Browsing by Author "Tiwari, Sarika"

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    Class I histone deacetylases in retinal progenitors and differentiating ganglion cells
    (Elsevier, 2018-12) Saha, Ankita; Tiwari, Sarika; Dharmarajan, Subramanian; Otteson, Deborah C.; Belecky-Adams, Teri L.; Biology, School of Science
    Background The acetylation state of histones has been used as an indicator of the developmental state of progenitor and differentiating cells. The goal of this study was to determine the nuclear localization patterns of Class I histone deacetylases (HDACs) in retinal progenitor cells (RPCs) and retinal ganglion cells (RGCs), as the first step in understanding their potential importance in cell fate determination within the murine retina. Results The only HDAC to label RPC nuclei at E16 and P5 was HDAC1. In contrast, there was generally increased nuclear localization of all Class I HDACs in differentiating RGCs. Between P5 and P30, SOX2 expression becomes restricted to Müller glial, cholinergic amacrine cells, and retinal astrocytes. Cholinergic amacrine showed a combination of changes in nuclear localization of Class I HDACs. Strikingly, although Müller glia and retinal astrocytes express many of the same genes, P30 Müller glial cells showed nuclear localization only of HDAC1, while retinal astrocytes were positive for HDACs 1, 2, and 3. Conclusion These results indicate there may be a role for one or more of the Class I HDACs in retinal cell type-specific differentiation.
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    THE EFFECT OF HDAC INHIBITORS ON RETINAL DEVELOPMENT OF CHICK EMBRYO
    (Office of the Vice Chancellor for Research, 2016-04-08) Morris, Kayla M.; Tiwari, Sarika; Belecky-Adams, Teri
    Introduction: Gene expression is regulated by the accessibility of regulatory cis-acting DNA elements as well as availability of transcription factors. Histone deacetylase (HDAC) can regulate gene expression by deacetylating histone tails, which leads to a closed conformation of the DNA/histone complex and generally a reduction in expression. HDACs have been proposed to play a key role in cell survival, proliferation and differentiation; however, fewer studies have been focusing on the role of HDACs in the developing vertebrate retina. Methods: Chick retinal explants were treated with vehicle (dimethyl sulphoxide(DMSO)) or 1.0 M Trichstatin A (TSA), a known inhibitor of class 1 and 2 HDACs. Immunohistochemistry (IHC) was performed to analyze the levels of cleaved caspase 3, a protein activated during apoptosis, phospho-histone 3 (pH3) marker for mitotic phase, SOX2 which marks progenitor cells, and Islet-1 which marks differentiated cells. Digital images were analyzed using Image J/FIJI software for numbers of labeled cells. Results: After treatment with control or TSA, numbers of progenitor and differentiating cells were quantified. TSA-treated samples showed a statistically significant increase in SOX2+ (progenitors) and an increase in islet-1+ (differentiating) cells. To assess if any differences in proliferation and/or cell death that might lead to an increase in the number of progenitor and differentiating cells, samples were labeled for pH3 or cleaved caspase 3. Treatment with TSA led to increases in cells positive for pH3 and a statistically significant increase in cells positive for cleaved caspase 3 compared to controls. Conclusions: HDAC inhibitor, TSA, increased the number of progenitor and differentiating cells by increasing proliferation within the developing retina. However, there was also an increase in the number of cells undergoing apoptosis. Ongoing studies will determine which HDACs may be responsible for these results.
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    Expression of histone deacetylase enzymes in murine and chick optic nerve
    (2013) Tiwari, Sarika; Belecky-Adams, Teri; Meyer, Jason S.; Marrs, James; Atkinson, Simon
    Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.
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    The Role of Transforming Growth Factorβ (TGF-β)-activated Kinase 1 (TAK1) in Retinal Development
    (Office of the Vice Chancellor for Research, 2015-04-17) Altara, Ira; Tiwari, Sarika; Belecky-Adams, Teri
    Purpose: The formation of the retina is dependent on multiple transcription factors being expressed in the correct time and place. Transforming growth factor-β-activated kinase1 (TAK1), a serine threonine kinase, has been increasingly associated with regulation of proliferation, differentiation and apoptosis of many cell types both within and outside of the central nervous system. However, little is known about its role in development of the retina. Previous results from our lab have indicated that TAK1 is expressed throughout the developing retina; however activated TAK1 is found predominantly in the dividing progenitors of the early developing chick retina. Retinas injected with TAK1 inhibitor appeared to have an increase in progenitor population and a decrease in differentiating retinal ganglion cells. The present study evaluated the potential role of TAK1 in inducing apoptosis in the developing chick retina. Methods: Embryonic day 3(E3) chick retina were injected with vehicle and 1.0 M or 2.0 M concentration (5Z)-7-Oxozeaenol, an irreversible inhibitor of TAK1. 24 hours post inhibition the tissue was harvested. Immunohistochemistry (IHC) was performed to analyze the levels of cleaved caspase 3 expression, a protein activated during apoptosis. Nuclei stained with DAPI were used to quantify the number of cells expressing the caspase3. Lipopolysaccharide (LPS) treated adult, postnatal 30 (P30), mouse retina was used as a positive control for our IHC. Results: No difference in the level of cleaved (activated) caspase 3 immunolabel was found in vehicle-, 1.0 and 2.0 M inhibitor-injected retinas. Conclusion: Lack of cleaved caspase 3 immunolabel in TAK1-inhibited retinas indicates that TAK1 may not be playing any role in inducing cell death through apoptosis in the developing chick retina used in our study. These preliminary results suggest further research should be done to better understand its role in retinal development. Mentors: Teri Belecky-Adams and Sarika Tiwari, Center for Regenerative Biology and Medicine, IUPUI, Department of Biology, IUPUI
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    TAK1 inhibition increases proliferation and differentiation of chick retinal cells
    (Frontiers Media, 2022-09-13) Carrillo, Casandra; Ravi, Vagisha; Tiwari, Sarika; Chernoff, Ellen A.; Belecky-Adams, Teri L.; Biology, School of Science
    The factors necessary for the differentiation of cell types within the retina are incompletely understood. The transforming growth factor beta (TGF-β) superfamily, including TGF-β1 and 2, the bone morphogenetic proteins, and the activins have all been implicated in differentiation; however, the mechanisms by which these factors affect differentiation are only partially understood. The studies herein focus on a potential role for transforming growth factor β-activated kinase 1 (TAK1), a hub kinase that lies at the intersection of multiple signaling pathways, in the differentiation of cell types within the chick retina. Previous studies have focused predominantly on the role this kinase plays in the inflammation process and axonal growth. TAK1 is downstream of multiple signaling pathways that are critical to development of the central nervous system, including transforming growth factor β (TGFβ), bone morphogenetic proteins (BMPs), and activins. The present study indicates that activated TAK1 is found throughout the developing retina; however, it is localized at higher levels in dividing and differentiating cells. Further, ex ovo retinal studies using TAK1 inhibitor 5Z-7-oxozeaenol increased both progenitor and differentiating cell populations, accompanied by a substantial increase in proliferation and a smaller increase in cell death. These results indicate a unique role for TAK1 in differentiating and proliferating retinal cells.