Dissecting the spatiotemporal regulation of nodal signaling and its role as a morphogenetic cue

Abstract

The TGF-â ligand Nodal is a key regulator of body axis formation and patterning in the developing vertebrate embryo. How the Nodal signaling pathway is activated and regulated, and how precise spatiotemporal control of the pathway is translated into morphogenetic cues for asymmetric morphogenesis is unclear. Attempts to gain insight into the dynamics of active Nodal signaling have been hindered by the inability to detect the endogenous Nodal ligand. Antibodies against the signal transducer phospho-Smad2 (pSmad2) have been used to detect active TGF-â signaling, but are of poor quality and cannot accurately depict Nodal signaling in situ. We generated a new pSmad2 antibody, with the goal of using it to create a spatiotemporal map of Nodal signaling during development. We planned to integrate such maps with our data showing increased F-actin labeling in the R LPM, and possible L vs. R cell shape differences, just prior to organ looping to aid in understanding how Nodal signaling directly translates into morphogenetic cues. Fully understanding the role of Nodal signaling in morphogenesis fundamentally includes understanding its regulation at the transcriptional level. Current models have the winged-helix transcription factor Foxh1 bound with pSmad2 as a central transcriptional activator of Nodal. However, a conserved Engrailed-homology-1 (EH1) motif, which is recognized by Groucho co-repressors, in Foxh1 suggests that Foxh1 functions as a transcriptional switch, toggling between transcriptional on and off states via pSmad2-Grg switching, to ensure properly timed initiation and suppression, and/or amplitude, of Nodal. We minimally mutated the Foxh1 EH1 motif, creating a novel Foxh1mEH1 allele to test the contribution of Foxh1-Grg–mediated repression on the transient, dynamic pattern of Nodal signaling in mice. We find that Foxh1-Grg–mediated repression is not essential for Nodal expression during mouse embryogenesis. This suggests that other regulators compensate for the loss of Foxh1-Grg–mediated repression, and that Nodal signaling exists within the context of a strongly buffered regulatory system that contributes to resilience and accuracy of its dynamic expression pattern.