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PubMed2026

Biallelic pathogenic variants in FLNB are associated with paediatric steroid-resistant nephrotic syndrome via podocyte cytoskeletal dysfunction.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a severe paediatric kidney disease and a leading cause of end-stage kidney disease in children, with a high genetic contribution. While over 80 monogenic causes of SRNS have been identified, a significant proportion of affected patients still lack a clear genetic diagnosis, indicating that additional causative genes remain to be discovered. METHODS: Through whole-exome sequencing of a paediatric SRNS cohort, we identified three probands carrying biallelic FLNB pathogenic variants. Sanger sequencing was performed for familial cosegregation verification and ACMG classification. Expression of Filamin B, Nephrin and Synaptopodin in renal tissues was assessed by immunohistochemistry/immunofluorescence. Wild-type and patient-derived variant FLNB plasmids were constructed and transfected into HEK293T cells and immortalised human podocytes (HPCs). The effects of these variants on protein expression, localisation and cytoskeletal organisation were assessed by western blotting and immunofluorescence. FLNB expression in HPCs was silenced using shRNA to evaluate the impact on podocyte marker proteins, cytoskeletal integrity and migratory capacity. A zebrafish flnb knockdown model was employed to validate its effects on renal development. RESULTS: All three probands presented with isolated SRNS without skeletal developmental abnormalities, and renal tissues showed significantly reduced Filamin B protein expression. In vitro, p.L117P and p.M1803L variants led to markedly reduced protein expression, while p.R470L and p.K2586R induced perinuclear aggregation of Filamin B accompanied by F-actin rearrangement. FLNB silencing led to downregulation of Nephrin and Synaptopodin, cytoskeletal disorganisation and impaired cell migration. Zebrafish flnb knockdown exhibited pericardial oedema, defective nephron development and abnormal podocyte foot processes. CONCLUSION: We report for the first time that biallelic FLNB pathogenic variants are associated with paediatric SRNS by disrupting Filamin B expression, cytoskeletal integrity and podocyte function, providing evidence that FLNB is a novel monogenic cause of SRNS.

Humans↗
PubMed2003

The pro-opiomelanocortin gene of the zebrafish (Danio rerio).

The cDNA and the gene for pro-opiomelanocortin (POMC) in the zebrafish (Danio rerio) were isolated and analyzed. The gene consists of three exons and two short introns and has a similar overall structural organization as in Homo sapiens. Intron 1 (339 bp) divides the 5(') untranslated region from the coding region while intron 2 (1522 bp) is located between the signal peptide and the sequence encoding ACTH. Transcription starts 26 bp downstream of a TATA box and there is one polyadenylation signal in the 3(') untranslated region. The cDNA comprises of 964 bp with an open reading frame encoding a 222 amino acid hormone prepropeptide that is split into six putative hormones. Sequence comparison of zebrafish POMC to sequences of various other vertebrate species reveals four regions that are highly conserved during the evolution of vertebrates-the N-terminal region, ACTH, beta-MSH, and beta-endorphin, whereas the connecting peptides show a much higher degree of variability. Phylogenetic analysis of the POMC sequences of various vertebrate species resulted in the expected pattern of species evolution. In situ hybridization demonstrated POMC expression in a cluster of cells (corticotrophs) in the pituitary of the zebrafish as early as 23 h after fertilization. These findings will facilitate the use of the zebrafish as a model organism in the study of the physiological role of POMC-derived peptides.

Amino Acid Sequence↗
PubMed1998

Isolation of the zebrafish homologues for the tie-1 and tie-2 endothelium-specific receptor tyrosine kinases.

Several characteristics of the zebrafish embryo make it an attractive model in which to study the development of the cardiovascular system. The utility of the zebrafish as a model of mammalian vascular development will depend on the conservation of molecular and morphogenetic mechanisms of vessel growth. Here, we report the cloning of the zebrafish homologues of the endothelium-specific receptor tyrosine kinases tie-1 and tie-2. The Z tie-2 clone represents the first report of a full-length zebrafish endothelium-specific gene. The zebrafish tie family members have significant structural homology with their murine and human counterparts. In addition, like the murine tie-1 and tie-2 genes, expression was found predominantly in endothelial cells. At 24-hr postfertilization (HPF), Z tie-1 was expressed in all observed populations of endothelial cells. Interestingly, Z tie-2 exhibited a similar, although slightly more restricted, expression pattern. Taken together, these data strongly suggest that mechanisms of vascular development are highly conserved across species and that zebrafish will continue to be a useful model for the investigation of vertebrate embryonic vascular development.

