Cellular, Molecular And Genetic Analysis Of Neural Fate

Child Health And Human Development

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Abstract

Early neurons in zebrafish are distributed in a simple pattern at the neural plate stage. We use cellular, molecular, genetic and computational tools for understanding molecular mechanisms that determine how neurons are made in the correct number and location in the nervous system. Our broader goal is to understand how neurogenesis is regulated in the vertebrate embryo, a process that is essential for the growth and development of a healthy nervous system. Lateral inhibition mediated by Delta-Notch signaling limits the number of cells that are permitted to become early neurons. Failure of Notch signaling in the zebrafish mind bomb (mib) mutants results in the production of an excess of early neurons. Mib was identified as an ubiquitin ligase that is required for endocytosis of Delta. In this context ubiquitylation of Delta does not serve its traditional role as a degradation signal but rather as a signal for internalization. Analysis of the mind bomb (mib) mutant revealed that internalization of the Notch ligand, Delta is essential for effective lateral inhibition mediated by Notch signaling. An important question is to understand how endocytosis of Delta contributes to activation of its receptor in neighboring cells. There are at least two broad models that account for how Delta endocytosis in one cell might contribute to activation of Notch in the neighboring cell. The first class of models is related to the possibility that Delta endocytosis facilitates cleavage and removal of the Notch Extracellular domain (NECD), a step that is critical for activation of the Notch receptor. A second class of models is related to the possibility that Delta ubiquitylation and endocytosis facilitates interactions between Delta and Notch. In the second set of models, Delta undergoes endocytosis, and its subsequent recycling back to the surface, following modifications or some change in the context in which it is presented, makes Delta a more attractive ligand. This includes the possibility that Delta is returned to the surface in a clustered state, with modifications of its extracellular domain, or in association with cofactors that allow it to bind more easily to Notch. In addition, specifically in the context of polarized epithelial cells, recycled Delta may be presented on the basolateral membrane where interactions with Notch are facilitated with adjacent cells. To understand the mechanism by which Delta internalization and ubiquitylation promotes activation of its receptor, Notch, we have investigated trafficking of fluorescent-tagged forms of deltaD in cell culture. A monoclonal antibody, which recognizes the deltaD extracellular domain was used to monitor Notch-independent deltaD trafficking, while a soluble form of the Notch2 receptor (N2ECD-Fc), including the Notch2 extracellular domain (N2ECD) fused to an Fc fragment, was used to monitor Delta trafficking following interaction with its receptor. We found that pre-clustering of NECD-Fc is required for effective interaction with DeltaD and the interaction promotes Delta ubiquitylation in COS7 cells. Analysis of DeltaD trafficking revealed that manipulations that are predicted to reduce recycling reduce availability of DeltaD on the cell surface. In contrast to the effects of interfering with recycling, inhibition of protein synthesis for 2 hours does not significantly reduce surface DeltaD. Together these observations suggest DeltaD internalization and recycling may be a critical determinant of Delta availability at the cell surface. It is not yet clear what variables determine if Delta is recycled or degraded following internalization. Future studies will determine to what degree Ubiquitylation influences this choice.