D. Ferrandon, L. Elphick, C. Nüsslein-Volhard and D. St Johnston (1994). Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 79, 1221-32.
Staufen protein is required in order to anchor bicoid (bcd) mRNA at the anterior pole of the Drosophila egg. Here we show that staufen protein colocalizes with bcd mRNA at the anterior, and that this localization depends upon its association with the mRNA. Upon injection into the embryo, bcd transcripts specifically interact with staufen, and we have mapped the sequences required to three regions of the 3'UTR, each of which is predicted to form a long stem-loop. The resulting staufen-bcd 3'UTR complexes form particles that show a microtubule-dependent localization. Since staufen is also transported with oskar (osk) mRNA during oogenesis, staufen associates specifically with both osk and bcd mRNAs to mediate their localizations, but at two distinct stages of development.
J. Großhans, A. Bergmann, P. Haffter and C. Nüsslein-Volhard (1994). Activation of the kinase Pelle by Tube in the dorsoventral signal transduction pathway of Drosophila embryo [see comments]. Nature 372, 563-6.
The concentration of Dorsal protein in the nucleus determines cell fate along the dorsoventral axis of the Drosophila embryo. The dorsal-group genes and the cactus gene are required for production and transmission of a localized signal on the ventral side of the embryo which determines the position of the highest nuclear concentration of Dorsal protein. The ventralizing signal produced in somatic cells is transmitted through the perivitelline space to the integral membrane protein Toll. Inside the embryo it leads to dissociation of the cytoplasmic Dorsal-Cactus complex and subsequent nuclear localization of Dorsal protein. Two components are known to mediate the signal transduction between Toll and Dorsal-Cactus: Pelle, a serine/threonine protein kinase, and Tube, a protein with an unknown biochemical activity. Here we construct gain-of-function alleles of pelle and tube and show that pelle functions downstream of tube. In addition, Pelle and Tube interact directly with one another. We propose that Tube is a direct activator of the protein kinase Pelle.
K. Isoda and C. Nüsslein-Volhard (1994). Disulfide cross-linking in crude embryonic lysates reveals three complexes of the Drosophila morphogen dorsal and its inhibitor cactus. Proc Natl Acad Sci U S A 91, 5350-4.
In Drosophila embryos dorsoventral polarity is determined by a concentration gradient of dorsal (dl) protein in the nuclei formed by the differential regulation of nuclear localization of dl protein. cactus (cact) represses the nuclear localization of dl protein. By introducing intermolecular disulfide bonds in homogenates of embryos, we detected three complexes of dl and/or cact proteins. Complex 1 (190 kDa) is a dl protein homodimer (dl2). Complex 2 (270 kDa) consists of one complex 1 and one cact molecule (dl2cact). Complex 3 (200 kDa) is a cact protein complex that does not contain dl protein. In wild-type embryos dl2cact was detected as the major form of dl protein, and dl2 was minor. With this assay virtually no dl monomer is detected. Analysis of the dl protein complexes in ventralized and dorsalized mutant embryos indicates that dl2cact is a cytoplasmic form, whereas dl2 is localized mainly in the nuclei. It seems that a small amount of dl2 is also present in the cytoplasm.
M. C. Mullins, M. Hammerschmidt, P. Haffter and C. Nüsslein-Volhard (1994). Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Curr Biol 4, 189-202.
BACKGROUND: In Drosophila melanogaster and Caenorhabditis elegans, the elucidation of developmental mechanisms has relied primarily on the systematic induction and isolation of mutations in genes with specific functions in development. Such an approach has not yet been possible in a vertebrate species, owing to the difficulty of analyzing and keeping a sufficiently high number of mutagenized lines of animals. RESULTS: We have developed the methods necessary to perform large-scale saturation screens for mutations affecting embryogenesis in the zebrafish, Danio (Brachydanio) rerio. Firstly, a new aquarium system was developed to raise and keep large numbers of strains of genetically different fish safely and with little maintenance care. Secondly, by placing adult male fish in water containing the chemical mutagen, ethylnitrosourea, we induced point mutations in premeiotic germ cells with a rate of one to three mutations per locus per 1,000 mutagenized haploid genomes. This rate, which is similar to the mutagenesis rates produced by ethylmethanesulfonate in Drosophila, was determined for alleles at four different pigmentation genes. Finally, in a pilot screen in which mutagenized fish were inbred for two generations and scored for embryonic mutants, we isolated 100 recessive mutations with phenotypes visible in the homozygous embryos. CONCLUSION: The high rate of induction and recovery of point mutations, in addition to an efficient aquarium system to house large numbers of mutagenized lines, means that it is now possible to perform saturation mutagenesis screens in a vertebrate, similar to those done in invertebrates.
C. Nüsslein-Volhard (1994). Of flies and fishes. Science 266, 572-4.
S. Schulte-Merker, F. J. van Eeden, M. E. Halpern, C. B. Kimmel and C. Nüsslein-Volhard (1994). no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 120, 1009-15.
The mouse T (Brachyury) gene is required for normal mesoderm development and the extension of the body axis. Recently, two mutant alleles of a zebrafish gene, no tail (ntl), have been isolated (Halpern, M. E., Ho., R. K., Walker, C. and Kimmel, C. B. (1993) Cell 75, 99-111). ntl mutant embryos resemble mouse T/T mutant embryos in that they lack a differentiated notochord and the caudal region of their bodies. We report here that this phenotype is caused by mutation of the zebrafish homologue of the T gene. While ntl embryos express mutant mRNA, they show no nuclear protein product. Later, expression of mRNA in mutants, but not in wild types, is greatly reduced along the dorsal midline where the notochord normally forms. This suggests that the protein is required for maintaining transcription of its own gene.
GenBank accession no.: X71596
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S. Schulte-Merker, M. Hammerschmidt, D. Beuchle, K. W. Cho, E. M. De Robertis and C. Nüsslein-Volhard (1994a). Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos. Development 120, 843-52.
goosecoid is an immediate early gene expressed at the dorsal blastoporal lip of the Xenopus gastrula. Microinjection experiments have suggested a direct role for goosecoid in organizing the dorsoventral axis of the frog embryo. Here we characterize the zebrafish homologue of goosecoid (gsc) and compare its expression to that of Brachyury or no tail (ntl), another immediate early gene required in developing mesoderm. We show that gsc exhibits two independent phases of expression: an early one in cells anterior to the presumptive notochord, but not in cells of the notochord itself, and a later one in neural crest derivatives in the larval head. Zygotic gsc transcripts are detected soon after the midblastula transition, and at the blastula stage form a gradient with a maximum at the dorsal side. Use of gsc as a dorsal marker allowed us to demonstrate that ntl expression is initially activated at the dorsal side of the blastula. At this early stage, gsc and ntl show overlapping domains of expression and are co-expressed in cells at the dorsal midline of the early gastrula. However, gsc- and ntl-expressing cells become separated in the course of gastrulation, with gsc being expressed in the axial hypoblast (prechordal plate) anterior to the ntl-expressing presumptive notochord cells. Studies with mutant embryos suggest that gsc is independent of ntl function in vivo.
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