Publications by authors named "Chrissa Kioussi"

46 Publications

Xanthohumol ameliorates Diet-Induced Liver Dysfunction via Farnesoid X Receptor-Dependent and Independent Signaling.

Front Pharmacol 2021 20;12:643857. Epub 2021 Apr 20.

Linus Pauling Institute, Oregon State University, Corvallis, OR, United States.

The farnesoid X receptor (FXR) plays a critical role in the regulation of lipid and bile acid (BA) homeostasis. Hepatic FXR loss results in lipid and BA accumulation, and progression from hepatic steatosis to nonalcoholic steatohepatitis (NASH). This study aimed to evaluate the effects of xanthohumol (XN), a hop-derived compound mitigating metabolic syndrome, on liver damage induced by diet and FXR deficiency in mice. Wild-type (WT) and liver-specific FXR-null mice (FXR) were fed a high-fat diet (HFD) containing XN or the vehicle formation followed by histological characterization, lipid, BA and gene profiling. HFD supplemented with XN resulted in amelioration of hepatic steatosis and decreased BA concentrations in FXR mice, the effect being stronger in male mice. XN induced the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and glucocorticoid receptor (GR) gene expression in the liver of FXR mice. These findings suggest that activation of BA detoxification pathways represents the predominant mechanism for controlling hydrophobic BA concentrations in FXR mice. Collectively, these data indicated sex-dependent relationship between FXR, lipids and BAs, and suggest that XN ameliorates HFD-induced liver dysfunction via FXR-dependent and independent signaling.
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http://dx.doi.org/10.3389/fphar.2021.643857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093804PMC
April 2021

Pitx genes in development and disease.

Cell Mol Life Sci 2021 Apr 12. Epub 2021 Apr 12.

Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, 97331, USA.

Homeobox genes encode sequence-specific transcription factors (SSTFs) that recognize specific DNA sequences and regulate organogenesis in all eukaryotes. They are essential in specifying spatial and temporal cell identity and as a result, their mutations often cause severe developmental defects. Pitx genes belong to the PRD class of the highly evolutionary conserved homeobox genes in all animals. Vertebrates possess three Pitx paralogs, Pitx1, Pitx2, and Pitx3 while non-vertebrates have only one Pitx gene. The ancient role of regulating left-right (LR) asymmetry is conserved while new functions emerge to afford more complex body plan and functionalities. In mouse, Pitx1 regulates hindlimb tissue patterning and pituitary development. Pitx2 is essential for the development of the oral cavity and abdominal wall while regulates the formation and symmetry of other organs including pituitary, heart, gut, lung among others by controlling growth control genes upon activation of the Wnt/ß-catenin signaling pathway. Pitx3 is essential for lens development and migration and survival of the dopaminergic neurons of the substantia nigra. Pitx gene mutations are linked to various congenital defects and cancers in humans. Pitx gene family has the potential to offer a new approach in regenerative medicine and aid in identifying new drug targets.
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http://dx.doi.org/10.1007/s00018-021-03833-7DOI Listing
April 2021

Vitamin E Deficiency Disrupts Gene Expression Networks during Zebrafish Development.

Nutrients 2021 Jan 30;13(2). Epub 2021 Jan 30.

Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA.

Vitamin E (VitE) is essential for vertebrate embryogenesis, but the mechanisms involved remain unknown. To study embryonic development, we fed zebrafish adults (>55 days) either VitE sufficient (E+) or deficient (E-) diets for >80 days, then the fish were spawned to generate E+ and E- embryos. To evaluate the transcriptional basis of the metabolic and phenotypic outcomes, E+ and E- embryos at 12, 18 and 24 h post-fertilization (hpf) were subjected to gene expression profiling by RNASeq. Hierarchical clustering, over-representation analyses and gene set enrichment analyses were performed with differentially expressed genes. E- embryos experienced overall disruption to gene expression associated with gene transcription, carbohydrate and energy metabolism, intracellular signaling and the formation of embryonic structures. mTOR was apparently a major controller of these changes. Thus, embryonic VitE deficiency results in genetic and transcriptional dysregulation as early as 12 hpf, leading to metabolic dysfunction and ultimately lethal outcomes.
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http://dx.doi.org/10.3390/nu13020468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912379PMC
January 2021

Vitamin E is necessary for zebrafish nervous system development.

