Multiple osteochondromas (MO) syndrome is an autosomal dominant disease characterized by the formation of benign bone tumors (osteochondromas or exostoses) at the epiphyses of endochondral bones. Histologically, osteochondromas are peduncolate or sessile cartilage capped bony projections, usually growing until the end of puberty (Hennekam, 1991). MO has been associated to loss of function mutations in EXT genes, with a more severe phenotype associated to EXT1 mutations rather than EXT2 (Lüdecke et al., 1995; Porter et al., 2004; Wuyts et al., 1995). EXT1 and EXT2 encode for glycosyltransferases responsible for elongation of Heparan Sulfate (HS) chain. HS are polysaccharides decorating the plasma membrane of all animal cells and a major component of the extracellular matrix. They play a key role in mediating the activation of different signaling pathways, like FGF and VEGF, and regulation spatial distribution of morphogens like Ihh and BMPs. Two fundamental questions remain open about osteochondromas development. 1. Which is the cellular origin of osteochondromas? It has been proposed that the first event that leads to development of the disease could happen either in a physeal chondrocyte or in a cell of osteoblastic lineage from the adjacent perichondrium. 2. The molecular mechanism responsible for osteochondromas development is still unclear. As MO is an autosomal dominant syndrome it might be either that haploinsufficiency could be sufficient to induce misregulation of HS-dependent signaling pathways, or that loss of heterozygosity is necessary for exostoses to develop. In this study different mouse models were analyzed to find the molecular function of HS during osteochondromas development. In contrast to Ext1-/- mice which die during gastrulation Ext1Gt/Gt mice survive until midgestation. Ext1Gt/Gt produce about 20% of HS content compared to wild type. Previous data from these mice revealed a general delay in chondrocyte differentiation due to the reduced amount of HS (Koziel et al., 2004). In additional to an effect on chondrogenesis, loss of HS leads to failure in vascular development. In cell culture experiments I identified a defect in the production/release of VEGF-A protein, which regulates the correct activation of vascularization. Correlated to the perturbed vascularization, osteoclast invasion and bone formation was also delayed in Ext1Gt/Gt mutant. As Ext1Gt/Gt die at embryonic stage of development, rendering prohibitive to study osteochondromas development I analyzed a conditional mouse model for Ext1 (Col2-rtTA-Cre;Ext1e2fl/e2fl). In these mice, allelic recombination leads to disruption of Ext1 function, similarly to Ext1-/- and Ext2-/- mice. Col2-rtTA-Cre;Ext1e2fl/e2fl mice developed osteochondromas at endochondral bones, both in the axial and appendicular skeleton. Femurs of Col2-rtTA-Cre;Ext1e2fl/e2fl mutant mice were analyzed at different stages (between 4 and 8 weeks old) to observe osteochondromas development at the side of the growth plate. At early stages, in 4 to 6 weeks old mice, osteochondromas are mostly chondrogenic, developing from a small chondrocyte cluster into a more complex structure, in which chondrocytes have differentiated into different cell population, resembling the organization of the growth plate. Cells from osteochondroma tissue were isolated by laser microdissection for genetic analyses and the HS amount was analyzed by immunohistochemistry. In addition Col2-rtTA-Cre;Ext1e2fl/e2fl mice show signs of early osteoarthritis, which lead to enzymatic proteoglycan loss as shown by immunohistochemistry. In conclusion my data support the hypothesis that MO syndrome is generated by Loss of heterozygosity in a distal chondrocyte of the growth plate, which proliferates and differentiates into a structure that resemble the growth plate organization. Moreover, HS regulate vascularization of the cartilage by modification of VEGF-A protein production or release, which influences in turn osteoblast differentiation.