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Arising from: A. V. Gore et al. Nature 438, 1030-1035 (2005). In fish and amphibians, the dorsal axis is specified by the asymmetric localization of maternally provided components of the [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.01.014 Byline: Srinivas Ramasamy (a)(b), Hui Wang (a), Helen Ngoc Bao Quach (a), Karuna Sampath (a)(b) Keywords: Antisense morpholino oligonucleotides; Cell death; Dominant negative proteins; Germ line; Double stranded RNA binding domain; Cell migration; nanos1; Neurons; Primordial germ cells; staufen; stau1; stau2; vasa; Zebrafish Abstract: In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690-700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration. Author Affiliation: (a) Vertebrate Development Group, Temasek Life Sciences Laboratory, 1 Research link, National University of Singapore, 117604, Singapore (b) Department of Biological Sciences, National University of Singapore, Singapore Article History: Received 27 July 2005; Revised 11 January 2006; Accepted 12 January 2006

Zebrafish cyclops (cyc) mutations lead to deficiencies in the dorsal mesendoderm and ventral neural tube, resulting in neural defects and cyclopia. It is reported that cyc encodes a transforming growth factor-beta (TGF-beta) related intercellular signalling molecule, similar to mouse nodal. Sonic hedgehog-expressing cells of the ventral brain and floorplate are restored with injection of cyc RNA. It is suggested that cyc offers an important non-cell-autonomous signal at gastrulation.

The nodal gene is asymmetrically expressed around the node and in the lateral plate mesoderm (LPM) in mouse embryo at a stage similar to that in Xenopus nodal related-1 gene in frogs. Whole-mount in situ hybridization is used to express the nodal in mutant situs inversus viscerum (iv) and the inversion of embryo turning (inv). The nodal is consistently expressed on the opposite side of LPM in inv, whereas the expression of nodal in iv is complex. Both the iv and inv mutants significantly affect the left-right development.

Author(s): Aniket V. Gore [1, 4]; Albert Cheong [1, 4]; Patrick C. Gilligan [1]; Karuna Sampath [1, 2, 3] Replying to: J. T. Bennett et al. Nature 450, doi: 10.1038/nature06314 [...]

Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the β- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton. [ABSTRACT FROM AUTHOR]