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1.
Int J Mol Sci ; 25(13)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-39000367

ABSTRACT

Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE.


Subject(s)
Vesicular Transport Proteins , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Mutation , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/metabolism , Disease Models, Animal , Myelin Sheath/metabolism , Behavior, Animal
2.
Front Cell Dev Biol ; 11: 1233269, 2023.
Article in English | MEDLINE | ID: mdl-37745292

ABSTRACT

Background: Adult zebrafish are capable of photoreceptor (PR) regeneration following acute phototoxic lesion (AL). We developed a chronic low light (CLL) exposure model that more accurately reflects chronic PR degeneration observed in many human retinal diseases. Methods: Here, we characterize the morphological and transcriptomic changes associated with acute and chronic models of PR degeneration at 8 time-points over a 28-day window using immunohistochemistry and 3'mRNA-seq. Results: We first observed a differential sensitivity of rod and cone PRs to CLL. Next, we found no evidence for Müller glia (MG) gliosis or regenerative cell-cycle re-entry in the CLL model, which is in contrast to the robust gliosis and proliferative response from resident MG in the AL model. Differential responses of microglia between the models was also observed. Transcriptomic comparisons between the models revealed gene-specific networks of PR regeneration and degeneration, including genes that are activated under conditions of chronic PR stress. Finally, we showed that CLL is at least partially reversible, allowing for rod and cone outer segment outgrowth and replacement of rod cell nuclei via an apparent upregulation of the existing rod neurogenesis mechanism. Discussion: Collectively, these data provide a direct comparison of the morphological and transcriptomic PR degeneration and regeneration models in zebrafish.

3.
Mol Neurobiol ; 59(5): 2910-2931, 2022 May.
Article in English | MEDLINE | ID: mdl-35246819

ABSTRACT

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in situ hybridization, and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.


Subject(s)
MicroRNAs , Zebrafish , Animals , Bromodeoxyuridine/metabolism , Cell Proliferation/physiology , Inflammation/metabolism , Kinetics , Macrophages , Mammals , MicroRNAs/genetics , MicroRNAs/metabolism , Microglia , Retina/metabolism , Zebrafish/metabolism
4.
Sci Rep ; 12(1): 3511, 2022 03 03.
Article in English | MEDLINE | ID: mdl-35241734

ABSTRACT

Genetic Leukoencephalopathies (gLEs) are heritable white matter disorders that cause progressive neurological abnormalities. A founder mutation in the human endolysosomal trafficking protein VPS11 has been identified in Ashkenazi Jewish patients manifesting classic gLE symptoms of hypomyelination, developmental delay, motor and systemic deficits. In this study, we characterized the visual and sensorimotor function of two zebrafish vps11 mutant lines: the previously reported vps11(plt), and a new vps11(-/-) null mutant line, using behavioral analysis to track larval motor responses to visual and acoustic stimuli. We found that mutant larvae from both vps11(plt) and vps11(-/-) lines were able to visually distinguish light and dark, but showed a progressive loss of a normal sensorimotor response to visual stimuli from 5 days post fertilization (dpf) to 7dpf. Additionally, optokinetic response analysis performed at 5dpf indicated that the mutants were significantly visually impaired. Both mutant lines also displayed a progressively lower sensorimotor response to a singular acoustic stimulus from 5-7dpf. Next, we tested the habituation response of the mutant lines to series of acoustic taps. We found both mutant lines habituated faster than their siblings, and that vps11(plt) mutants habituated faster than the vps11(-/-) mutants. Together, these data suggest that loss of Vps11 function results in progressive visual and sensorimotor abnormalities in the zebrafish vps11(plt) and vps11(-/-) mutant lines. This is the first study to characterize behavioral deficits in a vertebrate model of Vps11-dependent gLE. The mutants and behavioral assays described here could be a valuable model system in which to test potential pharmacological interventions for gLE.


