Publications by authors named "Madhuri Kango-Singh"

41 Publications

A Two-Clone Approach to Study Signaling Interactions among Neuronal Cells in a Pre-clinical Alzheimer's Disease Model.

iScience 2020 Dec 18;23(12):101823. Epub 2020 Nov 18.

Department of Biology, University of Dayton, Dayton, OH 45469, USA.

To understand the progression of Alzheimer's disease, studies often rely on ectopic expression of amyloid-beta 42 (Aβ42) throughout an entire tissue. Uniform ectopic expression of Aβ42 may obscure cell-cell interactions that contribute to the progression of the disease. We developed a two-clone system to study the signaling cross talk between GFP-labeled clones of Aβ42-expressing neurons and wild-type neurons simultaneously generated from the same progenitor cell by a single recombination event. Surprisingly, wild-type clones are reduced in size as compared with Aβ42-producing clones. We found that wild-type cells are eliminated by the induction of cell death. Furthermore, aberrant activation of c-Jun-N-terminal kinase (JNK) signaling in Aβ42-expressing neurons sensitizes neighboring wild-type cells to undergo progressive neurodegeneration. Blocking JNK signaling in Aβ42-producing clones restores the size of wild-type clones.
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http://dx.doi.org/10.1016/j.isci.2020.101823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724150PMC
December 2020

Tep1 Regulates Yki Activity in Neural Stem Cells in Glioma Model.

Front Cell Dev Biol 2020 8;8:306. Epub 2020 May 8.

Department of Biology, University of Dayton, Dayton, OH, United States.

Glioblastoma Multiforme (GBM) is the most common form of malignant brain tumor with poor prognosis. Amplification of Epidermal Growth Factor Receptor (EGFR), and mutations leading to activation of Phosphatidyl-Inositol-3 Kinase (PI3K) pathway are commonly associated with GBM. Using a previously published glioma model generated by coactivation of PI3K and EGFR pathways [by downregulation of Pten and overexpression of oncogenic Ras] in glial cells, we showed that the Tep1 gene (ortholog of human CD109) regulates Yki (the ortholog of human YAP/TAZ) via an evolutionarily conserved mechanism. Oncogenic signaling by the YAP/TAZ pathway occurs in cells that acquire CD109 expression in response to the inflammatory environment induced by radiation in clinically relevant models. Further, downregulation of Tep1 caused a reduction in Yki activity and reduced glioma growth. A key function of Yki in larval CNS is stem cell renewal and formation of neuroblasts. Other reports suggest different upstream regulators of Yki activity in the optic lobe versus the central brain regions of the larval CNS. We hypothesized that Tep1 interacts with the Hippo pathway effector Yki to regulate neuroblast numbers. We tested if Tep1 acts through Yki to affect glioma growth, and if in normal cells Tep1 affects neuroblast number and proliferation. Our data suggests that Tep1 affects Yki mediated stem cell renewal in glioma, as reduction of Tep significantly decreases the number of neuroblasts in glioma. Thus, we identify Tep1-Yki interaction in the larval CNS that plays a key role in glioma growth and progression.
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http://dx.doi.org/10.3389/fcell.2020.00306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225285PMC
May 2020

A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration.

Front Cell Dev Biol 2020 13;8:117. Epub 2020 Mar 13.

Department of Biology, University of Dayton, Dayton, OH, United States.

Alzheimer's disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aβ42-mediated neurodegeneration. This deficiency uncovers () gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aβ42-mediated neurodegeneration.
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http://dx.doi.org/10.3389/fcell.2020.00117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082232PMC
March 2020

Inactivation of Hippo and cJun-N-terminal Kinase (JNK) signaling mitigate FUS mediated neurodegeneration in vivo.

Neurobiol Dis 2020 07 19;140:104837. Epub 2020 Mar 19.

Department of Biology, University of Dayton, Dayton, OH 45469, USA; Premedical Program, University of Dayton, Dayton, OH 45469, USA; Center for Tissue Regeneration and Engineering at Dayton (TREND), University of Dayton, Dayton, OH 45469, USA; The Integrative Science and Engineering Center, University of Dayton, Dayton, OH 45469, USA; Center for Genomic Advocacy (TCGA), Indiana State University, Terre Haute, IN, USA. Electronic address:

Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. We screened for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. We identified hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpo triggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH) Kinase (JNK) mediated cell death. We found that downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. Our study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders.
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http://dx.doi.org/10.1016/j.nbd.2020.104837DOI Listing
July 2020

Hippo Signaling in Cancer: Lessons From Models.

