Functional characterization of Nodal receptors during early embryonic development in zebrafish

Abstract

Understanding the robust control mechanisms that steer the development of a single cell into a complex organism remains one of the most central challenges in developmental biology. A crucial factor for the coordination of cell fates in the developing embryo is the intercellular communication mediated by different signaling pathways and their respective activity levels. The complex interplay of these pathways successively determines distinct cell fates. However, the mechanisms that control signal dispersal in the embryo are not fully understood. In vertebrates, the Nodal signaling pathway is crucial for mediating germ layer patterning. The secreted transforming growth factor beta (TGF-β) ligand Nodal activates downstream transcriptional regulation by recruiting a receptor complex comprised of Type I and Type II Activin receptors and an EGF-CFC co-receptor. Previous studies in human, mouse, Xenopus and zebrafish have identified several of these receptors that mediate Nodal signaling. While in mouse several Nodal receptor mutants show severe developmental defects, in zebrafish only mutants of the co-receptor one-eyed pinhead (oep) recapitulate a Nodal loss-of-function phenotype. Here, I systematically identified and characterized three Type I and four Type II Activin receptor homologs in zebrafish. Temporal and spatial expression analysis demonstrated that, except for the Type I receptor acvr1c, all investigated putative Nodal receptors are maternally deposited and continuously expressed during germ layer patterning. To assess the role of the putative Nodal receptors during germ layer patterning in zebrafish, I generated receptor mutants and used them in a combinatorial knockdown assay with receptor targeting morpholinos. Using this approach, I could show that the two Type I receptors acvr1b-a and acvr1b-b redundantly mediate early Nodal signaling in zebrafish. Measurements in zebrafish suggest that the secreted Nodal ligands have a lower diffusivity and a shorter range than their long-range antagonist Lefty. Both, Nodal and Lefty belong to the TGF β superfamily and are similar in size and structure. Interaction of Nodal with extracellular binding partners, so called diffusion regulators, has been proposed to explain their different mobility. To elucidate whether the Nodal receptors can function as such diffusion regulators, I used quantitative approaches to analyze their effect on Nodal distribution and diffusion. My results show that range and shape of the Nodal distribution gradient can be influenced by receptor and co-receptor levels. These findings highlight the potential of Nodal receptor interaction as a mechanism for restricting Nodal dispersal during germ layer patterning. In summary, my thesis contributes to a more detailed understanding of the role of Nodal receptors during early germ layer patterning in zebrafish. My findings emphasize the importance of receptor redundancy in zebrafish and reinforce the ability of receptors to influence signal propagation in ways that go beyond signal transduction.