Subject(s)
Leukoencephalopathies , Vesicular Transport Proteins , Zebrafish Proteins , Zebrafish , Animals , Leukoencephalopathies/genetics , Leukoencephalopathies/metabolism , Nystagmus, Optokinetic , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , Vision, Ocular , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
5.
Aging Cell ; 21(4): e13597, 2022 04.
Article in English | MEDLINE | ID: mdl-35315590

ABSTRACT

Ageing is a significant risk factor for degeneration of the retina. Müller glia cells (MG) are key for neuronal regeneration, so harnessing the regenerative capacity of MG in the retina offers great promise for the treatment of age-associated blinding conditions. Yet, the impact of ageing on MG regenerative capacity is unclear. Here, we show that the zebrafish retina undergoes telomerase-independent, age-related neurodegeneration but that this is insufficient to stimulate MG proliferation and regeneration. Instead, age-related neurodegeneration is accompanied by MG morphological aberrations and loss of vision. Mechanistically, yes-associated protein (Yap), part of the Hippo signalling, has been shown to be critical for the regenerative response in the damaged retina, and we show that Yap expression levels decline with ageing. Despite this, morphologically and molecularly altered aged MG retain the capacity to regenerate neurons after acute light damage, therefore, highlighting key differences in the MG response to high-intensity acute damage versus chronic neuronal loss in the zebrafish retina.


Subject(s)
Retina , Zebrafish , Animals , Animals, Genetically Modified , Cell Proliferation/physiology , Ependymoglial Cells , Neuroglia/metabolism , Retina/metabolism
6.
Infect Immun ; 90(3): e0046621, 2022 03 17.
Article in English | MEDLINE | ID: mdl-35072520

ABSTRACT

Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a proinflammatory response followed by a noninflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, interleukin 8 (IL-8), and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), but growth was recovered by the addition of the transition metals zinc and manganese. The expression of downstream calprotectin targets was also significantly increased in the zebrafish. These findings illuminate the role of host calprotectin in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.


Subject(s)
Cholera , Vibrio cholerae , Animals , Cholera/microbiology , Leukocyte L1 Antigen Complex , Neutrophils , Vibrio cholerae/physiology , Zebrafish
7.
Front Cell Dev Biol ; 9: 741514, 2021.
Article in English | MEDLINE | ID: mdl-34790663

ABSTRACT

Following photoreceptors ablation by intense light exposure, adult zebrafish are capable of complete regeneration due to the ability of their Müller glia (MG) to re-enter the cell cycle, creating progenitors that differentiate into new photoreceptors. The majority of previous reports on retinal regeneration focused on the first few days of the regenerative response, which include MG cell-cycle re-entry and progenitor cell proliferation. With this study, we analyzed the full 28-day time-course of regeneration by pairing a detailed morphological/immunological analysis with RNA-seq transcriptional profiling at 8 key time points during retinal regeneration. We observed several novel findings. First, we provide evidence for two separate peaks of MG gliosis, with the secondary gliotic peak occurring after MG cell-cycle re-entry. Second, we highlight a distinct transcriptional shift between 5- and 10-days post lesion that highlights the transition from progenitor proliferation to differentiation into new photoreceptors. Third, we show distinctly different patterns of transcriptional recovery of the photoreceptor opsins at 28 days post lesion. Finally, using differential gene expression analysis, we revealed that the established functional recovery of the retina at 28 days post lesion does not, in fact, return to an undamaged transcriptional state, potentially redefining what the field considers complete regeneration. Together, to our knowledge, this work represents the first histological and transcriptomic map of a 28-day time-course of retinal regeneration in adult zebrafish.