Front Cell Dev Biol 2019 24;7:85. Epub 2019 May 24.

Department of Biology, University of Dayton, Dayton, OH, United States.

Hippo pathway was initially identified through genetic screens for genes regulating organ size in fruitflies. Recent studies have highlighted the role of Hippo signaling as a key regulator of homeostasis, and in tumorigenesis. Hippo pathway is comprised of genes that act as tumor suppressor genes like () and (), and oncogenes like (). YAP and TAZ are two related mammalian homologs of Yki that act as effectors of the Hippo pathway. Hippo signaling deficiency can cause YAP- or TAZ-dependent oncogene addiction for cancer cells. YAP and TAZ are often activated in human malignant cancers. These transcriptional regulators may initiate tumorigenic changes in solid tumors by inducing cancer stem cells and proliferation, culminating in metastasis and chemo-resistance. Given the complex mechanisms (e.g., of the cancer microenvironment, and the extrinsic and intrinsic cues) that overpower YAP/TAZ inhibition, the molecular roles of the Hippo pathway in tumor growth and progression remain poorly defined. Here we review recent findings from studies in whole animal model organism like on the role of Hippo signaling regarding its connection to inflammation, tumor microenvironment, and other oncogenic signaling in cancer growth and progression.
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http://dx.doi.org/10.3389/fcell.2019.00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558396PMC
May 2019

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation.

Cell Rep 2019 02;26(7):1893-1905.e7

Department of Translational Molecular Pathology and Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Electronic address:

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133 PN signature, transition to a CD109 MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133/CD109 cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.
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http://dx.doi.org/10.1016/j.celrep.2019.01.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594377PMC
February 2019

A soy protein Lunasin can ameliorate amyloid-beta 42 mediated neurodegeneration in Drosophila eye.

Sci Rep 2018 09 10;8(1):13545. Epub 2018 Sep 10.

Department of Biology, University of Dayton, Dayton, OH, 45469, USA.

Alzheimer's disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (Aβ42) plaques. These Aβ42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of human Aβ42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer's like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. We show that misexpression of Lunasin by transgenic approach can rescue Aβ42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in Aβ42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human Aβ42 in flies. Our studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate Aβ42 mediated neurodegeneration by downregulating JNK signaling.
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http://dx.doi.org/10.1038/s41598-018-31787-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131139PMC
September 2018

Markers and Methods to Study Adult Midgut Stem Cells.

Methods Mol Biol 2018 ;1842:123-137

Basic Research Laboratory, Stem Cell Regulation and Animal Aging Section, National Cancer Institute, Frederick, MD, USA.

Stem cells have emerged as a promising cell source to heal, replace or regenerate tissue and organs damaged by aging, injury or diseases. The intestinal epithelium is the most rapidly renewing tissue in our body, which is maintained by intestinal stem cells (ISCs), located at the bottom of the crypts. ISCs continuously replace lost or injured intestinal epithelial cells in organisms ranging from Drosophila to humans. The adult Drosophila midgut provides an excellent in vivo model system to study ISC behavior during stress, regeneration, aging and infection. There are several signaling pathways/genes have been identified to regulate ISCs self-renewal and differentiation during normal and pathological conditions. A significant number of genetic tools and markers have been developed in the last one decade to study Drosophila ISCs behavior. Here, we describe some of the markers and methods used to study ISCs behavior in adult midgut of Drosophila.
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http://dx.doi.org/10.1007/978-1-4939-8697-2_9DOI Listing
April 2019

Cullin-4 regulates Wingless and JNK signaling-mediated cell death in the Drosophila eye.

Cell Death Dis 2016 12 29;7(12):e2566. Epub 2016 Dec 29.

Department of Biology, University of Dayton, Dayton, OH, USA.

