Search Results (20)

The common carp Cyprinus carpio L. is a cornerstone aquaculture species, yet transcriptome interpretation is complicated by its paleotetraploid genome and extensive alternative splicing. A species-tailored oligonucleotide microarray was developed to deliver reproducible, gene-level expression profiling. Probe design was anchored to the SPL01 reference and implemented on an Agilent platform using a gene-level strategy that collapsed transcripts to genes, selected the longest isoform, and placed 3′-anchored 60-mer probes. The workflow incorporated embedded technical controls and a standardized two-color pipeline to ensure stable measurements across arrays. Baseline functional organization of the head kidney was defined using 614 C. carpio L. -Danio rerio orthologs and complementary enrichment tools. Coherent signatures emerged for hemoglobin-mediated oxygen transport, heme and porphyrin metabolism, antioxidant defense driven by peroxiredoxin and thioredoxin systems, including hydrogen peroxide detoxification, protease regulation through SERPIN, SPINK, and WFDC families, and elements of innate and humoral immunity. Targets bearing c-Myc motifs showed a modest positive bias consistent with ongoing hematopoiesis. These resolved baseline modules provide a reference against which infection- or exposure-induced programs such as interferon-stimulated genes, chemokines and chemotaxis, complement activation, and degranulation can be detected and quantified. The platform complements RNA-seq by offering cost-efficient, rapid, and comparable measurements suited to large cohorts and longitudinal designs. Anticipated applications include host–pathogen studies for viral and bacterial agents and the assessment of chemical contaminants in aquaculture surveillance, supporting standardized, cross-study decision-making in research and health monitoring. Full article

Nephronophthisis (NPH) is the leading genetic cause of end-stage renal disease in children and young adults, but no effective disease-modifying therapies are currently available. Here, we identify glucagon-like peptide-1 (GLP-1) signaling as a novel therapeutic target for NPH through a systematic drug repurposing screen in zebrafish. By simultaneously depleting nphp1 and nphp4, we developed a robust zebrafish model that reproduces key features of human NPH, including glomerular cyst formation. Our screen revealed that dipeptidyl peptidase-4 (DPP4) inhibitors (Omarigliptin and Linagliptin) and GLP-1 receptor agonists (Semaglutide) significantly reduce cystogenesis in a dose-dependent manner. Genetic analysis demonstrated that GLP-1 receptor signaling is important for maintaining pronephros integrity, with gcgra and gcgrb (GLP-1 receptor genes) playing a particularly important role. Transcriptomic profiling identified adenosine receptor A2ab (adora2ab) as a key downstream effector of GLP-1 signaling, which regulates ciliary morphology and prevents cyst formation. Notably, nphp1/nphp4 double mutant zebrafish exhibited the upregulation of gcgra as a compensatory mechanism, which might explain their resistance to cystogenesis. This compensation was disrupted by the targeted depletion of GLP-1 receptors or the inhibition of adenylate cyclase, resulting in enhanced cyst formation, specifically in the mutant background. Our findings establish a signaling cascade from GLP-1 receptors to adora2ab in terms of regulating ciliary organization and preventing cystogenesis, offering new therapeutic opportunities for NPH through the repurposing of FDA-approved medications with established safety profiles. Full article

Endocannabinoid signaling plays a significant role in neurogenesis and nervous system physiology, but its roles in the development of other tissues are just beginning to be appreciated. Previous reports have shown the presence of the key endocannabinoid receptor Cannabinoid receptor 1 (CB1 or Cnr1) in multiciliated (MCC) tissues and its upregulation in kidney diseases, yet the relationship between Cnr1 and renal MCC development is unknown. Here, we report that Cnr1 is essential for cilia development across tissues and regulates renal MCCs via cyclic AMP (cAMP) signaling during zebrafish embryogenesis. Using a combination of genetic and pharmacological studies, we found that the loss of function, agonism and antagonism of cnr1 all lead to reduced mature renal MCC populations. cnr1 deficiency also led to reduced cilia development across tissues, including the pronephros, ear, Kupffer’s vesicle (KV), and nasal placode. Interestingly, treatment with the cAMP activator Forskolin (FSK) restored renal MCC defects in agonist-treated embryos, suggesting that cnr1 mediates cAMP signaling in renal MCC development. Meanwhile, treatment with the cAMP inhibitor SQ-22536 alone or with cnr1 deficiency led to reduced MCC populations, suggesting that cnr1 also mediates renal MCC development independently of cAMP signaling. Our findings indicate that cnr1 has a critical role in controlling renal MCC development both via cAMP signaling and an independent pathway, further revealing implications for ciliopathies and renal diseases. Full article

