Transgenic labeling of the zebrafish pronephric duct and tubules using a promoter from the enpep gene.
Ontology highlight
ABSTRACT:
SUBMITTER: Seiler C
PROVIDER: S-EPMC4670236 | biostudies-literature | 2011 Jan-Feb
REPOSITORIES: biostudies-literature
Publications
Transgenic labeling of the zebrafish pronephric duct and tubules using a promoter from the enpep gene.
Seiler Christoph C Pack Michael M
Gene expression patterns : GEP 20101020 1-2
In recent years the zebrafish has become a popular model system to study organ development and disease. To facilitate these studies, genetic tools are required which allow to modify and manipulate gene expression in organs of interest. Here we describe a zebrafish 2kb glutamyl aminopeptidase (enpep) promoter fragment, and show that it can drive gene expression specifically in the kidney during early and late development. We established a stable transgenic line using this promoter fragment that h ...[more]
Similar Datasets
Project description:Pronephros, a developmental model for adult mammalian kidneys (metanephros) and a functional kidney in early teleosts, consists of glomerulus, tubule, and duct. These structural and functional elements are responsible for different kidney functions, e.g., blood filtration, waste extraction, salt recovery, and water balance. During pronephros organogenesis, cell differentiation is a key step in generating different cell types in specific locations to accomplish designated functions. However, it is poorly understood what molecules regulate the differentiation of different cell types in different parts of the kidney. Two types of epithelial cells, multi-cilia cells and principal cells, are found in the epithelia of the zebrafish distal pronephric duct. While the former is characterized by at least 15 apically localized cilia and expresses centrin2 and rfx2, the latter is characterized by a single primary cilium and sodium pumps. Multi-cilia cells and principal cells differentiate from 17.5 hours post-fertilization onwards in a mosaic pattern. Jagged2a-Notch1a/Notch3-Her9 is responsible for specification and patterning of these two cell types through a lateral inhibition mechanism. Furthermore, multi-cilia cell hyperplasia was observed in mind bomb mutants and Mind bomb was shown to interact with Jagged2a and facilitate its internalization. Taken together, our findings add a new paradigm of Notch signaling in kidney development, namely, that Jagged2a-Notch signaling modulates cell fate choice in a nephric segment, the distal pronephric duct.
| S-EPMC1781496 | biostudies-literature
Project description:The aim of this study was to isolate cis-acting regulatory elements for the generation of transgenic zebrafish models of neurodegeneration. Zebrafish enolase-2 (eno2) showed neuronal expression increasing from 24 to 72 h post-fertilization (hpf) and persisting through adulthood. A 12 kb eno2 genomic fragment, extending from 8 kb upstream of exon 1 to exon 2, encompassing intron 1, was sufficient to drive neuronal reporter gene expression in vivo over a similar time course. Five independent lines of stable Tg(eno2 : GFP) zebrafish expressed GFP widely in neurons, including populations with relevance to neurodegeneration, such as cholinergic neurons, dopaminergic neurons and cerebellar Purkinje cells. We replaced the exon 2-GFP fusion gene with a cDNA encoding the 4-repeat isoform of the human microtubule-associated protein Tau. The first intron of eno2 was spliced with high fidelity and efficiency from the chimeric eno2-Tau transcript. Tau was expressed at approximately 8-fold higher levels in Tg(eno2 : Tau) zebrafish brain than normal human brain, and localized to axons, neuropil and ectopic neuronal somatic accumulations resembling neurofibrillary tangles. The 12 kb eno2 promoter drives high-level transgene expression in differentiated neurons throughout the CNS of stable transgenic zebrafish. This regulatory element will be useful for the construction of transgenic zebrafish models of neurodegeneration.
| S-EPMC2095798 | biostudies-other
Project description:The zebrafish provides a useful experimental system for investigations of aural development. To permit the controlled expression of transgenes in developing hair cells, we isolated the genomic control regions of the parvalbumin 3a (pvalb3a) and parvalbumin 3b (pvalb3b) genes. Deletion analysis and somatic-cell transgenesis restricted the cis-acting control regions for hair cells to as little as 484base pairs for pvalb3a and 650base pairs for pvalb3b. Using both meganuclease-mediated and standard methods, we produced transgenic animals that transmit transgenes through their germ lines. These fish express GFP in hair cells in the inner ear and lateral line. Two stable transgenic lines express GFP prior to hair-bundle formation, so the associated promoter constructs are suitable for manipulating gene expression during bundle development. We additionally identified a transgenic line that offers variable labeling of supporting cells.
| S-EPMC2863287 | biostudies-literature
Project description:Vertebrate glomerular podocytes possess a highly sialylated transmembrane glycoprotein, Podocalyxin. In mammals, the sialic acid of Podocalyxin plays a crucial role in the formation of the characteristic podocyte architecture required for glomerular filtration. We examined the function of Podocalyxin in the developing zebrafish pronephros by disrupting the expression of podocalyxin through the use of morpholino antisense oligonucleotides. Podocalyxin was localized at the apical membrane of podocytes throughout pronephric glomerular development in zebrafish. Translational blocking of podocalyxin expression resulted in pericardial edema and a hypoplastic glomerulus. Whereas regular foot processes with a slit diaphragm covered 66.7 ± 7.8% of the urinary surface of glomerular basement membrane in control fish, only 14.4 ± 7.5% of this area was covered with regular foot processes in the translationally-blocked morphants. Splice blocking of podocalyxin exon 2, which partially encodes the bulky mucin domain with extensive sialic acid-containing sugar chains, resulted in the deletion of 53% of mucin domain-coding sequence from podocalyxin mRNA. Approximately 40% of these splice-blocked morphants had mild pericardial edema. Although the pronephric glomerulus in the splice-blocked morphants exhibited almost normal appearance with developed glomerular capillaries and mesangium, they had only 36.3 ± 6.9% of the area covered with regular foot processes. In conclusion, Podocalyxin is predominantly expressed in the podocytes and plays a distinct role in the formation of the podocyte foot processes with a slit diaphragm during zebrafish pronephric development.
| S-EPMC3819108 | biostudies-literature
Project description:Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.
| S-EPMC5333796 | biostudies-literature
Project description:The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.
| S-EPMC3265064 | biostudies-literature
Project description:Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.
| S-EPMC3142191 | biostudies-literature
Project description:The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706?C?>?T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.
| S-EPMC5309805 | biostudies-literature
Project description:Endocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the scavenger receptor megalin and its co-receptor cubilin mediate endocytosis of low molecular weight proteins from the renal filtrate. However, the extent to which megalin endocytosis relies on different components of the trafficking machinery remains relatively poorly defined in vivo. In this study, we identify a functional requirement for the F-BAR protein pacsin2 in endocytosis in the renal proximal tubule of zebrafish larvae. Pacsin2 is expressed throughout development and in all zebrafish tissues, similar to the mammalian orthologue. Within renal tubular epithelial cells, pacsin2 is enriched at the apical pole where it is localised to endocytic structures. Loss of pacsin2 results in reduced endocytosis within the proximal tubule, which is accompanied by a reduction in the abundance of megalin and endocytic organelles. Our results indicate that pacsin2 is required for efficient endocytosis in the proximal tubule, where it likely cooperates with other trafficking machinery to maintain endocytic uptake and recycling of megalin.
| S-EPMC9235069 | biostudies-literature
Project description:BACKGROUND: How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. RESULTS: We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. CONCLUSION: These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation.
| S-EPMC1877083 | biostudies-other