8.
Exp Eye Res ; 209: 108630, 2021 08.
Article in English | MEDLINE | ID: mdl-34029596

ABSTRACT

Zebrafish (Danio rerio) have become a highly-utilized model system in the field of regenerative biology because of their endogenous ability to regenerate many tissues and organs, including the retina. The vast majority of previous research on retinal regeneration in adult zebrafish utilizes acute methodologies for retinal damage. Acute retinal cell death triggers a reactive gliosis response of Müller glia (MG), the resident macroglia of the retina. In addition, each activated MG undergoes asymmetric cell division to produce a neuronal progenitor, which continues to divide and ultimately gives rise to new retinal neurons. Studies using these approaches have uncovered many crucial mechanisms by which MG respond to acute damage. However, they may not adequately mimic the chronic neuronal degeneration observed in many human retinal degenerative diseases. The current study aimed to develop a new long-term, chronic photoreceptor damage and degeneration model in adult zebrafish. Comparing the subsequent cellular responses to that of the commonly-used acute high-intensity model, we found that low, continuous light exposure damaged the outer segments of both rod and cone photoreceptors, but did not result in significant apoptotic cell death, MG gliosis, or MG cell-cycle re-entry. Instead, chronic light nearly completely truncated photoreceptor outer segments and resulted in a recruitment of microglia to the area. Together, these studies present a chronic photoreceptor model that can be performed in a relatively short time frame (21 days), that may lend insight into the cellular events underlying non-regenerative photoreceptor degeneration observed in other model systems.


Subject(s)
Nerve Regeneration/physiology , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/diagnosis , Retinal Neurons/pathology , Animals , Animals, Genetically Modified , Apoptosis , Cell Proliferation , Chronic Disease , Disease Models, Animal , Ependymoglial Cells/pathology , Retinal Degeneration/physiopathology , Zebrafish
9.
ASN Neuro ; 13: 17590914211009851, 2021.
Article in English | MEDLINE | ID: mdl-33874780

ABSTRACT

A founder mutation in human VPS11 (Vacuolar Protein Sorting 11) was recently linked to a genetic leukoencephalopathy in Ashkenazi Jews that presents with the classical features of white matter disorders of the central nervous system (CNS). The neurological deficits include hypomyelination, hypotonia, gradual loss of vision, and seizures. However, the cells expressing the mutation were not identified. Here we describe, using immunocytochemistry, the strong expression of Vps11 in mouse oligodendrocytes and, specifically, its localization with Myelin Associated Glycoprotein (MAG) in the inner tongue of myelin. In longitudinal sections of myelin, it forms a bead-like structure, alternating with Myelin Basic Protein (MBP). Immunofluorescent staining with Vps11 and neurofilament proteins indicates the absence of Vps11 in axons in vivo. Finally, changes in Vps11 expression are associated with altered proteolipid protein (PLP) levels based upon mice with duplications or deletions of the Plp1 gene. To determine potential functional contributions of Vps11, we combined Vps11 with Platelet Derived Growth Factor Receptor-α (PDGFRα) in vitro and in vivo: in both conditions, co-localization of the two proteins was frequently found in round vesicles of OPCs/oligodendrocytes, suggesting retrograde transport for degradation by the endolysosomal system. Neuron-to-glial communication has been invoked to explain degenerative changes in myelin followed by degenerative changes in axons, and vice versa; but to our knowledge, no specific proteins in retrograde transport from the myelin inner tongue to oligodendrocyte perikarya have been identified. The identification of mutations in VPS11 and its localization at the axon-myelin interface should open new avenues of research.


Subject(s)
Oligodendroglia/metabolism , Vesicular Transport Proteins/biosynthesis , Vesicular Transport Proteins/genetics , Animals , Cells, Cultured , Gene Expression , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Myelin Sheath/genetics , Myelin Sheath/metabolism
10.
PLoS One ; 15(6): e0232308, 2020.
Article in English | MEDLINE | ID: mdl-32530962

ABSTRACT

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.


Subject(s)
Midkine/metabolism , Regeneration/physiology , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animal Fins/physiology , Animals , Animals, Genetically Modified/metabolism , Cell Differentiation , Cell Proliferation , Midkine/genetics , Mutagenesis , Neuroglia/cytology , Neuroglia/metabolism , Oculomotor Muscles/physiology , Retinal Neurons/physiology , Zebrafish Proteins/genetics
11.
Pathogens ; 8(4)2019 Oct 26.
Article in English | MEDLINE | ID: mdl-31717750