Background: The current gold standards for diagnosing acute kidney injury (AKI) are an increase in serum creatinine and a decrease in urine output, which are inadequate for rapid diagnosis. Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein produced and secreted by injured kidney tubule epithelial cells, and can serve as an early urinary biomarker for AKI. ProNephro AKI (NGAL) is an immunoassay for the quantitative determination of NGAL in urine (uNGAL) that recently received FDA clearance. A multisite, cross-sectional study was conducted to establish reference intervals for uNGAL in apparently healthy individuals. Methods: Urine samples were collected from apparently healthy individuals aged ≥3 months who met all inclusion criteria and no exclusion criteria. Specimens were temporarily stored at room temperature or 2–8 °C, then transferred into urinalysis tubes before being frozen and shipped for testing. uNGAL testing was performed using the ProNephro AKI (NGAL) immunoassay on a Roche cobas c501 analyzer. Results: Of the 688 individuals screened, 677 were eligible, and 629 (91.4%) of those were deemed evaluable. The 95th and 97.5th percentile uNGAL values for all pediatric participants were below the clinical cutoff of 125 ng/mL. uNGAL values were statistically significantly higher for female vs. male participants in both adult (p = 0.003) and pediatric groups (p < 0.001), while differences were not statistically significant for age, site location, race, or ethnicity. Conclusions: This study provides normal reference intervals for uNGAL with the ProNephro AKI (NGAL) clinical chemistry immunoassay that may be useful for interpreting patient results. Full article

The Chinese sturgeon (Acipenser sinensis) is an ancient, complex autooctoploid fish species that is currently facing conservation challenges throughout its distribution. To comprehensively characterize the expression profiles of genes and their associated biological functions across different tissues, we performed a transcriptome-scale gene expression analysis, focusing on housekeeping genes (HKGs), tissue-specific genes (TSGs), and co-expressed gene modules in various tissues. We collected eleven tissues to establish a transcriptomic repository, including data from Pacific Biosciences isoform sequencing (PacBio Iso-seq) and RNA sequencing (RNA-seq), and then obtained 25,434 full-length transcripts, with lengths from 307 to 9515 bp and an N50 of 3195 bp. Additionally, 20,887 transcripts were effectively identified and classified as known homologous genes. We also identified 787 HKGs, and the number of TSGs varied from 25 in the liver to 2073 in the brain. TSG functions were mainly enriched in certain signaling pathways involved in specific physiological processes, such as voltage-gated potassium channel activity, nervous system development, glial cell differentiation in the brain, and leukocyte transendothelial migration in the spleen and pronephros. Meanwhile, HKGs were highly enriched in some pathways involved in ribosome biogenesis, proteasome core complex, spliceosome activation, elongation factor activity, and translation initiation factor activity, which have been strongly implicated in fundamental biological tissue functions. We also predicted five modules, with eight hub genes in the brown module, most of which (such as rps3a, rps7, rps23, rpl11, rpl17, rpl27, and rpl28) were linked to ribosome biogenesis. Our results offer insights into ribosomal proteins that are indispensable in ribosome biogenesis and protein synthesis, which are crucial in various cell developmental processes and neural development of Chinese sturgeon. Overall, these findings will not only advance the understanding of fundamental biological functions in Chinese sturgeon but also supply a valuable genetic resource for characterizing this extremely important species. Full article

Multiciliated cells (MCCs) serve many important functions, including fluid propulsion and chemo- and mechanosensing. Diseases ranging from rare conditions to the recent COVID-19 global health pandemic have been linked to MCC defects. In recent years, the zebrafish has emerged as a model to investigate the biology of MCCs. Here, we review the major events in MCC formation including centriole biogenesis and basal body docking. Then, we discuss studies on the role of MCCs in diseases of the brain, respiratory, kidney and reproductive systems, as well as recent findings about the link between MCCs and SARS-CoV-2. Next, we explore why the zebrafish is a useful model to study MCCs and provide a comprehensive overview of previous studies of genetic components essential for MCC development and motility across three major tissues in the zebrafish: the pronephros, brain ependymal cells and nasal placode. Taken together, here we provide a cohesive summary of MCC research using the zebrafish and its future potential for expanding our understanding of MCC-related disease states. Full article

Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory mediators leads to the recruitment and infiltration of immune cells, particularly neutrophils, at the site of inflammation. Cisplatin is often used as an inducer of acute kidney injury (AKI) in experimental models, including zebrafish (Danio rerio), due to its accumulation in kidney cells. Current protocols in larval zebrafish focus on studying its effect as an AKI inducer but ignore other systematic outcomes. In this study, cisplatin was added directly to the embryonic medium to assess its toxicity and impact on systemic inflammation using locomotor activity analysis, qPCR, microscopy, and flow cytometry. Our data showed that larvae exposed to cisplatin at 7 days post-fertilization (dpf) displayed dose-dependent mortality and morphological changes, leading to a decrease in locomotion speed at 9 dpf. The expression of pro-inflammatory cytokines such as interleukin (il)-12, il6, and il8 increased after 48 h of cisplatin exposure. Furthermore, while a decrease in the number of neutrophils was observed in the glomerular region of the pronephros, there was an increase in neutrophils throughout the entire animal after 48 h of cisplatin exposure. We demonstrate that cisplatin can have systemic effects in zebrafish larvae, including morphological and locomotory defects, increased inflammatory cytokines, and migration of neutrophils from the hematopoietic niche to other parts of the body. Therefore, this protocol can be used to induce systemic inflammation in zebrafish larvae for studying new therapies or mechanisms of action involving neutrophils. Full article

