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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 -
Cells May 2020Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional...
Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.
Topics: Animals; Biological Assay; Embryo, Nonmammalian; Fluorescein-5-isothiocyanate; Heart Function Tests; Heart Rate; Kidney; Larva; Pronephros; Zebrafish
PubMed: 32443839
DOI: 10.3390/cells9051269 -
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 -
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 -
BMC Developmental Biology Dec 2019Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate...
BACKGROUND
Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking.
RESULTS
Here, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf.
CONCLUSIONS
Our results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos.
Topics: Animals; Central Nervous System; Conserved Sequence; Female; Gastrointestinal Tract; Gene Expression Regulation, Developmental; Maternal Inheritance; Multigene Family; Protein Domains; Tissue Distribution; Zebrafish; Zebrafish Proteins; rab GTP-Binding Proteins
PubMed: 31884948
DOI: 10.1186/s12861-019-0207-7 -
Journal of the American Society of... Jan 2020Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in , which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction...
BACKGROUND
Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in , which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant causes these symptoms isn't clear.
METHODS
We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 knockout cells, multiple PT cell lines treated with -targeting siRNA, and in -mutant zebrafish.
RESULTS
OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both mutant zebrafish and zebrafish injected with morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment.
CONCLUSIONS
Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.
Topics: Cell Line; Humans; Kidney Tubules, Proximal; Models, Biological; Mutation; Oculocerebrorenal Syndrome; Phosphoric Monoester Hydrolases; Proteins
PubMed: 31676724
DOI: 10.1681/ASN.2019020125 -
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 -
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 -
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 -
Genes Jun 2019Ankyrin repeats, the most common protein-protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin...
Ankyrin repeats, the most common protein-protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin repeat-containing proteins play diverse biological functions. Here, we identified the gene ankrd45, which encodes a novel, two ankyrin repeat-containing protein. Zebrafish ankrd45 displayed a tissue specific expression pattern during early development, with high expression in ciliated tissues, including otic vesicles, Kupffer's vesicles, pronephric ducts, and floor plates. Surprisingly, zebrafish ankrd45 mutants were viable and developed grossly normal cilia. In contrast, mutant larvae developed enlarged livers when induced with liver specific expression of Kras, one of the common mutations of KRAS that leads to cancer in humans. Further, histological analysis suggested that multiple cysts developed in the mutant liver due to cell apoptosis. Similarly, knockdown of ANKRD45 expression with either siRNA or CRISPR/Cas9 methods induced apoptosis in cultured cells, similar to those in zebrafish ankrd45 mutant livers after induction. Using different cell lines, we show that the distribution of ANKRD45 protein was highly dynamic during mitosis. ANKRD45 is preferably localized to the midbody ring during cytokinesis. Together, our results suggest that Ankrd45 is a novel ankyrin repeat protein with a conserved role during cell proliferation in both zebrafish embryos and mammalian cells.
Topics: Animals; Ankyrin Repeat; Body Patterning; CRISPR-Cas Systems; Cell Proliferation; Cilia; Embryonic Development; Gene Expression Regulation, Developmental; HeLa Cells; Humans; Larva; Mitosis; Zebrafish; Zebrafish Proteins
PubMed: 31208154
DOI: 10.3390/genes10060462