages of development in a complex process called endochondral ossification. Elucidation of the cellular and molecular events regulating cartilage-bone turnover during endochondral ossification are important not only for understanding mechanisms of skeletal development, but also the pathology of genetic skeletal disorders such as dwarfism or chondrodysplasias, and for improved management of bone fracture healing. The aim of this project was to elucidate the role of selected genes involved in endochondral ossification by specific overexpression or deletion in hypertrophic chondrocytes using BACCol10 transgenic mice. During endochondral bone formation, hypertrophic chondrocytes play key role in regulating pace and site of cartilage –bone turnover in the growth plates of long bones, ribs and vertebrae during fetal and juvenile stages of skeletal development. After undergoing a series of strictly controlled differentiation stages, hypertrophic chondrocytes express a distinct pattern of genes which induce cartilage calcification, matrix degradation, and invasion of bone marrow capillaries. Col10a1 is the gene coding for collagen X which is almost exclusively expressed in hypertrophic chondrocytes. For that reason we had generated transgenic mice bearing a Col10a1–containing BAC (bacterial artificial chromosome) which allowed us to express any gene specifically in hypertrophic chondrocytes under the Col10a1 promoter. In the preceding part of this project we have shown that specific overexpression of the chondrogenic transcription factor Sox9 in hypertrophic chondrocytes of BACCol10Sox9 transgenic mice severely impaired cartilage resorption and bone marrow formation, causing growth retardation. This was caused by suppressing terminal differentiation of chondrocytes and the expression of genes which regulate cartilage resorption and bone marrow invasion such as Mmp13, Vegfa and others. One subject of the current proposal was to verify whether the suppression of Mmp13 expression by Sox9 occurs directly at transcriptional level. In fact, using Mmp13/Luciferase reporter genes we demonstrated that Sox9 directly binds to Sox9 consensus sequences in the Mmp13 promoter. This explains why Mmp13 is not expressed in resting and proliferating chondrocytes of the growth late which express high Sox9 levels, but starts to be expressed in the late hypertrophic zone where Sox9 is no longer present. Affymetrix expression array experiments revealed numerous further genes to be up- or downreguated in hypertrophic cartilage by Sox9 overexpression. Most interestingly, we found strong upregulation of myelin basic protein (mbp) in hypertrophic chondrocytes, a protein normally found only associated with neural tissues where is it regulated by Sox10. The second goal of this proposal to was to elucidate the specific role of β-catenin in hypertrophic chondrocytes ossification. In numerous studies it has been shown that during endochondral ossification and skeletal development canonical Wnt signaling plays a central, but complex role in the regulation of chondrogenic and osteogenic differentiation. In vivo and in vitro gain- and loss-of-function studies have indicated that β-catenin is essential for osteogenic differentiation in early limb and head mesenchyme, while it represses differentiation of mesenchymal precursor cells to chondrocytes. Since hypertrophic chondrocytes express a number of osteogenic transcription factors such as Runx2 and osterix which are regulate by β-catenin, we decided to delete the β-catenin gene ctnnb1 specifically in hypertrophic chondrocytes by mating our BACCol10Cre deleter mice with a floxed β-ctnnb1 fl/fl mouse, kindly provided by Dr.R.Kemler, MPI, Freiburg. Interestingly, histological analysis of the skeleton of newborns of the F2 generation revealed that homozygous BACCol10Cre; ctnnb1 fl/fl mice lacked almost completely trabecular bone in the primary spongiosa. The aim this proposal was to clarify whether this deficiency was due to impaired osteoblast differentiation and bone formation, or due to enhanced bone resorption. Using immunohistological and molecular biological tools, we demonstrated that the lack of trabecular bone was caused by enhanced osteoclast activity caused by increased rankl expression in βcatenin deficient hypertrophic chondrocytes. These results indicate that β-catenin levels in hypertrophic chondrocytes play a key role in controlling osteoclast activity and trabecular bone formation. The origin of trabecular osteoblasts inside long bones