-
Fish & Shellfish Immunology Jan 2019This study investigates the development of lymphoid organs and mucosal tissues in larval and juvenile meagre, Argyrosomus regius. For this purpose, meagre larvae were... (Review)
Review
This study investigates the development of lymphoid organs and mucosal tissues in larval and juvenile meagre, Argyrosomus regius. For this purpose, meagre larvae were reared from hatch to the juvenile stage, under mesocosm conditions at 18-19 °C, using standard feeding sequences with live prey and artificial food. The kidney was evident upon hatch and included a visible pronephros, with undifferentiated stem cells and excretory tubules at 1 dph (3.15 ± 0.1 mm SL). The thymus was first detected 8 dph (4.49 ± 0.39 mm SL) and was clearly visible 12 dph (5.69 ± 0.76 mm SL), 33 dph (15.69 ± 1.81 mm SL) an outer thymocytic zone and inner epithelial zone were visible. The spleen was present 12 dph, located between exocrine pancreas and intestine and by 26 dph (11.84 ± 1.3 mm SL) consisted of a mass of sinusoids filled with red blood cells. Melanomacrophage centers were found 83 dph (66.25 ± 4.35 mm SL) in the spleen. Between 14-15 dph (6.9 ± 1.1 mm SL), goblet and rodlet cells appear in the gill and intestinal epithelium. The lymphoid organs, which appear in the order of pronephric kidney (1 dph), thymus (8 dph) and spleen (12 dph) remarkably increase in size during the post-flexion stage. While functional studies are needed to confirm the activity of the immune response, the morphology of the lymphoid organs suggest that meagre is not immuno-competent until 83 dph.
Topics: Animals; Lymphoid Tissue; Mucous Membrane; Perciformes
PubMed: 30227257
DOI: 10.1016/j.fsi.2018.09.033 -
Journal of Cellular Biochemistry Dec 2018Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca -dependent cation channel with versatile functions including control of extracellular calcium...
Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca -dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca ]i) from internal stores of endoplasmic reticulum, and calcium-dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left-right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca ]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase-D1, and disheveled-2 has been confirmed by immunoblotting analysis of TRPP2-coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase-D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells.
Topics: Animals; Calcium; Cilia; Dishevelled Proteins; Endoplasmic Reticulum; Epithelial Cells; Gastrula; Gene Expression Regulation, Developmental; Kidney; Signal Transduction; TRPP Cation Channels; Xenopus Proteins; Xenopus laevis
PubMed: 30171710
DOI: 10.1002/jcb.27376 -
Journal of Molecular Cell Biology May 2019Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that...
Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that drive ciliary beat have been extensively studied in unicellular Chlamydomonas, to what extent such knowledge can be applied to vertebrate is poorly known. In Chlamydomonas, Dynein-f controls flagellar waveforms but is dispensable for beating. The flagellar assembly of its heavy chains (HCs) requires its intermediate chain (IC) IC140 but not IC138. Here we show that, unlike its Chlamydomonas counterpart, vertebrate Dynein-f is essential for ciliary beat. We confirmed that Wdr78 is the vertebrate orthologue of IC138. Wdr78 associated with Dynein-f subunits such as Dnah2 (a HC) and Wdr63 (IC140 orthologue). It was expressed as a motile cilium-specific protein in mammalian cells. Depletion of Wdr78 or Dnah2 by RNAi paralyzed mouse ependymal cilia. Zebrafish Wdr78 morphants displayed ciliopathy-related phenotypes, such as curved bodies, hydrocephalus, abnormal otolith, randomized left-right asymmetry, and pronephric cysts, accompanied with paralyzed pronephric cilia. Furthermore, all the HCs and ICs of Dynein-f failed to localize in the Wdr78-depleted mouse ependymal cilia. Therefore, both the functions and subunit dependency of Dynein-f are altered in evolution, probably to comply with ciliary roles in higher organisms.
Topics: Animals; Axonemal Dyneins; Axoneme; Chlamydomonas; Cilia; Cytoskeletal Proteins; Dyneins; Mice; Mice, Inbred C57BL; Morpholinos; Plant Proteins; Protein Subunits; RNA Interference; RNA, Small Interfering; Zebrafish; Zebrafish Proteins
PubMed: 30060180
DOI: 10.1093/jmcb/mjy043 -
Gene Expression Patterns : GEP Sep 2018There are three isoforms of natriuretic peptide (NP) specific cell surface receptor: NP receptor-A (NPRA), receptor-B (NPRB), and receptor-C (NPRC). They are also known...
