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Scientific Reports Nov 2021The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads... (Comparative Study)
Comparative Study
The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.
Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Genotype; Kidney Diseases; Larva; MicroRNAs; Phenotype; Podocytes; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish
PubMed: 34819534
DOI: 10.1038/s41598-021-00075-2 -
PLoS Genetics Mar 2020E2f5 is a member of the E2f family of transcription factors that play essential roles during many cellular processes. E2f5 was initially characterized as a...
E2f5 is a member of the E2f family of transcription factors that play essential roles during many cellular processes. E2f5 was initially characterized as a transcriptional repressor in cell proliferation studies through its interaction with the Retinoblastoma (Rb) protein for inhibition of target gene transcription. However, the precise roles of E2f5 during embryonic and post-embryonic development remain incompletely investigated. Here, we report that zebrafish E2f5 plays critical roles during spermatogenesis and multiciliated cell (MCC) differentiation. Zebrafish e2f5 mutants develop exclusively as infertile males. In the mutants, spermatogenesis is arrested at the zygotene stage due to homologous recombination (HR) defects, which finally leads to germ cell apoptosis. Inhibition of cell apoptosis in e2f5;tp53 double mutants rescued ovarian development, although oocytes generated from the double mutants were still abnormal, characterized by aberrant distribution of nucleoli. Using transcriptome analysis, we identified dmc1, which encodes an essential meiotic recombination protein, as the major target gene of E2f5 during spermatogenesis. E2f5 can bind to the promoter of dmc1 to promote HR, and overexpression of dmc1 significantly increased the fertilization rate of e2f5 mutant males. Besides gametogenesis defects, e2f5 mutants failed to develop MCCs in the nose and pronephric ducts during early embryonic stages, but these cells recovered later due to redundancy with E2f4. Moreover, we demonstrate that ion transporting principal cells in the pronephric ducts, which remain intercalated with the MCCs, do not contain motile cilia in wild-type embryos, while they generate single motile cilia in the absence of E2f5 activity. In line with this, we further show that E2f5 activates the Notch pathway gene jagged2b (jag2b) to inhibit the acquisition of MCC fate as well as motile cilia differentiation by the neighboring principal cells. Taken together, our data suggest that E2f5 can function as a versatile transcriptional activator and identify novel roles of the protein in spermatogenesis as well as MCC differentiation during zebrafish development.
Topics: Animals; Cell Cycle Proteins; Cell Differentiation; Cilia; DNA-Binding Proteins; E2F5 Transcription Factor; Male; Receptors, Notch; Signal Transduction; Spermatogenesis; Zebrafish; Zebrafish Proteins
PubMed: 32196499
DOI: 10.1371/journal.pgen.1008655 -
Aquatic Toxicology (Amsterdam,... Aug 2020In the present research, the effects of exposure to a sublethal concentration of zinc (Zn) on metal and ion homeostasis, and the regulation and the localization of...
In the present research, the effects of exposure to a sublethal concentration of zinc (Zn) on metal and ion homeostasis, and the regulation and the localization of various Zn transporters (i.e., the Zrt-Irt Like Protein (ZIP) family of Zn transporters), were investigated in zebrafish (Danio rerio) during early development. Exposure to an elevated level of Zn [4 μM (high) vs. 0.25 μM (control)] from 0 day post-fertilization (dpf) resulted in a significant increase in the whole body content of Zn at 5 dpf. A transient decrease in the whole body calcium (Ca) level was observed in 3 dpf larvae exposed to high Zn. Similarly, whole body nickel (Ni) and copper (Cu) contents were also reduced in 3 dpf larvae exposed to high Zn. Importantly, the magnitude of reduction in whole body Ni and Cu contents following Zn exposure was markedly higher than that in Ca content, suggesting that internal Ni and Cu balance were likely more sensitive to Zn exposure in developing zebrafish. Exposure to high Zn altered the mRNA expression levels of specific zip transporters, with an increase in zip1 (at 3 dpf) and zip8 (at 5 dpf), and a decrease in zip4 (at 5 dpf). The expression levels of most zip transporters tended to decrease from 3 dpf to 5 dpf with the exception of zip4 and zip8. Results from in situ hybridization revealed that several zip transporters exhibited distinct spatial distribution (e.g., zip8 in the intestinal tract, zip14 in the pronephric tubules). Overall, our findings suggested that exposure to sublethal concentrations of Zn disrupts the homeostasis of essential metals during early development and that different ZIP transporters may play unique roles in regulating Zn homeostasis in various organs in developing zebrafish.
