Morphogen-mediated patterning is a highly dynamic developmental process. To obtain an accurate understanding of morphogen gradients, biophysical parameters such as protein diffusivities must be quantified
in vivo. The dorsal-ventral (DV) patterning of early Drosophilaembryos by the NF-B homolog Dorsal (Dl) is an excellent system for understanding morphogen gradient formation. Dl gradient formation is controlled by the inhibitor Cactus/IB (Cact), which regulates the nuclear import and diffusion of Dl protein. However, quantitative measurements of spatiotemporal Dl movement are currently lacking. Here, we use scanning fluorescence correlation spectroscopy to quantify the mobility of Dl. We find that the diffusivity of Dl varies along the DV axis, with lowest diffusivities on the ventral side, and the DV asymmetry in diffusivity is exclusive to the nuclei. Moreover, we also observe that nuclear export rates are lower in the ventral and lateral regions of the embryo. Both cross correlation spectroscopy measurements and a computational model of Dl/DNA binding suggest that DNA binding of Dl, which is more prevalent on the ventral side of the embryo, is correlated to a lower diffusivity and nuclear export rate. We propose that the variation in Dl/DNA binding along the DV axis is dependent on Cact binding Dl, which prevents Dl from binding DNA in dorsal and lateral regions of the embryo. Thus, our results highlight the complexity of morphogen gradient dynamics and the need for quantitative measurements of biophysical interactions in such systems.