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Gene Expression Patterns : GEP Sep 2019UNC119 proteins are required for ciliary trafficking in a process called lipidated protein intraflagellar targeting (LIFT) or through vesicle transport. However,...
UNC119 proteins are required for ciliary trafficking in a process called lipidated protein intraflagellar targeting (LIFT) or through vesicle transport. However, although unc119 has been studied in a variety of contexts, either organismal constraints or genetic redundancy has largely restricted their study in ciliary contexts. One possible solution for this is to use the zebrafish, however, the unc119 genes have not been well described in this species. In our study, we show in a condensed species tree that the presence of unc119 genes correlates with the presence of cilia across eukaryotes and that phylogenetic evidence suggests there are three subgroups of UNC119 proteins. Zebrafish contain all three of these subgroups: two vertebrate-specific UNC119A proteins, one vertebrate-specific UNC119B protein, and one UNC119. Expression analyses show that each of the zebrafish unc119 genes are maternally-expressed and have overlapping but distinct expression in ciliated tissues, such as the eye, pronephric duct, and spinal cord. Overall, these findings set the foundation for future studies into the use of the zebrafish to study unc119 gene knock-outs, particularly from a ciliary perspective.
Topics: Animals; Cilia; Conserved Sequence; Evolution, Molecular; Eye; Nephrons; Phylogeny; Spinal Cord; Synteny; Zebrafish; Zebrafish Proteins
PubMed: 31055152
DOI: 10.1016/j.gep.2019.04.003 -
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 -
Current Molecular Medicine 2019In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh)...
BACKGROUND
In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh) signaling in many cases. However, the role of FoxF1 in cilium remains unknown. Here, we showed an essential role of FoxF1 in the regulation of ciliogenesis and in the distribution of Shh signaling components in cilium.
METHODS
NIH/3T3 cells were serum starved for 24h to induce cilium. Meanwhile, shRNA was used to knockdown the FoxF1 expression in the cells and CRISPR/Cas9 was used to generate the FoxF1 zebrafish mutant. The mRNA and protein expression of indicated genes were detected by the qRT-PCR and western blot, respectively. Immunofluorescence staining was performed to detect the cilium and Shh components distribution.
RESULTS
FoxF1 knockdown decreased the cilium length in NIH/3T3 cells. Meanwhile, the disruption of FoxF1 function inhibited the expression of cilium-related genes and caused an abnormal distribution of Shh components in the cilium. Furthermore, homozygous FoxF1 mutants exhibited defective development of pronephric cilium in early zebrafish embryos.
CONCLUSION
Together, our data illustrated that FoxF1 is required for ciliogenesis in vitro and in vivo and for the proper localization of Shh signaling components in cilium.
Topics: Animals; Cell Line; Cilia; Forkhead Transcription Factors; Hedgehog Proteins; Humans; Mice; NIH 3T3 Cells; Organogenesis; Signal Transduction; Zebrafish
PubMed: 30950350
DOI: 10.2174/1566524019666190405115420 -
International Journal of Molecular... Mar 2019The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible...
The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift17 in embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.
Topics: Adaptor Proteins, Signal Transducing; Algorithms; Animals; Carrier Proteins; Cytoskeletal Proteins; Disease Models, Animal; Embryo, Nonmammalian; Gene Knockdown Techniques; Humans; Image Processing, Computer-Assisted; Kidney; Organ Specificity; Phenotype; Polycystic Kidney Diseases; Small Molecule Libraries; Software; Workflow; Zebrafish
PubMed: 30875791
DOI: 10.3390/ijms20061290 -
Frontiers in Physiology 2019The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and...
