The formation of a healthy nervous system involves the regulated production of neurons in appropriate numbers and in appropriate locations in the neural tissue during the course of embryonic development. To understand the cellular and molecular interactions that determine where neurons are made and how many cells are permitted to become neurons we have studied how a relatively simple pattern of early neurons is established in the zebrafish neural plate and used genetics to identify essential molecular mechanisms involved in this process. Previous studies have shown that proneural genes, related to atonal in Drosophila, define domains in the neural plate where cells can become neurons and that lateral inhibition, mediated by Notch signaling, limits the number of cells that are allowed to become neurons within these domains. In the past year we have shown that two zebrafish atonal homologues, neurogenin1 and zath3 have partially redundant roles in determining neuronal fate in specific cranial ganglia. We also showed that HER5 (Hairy Enhancer of split Related gene- 5) has a partially redundant role in preventing neurogenesis at the midbrain-hindbrain boundary. Previous screens for mutants with an aberrant pattern of early neurons led to the identification of headless (hdl) mutants that have a rostral expansion of trigeminal ganglia and mind bomb (mib) mutants that have an unusually high density of neurons in otherwise normal locations. Analysis of the hdl mutant has provided novel insights about how the neural tube is divided into discrete compartments along the rostral caudal axis and analysis of mib has led to the identification of a novel component of the Notch signaling pathway. HEADLESS hdl is a loss-of-function mutation in a zebrafish homologue of T-Cell Factor 3 (TCF3), a member of the tcf/lef transcription factor family that has an essential role in regulating expression of genes activated by Wnt signaling. Analysis of the hdl mutant showed that the rostral expansion of trigeminal neurons is just one consequence of a more general posteriorization defect, where loss of relatively rostral neural tissues, like the forebrain and eyes, is accompanied by expansion of relatively caudal domains, like the midbrain-hindbrain boundary region and adjacent trigeminal neurons. Furthermore it was shown that the mutant phenotype results from the loss of the essential function of Hdl as a transcriptional repressor. Loss of hdl function leads to exaggerated Wnt signaling in a manner that specifically affects patterning events at the shield stage. We interpreted the posteriorized phenotype of MZ hdl mutants to suggest that, in the absence of repression provided by Hdl, posteriorizing factors drive expression of """"""""caudal"""""""" transcription factors in relatively rostral compartments, causing their transformation to more caudal fates. The role of Wnts as essential posteriorizing factors is supported by the observation that a knock-down of wnt8 by morpholinos leads to a phenotype that is exactly the opposite of hdl, where a reduction of caudal domains is accompanied by an expansion of relatively rostral domains. However, inhibition of wnt8 function in a MZ hdl mutant background causes no obvious reduction in the rostral expansion of caudal domains. This suggests that Wnt8 is not directly required for driving expression of caudal genes, rather, its primary role is to relieve Hdl -mediated repression of caudal genes and indirectly facilitate their expression. We showed that Wnt/ ?-catenin signaling can inhibit function of Hdl as a repressor and this inhibition requires an intact ?-catenin binding domain. In MZ hdl mutants there are changes in the relative sizes of discrete compartments of the anterior neurectoderm including the midbrain-hindbrain boundary domain, however, there are no obvious changes in the size of more caudal compartments. We discovered why loss of hdl function interferes with patterning at the shield stage and leads to deficits that do not extend into more caudal domains in a collaboration with Richard Dosrky, who had identified an additional tcf3 homologue, tcf3b. hdl is expressed maternally and it continues to be expressed throughout gastrulation in the prospective anterior neuroectoderm. tcf3b is also expressed maternally but it is not expressed at the shield stage. As a consequence, loss of hdl function leads to a specific deficit in Tcf3-mediated repression at this stage. We think variable levels of maternal Tcf3b that survive till early gastrulation help limit the posteriorization defect caused by loss of hdl. Consistent with this interpretation, knock-down of tcf3b in a MZ hdl background leads to progressively greater posteriorization that now affects the hindbrain and more caudal domains. MINDBOMB Zebrafish mind bomb (mib) mutants are characterized by a severe neurogenic phenotype characterized by an excess of early neurons and a reduction of neurons that are generated relatively late in development. 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 signaling. 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. Binding to Delta makes the Notch receptor vulnerable to metalloproteases that cleave Notch at a site outside its transmembrane domain facilitating the removal of the Notch extracellular domain (NECD). The membrane-bound Notch fragment that remains on the adjacent cell following removal of the NECD is a constitutive substrate for gamma-secretases that are responsible for an intra-membranous cleavage. This cleavage releases the Notch intracellular domain (NICD), which is the active fragment of the Notch receptor. Though many aspects of Notch signaling have been studied for a number of years, the precise steps by which the Notch ligands activate the Notch receptor remain poorly defined. Positional cloning of mib revealed that it is a novel gene in the Notch pathway that encodes a RING E3 ubiquitin ligase, an enzyme plays a key role in ubiquitylation of specific substrates. Analysis of its role in Notch signaling has revealed that Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Paradoxically, Mib mediated internalization of the Notch ligand appears to be essential in the Delta-expressing cell for efficient activation of Notch in neighboring cells. The mechanism by which Delta internalization facilitates Notch activation in neighboring cells appears unclear at this time. We are now investigating if Mib mediated ubiquitylation and internalization of Delta is required primarily to facilitate access of metalloproteases to the Notch extracellular and/or to facilitate removal of the NECD following cleavage by proteases. Previous studies have shown that clustering of Delta dramatically increases its ability to interact with and activate Notch. In this context we are also investigating if Mib contributes to Notch signaling by promoting the clustering of Delta.
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