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During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model.

Correction to: Cell Death and Disease (2013) 4, e500; doi:10.1038/cddis.2013.32; published online 21 February 2013 Since the publication of this article the authors have noticed that the Authors Contribution section was omitted. The error has now been rectified. The article with the Authors Contribution appears online together with this corrigendum.

The transcription factor GHF1/Pit1, required for the expression of the prolactin (PRL) and other pituitary-specific genes, is highly conserved from fish to mammals but the mechanisms by which it activates transcription are poorly understood. The activity of the promoter (-627/+15 region) of the rainbow trout PRL (tPRL) gene fused to the luciferase reporter gene was studied using GHF1-expressing rat pituitary GC cells. Nuclear extracts of GC cells produced five GHF1-specific footprints in the tPRL promoter, with the position of the two most proximal ones being highly conserved in trout and mammalian GHF1-regulated genes. Deletional and mutational analyses of the tPRL promoter showed that the most proximal GHF1 site alone is sufficient to confer sub-maximal GHF1-dependent transcriptional activity and that a glucocorticoid response element-like motif mediates dexamethasone stimulation. It is suggested that GHF1 molecules bound to different sites of the tPRL promoter cannot interact simultaneously with the transcriptional apparatus. Moreover, GHF1 and the ligand-bound glucocorticoid receptor tethered to their cognate elements in the promoter could cooperate to enhance transcription by interacting simultaneously with different members of the basal transcriptional complex.

Background Teleost fish display widespread post-embryonic neurogenesis originating from many different proliferative niches that are distributed along the brain axis. During the development of the central nervous system (CNS) different cell types are produced in a strict temporal order from increasingly committed progenitors. However, it is not known whether diverse neural stem and progenitor cell types with restricted potential or stem cells with broad potential are maintained in the teleost fish brain. Results To study the diversity and output of neural stem and progenitor cell populations in the zebrafish brain the cerebellum was used as a model brain region, because of its well-known architecture and development. Transgenic zebrafish lines, in vivo imaging and molecular markers were used to follow and quantify how the proliferative activity and output of cerebellar progenitor populations progress. This analysis revealed that the proliferative activity and progenitor marker expression declines in juvenile zebrafish before they reach sexual maturity. Furthermore, this correlated with the diminished repertoire of cell types produced in the adult. The stem and progenitor cells derived from the upper rhombic lip were maintained into adulthood and they actively produced granule cells. Ventricular zone derived progenitor cells were largely quiescent in the adult cerebellum and produced a very limited number of glia and inhibitory inter-neurons. No Purkinje or Eurydendroid cells were produced in fish older than 3 months. This suggests that cerebellar cell types are produced in a strict temporal order from distinct pools of increasingly committed stem and progenitor cells. Conclusions Our results in the zebrafish cerebellum show that neural stem and progenitor cell types are specified and they produce distinct cell lineages and sub-types of brain cells. We propose that only specific subtypes of brain cells are continuously produced throughout life in the teleost fish brain. This implies that the post-embryonic neurogenesis in fish is linked to the production of particular neurons involved in specific brain functions, rather than to general, indeterminate growth of the CNS and all of its cell types.

A recent theory on the evolution of sexuality, has hypothesized heritable variation in the functional properties of centrosomes, leading to competition for the organization of the mitotic spindle when different centrosomes enter a common cytoplasm. We present here data on polyethylene glycol-induced polykaryocytes of cultured Chinese hamster fibroblasts indirectly supporting centrosome competition. On the assumption that sib centrosomes are similar and variation increases with cell generations, the frequencies of multipolar mitoses were compared in cultures fused under conditions favoring sib cell fusion or fusion of distantly related cells. Multipolar mitoses were considerably more frequent in the former, when the average difference between pairs of centrosomes was assumed to be too small for one centrosome to "outcompete" the other.

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left–right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.

Summary While endothelial cell (EC) function is influenced by mitochondrial metabolism, the role of mitochondrial dynamics in angiogenesis, the formation of new blood vessels from existing vasculature, is unknown. Here we show that the inner mitochondrial membrane mitochondrial fusion protein optic atrophy 1 (OPA1) is required for angiogenesis. In response to angiogenic stimuli, OPA1 levels rapidly increase to limit nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling, ultimately allowing angiogenic genes expression and angiogenesis. Endothelial Opa1 is indeed required in an NFκB-dependent pathway essential for developmental and tumor angiogenesis, impacting tumor growth and metastatization. A first-in-class small molecule-specific OPA1 inhibitor confirms that EC Opa1 can be pharmacologically targeted to curtail tumor growth. Our data identify Opa1 as a crucial component of physiological and tumor angiogenesis.