The retinoblastoma protein (pRb) is a tumor suppressor that is found mutated or dysregulated in most human cancers.  It is most commonly directly affected in retinoblastoma and osteosarcoma.  To appreciate why direct mutation of pRb is important in cancer development in certain tissues, understanding its role in normal development is essential.  Through various studies, the sponsor's lab found that pRb is necessary for lineage commitment of osteoblasts and that there are more bipotent progenitors in the calvarium of animals lacking pRb, which could contribute to malignant phenotypes.  The lab also found more recently that Indian hedgehog (Ihh) is downregulated in Rb1-/- calvarial cells during differentiation, implicating this factor as a potential mediator of pRb's function in bone development and cancer.  The purpose of this project is to determine the role of Ihh in lineage commitment of osteoblasts as well as the possible roles for Ihh as a mediator of pRb function in osteosarcoma.  Using both in vitro and in vivo tools, the bipotency/stemness of calvarial cells lacking Ihh will be assessed to determine if Ihh is necessary for osteoblast commitment.  This will be accomplished by first differentiating the cells into adipocytes and osteoblasts to determine if Ihh-/- calvarial cells are bipotent.  Stemness will be assessed by serially differentiating these cells, that is, differentiating with osteogenic media until full differentiation has been achieved, then replating and differentiating again.  In addition to these studies, the effect of adding Ihh to pRb- deficient calvarial cells during differentiation will be assessed utilizing recombinant Ihh (rIhh) and the adipoctye/osteoblast differentiation protocols.  It is expected that loss of Ihh will increase the pool of bipotent progenitor cells in the calvariu (similar to pRb loss) and that adding rIhh to Rb1-/- cultures will impair their bipotent capabilitis.  The effect of Ihh loss in an osteosarcoma model will also be assessed to determine a role for Ihh in bone tumorigenesis.  To this end, animals will be mated to carry conditional alleles of Trp53 and Ihh, after which both will be deleted in osteoblasts by introducing an Osterix-Cre allele into this population.  Animals will be monitored for osteosarcoma formation and tumor cells will be isolated and characterized to assess their adipocyte and osteoblast differentiation abilities.  In addition, the loss of both p53 and Ihh in osteoblast commitment and differentiation will be assessed using calvarial cells from E18.5 embryos and differentiating these cells with osteogenic media.  It is expected that Ihh loss will contribute to transformation of osteoblasts and promote tumorigenesis.  PUBLIC HEALTH RELEVANCE: This purpose of this project is to understand how a key factor in bone development contributes to this process as well as tumor formation.  As the incidence of cancer rises worldwide, determining ways in which various proteins interact to aid in tumorigenesis is necessary to work toward treating and curing the disease.  Although this project focuses on osteosarcoma, it will also contribute to the broader understanding of how developmental proteins affect cancer initiation and growth.