Amino Acid Sequence↗
PubMed2026

Pseudomonas aeruginosa mgtC gene is under the control of PhoP and CbrAB regulators, and its expression can be visualized in macrophages.

The MgtC virulence factor is important during the intramacrophage stage in both classical intracellular pathogens, such as Salmonella Typhimurium, and in extracellular bacteria that transiently encounter intracellular environments during infection, such as Pseudomonas aeruginosa. In these different pathogens, mgtC expression is induced in vitro by magnesium ion depletion, a condition reported to mimic the macrophage environment. Here, we developed an unstable GFP reporter system to monitor in real time the transcriptional activation of the P. aeruginosa mgtC promoter. After in vitro validation in magnesium-defined media, this reporter system allowed visualization of the mgtC promoter induction in a subset of bacteria when P. aeruginosa localized inside cultured macrophages. In addition, although rare under our experimental conditions, in vivo activation of the mgtC promoter was observed for the first time within macrophages of live, infected zebrafish larvae, a cutting-edge vertebrate model for real-time imaging. While MgtC regulation in Salmonella is mediated by the magnesium-responsive PhoPQ two-component system, its regulation in P. aeruginosa remained unknown. The use of mutant strains for two-component regulatory systems revealed that the PhoP regulator, but not by its cognate sensor PhoQ, was required to activate P. aeruginosa MgtC expression in vitro. Unexpectedly, CbrAB, a two-component system specific to Pseudomonas species, was also involved in P. aeruginosa MgtC regulation. Both PhoP and CbrB regulatory proteins were found to directly bind the mgtC promoter, supporting a dual transcriptional control. These findings reveal substantial differences in mgtC gene regulation in different bacterial pathogens, reflecting distinct strategies to drive appropriate expression of a shared virulence factor involved in macrophage adaptation.IMPORTANCEThe adaptation of bacterial pathogens to the host intracellular microenvironment requires tight and rapid regulation of specific genes, and investigating the in vivo transcriptional dynamics of such genes is a major challenge. Here, we focused on the expression of mgtC, a gene important for adaptation to the intramacrophage environment in classical intracellular pathogens, such as Salmonella Typhimurium, and bacteria with a transient intracellular lifestyle, such as Pseudomonas aeruginosa. An unstable GFP reporter system was designed to monitor the transcriptional dynamics of P. aeruginosa mgtC. The use of this reporter system in a state-of-the-art vertebrate model for live imaging, the zebrafish embryo, allowed in vivo tracking of P. aeruginosa mgtC promoter activation inside macrophages in a living host. Furthermore, the expression of P. aeruginosa mgtC was found to be regulated through a mechanism distinct from that of Salmonella MgtC, since it involves the PhoP regulatory protein, but not the PhoQ sensor, and the Pseudomonas-specific CbrAB two-component system, reflecting diverse, finely tuned strategies to control a virulence factor shared by several major human pathogens.

Pseudomonas aeruginosa↗
PubMed1999

Getting a-head of the organizer: anterior-posterior patterning of the forebrain.

The molecular mechanisms that drive the development of embryonic tissues are being uncovered rapidly. One such fascinating example is the development of the forebrain, the most anterior part of the nervous system. In this review, we will discuss the mechanisms that induce the formation of the forebrain in multiple vertebrate systems, placing emphasis on a recent article published by Grinblat et al. ((1)) Using zebrafish as a model system, these authors combine elegant embryological manipulations with the use of early markers of the presumptive forebrain, to show that initial induction and patterning of this tissue occurs near the onset of gastrulation. In addition, their results confirm observations made in other systems that planar signals, those traveling in the plane of the ectoderm, are involved in forebrain induction and patterning.

Animals↗
PubMed2001

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Acetylcholinesterase↗
PubMed1996

Class III POU genes of zebrafish are predominantly expressed in the central nervous system.

POU genes encode a family of transcription factors involved in a wide variety of cell fate decisions and in the regulation of differentiation pathways. We have searched for POU genes in the zebrafish, a popular model organism for the study of early development of vertebrates. Besides five putative pseudogenes we have identified five POU genes that are expressed during embryogenesis. Probes obtained by PCR were used to isolate full-length cDNAs. Four of the isolated genes encode proteins with class III POU domains. Analysis of genomic clones suggests that the fish genes in general do not contain introns, similar to class III genes of mammals. However, the C-termini of two of the encoded proteins vary due to facultative splicing of a short intervening sequence. These two genes show very strong similarities in their sequence. They have probably arisen by gene duplication, possibly as part of a larger scale duplication of part of the zebrafish genome. Analysis of the expression of the class III genes shows that they are predominantly expressed in the central nervous system and that they may play important roles in patterning the embryonic brain.

