Zebrafish Homologue Research Articles

Generation of a zebrafish neurofibromatosis model via inducible knockout of nf2.

Neurofibromatosis Type 2 (NF-2) is a dominantly inherited genetic disorder that results from mutations in the tumor suppressor gene, neurofibromin 2 (NF2) gene. Here, we report the generation of a conditional zebrafish model of neurofibromatosis established by an inducible genetic knockout of nf2a/b, the zebrafish homolog of human NF2. Analysis of nf2a and nf2b expression reveals ubiquitous expression of nf2b in the early embryo, with overlapping expression in the neural crest and its derivatives and in the cranial mesenchyme. In contrast, nf2a displays lower expression levels. Induction of nf2a/b knockout at early stages increases the proliferation of larval Schwann cells and meningeal fibroblasts. Subsequently, in adult zebrafish, nf2a/b knockout triggers the development of a spectrum of tumors, including vestibular Schwannomas, spinal Schwannomas, meningiomas, and retinal hamartomas, mirroring the tumor manifestations observed in patients with NF-2. Collectively, these findings highlight the generation of a novel zebrafish model that mimics the complexities of the human NF-2 disorder. Consequently, this model holds significant potential for facilitating therapeutic screening and elucidating key driver genes implicated in NF-2 onset.

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish where expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

The thiol methyltransferase activity of TMT1A (METTL7A) is conserved across species

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7%, and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170, and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.

Abcg2a is the functional homolog of human ABCG2 expressed at the zebrafish blood–brain barrier

BackgroundA principal protective component of the mammalian blood–brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs.MethodsTo determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish.ResultsWe found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature.ConclusionsThese results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.

SDF-1/CXCR4 signal is involved in the induction of Primordial Germ Cell migration in a model marine fish, Japanese anchovy (Engraulis japonicus)

Primordial germ cells (PGCs) are pivotal for gonadal development and reproductive success. Though artificial induction of sterility by targeting PGCs are gaining popularity due to its advantages in fish surrogacy and biodiversity management, it is often skill and time intensive. In this study, we have focused on understanding the role of PGCs and the chemotactic SDF-1/CXCR4 signaling on gonad development of Japanese anchovy (JA, Engraulis japonicus), an upcoming marine model organism with eco-commercial values, with an aim to develop a novel, easy, and versatile gonad sterilization method. Our data showed that PGC migration related genes, i.e., sdf-1a, sdf-1b, cxcr4a, cxcr4b and vasa, are phylogenetically closer relatives of respective herring (Clupea harengus) and zebrafish (Danio rerio) homolog. Subsequently, PGC marking and live tracing experiments confirmed that PGC migration in JA initiates from 16 hours post fertilization (hpf) followed by PGC settlement in the gonadal ridge at 44 hpf. We found that overexpression of zebrafish sdf-1a mRNA in the germ cell suppresses cxcr4a and increases cxcr4b transcription at 8 hpf, dose dependently disrupts PGC migration at 24–48 hpf, induces PGC death and upregulates sdf-1b at 5 days post hatching. 48 h of immersion treatment with CXCR4 antagonist (AMD3100, Abcam) also accelerated PGC mismigration and pushed the PGC away from gonadal ridge in a dose responsive manner, and further when grown to adulthood caused germ cell less gonad formation in some individuals. Cumulatively, our data, for the first time, suggests that JA PGC migration is largely regulated by SDF1/CXCR4 signaling, and modulation of this signaling has strong potential for sterile, germ cell less gonad preparation at a mass scale. However, further in-depth analysis is pertinent to apply this methodology in marine fish species to successfully catapult Japanese anchovy into a true marine fish model.

Characterization of type II IFNs and their receptors in a cyprinid fish, the blunt snout bream Megalobrama amblycephala

