This proposal addresses whether endochondral ossification (EO), involving hypertrophic cartilage replacement by bone and marrow, may establish the marrow stromal environment prerequisite for blood cell differentiation. This hypothesis stems from our analyses of transgenic (Tg) and null mice for collagen X, a matrix protein expressed in hypertrophic cartilage prior to EO. Resultant murine skeleto-hematopoietic defects include growth plate compressions, osteopenia, and marrow aplasia; the latter results in lethality or compromised immunity. Our data maintain that collagen X provides a structural framework reinforcing growth plates, the disruption of which alters subsequent bone and marrow formation. This proposal will address if structural defects in growth plates alter the marrow stroma and subsequent hematopoiesis by l) re-evaluating collagen X or transgene product expression in extra-skeletal organs via RT-PCR, northern blot, in situ hybridization, immunohistochemistry, and transgenesis (Aim 1). 2) Flow cytometry and immunohistochemistry will identify affected blood cell lineages and correlate the temporal onset of these defects to the skeletal changes.
(Aim 2). 3) Marrow analysis will involve flow cytometry, immunohistochemistry and cell culture; 4) marrow transplantation will establish if altered hematopoieis stems from defects in the Stroma or in blood progenitors; and 5) co-culture assays will identify the defective cellular component in the marrow milieu (Aim 3). 6) The animal's immune response will be tested by Toxoplasma gondii infections, and the phenotypic variability seen in both the Tg and KO mice will be assessed by 7) peripheral blood and marrow analysis for sepsis; 8) ELIZA for IL-12, IFN-g, and IL-10 serum levels; and 9) injections of cytokines and neutralizing antibodies will address the cause of marrow aplasia (Aim 4). 10) The involvement of a modifier gene will be tested by inbreeding mice into uniform backgrounds (Aim 4). Results will provide insights into hypertrophic cartilage and collagen X function in skeleto- hematopoietic development. Data may also establish mechanistic links between endochondral skeletogenesis and hematopoiesis, and provide clues into the pathogenesis of disorders with skeletal, immunologic, or metastatic involvement.