Amino Acid Sequence↗
PubMed2025

Studying the Role of HOX Genes in Thrombocyte Development.

In our laboratory, we study thrombopoiesis and hemostasis using zebrafish as a model organism to unravel the mechanisms of differentiation and development of thrombocytes. We have shown in our earlier work that thrombocytes are functional equivalents of platelets and have transcriptional machinery similar to megakaryocytes. We recently found evidence that hox genes play a role in their development. We used piggyback gene knockdown and thrombocyte quantification assays to understand the influence of these ancient developmental regulators on thrombopoiesis. In this chapter, we describe methods used to discover these hox genes.

Animals↗
PubMed2002

Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney.

Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.

Amino Acid Sequence↗
PubMed2002

Epidermal growth factor and TGFalpha promote zebrafish oocyte maturation in vitro: potential role of the ovarian activin regulatory system.

Epidermal growth factor (EGF) and TGFalpha are well known for their activities in the ovary. Both factors initiate signal transduction by binding to the common EGF receptor that has been demonstrated in the ovary across vertebrates from fish to humans. Using zebrafish as the model, we demonstrated in the present study that recombinant human EGF and TGFalpha significantly enhanced final maturation of the fully grown, follicle-enclosed oocytes (0.58-0.65 mm) in vitro in a clear time- and dose-dependent manner. The effect of EGF/TGFalpha was additive to that of hCG at low concentrations, but the additivity diminished when the concentration increased. Both actinomycin D and cycloheximide completely blocked the effect of EGF/TGFalpha, indicating that the promotion of oocyte maturation by EGF/TGFalpha requires de novo mRNA transcription and protein synthesis. Interestingly, the effect of EGF/TGFalpha could be blocked by cotreatment with follistatin, a potent binding protein for activin, an ovarian growth factor belonging to the TGFbeta superfamily. Semiquantitative RT-PCR assays showed that both EGF and TGFalpha significantly stimulated the expression of activin betaA and activin type II receptor in the cultured zebrafish ovarian follicle cells in a clear time- and dose-dependent manner. This together with our previous report that activin had a potent stimulatory effect on zebrafish oocyte maturation strongly suggests that the intrinsic ovarian activin system is probably a downstream mediator of EGF/TGFalpha actions in the zebrafish ovary.

Activin Receptors, Type II↗
PubMed1997

Jak1 kinase is required for cell migrations and anterior specification in zebrafish embryos.

Establishment of the vertebrate body plan requires a variety of signaling molecules. In a search for tyrosine kinases expressed in early zebrafish embryos, a model system for the study of vertebrate development, we discovered Jak1 kinase to be maternally encoded and the mRNA evenly distributed among the cells of blastula-stage embryos. Injection of RNA-encoding dominant-negative Jak1 kinases reduces a specific cell migration, epiboly, and results in the reduction of goosecoid expression and of anterior structures. This work establishes that, in addition to its role in signal transduction of cytokines in adult tissues, Jak1 kinase has a role in early vertebrate development.

Animals↗
PubMed1996

Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, all-trans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

Amino Acid Sequence↗
PubMed2001

Rod contributions to the electroretinogram of the dark-adapted developing zebrafish.

Anatomical studies of the developing zebrafish retina have shown that rods approach maturity at about 15 days postfertilization (dpf). Past work has examined the photopic spectral sensitivity function of the developing zebrafish, but not spectral sensitivity under dark-adapted conditions. This study examined rod contributions to the dark-adapted spectral sensitivity function of the ERG b-wave component in developing zebrafish. ERG responses to stimuli of various wavelengths and irradiances were obtained from dark-adapted fish at 6-8, 13-15, 21-24, and 27-29 dpf. The results show that dark-adapted spectral sensitivity varied with age. Spectral sensitivity functions of the 6-8 and 13-15 dpf groups appeared to be cone dominated and contained little or no rod contributions. Spectral sensitivity functions of the 21-24 and 27-29 dpf groups appeared to have both rod and cone contributions. Even at the oldest age group tested, the dark-adapted spectral sensitivity function did not match the adult function. Thus, consistent with anatomical findings, the rod contributions to the ERG spectral sensitivity function appear to develop with age; however, these contributions are still not adult-like by 29 dpf, which is contrary to anatomical work. These results illustrate that the zebrafish is an excellent model for visual development.