ABSTRACT

Gram-positive bacteria remain the leading cause of endophthalmitis, a blinding infectious disease of the eye. Murine models have been widely used for understanding the pathogenesis of bacterial endophthalmitis. In this study, we sought to develop an alternative zebrafish (Danio rerio) model for Staphylococcus aureus and compare the disease pathobiology to a murine model. Endophthalmitis was induced in zebrafish and C57BL/6 mice through the intravitreal injection of S. aureus. Disease progression was monitored by assessing corneal haze, opacity, bacterial burden, and retinal histology. Our results demonstrated that, unlike the murine models, zebrafish maintained ocular integrity, corneal transparency, and retinal architecture. We found that the zebrafish was capable of clearing S. aureus from the eye via transport through retinal vessels and the optic nerve and by mounting a monocyte/macrophage response beginning at 8 hour post-infection (hpi). The bacterial burden increased up to 8 hpi and significantly decreased thereafter. An assessment of the innate retinal response revealed the induced expression of Il-1ß and Il-6 transcripts. Collectively, our study shows that unlike the murine model, zebrafish do not develop endophthalmitis and rapidly clear the pathogen. Hence, a better understanding of the zebrafish protective ocular innate response may provide new insights into the pathobiology of bacterial endophthalmitis.

12.
Toxics ; 7(4)2019 Sep 21.
Article in English | MEDLINE | ID: mdl-31546644

ABSTRACT

Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed.

13.
New Phytol ; 221(4): 2273-2285, 2019 03.
Article in English | MEDLINE | ID: mdl-30347428

ABSTRACT

Anecdotal evidence indicating substantial silica accumulation in tissues of bryophytes suggests that silica (phytolith) deposition evolved early on in embryophytes. To test this hypothesis, we conducted the first survey of phytolith content representing the major liverwort, moss and hornwort clades. We also assessed the diagnostic value of bryophyte phytoliths. Silica extracted from bryophyte material through wet-ashing was described, focusing on abundance, classifying taxa as nonproducers, light producers and higher producers; and phytolith morphotypes. Ancestral state reconstruction of these characters was performed for mosses and liverworts using published phylogenies. Phytoliths are present in multiple subclades within liverworts, mosses and hornworts, but these phyla were not ancestrally high silica-producers. Higher deposition occurs in liverworts and mosses with specialized water-conducting cells. We hypothesize that active, high silica accumulation was not ancestral for embryophytes, but became possible in clades with increased water conductance. Phytoliths of diagnostic structures (e.g. pegged rhizoids) could help track bryophyte clades or water conductance evolution in the fossil record.


Subject(s)
Biological Evolution , Bryophyta/physiology , Fossils , Silicon Dioxide/chemistry , Germ Cells, Plant/physiology
14.
PeerJ ; 6: e5646, 2018.
Article in English | MEDLINE | ID: mdl-30258730

ABSTRACT

Müller glia in the zebrafish retina respond to retinal damage by re-entering the cell cycle, which generates large numbers of retinal progenitors that ultimately replace the lost neurons. In this study we compared the regenerative outcomes of adult zebrafish exposed to one round of phototoxic treatment with adult zebrafish exposed to six consecutive rounds of phototoxic treatment. We observed that Müller glia continued to re-enter the cell cycle to produce clusters of retinal progenitors in zebrafish exposed to multiple rounds of phototoxic light. Some abnormalities were noted, however. First, we found that retinas exposed to multiple rounds of damage exhibited a greater loss of photoreceptors at 36 hours of light damage than retinas that were exposed to their first round of light damage. In addition, we found that Müller glia appeared to have an increase in the acute gliotic response in retinas exposed to multiple rounds of light treatment. This was evidenced by cellular hypertrophy, changes in GFAP cellular localization, and transient increases in stat3 and gfap expression. Finally, following the sixth round of phototoxic lesion, we observed a significant increase in mis-localized HuC/D-positive amacrine and ganglion cells in the inner plexiform layer and outer retina, and a decreased number of regenerated blue cone photoreceptors. These data add to recent findings that retinal regeneration in adult zebrafish occurs concomitant with Müller glia reactivity and can result in the generation of aberrant neurons. These data are also the first to demonstrate that Müller glia appear to modify their phenotype in response to multiple rounds of phototoxic lesion, exhibiting an increase in acute gliosis while maintaining a remarkable capacity for long-term regeneration of photoreceptors.

