Toward an understanding of the epithelial requirement for osteogenesis in scleral mesenchyme of the embryonic chick

Explants of scleral tissue from chick embryos of H.H. stage 29-36 (6-10 days of incubation) were used to determine if the epithelial-mesenchymal interaction which initiates scleral bone formation is cell contact, extracellular matrix, or diffusion mediated. Transfilter tissue recombinations, in which explanted interacting tissues are associated across interposing Nuclepore filters of various pore sizes and thicknesses, were performed with scleral mesenchyme and epithelium. When filters with pore sizes which would allow the passage of cell processes and diffusible substances were used, osteogenesis was initiated in the scleral mesenchyme. When cell processes were blocked with thicker filters or smaller pore sizes, bone formation still occurred, indicating that a diffusible substance mediates this tissue interaction. Further support for a diffusion-mediated interaction came from transfilter experiments using dialysis membranes to discriminate the size of the molecule(s), and Millipore filters to determine the distance over which these molecules travel. These experiments revealed that the scleral epithelial diffusible factor has a molecular weight of between 3500 and 6000 daltons, and acts over distances between 150 and 300 microns.