The Bcd Morphogenetic Concentration Gradient is Formed by Diffusion

Abstract

The morphogenic protein Bicoid is an essential activator of cellular differentiation and pattern formation in the fruit fly Drosophila melanogaster. It forms an exponential concentration gradient along the anterior-posterior axis of fly embryo and acts as a transcription factor that activates a cascade of target genes. The currently accepted model, known as the Synthesis, Diffusion & Degradation (SDD) model, assumes that the protein spreads across the embryo by simple diffusion, as was initially proposed by Francis Crick in 1970. This Model, however, has been called into question by several recent studies. To test the validity of the SDD model, we studied the localization and dynamics of a Bcd-EGFP fusion protein in live embryos using complementary fluorescence techniques: Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Correlation spectroscopy (FCS). We observed that Bcd-EGFP concentration decayed exponentially along the anterior-posterior axis of the embryo with a characteristic length of ∼100 μm, as previously reported by other groups, and we estimated the absolute nuclear and cytoplasmic Bcd-EGFP concentrations at the anterior pole to be 120 nM and 15 nM, respectively. In the cytoplasm, we found that the overwhelming majority of Bcd molecules were undergoing diffusive motion, with an average diffusion coefficient D∼5 μm2/s. This is an important result, because it provides the first experimental evidence that the mobility of cytoplasmic Bcd is high enough to support the establishment of a concentration gradient across the embryo before the beginning of cellularization, as envisioned in the SDD model. We also observed that 35% of the nuclear Bcd population was engaged in transient binding to immobile structures, with an average binding time τB=1/koff=120 ms, a result consistent with the fact that Bcd functions as a transcription factor.