A Role for SCL in Developing Bone? the SCL +19 Core Enhancer Targets Expression to Cells of Developing Bone, Osteocytes, Chondrocytes and Bone Lining Cells.

The transcription factor SCL/TAL1 is essential for haematopoiesis and vascular development in the embryo. It is also expressed in the developing skeletal and central nervous systems, but lethality of SCL null mice has precluded detailed analysis of the role of this gene in these tissues.We have previously demonstrated that the SCL +18/19 enhancer directs reporter gene expression to haemangioblasts, endothelium and haematopoietic progenitors. Using human placental alkaline phosphatase as a reporter gene, we now report that the +19 core enhancer also directs expression to osteocytes, articular chondrocytes and bone lining cells in adult mice. In E14.5 embryos, the transgene is expressed in cells within the cartilaginous template of future bone and in the perichondrium. The pattern of expression of the transgene resembles that of endogenous SCL RNA and protein expression in age matched embryos. At E16.5, transgene-positive cells are seen within ossification centres. Activity of the enhancer during osteogenesis was observed both in bones formed through intra-membranous (e.g. cranial vault) and endochondral ossification (limbs and axial skeleton). We found that the murine pre-osteoblast cell line, MC3T3, expresses endogenous SCL. Stable transfection of these cells with luciferase reporter constructs that include the +19 core enhancer produce a 5-fold increase in luciferase activity. There is a progressive reduction in SCL RNA expression in these cells during osteogenic differentiation. This is consistent with a previous report that expression of SCL protein is down-regulated in skeletal tissues of more advanced stage embryos, and with our finding that the MLO-Y4 cell line, which phenotypically and functionally resembles mature osteocytes, does not express SCL. We addressed the issue of SCL function in bone formation using in-vitro differentiation of embryonic stem (ES) cells. Wild type and SCL^−/− J1 ES cells did not differ in their ability to form bone nodules. These data demonstrate that SCL is not required for bone specification, but do not exclude a role for SCL in regulating bone formation in vivo. The SCL +19 core enhancer directs expression to haematopoietic progenitors and endothelium together with their putative precursors, haematopoietic endothelium and haemangioblasts. Our demonstration that the +19 core enhancer also targets expression to osteogenic cells is consistent with recent data suggesting that haematopoietic and osteogenic cells share a common precursor (Olmsted-Davis, EA et al PNAS 2003, Dominici M et al PNAS 2004) and that osteoblastic bone lining cells are intimately associated with haematopoietic stem cells (Calvi LM et al Nature 2003; Zhang J et al Nature 2003).