mtor Haploinsufficiency Ameliorates Renal Cysts and Cilia Abnormality in Adult Zebrafish tmem67 Mutants

2021 ◽  
pp. ASN.2020070991
Author(s):  
Ping Zhu ◽  
Qi Qiu ◽  
Peter C. Harris ◽  
Xiaolei Xu ◽  
Xueying Lin
Keyword(s):  
Renal Cysts ◽  
Cyst Formation ◽  
Adult Zebrafish ◽  
Transcription Activator ◽  
Primary Defect ◽  
Causative Gene ◽  
Mtor Inhibition ◽  
Adaptive Action ◽  
Single Nephron ◽  
Vertebrate Model

BackgroundAlthough zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.MethodsTranscription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67, 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 mtor strain or rapamycin was used to inhibit mTOR activity.ResultsAdult tmem67 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.ConclusionsAdult zebrafish tmem67 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.

scholarly journals mtor Haploinsufficiency Ameliorates Renal Cyst Formation in Adult Zebrafish tmem67 Mutants

2019 ◽  
Author(s):  
Ping Zhu ◽  
Qi Qiu ◽  
Peter C. Harris ◽  
Xiaolei Xu ◽  
Xueying Lin
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Therapeutic Strategy ◽  
Cyst Formation ◽  
Zebrafish Embryos ◽  
Adult Fish ◽  
Adult Zebrafish ◽  
Polycystic Kidney ◽  
Mtor Inhibition ◽  
Fish Model

AbstractAlthough zebrafish embryos have been utilized to study ciliogenesis and to model polycystic kidney disease (PKD), adult zebrafish remain unexplored. Here, we report the generation and characterization of a zebrafish mutant of tmem67, a homologue of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Although a small population of homozygous embryos exhibited pronephric cysts, all mutants were able to survive to adulthood and developed progressive mesonephric cysts with full penetrance. The cysts in the adult zebrafish kidneys manifested features of mammalian PKD, including switching of cyst origin from the proximal tubules to the collecting ducts, increased proliferation of cyst-lining epithelial cells, and hyperactive mTOR signaling. Consistent ciliary abnormalities were observed in both the embryonic and adult zebrafish mutants compared with the wild-type fish, including shorter and fewer single cilia in the distal pronephros and all segments of the mesonephros and greater numbers of multiciliated cells (MCCs). Lack of single cilium preceded cystogenesis, suggestive of a primary defect. Expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of cystogenesis in young adult zebrafish, suggesting an adaptive action. Interestingly, mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. In summary, we have established a tmem67 mutant as the first adult zebrafish PKD model, revealed a novel aspect of cilium regulation, and identified sustained mTOR inhibition as a candidate therapeutic strategy for tmem67-based PKD.Significance StatementWhile zebrafish embryos are well recognized for their value in studying ciliogenesis and polycystic kidney disease (PKD), adult zebrafish have not commonly been used. Here, we report the establishment of the first adult zebrafish model for PKD, which exhibits characteristics of mammalian PKD and shows kidney ciliary abnormalities consistent with those observed in an embryonic model. We also provide evidence for mTOR inhibition as a therapeutic strategy for this particular type of cystogenesis. Compared to the embryonic model, the adult fish model exhibits a spectrum of progressive pathogeneses and enables ciliary abnormalities to be discerned as either primary or secondary to cystogenesis. We believe that this novel adult fish model will facilitate mechanistic studies and therapeutic development for PKD.

Abstract 108: TALEN-mediated Gene-Editing in Zebrafish for Studying Titin Biology

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
YU-HUAN SHIH ◽  
Xiaolei Xu
Keyword(s):  
Gene Editing ◽  
Muscle Development ◽  
Human Diseases ◽  
Transcription Activator ◽  
Phenotypic Analysis ◽  
Causative Gene ◽  
Zebrafish Model ◽  
Powerful Research Tool ◽  
Vertebrate Model ◽  
A Domains

Background: Titin, the largest protein found in nature, has been found to be crucial for heart and muscle development and mutations on titin caused human diseases. Especially, truncated mutations on TITIN were found to be a major causative gene (more than 25%) for familiar dilated cardiomyopathy (DCM) but the mechanism is lacking. Due to the gigantic size and number of exons, genetic study of titin has been challenging. To facilitate genetic analysis of titin biology, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to edit the titin gene directly. Results: We generated TALEN pairs targeting different domains of zebrafish titin, including Z-disc domain, N2B domain, and A domains. Because zebrafish contain two titin homologues, ttna and ttnb , TALEN pairs targeting the corresponding loci for both homologues were made. The mutations are efficiently introduced on the titin using TALENs. In addition to these truncated mutants, TALEN technology can also create internal deletions. The titin mutant fish were identified and further phenotypic analysis has been carried out. Conclusions: Combined with TALEN technology, zebrafish can provide a convenient vertebrate model for genetic studies of titin biology. Moreover, this gene-editing technology will facilitate the use of the zebrafish model as a powerful research tool for modeling human diseases and investigating their pathogenesis.

scholarly journals Cyst fluid from human autosomal dominant polycystic kidneys promotes cyst formation and expansion by renal epithelial cells in vitro.

