Dynamic Regulation of Notch Signaling During Zebrafish Neurogenesis

Ajay Chitnis, MBBS, PhD, Head, Section on Neural Developmental Dynamics
Damian E. Dalle Nogare, PhD, Postdoctoral Fellow
Hiromi Ikeda, PhD, Postdoctoral Fellow
Miho Matsuda, PhD, Postdoctoral Fellow
Kinneret Rand, PhD, Postdoctoral Fellow
Kyeong-Won Yoo, PhD, Postdoctoral Fellow
Gregory Palardy, BS, Research Technician
Chongmin Wang, MS, Research Technician
Christopher M. Worsham, Summer Student

Our goal is to understand how, during early development, the architecture of the mature nervous system emerges as a consequence of local interactions between cells. We use a combination of cellular, molecular, genetic, and computational tools to understand how neurons differentiate in distinct patterns in the various compartments of the zebrafish nervous system. We analyze zebrafish mutants and embryos microinjected with morpholinos or mRNA for the purpose of altering gene function, allowing us (1) to examine mechanisms involved in the division of the prospective neural tissue into compartments with distinct fate and (2) to investigate how neuronal differentiation is regulated within each compartment. Genetic analysis in zebrafish leads to identification of regulatory networks essential for specific aspects of neural patterning while cell-biological experiments identify trafficking events essential for regulating signaling. Delta-Notch signaling regulates the number of neurons. Our recent studies have focused on the role of Delta endocytosis in activating its receptor Notch in neighboring cells and revealed that interactions with Notch play a critical role in regulating Delta endocytosis.

Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue

Matsuda, Chitnis

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 Drosophila, 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 neighboring cells, prevents early differentiation of some progenitors within these domains and allows them to be maintained or to differentiate later with other fates.

Both Notch and its ligands are single-pass membrane proteins expressed on the cell surface; their interactions allow the ligand-expressing cell to regulate cell fate in a neighboring Notch-expressing cell. The Notch receptor has an intracellular domain (NICD) that can function as part of a transcriptional activator complex. Removal of an extracellular fragment of Notch (NECD) regulates release of the NICD domain and its translocation to the nucleus. The NECD fragment is cleaved during synthesis of the Notch receptor but 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 cleave the Notch receptor sequentially—first in the extracellular stub and then in the intramembranous domain. The result is the 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 to regulatory sites in target genes.

Removal of the NECD fragment and subsequent activation of Notch is facilitated by the former’s binding to ligands on the surface of neighboring cells. Recent studies have suggested that, once Notch ligands such as Delta and Jagged/Serrate bind to Notch in a neighboring cell, the ligand-expressing cell internalizes the ligand bound to the NECD fragment. The result is the separation of the NECD fragment from the remaining membrane-bound Notch receptor, thereby allowing proteolytic cleavages that activate Notch by releasing the NICD fragment into the cell. Previously, we showed that endocytosis of Delta is triggered by Mind bomb (Mib), a RING ubiquitin ligase that facilitates ubiquitylation of Delta. The process results in the covalent linkage of a single or several molecules of the 76–amino acid protein ubiquitin to lysines in Delta’s intracellular domain. Ubiquitin recruits components of the endocytic machinery, which, in turn, facilitate endocytosis of ubiquitylated Delta.

In the context of the above studies, we were unable to determine whether ubiquitylation and endocytosis of Notch ligands were triggered by the ligands’ interaction with Notch in a neighboring cell or whether Mib-mediated endocytosis of Notch ligands such as Delta is a constitutive process that occurs independently of the ligands’ interaction with Notch. We have now shown that interactions with Notch play an important role in determining the endocytosis of two Delta homologues, DeltaD and DeltaA, but not of 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 endocytosis.

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). We thus investigated whether interactions either in cis or trans are critical for Delta endocytosis. To that end, we transplanted cells from an embryo in which Notch had been knocked down with morpholinos into wild-type embryos with Notch and, conversely, we transplanted wild-type cells with Notch into embryos without Notch. Changes in the cellular distribution of Delta in the transplanted cells and their neighbors helped us determine whether 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 in both cis and trans contribute to DeltaD endocytosis.

As discussed, 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 in that same 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 the availability of both ligand and receptor at the cell surface. Together, our 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.

Matsuda M, Chitnis AB. Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue. Development 2008; [E-pub ahead of print].

Publications Related to Other Work

Chitnis A. Why is delta endocytosis required for effective activation of notch? Dev Dyn 2006;235:886-894.
Chitnis AB. Keeping single minded expression on the straight and narrow. Mol Cell 2006;21:450-452.
Diks SH, Sartori da Silva MA, Hillebrands JL, Bink RJ, Versteeg HH, van Rooijen C, Brouwers A, Chitnis AB, Peppelenbosch MP, Zivkovic D. d-Asb11 is an essential mediator of canonical Delta-Notch signalling. Nat Cell Biol 2008;10:1190-1198.
Keller MJ, Chitnis AB. Insights into the evolutionary history of the vertebrate zic3 locus from a teleost-specific zic6 gene in the zebrafish, Danio rerio. Dev Genes Evol 2007;217:541-547.
Yeo SY, Chitnis AB. Jagged-mediated Notch signaling maintains proliferating neural progenitors and regulates cell diversity in the ventral spinal cord. Proc Natl Acad Sci USA 2007;104:5913-5918.
Yeo SY, Kim M, Kim HS, Huh TL, Chitnis AB. Fluorescent protein expression driven by her4 regulatory elements reveals the spatiotemporal pattern of Notch signaling in the nervous system of zebrafish embryos. Dev Biol 2007;301:555-567.

For further information, contact chitnisa@mail.nih.gov.