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye.
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http://dx.doi.org/10.1038/cddis.2016.338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5261020PMC
December 2016

FOXD1-ALDH1A3 Signaling Is a Determinant for the Self-Renewal and Tumorigenicity of Mesenchymal Glioma Stem Cells.

Cancer Res 2016 12 28;76(24):7219-7230. Epub 2016 Aug 28.

Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama.

Glioma stem-like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1-ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1-ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219-30. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161538PMC
December 2016

Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells.

PLoS One 2016 21;11(6):e0158081. Epub 2016 Jun 21.

Department of Biology, University of Dayton, Dayton, Ohio, United States of America.

Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib) belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib-) surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158081PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915667PMC
July 2017

The Hippo pathway effector Yki downregulates Wg signaling to promote retinal differentiation in the Drosophila eye.

Development 2015 Jun 14;142(11):2002-13. Epub 2015 May 14.

Department of Biology, University of Dayton, Dayton, OH 45469, USA Premedical Program, University of Dayton, Dayton, OH 45469, USA Center for Tissue Regeneration and Engineering at Dayton (TREND), University of Dayton, Dayton, OH 45469, USA

The evolutionarily conserved Hippo signaling pathway is known to regulate cell proliferation and maintain tissue homeostasis during development. We found that activation of Yorkie (Yki), the effector of the Hippo signaling pathway, causes separable effects on growth and differentiation of the Drosophila eye. We present evidence supporting a role for Yki in suppressing eye fate by downregulation of the core retinal determination genes. Other upstream regulators of the Hippo pathway mediate this effect of Yki on retinal differentiation. Here, we show that, in the developing eye, Yki can prevent retinal differentiation by blocking morphogenetic furrow (MF) progression and R8 specification. The inhibition of MF progression is due to ectopic induction of Wingless (Wg) signaling and Homothorax (Hth), the negative regulators of eye development. Modulating Wg signaling can modify Yki-mediated suppression of eye fate. Furthermore, ectopic Hth induction due to Yki activation in the eye is dependent on Wg. Last, using Cut (Ct), a marker for the antennal fate, we show that suppression of eye fate by hyperactivation of yki does not change the cell fate (from eye to antenna-specific fate). In summary, we provide the genetic mechanism by which yki plays a role in cell fate specification and differentiation - a novel aspect of Yki function that is emerging from multiple model organisms.
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http://dx.doi.org/10.1242/dev.117358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919294PMC
June 2015

Drosophila C-terminal Src kinase regulates growth via the Hippo signaling pathway.

Dev Biol 2015 Jan 22;397(1):67-76. Epub 2014 Oct 22.

Department of Biology, University of Dayton, Dayton, OH 45469, USA; Center for Tissue Regeneration and Engineering at Dayton, Dayton, OH 45469, USA; Premedical Programs, University of Dayton, Dayton, OH 45469, USA. Electronic address:

The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase (d-Csk) is a genetic modifier of warts (wts), a tumor-suppressor gene in the Hippo pathway, and interacts with the Src oncogene. Reduction in d-Csk expression and the consequent activation of Src are frequently seen in several cancers including hepatocellular and colorectal tumors. Previous studies show that d-Csk regulates cell proliferation and tissue size during development. Given the similarity in the loss-of-function phenotypes of d-Csk and wts, we have investigated the interactions of d-Csk with the Hippo pathway. Here we present multiple lines of evidence suggesting that d-Csk regulates growth via the Hippo signaling pathway. We show that loss of dCsk caused increased Yki activity, and our genetic epistasis places dCsk downstream of Dachs. Furthermore, dCsk requires Yki for its growth regulatory functions, suggesting that dCsk is another upstream member of the network of genes that interact to regulate Wts and its effector Yki in the Hippo signaling pathway.
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http://dx.doi.org/10.1016/j.ydbio.2014.10.010DOI Listing
January 2015

Intercellular cooperation and competition in brain cancers: lessons from Drosophila and human studies.

Stem Cells Transl Med 2014 Nov 17;3(11):1262-8. Epub 2014 Sep 17.