1992 ◽  
Vol 3 (4) ◽  
pp. 984-994
Author(s):  
M Ye ◽  
M Grant ◽  
M Sharma ◽  
L Elzinga ◽  
S Swan ◽  
...  
Keyword(s):  
Growth Factor ◽  
Autosomal Dominant ◽  
Renal Cysts ◽  
Secretory Activity ◽  
Collagen Matrix ◽  
Fluid Secretion ◽  
Cyst Formation ◽  
Cyst Fluid ◽  
Epidermal Growth

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive renal enlargement, culminating in renal insufficiency in over one half of affected individuals. The highly variable onset and clinical course of ADPKD may be due to factors extrinsic to the genetically defined renal cysts. In this study, cyst fluid samples from 12 nonazotemic and 18 azotemic ADPKD subjects were examined for in vitro biologic activity that promotes cellular proliferation and the secretion of fluid by renal epithelial monolayers, two pathogenetic mechanisms that have critical roles in the formation and the rate of expansion of renal cysts. Cyst fluid added to culture medium (final concentrations, 1 to 20%) caused Madin-Darby canine kidney cells and human kidney cortex (HKC) cells derived from primary cultures to form cysts in Type I collagen matrix. Cyst fluid stimulated the net transepithelial secretion of fluid by polarized monolayers composed of these same cells. Absolute levels of fluid secretory activity determined by MDCK bioassay were correlated directly with the rate of fluid secretion by HKC cell monolayers and with the extent of cyst formation by MDCK and HKC cells embedded in collagen matrix. The secretory activity of urine was negligible; secretory activity was detectable in the serum of normal and ADPKD subjects, but the levels were much lower than in cyst fluid. cAMP agonists prostaglandins E1 and E2, arginine vasopressin, and 8-Br-cAMP stimulated fluid secretion by MDCK and HKC monolayers, but these substances did not cause HKC cells to form cysts in collagen matrix, whereas cyst fluid did. Among other naturally occurring growth factors and autacoids, only epidermal growth factor and transforming growth factor alpha stimulated cyst formation by HKC cells; however, the capacity of cyst fluid to stimulate fluid secretion was not affected by treatment with antiserum to epidermal growth factor. It was concluded that potent, and possibly unique, substances in the cyst fluids of individuals with ADPKD support and augment biologic processes in renal epithelial cells that may be important in the promotion of progressive cyst expansion.

The molecular basis of ciliopathies and cyst formation

2015 ◽  
Author(s):  
Wolfgang Kühn ◽  
Gerd Walz
Keyword(s):  
Planar Cell Polarity ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Renal Cysts ◽  
Cyst Formation ◽  
Cystic Kidney Disease ◽  
Cystic Kidney ◽  
Convergent Extension ◽  
Diverse Range ◽  
Cell Intercalation

Abnormalities of the cilium, termed ‘ciliopathies’, are the prime suspect in the pathogenesis of renal cyst formation because the gene products of cystic disease-causing genes localize to them, or near them. However, we only partially understand how cilia maintain the geometry of kidney tubules, and how abnormal cilia lead to renal cysts, and the diverse range of diseases attributed to them. Some non-cystic diseases share pathology of the same structures. Although still incompletely understood, cilia appear to orient cells in response to extracellular cues to maintain the overall geometry of a tissue, thereby intersecting with the planar cell polarity (PCP) pathway and the actin cytoskeleton. The PCP pathway controls two morphogenetic programmes, oriented cell division (OCD) and convergent extension (CE) through cell intercalation that both seem to play a critical role in cyst formation. The two-hit theory of cystogenesis, by which loss of the second normal allele causes tubular epithelial cells to form kidney cysts, has been largely borne out. Additional hits and influences may better explain the rate of cyst formation and inter-individual differences in disease progression. Ciliary defects appear to converge on overlapping signalling modules, including mammalian target of rapamycin and cAMP pathways, which can be targeted to treat human cystic kidney disease irrespective of the underlying gene mutation.

