-
Nature Communications Oct 2015Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF...
Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.
Topics: Animals; Blotting, Western; Carrier Proteins; Cell Polarity; Circular Dichroism; Embryo, Nonmammalian; Female; Fluorescent Antibody Technique; Gene Knockout Techniques; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Immunoprecipitation; Kidney Diseases, Cystic; Male; Microphthalmos; Microtubule-Associated Proteins; Microtubules; Mutation; Pedigree; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish; Zebrafish Proteins
PubMed: 26487268
DOI: 10.1038/ncomms9666 -
The International Journal of... 2021The molecular expression profiles of zebrafish and have not been defined to date. Phylogenetic trees of EP2a and EP4b in zebrafish and other species revealed that...
The molecular expression profiles of zebrafish and have not been defined to date. Phylogenetic trees of EP2a and EP4b in zebrafish and other species revealed that human EP4 and zebrafish EP4b were more closely related than EP2a. Zebrafish EP2a is a 281 amino acid protein which shares high identity with that of human (43%), mouse (44%), rat (43%), dog (44%), cattle (41%), and chicken (41%). Zebrafish EP4b encoded a 497 amino acid precursor with high amino acid identity to that of mammals, including human (57%), mouse (54%), rat (55%), dog (55%), cattle (56%), and chicken (54%). Whole-mount hybridization revealed that was robustly expressed in the anterior four somites at the 10-somites stages, but was absent in the somites at 19 hpf. It was observed again in the pronephric duct at 24 hpf, in the intermediate cell mass located in the trunk, and in the rostral blood island at 30 hpf. was also expressed in the notochord at 48 hpf. During somitogenesis, was highly expressed in the eyes, somites, and the trunk neural crest. From 30 to 48 hpf, could be detected in the posterior cardinal vein and the neighboring inner cell mass. From these data we conclude that and are conserved in vertebrates and that the presence of and transcripts during developmental stages infers their role during early zebrafish larval development. In addition, the variable expression of the two receptor isoforms was strongly suggestive of divergent roles of molecular regulation.
Topics: Amino Acids; Animals; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Phylogeny; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP4 Subtype; Zebrafish; Zebrafish Proteins
PubMed: 34549801
DOI: 10.1387/ijdb.210003wh -
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 -
Journal of Visualized Experiments : JoVE Feb 2015The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and...
The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria 'dipstick', which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function.
Topics: Animals; Disease Models, Animal; Female; Fluorescent Dyes; Kidney; Male; Renal Insufficiency, Chronic; Zebrafish
PubMed: 25742415
DOI: 10.3791/52540 -
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 -
International Journal of Molecular... Mar 2019The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible...
The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift17 in embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.
Topics: Adaptor Proteins, Signal Transducing; Algorithms; Animals; Carrier Proteins; Cytoskeletal Proteins; Disease Models, Animal; Embryo, Nonmammalian; Gene Knockdown Techniques; Humans; Image Processing, Computer-Assisted; Kidney; Organ Specificity; Phenotype; Polycystic Kidney Diseases; Small Molecule Libraries; Software; Workflow; Zebrafish
PubMed: 30875791
DOI: 10.3390/ijms20061290 -
American Journal of Physiology. Renal... Dec 2014The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular...
The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish.
Topics: Animals; Apolipoproteins; Carbocyanines; Dextrans; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Glomerular Filtration Barrier; Glomerular Filtration Rate; Larva; Microscopy, Confocal; Microscopy, Fluorescence; Morpholinos; Myosin Heavy Chains; Renal Elimination; Time Factors; Zebrafish; Zebrafish Proteins
PubMed: 25298528
DOI: 10.1152/ajprenal.00029.2014 -
PloS One 2015Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT), consisting mainly of protein complexes IFT-A, IFT-B,...
Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT), consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. Tetratricopeptide repeat-containing (TTC) proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs) into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, ttc4, -9c, -36, and -39c, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of ttc4 and ttc25, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia.
Topics: Animals; Carrier Proteins; Cell Differentiation; Cells, Cultured; Cilia; Epithelial Cells; Humans; Mice; Multiprotein Complexes; Oligonucleotide Array Sequence Analysis; Phenotype; Trachea; Up-Regulation; Zebrafish; Zebrafish Proteins
PubMed: 25860617
DOI: 10.1371/journal.pone.0124378 -
Developmental Biology May 2017During zebrafish embryogenesis the pronephric kidney arises from a small population of posterior mesoderm cells that then undergo expansion during early stages of renal...
