Postnatal bone and bone marrow (BM) share a specialized vascular networks in which Skeletal Stem Cells (SSCs) reside as perivascular cells. These cells generate in vivo bone, cartilage, adipocytes and BM stroma. Although these lineages coincide with the assayable differentiation potential of SSCs, they emerge at different times during skeletal development. For example, both in mice and humans, BM adipocytes (BMAs) develop postnatally and fill completely specific skeletal sites at an early postnatal age.
Fibrous Dysplasia (FD) is a crippling and therapeutically orphan postnatal skeletal disease caused by postzygotic activating mutations of the Gsa gene. The defining features of FD pathology include BM fibrosis, osteomalacia, and osteolysis, that make the affected bone soft and prompt to fractures and deformities.
Key questions as to the natural history of the FD lesions, for example why and from which lineage they develop postnatally, have required modeling of the disease at organ/organism level and led to the development of specific transgenic models. In this context, mice with constitutive and ubiquitous (i.e. in all SSC-derived lineages) expression of GsaR201C, one of the mutations that cause FD in humans, replicate faithfully the human disease. However, if the same mutation is specifically targeted to osteoblasts, mice develop a high bone mass phenotype but not FD, indicating that cells other than osteoblasts are the key to the development of FD lesions.
Since, both in mice with constitutive expression of GsaR201C and humans, BMAs and FD lesions occur postnatally and earliest and most prevalent FD involved sites are those normally filled with adipocytes, we generated through a Cre-Lox approach two mouse lines in which the Cre expression was driven by Adipocyte Protein-2 or AdipoQ promoters, genes expressed in early and mature adipocytes respectively. The analysis of these mice will contribute to understand the pathogenetic role of BMAs in the development of FD.
During the last two decades, the use of site-specific recombinases have greatly improve the ability to manipulate cells and gene expression. These site-specific recombinases bind to and recombine specific sequences of DNA, allowing researchers to heritably label cells, conditionally inactivate or activate genes, and even ablate cells based on their gene expression. This methodology will be used in this research proposal the aim of which is to investigate the role of bone marrow adipocytes (BMAs) in the development of Fibrous Dysplasia (FD). To do this, previously generated Rosa26LSL-Gs¿R201C mice were crossed with mice expressing Cre-recombinase under the control of two adipose tissue specific gene promoters. This crossing allows to activate the expression of GsaR201C in cell of the adipose tissue lineage in the offspring. The chosen adipocyte gene promoters, the Fatty acid binding protein-4 (Fabp4, also known as Adipocyte Protein) and Adipo Q (also known as Adiponectin), are highly expressed during early adipocyte differentiation and in well differentiated mature adipocytes respectively. The phenotypic characterization of these mice should allow to evaluate the role of the selective expression of the Gsa201C in the early and mature BMAs and, as a consequence, to probe a mechanism for the development of FD lesions entirely ignored to date. In fact, a direct involvement of BMAs in the development of FD lesions has never been suspected. If proved, this mechanism should validate our hypothesis according to which FD lesions are the consequences of a mutation-dependent fate shift of BMAs into abnormal (fibrodysplastic) osteoblasts according to the following sequence of events: a) Gsa¿mutation leads to the intra-cellular increase of cAMP in adipocyte; b) the effect of intra-cellular increase of cAMP in adipocytes is their brownization; c) in the bone-BM microenvironment, brownization of adipocytes is associated to their conversion into abnormal (fibrodysplastic) osteogenic cells (precursors and then mature osteoblasts) that replace the yellow marrow by producing the FD tissue with concomitant disappearance of BMAs and haematopoiesis.
More in general, the analysis of these mice could also contribute to expand the relationship between the adipogenic and the osteogenic lineages (1-5) that could rely not only on the the existence, as actually well established in the literature (6-10), of a common bipotent progenitor (the Skeletal Stem Cell) but also on the plastic capability to convert into each other, even when terminally differentiated and mature.
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