15.
Exp Eye Res ; 166: 106-115, 2018 01.
Article in English | MEDLINE | ID: mdl-29030175

ABSTRACT

In contrast to the mammalian retina, the zebrafish retina possesses the ability to regenerate. This is primarily accomplished through Müller glial cells, which, upon damage, re-enter the cell cycle to form retinal progenitors. The progenitors continue to proliferate as they migrate to the area of damage and ultimately differentiate into new neurons. The purpose of this study was to characterize the expression and function of Sonic Hedgehog (Shh) during regeneration of the adult zebrafish retina. Expression profiling of Shh pathway genes showed a significant upregulation of expression associated with stages of progenitor proliferation and neuronal differentiation. Activation of Shh signaling during early stages of retinal regeneration using intraocular injections of the recombinant human SHH (SHH-N) resulted in increased Müller cell gliosis, proliferation, and neuroprotection of damaged retinal neurons. Continued activation of Shh resulted in a greater number of differentiated amacrine and ganglion cells in the fully regenerated retina. Conversely, inhibition of Shh signaling using intraocular injections of cyclopamine resulted in decreased Müller glial cell proliferation and a fewer number of regenerated amacrine and ganglion cells. These data suggest that Shh signaling plays pleiotropic roles in proliferation and differentiation during adult zebrafish retinal regeneration.


Subject(s)
Hedgehog Proteins/physiology , Regeneration/physiology , Retina/physiology , Signal Transduction/physiology , Animals , Cell Differentiation/physiology , Cell Proliferation/physiology , Ependymoglial Cells , Hedgehog Proteins/metabolism , Retina/metabolism , Retinal Neurons/metabolism , Zebrafish
16.
Dev Biol ; 428(1): 148-163, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28579318

ABSTRACT

The zebrafish kidney is conserved with other vertebrates, making it an excellent genetic model to study renal development. The kidney collects metabolic waste using a blood filter with specialized epithelial cells known as podocytes. Podocyte formation is poorly understood but relevant to many kidney diseases, as podocyte injury leads to progressive scarring and organ failure. zeppelin (zep) was isolated in a forward screen for kidney mutants and identified as a homozygous recessive lethal allele that causes reduced podocyte numbers, deficient filtration, and fluid imbalance. Interestingly, zep mutants had a larger interrenal gland, the teleostean counterpart of the mammalian adrenal gland, which suggested a fate switch with the related podocyte lineage since cell proliferation and cell death were unchanged within the shared progenitor field from which these two identities arise. Cloning of zep by whole genome sequencing (WGS) identified a splicing mutation in breast cancer 2, early onset (brca2)/fancd1, which was confirmed by sequencing of individual fish. Several independent brca2 morpholinos (MOs) phenocopied zep, causing edema, reduced podocyte number, and increased interrenal cell number. Complementation analysis between zep and brca2ZM_00057434 -/- zebrafish, which have an insertional mutation, revealed that the interrenal lineage was expanded. Importantly, overexpression of brca2 rescued podocyte formation in zep mutants, providing critical evidence that the brca2 lesion encoded by zep specifically disrupts the balance of nephrogenesis. Taken together, these data suggest for the first time that brca2/fancd1 is essential for vertebrate kidney ontogeny. Thus, our findings impart novel insights into the genetic components that impact renal development, and because BRCA2/FANCD1 mutations in humans cause Fanconi anemia and several common cancers, this work has identified a new zebrafish model to further study brca2/fancd1 in disease.


Subject(s)
BRCA2 Protein/genetics , Gene Expression Regulation, Developmental , Organogenesis/genetics , Podocytes/cytology , Pronephros/embryology , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Animals, Genetically Modified , Cell Differentiation , Cell Proliferation , Cloning, Molecular , Disease Models, Animal , In Situ Hybridization, Fluorescence , Morpholinos/genetics , Pronephros/cytology , Zebrafish/genetics
17.
Methods Mol Biol ; 1565: 79-85, 2017.
Article in English | MEDLINE | ID: mdl-28364235

ABSTRACT

In the zebrafish regenerating fin, specific gene-targeting morpholinos have been widely utilized to assess gene function. Unlike in embryos, injection of standard morpholinos in the adult regenerating fin is not sufficient for cellular uptake. Rather, morpholinos are first injected extracellularly into the blastemal compartment, followed by electroporation for cellular uptake. Knockdown phenotypes are evaluated 1-4 days post electroporation. This chapter provides a description of the reagents, equipment, and procedure for successful injection and electroporation of morpholinos into the regenerating fin.


