ABSTRACT
Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.
Subject(s)
RNA Splicing Factors , Zebrafish Proteins , Zebrafish , Animals , Embryonic Development , Mutation , RNA Precursors/metabolism , RNA Splicing Factors/metabolism , Stem Cells/metabolism , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.
Subject(s)
Alternative Splicing , Zebrafish , Alternative Splicing/genetics , Animals , Hematopoiesis/genetics , Hematopoietic Stem Cells , Myeloid Progenitor Cells , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.
Subject(s)
Retinal Degeneration , Zebrafish , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Eye Proteins/metabolism , Mammals/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.
Subject(s)
Alternative Splicing , Neural Stem Cells/metabolism , Neurogenesis/genetics , Retina/embryology , Zebrafish Proteins/physiology , Animals , Apoptosis , CRISPR-Cas Systems , Cell Survival , DNA Damage , DNA Repair , Exons , Gene Knockout Techniques , M Phase Cell Cycle Checkpoints , Neural Stem Cells/cytology , Retinal Neurons/cytology , Retinal Neurons/metabolism , Spindle Apparatus/ultrastructure , Tumor Suppressor Protein p53/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The bistable flow is attractive as it can be analogous to a switch to realize flow control. Based on the previous studies on actuation technique, the present study first proposed temperature-driven switching of bistable slit flow. A two-dimensional numerical simulation was conducted to investigate the flow deflection characteristics and switching mechanism. It was concluded that the temperature gradient not only biases the slit flow but also locks it to the high-temperature side. The flow deflection angle became larger with the increase in temperature gradient. Being driven by the temperature, the flow can be switched from one side to the other. Furthermore, the fluid viscosity, which varies with temperature, determines the degree of flow deflection and the entire switching time. This research can enrich the active regulation of flow and has significant potential applications in thermal sensors, thermal detectors, microelectromechanical systems, biomedicine, and other equivalent fields.
ABSTRACT
Hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium via the endothelial-to-hematopoietic transition, are self-renewing, and replenish all lineages of blood cells throughout life. BCAS2 (breast carcinoma amplified sequence 2) is a component of the spliceosome and is involved in multiple biological processes. However, its role in hematopoiesis remains unknown. We established a bcas2 knockout zebrafish model by using transcription activator-like effector nucleases. The bcas2 -/- zebrafish showed severe impairment of HSPCs and their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the caudal hematopoietic tissue of bcas2 -/- zebrafish, which may be rescued by suppression of p53. Furthermore, we show that the bcas2 deletion induces an abnormal alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis, which provides a mechanistic explanation of the deficiency observed in HSPCs. Our findings revealed a novel and vital role for BCAS2 during HSPC maintenance in zebrafish.
Subject(s)
Embryo, Nonmammalian/embryology , Embryonic Development , Hematopoietic Stem Cells/metabolism , Neoplasm Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Animals, Genetically Modified/embryology , Animals, Genetically Modified/genetics , Gene Knockdown Techniques , Neoplasm Proteins/genetics , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated a brg1 knockout zebrafish model using TALEN technology. We found that in brg1-/- embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment in brg1-/- embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter of KLF2 and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotes klf2a expression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Embryo, Nonmammalian/embryology , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Stem Cell Niche , Zebrafish Proteins/metabolism , Zebrafish/embryology , Adaptor Proteins, Signal Transducing/genetics , Animals , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Hematopoietic Stem Cells/cytology , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Nitric Oxide/genetics , Nitric Oxide/metabolism , Response Elements , Transcription, Genetic , Zebrafish/genetics , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/geneticsABSTRACT
Mutations in human prominin 1 (PROM1), encoding a transmembrane glycoprotein localized mainly to plasma membrane protrusions, have been reported to cause retinitis pigmentosa, macular degeneration, and cone-rod dystrophy. Although the structural role of PROM1 in outer-segment (OS) morphogenesis has been demonstrated in Prom1-knockout mouse, the mechanisms underlying these complex disease phenotypes remain unclear. Here, we utilized a zebrafish model to further investigate PROM1's role in the retina. The Prom1 orthologs in zebrafish include prom1a and prom1b, and our results showed that prom1b, rather than prom1a, plays an important role in zebrafish photoreceptors. Loss of prom1b disrupted OS morphogenesis, with rods and cones exhibiting differences in impairment: cones degenerated at an early age, whereas rods remained viable but with an abnormal OS, even at 9 months postfertilization. Immunofluorescence experiments with WT zebrafish revealed that Prph2, an ortholog of the human transmembrane protein peripherin 2 and also associated with OS formation, is localized to the edge of OS and is more highly expressed in the cone OS than in the rod OS. Moreover, we found that Prom1b deletion causes mislocalization of Prph2 and disrupts its oligomerization. We conclude that the variation in Prph2 levels between cones and rods was one of the reasons for the different PROM1 mutation-induced phenotypes of these retinal structures. These findings expand our understanding of the phenotypes caused by PROM1 mutations and provide critical insights into its function.
