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Developmental Biology Aug 2017The zebrafish kidney is conserved with other vertebrates, making it an excellent genetic model to study renal development. The kidney collects metabolic waste using a...
The zebrafish kidney is conserved with other vertebrates, making it an excellent genetic model to study renal development. The kidney collects metabolic waste using a blood filter with specialized epithelial cells known as podocytes. Podocyte formation is poorly understood but relevant to many kidney diseases, as podocyte injury leads to progressive scarring and organ failure. zeppelin (zep) was isolated in a forward screen for kidney mutants and identified as a homozygous recessive lethal allele that causes reduced podocyte numbers, deficient filtration, and fluid imbalance. Interestingly, zep mutants had a larger interrenal gland, the teleostean counterpart of the mammalian adrenal gland, which suggested a fate switch with the related podocyte lineage since cell proliferation and cell death were unchanged within the shared progenitor field from which these two identities arise. Cloning of zep by whole genome sequencing (WGS) identified a splicing mutation in breast cancer 2, early onset (brca2)/fancd1, which was confirmed by sequencing of individual fish. Several independent brca2 morpholinos (MOs) phenocopied zep, causing edema, reduced podocyte number, and increased interrenal cell number. Complementation analysis between zep and brca2 -/- zebrafish, which have an insertional mutation, revealed that the interrenal lineage was expanded. Importantly, overexpression of brca2 rescued podocyte formation in zep mutants, providing critical evidence that the brca2 lesion encoded by zep specifically disrupts the balance of nephrogenesis. Taken together, these data suggest for the first time that brca2/fancd1 is essential for vertebrate kidney ontogeny. Thus, our findings impart novel insights into the genetic components that impact renal development, and because BRCA2/FANCD1 mutations in humans cause Fanconi anemia and several common cancers, this work has identified a new zebrafish model to further study brca2/fancd1 in disease.
Topics: Animals; Animals, Genetically Modified; BRCA2 Protein; Cell Differentiation; Cell Proliferation; Cloning, Molecular; Disease Models, Animal; Gene Expression Regulation, Developmental; In Situ Hybridization, Fluorescence; Morpholinos; Organogenesis; Podocytes; Pronephros; Zebrafish; Zebrafish Proteins
PubMed: 28579318
DOI: 10.1016/j.ydbio.2017.05.025 -
Developmental Biology Jan 2015The respective role of Pax2 and Pax8 in early kidney development in vertebrates is poorly understood. In this report, we have studied the roles of Pax8 and Pax2 in...
The respective role of Pax2 and Pax8 in early kidney development in vertebrates is poorly understood. In this report, we have studied the roles of Pax8 and Pax2 in Xenopus pronephros development using a loss-of-function approach. Our results highlight a differential requirement of these two transcription factors for proper pronephros formation. Pax8 is necessary for the earliest steps of pronephric development and its depletion leads to a complete absence of pronephric tubule. Pax2 is required after the establishment of the tubule pronephric anlage, for the expression of several terminal differentiation markers of the pronephric tubule. Neither Pax2 nor Pax8 is essential to glomus development. We further show that Pax8 controls hnf1b, but not lhx1 and Osr2, expression in the kidney field as soon as the mid-neurula stage. Pax8 is also required for cell proliferation of pronephric precursors in the kidney field. It may exert its action through the wnt/beta-catenin pathway since activation of this pathway can rescue MoPax8 induced proliferation defect and Pax8 regulates expression of the wnt pathway components, dvl1 and sfrp3. Finally, we observed that loss of pronephros in Pax8 morphants correlates with an expanded vascular/blood gene expression domain indicating that Pax8 function is important to delimit the blood/endothelial genes expression domain in the anterior part of the dorso-lateral plate.
Topics: Animals; Bromodeoxyuridine; DNA Primers; Gene Expression Regulation, Developmental; In Situ Hybridization; PAX2 Transcription Factor; PAX8 Transcription Factor; Paired Box Transcription Factors; Polymerase Chain Reaction; Pronephros; Real-Time Polymerase Chain Reaction; Wnt Signaling Pathway; Xenopus; Xenopus Proteins
PubMed: 25446030
DOI: 10.1016/j.ydbio.2014.10.022 -
Developmental Biology Jan 2022The corpuscles of Stannius (CS) represent a unique endocrine organ of teleostean fish that secrets stanniocalcin-1 (Stc1) to maintain calcium homeostasis. Appearing at...
The corpuscles of Stannius (CS) represent a unique endocrine organ of teleostean fish that secrets stanniocalcin-1 (Stc1) to maintain calcium homeostasis. Appearing at 20-25 somite stage in the distal zebrafish pronephros, stc1-expressing cells undergo apical constriction, and are subsequently extruded to form a distinct gland on top of the distal pronephric tubules at 50 h post fertilization (hpf). Several transcription factors (e.g. Hnf1b, Irx3b, Tbx2a/b) and signaling pathways (e.g. Notch) control CS development. We report now that Fgf signaling is required to commit tubular epithelial cells to differentiate into stc1-expressing CS cells. Inhibition of Fgf signaling by SU5402, dominant-negative Fgfr1, or depletion of fgf8a prevented CS formation and stc1 expression. Ablation experiments revealed that CS have the ability to partially regenerate via active cell migration involving extensive filopodia and lamellipodia formation. Activation of Wnt signaling curtailed stc1 expression, but had no effect on CS formation. Thus, our observations identify Fgf signaling as a crucial component of CS cell fate commitment.
