A constriction in the neural tube at the junction of the midbrain and hindbrain is a conserved feature of vertebrate embryos. The constriction is a defining feature of the midbrain-hindbrain boundary (MHB), a signaling center that patterns the adjacent midbrain and rostral hindbrain and forms at the junction of two gene expression domains in the early neural plate: an anterior otx2/wnt1 positive domain and a posterior gbx/fgf8 positive domain. otx2 and gbx genes encode mutually repressive transcription factors that create a lineage restriction boundary at their expression interface. Wnt and Fgf genes form a mutually dependent feedback system that maintains their expression domains on the otx2 or gbx side of the boundary, respectively. Constriction morphogenesis occurs after these conserved gene expression domains are established and while their mutual interactions maintain their expression pattern; consequently, mutant studies in zebrafish have led to the suggestion that constriction morphogenesis should be considered a unique phase of MHB development. We analyzed MHB morphogenesis in fgf8 loss of function zebrafish embryos using a reporter driven by the conserved wnt1 enhancer to visualize anterior boundary cells. We found that fgf8 loss of function results in a re-activation of wnt1 reporter expression posterior to the boundary simultaneous with an inactivation of the wnt1 reporter in the anterior boundary cells, and that these events correlate with relaxation of the boundary constriction. In consideration of other results that correlate the boundary constriction with Wnt and Fgf expression, we propose that the maintenance of an active Wnt-Fgf feedback loop is a key factor in driving the morphogenesis of the MHB constriction.

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Mutations of the protein Superoxide Dismutase 1 (SOD1) are implicated in a subset of Amyotrophic Lateral Sclerosis (ALS) cases. The C-terminal domain of TNF Receptor-Associated Factor 6 (TRAF6) is a newly discovered interactor of misfolded mutants of SOD1, but not wild-type SOD1. However no structural information is available yet to characterize how the proteins might directly interact. We sought to investigate possible mechanisms of this interaction through molecular dynamics (MD) and metadynamics simulations of a dimeric model system, coarse-grained using the AWSEM force field. We used direct MD simulations to predict possible binding poses by subjecting the trajectory to hierarchical clustering. Metadynamics simulations were used to deduce the preferred binding regions on the protein surfaces from the potential of the mean force in the spherical (q, 4) orientation space around each protein. We found that the SOD1-interacting surface of TRAF6 is broadly distributed about the equatorial region of the beta barrel, while the TRAF6 interacting surface of SOD1 is relatively localized. When SOD1 is wellfolded, the TRAF6 heterodimer interaction surface involves a co-option of the native SOD1 homodimer interface, while if SOD1 contains disordered loops IV and VII, as typically occurs in the absence of stabilizing Zn 2þ ion binding, the disordered loops now partake in novel interactions with TRAF6. Using cDNA constructs, we expressed variants of TRAF6 with mutations in the predicted binding sites and found that TRAF6 T475D no longer interacts with G93A and E100G mutants of SOD1 and has reduced interaction with SOD1 A4V . These observations suggest a structural mechanism for the SOD1-TRAF6 interaction and may have implications for developing ALS therapeutics in the future.

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