Subject(s)
AC133 Antigen/metabolism , Photoreceptor Cells/metabolism , Rod Cell Outer Segment/metabolism , AC133 Antigen/genetics , Animals , Cone-Rod Dystrophies/genetics , Disease Models, Animal , HeLa Cells , Humans , Macular Degeneration/metabolism , Membrane Proteins/metabolism , Morphogenesis , Mutation , Peripherins/genetics , Retina/metabolism , Retina/physiology , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/physiopathology , Retinal Rod Photoreceptor Cells/metabolism , Retinitis Pigmentosa/genetics , Sequence Deletion , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Aging is regulated by complex signaling networks, the details of which remain poorly understood. Here, we demonstrate that VPS-22/SNF8, a component of endosomal sorting complex required for transport-II (ESCRT-II), regulates the lifespan of C. elegans. In this study we show that worms with vps-22/snf8 gene knockdown had a shorter lifespan than wild-type worms. The expression pattern of VPS-22/SNF8 in C. elegans was highly similar to that of DAF-16. Knockout of daf-16 in C. elegans shortened the worms' lifespan; however, reducing the expression of vps-22/snf8 in daf-16 null worms did not further shorten their lifespan, indicating that vps-22/snf8 and daf-16 may act in the same signaling pathway to regulate longevity. Over-expression of daf-16 rescued the short-lived phenotype of vps-22/snf8 knockdown worms. Moreover, down-regulation of vps-22/snf8 decreased the nuclear localization of DAF-16 and modulated the expression of daf-16 downstream genes that regulate longevity in C. elegans. In summary, our results indicate that vps-22/snf8 can regulate the longevity of C. elegans by partially modulating the activity of daf-16. These findings may help us to better understand the mechanisms of aging.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Endosomal Sorting Complexes Required for Transport/physiology , Forkhead Transcription Factors/physiology , Longevity/physiology , Active Transport, Cell Nucleus , Aging/genetics , Aging/physiology , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Down-Regulation , Endosomal Sorting Complexes Required for Transport/deficiency , Endosomal Sorting Complexes Required for Transport/genetics , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/genetics , Gene Knockdown Techniques , Genes, Helminth , Longevity/genetics , PhenotypeABSTRACT
Most cases of Usher syndrome type II (USH2) are due to mutations in the USH2A gene. There are no effective treatments or ideal animal models for this disease, and the pathological mechanisms of USH2 caused by USH2A mutations are still unknown. Here, we constructed a ush2a knockout (ush2a-/-) zebrafish model using TALEN technology to investigate the molecular pathology of USH2. An early onset auditory disorder and abnormal morphology of inner ear stereocilia were identified in the ush2a-/- zebrafish. Consequently, the disruption of Ush2a in zebrafish led to a hearing impairment, like that in mammals. Electroretinography (ERG) test indicated that deletion of Ush2a affected visual function at an early stage, and histological analysis revealed that the photoreceptors progressively degenerated. Rod degeneration occurred prior to cone degeneration in ush2a-/- zebrafish, which is consistent with the classical description of the progression of retinitis pigmentosa (RP). Destruction of the outer segments (OSs) of rods led to the down-regulation of phototransduction cascade proteins at late stage. The expression of Ush1b and Ush1c was up-regulated when Ush2a was null. We also found that disruption of fibronectin assembly at the retinal basement membrane weakened cell adhesion in ush2a-/- mutants. In summary, for the first time, we generated a ush2a knockout zebrafish line with auditory disorder and retinal degeneration which mimicked the symptoms of patients, and revealed that disruption of fibronectin assembly may be one of the factors underlying RP. This model may help us to better understand the pathogenic mechanism and find treatment for USH2 in the future.