Topics: Animals; Cell Differentiation; Endocrine Glands; Fibroblast Growth Factors; Glycoproteins; Pronephros; Wnt Signaling Pathway; Zebrafish; Zebrafish Proteins
PubMed: 34666023
DOI: 10.1016/j.ydbio.2021.10.005 -
Journal of Visualized Experiments : JoVE Aug 2011Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure....
Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys. The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.
Topics: Animals; Epithelium; Kidney; Laser Therapy; Pronephros; Regeneration; Zebrafish
PubMed: 21897358
DOI: 10.3791/2845 -
The Journal of Biological Chemistry Jan 2015Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa...
Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and β1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development.
Topics: Animals; Embryo, Nonmammalian; Endoplasmic Reticulum Chaperone BiP; Female; Gene Expression Regulation, Developmental; Heat-Shock Proteins; Organogenesis; Pronephros; Signal Transduction; Tretinoin; Xenopus Proteins; Xenopus laevis
PubMed: 25398881
DOI: 10.1074/jbc.M114.591628 -
Biochemical and Biophysical Research... Jul 2015Claudins are tetratransmembrane tight junction proteins and play important roles in regulating paracellular permeability of different nephron segments of the kidney....
Claudins are tetratransmembrane tight junction proteins and play important roles in regulating paracellular permeability of different nephron segments of the kidney. However, the roles of claudins in kidney development remain largely unknown. Here we studied the expression and functions of claudin-6 in Xenopus pronephros development. Xenopus claudin-6 is expressed in the developing pronephric tubule and duct but not glomus. Knockdown of claudin-6 by specific morpholino led to severe defects in pronephros tubular morphogenesis and blocked the terminal differentiation of the tubule cells. The claudin-6 morpholino targeted tubule cells showed failure of apical accumulation of actin and reduced lateral expression of tight junction protein Na/K-ATPase, suggesting an incomplete epithelization likely due to defected cell adhesions and apical-lateral polarity. Our work uncovered a novel role for claudin-6 in embryonic kidney development.
Topics: Animals; Claudins; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Immunohistochemistry; Organogenesis; Pronephros; RNA, Messenger; Tight Junctions; Xenopus Proteins; Xenopus laevis
PubMed: 25979361
DOI: 10.1016/j.bbrc.2015.04.065 -
Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish.Molecular Biology of the Cell Aug 2012In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From...
In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.
Topics: Animals; Cell Line; Cilia; Gene Knockdown Techniques; Larva; Male; Mice; Morpholinos; Phenotype; Photoreceptor Cells; Pronephros; Protein Transport; RNA Interference; Rats; Zebrafish; Zebrafish Proteins
PubMed: 22718903
DOI: 10.1091/mbc.E12-01-0019 -
Clinical Science (London, England :... Dec 2018The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since... (Review)
Review
The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides.
Topics: Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Animals, Genetically Modified; Gene Expression Regulation, Developmental; Kidney Diseases; Luminescent Proteins; Pronephros; Renin-Angiotensin System; Signal Transduction; Zebrafish; Zebrafish Proteins
PubMed: 30518571
DOI: 10.1042/CS20180721 -
Vaccine Sep 2012DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have...
DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.
Topics: Animals; Antigens, Viral; Cell Culture Techniques; Cell Line; Green Fluorescent Proteins; Hemorrhagic Septicemia, Viral; Leukocytes; Novirhabdovirus; Plasmids; Pronephros; Sequence Analysis, DNA; Transfection; Trout; Up-Regulation; Vaccination; Vaccines, DNA; Viral Envelope Proteins; Viral Vaccines
PubMed: 22824344
DOI: 10.1016/j.vaccine.2012.07.013 -
Developmental and Comparative Immunology Jan 2017The head kidney, analogous to the mammalian adrenal gland, is an organ unique for teleost fish. It comprises cytokine-producing lymphoid cells from the immune system and... (Review)
Review
The head kidney, analogous to the mammalian adrenal gland, is an organ unique for teleost fish. It comprises cytokine-producing lymphoid cells from the immune system and endocrine cells secreting cortisol, catecholamines, and thyroid hormones. The intimate organization of the immune system and endocrine system in one single organ makes bidirectional signalling between these possible. In this review we explore putative interactions between the thyroid and immune system in the head kidney. We give a short overview of the thyroid system, and consider the evidence for the presence of thyroid follicles in the head kidney as a normal, healthy trait in fishes. From mammalian studies we gather data on the effects of three important pro-inflammatory cytokines (TNFα, IL-1β, IL-6) on the thyroid system. A general picture that emerges is that pro-inflammatory cytokines inhibit the activity of the thyroid system at different targets. Extrapolating from these studies, we suggest that the interaction of the thyroid system by paracrine actions of cytokines in the head kidney is involved in fine-tuning the availability and redistribution of energy substrates during acclimation processes such as an immune response or stress response.
Topics: Animals; Cytokines; Energy Metabolism; Fishes; Head Kidney; Humans; Immunity; Neuroimmunomodulation; Paracrine Communication; Signal Transduction; Stress, Physiological; Thyroid Gland
PubMed: 27387152
DOI: 10.1016/j.dci.2016.06.025