Sci Rep 2020 09 21;10(1):15028. Epub 2020 Sep 21.

Linus Pauling Institute, Oregon State University, 307 LPSC, Corvallis, OR, USA.

Vitamin E (VitE) deficiency results in embryonic lethality. Knockdown of the gene ttpa encoding for the VitE regulatory protein [α-tocopherol transfer protein (α-TTP)] in zebrafish embryos causes death within 24 h post-fertilization (hpf). To test the hypothesis that VitE, not just α-TTP, is necessary for nervous system development, adult 5D strain zebrafish, fed either VitE sufficient (E+) or deficient (E-) diets, were spawned to obtain E+ and E- embryos, which were subjected to RNA in situ hybridization and RT-qPCR. Ttpa was expressed ubiquitously in embryos up to 12 hpf. Early gastrulation (6 hpf) assessed by goosecoid expression was unaffected by VitE status. By 24 hpf, embryos expressed ttpa in brain ventricle borders, which showed abnormal closure in E- embryos. They also displayed disrupted patterns of paired box 2a (pax2a) and SRY-box transcription factor 10 (sox10) expression in the midbrain-hindbrain boundary, spinal cord and dorsal root ganglia. In E- embryos, the collagen sheath notochord markers (col2a1a and col9a2) appeared bent. Severe developmental errors in E- embryos were characterized by improper nervous system patterning of the usually carefully programmed transcriptional signals. Histological analysis also showed developmental defects in the formation of the fore-, mid- and hindbrain and somites of E- embryos at 24 hpf. Ttpa expression profile was not altered by the VitE status demonstrating that VitE itself, and not ttpa, is required for development of the brain and peripheral nervous system in this vertebrate embryo model.
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http://dx.doi.org/10.1038/s41598-020-71760-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506018PMC
September 2020

Targeting the Liver-Brain Axis with Hop-Derived Flavonoids Improves Lipid Metabolism and Cognitive Performance in Mice.

Mol Nutr Food Res 2020 08 6;64(15):e2000341. Epub 2020 Jul 6.

Linus Pauling Institute, Oregon State University, Corvallis, OR, 97331, USA.

Scope: Sphingolipids including ceramides are implicated in the pathogenesis of obesity and insulin resistance. Correspondingly, inhibition of pro-inflammatory and neurotoxic ceramide accumulation prevents obesity-mediated insulin resistance and cognitive impairment. Increasing evidence suggests the farnesoid X receptor (FXR) is involved in ceramide metabolism, as bile acid-FXR crosstalk controls ceramide levels along the gut-liver axis. The authors previously reported that FXR agonist xanthohumol (XN), the principal prenylated flavonoid in hops (Humulus lupulus), and its hydrogenated derivatives, α,β-dihydroxanthohumol (DXN), and tetrahydroxanthohumol (TXN), ameliorated obesity-mediated insulin resistance, and cognitive impairment in mice fed a high-fat diet.

Methods And Results: To better understand how the flavonoids improve both, lipid and bile acid profiles in the liver are analyzed, sphingolipid relative abundance in the hippocampus is measured, and linked them to metabolic and neurocognitive performance. XN, DXN, and TXN (30 mg kg BW per day) decrease ceramide content in liver and hippocampus; the latter is linked to improvements in spatial learning and memory. In addition, XN, DXN, and TXN decrease hepatic cholesterol content by enhancing de novo synthesis of bile acids.

