Abstract
A complex system of multiple signaling molecules often produce differential gene expression patterns in animal embryos. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce Otx expression in the neural lineage at the 32-cell stage. However, it has not been determined whether differential inputs of all of these signaling pathways are really necessary. It is possible that differential activation of one of these signaling pathways is sufficient and the remaining signaling pathways are activated in all cells at similar levels. To address this question, we developed a parameter-free method for determining a Boolean function for Otx expression in the present study. We treated activities of signaling pathways as Boolean values, and we also took all possible patterns of signaling gradients into consideration. We successfully determined a Boolean function that explains Otx expression in the animal hemisphere of wild-type and morphant embryos at the 32-cell stage. This Boolean function was not inconsistent with three sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. These sensing patterns all indicated that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r signaling. To confirm this hypothesis experimentally, we simultaneously knocked-down Admp, Gdf1/3-r, and Fgf9/16/20, and treated this triple morphant with recombinant bFGF and BMP4 proteins, which mimic Fgf9/16/20 and Admp/Gdf1/3-r activity, respectively. Although no differential inputs of Admp, Gdf1/3-r and Fgf9/16/20 signaling were expected under this experimental condition, Otx was expressed specifically in the neural lineage. Thus, direct cell–cell interactions through Efna.d play a critical role in patterning the ectoderm of the early ascidian embryo.
Highlights
In animal embryos, cell-cell interactions directed by secreted and membrane-bound signaling ligands play an important role in establishing specific gene expression patterns
We developed a parameter-free method for determining a Boolean function for Otx expression in the present study
We developed a parameter-free method for determining a Boolean function for understanding a complex signaling system using gene expression patterns of signaling molecules and geometrical configurations of individual cells within the embryo
Summary
Cell-cell interactions directed by secreted and membrane-bound signaling ligands play an important role in establishing specific gene expression patterns. Another study indicated that a differential input of Fgf9/16/20 signaling could direct differential Otx expression in the ectoderm [7]. It has not yet been established which of these factors is critical for patterning of the ectoderm of normal embryos. In other words, it has not been determined whether differential inputs of all of these signaling pathways are really necessary. Because our previous experiments [4] did not necessarily give an answer to this question, we took an advantage of theoretical analysis in the present study
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