has long been debated. According to the general understanding, the chondrocyte lineage terminates with elimination of late hypertrophic cells by apoptosis in the growth plate. Several recent studies, however, have provided experimental evidence that hypertrophic chondrocytes may also undergo transdifferentiation to osteoblasts. In order to answer this question, we performed genetic lineage tracing experiments using BACCol10Cre;R26R and BACCol10Cre;R26YFP reporter mice to follow the cell fate of hypertrophic chondrocytes during endochondral ossification. The results of this study provide evidence for a second pool of trabecular osteoblasts in the spongiosa originating from hypertrophic chondrocytes. Analyzing the skeleton of newborn BACCol10Cre; R26R mice revealed X-gal positive cells with typical epitheloid, osteoblastic morphology lining trabecular bone spicules in the primary spongiosa of long bones, ribs, and vertebrae. In 3 -4 week old mice, about 40 % of all osteocytes in cortical bone were also X-gal positive. Immunofluorescence analysis of BACCol10Cre; ROSA YFP mice for osteoblast markers and YFP revealed that in long bones at E18.5 about 30% of all Osterix (Osx)-positive cells associated with subchondral bone trabeculae and endosteum were YFP positive. This confirms the notion that a hypertrophic chondrocytederived progeny of cells differentiates into trabecular osteoblasts and challenges the common view that all terminal hypertrophic chondrocytes in the growth plate are eliminated by apoptosis. In searching for a cellular mechanism of chondrocyte-osteoblast transition, we identified by confocal microscopy a novel small YFP + Osx + cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseus junction. We propose that this cell type termed CDOP (chondrocyte derived osteoprogenitor) cell marks the transitional state between late hypertrophic chondrocytes and chondrocyte-derived trabecular osteoblasts. When isolated from growth plates by fractional enzymatic digestion, CDOP cells expressed bone typical genes as well as stem cell markers. These findings revise current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilagebone transition process in the growth plate. Our studies on chondrocyte transdifferentiation were supplemented and confirmed in a collaborative study with Drs. Xin Zhou and B. de Crombrugghe, MDAnderson Cancer Center, Houston, using a tamoxifen- inducible Aggrecan-Cre mouse in addition to the non-inducible BACoCol10Cre mouse. While the specificity the BACCol10Cre mouse has frequently been questioned, although we have documented the restriction of Cre expression to hypertrophic chondrocytes in several places by in situ hybridization and RT-PCR, the lineage tracing system using TAM-inducible Agcn-Cre expression allowed the follow the cell fate of reporter-gene expressing chondrocytes more convincingly after various pulse-chase periods, starting at stages before appearance of the first trabecular osteoblasts in the spongiosa. Furthermore, X.Zhou confirmed the capacity of chondrocytes to convert into osteoblasts during endochondral bone formation in experimentally induce fracture callus healing.
Publications
- Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status. J Bone Miner Res. 2010 Jun;25(6):1267-81
Belluoccio D, Etich J, Rosenbaum S, Frie C, Grskovic I, Stermann J, Ehlen H, Vogel S, Zaucke F, von der Mark K, Bateman JF, Brachvogel B
- Ucma is not necessary for normal development of the mouse skeleton. Bone. 2012 Mar;50(3):670-80
Eitzinger N, Surmann-Schmitt C, Bösl M, Schett G, Engelke K, Hess A, von der Mark K, Stock M
(See online at https://doi.org/10.1016/j.bone.2011.11.017) - Deletion of beta catenin in hypertrophic growth plate chondrocytes impairs trabecular bone formation. Bone. 2013 Jul;55(1):102-12
Golovchenko S, Hattori T, Hartmann C, Gebhardt M, Gebhard S, Hess A, Pausch F, Schlund B, von der Mark K
(See online at https://doi.org/10.1016/j.bone.2013.03.019) - Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice. PLOS Genetics 10 (12) (2014) e1004820
Zhou Xin, K. , von der Mark,K., Henry S., Norton,W, Adams,H., deCrombrugghe, B.
(See online at https://doi.org/10.1371/journal.pgen.1004820) - Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage. Biology Open, 2015, 4: 608-621
Park J, Gebhardt M, Svitlana Golovchenko, Francesc Perez Branguli, Takako Hattori, Christine Hartmann, Xin Zhou, Benoit deCrombrugghe, Michael Stock, Holm Schneider, Klaus von der Mark
(See online at https://doi.org/10.1242/bio.201411031)
