Quantifying the Bicoid Morphogen Gradient in Living Fly Embryos

Abstract

In multicellular organisms, patterns of gene expression are established in response to gradients of signaling molecules. During fl y development in early Drosophila embryos, the Bicoid (Bcd) morphogen gradient is established within the fi rst hour after fertilization. Bcd acts as a transcription factor, initiating the expression of a cascade of genes that determine the segmentation pattern of the embryo, which serves as a blueprint for the future adult organism. A robust understanding of the mechanisms that govern this segmentation cascade is still lacking, and a new generation of quantitative measurements of the spatiotemporal concentration dynamics of the individual players in this cascade is necessary for further progress. Here we describe a series of methods that represent the beginning of the use of Bcd as a quanti fi cation example. We describe the generation of a transgenic fl y line expressing a Bcd-enhanced green fl uorescent protein fusion protein. Using two-photon microscopy, we analyze the Bcd concentration dynamics and measure absolute Bcd expression levels in living fl y embryos. These experiments have proven to be fruitful, generating new insights into the mechanisms that lead to the establishment and readout of the Bcd gradient. Generalization of these methods to other genes in the Drosophila segmentation cascade is straightforward and should further our understanding of the early patterning processes and the architecture of the underlying genetic network structure.