Cellular, Molecular And Genetic Analysis Of Neural Fate In Zebrafish Embryos

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Neurons in zebrafish embryos are generated in discrete neurogenic domains where expression of basic helix loop helix transcription factors, related to the proneural genes in Drososphila, gives cells the potential to differentiate as neurons. However, activation of the Notch receptor in progenitors by its ligands, Delta and Jagged, expressed on the surface of in neighboring cells, prevents early differentiation of some progenitors within these domains and allows them to be maintained or to differentiate later with alternate fates. [unreadable] Both Notch and its ligands are single pass membrane proteins expressed on the cell surface and their interactions allow the ligand-expressing cell to regulate cell fate in the neighboring Notch-expressing cell. The Notch receptor has an intracellular domain (NICD) that can function as part of a transcriptional activator complex. Release of the NICD domain and its translocation to the nucleus is regulated by removal of an extracellular fragment of Notch (NECD). The NECD fragment is cleaved off during synthesis of the Notch receptor, however, it remains bound to the extracellular stub of the remaining membrane bound fragment of Notch where it prevents access to critical proteolytic cleavage sites. Removal of the NECD fragment allows proteases to sequentially cleave the Notch receptor, first in the extracellular stub and then in the intra-membranous domain. This results in release of the NICD fragment, which can then translocate to the nucleus and activate target genes after forming a complex with co-factors that bind regulatory sites in target genes.[unreadable] Removal of the NECD fragment and activation of Notch is facilitated by its binding to ligands on the surface of neighboring cells. Recent studies have suggested that once Notch ligands, like Delta and Jagged/Serrate bind Notch in a neighboring cell, the ligand bound to the NECD fragment is internalized by the ligand-expressing cell. This results in separation of the NECD fragment from the remaining membrane-bound Notch receptor and allows proteolytic cleavages that activate Notch by releasing the NICD fragment into the cell. Previously we showed that endocytosis of Delta is triggered by a RING ubiquitin ligase, Mind bomb (Mib), which facilitates ubiquitylation of Delta. This process results in covalent linkage of a single or multiple molecules of the 76 amino acid protein, Ubiquitin, to lysines in the intracellular domain of Delta. Ubiquitin recruits components of the endocytic machinery and these in turn facilitate endocytosis of ubiquitylated Delta.[unreadable] In the context of these studies it was not clear if ubiquitylation and endocytosis of Notch ligands was triggered by their interaction with Notch in a neighboring cell or whether Mib-mediated endocytosis of Notch ligands, like Delta, is a constitutive process that takes place independent of their interaction with Notch. We have now shown that interactions with Notch have an important role in determining endocytosis of two Delta homologues, DeltaD and DeltaA, but not a third homologue, DeltaC. First we showed that in the central nervous system most of the Notch ligand, DeltaD, is located in cytoplasmic vesicles. However, in mib mutants, DeltaD and DeltaA accumulate on the cell surface suggesting that Mib-mediated DeltaD endocytosis plays an important role in determining the cellular distribution of Delta. We then showed that in Notch morphants, in which Notch receptors are reduced by injecting embryos with anti-sense morpholinos, DeltaD and DeltaA accumulate on the cell surface just as they do in Mib mutants, suggesting that interaction with Notch is also an important determinant of DeltaA and DeltaD endocytosis but not of DeltaC. [unreadable] Delta has the potential to interact with Notch both on the surface of a neighboring cell (in trans) and on the surface of the same cell (in cis) so we then investigated whether interactions in cis or in trans are critical for Delta endocytosis. To address this issue cells from an embryo in which Notch had been knocked-down with morpholinos were transplanted into wild-type embryos with Notch, and conversely wild-type cells with Notch were transplanted into embryos without Notch. Changes in the cellular distribution of Delta in the transplanted cells and in their neighbors in these specific contexts helped us determine if interactions with Notch within the same cell (in cis) or with Notch in a neighboring cell (in trans) were critical for Delta endocytosis. Interestingly, our experiments demonstrated that interactions with Notch both in cis and in trans contribute to DeltaD endocytosis.[unreadable] As discussed earlier, interactions with Notch in trans help activate Notch in a neighboring cell. In contrast, it is thought that interaction of Delta with Notch in cis interferes with Notch activation within that cell. Our observation that Delta-Notch interactions in cis contribute to Delta endocytosis suggests that, following an interaction in cis, endocytosis of the ligand-receptor complex reduces availability of both at the cell surface. Together these observations show how interactions between Delta and Notch within the same cell and between adjacent cells play a critical role in the dynamic regulation of Notch signaling during development.