Abstract
Morphogen-mediated patterning is a highly dynamic developmental process. To obtain an accurate understanding of morphogen gradient formation and downstream gene expression, biophysical parameters such as protein mobilities must be quantified in vivo. The dorsal-ventral (DV) patterning of early Drosophila embryos by the NF-κB homolog Dorsal (Dl) is an excellent system for understanding morphogen gradient formation. Dl gradient formation is controlled by the inhibitor Cactus/IκB (Cact), which regulates the nuclear import and diffusion of Dl protein. However, quantitative measurements of Dl mobility and binding are currently lacking. Here, we use scanning fluorescence correlation spectroscopy to quantify the mobility of GFP-tagged Dl. We find that the DNA binding of Dl-GFP, which affects its mobility, varies along the DV axis, with highest DNA binding on the ventral side. Moreover, we also observe that the time scale for Dl-GFP to exit the nucleus is longer in the ventral and lateral regions of the embryo, which is consistent with stronger DNA binding. Using analysis of mutant alleles of dl tagged with GFP, we conclude that Dl-GFP/Cact interactions in the nuclei are responsible for the variation in Dl-GFP/DNA binding along the DV axis, which impacts our understanding of the spatial range of the Dl gradient and the robustness and precision of downstream gene expression. Thus, our results highlight the complexity of morphogen gradient dynamics and the ability of quantitative measurements of biophysical interactions to drive biological discovery.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We generated four new Dorsal-GFP constructs: one control construct, one that reduces Dorsal/DNA binding, one that reduces phosphorylation of Dorsal by Toll, and one that reduces Dorsal/Cactus binding. Analysis of the first two validates our methods for measuring DNA binding. The remaining two, when compared to the control, resulted in rejection of the Toll hypothesis, but not of the Cactus hypothesis. Additionally, we re-plotted some of the results as continuous vs the nuclear-to-cytoplasmic ratio (NCR) of Dl-GFP intensity, rather than binning the results into three bins of NCR.