Department of Neurological Surgery and James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Glioblastoma (GBM) is a primary brain cancer with an extremely poor prognosis. GBM tumors contain heterogeneous cellular components, including a small subpopulation of tumor cells termed glioma stem cells (GSCs). GSCs are characterized as chemotherapy- and radiotherapy-resistant cells with prominent tumorigenic ability. Studies in Drosophila cancer models demonstrated that interclonal cooperation and signaling from apoptotic clones provokes aggressive growth of neighboring tumorigenic clones, via compensatory proliferation or apoptosis induced proliferation. Mechanistically, these aggressive tumors depend on activation of Jun-N-terminal kinase (upstream of c-JUN), and Drosophila Wnt (Wg) in the apoptotic clones. Consistent with these nonmammalian studies, data from several mammalian studies have shown that c-JUN and Wnt are hyperactivated in aggressive tumors (including GBM). However, it remains elusive whether compensatory proliferation is an evolutionarily conserved mechanism in cancers. In the present report, we summarize recent studies in Drosophila models and mammalian models (e.g., xenografts of human cancer cells into small animals) to elucidate the intercellular interactions between the apoptosis-prone cancer cells (e.g., non-GSCs) and the hyperproliferative cancer cells (e.g., GSCs). These evolving investigations will yield insights about molecular signaling interactions in the context of post-therapeutic phenotypic changes in human cancers. Furthermore, these studies are likely to revise our understanding of the genetic changes and post-therapeutic cell-cell interactions, which is a vital area of cancer biology with wide applications to many cancer types in humans.
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http://dx.doi.org/10.5966/sctm.2014-0086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214844PMC
November 2014

Toxicity and localization studies of a potential photodynamic therapy agent in Drosophila.

Genesis 2014 Apr 12;52(4):309-14. Epub 2014 Mar 12.

Department of Chemistry, University of Dayton, Dayton, Ohio, 45469; Department of Biology, University of Dayton, Dayton, Ohio, 45469.

Photodynamic therapy utilizes light, a photosensitizer, and molecular oxygen as a treatment modality for a variety of cancers. We have recently combined ruthenium(II) polypyridyl groups with a zinc(II) centered porphyrin as a new photosensitizer for the treatment of melanoma. In-vitro studies have indicated that this photosensitizer is toxic to melanoma cells when irradiated with low energy light; however, it is nontoxic to normal cells under similar conditions. To determine the toxicity and cell viability of this compound in-vivo we present, herein, a study using Drosophila melanogaster. In the absence of light, the new photosensitizer shows no discernible effects to fly larvae at various concentrations of compound and stages of larval development. When the larvae were fed the photosensitizer it was observed, by fluorescence microscopy, that the compound passes through the cell membrane and localizes in the cytosol at lower concentrations and the nucleus at slightly higher concentrations indicating that the compound is not immediately metabolized.
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http://dx.doi.org/10.1002/dvg.22760DOI Listing
April 2014

Homeotic Gene teashirt (tsh) has a neuroprotective function in amyloid-beta 42 mediated neurodegeneration.

PLoS One 2013 25;8(11):e80829. Epub 2013 Nov 25.

Department of Biology, University of Dayton, Dayton, Ohio, United States of America.

Background: Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive.

Methodology/ Principle Findings: We have generated a transgenic Drosophila eye model where high levels of human Aβ42 is misexpressed in the differentiating photoreceptor neurons of the developing eye, which phenocopy Alzheimer's like neuropathology in the neural retina. We have utilized this model for a gain of function screen using members of various signaling pathways involved in the development of the fly eye to identify downstream targets or modifiers of Aβ42 mediated neurodegeneration. We have identified the homeotic gene teashirt (tsh) as a suppressor of the Aβ42 mediated neurodegenerative phenotype. Targeted misexpression of tsh with Aβ42 in the differentiating retina can significantly rescue neurodegeneration by blocking cell death. We found that Tsh protein is absent/ downregulated in the neural retina at this stage. The structure function analysis revealed that the PLDLS domain of Tsh acts as an inhibitor of the neuroprotective function of tsh in the Drosophila eye model. Lastly, we found that the tsh paralog, tiptop (tio) can also rescue Aβ42 mediated neurodegeneration.

