-
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 -
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 -
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 -
Journal of Developmental Biology Oct 2022The Wilms' tumor suppressor gene, , encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes....
The Wilms' tumor suppressor gene, , encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes. was first identified as a tumor suppressor gene in Wilms' tumor, a pediatric kidney tumor, and has been implicated in normal kidney development. The WT1 protein has transcriptional activation and repression domains and acts as a transcriptional activator or repressor, depending on the target gene and context. In , an ortholog of has been isolated and shown to be expressed in the developing embryonic pronephros. To investigate the role of in pronephros development in embryos, we mutated by CRISPR/Cas9 and found that the expression of pronephros marker genes was reduced. In reporter assays in which known WT1 binding sequences were placed upstream of the gene, WT1 activated transcription of the gene. The injection of wild-type or artificially altered transcriptional activity of mRNA disrupted the expression of pronephros marker genes in the embryos. These results suggest that the appropriate amounts and activity of WT1 protein are required for normal pronephros development in embryos.
PubMed: 36412640
DOI: 10.3390/jdb10040046 -
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 -
Frontiers in Immunology 2021CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its...
CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated . consists of four exons and three introns, and the full-length cDNA of was 675-bp encoded 224 amino acids. The conserved motif (TFPPPF) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of and in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.
Topics: Amino Acid Sequence; Animals; Antigens; Base Sequence; CD28 Antigens; Cell Line; Cells, Cultured; Fish Proteins; Flounder; Gills; Head Kidney; Hemocyanins; Immunity; Interleukin-2; Leukocytes; Lymphocyte Activation; Phylogeny; Sequence Homology, Amino Acid; Spleen; Thymus Gland; Transcriptome
PubMed: 34858416
DOI: 10.3389/fimmu.2021.765036 -
Frontiers in Immunology 2021In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F)...
In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F) genes have been identified to be homologous to mammalian IL-17A and IL-17F, but little is known about their functional activity. In this study, _IL-17A/F1, 2 and 3 genes were cloned from yellow catfish () and they differed in protein structure and exon length, implying that they may have divergent bioactivity. Real-time quantitative PCR analyses revealed that three _IL-17A/F genes were highly expressed in blood and mucosal tissues (skin+mucus and gill) from healthy adult fish. The mRNA expressions of _IL-17A/F1, 2 and 3 genes were significantly up-regulated in the gill, skin+mucus, head kidney and spleen after challenge with and in the isolated peripheral blood leucocytes (PBLs) of yellow catfish after stimulation with phytohaemagglutinin (PHA), lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). These results indicate that _IL-17A/F1, 2 and 3 genes may play a vital role in the regulation of immune against pathogens. Additionally, the recombinant (r) _IL-17A/F1, 2 and 3 proteins significantly induced the mRNA expressions of proinflammatory cytokines, chemokines and antibacterial peptides genes, and the r_IL-17A/F 2 and 3 proteins promoted phagocytosis of PBLs more powerfully than the r_IL-17A/F1. Furthermore, the r_IL-17A/F1, 2 and 3 proteins might activate the NF-κB and MAPK signal pathways by IL-17RA, ACT1, TRAF6, TRAF2, TRAF5 and TAK1, indicating that the three _IL-17A/F proteins may play different roles in promoting inflammatory response.
Topics: Animals; Catfishes; Fish Proteins; Head Kidney; Interleukin-17; Leukocytes; Lipopolysaccharides; Peptidoglycan; Phytohemagglutinins; Poly I-C; Spleen
PubMed: 34267744
DOI: 10.3389/fimmu.2021.626895 -
Pathogens & Immunity 2022Uropathogenic (UPEC) infections are common and when they disseminate can be of high morbidity.
BACKGROUND
Uropathogenic (UPEC) infections are common and when they disseminate can be of high morbidity.
METHODS
We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed.
RESULTS
Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a , Tribbles Pseudokinase 3 (, Arginase 2 ( and Polo Like Kinase 3 (
CONCLUSIONS
Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.
PubMed: 35178490
DOI: 10.20411/pai.v7i1.479 -
IScience Dec 2020Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development...
Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development of several metabolic diseases. Here, we report on a zebrafish mutant for Aldo-keto-reductase 1a1b () as a regulator of gluconeogenesis. Adult mutant zebrafish developed fasting hypoglycemia, which was caused by inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression as rate-limiting enzyme of gluconeogenesis. Subsequently, glucogenic amino acid glutamate as substrate for gluconeogenesis accumulated in the kidneys, but not in livers, and induced structural and functional pronephros alterations in 48-hpf embryos. mutants displayed increased nitrosative stress as indicated by increased nitrotyrosine, and increased protein-S-nitrosylation. Inhibition of nitrosative stress using the NO synthase inhibitor L-NAME prevented kidney damage and normalized PEPCK expression in mutants. Thus, the data have identified Akr1a1b as a regulator of gluconeogenesis in zebrafish and thereby controlling glucose homeostasis.
PubMed: 33251496
DOI: 10.1016/j.isci.2020.101763