The vertebrate nervous system is divided along the anterior-posterior (AP) axis into compartments that form the forebrain, midbrain, hindbrain, and spinal cord. Within each of these specialized compartments, neurons and glia are produced in a stereotyped pattern by neural progenitors. Our laboratory is investigating fundamental patterning mechanisms that are responsible for dividing the nervous system into discrete compartments and ensuring that neurons are made in the appropriate number and location within each compartment. To study molecular mechanisms that are responsible for these early patterning events, we use genetics in zebrafish to identify mutants with aberrant neural patterning. The identification and analysis of headless mutants, characterized by the absence of a forebrain and eyes, has allowed us to investigate how a morphogen gradient helps define discrete compartments along the AP axis during early development. Analysis of another mutant, mind bomb, which is characterized by the production of too many neurons, has revealed how lateral inhibition, mediated by Notch signalling, helps single out neural progenitors that are permitted to become neurons in the nervous system.

Cooperative Head Patterning by Two Tcf3 Genes
Itoh, Chitnis; in collaboration with Dorsky, Moon
The blastoderm margin is the source of secreted factors that activate gene expression in a dose-dependent manner along the AP axis of the zebrafish gastrula. These factors, including the Wnts and FGFs, are expressed around the blastoderm margin during early gastrulation. They cooperate to establish a gradient of posteriorizing activity in the zebrafish gastrula with its high end around the blastoderm margin and its low end near the animal pole.

Analysis of maternal zygotic (MZ) headless (hdl) mutants (Kim C-H et al., 2000) has suggested that posteriorizing factors, in particular Wnts, operate in the context of basal repression provided by hdl, a homolog of T-cell factor-3 (TCF3). Canonical Wnt signalling induces the expression of downstream target genes through the transcriptional activator b-catenin, which associates in the nucleus with Lef/Tcf proteins that bind to DNA-regulatory elements. When b-catenin levels are low, Tcf proteins associate with the co-repressors Groucho and CtBP to maintain target genes in a repressed state.

Loss of repression provided by hdl leads to a loss of rostral compartments and expansion of relatively caudal compartments of the neural tube, whose specification depends on posteriorizing factors. We have shown that a second zebrafish tcf3 homolog, tcf3b, limits posteriorization caused by loss of Hdl function. While loss of hdl function leads to loss of forebrain and eyes and expansion of the midbrain-hindbrain domain, it leaves the hindbrain relatively unaffected. On the other hand, the additional loss of tcf3b function in an MZ hdl mutant background leads to further loss of anterior structures and expansion of the hindbrain domain, indicating further posteriorization of the neural tube. Our studies also indicate that, while Hdl and Tcf3b are primarily responsible for repression, a third Lef/Tcf family member, Lef1, may have a primary role in activation of Wnt target genes: injection of lef1 mRNA does not lead to a recovery of anterior structures in MZ hdl mutant embryos as does injection of hdl or tcf3b mRNA.

By comparing systematic changes in gene expression observed as a consequence of progressive loss of Hdl and Tcf3b function with predictions of computer simulations, we have been able to make specific predictions about the likely shape of the posteriorizing morphogen gradient during early gastrulation. Finally, we have shown that tcf3b has a second and unique role in the morphogenesis of rhombomere boundaries, indicating that it controls multiple aspects of brain development.

We are now investigating how the posteriorizing gradient is established and how cells respond to the gradient of posteriorizing factors to define discrete domains of gene expression in the neurectoderm.

Ubiquitin Ligase Mib Essential for Efficient Activation of Notch Signalling by Delta
Itoh, Kim, Palardy, Maust, Yeo, Chitnis; in collaboration with Oda, Jiang, Lorick, Wright, Ariza-McNaughton, Weissman, Lewis, Chandrasekharappa
Neurons are distributed in a simple pattern in the zebrafish neural plate. They are formed in three bilateral longitudinal “proneuronal” domains where cells acquire the potential to become neurons by expressing the proneural gene neurogenin (ngn1). Ngn1 drives the expression of Delta, a membrane-bound ligand that interacts with its receptor Notch in neighboring cells. Activation of Notch by Delta inhibits proneural function. Through this simple feedback loop, each cell within a “proneuronal” domain tends to prevent its neighbors from adopting a similar fate, thus creating a competi-tive situation. As a consequence of lateral inhibition, a subset of cells eventually emerges as winners and, through auto-regulation, acquires high enough levels of proneural gene expression to permit adoption of a neural fate.

Zebrafish mind bomb (mib) mutants are characterized by a severe neurogenic phenotype; they also exhibit a wide range of defects in the development of somites, neural crest, and vasculature, which have been interpreted as consequences of deficits in Notch signalling in all these tissues. Though previous studies have suggested that mib is likely to encode an essential component of the Notch pathway, the molecular nature of mib had remained elusive, and it was not known how it contributes to Notch signalling.

Notch is a one-pass transmembrane receptor that is synthesized as a single peptide. Furin-mediated cleavage of the peptide creates two fragments that are held together in the mature receptor as a heterodimer. The extracellular fragment mediates interactions with the ligand, Delta. An important step in the activation of Notch is the removal of its extracellular fragment. Binding to Delta makes the receptor vulnerable to metallopro-teases that cleave Notch at a second site outside its transmembrane domain. In Drosophila, it has been shown that the Delta-Notch interaction is accompanied by endocytosis of Delta by the signalling cell, which carries with it the bound Notch extracellular domain. The membrane-bound Notch fragment that remains on the adjacent cell after the cleavage by metallopro-teases is a substrate for g-secretases. The enzymes cleave the fragment at a third site, within the membrane, which releases an intracellular fragment that functions in a transcriptional activator complex with Su(H)/CBF1/RBP-J to activate Notch target genes that inhibit ngn1 function.

Our analysis has shown that in mib mutants reduced lateral inhibition mediated by Notch, permitting too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a novel gene in the Notch pathway, which encodes a RING E3 ubiquitin ligase, an enzyme that plays a central role in ubiquitylation. Ubiquitylation is a multi-step process that results in the addition of a polypeptide, ubiquitin, to a substrate protein. First, a ubiquitin-activating enzyme (E1) activates ubiquitin in an ATP-dependent manner; then, a ubiquitin-conjugating enzyme (E2) receives its ubiquitin from an E1. Finally, a ubiquitin ligase (E3), which contains a substrate recognition domain and provides a docking site for an E2, facilitates transfer of ubiquitin from the E2 to its specific substrate. Ubiquitylation was originally recognized for its role in tagging proteins for destruction in proteosomes. More recently, it has been shown that the addition of ubiquitin to proteins can play a pivotal role in changing the behavior or distribution of a protein and can affect a variety of events, including endocytosis.

We have shown that Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in signalling cells for efficient activation of Notch in neighboring cells. Our observations provide support for a model in which Mib promotes the trans-endocytosis of the Notch extracellular domain by promoting endocytosis of Delta and, in doing so, facilitates proteolytic events that generate the transcriptionally active Notch intracellular fragment.