Type II interferons (IFNs) are a key class of molecules regulating innate and adaptive immunity in vertebrates. In the present study, two members of the type II IFNs, IFN-γ and IFNγ-rel, were identified in the blunt snout bream (Megalobrama amblycephala). The open reading frame (ORF) of IFN-γ and IFNγ-rel was found to have 564 bp and 492 bp, encoding 187 and 163 amino acids, with the first 26 and 24 amino acids being the signal peptide, respectively. IFN-γ and IFNγ-rel genes showed a high degree of similarity to their zebrafish homologues, being 76.9 % and 58.9 %, respectively. In the phylogenetic tree, IFN-γ and IFNγ-rel were clustered with homologous genes in cyprinids. In blunt snout bream, IFN-γ and IFNγ-rel were constitutively expressed in trunk kidney, head kidney, spleen, liver, heart, muscle, gill, intestine and brain and were significantly up-regulated by poly (I:C) induction in head kidney, spleen, liver, gill and intestine. Using recombinant proteins of IFN-γ and IFNγ-rel, the surface plasmon resonance (SPR) results showed that IFN-γ was bound to CRFB6, CRFB13 and CRFB17, but mainly to CRFB6 and CRFB13, whereas IFN-γrel bound mainly to CRFB17 and had no affinity with CRFB6. These results contribute to a better understanding on type II IFNs and their receptor usage in teleost fish.

Computational prediction and experimental validation identify functionally conserved lncRNAs from zebrafish to human

Functional studies of long noncoding RNAs (lncRNAs) have been hindered by the lack of methods to assess their evolution. Here we present lncRNA Homology Explorer (lncHOME), a computational pipeline that identifies a unique class of long noncoding RNAs (lncRNAs) with conserved genomic locations and patterns of RNA-binding protein (RBP) binding sites (coPARSE-lncRNAs). Remarkably, several hundred human coPARSE-lncRNAs can be evolutionarily traced to zebrafish. Using CRISPR–Cas12a knockout and rescue assays, we found that knocking out many human coPARSE-lncRNAs led to cell proliferation defects, which were subsequently rescued by predicted zebrafish homologs. Knocking down coPARSE-lncRNAs in zebrafish embryos caused severe developmental delays that were rescued by human homologs. Furthermore, we verified that human, mouse and zebrafish coPARSE-lncRNA homologs tend to bind similar RBPs with their conserved functions relying on specific RBP-binding sites. Overall, our study demonstrates a comprehensive approach for studying the functional conservation of lncRNAs and implicates numerous lncRNAs in regulating vertebrate physiology.

THU079 Danio Rerio (Zebrafish): A New Model Of AIP Loss Of Function

Abstract Disclosure: X. Wang: None. A. Leggieri: None. S. Anagianni: None. C.H. Brennan: None. M. Korbonits: None. Background: Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene account for 10% of cases with familial isolated pituitary adenoma (FIPA). The main features of patients carrying heterozygous AIP mutations include young-onset, large and invasive growth hormone and/or prolactin-secreting pituitary tumours, although a milder spectrum of presentation has been also described. In several animal models (mouse, fruit fly, round worm), homozygous deletion of AIP results in embryonic mortality. In mice abnormal cardiac development has been observed. Objective: The aim of this study was to generate a zebrafish loss-of-function model to explore the cellular biological processes affected by heterozygous or homozygous aip mutations. Methods: Zebrafish homologue of human AIP was identified by EMBOSS Needle program. We used CRISPR/Cas9 to generate a line of fish carrying loss-of-function mutations targeting zebrafish aip exon 2. aip RNA expression was examined using in situ hybridisation. The growth rate and cardiac function were assessed using a stereo microscope with an integrated camera. The videos and pictures were analysed in ImageJ. Antisense in situ hybridisation with probes to growth hormone 1 (gh1), prolactin (prl) and proopiomelanocortin a (pomca) was used to assess pituitary phenotypes. Results: Zebrafish aip shows 78.8% of homology to human AIP. aip mRNA is expressed throughout the developing zebrafish embryo until 28 hours post fertilisation (hpf) stage when it becomes more strongly expressed in the head. We generated a 29 bp deletion in exon 2 of the zebrafish aip gene. These mutant animals showed reduced aip expression. No significant gross morphology differences were observed at 3 to 7 days post fertilisation (dpf) between wildtype (WT) and heterozygote (Het) animals. At 5dpf, WTs showed an increased heart rate than Hets (P=0.033). Conclusion: We have generated an aip loss of function zebrafish line that provides an ideal opportunity to analyse the role of aip in development. Presentation: Thursday, June 15, 2023

Disrupting the Repeat Domain of Premelanosome Protein (PMEL) Produces Dysamyloidosis and Dystrophic Ocular Pigment Reflective of Pigmentary Glaucoma.

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.

Identification of the zebrafish homologues of IMPG2, a retinal proteoglycan.

