Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

The product of the Delta gene, acting as ligand, and that of the Notch gene, acting as receptor, are key components in a lateral-inhibition signalling pathway that regulates the detailed patterning of many different tissues in Drosophila. During neurogenesis in particular, neural precursors, by expressing Delta, inhibit neighbouring Notch-expressing cells from becoming committed to a neural fate. Vertebrates are known to have several Notch genes, but their functions are unclear and their ligands hitherto unidentified. Here we identify and describe a chick Delta homologue, C-Delta-1. We show that C-Delta-1 is expressed in prospective neurons during neurogenesis, as new cells are being born and their fates decided. Our data from the chick, combined with parallel evidence from Xenopus, suggest that both the Delta/Notch signalling mechanism and its role in neurogenesis have been conserved in vertebrates.

X-Delta-1, a Xenopus homologue of the Drosophila Delta gene, is expressed in the early embryonic nervous system in scattered cells that appear to be the prospective primary neurons. Ectopic X-Delta-1 activity inhibits production of primary neurons and interference with endogenous X-Delta-1 activity results in overproduction of primary neurons. These results indicate that the X-Delta-1 protein mediates lateral inhibition delivered by prospective neurons to adjacent cells, and that commitment to a neural fate in vertebrates is regulated by Delta-Notch signalling as in Drosophila.

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo 1 by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers [2][3][4] . Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation [5][6][7] . These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals 8 . Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in Tcell factor-3 (Tcf3), a member of the Tcf/Lef family 9,10 . Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.The hdl mutant was isolated as part of a screen for ethyl nitrosourea (ENU)-induced mutations that disrupt early neurogenesis in zebrafish 11 . Mutant embryos obtained from hdl heterozygous parents, however, display a weak phenotype and are characterized by a slight reduction in eye size. Their weak phenotype allowed a subset of homozygous hdl fish to be grown to adulthood. Here, hdl mutants refers to maternally and zygotically homozygous mutant embryos. Our examination of hdl mutants with the early neuronal marker, huC 12 , revealed an aberrant pattern of trigeminal neurons (Fig. 1a, b). The mutation, however, derives its name -headless -from the head defect in embryos that is characterized by complete loss of eyes, forebrain and part of the midbrain (Fig. 1c, d, e). Analysis of the head skeleton reveals cranial-specific defects (Fig. 1f, g); the pharyngeal arches appear relatively unaffected (Fig. 1h, i Hesx1, normally expressed in the anterior neural plate (Fig. 2m), is almost absent in mutants (Fig. 2s). Mutational analysis has shown that Hesx1 is required for normal forebrain development in mice and humans 13 . Transcripts of six3, another anterior brain marker 14 , are also reduced in the anterior neural plate, but its expression in the underlying prechordal plate is not changed (Fig. 2n, t). This suggests that the hdl phenotype is not related to loss of the underlying prechordal plate. Expression of rx3, a marker for the presumptive retina and ventral forebrain, is also strongly reduced in mutants (Fig. 2o, u). Mouse embryos carrying a null allele of the Rx gene have severe defects in eye and forebrain formation 15 . The reduction in expression of these anterior neural-specific genes is accompanied by a rostral expansion of midbrain-hindbrain boundary (MHB) genes such as pax2 (Fig. 2p, v) and engrailed2 (eng2) (Fig. 2q, w) but not krox20 (krx20), expressed in rhombomeres 3 and 5 (Fig. 2w).To investigate how early changes in the vertebrate organizer contri...