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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 -
Journal of the American Society of... Mar 2021Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic...
BACKGROUND
Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease.
METHODS
Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. and studies determined the functional significance of the mutations identified.
RESULTS
Three biallelic variants of the transcriptional regulator were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in embryos disrupted pronephric development. Human wild-type RNA rescued the disruption, but the three variants did not. Finally, CRISPR-mediated knockout of in human podocytes led to dysregulation of several renal developmental genes.
CONCLUSIONS
Variants in can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.
Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cell Line; Child, Preschool; DNA-Binding Proteins; Female; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Gene Knockout Techniques; Hernia, Hiatal; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Male; Microcephaly; Models, Molecular; Mutation, Missense; Nephrosis; Nephrotic Syndrome; Podocytes; Polymorphism, Single Nucleotide; Pronephros; Protein Stability; Transcription Factors; Xenopus laevis; Zinc Fingers
PubMed: 33593823
DOI: 10.1681/ASN.2020040490 -
Journal of the American Society of... Apr 2021Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.
BACKGROUND
Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.
METHODS
Transcription activator-like effector nucleases (TALEN) technology was used to generate mutant for , the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic strain or rapamycin was used to inhibit mTOR activity.
RESULTS
Adult zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants.
CONCLUSIONS
Adult zebrafish mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.
PubMed: 33574160
DOI: 10.1681/ASN.2020070991 -
Fish & Shellfish Immunology Apr 2021The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of...
The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.
Topics: Amino Acid Sequence; Animals; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Immunity; Novirhabdovirus; Phylogeny; Rhabdoviridae Infections; Sequence Alignment; Tetraspanin 30; Zebrafish
PubMed: 33556552
DOI: 10.1016/j.fsi.2021.01.016 -
Journal of Genetics and Genomics = Yi... Oct 2020The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various...
The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.
Topics: 1-Phosphatidylinositol 4-Kinase; Animals; CRISPR-Cas Systems; Cell Movement; Cilia; Embryonic Development; Gene Expression Regulation, Developmental; Humans; Phosphatidylinositol Phosphates; Sequence Deletion; Signal Transduction; Zebrafish; Zebrafish Proteins
PubMed: 33358778
DOI: 10.1016/j.jgg.2020.07.007 -
Aging Sep 2020Cellular senescence is considered a stress response imposing a stable cell cycle arrest to restrict the growth of damaged cells. More recently however, cellular...
Cellular senescence is considered a stress response imposing a stable cell cycle arrest to restrict the growth of damaged cells. More recently however, cellular senescence was identified during mouse embryo development at particular structures during specific periods of time. This programmed cell senescence has been proposed to serve developmental and morphogenetic functions and to potentially represent an evolutionary origin of senescence. Cellular senescence has also been described to take place during bird (chick and quail) and amphibian (xenopus and axoltl) development. Fish however, have been described to show a very narrow and restricted pattern of developmental cell senescence. Here we carried out a detailed characterization of senescence during zebrafish development and found it to be conserved and widespread. Apart from yolk and cloaca, previously described structures, we also identified senescence in the developing central nervous system, intestine, liver, pronephric ducts, and crystalline. Interestingly, senescence at these developing structures disappeared upon treatment with senolytic compound ABT-263, supporting their senescent identity and opening the possibility of studying the contribution of this process to development. In summary, our findings extend the description of developmentally-programmed cell senescence to lower vertebrates contributing to the notion of the relevance of this process for embryo development.
PubMed: 32991320
DOI: 10.18632/aging.103968 -
Frontiers in Cell and Developmental... 2020Previous studies in developing and zebrafish reported that the phosphate transporter is expressed in pronephric kidneys. The recent identification of as a monoallelic...
Previous studies in developing and zebrafish reported that the phosphate transporter is expressed in pronephric kidneys. The recent identification of as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog . This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest is involved in urinary tract and urorectal development and implicate as a disease-gene for BEEC.