There are three isoforms of natriuretic peptide (NP) specific cell surface receptor: NP receptor-A (NPRA), receptor-B (NPRB), and receptor-C (NPRC). They are also known as NPR1, NPR2 and NPR3, respectively. NPs and their receptors were revealed to involve in diverse cellular and physiological processes including renal, cardiovascular, neuronal, and immunological aspects. However, the systematic analysis of the expression of these receptors in non-mammalian vertebrates is thus far lacking. In this study, two versions of the npr1 gene (npr1a and npr1b) in zebrafish was identified. Multiple sequences alignment analysis showed that zebrafish NPRs shared high homologies with NPRs of other species and possessed a typical signature domain of NPRs. The results of whole mount in situ hybridization and reverse transcription polymerase chain reaction analysis revealed that at embryonic stages, npr1a was mainly expressed in tectal ventricle, brian, heart and retina, whereas npr1b was broadly present in anterior pronephric duct. Unlike npr1, npr2 mainly expressed in branchial arches and neural tube during embryonic development. However, npr3 was expressed in pronephric ducts and corpuscle of stannius in zebrafish embryos at 72 hpf. In adults, we demonstrated that all the three NP receptors were highly existed in brain and kidney. Overall, these findings will provide an important basis for the functional analysis of NPs and its receptor during embryonic development.
Topics: Animals; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Phylogeny; Receptors, Atrial Natriuretic Factor; Zebrafish; Zebrafish Proteins
PubMed: 30018004
DOI: 10.1016/j.gep.2018.07.001 -
The International Journal of... 2018The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell...
The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. The pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. In order to obtain insights into the potential conservation of this class of transcription factors in vertebrates, we carried out a phylogenetic analysis and a comprehensive comparative study of pou3f expression in the frog Xenopus laevis. All vertebrates examined possessed members of the four pou3f subfamilies, excepting the zebrafish, which lacked a pou3f4 gene. Whole mount in situ hybridization and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that Xenopus pou3f genes were expressed in the forming neural tube and their expression was maintained in the brain, mostly in the dorsal part, at tailbud stages. The pou3f2, pou3f3, and pou3f4 genes were also expressed in the developing otic vesicle, and pou3f1 in some cells of the epidermis. Besides ectodermal derivatives, pou3f3 and pou3f4 were expressed in the developing kidney. Their expression started at the early tailbud stage in the pronephric anlage and partly overlapped. In the mature pronephric tubule, pou3f3 was restricted to the intermediate tubule, while pou3f4 was also expressed in the distal and connecting tubule. Together, our results highlight a significant conservation of pou3f gene expression in vertebrates and indicate that they may have distinct but also redundant functions during neural and renal development.
Topics: Animals; Brain; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Kidney; Organogenesis; POU Domain Factors; Xenopus Proteins; Xenopus laevis
PubMed: 29877572
DOI: 10.1387/ijdb.170260RL -
The International Journal of... 2018LRPAP1, also known as receptor associated protein (RAP) is a small protein of 40 kDa associated with six of the seven members of the evolutionary conserved family of LDL...
LRPAP1, also known as receptor associated protein (RAP) is a small protein of 40 kDa associated with six of the seven members of the evolutionary conserved family of LDL receptors. Numerous studies showed that LRPAP1 has a dual function, initially as a chaperone insuring proper formation of intermolecular disulfide bonds during biogenesis of low density lipoprotein (LDL) receptors and later as an escort protein during trafficking through the endoplasmic reticulum and the early Golgi compartment, preventing premature interaction of receptor and ligand. Because of the general influence of LRPAP1 protein on lipid metabolism, we analyzed the temporal and spatial expression of the Xenopus laevis ortholog of lrpap1. Here, we show that lrpap1 was expressed in the developing neural system, the eye and ear anlagen, the branchial arches, the developing skin and the pronephric kidney. The very high expression level of lrpap1 specifically in the proximal tubules of the developing pronephros establishes this gene as a novel marker for the analysis of pronephros formation.
Topics: Animals; Biomarkers; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Kidney Tubules, Proximal; LDL-Receptor Related Protein-Associated Protein; Organogenesis; Xenopus Proteins; Xenopus laevis
PubMed: 29877571
DOI: 10.1387/ijdb.170295hn -
Genes Apr 2018embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between and...
embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between and humans, allowing for the study of human disease-causing genes. embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this :eGFP line, showing green fluorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of :eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes.
PubMed: 29642376
DOI: 10.3390/genes9040197 -
Developmental Dynamics : An Official... Jun 2018Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca -conducting TRP...
BACKGROUND
Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca -conducting TRP channel enriched in ciliated cells and gated by extracellular signals, is necessary to activate the multifunctional Ca calmodulin-dependent protein kinase type 2 (CaMK-II), enabling kidney morphogenesis and cilia stability.
RESULTS
In this study, antisense morpholino oligonucleotides and pharmacological compounds were employed to investigate the roles of class II HDAC family members (HDAC 4, 5, and 6) in Zebrafish kidney development. While all three class II HDAC genes were expressed throughout the embryo during early development, HDAC5-morphant embryos exhibited anterior cysts and destabilized cloacal cilia, similar to PKD2 and CaMK-II morphants. In contrast, HDAC4-morphant embryos exhibited elongated cloacal cilia and lacked anterior kidney defects. Suppression of HDAC4 partially reversed the cilia shortening and anterior convolution defects caused by CaMK-II deficiency, whereas HDAC5 loss exacerbated these defects. EGFP-HDAC4, but not EGFP-HDAC5, translocated into the nucleus upon CaMK-II suppression in pronephric kidney cells.