Subject(s)
Extracellular Matrix Proteins , Gene Knockout Techniques , Usher Syndromes , Zebrafish Proteins , Zebrafish , Animals , Disease Models, Animal , Electroretinography , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Humans , Usher Syndromes/genetics , Usher Syndromes/metabolism , Usher Syndromes/pathology , Usher Syndromes/physiopathology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismSubject(s)
Retinal Degeneration/genetics , Zebrafish , Animals , Humans , Morphogenesis , Sequence DeletionABSTRACT
Graphene foam prepared by the chemical vapor deposition method is a promising thermal interfacial material. However, the thermal properties of graphene foam highly depend on the experimental fabrication conditions during the chemical vapor deposition process. Aiming to reveal how to prepare the appropriate graphene foam for the various thermal management scenarios, the influence of experimental conditions on thermal properties of graphene foam was investigated. Furthermore, the contribution of thermal conductivity and thermal radiation to the effective thermal coefficient of graphene foam was carried out for comparison. The research results showed that the porosity and the cross-section shape of the struts of the growth template were two critical factors affecting the thermal transport of graphene foam, especially with the increase of temperature. In addition, the deposition time of graphene determined the wall thickness and affected the thermal conductivity directly. The thermal radiation contributed more than thermal conductivity when the temperature climbed continuously. Comparatively, the effective thermal coefficient of graphene foam composite with high porosity and circular-shape struts was much superior to that of others at high temperature. The research findings provide important guidance for graphene foam fabrication and its applications in the field of thermal management.
ABSTRACT
Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27's integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.
Subject(s)
Retinitis Pigmentosa , Zebrafish Proteins , Zebrafish , Animals , Humans , Biological Transport , Cilia/genetics , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/pathology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
OBJECTIVE: To investigate the prenatal imaging characteristics, genetic characteristics and pregnancy outcome of fetuses with cardiac rhabdomyoma. STUDY DESIGN: The prenatal ultrasound, cranial MRI imaging information and genetic test results of 35 fetuses prenatally diagnosed with cardiac rhabdomyoma were collected and retrospectively analyzed, and the pregnancy outcome was followed up. RESULT: Cardiac rhabdomyomas mainly occurred in left ventricular wall and ventricular septum; cranial MRI imaging was found abnormal in 38.1% (8/21) of the fetuses; genetic test was found abnormal in 58.82% (10/17) of the fetuses; the fetus was born in 12 cases and the pregnancy was terminated in 23 cases. CONCLUSION: TRIO whole exome sequencing (TrioWES) is recommended as the genetic test regime for cardiac rhabdomyoma. The comprehensive evaluation of prognosis of fetuses needs to consider the genetic results and whether the brain is involved; the prognosis of fetuses with simple cardiac rhabdomyoma is good.