Conclusion: These observations suggest that XN, DXN, and TXN may alleviate obesity-induced metabolic and neurocognitive impairments by targeting the liver-brain axis.
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http://dx.doi.org/10.1002/mnfr.202000341DOI Listing
August 2020

Culturing and Manipulating Mouse Embryonic Stem Cells.

Authors:
Chrissa Kioussi

Methods Mol Biol 2020 ;2155:1-9

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.

Mouse embryonic stem cells (mESC) have the ability to self-renew due to their rapid proliferation and high telomerase activity while maintaining their pluripotency. Depending on the environment, mESC can differentiate into a broad range of cell types. These characteristics have established mESC as a tool for modeling human disease, genetic engineering, lineage specificity, stem cell-based therapies, and tissue regeneration. Here we describe a protocol for mESC expansion and differentiation.
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http://dx.doi.org/10.1007/978-1-0716-0655-1_1DOI Listing
March 2021

Requirement of Pitx2 for skeletal muscle homeostasis.

Dev Biol 2019 01 8;445(1):90-102. Epub 2018 Nov 8.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA. Electronic address:

Skeletal muscle is generated by the successive incorporation of primary (embryonic), secondary (fetal), and tertiary (adult) fibers into muscle. Conditional excision of Pitx2 function by an MCK driver resulted in animals with histological and ultrastructural defects in P30 muscles and fibers, respectively. Mutant muscle showed severe reduction in mitochondria and FoxO3-mediated mitophagy. Both oxidative and glycolytic energy metabolism were reduced. Conditional excision was limited to fetal muscle fibers after the G1-G0 transition and resulted in altered MHC, Rac1, MEF2a, and alpha-tubulin expression within these fibers. The onset of excision, monitored by a nuclear reporter gene, was observed as early as E16. Muscle at this stage was already severely malformed, but appeared to recover by P30 by the expansion of adjoining larger fibers. Our studies demonstrate that the homeodomain transcription factor Pitx2 has a postmitotic role in maintaining skeletal muscle integrity and energy homeostasis in fetal muscle fibers.
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http://dx.doi.org/10.1016/j.ydbio.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289786PMC
January 2019

Location, Location, Location: Signals in Muscle Specification.

J Dev Biol 2018 May 18;6(2). Epub 2018 May 18.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.

Muscles control body movement and locomotion, posture and body position and soft tissue support. Mesoderm derived cells gives rise to 700 unique muscles in humans as a result of well-orchestrated signaling and transcriptional networks in specific time and space. Although the anatomical structure of skeletal muscles is similar, their functions and locations are specialized. This is the result of specific signaling as the embryo grows and cells migrate to form different structures and organs. As cells progress to their next state, they suppress current sequence specific transcription factors (SSTF) and construct new networks to establish new myogenic features. In this review, we provide an overview of signaling pathways and gene regulatory networks during formation of the craniofacial, cardiac, vascular, trunk, and limb skeletal muscles.
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http://dx.doi.org/10.3390/jdb6020011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027348PMC
May 2018

FACS-Seq analysis of Pax3-derived cells identifies non-myogenic lineages in the embryonic forelimb.

Sci Rep 2018 05 16;8(1):7670. Epub 2018 May 16.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, 97331, USA.

Skeletal muscle in the forelimb develops during embryonic and fetal development and perinatally. While much is known regarding the molecules involved in forelimb myogenesis, little is known about the specific mechanisms and interactions. Migrating skeletal muscle precursor cells express Pax3 as they migrate into the forelimb from the dermomyotome. To compare gene expression profiles of the same cell population over time, we isolated lineage-traced Pax3 cells (Pax3 ) from forelimbs at different embryonic days. We performed whole transcriptome profiling via RNA-Seq of Pax3 cells to construct gene networks involved in different stages of embryonic and fetal development. With this, we identified genes involved in the skeletal, muscular, vascular, nervous and immune systems. Expression of genes related to the immune, skeletal and vascular systems showed prominent increases over time, suggesting a non-skeletal myogenic context of Pax3-derived cells. Using co-expression analysis, we observed an immune-related gene subnetwork active during fetal myogenesis, further implying that Pax3-derived cells are not a strictly myogenic lineage, and are involved in patterning and three-dimensional formation of the forelimb through multiple systems.
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http://dx.doi.org/10.1038/s41598-018-25998-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956100PMC
May 2018

To roll the eyes and snap a bite - function, development and evolution of craniofacial muscles.