CONCLUSIONS
These results support a model by which activated CaMK-II sequesters HDAC4 in the cytosol to enable primary cilia formation and kidney morphogenesis. Developmental Dynamics 247:807-817, 2018. © 2018 Wiley Periodicals, Inc.
Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Gene Expression Regulation, Developmental; Histone Deacetylases; Kidney; Organogenesis; Zebrafish; Zebrafish Proteins
PubMed: 29633426
DOI: 10.1002/dvdy.24632 -
Development (Cambridge, England) Mar 2018The development of the kidney relies on the establishment and maintenance of a precise tubular diameter of its functional units, the nephrons. This process is disrupted...
The development of the kidney relies on the establishment and maintenance of a precise tubular diameter of its functional units, the nephrons. This process is disrupted in polycystic kidney disease (PKD), resulting in dilations of the nephron and renal cyst formation. In the course of exploring G-protein-coupled signaling in the pronephric kidney, we discovered that loss of the G-protein α subunit, Gnas, results in a PKD phenotype. Polycystin 1, one of the genes mutated in human PKD, encodes a protein resembling a G-protein-coupled receptor. Furthermore, deletion of the G-protein-binding domain present in the intracellular C terminus of polycystin 1 impacts functionality. A comprehensive analysis of all the G-protein α subunits expressed in the pronephric kidney demonstrates that polycystin 1 recruits a select subset of G-protein α subunits and that their knockdown - as in the case of Gnas - results in a PKD phenotype. Mechanistically, the phenotype is caused by increased endogenous G-protein β/γ signaling and can be reversed by pharmacological inhibitors as well as knocking down Gnb1. Together, our data support the hypothesis that G proteins are recruited to the intracellular domain of PKD1 and that this interaction is crucial for its function in the kidney.
Topics: Animals; Cell Culture Techniques; GTP-Binding Proteins; Humans; In Situ Hybridization; Kidney; Polycystic Kidney Diseases; Signal Transduction; Surface Plasmon Resonance; TRPP Cation Channels; Xenopus laevis
PubMed: 29530879
DOI: 10.1242/dev.158931 -
FASEB Journal : Official Publication of... Aug 2018Polarity complexes, including the PAR (Partitioning-defective), CRB (Crumbs) and SCRIB (Scribble) complexes, are required for the physiologic establishment,...
Polarity complexes, including the PAR (Partitioning-defective), CRB (Crumbs) and SCRIB (Scribble) complexes, are required for the physiologic establishment, stabilization, and maintenance of a functional apical-basolateral polarity. Inactivation of some of the polarity complexes results in cystic kidneys, and apical-basolateral polarity defects are commonly observed in autosomal-dominant polycystic kidney disease (ADPKD); however, little is known about the role that polarity complexes play in ADPKD. Here, we demonstrate that Scribble, a core protein of the SCRIB complex, is down-regulated in ADPKD cell lines and the zebrafish model of this disease ( pkd2 morphants). Overexpression of Scribble could reduce cyst formation in pkd2 morphants, and loss of scrib led to a dilated pronephric duct in zebrafish. Furthermore, the Hippo signaling pathway was inactivated in scrib mutants and pkd2 morphants in which Yes-associated protein (YAP), which is physiologically located in the cytoplasm, was translocated to the nucleus. Of note, overexpression of cytoplasmic YAP, instead of nuclear YAP, could reduce cyst formation in pkd2 morphants. Consistently, knockout of yap resulted in cystic kidneys in zebrafish, which was rescued by the overexpression of cytoplasmic YAP, but not nuclear YAP. Finally, scrib and yap had a genetic interaction with pkd2 in cyst formation, and the overexpression of Scribble attenuated the down-regulation of cytoplasmic YAP in ADPKD. Altogether, our data indicate that Scribble induces the phosphorylation of YAP and, consequently, influences cyst formation in ADPKD by mediating YAP nucleocytoplasmic shuttling.-Xu, D., Lv, J., He, L., Fu, L., Hu, R., Cao, Y., Mei, C. Scribble influences cyst formation in autosomal-dominant polycystic kidney disease by regulating Hippo signaling pathway.
Topics: Animals; Cell Line; Cysts; Down-Regulation; HEK293 Cells; Humans; Kidney; Mice; Phosphorylation; Polycystic Kidney, Autosomal Dominant; Protein Kinase D2; Protein Kinases; Protein Serine-Threonine Kinases; Signal Transduction; Tumor Suppressor Proteins; Zebrafish
PubMed: 29529391
DOI: 10.1096/fj.201701376RR