Subject(s)
Animal Fins/growth & development , Gene Expression Regulation , Morpholinos/genetics , Regeneration/genetics , Animals , Electroporation/methods , Gene Knockdown Techniques , Microinjections , Morpholinos/administration & dosage , Phenotype , Zebrafish/genetics , Zebrafish Proteins/genetics
18.
Elife ; 62017 02 28.
Article in English | MEDLINE | ID: mdl-28244868

ABSTRACT

Perilipin (PLIN) proteins constitute an ancient family important in lipid droplet (LD) formation and triglyceride metabolism. We identified an additional PLIN clade (plin6) that is unique to teleosts and can be traced to the two whole genome duplications that occurred early in vertebrate evolution. Plin6 is highly expressed in skin xanthophores, which mediate red/yellow pigmentation and trafficking, but not in tissues associated with lipid metabolism. Biochemical and immunochemical analyses demonstrate that zebrafish Plin6 protein targets the surface of pigment-containing carotenoid droplets (CD). Protein kinase A (PKA) activation, which mediates CD dispersion in xanthophores, phosphorylates Plin6 on conserved residues. Knockout of plin6 in zebrafish severely impairs the ability of CD to concentrate carotenoids and prevents tight clustering of CD within carotenoid bodies. Ultrastructural and functional analyses indicate that LD and CD are homologous structures, and that Plin6 was functionalized early in vertebrate evolution for concentrating and trafficking pigment.


Subject(s)
Lipid Droplets/metabolism , Perilipin-1/metabolism , Zebrafish , Animals , Carotenoids/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Profiling , Gene Knockout Techniques , Perilipin-1/genetics , Phosphorylation , Protein Processing, Post-Translational
19.
Front Immunol ; 8: 95, 2017.
Article in English | MEDLINE | ID: mdl-28280493

ABSTRACT

On the basis of mouse I-Ab-binding motifs, two sequences of the murine apolipoprotein B-100 (mApoB-100), mApoB-1003501-3515 (designated P3) and mApoB-100978-992 (designated P6), were found to be immunogenic. In this report, we show that P6 is also atherogenic. Immunization of Apoe-/- mice fed a high-fat diet (HFD) with P6 resulted in enhanced development of aortic atheroma as compared to control mice immunized with an irrelevant peptide MOG35-55 or with complete Freund's adjuvant alone. Adoptive transfer of lymph node cells from P6-immunized donor mice to recipients fed an HFD caused exacerbated aortic atheromas, correlating P6-primed cells with disease development. Finally, P6-specific T cell clones were generated and adoptive transfer of T cell clones into recipients fed an HFD led to significant increase in aortic plaque coverage when compared to control animals receiving a MOG35-55-specific T cell line. Recipient mice not fed an HFD, however, did not exhibit such enhancement, indicating that an inflammatory environment facilitated the atherogenic activity of P6-specific T cells. That P6 is identical to or cross-reacts with a naturally processed peptide of ApoB-100 is evidenced by the ability of P6 to stimulate the proliferation of T cells in the lymph node of mice primed by full-length human ApoB-100. By identifying an atherogenic T cell epitope of ApoB-100 and establishing specific T cell clones, our studies open up new and hitherto unavailable avenues to study the nature of atherogenic T cells and their functions in the atherosclerotic disease process.

20.
Proc Natl Acad Sci U S A ; 114(5): E717-E726, 2017 01 31.
Article in English | MEDLINE | ID: mdl-28096348

ABSTRACT

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.


Subject(s)
Animal Fins/physiology , Zebrafish , Animals , Female , Male , Metabolomics , Proteomics , Regeneration/physiology , Transcriptome , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish/physiology
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