The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and water absorption and waste excretion. The embryonic kidney of consists of a single functional nephron composed of regions that are analogous to those found in the human nephron, making it a simple model for the study of nephrogenesis. The exocyst complex, which traffics proteins to the cell membrane in vesicles via CDC42, is essential for normal kidney development. Here, we show that the CDC42-GEF, dynamin binding protein (Dnmbp/Tuba), is essential for nephrogenesis in . is expressed in embryo kidneys during development, and knockdown of Dnmbp using two separate morpholino antisense oligonucleotides results in reduced expression of late pronephric markers, whereas the expression of early markers of nephrogenesis remains unchanged. A greater reduction in expression of markers of differentiated distal and connecting tubules was seen in comparison to proximal tubule markers, indicating that Dnmbp reduction may have a greater impact on distal and connecting tubule differentiation. Additionally, Dnmbp reduction results in glomus and ciliary defects. knockout using CRISPR results in a similar reduction of late markers of pronephric tubulogenesis and also results in edema formation in later stage embryos. Overexpression of in the kidney also resulted in disrupted pronephric tubules, suggesting that levels in the developing kidney are tightly regulated, with either increased or decreased levels leading to developmental defects. Together, these data suggest that Dnmbp is required for nephrogenesis.
PubMed: 30863317
DOI: 10.3389/fphys.2019.00143 -
Aquatic Toxicology (Amsterdam,... Apr 2019Brominated flame retardants are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, but the molecular mechanisms underlying this process and...
Brominated flame retardants are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, but the molecular mechanisms underlying this process and their effects on TH-sensitive tissues during the stages of early development are not well characterised. In this study, we exposed zebrafish (Danio rerio) embryo-larvae to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and tetrabromobisphenol A (TBBPA) via the water for 96 h from fertilisation and assessed for lethality, effects on development and on the expression of a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis via both real time quantitative PCR (qRT-PCR) on whole body extracts and whole mount in situ hybridisation (WISH) to identify tissue targets. The 96-h lethal median concentration (96h-LC) for TBBPA was 0.9 μM and mortality was preceded by retardation of development (smaller animals) and morphological deformities including, oedemas in the pericardial region and tail, small heads, swollen yolk sac extension. Exposure to BDE-47 did not affect zebrafish embryo-larvae survival at any of the concentrations tested (1-100 μM) but caused yolk sac and craniofacial deformities, a curved spine and shorter tail at the highest exposure concentration. TBBPA exposure resulted in higher levels of mRNAs for genes encoding deiodinases (dio1), transport proteins (ttr), the thyroid follicle synthesis protein paired box 8 (pax8) and glucuronidation enzymes (ugt1ab) and lower levels of dio3b mRNAs in whole body extracts, with responses varying with developmental stage. BDE-47 exposure resulted in higher levels of thrb, dio1, dio2, pax8 and ugt1ab mRNAs and lower levels of ttr mRNAs in whole body extracts. TBBPA and BDE-47 therefore appear to disrupt the TH system at multiple levels, increasing TH conjugation and clearance, disrupting thyroid follicle development and altering TH transport. Compensatory responses in TH production/ metabolism by deiodinases were also evident. WISH analyses further revealed that both TBBPA and BDE-47 caused tissue-specific changes in thyroid receptor and deiodinase enzyme expression, with the brain, liver, pronephric ducts and craniofacial tissues appearing particularly responsive to altered TH signalling. Given the important role of TRs in mediating the actions of THs during key developmental processes and deiodinases in the control of peripheral TH levels, these transcriptional alterations may have implications for TH sensitive target genes involved in brain and skeletal development. These findings further highlight the potential vulnerability of the thyroid system to disruption by BFRs during early developmental windows.
Topics: Animals; Embryo, Nonmammalian; Flame Retardants; Gene Expression Regulation, Developmental; Halogenated Diphenyl Ethers; Iodide Peroxidase; Larva; Organ Specificity; Polybrominated Biphenyls; RNA, Messenger; Toxicity Tests, Acute; Water Pollutants, Chemical; Zebrafish; Zebrafish Proteins
PubMed: 30763833
DOI: 10.1016/j.aquatox.2019.01.022 -
Fish & Shellfish Immunology Apr 2019The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds...
The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot.
Topics: Animals; Embryonic Development; Female; Fish Proteins; Flatfishes; Organ Specificity; Receptors, Polymeric Immunoglobulin
PubMed: 30633962
DOI: 10.1016/j.fsi.2019.01.010 -
Frontiers in Endocrinology 2018Prolactin (PRL) has been considered a key regulator of ion uptake in zebrafish. The genes and , which are Na and chloride Cl co-transporters, have been reported to be...