Conclusions/significance: We have identified tsh and tio as new genetic modifiers of Aβ42 mediated neurodegeneration. Our studies demonstrate a novel neuroprotective function of tsh and its paralog tio in Aβ42 mediated neurodegeneration. The neuroprotective function of tsh is independent of its role in retinal determination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840013PMC
August 2014

Novel neuroprotective function of apical-basal polarity gene crumbs in amyloid beta 42 (aβ42) mediated neurodegeneration.

PLoS One 2013 18;8(11):e78717. Epub 2013 Nov 18.

Premedical Program, University of Dayton, Dayton, Ohio, United States of America.

Alzheimer's disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophila eye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. We demonstrate a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078717PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832507PMC
July 2014

Genetic, immunofluorescence labeling, and in situ hybridization techniques in identification of stem cells in male and female germline niches.

Methods Mol Biol 2013 ;1035:9-23

Mouse Cancer Genetics Program, National Cancer Institute, NIH, Frederick, MD, USA.

Stem cells have an enormous capacity of self-renewal, as well as the ability to differentiate into specialized cell types. Proper control of these two properties of stem cells is crucial for animal development, growth control, and reproduction. Germline stem cells (GSCs) are a self-renewing population of germ cells, which generate haploid gametes (sperms or oocyte) that transmit genetic information from generation to generation. In Drosophila testis and ovary, GSCs are anchored around the niche cells. The cap cells cluster in females and hub cells in males act as a niche to control GSC behavior. With highly sophisticated genetic techniques in Drosophila, tremendous progress has been made in understanding the interactions between stem cells and niches at cellular and molecular levels. Here, we provide details of genetic, immunofluorescence labeling, and in situ hybridization techniques in identification and characterization of stem cells in Drosophila male and female germline niches.
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http://dx.doi.org/10.1007/978-1-62703-508-8_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571255PMC
March 2014

Scribble acts in the Drosophila fat-hippo pathway to regulate warts activity.

PLoS One 2012 5;7(11):e47173. Epub 2012 Nov 5.

Department of Biology, University of Dayton, Dayton, Ohio, United States of America.

Epithelial cells are the major cell-type for all organs in multicellular organisms. In order to achieve correct organ size, epithelial tissues need mechanisms that limit their proliferation, and protect tissues from damage caused by defective epithelial cells. Recently, the Hippo signaling pathway has emerged as a major mechanism that orchestrates epithelial development. Hippo signaling is required for cells to stop proliferation as in the absence of Hippo signaling tissues continue to proliferate and produce overgrown organs or tumors. Studies in Drosophila have led the way in providing a framework for how Hippo alters the pattern of gene transcription in target cells, leading to changes in cell proliferation, survival, and other behaviors. Scribble (Scrib) belongs to a class of neoplastic tumor suppressor genes that are required to establish apical-basal cell polarity. The disruption of apical-basal polarity leads to uncontrolled cell proliferation of epithelial cells. The interaction of apical basal polarity genes with the Hippo pathway has been an area of intense investigation. Loss of scrib has been known to affect Hippo pathway targets, however, its functions in the Hippo pathway still remain largely unknown. We investigated the interactions of Scrib with the Hippo pathway. We present data suggesting that Drosophila scrib acts downstream of the Fat (Ft) receptor, and requires Hippo signaling for its growth regulatory functions. We show that Ft requires Scrib to interact with Expanded (Ex) and Dachs (D), and for regulating Warts (Wts) levels and stability, thus placing Scrib in the Hippo pathway network.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047173PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489842PMC
April 2013

Domain specific genetic mosaic system in the Drosophila eye.

Genesis 2013 Jan 26;51(1):68-74. Epub 2012 Nov 26.

Department of Biology, University of Dayton, Dayton, Ohio 45469, USA.

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss-of-function as well as gain-of-function clones on the dorsal and ventral eye margins. We used the bifid-Gal4 driver to drive expression of UAS-FLP. This reagent can have multiple applications in (i) studying spatio-temporal function of a gene during dorso-ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner.
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http://dx.doi.org/10.1002/dvg.22355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547150PMC
January 2013

Generation and staining of intestinal stem cell lineage in adult midgut.

Methods Mol Biol 2012 ;879:47-69

Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD, USA.

