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Communications Biology Oct 2021The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although...
The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus. Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis.
Topics: Animals; Body Patterning; Embryo, Nonmammalian; Embryonic Development; Gene Regulatory Networks; Kidney; Phosphoric Diester Hydrolases; Signal Transduction; Xenopus Proteins; Xenopus laevis
PubMed: 34620987
DOI: 10.1038/s42003-021-02688-9 -
Biochemical and Biophysical Research... Sep 2023Nephronophthisis (NPH), an autosomal recessive ciliopathy, results from mutations in more than 20 different genes (NPHPs). These gene products form protein complexes...
Nephronophthisis (NPH), an autosomal recessive ciliopathy, results from mutations in more than 20 different genes (NPHPs). These gene products form protein complexes that regulate trafficking within the cilium, a microtubular structure that plays a crucial role in developmental processes. Several NPHPs, including NPHP2/Inversin, have been linked to extraciliary functions. In addition to defining a specific segment of primary cilia (Inversin compartment), NPHP2 participates in planar cell polarity (PCP) signaling along with Dishevelled and Vangl family members. We used the mutant zebrafish line invs, containing a stop codon at amino acid 314, to characterize tissue-specific functions of zebrafish Nphp2. The invs line exhibits mild ciliopathy phenotypes and increased glomerular and cloaca cyst formation. These mutants showed enhanced susceptibility to the simultaneous depletion of the nphp1/nphp2/nphp8 module, known to be involved in the cytoskeletal organization of epithelial cells. Notably, simultaneous depletion of zebrafish nphp1 and vangl2 led to a pronounced increase in cloaca malformations in the invs mutant embryos. Time-lapse imaging showed that the pronephric cells correctly migrated towards the ectodermal cells in these embryos, but failed to form the cloaca opening. Despite these abnormal developments, cellular fate does not seem to be affected in nphp1 and vangl2 MO-depleted invs mutants, as shown by in situ hybridizations for markers of pronephros and ectodermal cell development. However, significantly reduced apoptotic activity was observed in this double knockdown model, signifying the role of apoptosis in cloacal morphogenesis. Our findings underscore the critical interplay of nphp1, nphp2/Inversin, and vangl2 in orchestrating normal cloaca formation in zebrafish, shedding light on the complex molecular mechanisms underlying ciliopathy-associated phenotypes.
Topics: Animals; Zebrafish; Cloaca; Cell Polarity; Membrane Proteins; Zebrafish Proteins
PubMed: 37352572
DOI: 10.1016/j.bbrc.2023.06.058 -
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 -
Cell Cycle (Georgetown, Tex.) Oct 2019Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified being...
Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified being essential for physiological pronephros development in zebrafish, which is driven by blocking apoptosis in developing kidneys. Yet, zebrafish mutants for have not been described so far. In order to compare a transient knockdown vs. permanent knockout for in zebrafish on pronephros development, we used CRISPR/Cas9 technology to generate knockout zebrafish mutants and we performed structural and functional studies on pronephros development. In contrast to morphants, zebrafish mutants showed normal pronephros structure; however, a previously uncharacterized gene in zebrafish, named , was identified and upregulated in mutants. knockdown altered pronephros development, which was further aggravated in mutants. Likewise, and morphants regulated similar and differential gene signatures related to kidney development in zebrafish. In conclusion, stable loss of during embryonic development can be compensated by leading to phenotypical discrepancies in knockdown and knockout zebrafish embryos.
Topics: Animals; CRISPR-Cas Systems; Embryo, Nonmammalian; Erythropoietin; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Gene Knockout Techniques; Heterozygote; Homozygote; Microscopy, Electron; Morpholinos; Organogenesis; Pronephros; Recombinant Proteins; Zebrafish; Zebrafish Proteins
PubMed: 31451030
DOI: 10.1080/15384101.2019.1656019 -
Frontiers in Immunology 2020Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to...
Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals.
Topics: Animals; Arginase; Carboxy-Lyases; Carps; Cell Polarity; Cyclic AMP; Gene Expression; Glycolysis; Head Kidney; Lipopolysaccharides; Macrophage Activation; Macrophages; Mitochondria; Nitric Oxide; Oxidative Phosphorylation; Phenotype; Transcriptome
PubMed: 32158446
DOI: 10.3389/fimmu.2020.00152 -
Gene Expression Patterns : GEP Jan 2020Nucleosome assembly protein 1-like (Nap1l) family plays numerous biological roles including nucleosome assembly, transcriptional regulation, and cell cycle progression....
