molecular mechanisms of skeletal development and blood cell differentiation.
Description of Research
Hematopoietic disorders such as bone marrow failure and immune dysfunction (e.g. aplastic anemia, lymphopenia, rheumatoid arthritis), as well as certain cancers (e.g. leukemia, lymphoma, myeloma), may involve a link between skeletal formation and establishment of the marrow. Dr. Jacenko's laboratory is exploring a mechanistic basis for this skeleto-hematopoietic link by addressing how the marrow microenvironment that is needed for blood cell differentiation may be established by the replacement of cartilage by bone and marrow during skeletal formation by endochondral ossification (EO).
This novel and previously unforeseen hypothesis linking skeletal development and blood cell differentiation stems from the generation of several transgenic mouse models in Dr. Jacenko’s lab where either the cartilage structure (via mutations in collagen X), or cell cycle (via targeted expression of c-myc) has been altered in hypertrophic cartilage (the cartilage zone that is replaced by bone and marrow in EO). The resultant murine phenotypes include both skeletal deformities and altered blood cell differentiation; all skeletal tissues formed by EO are affected, and hematopoietic defects manifest in marrows as either hypoplasia resembling aplastic anemia (e.g. collagen X mice), or hematopoietic hyperproliferation resembling chronic myelogenous leukemia (c-myc mice). One goal of this laboratory is to provide a mechanism for this skeleto-hematopoietic link through analysis of mouse models with altered skeletal development. The multidisciplinary approach of the laboratory relies heavily on transgenesis, and involves molecular biology, biochemistry, immunology, hematology and histology. Short-term goals are summarized under potential projects.
1. Identification of defective cellular component in the marrow environment of mice with disrupted collagen X function by co-culture assays. We anticipate an inappropriate hematopoietic environment in the marrow arising as a consequence of the disruption of collagen X function. Studies will ascertain if the marrow defects originate in the marrow stroma, or involve blood cell progenitors by co-culturing hematopoietic stem cells with stromal cells, hypertrophic chondrocytes, and osteoblasts in liquid and methylcellulose.
2. Bone marrow transplants. This in vivo approach will test the hypothesis that the defect in collagen X mice resides in the stroma.
3. Identification of proteoglycans (PGs)/glycosaminoglycans (GAGs) that are altered in the growth plate/bone marrow junction, and testing if these molecules bind collagen X. Our data indicate a reduction of heparan sulfate (HS)-containing PG(s) in hypertrophic cartilage of the collagen X mice. HSPGs have been implicated in orchestrating hematopoietic niches by sequestering cytokines and juxtaposing them with hematopoietic stem cells and the stroma. We propose that the collagen X network is stabilized by associations with these molecules, and that its disruption leads to GAG/PG decompartmentalization in the growth plate-marrow junction. Studies will include: a) localization of selected PGs in the chondro-osseous junction by immunohistochemistry; b) co-localization of affected molecule(s) with collagen X by immunoelectron microscopy; c) determination if these PGs bind collagen X by affinity coelectrophoresis (ACE); and d) co-culture of hematopoietic stem cells with hypertrophic cartilage and collagen X/PG/GAG-containing matrices, +/- modulators of GAG biosynthesis.
4. Examination of the relationship between the severity of the skeleto-hematopoietic disease phenotype and abnormalities in cytokine expression. We propose that in mice with disrupted collagen X function, dysregulation of cytokine production and localization contributes to the skeleto-hematopoietic defects and influences the susceptibility of the affected animals to disease. Studies will include: a) identification of the defect in the animals’ ability to initiate and regulate an immune response by endotoxin challenge; b) characterization via blot arrays which cytokines are dysregulated; c) identification of tissue source with aberrant cytokine production by RT-PCR/lightcycler; d) induction/blocking of disease phenotype by injection of cytokines or neutralizing antibodies.
5. Testing the hypothesis that other murine disease phenotypes characterized by altered hypertrophic cartilage function are also associated with hematopoietic defects caused by aberrant cytokine homeostasis. We speculate that the disease phenotypes seen in several existing mouse models with skeletal abnormalities are identical to those of the collagen X mice, implying the involvement of the same molecular cascade of events. Studies will involve a) characterization of these models by selected assays used for the collagen X mice, with emphasis on PGs and cytokines; b) gene array profiling of growth plates and marrows. Future goals may include genetic manipulation in mice of key molecular players altered in each set of these models.
6. Characterization of skeleto-hematopoietic changes in the c-myc transgenic mice. Our recently generated mice express c-myc in the post-mitotic hypertrophic cartilage, and as a result, develop skeletal defects and marrow hyperproliferation. Studies would address the relationship between cartilage cell proliferation/apoptosis, and transition from hypertrophic cartilage to bone and marrow using similar assays to those outlines above.
***Lab rotation projects would be designed around the interests of the student, and may include a part of any of the above possibilities***
Elizabeth Sweeney, Ph.D., Postdcotoral Fellow
Projects involve: Hematooietic co-culure assays and bone marrow transplants to identify defective cellular component that leads to altered hematopoiesis in the collagen X Tg and null mice.
Kathryn Rodgers, Ph.D., Postdoctoral Fellow
Projects involve: Analysis of transgenic mice with disrupted perlecan function (mouse model for Schwartz-Jampel syndrome).
Doug Roberts, M.A. Lab Manager
Projects involve: 1. Analysis of aplastic or hyperplastic marrows and lymphatic organs from collagen X transgenic or c-myc mice by flow cytometry, immunohistochemistry, and RT-PCR; 2. Co-culture assays of hematopoietic stem cells, stromal cells, hypertrophic chondrocytes, and osteoblasts from control and mutant mice to identify defective cellular component in the marrow environment of the collagen X mice.
Krystal Watkins, B.S., Technician and Master Student
Projects involve: 1. Genotyping, analyzing, and maintaining collagen X transgenic mouse colony; 2. flow cytometric analysis of hematopoietic defects in collagen X and c-myc mice.
Christine Credidio, B.S., Technician and Nursing Student
Projects involve: 1. Analysis of cytokine status in collagen X mice; 2. Immune function challenge assays by infection of transgenic mice with parasites;
Kevin Kedra, Independent study
Projects involve: RT-PCR and light cycler analysis of cytokine levels in tissues from collagen X mice.
Jacenko, O., Campbell, M.R., Roberts, D.W. Linking endochondral ossification to hematopoiesis The Growth Plate : 159-73, 2002.Jacenko, O., Roberts, D.W., Campbell, M.R., McManus, P.M., Gress, C.J., and Tao, Z. Linking hematopoiesis to endochondral skeletogenesis through analysis of mice transgenic for collagen X Am. J. Pathol. : 2019-34, 2002.Jacenko, O., Chan, D., Franklin, A., Ito, S., Bateman, J., Campbell, M.R. A dominant interference collagen X mutation disrupts hypertrophic chondrocyte pericellular matrix, and glycosaminoglycan and proteoglycan distribution in transgenic mice Am. J. Pathol. 159: 2257-69, 2001.Gress, C.J. and Jacenko, O. Growth plate compressions and altered hematopoiesis in collagen X null mice J. Cell Biol. 149: 983-93, 2000.Chan, D. and Jacenko, O. Phenotypic and biochemical consequences of collagen X mutations in mice and humans Matrix Biol 17: 169-84, 1998.