Topics: Animals; Calcium; Cation Transport Proteins; Copper; Dose-Response Relationship, Drug; Homeostasis; Larva; No-Observed-Adverse-Effect Level; Water Pollutants, Chemical; Zebrafish; Zebrafish Proteins; Zinc
PubMed: 32610223
DOI: 10.1016/j.aquatox.2020.105524 -
Environmental Science and Pollution... Feb 2023The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any...
The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any defects in kidney development and function are likely associated with a decreased ability to osmoregulate. Previous work has shown that early-life stage (ELS) zebrafish (Danio rerio) acutely exposed to Deepwater Horizon (DWH) crude oil exhibit transcriptional changes in key genes involved in pronephros development and function, as well as pronephric morphological defects and whole-animal osmoregulatory impairment. The objective of this study was to examine the acute effects of crude oil exposure during zebrafish ELS on pronephros function by assessing its fluid clearance capacity and glomerular filtration integrity. Following a 72-h exposure to control conditions, 20% or 40% dilutions of high-energy water-accommodated fractions (HEWAF) of DWH crude oil, zebrafish were injected into the common cardinal vein either with fluorescein-labeled (FITC) 70-kDa dextran to assess glomerular filtration integrity or with FITC-inulin to assess pronephric clearance capacity. Fluorescence was quantified after the injections at predetermined time intervals by fluorescence microscopy. The results demonstrated a diminished pronephric fluid clearance capacity and failed glomerular perfusion when larvae were exposed to 40% HEWAF dilutions, whereas only a reduced glomerular filtration selectivity was observed in zebrafish previously exposed to the 20% HEWAF dilution.
Topics: Animals; Zebrafish; Petroleum; Kidney; Larva; Petroleum Pollution; Water Pollutants, Chemical
PubMed: 36280635
DOI: 10.1007/s11356-022-23805-z -
International Journal of Molecular... Mar 2024Mutations of coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many...
Mutations of coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish () as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human paralogs, and . Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in -morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.
Topics: Animals; Humans; Polycystic Kidney, Autosomal Dominant; Zebrafish; Leucine; TRPP Cation Channels; Polycystic Kidney Diseases; Laminin; Kidney
PubMed: 38474131
DOI: 10.3390/ijms25052886 -
BioMed Research International 2019The 6-O-endosulfatases (sulfs) are important enzymatic components involved in the regulation of heparan sulfate by altering the sulfatation pattern. Specifically in the...
The 6-O-endosulfatases (sulfs) are important enzymatic components involved in the regulation of heparan sulfate by altering the sulfatation pattern. Specifically in the kidney, sulfs have been implicated in the glomerular podocyte-endothelial cell crosstalk and in the preservation of the glomerular filtration barrier (GFB) in different mouse models. Since it has been shown that in zebrafish larvae, Sulf1, Sulf2a, and Sulf2b are expressed in the pronephric kidney we set out to establish if a reduction in sulf expression leads to GFB dysfunction. Here, we show that a reduced sulf expression following morpholino (MO) induced knockdown in zebrafish larvae promotes damage to the GFB leading to renal plasma protein loss from the circulation. Moreover, a combined knockdown of Sulf1, Sulf2a, and Sulf2b is associated with severe morphologic changes including narrowing of the fenestration between glomerular endothelial cells as well as thickening of the glomerular basement membrane and podocyte foot process effacement, suggesting that glomerular damage is an underlying cause of the circulatory protein loss observed after MO injection. Additionally, we show that a decrease in sulf expression reduces the bioavailability of VegfA in the glomerulus of the pronephros, which may contribute to the structural changes observed in the glomeruli of morphant fish. Furthermore, consistent with previous results, knockdown of the sulfs is associated with arteriovenous malformations in particular in the tail region of the larvae. Overall, taken together our results suggest that 6-O-endosulfatases are important in the preservation of GFB integrity and a reduction in their expression levels induces phenotypic changes that are indicative of renal protein loss.