scholarly journals Phenotyping an adult zebrafish lamp2 cardiomyopathy model identifies mTOR inhibition as a candidate therapy

2019 ◽  
Vol 133 ◽  
pp. 199-208 ◽  
Author(s):  
Alexey V. Dvornikov ◽  
Mingmin Wang ◽  
Jingchun Yang ◽  
Ping Zhu ◽  
Tai Le ◽  
...  
Keyword(s):  
Adult Zebrafish ◽  
Mtor Inhibition

scholarly journals An NMDA-R mediated short-term memory resistant to anesthesia in adult Danio rerio

2019 ◽  
Author(s):  
Gokul Rajan ◽  
Joby Joseph
Keyword(s):  
Fear Conditioning ◽  
Danio Rerio ◽  
Early Phase ◽  
Short Term Memory ◽  
Green Light ◽  
Long Term Memory ◽  
Adult Zebrafish ◽  
Term Memory ◽  
Vertebrate Model

ABSTRACTMemory in animals is labile in the early phase post-training. Memory in the early phase has been shown to be disrupted by treatments such as electroconvulsive shock or cold-shock (Quinn and Dudai, 1976). Using hypothermic shock and other pharmacological interventions, the various underlying memory pathways in Drosophila can be identified as an immediate short-lasting anesthesia sensitive memory and a delayed anesthesia resistant memory which is followed by a more stable protein synthesis dependent long-term memory (Margulies et al., 2005). In another ectothermic animal, Danio rerio, a popular vertebrate model, we ask if such a memory component exists which is sensitive to hypothermic disruption. To test this, we developed a fear conditioning assay with a green light at the bottom of the tank as the conditioned stimulus (CS) and electric shock as the unconditioned stimulus (US). We also standardized a cold anesthesia protocol in adult zebrafish to induce stage V anesthesia. The learning/memory was found to be NMDA-R mediated. Cold anesthesia as well as tricaine mediated anesthesia did not significantly affect the early-acting memory trace induced by a fear-conditioning protocol in adult zebrafish. We suggest future directions to tease out the underlying memory components in the early phase of memory in zebrafish.

scholarly journals Genesis of renal cysts is associated with clusterin expression in experimental cystic disease.

1995 ◽  
Vol 5 (9) ◽  
pp. 1669-1674
Author(s):  
M E Rosenberg ◽  
J C Manivel ◽  
F A Carone ◽  
Y S Kanwar
Keyword(s):  
Time Course ◽  
Drug Withdrawal ◽  
Renal Cysts ◽  
Cyst Formation ◽  
Sprague Dawley ◽  
Polycystic Kidney ◽  
Tubular Basement Membrane ◽  
Sprague Dawley Rats ◽  
Chemically Induced ◽  
Clusterin Expression

Phenol II is a cystogenic chemical that rapidly induces renal cysts, which regress after drug withdrawal. Cyst formation in this model parallels changes in the tubular basement membrane. Clusterin is a potent cohesive factor induced in states of tissue remodeling. The purpose of this study was to determine if renal clusterin was increased in the Phenol II model and to define the time course and distribution of its induction. Male Sprague-Dawley rats were given, by daily gavage, Phenol II (1.2 mg/kg per day) or vehicle (control). The kidneys were harvested after 1, 2, or 4 days of Phenol II treatment or 3 or 7 days after drug withdrawal. An increase in immunoreactive clusterin was seen in the kidneys of Phenol II-treated rats but not in controls. The appearance of clusterin followed a time course similar to that for cyst formation, with expression confined to the epithelial lining and intratubular casts of dilated or cystic tubules. After Phenol II withdrawal, renal cysts regressed and clusterin staining disappeared. The development of cysts was associated with an increase in clusterin mRNA that decreased after drug withdrawal. In conclusion, a marked, yet reversible induction of clusterin occurred in chemically induced polycystic kidney disease. The function of clusterin in this setting remains enigmatic.

scholarly journals Discovery Sulfoglycomics and Identification of the Characteristic Fragment Ions for High-Sensitivity Precise Mapping of Adult Zebrafish Brain–Specific Glycotopes

2021 ◽  
Vol 8 ◽  
Author(s):  
Huan-Chuan Tseng ◽  
Cheng-Te Hsiao ◽  
Nao Yamakawa ◽  
Yann Guérardel ◽  
Kay-Hooi Khoo
Keyword(s):  
Full Range ◽  
High Sensitivity ◽  
Structural Features ◽  
Mode Analysis ◽  
Adult Zebrafish ◽  
Organ Specific ◽  
Vertebrate Model ◽  
Functional Relevance ◽  
Diagnostic Ions ◽  
The Brain