Stem cell-mediated tissue repair is a promising approach in regenerative medicine. Intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. Recently, using lineage tracing and molecular marker labeling, intestinal stem cells (ISCs) have been identified in Drosophila adult midgut. ISCs reside at the basement membrane and are multipotent as they produce both enterocytes and enteroendocrine cells. The adult Drosophila midgut provides an excellent in vivo model organ to study ISC behavior during aging, stress, regeneration, and infection. It has been demonstrated that Notch, Janus kinase/signal transducer and activator of transcription, epidermal growth factor receptor/mitogen-activated protein kinase, Hippo, and wingless signaling pathways regulate ISCs proliferation and differentiation. There are plenty of genetic tools and markers developed in recent years in Drosophila stem cell studies. These tools and markers are essential in the precise identification of stem cells as well as manipulation of genes in stem cell regulation. Here, we describe the details of genetic tools, markers, and immunolabeling techniques used in identification and characterization of adult midgut stem cells in Drosophila.
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http://dx.doi.org/10.1007/978-1-61779-815-3_4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461621PMC
September 2012

Annual Drosophila Research Conference, 2012.

Dev Dyn 2012 Jul 11;241(7):1227-36. Epub 2012 Jun 11.

Department of Pharmacology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, Arizona, USA.

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http://dx.doi.org/10.1002/dvdy.23806DOI Listing
July 2012

Tumor suppression by cell competition through regulation of the Hippo pathway.

Proc Natl Acad Sci U S A 2012 Jan 21;109(2):484-9. Epub 2011 Dec 21.

Department of Biochemistry and Molecular Biology and Program in Genes and Development, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. Here we investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib(-) cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib(-) cells are prevented from overproliferating. We report that cell competition between scrib(-) and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib(-) cells. Suppressing Yki activation is critical for scrib(-) clone elimination by cell competition, and experimental elevation of Yki activity in scrib(-) cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib(-) cells.
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http://dx.doi.org/10.1073/pnas.1113882109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258595PMC
January 2012

A glimpse into dorso-ventral patterning of the Drosophila eye.

Dev Dyn 2012 Jan 27;241(1):69-84. Epub 2011 Oct 27.

Department of Biology, University of Dayton, Dayton, Ohio 45469, USA.

During organogenesis in all multi-cellular organisms, axial patterning is required to transform a single layer organ primordium into a three-dimensional organ. The Drosophila eye model serves as an excellent model to study axial patterning. Dorso-ventral (DV) axis determination is the first lineage restriction event during axial patterning of the Drosophila eye. The early Drosophila eye primordium has a default ventral fate, and the dorsal eye fate is established by onset of dorsal selector gene pannier (pnr) expression in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments called the equator is the site for Notch (N) activation, which triggers cell proliferation and differentiation. This review will focus on (1) chronology of events during DV axis determination; (2) how early division of eye into dorsal and ventral compartments contributes towards the growth and patterning of the fly retina, and (3) functions of DV patterning genes.
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http://dx.doi.org/10.1002/dvdy.22764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538059PMC
January 2012

Activation of JNK signaling mediates amyloid-ß-dependent cell death.

PLoS One 2011 14;6(9):e24361. Epub 2011 Sep 14.

Department of Biology, University of Dayton, Dayton, Ohio, United States of America.

Background: Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood.

Methodology/principal Findings: We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aß42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aß42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aß42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone.

Conclusions/significance: Our data suggests that (i) accumulation of Aß42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to Aß42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024361PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173392PMC
February 2012

Opposing interactions between homothorax and Lobe define the ventral eye margin of Drosophila eye.

Dev Biol 2011 Nov 2;359(2):199-208. Epub 2011 Sep 2.

Department of Biology, University of Dayton, OH 45469, USA.

