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Scientific Reports Oct 2023The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary...
The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) interacts with the Ldb1-Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1-Lhx1-Ssbp transcriptional complexes in embryonic development.
Topics: Animals; Xenopus laevis; LIM-Homeodomain Proteins; Gene Expression Regulation, Developmental; Transcription Factors; Kidney; Embryonic Development; Morphogenesis; Pronephros; Xenopus Proteins; Mammals
PubMed: 37794075
DOI: 10.1038/s41598-023-43662-1 -
BioRxiv : the Preprint Server For... Sep 2023Developmental studies have revealed the importance of the transcription factor Hand2 in cardiac development. Hand2 promotes cardiac progenitor differentiation and...
Developmental studies have revealed the importance of the transcription factor Hand2 in cardiac development. Hand2 promotes cardiac progenitor differentiation and epithelial maturation, while repressing other tissue types. The mechanisms underlying the promotion of cardiac fates are far better understood than those underlying the repression of alternative fates. Here, we assess Hand2-dependent changes in gene expression and chromatin remodeling in cardiac progenitors of zebrafish embryos. Cell-type specific transcriptome analysis shows a dual function for Hand2 in activation of cardiac differentiation genes and repression of pronephric pathways. We identify functional regulatory elements whose chromatin accessibility are increased in mutant cells. These regulatory elements associate with non-cardiac gene expression, and drive reporter gene expression in tissues associated with Hand2-repressed genes. We find that functional Hand2 is sufficient to reduce non-cardiac reporter expression in cardiac lineages. Taken together, our data support a model of Hand2-dependent coordination of transcriptional programs, not only through transcriptional activation of cardiac and epithelial maturation genes, but also through repressive chromatin remodeling at the DNA regulatory elements of non-cardiac genes.
PubMed: 37790542
DOI: 10.1101/2023.09.23.559156 -
Journal of the American Society of... Apr 2021Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.
BACKGROUND
Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.
METHODS
Transcription activator-like effector nucleases (TALEN) technology was used to generate mutant for , the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic strain or rapamycin was used to inhibit mTOR activity.
RESULTS
Adult zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants.
CONCLUSIONS
Adult zebrafish mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.
PubMed: 33574160
DOI: 10.1681/ASN.2020070991 -
Disease Models & Mechanisms Nov 2021The Hippo signaling pathway is a kinase cascade that plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription...
The Hippo signaling pathway is a kinase cascade that plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated the Hippo pathway in kidney development and kidney diseases. To further illustrate its roles in vertebrate kidney, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes. The stk3-/- mutant exhibited edema, formation of glomerular cysts and pronephric tubule dilation during the larval stage. Interestingly, disruption of wwtr1, but not yap1, significantly alleviated the renal phenotypes of the stk3-/- mutant, and overexpression of Wwtr1 with the CMV promoter also induced pronephric phenotypes, similar to those of the stk3-/- mutant, during larval stage. Notably, adult fish with Wwtr1 overexpression developed phenotypes similar to those of human polycystic kidney disease (PKD). Overall, our analyses revealed roles of Stk3 and Wwtr1 in renal cyst formation. Using a pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development.
Topics: Animals; Hippo Signaling Pathway; Kidney; Mammals; Polycystic Kidney Diseases; Signal Transduction; Zebrafish
PubMed: 34545930
DOI: 10.1242/dmm.049027 -
BMC Developmental Biology Dec 2019Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate...
BACKGROUND
Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking.
RESULTS
Here, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf.
CONCLUSIONS
Our results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos.
Topics: Animals; Central Nervous System; Conserved Sequence; Female; Gastrointestinal Tract; Gene Expression Regulation, Developmental; Maternal Inheritance; Multigene Family; Protein Domains; Tissue Distribution; Zebrafish; Zebrafish Proteins; rab GTP-Binding Proteins
PubMed: 31884948
DOI: 10.1186/s12861-019-0207-7 -
Journal of the American Society of... Mar 2021Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic...
BACKGROUND
Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease.
METHODS
Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. and studies determined the functional significance of the mutations identified.
RESULTS
Three biallelic variants of the transcriptional regulator were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in embryos disrupted pronephric development. Human wild-type RNA rescued the disruption, but the three variants did not. Finally, CRISPR-mediated knockout of in human podocytes led to dysregulation of several renal developmental genes.
CONCLUSIONS
Variants in can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.
Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cell Line; Child, Preschool; DNA-Binding Proteins; Female; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Gene Knockout Techniques; Hernia, Hiatal; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Male; Microcephaly; Models, Molecular; Mutation, Missense; Nephrosis; Nephrotic Syndrome; Podocytes; Polymorphism, Single Nucleotide; Pronephros; Protein Stability; Transcription Factors; Xenopus laevis; Zinc Fingers
PubMed: 33593823
DOI: 10.1681/ASN.2020040490 -
Nephron 2018von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal...
BACKGROUND
von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene.
METHODS
We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays.
RESULTS
Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL.
CONCLUSIONS
Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.
Topics: Animals; Embryonic Development; Kidney Glomerulus; Kidney Tubules, Proximal; Larva; Mutation; Receptors, Vascular Endothelial Growth Factor; Tumor Suppressor Proteins; Zebrafish; Zebrafish Proteins
PubMed: 29342457
DOI: 10.1159/000484096 -
Frontiers in Cell and Developmental... 2020Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood....
Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.
PubMed: 32754590
DOI: 10.3389/fcell.2020.00583 -
American Journal of Human Genetics May 2019Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About...
Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.
Topics: Adult; Animals; Child; Chromosome Aberrations; DNA-Binding Proteins; Female; Fetal Diseases; Genes, Dominant; Gestational Age; Humans; Male; Mice; Middle Aged; Mutation; Pedigree; Pregnancy; Urinary Bladder Neck Obstruction; Zebrafish
PubMed: 31051115
DOI: 10.1016/j.ajhg.2019.03.023 -
Scientific Reports Aug 2017Autosomal dominant polycystic kidney disease (ADPKD) is a common kidney disease caused by mutations in PKD1 or PKD2. Metformin reduces cyst growth in mouse models of...
Autosomal dominant polycystic kidney disease (ADPKD) is a common kidney disease caused by mutations in PKD1 or PKD2. Metformin reduces cyst growth in mouse models of PKD1. However, metformin has not been studied in animal models of PKD2, and the cellular mechanism underlying its effectiveness is not entirely clear. This study investigated the effects of metformin on cyst formation in a zebrafish model of polycystin-2 deficiency resulting from morpholino knockdown of pkd2. We added metformin (2.5 to 20 mM) to the embryo media between 4 and 48 hours post fertilisation and observed pronephric cyst formation by using the wt1b promoter-driven GFP signal in Tg(wt1b:GFP) pkd2 morphants. Metformin inhibited pronephric cyst formation by 42-61% compared with the untreated controls. Metformin also reduced the number of proliferating cells in the pronephric ducts, the degree of dorsal body curvature, and the infiltration of leukocytes surrounding the pronephros. Moreover, metformin treatment increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced autophagy in the pronephros. Our data suggest that metformin reduces cyst formation through activation of the AMPK pathway and modulation of defective cellular events such as proliferation and autophagy. These results also imply that metformin could have therapeutic potential for ADPKD treatment.
Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Cell Proliferation; Cloaca; Cysts; Disease Models, Animal; Kidney Tubules; Leukocytes; Metformin; Polycystic Kidney, Autosomal Dominant; Spinal Curvatures; TRPP Cation Channels; Zebrafish
PubMed: 28769124
DOI: 10.1038/s41598-017-07300-x