Semin Cell Dev Biol 2019 07 10;91:31-44. Epub 2018 Jan 10.

Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, UK. Electronic address:

Craniofacial muscles, muscles that move the eyes, control facial expression and allow food uptake and speech, have long been regarded as a variation on the general body muscle scheme. However, evidence has accumulated that the function of head muscles, their developmental anatomy and the underlying regulatory cascades are distinct. This article reviews the key aspects of craniofacial muscle and muscle stem cell formation and discusses how this differs from the trunk programme of myogenesis; we show novel RNAseq data to support this notion. We also trace the origin of head muscle in the chordate ancestors of vertebrates and discuss links with smooth-type muscle in the primitive chordate pharynx. We look out as to how the special properties of head muscle precursor and stem cells, in particular their competence to contribute to the heart, could be exploited in regenerative medicine.
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http://dx.doi.org/10.1016/j.semcdb.2017.12.013DOI Listing
July 2019

Non-estrogenic Xanthohumol Derivatives Mitigate Insulin Resistance and Cognitive Impairment in High-Fat Diet-induced Obese Mice.

Sci Rep 2018 01 12;8(1):613. Epub 2018 Jan 12.

Linus Pauling Institute, Oregon State University, Corvallis, OR, 97331, USA.

Xanthohumol (XN), a prenylated flavonoid from hops, improves dysfunctional glucose and lipid metabolism in animal models of metabolic syndrome (MetS). However, its metabolic transformation into the estrogenic metabolite, 8-prenylnaringenin (8-PN), poses a potential health concern for its use in humans. To address this concern, we evaluated two hydrogenated derivatives, α,β-dihydro-XN (DXN) and tetrahydro-XN (TXN), which showed negligible affinity for estrogen receptors α and β, and which cannot be metabolically converted into 8-PN. We compared their effects to those of XN by feeding C57BL/6J mice a high-fat diet (HFD) containing XN, DXN, or TXN for 13 weeks. DXN and TXN were present at higher concentrations than XN in plasma, liver and muscle. Mice administered XN, DXN or TXN showed improvements of impaired glucose tolerance compared to the controls. DXN and TXN treatment resulted in a decrease of HOMA-IR and plasma leptin. C2C12 embryonic muscle cells treated with DXN or TXN exhibited higher rates of uncoupled mitochondrial respiration compared to XN and the control. Finally, XN, DXN, or TXN treatment ameliorated HFD-induced deficits in spatial learning and memory. Taken together, DXN and TXN could ameliorate the neurocognitive-metabolic impairments associated with HFD-induced obesity without risk of liver injury and adverse estrogenic effects.
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http://dx.doi.org/10.1038/s41598-017-18992-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766630PMC
January 2018

Differential gene regulatory networks in development and disease.

Cell Mol Life Sci 2018 03 10;75(6):1013-1025. Epub 2017 Oct 10.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, 97331, USA.

