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.

Stereotypical patterns of vascular and neuronal networks suggest that specific genetic programs tightly control path determination and, consequently, angiogenesis and axon-guidance mechanisms. Our study focuses on one member of the roundabout family of receptors, which traditionally mediate repulsion from the midline. Here, we characterize a fourth member of this family, roundabout4 (robo4), which is the predominant roundabout (robo) that is expressed in embryonic zebrafish vasculature. Gene knockdown and overexpression approaches show that robo4 is essential for coordinated symmetric and directed sprouting of intersomitic vessels and provide mechanistic insights into this process. Also, human robo4 gene functionally compensates for loss of robo4 gene function, suggesting evolutionary conservation. This article reports an endothelial-specific function for a robo gene in vertebrates in vivo.axon guidance ͉ endothelial cell ͉ zebrafish V ascular and neural networks established by angiogenesis and neurogenesis are essential for the regulation of physiological processes. Whether the processes of blood-vessel and peripheralnerve formation are intimately associated at a mechanistic level is a subject of active investigation (1). Recently, description of cellsurface molecules that are shared by neuronal and endothelial cells has suggested that this association may be more prevalent than previously thought. Neuropilin (2), ephrin (3), and plexin (4) are a few examples of molecules that are implicated in both processes. Recently, roundabout (robo), a class of neural guidance receptors that bind slit ligands have joined this group (5). slit-robo signaling mediates axonal repulsion (6) and inhibition of leukocyte migration (7). In vertebrate systems, three robo receptor family members were identified, all with prominent neural expression (8, 9). More recently, a fourth member of the robo gene family, roundabout4 (robo4) was identified (10). Compared with the canonical robo structure, robo4 is smaller in that it possesses only two of the five Ig and two of the three fibronectin domains present in the extracellular component of robo1, robo2, and robo3 (11). robo4 has been described as endothelial-specific, and it binds to slit and inhibits the migration of heterologous cells that express robo4 and primary endothelial cells (12). Although studies of robo4 in endothelial cells suggest that robo signaling is important for regulating endothelial cell migration, there are no reports that document the role of robo signaling in vascular development in vivo. Here, we cloned a zebrafish (Danio rerio) ortholog of human robo4 (hrobo4) and studied its expression and role in vascular development by gene knockdown and overexpression approaches. In contrast to the endothelial-specific expression of murine robo4, zebrafish robo4 is expressed in both endothelial and neural tissues with vascular expression seen in angioblasts, the dorsal aorta (DA), the posterior cardinal vein, and intersomitic vessels (ISV). Morpholino (MO) knockdown of robo...

Asymmetric division of progenitor/stem cells generates both self-renewing and differentiating progeny and is fundamental to development and regeneration. How this process is regulated in the vertebrate brain remains incompletely understood. Here we use time-lapse imaging to track radial glia progenitor behavior in the developing zebrafish brain. We find that asymmetric division invariably generates a basal self-renewing daughter and an apical differentiating sibling. Gene expression and genetic mosaic analysis further show that the apical daughter is the source of Notch ligand that is essential to maintain higher Notch activity in the basal daughter. Notably, establishment of this intra-lineage and directional Notch signaling requires the intrinsic polarity regulator Partitioning defective protein-3 (Par-3), which segregates the fate determinant Mind bomb unequally to the apical daughter, thereby restricting the self-renewal potential to the basal daughter. These findings reveal with single-cell resolution how self-renewal and differentiation become precisely segregated within asymmetrically dividing neural progenitor/stem lineages.