INVESTIGATION OF DYNAMICS OF MIDKINE AND PLEIOTROPHIN DURING ZEBRAFISH DEVELOPMENT USING FLUORESCENCE CORRELATION SPECTROSCOPY

Abstract

Midkine and pleiotrophin are low molecular weight secreted proteins with a high degree of structural and functional similarity constituting a family of heparin binding growth factors. Their roles during embryogenesis, particularly neural development has been widely studied. In adults however, expression is induced in several types of cancer and in tissue injuries. Zebrafish have two midkine genes (mdka and mdkb) and one pleiotrophin gene (ptn). In at least the first 24 hours of development, there is significant mRNA expression of mdka, mdkb and ptn with a maternal contribution suggesting their importance as signaling molecules for initiation of gastrulation. In addition, overexpression of a fluorescently labelled Ptn displayed a descending gradient of fluorescence intensity from the point of injection, representing the graded concentrations of Ptn. The current study aims at understanding the factors involved in the formation of concentration gradients for Mdka, Mdkb and Ptn. This was approached by studying the dynamics of free diffusion in the extracellular space and membrane restricted diffusion on the plasma membrane of individual cells in the embryo by employing fluorescence correlation spectroscopy (FCS) implemented on confocal microscope and selective plane illumination microscopy (SPIM). First both systems were calibrated with fluorescent proteins in various locations of the embryo (intracellular, membrane bound and extracellular) to determine the correct fit models expected in the various cases and ensure consistency between the measurements. Second, the distribution of Mdka, Mdkb and Ptn was mapped and the dynamics corresponding to membrane and extracellular diffusion were determined. Next, the role of heparan sulphate and chondroitin sulphate proteoglycans as concentration binders for gradient formation were investigated. Lastly, the interaction of ligands with a common receptor Rptpz1b was examined using fluorescence cross correlation spectroscopy (FCCS) and the effects of membrane localization in dependence of Rptpz1b was investigated. Overall, the results from this study constitute an important step in understanding the mechanisms involved in the formation of concentration gradient for Mdka, Mdkb and Ptn that influence gastrulation in zebrafish embryos.