Prolactin (PRL) has been considered a key regulator of ion uptake in zebrafish. The genes and , which are Na and chloride Cl co-transporters, have been reported to be regulated by PRL in freshwater fish. The integrative network of PRL signaling dissected from the knockout of tissue-specific downstream PRL ion transporters remains poor. In the present study, zebrafish models with increased endogenous levels of PRL were generated through the knockout of or , and the developmental consequences were analyzed. The increased levels of pituitary PRL were observed in both - and -deficient fish. Unlike the -deficient fish, which could survive to adulthood, the -deficient fish began to die at 9 days post-fertilization (dpf) and did not survive beyond 17 dpf. This survival defect is a result of defective Cl uptake in this mutant, indicating that Slc12a10.2 plays an essential role in Cl uptake. Intriguingly, compared to the levels in control fish, no significant differences in the levels of Na in the body were observed in - or -deficient zebrafish. The upregulations of the PRL downstream transporters, , and were observed in -deficient fish in both the gills/skin and the pronephric duct. However, this type of response was not observed in the pronephric duct of -deficient fish, except under Na-deprived conditions. Our results show that PRL is susceptible to deficiencies in downstream ion transporters. Moreover, both the gills/skin and pronephric duct show differential expression of downstream PRL targets in response to increased levels of pituitary PRL caused by the depletion of tissue-specific ion transporters.
PubMed: 30515132
DOI: 10.3389/fendo.2018.00683 -
FASEB Journal : Official Publication of... Mar 2019Centrosomal proteins play critical roles in ciliogenesis. Mutations in many centrosomal proteins have been documented to contribute to developmental defects and...
Centrosomal proteins play critical roles in ciliogenesis. Mutations in many centrosomal proteins have been documented to contribute to developmental defects and cilium-related diseases. Centrosomal protein fibroblast growth factor receptor 1 oncogene partner-related protein of 20 kDa (FOR20) is crucial for ciliogenesis in mammalian cells and the unicellular eukaryote Paramecium; however, the biologic significance of FOR20 in vertebrate development remains unclear. We cloned the zebrafish homolog of the for20 gene and found that for20 mRNA is enriched in ciliated tissues during early zebrafish development. Knockdown of for20 by morpholino oligonucleotides in zebrafish results in multiple ciliary phenotypes, including curved body, hydrocephaly, pericardial edema, kidney cysts, and left-right asymmetry defects. for20 morphants show reduced number and length of cilia in Kupffer's vesicle and pronephric ducts. High-speed video microscopy reveals that cilia in most for20 morphants are consistently paralyzed or beat arrhythmically. To confirm the ciliary phenotypes of for20 morphants, we used the CRISPR/Cas9 system to disrupt for20 gene in zebrafish. for20 mutants exhibit multiple ciliary phenotypes resembling the defects in for20 morphants. All of these phenotypes in for20 morphants and mutants are significantly reversed by exogenous expression of for20 mRNA. Taken together, these data suggest that FOR20 is required for cilium-mediated processes during zebrafish embryogenesis.-Xie, S., Jin, J., Xu, Z., Huang, Y., Zhang, W., Zhao, L., Lo, L. J., Peng, J., Liu, W., Wang, F., Shu, Q., Zhou, T. Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos.
Topics: Animals; Body Patterning; Centrosome; Cilia; Embryonic Development; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Morpholinos; Mutation; RNA, Messenger; Zebrafish; Zebrafish Proteins
PubMed: 30475641
DOI: 10.1096/fj.201801235RR -
Scientific Reports Oct 2018The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and...
The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.
Topics: Animals; Body Patterning; Cell Line; Chromatography, Liquid; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Gene Order; Genetic Vectors; Kidney; LIM-Homeodomain Proteins; MicroRNAs; Multiprotein Complexes; Organogenesis; Protein Binding; Repressor Proteins; Tandem Mass Spectrometry; Transcription Factors; Xenopus Proteins; Xenopus laevis
PubMed: 30375416
DOI: 10.1038/s41598-018-34038-x