Gateway compatible vectors for analysis of gene function in the zebrafish

The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos

Contribution of volcanic ashes to the regional geochemical balance: The 2008 eruption of Chaitén volcano, Southern Chile

The environmental geochemical behaviour of the rhyolitic ashes from the 2008 eruption of Chaitén volcano, Southern Chile, has been studied. After the bulk characterisation, the potential contribution to the regional geochemical fluxes was examined using: i) single batch leaching tests to provide a rapid screening of the implied major and trace elements; and ii) column experiments to evaluate the temporal mobility of leached elements. The environmental concerns of these ashes are related to the fine grained component present in each sample (independent of distance from the source), in particular the presence of cristobalite, and the geochemical hazards posed by ash-water interaction. Leaching experiments show the fast dissolution of surface salts and aerosols, which dominate over glass dissolution during the first steps of the ash-water interaction. Chaitén ashes could transfer to the environment more than 1×10 10g or 10,000metric tonnes (mt) of Cl, S, Ca, Na, Si, and K; between 1000 and 10,000mt of F, Mg, and Al; between 100 and 1000mt of As, Pb, P, Fe, Sr, Zn, Mn, and Br; between 10 and 100mt of Ba, Li, Ti, Ni, Nb, Cu, Rb, Zr, V, Mo, Co, and Sc; and less than 10mt of Cr, Sb, Ce, Ga, Cs, and Y. These results show the fertilising potential of the ashes (e.g., providing Ca and Fe) but also the input of potentially toxic trace elements (e.g., F and As) in the regional geochemical mass balance. The Chaitén results evidence lower potentials for poisoning and fertilising than low silica ashes due to the lower contents released of practically all elements. © 2012 Elsevier B.V.We acknowledge the technical support of ICTJA-CSIC labGEOTOP and DRX Surveys (M. Rejas and J. Elvira) and the Scientific-Technical Surveys of the University of Barcelona in the analytical work. This study was carried out in the framework of the PEGEFA Working Group (Catalonian Government “Grup de Recerca Consolidat” 2009-SGR-972), and was partly funded by the Projects ASH and QUECA of the Spanish Ministry of Science and Technology (CGL2008-00099 and CGL2011-23307) and the FPU Grant of the Spanish Ministry of Education of one of the authors (F. Ruggieri, Ref. AP2006-04592).Peer Reviewe

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene

Expression of RPRM/rprm in the olfactory system of embryonic zebrafish (Danio rerio)

The Reprimo (RPRM) family is composed of highly conserved single-exon genes. The expression pattern of this gene family has been recently described during zebrafish (Danio rerio) embryogenesis, and primarily locates in the nervous system. Its most characterized member, RPRM, which duplicated to give rise rprma and rprmb in the fish lineage, is known to act as a tumor-suppressor gene in mammalian models. Here, we describe in detail the spatiotemporal expression of three rprm genes (rprma, rprmb, and rprml) within distinct anatomical structures in the developing peripheral and central nervous system. In the zebrafish, rprma mRNA is expressed in the olfactory placodes (OP) and olfactory epithelium (OE), rprmb is observed in the tectum opticum (TeO) and trigeminal ganglion (Tg), whereas rprml is found primarily in the telencephalon (Tel). At protein level, RPRM is present in a subset of cells in the OP, and neurons in the OE, TeO, hindbrain and sensory peripheral structures. Most importantly, the expression of RPRM has been conserved between teleosts and mammals. Thus, we provide a reference dataset describing the expression patterns of RPRM gene products during zebrafish and mouse development as a first step to approach the physiological role of the RPRM gene family.BMBF-CONICYT 20140027 CONICYT-ANILLO ACT1402 CRP-ICGEB CHL15-01 FONDECYT 1160627 1140970 1151411 IMII P09/016-F CONICYT-FONDAP 1513001

Reprimo tissue-specific expression pattern is conserved between zebrafish and human

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRMand RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRMtranscripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.BMBF-CONICYT 20140027 / CONICYT-ANILLO ACT1402 / CRP-ICGEB, CH15-01/ FONDECYT, 1160627, 1140970, 1151411 / BMRC, 13CTI-21526-P6, IMII P09/016-F / CONICYT-FONDAP, 1513001

Zebrafish ae2.2 encodes a second slc4a2 anion exchanger

The genome of zebrafish (Danio rerio) encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish ae2 gene (Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. Am J Physiol Renal Physiol Renal Physiol 289: F835-F849, 2005), now called ae2.1. We now report the structural and functional characterization of Ae2.2, the product of the second zebrafish Ae2 gene, ae2.2. The ae2.2 gene of zebrafish linkage group 24 encodes a polypeptide of 1,232 aa in length, sharing 70% amino acid identity with zebrafish Ae2.1 and 67% identity with mouse AE2a. Zebrafish Ae2.2 expressed in Xenopus oocytes encodes a 135-kDa polypeptide that mediates bidirectional, DIDS-sensitive Cl(-)/Cl(-) exchange and Cl(-)/HCO(3)(-) exchange. Ae2.2-mediated Cl(-)/Cl(-) exchange is cation independent, voltage insensitive, and electroneutral. Acute regulation of anion exchange mediated by Ae2.2 includes activation by NH(4)(+) and independent inhibition by acidic intracellular pH and by acidic extracellular pH. In situ hybridization reveals low-level expression of Ae2.2 mRNA in zebrafish embryo, most notably in posterior tectum, eye, pharynx, epidermal cells, and axial vascular structures, without notable expression in the Ae2.1-expressing pronephric duct. Knockdown of Ae2.2 mRNA, of Ae2.1 mRNA, or of both with nontoxic or minimally toxic levels of N-morpholino oligomers produced no grossly detectable morphological phenotype, and preserved normal structure of the head and the pronephric duct at 24 h postfertilization

<i>RPRM</i> expression patterns are conserved between zebrafish and human brain.

<p>(A-D) <i>RPRM</i> expression patterns were examined using whole-mount in situ hybridization in wild-type embryos at 28 and (E-H) 72 hours post-fertilization [hpf]. (A-D) Frontal views of the embryo heads. (E-H) Dorsal views of the embryo heads. (I-L) Retina cross-sections. (A-C) At 28 hpf <i>rprma</i>, <i>rprmb</i> and <i>rprml</i> transcripts are expressed in neuronal populations such as dorsal thalamus (DT), preoptic region (Po) and ventral thalamus, respectively (black arrows). (D) <i>rprm3</i> is ubiquitously expressed throughout the brain. (E-G) At 72hpf <i>rprma</i> and <i>rprml</i> are expressed in the DT and the VT, respectively (white arrows), while <i>rprmb</i> is not expressed in those regions (asterisks). (H) At the same developmental stage, <i>rprm3</i> mRNA is expressed in the Po and the optic chiasma (Oc). (I-L) Cross sections of the retina. (I, J, L) <i>rprma</i>, <i>rprmb</i> and <i>rprm3</i> expression are restricted in the retina to the retinal ganglion cell layer (RGL, black arrow). (K) In contrast, <i>rprml</i> transcript expression is absent in the RGL (asterisk). (M, M’, N, N’) IHC staining for <i>RPRM</i> of white and grey matter sections from adult human samples (400x; inset magnifications 600x). (M-N) <i>RPRM</i> protein is expressed in the cytoplasm and axons of neurons (black arrowheads). (O-O’) <i>RPRM</i> protein is expressed in the nuclei of astrocytes.</p

miR-451 protects against erythroid oxidant stress by repressing 14-3-3ζ

The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3ζ, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451−/− erythroblasts, 14-3-3ζ accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3ζ in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3ζ protects miR-144/451−/− erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor