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Cell & Bioscience 2018The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is...
BACKGROUND
The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function.
RESULTS
mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development.
CONCLUSION
In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development.
PubMed: 30349665
DOI: 10.1186/s13578-018-0253-z -
Cells Feb 2023Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal...
Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17β-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, , revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, and its target . These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.
Topics: Animals; Zebrafish; Zebrafish Proteins; Kidney; Nephrons; Estrogens
PubMed: 36831333
DOI: 10.3390/cells12040666 -
Developmental Dynamics : An Official... Apr 2016Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric...
Proper Notch activity is necessary for the establishment of proximal cells and differentiation of intermediate, distal, and connecting tubule in Xenopus pronephros development.
BACKGROUND
Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric anlagen during the generation of pronephric components remains to be understood.
RESULTS
We investigated how components of pronephros (glomus, proximal tubule, intermediate tubule, distal tubule, and connecting tubule) were generated in Xenopus embryos by timed overactivation and suppression of Notch signaling. Notch activation resulted in expansion of the glomus and disruption of the proximal tubule formation. Inhibition of Notch signaling reduced expression of wt1 and XSMP-30. In addition, when Notch signaling was overactivated at stage 20 on, intermediate, distal, and connecting tubule markers, gremlin and clcnkb, were decreased while Notch down-regulation increased gremlin and clcnkb. Similar changes were observed with segmental markers, cldn19, cldn14, and rhcg on activation or inhibition of Notch. Although Notch did not affect the expression of pan-pronephric progenitor marker, pax2, its activation inhibited lumen formation in the pronephros.
CONCLUSIONS
Notch signal is essential for glomus and proximal tubule development and inhibition of Notch is critical for the differentiation of the intermediate, distal, and connecting tubule.
Topics: Animals; Antigens, Differentiation; Cell Differentiation; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Kidney Tubules, Proximal; Pronephros; Receptors, Notch; Signal Transduction; Xenopus laevis
PubMed: 26773453
DOI: 10.1002/dvdy.24386 -
International Journal of Molecular... May 2021Sustainability of aquaculture is tied to the origin of feed ingredients. In search of sustainable fish meal-free formulations for rainbow trout, we evaluated the effect...
Sustainability of aquaculture is tied to the origin of feed ingredients. In search of sustainable fish meal-free formulations for rainbow trout, we evaluated the effect of meal (H) and poultry by-product meal (P), singly (10, 30, and 60% of either H or P) or in combination (10% H + 50% P, H10P50), as partial replacement of vegetable protein (VM) on gut microbiota (GM), inflammatory, and immune biomarkers. Fish fed the mixture H10P50 had the best growth performance. H, P, and especially the combination H10P50 partially restored α-diversity that was negatively affected by VM. Diets did not differ in the Firmicutes:Proteobacteria ratio, although the relative abundance of Gammaproteobacteria was reduced in H and was higher in P and in the fishmeal control. H had higher relative abundance of chitin-degrading and , and . was also higher in H feed, suggesting feed-chain microbiome transmission. P increased the relative abundance of protein degraders and Bacteroidales. IL-1β, IL-10, TGF-β, COX-2, and TCR-β gene expression in the midgut and head kidney and plasma lipopolysaccharide (LPS) revealed that the diets did not compromise the gut barrier function or induce inflammation. H, P, and H10P50 therefore appear valid protein sources in fishmeal-free aquafeeds.
Topics: Animal Feed; Animal Proteins, Dietary; Animals; Aquaculture; Biomarkers; Diet; Gastrointestinal Microbiome; Head Kidney; Inflammation; Insecta; Oncorhynchus mykiss; Poultry; Poultry Products
PubMed: 34064267
DOI: 10.3390/ijms22115454 -
The International Journal of... 2016Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in...
Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.
Topics: Animals; Carrier Proteins; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Genetic Complementation Test; Humans; In Situ Hybridization; Morphogenesis; Mutation; Pronephros; RING Finger Domains; RNA, Messenger; Xenopus Proteins; Xenopus laevis; Zebrafish Proteins
PubMed: 26934292
DOI: 10.1387/ijdb.150381ld -
Scientific Reports Nov 2022Xenopus provides a simple and efficient model system to study nephrogenesis and explore the mechanisms causing renal developmental defects in human. Hnf1b (hepatocyte...