During zebrafish embryogenesis the pronephric kidney arises from a small population of posterior mesoderm cells that then undergo expansion during early stages of renal organogenesis. While wnt8 is required for posterior mesoderm formation during gastrulation, it is also transiently expressed in the post-gastrula embryo in the intermediate mesoderm, the precursor to the pronephros and some blood/vascular lineages. Here, we show that knockdown of wnt8a, using a low dose of morpholino that does not disrupt early mesoderm patterning, reduces the number of kidney and blood cells. For the kidney, wnt8a deficiency decreases renal progenitor growth during early somitogenesis, as detected by EdU incorporation, but has no effect on apoptosis. The depletion of the renal progenitor pool in wnt8a knockdown embryos leads to cellular deficits in the pronephros at 24 hpf that are characterised by a shortened distal-most segment and stretched proximal tubule cells. A pulse of the canonical Wnt pathway agonist BIO during early somitogenesis is sufficient to rescue the size of the renal progenitor pool while longer treatment expands the number of kidney cells. Taken together, these observations indicate that Wnt8, in addition to its well-established role in posterior mesoderm patterning, also plays a later role as a factor that expands the renal progenitor pool prior to kidney morphogenesis.
Topics: Animals; Apoptosis; Blood Cells; Body Patterning; Cell Count; Cell Proliferation; Cytoskeletal Proteins; Embryo, Nonmammalian; Gene Knockdown Techniques; Indoles; Kidney; Kidney Tubules; Mesoderm; Morpholinos; Oximes; Pronephros; Stem Cells; Wnt Proteins; Zebrafish; Zebrafish Proteins
PubMed: 28359809
DOI: 10.1016/j.ydbio.2017.03.027 -
Developmental Biology Mar 2015The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study...
The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study this renal development process because its nephrons contain segments akin to other vertebrates, including the proximal convoluted and straight tubules (PCT, PST). The zebrafish pronephros is also associated with the corpuscles of Stannius (CS), endocrine glands that regulate calcium and phosphate homeostasis, but whose ontogeny from renal progenitors is largely mysterious. Initial patterning of zebrafish renal progenitors in the intermediate mesoderm (IM) involves the formation of rostral and caudal domains, the former being reliant on retinoic acid (RA) signaling, and the latter being repressed by elevated RA levels. Here, using expression profiling to gain new insights into nephrogenesis, we discovered that the gene single minded family bHLH transcription factor 1a (sim1a) is dynamically expressed in the renal progenitors-first marking the caudal domain, then becoming restricted to the proximal segments, and finally exhibiting specific CS expression. In loss of function studies, sim1a knockdown expanded the PCT and abrogated both the PST and CS populations. Conversely, overexpression of sim1a modestly expanded the PST and CS, while it reduced the PCT. These results show that sim1a activity is necessary and partially sufficient to induce PST and CS fates, and suggest that sim1a may inhibit PCT fate and/or negotiate the PCT/PST boundary. Interestingly, the sim1a expression domain in renal progenitors is responsive to altered levels of RA, suggesting that RA regulates sim1a, directly or indirectly, during nephrogenesis. sim1a deficient embryos treated with exogenous RA formed nephrons that were predominantly composed of PCT segments, but lacked the enlarged PST observed in RA treated wild-types, indicating that RA is not sufficient to rescue the PST in the absence of sim1a expression. Alternately, when sim1a knockdowns were exposed to the RA inhibitor diethylaminobenzaldehyde (DEAB), the CS was abrogated rather than expanded as seen in DEAB treated wild-types, revealing that CS formation in the absence of sim1a cannot be rescued by RA biosynthesis abrogation. Taken together, these data reveal previously unappreciated roles for sim1a in zebrafish pronephric proximal tubule and CS patterning, and are consistent with the model that sim1a acts downstream of RA to mitigate the formation of these lineages. These findings provide new insights into the genetic pathways that direct nephron development, and may have implications for understanding renal birth defects and kidney reprogramming.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Body Patterning; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; In Situ Hybridization; Kidney Tubules, Proximal; Nephrons; Organogenesis; Repressor Proteins; Time Factors; Tretinoin; Zebrafish; Zebrafish Proteins
PubMed: 25542995
DOI: 10.1016/j.ydbio.2014.12.020