Photoreceptor outer segments are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix, necessary for physiological retinal function. Few roles for molecules characterizing the interphotoreceptor matrix have been clearly defined. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2) gene in patients affected by retinal dystrophies. IMPG2 encodes for a proteoglycan synthesized by photoreceptors and secreted in the interphotoreceptor matrix. Little is known about the structure and function of this protein, we thus decided to characterize zebrafish impg2. In zebrafish there are two Impg2 proteins, Impg2a and Impg2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among vertebrates, showing a significant similarity between humans and teleosts. The human and zebrafish proteins share conserved domains, as also shown by homology models. Expression analyses of impg2a and impg2b show a continued expression in the photoreceptor layer starting from developmental stages and continuing through adulthood. Between 1 and 6months post-fertilization, there is a significant shift of Impg2 expression toward the outer segment region, suggesting an increase in secretion. This raises intriguing hypotheses about its possible role(s) during retinal maturation, laying the groundwork for the generation of most needed models for the study of IMPG2-related inherited retinal dystrophies.

Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.

Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.

Kidney transcriptome and cystic kidney disease genes in zebrafish.

Introduction: Polycystic kidney disease (PKD) is a condition where fluid filled cysts form on the kidney which leads to overall renal failure. Zebrafish has been recently adapted to study polycystic kidney disease, because of its powerful embryology and genetics. However, there are concerns on the conservation of this lower vertebrate in modeling polycystic kidney disease. Methods: Here, we aim to assess the molecular conservation of zebrafish by searching homologues polycystic kidney disease genes and carrying transcriptome studies in this animal. Results and Discussion: We found that out of 82 human cystic kidney disease genes, 81 have corresponding zebrafish homologs. While 75 of the genes have a single homologue, only 6 of these genes have two homologs. Comparison of the expression level of the transcripts enabled us to identify one homolog over the other homolog with >70% predominance, which would be prioritized for future experimental studies. Prompted by sexual dimorphism in human and rodent kidneys, we studied transcriptome between different sexes and noted significant differences in male vs. female zebrafish, indicating that sex dimorphism also occurs in zebrafish. Comparison between zebrafish and mouse identified 10% shared genes and 38% shared signaling pathways. String analysis revealed a cluster of genes differentially expressed in male vs. female zebrafish kidneys. In summary, this report demonstrated remarkable molecular conservation, supporting zebrafish as a useful animal model for cystic kidney disease.

SERPINB7 mutation causes Nagashima-type palmoplantar keratosis and its spatiotemporal expression in zebrafish.

Serine protease inhibitor B7 (SERPINB7) mutations have been reported to cause Nagashima-type palmoplantar keratosis (NPPK), but their biological effects are largely unknown. We conducted whole-exome sequencing and identified a c.796C>T (p.Arg266Ter) mutation in SERPINB7 in a Chinese pedigree, which presented as an autosomal recessive inheritance pattern. We assessed the function of SERPINB7 in homozygous and heterozygous mutation carriers, and the results suggested that the single c.796C>T mutation may alter the subcellular localization of SERPINB7. One of the homozygous mutation patients (II-3) was treated with ixekizumab and showed moderate improvement in keratinization. In addition, we analysed the spatiotemporal expression of serpinb1l1 and serpinb1l3, the zebrafish homologue of human SERPINB7, which is expressed in larvae and adults. In larvae, both serpinb1l1 and serpinb1l3 were expressed in the digestive tract. Then, we performed RT-PCR on adult fins based on similarity to the site of NPPK expression in humans and found that the genes were expressed in five fins (pectoral, pelvic, dorsal, anal and caudal) of the zebrafish distal extremity. Taken together, our results demonstrated that the single c.796C>T (p.Arg266Ter) mutation may alter the location of SERPINB7-encoded protein in the skin, while zebrafish SERPINB7 homologue was expressed in adult fins. These findings will enable us to construct knock-out models to explore the pathogenesis of palmoplantar keratosis.

Nile tilapia (Oreochromis niloticus) Nanog co-expression with Pou5f3, transcriptional regulation and biological activity in embyonic development and embryonic cells.

