The Roles Of Plexina2-Semaphorin6a Bi-Directional Signaling In Zebrafish Eye Development

Caroline Dumas, University of Vermont


Semaphorin6A (Sema6A) and its main receptor PlexinA2 (PlxnA2) play critical roles as repulsive guidance molecules in various developmental processes including the development of the nervous and immune systems. Further Sema6a-PlxnA2 signaling is implicated in numerous disease conditions. Importantly, Sema6A can partner with PlxnA2 (or PlxnA4) to play important roles in many aspects of eye development. Eye development relies heavily on cells proliferating, differentiating, migrating and synapsing correctly in order to set up a proper structure-function relationship. It has been demonstrated that Sema6A-PlxnA2 signaling contributes to the establishment of appropriate eye field formation as well as the appropriate arrangement of retinal cell layers. Interestingly, Sema6A is a transmembrane protein and can signal bi-directionally. This means that not only can Sema6A act as a ligand to PlxnA2/A4-expressing cells in a forward signaling process, but it can also act as a receptor to signal intracellularly in a reverse signaling manner. While we have identified roles for Sema6A signaling in eye development, we have not investigated which aspects of eye development rely on Sema6A forward signaling compared to Sema6A reverse signaling. In order to understand what aspects of eye development rely on Sema6A forward or reverse signaling, we used knock-down and rescue experiments in zebrafish. We conducted a comprehensive analysis of eye development by comparing rescue experiments using a full-length Sema6A construct in comparison with a Sema6A construct that lacks the intracellular domain. From this, we were able to find a role for the Sema6A intracellular domain in zebrafish eye development. We were surprised to discover that the Sema6A intracellular domain does not play a role in the formation of the eye field, retinal precursor cell proliferation, or cell number in the retina. Additionally, it is not necessary for the proper layering of neurons within the retina. However, the Sema6A intracellular domain is essential for the appropriate structure or maintenance of the retina, development of Müller glia, and cell survival within the retina. This study was the first to explore the role of Sema6A reverse signaling in vertebrate development, providing us with a model output to consider for further investigation of other aspects of Sema6A reverse signaling. To gain a better understanding of the Sema6A signaling mechanisms associated with the zebrafish phenotypes seen with the lack of the Sema6A intracellular domain, we investigated the relationship between the Sema6A intracellular domain and the tyrosine kinase Abl. It was already shown that Abl can bind to the intracellular domain of Sema6A, and we aimed to identify if this interaction leads to tyrosine phosphorylation on Sema6A. Indeed, we identified three phosphorylation sites on the intracellular domain of Sema6A induced by Abl activity. We subsequently showed that two of those Abl induced phosphorylation sites are necessary for a Sema6A expressing cell to collapse in response to a PlxnA2 cue. This finding highlights the significance of these phosphorylation sites in the context of Sema6A reverse signaling. Future research will investigate the role of the Abl induced phosphorylation sites on Sema6A in the context of zebrafish eye development. Together, our findings demonstrate a critical role for the Sema6A intracellular domain in zebrafish eye development and identify one potential signaling mechanism through Abl that could play a role in vertebrate development. Furthermore, these findings provide a basis for future investigations into the mechanisms of Sema6A reverse signaling in relation to developmental processes and diseases.