Subject(s)
Fetal Diseases , Heart Neoplasms , Rhabdomyoma , Tuberous Sclerosis , Female , Pregnancy , Humans , Pregnancy Outcome , Rhabdomyoma/diagnostic imaging , Rhabdomyoma/genetics , Retrospective Studies , Tuberous Sclerosis/diagnosis , Tuberous Sclerosis/genetics , Fetal Diseases/diagnostic imaging , Fetal Diseases/genetics , Prenatal Diagnosis/methods , Fetus/diagnostic imaging , Heart Neoplasms/diagnostic imaging , Heart Neoplasms/genetics , Ultrasonography, PrenatalABSTRACT
Mutations in TUB-like protein 1 (TULP1) are associated with severe early-onset retinal degeneration in humans. However, the pathogenesis remains largely unknown. There are two homologous genes of TULP1 in zebrafish, namely tulp1a and tulp1b. Here, we generated the single knockout (tulp1a-/- and tulp1b-/-) and double knockout (tulp1-dKO) models in zebrafish. Knockout of tulp1a resulted in the mislocalization of UV cone opsins and the degeneration of UV cones specifically, while knockout of tulp1b resulted in mislocalization of rod opsins and rod-cone degeneration. In the tulp1-dKO zebrafish, mislocalization of opsins was present in all types of photoreceptors, and severe degeneration was observed at a very early age, mimicking the clinical manifestations of TULP1 patients. Photoreceptor cilium length was significantly reduced in the tulp1-dKO retinas. RNA-seq analysis showed that the expression of tektin2 (tekt2), a ciliary and flagellar microtubule structural component, was downregulated in the tulp1-dKO zebrafish. Dual-luciferase reporter assay suggested that Tulp1a and Tulp1b transcriptionally activate the promoter of tekt2. In addition, ferroptosis might be activated in the tulp1-dKO zebrafish, as suggested by the up-regulation of genes related to the ferroptosis pathway, the shrinkage of mitochondria, reduction or disappearance of mitochondria cristae, and the iron and lipid droplet deposition in the retina of tulp1-dKO zebrafish. In conclusion, our study establishes an appropriate zebrafish model for TULP1-associated retinal degeneration and proposes that loss of TULP1 causes defects in cilia structure and opsin trafficking through the downregulation of tekt2, which further increases the death of photoreceptors via ferroptosis. These findings offer insight into the pathogenesis and clinical treatment of early-onset retinal degeneration.
Subject(s)
Ferroptosis , Retinal Degeneration , Animals , Humans , Retinal Degeneration/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Eye Proteins/metabolism , Photoreceptor Cells, Vertebrate/metabolismABSTRACT
Purpose: Bietti crystalline dystrophy (BCD) is a progressive retinal degenerative disease primarily characterized by numerous crystal-like deposits and degeneration of retinal pigment epithelium (RPE) and photoreceptor cells. CYP4V2 (cytochrome P450 family 4 subfamily V member 2) is currently the only disease-causing gene for BCD. We aimed to generate a zebrafish model to explore the functional role of CYP4V2 in the development of BCD and identify potential therapeutic targets for future studies. Methods: The cyp4v7 and cyp4v8 (homologous genes of CYP4V2) knockout zebrafish lines were generated by CRISPR/Cas9 technology. The morphology of photoreceptor and RPE cells and the accumulation of lipid droplets in RPE cells were investigated at a series of different developmental stages through histological analysis, immunofluorescence, and lipid staining. Transcriptome analysis was performed to investigate the changes in gene expression of RPE cells during the progression of BCD. Results: Progressive retinal degeneration including RPE atrophy and photoreceptor loss was observed in the mutant zebrafish as early as seven months after fertilization. We also observed the excessive accumulation of lipid droplets in RPE cells from three months after fertilization, which preceded the retinal degeneration by several months. Transcriptome analysis suggested that multiple metabolism pathways, especially the lipid metabolism pathways, were significantly changed in RPE cells. The down-regulation of the peroxisome proliferator-activated receptor α (PPARα) pathway was further confirmed in the mutant zebrafish and CYP4V2-knockdown human RPE-1 cells. Conclusions: Our work established an animal model that recapitulates the symptoms of BCD patients and revealed that abnormal lipid metabolism in RPE cells, probably caused by dysregulation of the PPARα pathway, might be the main and direct consequence of CYP4V2 deficiency. These findings will deepen our understanding of the pathogenesis of BCD and provide potential therapeutic approaches.