Nephrons are the functional units which comprise the kidney. Each nephron contains a number of physiologically unique populations of specialized epithelial cells that are organized into discrete domains known as segments. The principles of nephron segment development have been the subject of many studies in recent years. Understanding the mechanisms of nephrogenesis has enormous potential to expand our knowledge about the basis of congenital anomalies of the kidney and urinary tract (CAKUT), and to contribute to ongoing regenerative medicine efforts aimed at identifying renal repair mechanisms and generating replacement kidney tissue. The study of the zebrafish embryonic kidney, or pronephros, provides many opportunities to identify the genes and signaling pathways that control nephron segment development. Here, we describe recent advances of nephron segment patterning and differentiation in the zebrafish, with a focus on distal segment formation. Full article

Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17β-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis. Full article

The Wilms’ tumor suppressor gene, wt1, encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes. wt1 was first identified as a tumor suppressor gene in Wilms’ tumor, a pediatric kidney tumor, and has been implicated in normal kidney development. The WT1 protein has transcriptional activation and repression domains and acts as a transcriptional activator or repressor, depending on the target gene and context. In Xenopus, an ortholog of wt1 has been isolated and shown to be expressed in the developing embryonic pronephros. To investigate the role of wt1 in pronephros development in Xenopus embryos, we mutated wt1 by CRISPR/Cas9 and found that the expression of pronephros marker genes was reduced. In reporter assays in which known WT1 binding sequences were placed upstream of the luciferase gene, WT1 activated transcription of the luciferase gene. The injection of wild-type or artificially altered transcriptional activity of wt1 mRNA disrupted the expression of pronephros marker genes in the embryos. These results suggest that the appropriate amounts and activity of WT1 protein are required for normal pronephros development in Xenopus embryos. Full article

Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that hdac6, hdac8, and nfkbiaa expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of hdac6, hdac8, or nfkbiaa with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in hdac6-depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease. Full article

Acute kidney injury (AKI) is a common complication of severe human diseases, resulting in increased morbidity and mortality as well as unfavorable long-term outcomes. Although the mammalian kidney is endowed with an amazing capacity to recover from AKI, little progress has been made in recent decades to facilitate recovery from AKI. To elucidate the early repair mechanisms after AKI, we employed the zebrafish pronephros injury model. Since damaged cells release large amounts of ATP and ATP-degradation products to signal apoptosis or necrosis to neighboring cells, we examined how depletion of purinergic and adenosine receptors impacts the directed cell migration that ensues immediately after a laser-induced tubular injury. We found that depletion of the zebrafish adenosine receptors adora1a, adora1b, adora2aa, and adora2ab significantly affected the repair process. Similar results were obtained after depletion of the purinergic p2ry2 receptor, which is highly expressed during zebrafish pronephros development. Released ATP is finally metabolized to inosine by adenosine deaminase. Depletion of zebrafish adenosine deaminases ada and ada2b interfered with the repair process; furthermore, combinations of ada and ada2b, or ada2a and ada2b displayed synergistic effects at low concentrations, supporting the involvement of inosine signaling in the repair process after a tubular injury. Our findings suggest that nucleotide-dependent signaling controls immediate migratory responses after tubular injury. Full article

The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including Hox genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers (Pax2 and Lim1), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of Hox4 genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek’s nephridium. This suggests that despite the conservation of Pax2 and Lim1 expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and Hox-dependent regulation is a gnathostome novelty. Full article

Brd2 belongs to the BET family of epigenetic transcriptional co-regulators that act as adaptor-scaffolds for the assembly of chromatin-modifying complexes and other factors at target gene promoters. Brd2 is a protooncogene and candidate gene for juvenile myoclonic epilepsy in humans, a homeobox gene regulator in Drosophila, and a maternal-zygotic factor and cell death modulator that is necessary for normal development of the vertebrate central nervous system (CNS). As two copies of Brd2 exist in zebrafish, we use antisense morpholino knockdown to probe the role of paralog Brd2b, as a comparative study to Brd2a, the ortholog of human Brd2. A deficiency in either paralog results in excess cell death and dysmorphology of the CNS, whereas only Brd2b deficiency leads to loss of circulation and occlusion of the pronephric duct. Co-knockdown of both paralogs suppresses single morphant defects, while co-injection of morpholinos with paralogous RNA enhances them, suggesting novel genetic interaction with functional antagonism. Brd2 diversification includes paralog-specific RNA variants, a distinct localization of maternal factors, and shared and unique spatiotemporal expression, providing unique insight into the evolution and potential functions of this gene. Full article

Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters. Full article