Using our quantitative imaging assay, we found that over the five last nuclear divisions, the level of MAPK phosphorylation is amplified at the poles and attenuated in the rest of the embryo (). Nuclear divisions lead to an exponential increase in the number of nuclei per unit volume; hence, there is a clear correlation between the spatial pattern of MAPK activation and nuclear density. The observed dynamics are consistent with a model whereby dpERK is trapped by the nuclei: an increase in the nuclear density increases the trapping of phosphorylated MAPK and prevents its diffusion from the poles towards the middle of embryo ().
Figure 4 (A) Dynamics of the wild-type the MAPK phosphorylation gradient: over the time course of the five last nuclear divisions, the level of MAPK phosphorylation is amplified at both poles and attenuated in the rest of the embryo; reproduced from Coppey et (more ...)
This model is based on the cell biological and biochemical studies that have established that phosphorylated MAPK rapidly translocates to the nucleus, which can also serve as a compartment of MAPK dephosphorylation.19–22
In combination with the progressive increase in nuclear density, these processes can amplify the dpERK levels at the poles and attenuate them in the rest of the embryo.23
Our subsequent experiments provided clear support for this model. In particular, we found pronounced disruptions in the spatial pattern of MAPK phosphorylation in embryos with defects in the spatial distribution of nuclei.17
Thus, syncytial nuclei not only sense the local level of MAPK phosphorylation that has been established by the upstream steps of MAPK activation, but actively control the spatial pattern of MAPK phosphorylation in the early embryo.
There is a strong biophysical analogy between this new intracellular diffusion-trapping module that controls the spatial distribution of phos-phorylated MAPK and the previously identified diffusion-trapping system that controls the spatial distribution of the extracellular ligand that activates Torso. Elegant genetic experiments by Casanova and Struhl established that removal of Torso receptors from the poles generates ectopic terminal structures in the middle of the embryo.24
Based on this, they proposed that uniformly expressed Torso both transduces the signal provided by Trunk and limits its diffusive spread. Indeed, ligand binding to Torso activates receptor tyrosine kinase signaling, but also leads to receptor-mediated ligand internalization.25
Casanova and Struhl have called this type of spatial regulation of receptor activation “ligand trapping”, an effect that has been subsequently identified in a large number of other patterning systems, both in Drosophila and other organisms.26,27
Since the spatial pattern of Torso occupancy on the plasma membrane provides an input that activates the MAPK cascade inside the embryo, it appears that the spatial pattern of MAPK activation is established by two sequentially acting diffusion-trapping systems. In the extracellular compartment, the Torso receptors limit the spatial spread of the Trunk ligand. Inside the embryo, nuclei limit the spread of diffusible dpERK. To assess the relative contributions of the extracellular and intracellular diffusion-trapping modules to establishing the final pattern of MAPK activation, we used a biophysical model for a cascade of diffusion trapping systems. A detailed biochemical model, like that discussed in the Introduction, contains a large number of species. Since only one of these species (dpERK) can be followed in the terminal system, we need the simplest possible model that is consistent with wild-type dynamics and can be used to predict the effects of genetic perturbations.