Xenopus provides a simple and efficient model system to study nephrogenesis and explore the mechanisms causing renal developmental defects in human. Hnf1b (hepatocyte nuclear factor 1 homeobox b), a gene whose mutations are the most commonly identified genetic cause of developmental kidney disease, is required for the acquisition of a proximo-intermediate nephron segment in Xenopus as well as in mouse. Genetic networks involved in Hnf1b expression during kidney development remain poorly understood. We decided to explore the transcriptional regulation of Hnf1b in the developing Xenopus pronephros and mammalian renal cells. Using phylogenetic footprinting, we identified an evolutionary conserved sequence (CNS1) located several kilobases (kb) upstream the Hnf1b transcription start and harboring epigenomic marks characteristics of a distal enhancer in embryonic and adult renal cells in mammals. By means of functional expression assays in Xenopus and mammalian renal cell lines we showed that CNS1 displays enhancer activity in renal tissue. Using CRISPR/cas9 editing in Xenopus tropicalis, we demonstrated the in vivo functional relevance of CNS1 in driving hnf1b expression in the pronephros. We further showed the importance of Pax8-CNS1 interaction for CNS1 enhancer activity allowing us to conclude that Hnf1b is a direct target of Pax8. Our work identified for the first time a Hnf1b renal specific enhancer and may open important perspectives into the diagnosis for congenital kidney anomalies in human, as well as modeling HNF1B-related diseases.
Topics: Humans; Adult; Mice; Animals; Hepatocyte Nuclear Factor 1-beta; Phylogeny; Kidney; Kidney Diseases; Regulatory Sequences, Nucleic Acid; Xenopus; Xenopus laevis; Mammals; PAX8 Transcription Factor
PubMed: 36402859
DOI: 10.1038/s41598-022-21171-x -
Gene Expression Patterns : GEP Sep 2018Extracellular matrix (ECM) remodeling by metalloproteinases is crucial during development. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I...
Extracellular matrix (ECM) remodeling by metalloproteinases is crucial during development. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling. The human family includes 19 members. In this study we identified the 19 members of the ADAMTS family in Xenopus laevis and Xenopus tropicalis. Gene identification and a phylogenetic study revealed strong conservation of the ADAMTS family and contributed to a better annotation of the Xenopus genomes. Expression of the entire ADAMTS family was studied from early stages to tadpole stages of Xenopus, and detailed analysis of ADAMTS9 revealed expression in many structures during organogenesis such as neural crest (NC) derivative tissues, the pronephros and the pancreas. Versican, a matrix component substrate of ADAMTS9 shows a similar expression pattern suggesting a role of ADAMTS9 in the remodeling of the ECM in these structures by degradation of versican.
Topics: ADAMTS9 Protein; Animals; Extracellular Matrix; Gene Expression Regulation, Developmental; Genome; Morphogenesis; Phylogeny; Xenopus Proteins; Xenopus laevis
PubMed: 29935379
DOI: 10.1016/j.gep.2018.06.001 -
Biology Open Jul 2021Early embryogenesis requires tightly controlled temporal and spatial coordination of cellular behavior and signaling. Modulations are achieved at multiple levels, from...
Early embryogenesis requires tightly controlled temporal and spatial coordination of cellular behavior and signaling. Modulations are achieved at multiple levels, from cellular transcription to tissue-scale behavior. Intracellularly, the endolysosomal system emerges as an important regulator at different levels, but in vivo studies are rare. In the frog Xenopus, little is known about the developmental roles of endosomal regulators, or their potential involvement in signaling, especially for late endosomes. Here, we analyzed a hypothesized role of Rab7 in this context, a small GTPase known for its role as a late endosomal regulator. First, rab7 showed strong maternal expression. Following localized zygotic transcript enrichment in the mesodermal ring and neural plate, it was found in tailbud-stage neural ectoderm, notochord, pronephros, eyes and neural crest tissues. Inhibition resulted in strong axis defects caused by a requirement of rab7 for mesodermal patterning and correct gastrulation movements. To test a potential involvement in growth factor signaling, we analyzed early Wnt-dependent processes in the mesoderm. Our results suggest a selective requirement for ligand-induced Wnt activation, implicating a context-dependent role of Rab7.
Topics: Animals; Embryo, Nonmammalian; Embryonic Development; Gastrulation; Gene Expression Regulation, Developmental; Mesoderm; Transcription Factors; Xenopus; Zygote; rab7 GTP-Binding Proteins
PubMed: 34096568
DOI: 10.1242/bio.056887 -
Scientific Reports Nov 2023Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the...
Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS kidney cells and injection into ctns zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns larvae, and restoration of the zebrafish pronephros function.
Topics: Animals; Cystinosis; Cystine; Zebrafish; RNA, Messenger; Models, Theoretical; Dietary Supplements; Amino Acid Transport Systems, Neutral
PubMed: 38016974
DOI: 10.1038/s41598-023-47085-w -
Scientific Reports Jul 2022Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also...
Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate β-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.
Topics: Animals; Diabetes Mellitus, Type 2; Hyperglycemia; Models, Theoretical; Pronephros; Thiosulfate Sulfurtransferase; Thiosulfates; Zebrafish
PubMed: 35840638
DOI: 10.1038/s41598-022-16320-1