Mammalian Nanog is critical in pluripotency acquisition and maintenance. Nonetheless, a recent report from zebrafish (Danio rerio) suggests that Nanog is not required for embryonic cells which is not like the mammalian homologs, but is necessary for the proper formation of the extra-embryonic yolk syncytial layer (YSL). However, whether its biological function in other fishes is conservative remains to be investigated. Our previous work shows that Nanog from Nile tilapia (Oreochromis niloticus) (termed as Ong thereafter) displays differential spatiotemporal expression patterns from the other teleost fishes including zebrafish. In this study, Ong co-expression with Pou5f3 (another core pluripotent transcription factor), transcriptional regulation and its biological functions during embryonic development and in the survival and proliferation of embryonic cells were investigated. At the blastula stage, both Ong and Pou5f3 were highly expressed in embryonic cells and co-located in the nucleus. After that, the expression of both Ong and Pou5f3 began to decrease at the gastrula stage (24 haf) and then exhibited a differential expression profile at the segmentation stage (28-36 haf). Ong disappeared in embryonic cells and was limited to YSL, whilst Pou5f3 was highly expressed in embryonic cells even some with obvious cytoplasmic distribution. Luciferase assay indicated that Ong was negatively regulated by Pou5f3 and positively regulated by androgen and itself. Ong depletion in fertilized one-cell embryos through CRISPR/Cas9 led to blastula blockage or death, and the survival and proliferation of blastula-derived embryonic cells in vitro failed. Collectively, Ong has similar expression and biological function to Pou5f3 at the blastula stage, which is similar to mammalian homolog but different from zebrafish homolog. These data suggest that the expression patterns and functions of Nanog are not conservative in fishes and vary from species to species. This study enriches our understanding about Nanog and its evolution.

A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience

Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through conserved homeostatic mechanisms. Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that mediates lipid trafficking to confer cold resilience. We identify lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7. LPD-3 bridges the endoplasmic reticulum (ER) and plasma membranes (PM), forming a structurally predicted hydrophobic tunnel for lipid trafficking. lpd-3 mutants exhibit abnormal phospholipid distribution, diminished FAT-7 abundance, organismic vulnerability to cold, and are rescued by Lecithin comprising unsaturated phospholipids. Deficient lpd-3 homologues in Zebrafish and mammalian cells cause defects similar to those observed in C. elegans. As mutations in BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, LPD-3 family proteins may serve as evolutionarily conserved highway bridges critical for ER-associated non-vesicular lipid trafficking and resilience to cold stress in eukaryotic cells.

The functional verification and analysis of Fugu promoter of cardiac gene tnni1a in zebrafish.

Troponin I type 1b (Tnni1b) is thought to be a novel isoform that is expressed only in the zebrafish heart. Knocking down of tnni1b can lead to cardiac defects in zebrafish. Although both the zebrafish tnni1b and human troponin I1 (TNNI1) genes are thought to be closely associated with fatal cardiac development, the regulatory molecular mechanisms of these genes are poorly understood. Analyzing the functionally conserved sequence, especially in the noncoding regulatory region involved in gene expression, clarified these mechanisms. In this study, we isolated a 3kb fragment upstream of Fugu tnni1a that can regulate green fluorescence protein (GFP) expression in a heart-specific manner, similar to the pattern of zebrafish homologue expression. Three evolutionarily conserved regions (ECRs) in the 5'-flanking sequence of Fugu tnni1a were identified by sequence alignment. Deletion analysis led to the identification of ECR2 as a core sequence that affects the heart-specific expression function of the Fugu tnni1a promoter. Interestingly, both the Fugu tnni1a promoter and ECR2 sequence were functionally conserved in zebrafish, although they shared no sequence similarity. Together, the findings of our study provided further evidence for the important role of tnni1a homologous in cardiac development and demonstrated that two functionally conserved sequences in the zebrafish and Fugu genomes may be ECRs, despite their lack of similarity.

Csf1rb regulates definitive hematopoiesis in zebrafish.

In vertebrates, hematopoietic stem and progenitor cells (HSPCs) are capable of self-renewal and continuously replenishing all mature blood lineages throughout life. However, the molecular signaling regulating the maintenance and expansion of HSPCs remains incompletely understood. Colony-stimulating factor 1 receptor (CSF1R) is believed to be the primary regulator for the myeloid lineage but not HSPC development. Here, we show a surprising role of Csf1rb, a zebrafish homolog of mammalian CSF1R, in preserving the HSPC pool by maintaining the proliferation of HSPCs. Deficiency of csf1rb leads to a reduction in both HSPCs and their differentiated progenies, including myeloid, lymphoid and erythroid cells at early developmental stages. Likewise, the absence of csf1rb conferred similar defects upon HSPCs and leukocytes in adulthood. Furthermore, adult hematopoietic cells from csf1rb mutants failed to repopulate immunodeficient zebrafish. Interestingly, loss-of-function and gain-of-function assays suggested that the canonical ligands for Csf1r in zebrafish, including Csf1a, Csf1b and Il34, were unlikely to be ligands of Csf1rb. Thus, our data indicate a previously unappreciated role of Csf1r in maintaining HSPCs, independently of known ligands.