Patterning in multi-cellular organisms involves progressive restriction of cell fates by generation of boundaries to divide an organ primordium into smaller fields. We have employed the Drosophila eye model to understand the genetic circuitry responsible for defining the boundary between the eye and the head cuticle on the ventral margin. The default state of the early eye is ventral and depends on the function of Lobe (L) and the Notch ligand Serrate (Ser). We identified homothorax (hth) as a strong enhancer of the L mutant phenotype of loss of ventral eye. Hth is a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD). Hth is known to bind Extradenticle (Exd) via its MH domain for its nuclear translocation. Loss-of-function of hth, a negative regulator of eye, results in ectopic ventral eye enlargements. This phenotype is complementary to the L mutant phenotype of loss-of-ventral eye. However, if L and hth interact during ventral eye development remains unknown. Here we show that (i) L acts antagonistically to hth, (ii) Hth is upregulated in the L mutant background, and (iii) MH domain of Hth is required for its genetic interaction with L, while its homeodomain is not, (iv) in L mutant background ventral eye suppression function of Hth involves novel MH domain-dependent factor(s), and (v) nuclear localization of Exd is not sufficient to mediate the Hth function in the L mutant background. Further, Exd is not a critical rate-limiting factor for the Hth function. Thus, optimum levels of L and Hth are required to define the boundary between the developing eye and head cuticle on the ventral margin.
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http://dx.doi.org/10.1016/j.ydbio.2011.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217235PMC
November 2011

Annual Drosophila research conference, 2011.

Dev Dyn 2011 Aug;240(8):2042-50

Department of Pharmacology, Midwestern University, Glendale, Arizona, USA.

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http://dx.doi.org/10.1002/dvdy.22689DOI Listing
August 2011

Dorsal eye selector pannier (pnr) suppresses the eye fate to define dorsal margin of the Drosophila eye.

Dev Biol 2010 Oct 5;346(2):258-71. Epub 2010 Aug 5.

University of Dayton, Dayton, OH 45469, USA.

Axial patterning is crucial for organogenesis. During Drosophila eye development, dorso-ventral (DV) axis determination is the first lineage restriction event. The eye primordium begins with a default ventral fate, on which the dorsal eye fate is established by expression of the GATA-1 transcription factor pannier (pnr). Earlier, it was suggested that loss of pnr function induces enlargement in the dorsal eye due to ectopic equator formation. Interestingly, we found that in addition to regulating DV patterning, pnr suppresses the eye fate by downregulating the core retinal determination genes eyes absent (eya), sine oculis (so) and dacshund (dac) to define the dorsal eye margin. We found that pnr acts downstream of Ey and affects the retinal determination pathway by suppressing eya. Further analysis of the "eye suppression" function of pnr revealed that this function is likely mediated through suppression of the homeotic gene teashirt (tsh) and is independent of homothorax (hth), a negative regulator of eye. Pnr expression is restricted to the peripodial membrane on the dorsal eye margin, which gives rise to head structures around the eye, and pnr is not expressed in the eye disc proper that forms the retina. Thus, pnr has dual function, during early developmental stages pnr is involved in axial patterning whereas later it promotes the head specific fate. These studies will help in understanding the developmental regulation of boundary formation of the eye field on the dorsal eye margin.
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http://dx.doi.org/10.1016/j.ydbio.2010.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945442PMC
October 2010

Regulation of organ size: insights from the Drosophila Hippo signaling pathway.

Dev Dyn 2009 Jul;238(7):1627-37

Division of Basic Sciences, Mercer University School of Medicine, Macon, Georgia 31207, USA.

Organ size control is a fundamental and core process of development of all multicellular organisms. One important facet of organ size control is the regulation of cell proliferation and cell death. Here we address the question, What are the developmental mechanisms that control intrinsic organ size? In several multicellular animals including humans and flies, organs develop according to an instructive model where proliferation is regulated by extracellular signals. However, the signals that regulate proliferation (and organ size) remain poorly understood. Recent data from flies have shed some light on the molecular mechanisms that regulate growth and size of organs. In this review, we will briefly discuss classic studies that revealed the mysteries of growth regulation. We will then focus on the recent findings from the Drosophila Hippo signaling pathway and its role in the regulation of organ size. Finally, we will discuss the mammalian Hippo pathway, and its implications in regulation of growth/proliferation during development and disease.
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http://dx.doi.org/10.1002/dvdy.21996DOI Listing
July 2009

Annual Drosophila research conference, 2008.

Dev Dyn 2008 Nov;237(11):3444-52

Mercer University School of Medicine, Division of Basic Sciences, Macon, Georgia, USA.

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http://dx.doi.org/10.1002/dvdy.21737DOI Listing
November 2008