548 research outputs found
Using the Tg(nrd:egfp)/albino Zebrafish Line to Characterize In Vivo Expression of neurod
In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults
PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration
Background: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.
Methodology: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ~100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.
Conclusions/Significance: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior
Characterization of retinal regeneration in adult zebrafish following multiple rounds of phototoxic lesion
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
Expression and regulation of caudal in the lower cyclorrhaphan fly Megaselia
The homeobox gene caudal (cad) regulates posterior development in Drosophila. In early embryos, the cad protein (CAD) is expressed in a posterior-to-anterior concentration gradient, which contributes polarity to the developing embryo. The CAD gradient is complementary to and dependent on the anterior pattern organizer Bicoid (BCD), which represses the translation of ubiquitous maternal cad transcripts in the anterior embryo through a direct interaction with the cad 3′ untranslated region (UTR). Here, we show that early embryos of the lower cyclorrhaphan fly Megaselia express the putative cad orthologue Mab-cad throughout the posterior three quarters of the blastoderm but lack maternal transcripts. In transgenic blastoderm embryos of Drosophila, Mab-cad cis-regulatory DNA drives the expression of a reporter gene in a similar pattern, while Mab-cad 3′ UTR fails to mediate translational repression of a ubiquitously transcribed reporter. For another lower cyclorrhaphan fly (Lonchoptera) and two related outgroup taxa of Cyclorrhapha (Empis, Haematopota), we report maternal cad expression in ovarian follicles. Together, our results suggest that BCD is not required for the translational repression of Mab-cad, and that maternal cad expression was lost in the Megaselia lineage
Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.
Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells
Complex Drug Interactions of the HIV Protease Inhibitors 3: Effect of Simultaneous or Staggered Dosing of Digoxin and Ritonavir, Nelfinavir, Rifampin or Bupropion
Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3
Background:
Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described.
Results:
Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates.
Conclusion:
We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution
Interpatient heterogeneity in expression of CYP3A4 and CYP3A5 in small bowel: Lack of prediction by the erythromycin breath test
The CYP3A subfamily of cytochromes P450 metabolize many medications and environmental contaminants. CYP3A4 and, in 25% of patients, CYP3A5 seem to be the major CYP3A genes expressed in adult liver. Hepatic levels of CYP3A4 can be estimated by the erythromycin breath test and vary at least 10-fold among patients. CYP3A4 has also been shown to be present in small bowel where it is responsible for significant "first-pass" metabolism of orally administered substrates. However, it is not known whether there is significant interindividual variability in the intestinal expression of CYP3A4, or whether the liver and intestinal catalytic activities of CYP3A4 correlate within an individual. It is also not known whether CYP3A5 is expressed in the small intestine. To address these questions, we administered the erythromycin breath test to 20 patients and obtained biopsies from their small bowel. There was a 6-fold variation in CYP3A catalytic activity (midazolam hydroxylation), an 11-fold variation in CYP3A4 protein content, and an 8-fold variation in CYP3A4 mRNA content in intestinal biopsies. There was an excellent correlation between intestinal CYP3A4 protein level and catalytic activity (r = 0.86; p = 0.0001); however, neither parameter significantly correlated with hepatic CYP3A4 activity as measured by the erythromycin breath test result (r = 0.27; p = 0.24 and r = 0.33; p = 0.15, respectively). We also found that CYP3A5 protein was readily detectable in biopsies from 14 (70%) of the patients, indicating that CYP3A5 is commonly expressed in human small intestine
The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: Implications for developing new model organisms
© 2015 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedNuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]
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