Nucleosome assembly protein 1-like (Nap1l) family plays numerous biological roles including nucleosome assembly, transcriptional regulation, and cell cycle progression. However, the tissue specific in vivo functions of the Nap1l family members remain largely unknown. In this study, we finished the complete expression patterns of nap1l1 and nap1l4a in zebrafish embryos by whole-mount in situ hybridization. We observed maternal existence of nap1l1 transcript and that its zygotic expression is abundant and not spatially restricted at 6 somite stage, while nap1l4a mRNA is not detectable until 6 somite stage when it is weakly transcribed throughout the embryo. At 24 h post-fertilization (hpf), nap1l1 is predominantly expressed in the central nervous system, neural tube, ventral mesoderm, branchial arches, and pectoral fins, while nap1l4a mRNA is throughout the embryo, enriched in the eyes, tectum, and myotomes. As the embryo develops, nap1l1 expression maintains throughout the head, with gradually enriched in the tectum, olfactory vesicle, lens, optic cups, heart, branchial arches, pectoral fins, axial vasculature, pronephros, and lateral line neuromasts, whereas nap1l4a expression is weak in the tectum, branchial arches, and pectoral fins. Overall, these expression analyses provide a valuable basis for the functional study of nap1l family in zebrafish development.
Topics: Animal Fins; Animals; Gene Expression Regulation, Developmental; Heart; Kidney; Mesoderm; Morphogenesis; Muscle, Skeletal; Myocardium; Neural Crest; Nucleosome Assembly Protein 1; Zebrafish; Zebrafish Proteins
PubMed: 31669493
DOI: 10.1016/j.gep.2019.119076 -
Developmental Biology Jan 2023Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field....
Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.
Topics: Animals; Xenopus laevis; Tretinoin; Xenopus Proteins; Paired Box Transcription Factors; Gene Expression Regulation, Developmental; Pronephros; Kidney; Aldehyde Dehydrogenase 1 Family; Retinal Dehydrogenase
PubMed: 36279927
DOI: 10.1016/j.ydbio.2022.10.009 -
Gene Expression Patterns : GEP Dec 2021Galloway-Mowat syndrome (GAMOS) is a rare developmental disease. Patients suffer from congenital brain anomalies combined with renal abnormalities often resulting in an...
Galloway-Mowat syndrome (GAMOS) is a rare developmental disease. Patients suffer from congenital brain anomalies combined with renal abnormalities often resulting in an early-onset steroid-resistant nephrotic syndrome. The etiology of GAMOS has a heterogeneous genetic contribution. Mutations in more than 10 different genes have been reported in GAMOS patients. Among these are mutations in four genes encoding members of the human KEOPS (kinase, endopeptidase and other proteins of small size) complex, including OSGEP, TP53RK, TPRKB and LAGE3. Until now, these components have been functionally mainly investigated in bacteria, eukarya and archaea and in humans in the context of the discovery of its role in GAMOS, but the KEOPS complex members' expression and function during embryogenesis in vertebrates is still unknown. In this study, in silico analysis showed that both gene localization and the protein sequences of the three core KEOPS complex members Osgep, Tp53rk and Tprkb are highly conserved across different species including Xenopus laevis. In addition, we examined the spatio-temporal expression pattern of osgep, tp53rk and tprkb using RT-PCR and whole mount in situ hybridization approaches during early Xenopus development. We observed that all three genes were expressed during early embryogenesis and enriched in tissues and organs affected in GAMOS. More precisely, KEOPS complex genes are expressed in the pronephros, but also in neural tissue such as the developing brain, eye and cranial cartilage. These findings suggest that the KEOPS complex plays an important role during vertebrate embryonic development.
Topics: Animals; Computational Biology; Embryonic Development; Hernia, Hiatal; Humans; Microcephaly; Nephrosis; Xenopus Proteins; Xenopus laevis
PubMed: 34619372
DOI: 10.1016/j.gep.2021.119215 -
Fish & Shellfish Immunology Sep 2020Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development....
Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development. Although two types of TNF-α have been identified in several teleost species, their functions in pathogen infection remain largely unexplored, especially in pathogen clearance. Herein, we cloned and characterized two types of TNF-α, termed shTNF-α1 and shTNF-α2, and their receptors, shTNFR1 and shTNFR2, from snakehead (Channa argus). These genes were constitutively expressed in all tested tissues, and were induced by Aeromonas schubertii and Nocardia seriolae in head kidney and spleen in vivo, and by lipoteichoic acid (LTA), lipopolysaccharides (LPS), and Polyinosinic-polycytidylic acid [Poly (I:C)] in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shTNF-α1 and shTNF-α2 upregulated the expression of endogenous shTNF-α1, shTNF-α2, shTNFR1, and shTNFR2, and enhanced intracellular bactericidal activity, with shTNF-α1 having a greater effect than shTNF-α2. These findings suggest important roles of fish TNFα1, TNFα2, and their receptors in bacterial infection and pathogen clearance, and provide a new insight into their function in antibacterial innate immunity.
Topics: Aeromonas; Animals; Fish Diseases; Fish Proteins; Fishes; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Leukocytes; Lipopolysaccharides; Nocardia; Nocardia Infections; Poly I-C; Receptors, Tumor Necrosis Factor; Spleen; Teichoic Acids; Tumor Necrosis Factor-alpha
PubMed: 32585357
DOI: 10.1016/j.fsi.2020.05.059 -
International Journal of Molecular... Aug 2022Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability...
Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that , , and expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of , , or with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in -depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease.
Topics: Acute Kidney Injury; Animals; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Mice; NF-kappa B; Pronephros; Repressor Proteins; Zebrafish; Zebrafish Proteins
PubMed: 36076983
DOI: 10.3390/ijms23179582