The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein ofvesicular While retroviral infection usually requires interaction between the viral envelope protein and specific cell surface receptor proteins, VSV-G interacts with a phospholipid component of the cell membrane to mediate viral entry by membrane fusion (6). Since viral entry seems not to be dependent on the presence of specific protein receptors, VSV has an extremely broad host-cell range (7). In addition, VSV can be concentrated by ultracentrifugation without loss of infectivity. We hypothesized that substitution of VSV-G for the MoMLV envelope protein might confer upon the pseudotyped particle the desirable properties of increased host-cell range and structural stability after ultracentrifugation. We report here the construction of a retroviral vector encapsidated in the envelope protein of VSV that has an extended host-cell range and can be concentrated to titers >109 colony-forming units (cfu)/ml with minimal loss of infectivity. As

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.

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Optokinetic and phototactic behaviors of zebrafish larvae were examined for their usefulness in screening for recessive defects in the visual system. The optokinetic response can be reliably and rapidly detected in 5-day larvae, whereas the phototactic response of larvae is variable and not robust enough to be useful for screening. We therefore measured optokinetic responses ofmutagenized larvae as a genetic screen for visual system defects. Third-generation larvae, representing 266 mutagenized genomes, were examined for abnormal optokinetic responses. Eighteen optokinetic-defective mutants were identified and two mutants that did not show obvious morphological defects, no optokinetic response a (noa) and partial optokinetic response a (poa), were studied further. We recorded the electroretinogram (ERG) to determine whether these two mutations affect the retina. The b-wave of noa larvae was grossly abnormal, being delayed in onset and significantly reduced in amplitude. In contrast, the ERG waveform of poa larvae was normal, although the b-wave was reduced in amplitude in bright light. Histologically, the retinas of noa and poa larvae appeared normal. We conclude that noa larvae have a functional defect in the outer retina, whereas the outer retina of poa larvae is likely to be normal. genetic dissection of the zebrafish visual system should be applicable to other vertebrates.Recently, two groups developed chemical mutagenesis procedures and methods for efficiently growing large numbers of zebrafish (9-12). These procedures have made it possible to conduct large-scale genetic screens in which zebrafish larvae from the third generation are analyzed for recessive mutations. Furthermore, a genetic linkage map in zebrafish is now available so mutant genes can be isolated by positional cloning (13).We first characterized two visual behaviors-phototaxis and optokinetic responses-in wild-type zebrafish larvae (3-19 days pf). Preliminary experiments on wild-type larvae (4) suggested that both of these assays would be useful. We then analyzed the optokinetic responses of mutagenized larvae as a primary screen for detecting recessive defects in the visual system. As a secondary screen, we recorded the electroretinogram (ERG) from larvae 5-7 days pf to identify mutations that specifically affect the retina. We describe here the feasibility of this approach for identifying mutations affecting the visual system and describe two mutants isolated on the basis of their abnormal optokinetic response.Benzer (1) was the first to report that mutant Drosophila could be identified by their phototactic behavior. Subsequently, a number of nonphototactic mutants were found to have specific molecular defects in their photoreceptors (2). A phototaxis mutant that failed to respond to UV light, sevenless, lacks UV-sensitive photoreceptor cells (3); analysis of this mutant has defined the role of cell-cell interactions in ommatidial development (for review, see ref. 6).Because there are significant differences between vertebrate and ...

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...

A gradient in concentration of the protein product of the bicoid gene is a determinant of the anterior-posterior axis of Drosophila embryos. By binding upstream of the segmentation gene hunchback the bicoid protein controls its transcription, thereby translating maternal pattern-generating information into differential activation of zygotic gene expression.

Essential components of animal behaviour are modulated by dopaminergic (DA) and noradrenergic circuitry. In this study, we reveal at cellular resolution the complete set of projections ('projectome') of every single type of DA and noradrenergio neurons in the central nervous system of zebrafish larvae. The most extensive DA projections are established by posterior tubercular otp-dependent neurons, with individual somata integrating the ascending DA system, the descending diencephalospinal, as well as the endohypothalamic circuitry. These findings suggest a major role in the modulation of physiology and behaviour for otp-dependent DA neurons, which correlate with the mammalian A11 group. We further identified an endogenous subpallial DA system that not only provides most of the local DA projections, but also connects to the ventral diencephalon. The catecholaminergic projectome map provides a framework to understand the evolution and function of these neuromodulatory systems.