Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte (ele) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mapping and cloning of the ele mutation showed that the affected gene is slbp, which encodes a conserved RNA stem-loop binding protein involved in replication dependent histone mRNA metabolism. Cells throughout the central nervous system remained in the cell cycle in ele mutant embryos at stages when, and locations where, post-mitotic cells have differentiated in wild-type siblings. Indeed, RNAseq analysis showed down-regulation of many genes associated with neuronal differentiation. This was coincident with changes in the levels and spatial localisation of expression of various genes implicated, for instance, in axon guidance, that likely underlie specific ele phenotypes. These results suggest that many of the cell and tissue specific phenotypes in ele mutant embryos are secondary to altered expression of modules of developmental regulatory genes that characterise, or promote transitions in, cell state and require the correct function of Slbp-dependent histone and chromatin regulatory genes

Funduscopy in Adult Zebrafish and Its Application to Isolate Mutant Strains with Ocular Defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals. A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out. Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas. In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible

The WD-repeats of Net2p Interact with Dnm1p and Fis1p to Regulate Division of Mitochondria

The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae. Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats. We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p. Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p. Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution. Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division. Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division

Zebrafish and mouse fundus images.

<p>(A–D) wild type zebrafish fundus at the level of the mid-peripheral retina (A), optic disc (B and C) and photoreceptor level, showing the presumed photoreceptor mosaic (D). Hypopigmented fundus of the zebrafish <i>albino</i> strain (E). Tortuous arteries and a darkened retina (F), black spots are visible in the retina (G and H). Fundus Image of a wild type mouse (I). Estimated scale bar: 50 µm.</p

Images of zebrafish eyes.

<p>(A) Overview of anaesthetized fish with cover glass, (B) shows a normal, transparent lens, (C–F) examples of opaque lenses (cataracts): (C) small inclusion are present in an otherwise normal lens, (D) massive cataract, (E) including cracks in the lens, and (F) membranous cataract. Scale bar: 500 µm.</p

Adult retina sections.

<p>(A) Wild type zebrafish. (B) Right eye and (C) left eye of a fish with black spots in the retina; the funduscopy of this fish is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015427#pone-0015427-g003" target="_blank">Figure 3</a> (H). Arrows indicate cells with black granules: likely macrophages filled with RPE cells or detached RPE cells. The detached RPE in (C) is a histological artefact. Scale bar: 50 µm.</p

Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits

<div><p>Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in <i>eisspalte</i> (<i>ele)</i> mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mapping and cloning of the <i>ele</i> mutation showed that the affected gene is <i>slbp</i>, which encodes a conserved RNA stem-loop binding protein involved in replication dependent histone mRNA metabolism. Cells throughout the central nervous system remained in the cell cycle in <i>ele</i> mutant embryos at stages when, and locations where, post-mitotic cells have differentiated in wild-type siblings. Indeed, RNAseq analysis showed down-regulation of many genes associated with neuronal differentiation. This was coincident with changes in the levels and spatial localisation of expression of various genes implicated, for instance, in axon guidance, that likely underlie specific <i>ele</i> phenotypes. These results suggest that many of the cell and tissue specific phenotypes in <i>ele</i> mutant embryos are secondary to altered expression of modules of developmental regulatory genes that characterise, or promote transitions in, cell state and require the correct function of Slbp-dependent histone and chromatin regulatory genes.</p></div