Gene regulatory networks, in which differential expression of regulator genes induce differential expression of their target genes, underlie diverse biological processes such as embryonic development, organ formation and disease pathogenesis. An archetypical systems biology approach to mapping these networks involves the combined application of (1) high-throughput sequencing-based transcriptome profiling (RNA-seq) of biopsies under diverse network perturbations and (2) network inference based on gene-gene expression correlation analysis. The comparative analysis of such correlation networks across cell types or states, differential correlation network analysis, can identify specific molecular signatures and functional modules that underlie the state transition or have context-specific function. Here, we review the basic concepts of network biology and correlation network inference, and the prevailing methods for differential analysis of correlation networks. We discuss applications of gene expression network analysis in the context of embryonic development, cancer, and congenital diseases.
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http://dx.doi.org/10.1007/s00018-017-2679-6DOI Listing
March 2018

Mapping the Chromatin State Dynamics in Myoblasts.

Gene Rep 2016 Jun 1;3:5-13. Epub 2016 Feb 1.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.

Background: Genome-wide mapping reveals chromatin landscapes unique to cell states. Histone marks of regulatory genes involved in cell specification and organ development provide a powerful tool to map regulatory sequences. H3K4me3 marks promoter regions; H3K27me3 marks repressed regions, and Pol II presence indicates active transcription. The presence of both H3K4me3 and H3K27me3 characterize poised sequences, a common characteristic of genes involved in pattern formation during organogenesis.

Results: We used genome-wide profiling for H3K27me3, H3K4me3, and Pol II to map chromatin states in mouse embryonic day 12 forelimbs in wild type (control) and -null mutant mice. We compared these data with previous gene expression studies from forelimb Lbx1 migratory myoblasts and correlated -dependent expression profiles and chromatin states. During forelimb development, several lineages including myoblast, osteoblast, neurons, angioblasts etc., require synchronized growth to form a functional limb. We identified 125 genes in the developing forelimb that are -dependent. Genes involved in muscle specification and cytoskeleton architecture were positively regulated, while genes involved in axonal path finding were poised.

Conclusion: Our results have established histone modification profiles as a useful tool for identifying gene regulatory states in muscle development, and identified the role of Pitx2 in extending the time of myoblast progression, promoting formation of sarcomeric structures, and suppressing attachment of neuronal axons.
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http://dx.doi.org/10.1016/j.genrep.2016.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786272PMC
June 2016

Phenotypic Screening of Drug Library in Actively Differentiating Mouse Embryonic Stem Cells.

J Biomol Screen 2016 Apr 8;21(4):399-407. Epub 2016 Jan 8.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA

Phenotypic screening enables the discovery of new drug leads with novel targets. ES cells differentiate into different lineages by successively making use of different subsets of the genome's possible macromolecular interactions. If a compound effectively targets just one of these interactions, it derails the developmental pathway to produce a phenotypical change. The OTRADI microsource spectrum library of 2000 approved drug components, natural products, and bioactive components was screened for compounds that can induce phenotypic changes in ES cell cultures at 10 µM after 3 days. Twenty-one compounds that induced specific morphologies also induced unique changes to an expression profile of a dozen markers of early embryonic development, indicating that each compound has derailed the molecular developmental process in a characteristic way. Phenotypic screens conducted with ES cultures differentiating along different lineages can be used to efficiently prescreen compounds able to regulate cell differentiation lineage.
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http://dx.doi.org/10.1177/1087057115624093DOI Listing
April 2016

Genome-Wide Mapping of Chromatin State of Mouse Forelimbs.

Open Access Bioinformatics 2014 Sep;6(2014):1-11

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.

Background: Cell types are defined at the molecular level during embryogenesis by a process called pattern formation and created by the selective utilization of combinations of sequence specific transcription factors. Developmental programs define the sets of genes that are available to each particular cell type, and real-time biochemical signaling interactions define the extent to which these sets are used at any given time and place. Gene expression is regulated through the integrated action of many -regulatory elements, including core promoters, enhancers, silencers, and insulators. The chromatin state in developing body parts provides a code to cellular populations that direct their cell fates. Chromatin profiling has been a method of choice for mapping regulatory sequences in cells that go through developmental transitions.

