64 research outputs found
Intermediate filaments of zebrafish retinal and optic nerve astrocytes and MΓΌller glia: differential distribution of cytokeratin and GFAP
<p>Abstract</p> <p>Background</p> <p>Optic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.</p> <p>Findings</p> <p>GFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the MΓΌller glial cells of the retina. Within MΓΌller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.</p> <p>Conclusions</p> <p>Studies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.</p
Identification of the molecular signatures integral to regenerating photoreceptors in the retina of the zebra fish
Investigating neuronal and photoreceptor regeneration in the retina of zebra fish has begun to yield insights into both the cellular and molecular means by which this lower vertebrate is able to repair its central nervous system. However, knowledge about the signaling molecules in the local microenvironment of a retinal injury and the transcriptional events they activate during neuronal death and regeneration is still lacking. To identify genes involved in photoreceptor regeneration, we combined light-induced photoreceptor lesions, laser-capture microdissection of the outer nuclear layer (ONL) and analysis of gene expression to characterize transcriptional changes for cells in the ONL as photoreceptors die and are regenerated. Using this approach, we were able to characterize aspects of the molecular signature of injured and dying photoreceptors, cone photoreceptor progenitors, and microglia within the ONL. We validated changes in gene expression and characterized the cellular expression for three novel, extracellular signaling molecules that we hypothesize are involved in regulating regenerative events in the retina
Genetics of photoreceptor degeneration and regeneration in zebrafish
Zebrafish are unique in that they provide a useful model system for studying two critically important problems in retinal neurobiology, the mechanisms responsible for triggering photoreceptor cell death and the innate stem cellβmediated regenerative response elicited by this death. In this review we highlight recent seminal findings in these two fields. We first focus on zebrafish as a model for studying photoreceptor degeneration. We summarize the genes currently known to cause photoreceptor degeneration, and we describe the phenotype of a few zebrafish mutants in detail, highlighting the usefulness of this model for studying this process. In the second section, we discuss the several different experimental paradigms that are available to study regeneration in the teleost retina. A model outlining the sequence of gene expression starting from the dedifferentiation of MΓΌller glia to the formation of rod and cone precursors is presented
Defects in ErbB-Dependent Establishment of Adult Melanocyte Stem Cells Reveal Independent Origins for Embryonic and Regeneration Melanocytes
Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish (Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a preβMSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration
Basal Jawed Vertebrate Phylogenomics Using Transcriptomic Data from Solexa Sequencing
The traditionally accepted relationships among basal jawed vertebrates have been challenged by some molecular phylogenetic analyses based on mitochondrial sequences. Those studies split extant gnathostomes into two monophyletic groups: tetrapods and piscine branch, including Chondrichthyes, Actinopterygii and sarcopterygian fishes. Lungfish and bichir are found in a basal position on the piscine branch. Based on transcriptomes of an armored bichir (Polypterus delhezi) and an African lungfish (Protopterus sp.) we generated, expressed sequences and whole genome sequences available from public databases, we obtained 111 genes to reconstruct the phylogenetic tree of basal jawed vertebrates and estimated their times of divergence. Our phylogenomic study supports the traditional relationship. We found that gnathostomes are divided into Chondrichthyes and the Osteichthyes, both with 100% support values (posterior probabilities and bootstrap values). Chimaeras were found to have a basal position among cartilaginous fishes with a 100% support value. Osteichthyes were divided into Actinopterygii and Sarcopterygii with 100% support value. Lungfish and tetrapods form a monophyletic group with 100% posterior probability. Bichir and two teleost species form a monophyletic group with 100% support value. The previous tree, based on mitochondrial data, was significantly rejected by an approximately unbiased test (AU test, pβ=β0). The time of divergence between lungfish and tetrapods was estimated to be 391.8 Ma and the divergence of bichir from pufferfish and medaka was estimated to be 330.6 Ma. These estimates closely match the fossil record. In conclusion, our phylogenomic study successfully resolved the relationship of basal jawed vertebrates based on transtriptomes, EST and whole genome sequences
Neural and Synaptic Defects in slytherin, a Zebrafish Model for Human Congenital Disorders of Glycosylation
Congenital disorder of glycosylation type IIc (CDG IIc) is characterized by mental retardation, slowed growth and severe immunodeficiency, attributed to the lack of fucosylated glycoproteins. While impaired Notch signaling has been implicated in some aspects of CDG IIc pathogenesis, the molecular and cellular mechanisms remain poorly understood. We have identified a zebrafish mutant slytherin (srn), which harbors a missense point mutation in GDP-mannose 4,6 dehydratase (GMDS), the rate-limiting enzyme in protein fucosylation, including that of Notch. Here we report that some of the mechanisms underlying the neural phenotypes in srn and in CGD IIc are Notch-dependent, while others are Notch-independent. We show, for the first time in a vertebrate in vivo, that defects in protein fucosylation leads to defects in neuronal differentiation, maintenance, axon branching, and synapse formation. Srn is thus a useful and important vertebrate model for human CDG IIc that has provided new insights into the neural phenotypes that are hallmarks of the human disorder and has also highlighted the role of protein fucosylation in neural development
Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by MΓΌller Cells in Adult Mammalian Retina
Background: Evidence emerging from a variety of approaches used in different species suggests that MΓΌller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched MΓΌller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells. Methodology/Principal Findings: We demonstrate that Notch and Wnt signaling activate MΓΌller cells through their canonical pathways and that a rare subset of activated MΓΌller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated MΓΌller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate MΓΌller cells. The number of activated MΓΌller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception. Conclusion/Significance: In summary, our results provide a proof of principle for non-neurotoxin-mediated activation o
Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish
Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current βhigh-contentβ (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of β₯0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current βhigh-contentβ whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform
Phenylthiourea Specifically Reduces Zebrafish Eye Size
Phenylthiourea (PTU) is commonly used for inhibiting melanization of zebrafish embryos. In this study, the standard treatment with 0.2 mM PTU was demonstrated to specifically reduce eye size in larval fish starting at three days post-fertilization. This effect is likely the result of a reduction in retinal and lens size of PTU-treated eyes and is not related to melanization inhibition. This is because the eye size of tyr, a genetic mutant of tyrosinase whose activity is inhibited in PTU treatment, was not reduced. As PTU contains a thiocarbamide group which is presented in many goitrogens, suppressing thyroid hormone production is a possible mechanism by which PTU treatment may reduce eye size. Despite the fact that thyroxine level was found to be reduced in PTU-treated larvae, thyroid hormone supplements did not rescue the eye size reduction. Instead, treating embryos with six goitrogens, including inhibitors of thyroid peroxidase (TPO) and sodium-iodide symporter (NIS), suggested an alternative possibility. Specifically, three TPO inhibitors, including those that do not possess thiocarbamide, specifically reduced eye size; whereas none of the NIS inhibitors could elicit this effect. These observations indicate that TPO inhibition rather than a general suppression of thyroid hormone synthesis is likely the underlying cause of PTU-induced eye size reduction. Furthermore, the tissue-specific effect of PTU treatment might be mediated by an eye-specific TPO expression. Compared with treatment with other tyrosinase inhibitors or bleaching to remove melanization, PTU treatment remains the most effective approach. Thus, one should use caution when interpreting results that are obtained from PTU-treated embryos
Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish
The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/beta-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/beta-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/beta-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/beta-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/beta-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species
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