Septins, a highly conserved family of GTP-binding proteins, were originally identified in a genetic screen for S. cerevisiae mutants defective in cytokinesis [1, 2]. In yeast, septins maintain the compartmentalization of the yeast plasma membrane during cell division by forming rings at the cortex of the bud neck, and these rings establish a lateral diffusion barrier. In contrast, very little is known about the functions of septins in mammalian cells [3, 4] including postmitotic neurons [5-7]. Here, we show that Septin 7 (Sept7) localizes at the bases of filopodia and at branch points in developing hippocampal neurons. Upon downregulation of Sept7, dendritic branching is impaired. In mature neurons, Sept7 is found at the bases of dendritic spines where it associates with the plasma membrane. Mature Sept7-deficient neurons display elongated spines. Furthermore, Sept5 and Sept11 colocalize with and coimmunoprecipitate with Sept7, thereby arguing for the existence of a Septin5/7/11 complex. Taken together, our findings show an important role for Sept7 in regulating dendritic branching and dendritic-spine morphology. Our observations concur with data from yeast, in which downregulation of septins yields elongated buds, suggesting a conserved function for septins from yeast to mammals.

Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, we found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (

Editor's Note: Toolboxes are intended to describe and evaluate methods that are becoming widely relevant to the neuroscience community or to provide a critical analysis of established techniques. For more information, see http://www.jneurosci.org/misc/ ifa_minireviews.dtl. Transfection Techniques for Neuronal CellsDaniela Karra 1 and Ralf Dahm 2,3 1 Institute for Neuropathology, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany, 2 Department of Biology, University of Padua, I-35121 Padua, Italy, and 3 Spanish National Cancer Research Centre (CNIO), E-28029 Madrid, Spain IntroductionThe transfection of nucleic acids into cells is crucial for the study of many aspects of neuronal cell biology. These include investigating gene and protein function by knocking down target proteins via RNA interference (RNAi) or microRNAs, expressing tagged proteins to track their subcellular localization, behavior, and turnover; and expressing mutant versions of proteins to study the functions of specific domains or mimic disease conditions. Moreover, reporter proteins can be used to detect intracellular ion concentrations or levels of gene expression.Despite efforts to optimize transfection techniques and protocols for neurons, no method has yet been developed that is suitable for all applications. Instead, the various established methods have their own advantages and drawbacks concerning transfection efficiency, expression levels, cell survival, and viability. Other considerations are the ease of use, reproducibility, cost, and applicability to a given experiment. Researchers therefore often face a bewildering roster of possibilities, making it difficult to decide which approach to take.In this review we provide a brief overview of methods used to transfect mam-

Gene duplication is a major driver of evolutionary divergence. In most vertebrates a single PAX6 gene encodes a transcription factor required for eye, brain, olfactory system, and pancreas development. In zebrafish, following a postulated whole-genome duplication event in an ancestral teleost, duplicates pax6a and pax6b jointly fulfill these roles. Mapping of the homozygously viable eye mutant sunrise identified a homeodomain missense change in pax6b, leading to loss of target binding. The mild phenotype emphasizes role-sharing between the co-orthologues. Meticulous mapping of isolated BACs identified perturbed synteny relationships around the duplicates. This highlights the functional conservation of pax6 downstream (3′) control sequences, which in most vertebrates reside within the introns of a ubiquitously expressed neighbour gene, ELP4, whose pax6a-linked exons have been lost in zebrafish. Reporter transgenic studies in both mouse and zebrafish, combined with analysis of vertebrate sequence conservation, reveal loss and retention of specific cis-regulatory elements, correlating strongly with the diverged expression of co-orthologues, and providing clear evidence for evolution by subfunctionalization.