Animals↗
PubMed2003

Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle.

In the present study, new applications of the transgenic technology in developing novel varieties of ornamental fish and bioreactor fish were explored in a model fish, the zebrafish (Danio rerio). Three "living color" fluorescent proteins, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a strong muscle-specific mylz2 promoter in stable lines of transgenic zebrafish. These transgenic zebrafish display vivid fluorescent colors (green, red, yellow, or orange) visible to unaided eyes under both daylight and ultraviolet light in the dark. The level of foreign protein expression is estimated between 3% and 17% of total muscle proteins, equivalent to 4.8-27.2mg/g wet muscle tissue. Thus, the fish muscle may be explored as another useful bioreactor system for production of recombinant proteins. In spite of the high level of foreign protein expression, the expression of endogenous mylz2 mRNAs was not negatively affected. Furthermore, compared to the wild-type fish, these fluorescent transgenic fish have no advantage in survival and reproduction.

Animals↗
PubMed2002

A simple spatial alternation task for assessing memory function in zebrafish.

A series of studies was initiated to examine learning and memory function in the zebrafish (Danio rerio) by using a simple spatial alternation paradigm for a food reward. Fish were fed on alternating sides of a divided fish tank, with a red card displayed on one side serving as a visual means of orientation. Although responses were recorded at cue (light tap on the tank), 5 s after cue (as food was delivered), and 5 s after food delivery, the learning test was choice of a correct side of the tank to receive food. Therefore, an accurate level of an animal's achievement of the spatial task was represented by responses at food delivery. Data collected from 11 separate experiments indicated that zebrafish learned to alternate for a food reward. Further, statistical analysis showed that the zebrafish learned the task in the first half of the experiment as exhibited by a calculated t1/2 of 13.9 trials. Zebrafish could recall the task after a short period of 10 days with no testing. The alternating behavior was extinguished by withholding the food reward. Thus, the spatial alternation task can be learned easily by zebrafish, and may be useful in addressing learning and memory functions in vertebrate animals using zebrafish as a model organism.

Journal Article↗
PubMed2001

Mechanistic considerations in small fish carcinogenicity testing.

Historically, small fish species have proven useful both as environmental sentinels and as versatile test animals in toxicity and carcinogenicity bioassays. They can be bred in large numbers, have low maintenance and bioassay costs, and have a low background incidence of tumors. However, more mechanistic information is needed to help validate the information garnered from these models and to keep pace with other more fully developed animal models. This paper focuses on mechanistic considerations when using small fish models for carcinogenicity testing. Several small aquarium fish species have proven useful. The Japanese medaka is perhaps the best characterized small fish model for carcinogenicity testing; however, the zebrafish is emerging as an important model because it is well characterized genetically. Both route and methodology of exposure may affect the outcome of the study. Most studies have been conducted by introducing the test compound into the ambient water, but dietary exposures and embryo microinjection have also been used. Other considerations in study design include use of an initiating carcinogen, such as diethlynitrosamine, and differences in xenobiotic metabolism, such as the fact that fish CYP2B is refractory to phenobarbital induction. The small size of these models has perhaps limited some types of mechanistic studies, such as formation and repair of DNA adducts in response to carcinogen exposure. However, improved analytical methods are allowing greater resolution and should be applied to small fish species. Slide-based methods such as immunohistochemistry are an important adjunct to routine histopathology and should be included in study design. However, there is a need for development of more species-specific antibodies for fish research. There is also a need for more fish-specific data on cytokines, serum biochemistry, and oncogenes to strengthen the use of these important test models.

Animals↗
PubMed2001

Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish.

The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.

Amino Acid Sequence↗
PubMed2001

Zebrafish mutants: behavioral genetic studies of visual system defects.

Zebrafish are a promising model for behavioral and genetic studies of vertebrate visual system development and retinal degeneration. In the past few years, numerous studies on zebrafish vision have been published. While most of the studies focus on the molecular and cellular characterization of mutations that disrupt zebrafish visual system structure in early development, others examine the mechanisms that underlie inherited visual system disorders in adults. Behavioral assays, along with morphologic and electrophysiological methods, are powerful tools for functional analyses of zebrafish visual development and performance.

Animals↗