Molecular Characterization, Evolution and Expression Analysis of TNFSF14 and Three TNFSF Receptors in Spotted Gar Lepisosteus oculatus

The tumor necrosis superfamily (TNFSF) and their receptors (TNFRs) play an essential role in inflammatory responses. In this study, tnfsf14, tnfrsf1a, tnfrsf1b and tnfrsf14 were identified in spotted gar. All the genes have conserved genomic organization and synteny with their respective homologs in zebrafish and humans. The putative TNFSF protein contains a typical TNF homology domain in the extracellular region. All three TNFRSFs possess characteristic cysteine-rich domains. TNFRSF1a has a death domain in the cytosolic region which is absent in the TNFRSF1b and TNFRSF14. Notably, TNFRSF14 lacks a transmembrane domain and is predicted to be secreted. Protein structure modeling revealed that the key residues involved in the interaction between TNFSF14 and TNFRSF14 are well conserved in spotted gar. All four genes were ubiquitously expressed in the spleen, liver, kidney, gills and intestine. Infection with Klebsiella pneumoniae resulted in remarkable downregulation of tnfsf14 and tnfrsf14 in tissues but upregulation of tnfrsf1a and tnfrsf1b. The results indicate that tnfsf14, tnfrsf1a, tnfrsf1b and tnfrsf14 are involved in the immune response to bacterial infection, and expand knowledge on the TNF system in the primitive ray-finned fish.

Repositioning of Lansoprazole as a Protective Agent Against Cisplatin-Induced Ototoxicity.

Cisplatin (CDDP) is a well-known chemotherapeutic drug approved for various cancers. However, CDDP accumulates in the inner ear cochlea via organic cation transporter 2 (OCT2) and causes ototoxicity, which is a major clinical limitation. Since lansoprazole (LPZ), a proton pump inhibitor, is known to inhibit OCT2-mediated transport of CDDP, we hypothesized that LPZ might ameliorate CDDP-induced ototoxicity (CIO). To test this hypothesis, we utilized in vivo fluorescence imaging of zebrafish sensory hair cells. The fluorescence signals in hair cells in zebrafish treated with CDDP dose-dependently decreased. Co-treatment with LPZ significantly suppressed the decrease of fluorescence signals in zebrafish treated with CDDP. Knockout of a zebrafish homolog of OCT2 also ameliorated the reduction of fluorescence signals in hair cells in zebrafish treated with CDDP. These in vivo studies suggest that CDDP damages the hair cells of zebrafish through oct2-mediated accumulation and that LPZ protects against CIO, possibly inhibiting the entry of CDDP into the hair cells via oct2. We also evaluated the otoprotective effect of LPZ using a public database containing adverse event reports. The analysis revealed that the incidence rate of CIO was significantly decreased in patients treated with LPZ. We then retrospectively analyzed the medical records of Mie University Hospital to examine the otoprotective effect of LPZ. The incidence rate of ototoxicity was significantly lower in patients co-treated with LPZ compared to those without LPZ. These retrospective findings suggest that LPZ is also protective against CIO in humans. Taken together, co-treatment with LPZ may reduce the risk of CIO.

Daw1 regulates the timely onset of cilia motility during development.

ABSTRACTMotile cilia generate cell propulsion and extracellular fluid flows that are crucial for airway clearance, fertility and left-right patterning. Motility is powered by dynein arm complexes that are assembled in the cytoplasm then imported into the cilium. Studies in Chlamydomonas reinhardtii showed that ODA16 is a cofactor which promotes dynein arm import. Here, we demonstrate that the zebrafish homolog of ODA16, Daw1, facilitates the onset of robust cilia motility during development. Without Daw1, cilia showed markedly reduced motility during early development; however, motility subsequently increased to attain close to wild-type levels. Delayed motility onset led to differential effects on early and late cilia-dependent processes. Remarkably, abnormal body axis curves, which formed during the first day of development due to reduced cilia motility, self-corrected when motility later reached wild-type levels. Zebrafish larva therefore possess the ability to survey and correct body shape abnormalities. This work defines Daw1 as a factor which promotes the onset of timely cilia motility and can explain why human patients harboring DAW1 mutations exhibit significant laterality perturbations but mild airway and fertility complications.