Mass spectrometry–based high-sensitivity mapping of terminal glycotopes relies on diagnostic MS2 and/or MS3 ions that can differentiate linkage and define the location of substituents including sulfates. Unambiguous identification of adult zebrafish glycotopes is particularly challenging due to the presence of extra β4-galactosylation on the basic building block of Galβ1-4GlcNAc that can be fucosylated and variably sialylated by N-acetyl, N-glycolyl, or deaminated neuraminic acids. Building on previous groundwork that have identified various organ-specific N- and O-glycans of adult zebrafish, we show here that all the major glycotopes of interest can be readily mapped by direct nano-LC-MS/MS analysis of permethylated glycans. Homing in on the brain-, intestine-, and ovary-derived samples, organ-specific glycomic reference maps based on overlaid extracted ion chromatograms of resolved glycan species, and composite charts of summed intensities of diagnostic MS2 ions representing the distribution and relative abundance of each of the glycotopes and sialic acid variants were established. Moreover, switching to negative mode analysis of sample fractions enriched in negatively charged glycans, we show, for the first time, that a full range of sulfated glycotopes is expressed in adult zebrafish. In particular, 3-O-sulfation of terminal Gal was commonly found, whereas terminal sulfated HexNAc as in GalNAcβ1-4GlcNAc (LacdiNAc), and 3-O-sulfated hexuronic acid as in HNK-1 epitope (SO3-3GlcAβ1-3Galβ1-4GlcNAc) were identified only in the brain and not in the intestine or ovaries analyzed in parallel. Other characteristic structural features of sulfated O- and N-glycans along with their diagnostic ions detected in this discovery mode sulfoglycomic work collectively expand our adult zebrafish glycome atlas, which can now allow for a more complete navigation and probing of the underlying sulfotransferases and glycosyltransferases, in search of the functional relevance of zebrafish-specific glycotopes. Of particular importance is the knowledge of glycomic features distinct from those of humans when using adult zebrafish as an alternative vertebrate model, rather than mouse, for brain-related glyco-neurobiology studies.

Abstract 232: Studies of Cardiac transcriptome and Dilated Cardiomyopathy-causative genes in zebrafish

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Yu-Huan Shih ◽  
Yuji Zhang ◽  
Tim Olson ◽  
Xiaolei Xu
Keyword(s):  
Dilated Cardiomyopathy ◽  
System Biology ◽  
Underlying Mechanism ◽  
The Novel ◽  
Rna Seq ◽  
Adult Zebrafish ◽  
Future System ◽  
Novel Variants ◽  
Vertebrate Model ◽  
Definition Of

Background: Rapid advance of genome technologies accelerate the discovery of genetic basis of cardiomyopathy and heart failure and also enable system biology studies to pinpoint underlying mechanism. However, the limited throughput of mammalian models restricted the number of genes that can be studied in a particular lab. Adult zebrafish has been recently pursued as a new model with higher throughput. However, as a non-mammalian model, its conservation is not tested. Objective: To assess the conservation of zebrafish for genetic studies of human dilated cardiomyopathy (DCM) via transcriptome analysis of 51 known DCM-causative genes. Methods and Results: By conducting RNA-sequencing (RNA-seq) analysis of larva and adult zebrafish, we identified genes with high expression level in the heart and fetal gene program using differential expression between embryonic and adult stages. We then searched zebrafish orthologues for 51 reported human DCM-causative or associated genes and identified zebrafish orthologues for 49 of them. While 30 genes have a single orthologue, 14 genes have two homologues and the remaining 5 genes have more than three. We then applied the transcriptome data to prioritize these homologues for the 19 DCM causative genes with more than one homologue. Based on the cardiac abundance and cardiac enrichment hypothesis, we are able to recommend a single zebrafish homologue of high priority for 12 out of 19 DCM genes, 2 zebrafish homologues of high priority for ACTC1. Interestingly, our expressional data suggested zebrafish othologues for human MYH6 and MYH7 , respectively. Similar to that in mammals, these two zebrafish othologues are oppositely expressed during zebrafish embryonic and adult stage. Conclusions: Orthologues for the majority of DCM causative genes can be found in Zebrafish, supporting its usage as a conserved vertebrate model for studying DCM. The definition of cardiac transcriptome in zebrafish will facilitate the future system biology studies. This vertebrate model with higher throughput can be further leveraged to validate the novel variants identified from human patients, to understand underlying signaling pathways and to develop novel therapeutics.

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