Subject(s)
Corneal Dystrophies, Hereditary , Retinal Degeneration , Retinal Diseases , Animals , Corneal Dystrophies, Hereditary/pathology , Cytochrome P450 Family 4/genetics , Humans , Lipid Droplets/metabolism , Lipid Droplets/pathology , Mutation , PPAR alpha/genetics , Retinal Degeneration/genetics , Retinal Diseases/diagnosis , ZebrafishABSTRACT
Zebrafish is an excellent model for exploring the development of the inner ear. Its inner ear has similar functions to that of humans, specifically in the maintenance of hearing and balance. Mafba is a component of the Maf transcription factor family. It participates in multiple biological processes, but its role in inner-ear development remains poorly understood. In this study, we constructed a mafba knockout (mafba-/-) zebrafish model using CRISPR/Cas9 technology. The mafba-/- mutant inner ear displayed severe impairments, such as enlarged otocysts, smaller or absent otoliths, and insensitivity to sound stimulation. The proliferation of p63+ epidermal stem cells and dlc+ ionocyte progenitors was inhibited in mafba-/- mutants. Moreover, the results showed that mafba deletion induces the apoptosis of differentiated K+-ATPase-rich (NR) cells and H+-ATPase-rich (HR) cells. The activation of p53 apoptosis and G0/G1 cell cycle arrest resulted from DNA damage in the inner-ear region, providing a mechanism to account for the inner ear deficiencies. The loss of homeostasis resulting from disorders of ionocyte progenitors resulted in structural defects in the inner ear and, consequently, loss of hearing. In conclusion, the present study elucidated the function of ionic channel homeostasis and inner-ear development using a zebrafish Mafba model and clarified the possible physiological roles.
ABSTRACT
Macroautophagy/autophagy is an important intracellular mechanism for the maintenance of cellular homeostasis. Here we show that the CERKL (ceramide kinase like) gene, a retinal degeneration (RD) pathogenic gene, plays a critical role in regulating autophagy by stabilizing SIRT1. In vitro and in vivo, suppressing CERKL results in impaired autophagy. SIRT1 is one of the main regulators of acetylation/deacetylation in autophagy. In CERKL-depleted retinas and cells, SIRT1 is downregulated. ATG5 and ATG7, 2 essential components of autophagy, show a higher degree of acetylation in CERKL-depleted cells. Overexpression of SIRT1 rescues autophagy in CERKL-depleted cells, whereas CERKL loses its function of regulating autophagy in SIRT1-depleted cells, and overexpression of CERKL upregulates SIRT1. Finally, we show that CERKL directly interacts with SIRT1, and may regulate its phosphorylation at Ser27 to stabilize SIRT1. These results show that CERKL is an important regulator of autophagy and it plays this role by stabilizing the deacetylase SIRT1.
Subject(s)
Autophagy/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Retinal Degeneration/genetics , Sirtuin 1/metabolism , Zebrafish Proteins/genetics , Acetylation , Animals , Autophagosomes/metabolism , Autophagy-Related Protein 5/chemistry , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 7/chemistry , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Cell Line , Disease Models, Animal , Humans , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Protein Processing, Post-Translational/genetics , Retina/pathology , Retinal Pigment Epithelium/metabolism , Retinitis Pigmentosa/genetics , Sirtuin 1/chemistry , Sirtuin 1/genetics , Up-Regulation , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1-2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7â¯dpf lca5-/- zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5-/- zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.
Subject(s)
Cone-Rod Dystrophies/genetics , Genetic Predisposition to Disease/genetics , Leber Congenital Amaurosis/genetics , Leber Congenital Amaurosis/metabolism , Protein Transport/physiology , Zebrafish/genetics , Animals , CRISPR-Cas Systems , Cilia/metabolism , Disease Models, Animal , Eye Proteins/metabolism , Gene Knockout Techniques , Humans , Leber Congenital Amaurosis/pathology , Microtubule-Associated Proteins , Phenotype , Retina/metabolism , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1â¯month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10â¯months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.