The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease using a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1–5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show this enhanced TALEN toolkit demonstrates a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino (MO)-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA (ssDNA) oligonucleotides (oligos) to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair (HDR), including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

We describe a conditional in vivo protein trap mutagenesis system that reveals spatio-temporal protein expression dynamics and assesses gene function in the vertebrate Danio rerio. Integration of pGBT-RP2 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript levels while simultaneously reporting protein expression of each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site blocking morpholinos, thus representing the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines including a diverse array of molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step towards a unified codex of protein expression and direct functional annotation of the vertebrate genome.

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...