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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 -
Frontiers in Cell and Developmental... 2020Previous studies in developing and zebrafish reported that the phosphate transporter is expressed in pronephric kidneys. The recent identification of as a monoallelic...
Previous studies in developing and zebrafish reported that the phosphate transporter is expressed in pronephric kidneys. The recent identification of as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog . This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest is involved in urinary tract and urorectal development and implicate as a disease-gene for BEEC.
PubMed: 32850778
DOI: 10.3389/fcell.2020.00567 -
International Journal of Biological... 2015The Hippo signaling pathway and its transcriptional co-activator Yap are known as essential regulators for cell proliferation and organ size. However, little is known...
The Hippo signaling pathway and its transcriptional co-activator Yap are known as essential regulators for cell proliferation and organ size. However, little is known about their roles in kidney development and ciliogenesis. We examined expression of Yap during zebrafish embryogenesis, and its transcripts were detected in pronephric duct, while Yap protein was found to be localized in the cytoplasm and apical membrane in kidney epithelium cells. By morpholino (MO) knockdown of yap expression in zebrafish, the injected larve exhibits pronephic cysts and many aspects of ciliopathy, which can be rescued by full-length yap mRNA, but not yap (S127A) mRNA. With transgenic Tg(Na(+)/K(+) ATPase:EGFP), we found that lacking Yap led to expansion and discontinuities of pronephric duct, as well as disorganization of cloaca during pronephros morphogenesis. Mis-located Na(+)/K(+) ATPase and ciliary abnormalities are also detected in pronephric duct of yap morphants. In addition, genetic analysis suggests that yap interacts with ift20, ift88 and arl13b in pronephric cyst formation. Taken together, our data reveals that Yap is required for pronephric duct integrity, maintenance of baso-lateral cell polarity, and ciliogenesis during zebrafish kidney development.
Topics: Animals; Ciliary Body; Gene Knockdown Techniques; Kidney; Morphogenesis; Sodium-Potassium-Exchanging ATPase; Subcellular Fractions; Trans-Activators; YAP-Signaling Proteins; Zebrafish; Zebrafish Proteins
PubMed: 26157348
DOI: 10.7150/ijbs.11346 -
Journal of Visualized Experiments : JoVE May 2016The embryonic kidney of Xenopus laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large,...
The embryonic kidney of Xenopus laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.
Topics: Animals; Gene Expression Regulation, Developmental; Microinjections; Pronephros; Xenopus; Xenopus laevis
PubMed: 27168375
DOI: 10.3791/53799 -
Genes Jun 2019Ankyrin repeats, the most common protein-protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin...
Ankyrin repeats, the most common protein-protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin repeat-containing proteins play diverse biological functions. Here, we identified the gene ankrd45, which encodes a novel, two ankyrin repeat-containing protein. Zebrafish ankrd45 displayed a tissue specific expression pattern during early development, with high expression in ciliated tissues, including otic vesicles, Kupffer's vesicles, pronephric ducts, and floor plates. Surprisingly, zebrafish ankrd45 mutants were viable and developed grossly normal cilia. In contrast, mutant larvae developed enlarged livers when induced with liver specific expression of Kras, one of the common mutations of KRAS that leads to cancer in humans. Further, histological analysis suggested that multiple cysts developed in the mutant liver due to cell apoptosis. Similarly, knockdown of ANKRD45 expression with either siRNA or CRISPR/Cas9 methods induced apoptosis in cultured cells, similar to those in zebrafish ankrd45 mutant livers after induction. Using different cell lines, we show that the distribution of ANKRD45 protein was highly dynamic during mitosis. ANKRD45 is preferably localized to the midbody ring during cytokinesis. Together, our results suggest that Ankrd45 is a novel ankyrin repeat protein with a conserved role during cell proliferation in both zebrafish embryos and mammalian cells.
Topics: Animals; Ankyrin Repeat; Body Patterning; CRISPR-Cas Systems; Cell Proliferation; Cilia; Embryonic Development; Gene Expression Regulation, Developmental; HeLa Cells; Humans; Larva; Mitosis; Zebrafish; Zebrafish Proteins
PubMed: 31208154
DOI: 10.3390/genes10060462 -
Molecular Metabolism Feb 2018Adult human kidneys produce erythropoietin (EPO), which regulates red blood cell formation; however, whether EPO also functions directly on kidney development and...
OBJECTIVE
Adult human kidneys produce erythropoietin (EPO), which regulates red blood cell formation; however, whether EPO also functions directly on kidney development and controls diabetic kidney disease remains unknown. Here we analyzed the role of EPO in kidney development and under hyperglycemic conditions in zebrafish and in humans.
METHODS
Diabetic patients and respective controls were enrolled in two cohorts. Serum EPO level and urine protein change upon human EPO administration were then analyzed. Transient knockdown and permanent knockout of EPO and EPOR in renal TG(WT1B:EGFP) zebrafish were established using the morpholino technology and CRISPR/Cas9 technology. Zebrafish embryos were phenotypically analyzed using fluorescence microscopy, and functional assays were carried out with the help of TexasRed labeled 70 kDa Dextran. Apoptosis was determined using the TUNEL assay and Annexin V staining, and caspase inhibitor zVADfmk was used for rescue experiments.
RESULTS
In type 2 diabetic patients, serum EPO level decreased with the duration of diabetes, which was linked to reduced kidney function. Human recombinant EPO supplementation ameliorated proteinuria in diabetic nephropathy patients. In zebrafish, loss-of-function studies for EPO and EPOR, showed morphological and functional alterations within the pronephros, adversely affecting pronephric structure, leading to slit diaphragm dysfunction by increasing apoptosis within the pronephros. Induction of hyperglycemia in zebrafish embryos induced pronephros alterations which were further worsened upon silencing of EPO expression.
CONCLUSIONS
EPO was identified as a direct renal protective factor, promoting renal embryonic development and protecting kidneys from hyperglycemia induced nephropathy.
Topics: Aged; Animals; Apoptosis; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Erythropoietin; Female; Humans; Kidney; Male; Middle Aged; Proteinuria; Recombinant Proteins; Zebrafish
PubMed: 29203238
DOI: 10.1016/j.molmet.2017.11.006 -
American Journal of Human Genetics Nov 2017Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing...
Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10 for novel LOF, increased to p = 4.1 × 10 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.
Topics: Alleles; Animals; Case-Control Studies; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Abnormalities; Exome; Female; Genetic Heterogeneity; Genome-Wide Association Study; Genotype; Heredity; Homozygote; Humans; Kidney; Kidney Diseases; Male; Membrane Proteins; Mice; Mutation; Neoplasm Proteins; Phenotype; RNA, Long Noncoding; Urinary Tract; Urogenital Abnormalities; Zebrafish
PubMed: 29100090
DOI: 10.1016/j.ajhg.2017.09.018 -
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 -
The Journal of Biological Chemistry Apr 2016Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for...
Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity.
Topics: Animals; Cilia; Cytoskeletal Proteins; Dogs; Gene Knockdown Techniques; Kidney; Madin Darby Canine Kidney Cells; Mice; Organogenesis; Zebrafish; Zebrafish Proteins; cdc42 GTP-Binding Protein
PubMed: 26895965
DOI: 10.1074/jbc.M115.688663 -
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