Topics: Animals; Endothelial Cells; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Knockdown Techniques; Glomerular Basement Membrane; Morpholinos; Podocytes; Sulfatases; Zebrafish; Zebrafish Proteins
PubMed: 31428635
DOI: 10.1155/2019/4508048 -
American Journal of Physiology. Renal... May 2021Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues...
Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues and organs. Little is known about the adjustments that kidneys undergo in response to these challenges. We performed single-cell RNA sequencing of zebrafish pronephric duct cells to understand how the developing kidney responds to changes in filtered substrates and intrinsic energy requirements. We found high levels of glucose transporters early in development and increased expression of monocarboxylate transporters at later times. This indicates that the zebrafish embryonic kidney displays a high glucose transporting capacity during early development, which is replaced by the ability to absorb monocarboxylates and amino acids at later stages. This change in transport capacity was accompanied by the upregulation of mitochondrial carriers, indicating a switch to increased oxidative phosphorylation to meet the increasing energy demand of a developing kidney. The zebrafish embryonic kidney has high levels of glucose transporters during early development, which are replaced by monocarboxylate and amino acid transporters later on. Inhibition of Na-glucose cotransporter-dependent glucose transport by sotagliflozin also increased expression, supporting the idea that the glucose transport capacity is dynamically adjusted during zebrafish pronephros development. Concurrent upregulation of mitochondrial SCL25 transporters at later stages supports the idea that the pronephros adjusts to changing substrate supplies and/or energy demands during embryonic development.
Topics: Animals; Energy Metabolism; Gene Expression Profiling; Gene Expression Regulation, Developmental; Pronephros; RNA, Messenger; RNA-Seq; Single-Cell Analysis; Solute Carrier Proteins; Transcriptome; Zebrafish; Zebrafish Proteins
PubMed: 33749326
DOI: 10.1152/ajprenal.00610.2020 -
Gene Expression Patterns : GEP Jun 2020The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in both cardiac and skeletal muscles, as well as in fetal...
The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in both cardiac and skeletal muscles, as well as in fetal inner ears, with suggested roles as mechanotransductor. Recently, several mutations in the SMPX gene have been associated with X-chromosomal progressive deafness in human. However, very little information is known concerning the roles of SMPX, and no in-vivo models are currently available. Therefore, we characterized the zebrafish ortholog of SMPX to pave the way towards the establishment of a biotool for future functional studies. Despite the genome duplication occurred in the ancestry of teleosts, zebrafish retain only one copy of smpx which shares a high degree of similarity with the mammalian counterpart in terms of genomic organization, syntenic map, and encoded protein. RT-PCR, as well as whole-mount in-situ hybridization and immunofluorescence analyses, revealed that smpx is expressed in several embryonic areas starting from the 4-somite stage. Specifically, smpx mRNA marked the Kupffer's vesicle (KV), the somites, the myocardium, the hair cells of the anterior and the posterior macula of the inner ear, the pronephric ducts, and the muscles of the branchial arches, eyes and pectoral fins. According to our data, zebrafish smpx expression pattern closely resembles that observed in mouse and human, supporting the notion that zebrafish might represent a suitable in-vivo model to disclose the cellular and molecular mechanisms underlying the involvement of SMPX in development and disease.