PubMed: 32850778
DOI: 10.3389/fcell.2020.00567 -
Frontiers in Cell and Developmental... 2020Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood....
Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.
PubMed: 32754590
DOI: 10.3389/fcell.2020.00583 -
European Journal of Human Genetics :... Dec 2020Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in...
Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis.
Topics: Adolescent; Adult; Animals; Cell Line; Child; Child, Preschool; Female; Growth Differentiation Factor 6; Heterozygote; Humans; Infant; Kidney Tubules; Male; Mice; Mutation; Urogenital Abnormalities; Vesico-Ureteral Reflux; Xenopus
PubMed: 32737436
DOI: 10.1038/s41431-020-0678-9 -
Aquatic Toxicology (Amsterdam,... Aug 2020Some chemicals in the environment disrupt thyroid hormone (TH) systems leading to alterations in organism development, but their effect mechanisms are poorly understood....
Expression dynamics of genes in the hypothalamic-pituitary-thyroid (HPT) cascade and their responses to 3,3',5-triiodo-l-thyronine (T3) highlights potential vulnerability to thyroid-disrupting chemicals in zebrafish (Danio rerio) embryo-larvae.
Some chemicals in the environment disrupt thyroid hormone (TH) systems leading to alterations in organism development, but their effect mechanisms are poorly understood. In fish, this has been limited by a lack of fundamental knowledge on thyroid gene ontogeny and tissue expression in early life stages. Here we established detailed expression profiles for a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) between 24-120 h post fertilisation (hpf) and quantified their responses following exposure to 3,3',5-triiodo-L-thyronine (T3) using whole mount in situ hybridisation (WISH) and qRT-PCR (using whole-body extracts). All of the selected genes in the HPT axis demonstrated dynamic transcript expression profiles across the developmental stages examined. The expression of thyroid receptor alpha (thraa) was observed in the brain, gastrointestinal tract, craniofacial tissues and pectoral fins, while thyroid receptor beta (thrb) expression occurred in the brain, otic vesicles, liver and lower jaw. The TH deiodinases (dio1, dio2 and dio3b) were expressed in the liver, pronephric ducts and brain and the patterns differed depending on life stage. Both dio1 and dio2 were also expressed in the intestinal bulb (96-120 hpf), and dio2 expression occurred also in the pituitary (48-120 hpf). Exposure of zebrafish embryo-larvae to T3 (30 and 100 μg L) for periods of 48, 96 or 120 hpf resulted in the up-regulation of thraa, thrb, dio3b, thyroid follicle synthesis proteins (pax8) and corticotropin-releasing hormone (crhb) and down-regulation of dio1, dio2, glucuronidation enzymes (ugt1ab) and thyroid stimulating hormone (tshb) (assessed via qRT-PCR) and responses differed across life stage and tissues. T3 induced thraa expression in the pineal gland, pectoral fins, brain, somites, gastrointestinal tract, craniofacial tissues, liver and pronephric ducts. T3 enhanced thrb expression in the brain, jaw cartilage and intestine, while thrb expression was suppressed in the liver. T3 exposure suppressed the transcript levels of dio1 and dio2 in the liver, brain, gastrointestinal tract and craniofacial tissues, while dio2 signalling was also suppressed in the pituitary gland. Dio3b expression was induced by T3 exposure in the jaw cartilage, pectoral fins and brain. The involvement of THs in the development of numerous body tissues and the responsiveness of these tissues to T3 in zebrafish highlights their potential vulnerability to exposure to environmental thyroid-disrupting chemicals.
Topics: Animals; Corticotropin-Releasing Hormone; Hypothalamus; Larva; Pituitary Gland; Thyroid Gland; Thyroid Hormones; Thyronines; Thyrotropin; Triiodothyronine; Water Pollutants, Chemical; Zebrafish; Zebrafish Proteins
PubMed: 32623180
DOI: 10.1016/j.aquatox.2020.105547