Topics: Animals; Fluorescent Antibody Technique; Gene Expression Regulation, Developmental; In Situ Hybridization; Mice; Muscle Proteins; Mutation; Zebrafish; Zebrafish Proteins
PubMed: 32197943
DOI: 10.1016/j.gep.2020.119110 -
Human Mutation Jan 2020Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high-genetic heterogeneity. Among the many genes...
Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high-genetic heterogeneity. Among the many genes involved, either in isolated or syndromic forms, eight have been implicated in postaxial polydactyly (PAP). Among those, IQCE has been recently identified in a single consanguineous family. Using whole-exome sequencing in patients with uncharacterized ciliopathies, including PAP, we identified three families with biallelic pathogenic variations in IQCE. Interestingly, the c.895_904del (p.Val301Serfs*8) was found in all families without sharing a common haplotype, suggesting a recurrent mechanism. Moreover, in two families, the systemic phenotype could be explained by additional pathogenic variants in known genes (TULP1, ATP6V1B1). RNA expression analysis on patients' fibroblasts confirms that the dysfunction of IQCE leads to the dysregulation of genes associated with the hedgehog-signaling pathway, and zebrafish experiments demonstrate a full spectrum of phenotypes linked to defective cilia: Body curvature, kidney cysts, left-right asymmetry, misdirected cilia in the pronephric duct, and retinal defects. In conclusion, we identified three additional families confirming IQCE as a nonsyndromic PAP gene. Our data emphasize the importance of taking into account the complete set of variations of each individual, as each clinical presentation could finally be explained by multiple genes.
Topics: Animals; Ciliopathies; Consanguinity; Fingers; Fluorescent Antibody Technique; Gene Expression Profiling; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; Homozygote; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Pedigree; Phenotype; Polydactyly; Signal Transduction; Toes; Transcriptome; Exome Sequencing; Zebrafish
PubMed: 31549751
DOI: 10.1002/humu.23924 -
Journal of Developmental Origins of... Feb 2020Several life-threatening diseases of the kidney have their origins in mutational events that occur during embryonic development. In this study, we investigate the role...
Several life-threatening diseases of the kidney have their origins in mutational events that occur during embryonic development. In this study, we investigate the role of the Wolffian duct (WD), the earliest embryonic epithelial progenitor of renal tubules, in the etiology of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with a germline mutation of one of the two Pkd1 alleles. For the disease to occur, a second event that disrupts the expression of the other inherited Pkd1 allele must occur. We postulated that this secondary event can occur in the pronephric WD. Using Cre-Lox recombination, mice with WD-specific deletion of one or both Pkd1 alleles were generated. Homozygous Pkd1-targeted deletion in WD-derived tissues resulted in mice with large cystic kidneys and serologic evidence of renal failure. In contrast, heterozygous deletion of Pkd1 in the WD led to kidneys that were phenotypically indistinguishable from control in the early postnatal period. High-throughput sequencing, however, revealed underlying gene and microRNA (miRNA) changes in these heterozygous mutant kidneys that suggest a strong predisposition toward developing ADPKD. Bioinformatic analysis of this data demonstrated an upregulation of several miRNAs that have been previously associated with PKD; pathway analysis further demonstrated that the differentially expressed genes in the heterozygous mutant kidneys were overrepresented in signaling pathways associated with maintenance and function of the renal tubular epithelium. These results suggest that the WD may be an early epithelial target for the genetic or molecular signals that can lead to cyst formation in ADPKD.
Topics: Alleles; Animals; Disease Models, Animal; Epithelium; Female; Germ-Line Mutation; Humans; Kidney Tubules; Mice; Mice, Knockout; Polycystic Kidney, Autosomal Dominant; Renal Insufficiency; Signal Transduction; TRPP Cation Channels; Wolffian Ducts
PubMed: 31412963
DOI: 10.1017/S2040174419000436