Results: We used antibodies against histone H3 lysine 4 trimethylations (H3K4me3) a modification associated with promoters and open/active chromatin, histone H3 lysine 27 trimethylations (H3K27me3) associated with Polycomb-repressed regions and RNA polymerase II (Pol2) associated with transcriptional initiation to identify the chromatin state signature of the mouse forelimb during mid-gestation, at embryonic day 12 (E12). The families of genes marked included those related to transcriptional regulation and embryogenesis. One third of the marked genes were transcriptionally active while only a small fraction were bivalent marked. Sequence specific transcription factors that were activated were involved in cell specification including bone and muscle formation.

Conclusion: Our results demonstrate that embryonic limb cells do not exhibit the plasticity of the ES cells but are rather programmed for a finer tuning for cell lineage specification.
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http://dx.doi.org/10.2147/OAB.S59043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271808PMC
September 2014

Culturing and differentiating mouse embryonic stem cells.

Methods Mol Biol 2014 ;1210:1-8

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 1601 SW Jefferson St., Corvallis, OR, 97331, USA.

Pluripotent embryonic stem (ES) cells have been used extensively for over 20 years for creating mouse mutants as models for developmental biology and humans diseases. The genetic manipulations of the ES cells have revolutionized our understanding of organ development and abilities to genetically manipulate the mouse embryo. Understanding the ES cell differentiation has provided new insights essential for establishing cell-based therapy and tissue regeneration.
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http://dx.doi.org/10.1007/978-1-4939-1435-7_1DOI Listing
May 2015

Grp1-associated scaffold protein regulates skin homeostasis after ultraviolet irradiation.

Photochem Photobiol Sci 2014 Mar 9;13(3):531-40. Epub 2014 Jan 9.

Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon 97331, USA.

Grp1-associated scaffold protein (Grasp), the product of a retinoic acid-induced gene in P19 embryonal carcinoma cells, is expressed primarily in brain, heart, and lung of the mouse. We report herein that Grasp transcripts are also found in mouse skin in which the Grasp gene is robustly induced following acute ultraviolet-B (UVB) exposure. Grasp(-/-) mice were found to exhibit delayed epidermal proliferation and a blunted apoptotic response after acute UVB exposure. Immunohistochemical analyses revealed that the nuclear residence time of the tumor suppressor protein p53 was reduced in Grasp(-/-) mice after UVB exposure. Taken together, our results suggest that a physiological role of Grasp may be to regulate skin homeostasis after UVB exposure, potentially by influencing p53-mediated apoptotic responses in skin.
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http://dx.doi.org/10.1039/c3pp50351hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128327PMC
March 2014

Pitx2-mediated cardiac outflow tract remodeling.

Dev Dyn 2013 May 12;242(5):456-68. Epub 2013 Mar 12.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, USA.

Background: Heart morphogenesis involves sequential anatomical changes from a linear tube of a single channel peristaltic pump to a four-chamber structure with two channels controlled by one-way valves. The developing heart undergoes continuous remodeling, including septation.

Results: Pitx2-null mice are characterized by cardiac septational defects of the atria, ventricles, and outflow tract. Pitx2-null mice also exhibited a short outflow tract, including unseptated conus and deformed endocardial cushions. Cushions were characterized with a jelly-like structure, rather than the distinct membrane-looking leaflets, indicating that endothelial mesenchymal transition was impaired in Pitx2(-/-) embryos. Mesoderm cells from the branchial arches and neural crest cells from the otic region contribute to the development of the endocardial cushions, and both were reduced in number. Members of the Fgf and Bmp families exhibited altered expression levels in the mutants.

Conclusions: We suggest that Pitx2 is involved in the cardiac outflow tract septation by promoting and/or maintaining the number and the remodeling process of the mesoderm progenitor cells. Pitx2 influences the expression of transcription factors and signaling molecules involved in the differentiation of the cushion mesenchyme during heart development.
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http://dx.doi.org/10.1002/dvdy.23934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673775PMC
May 2013

Selective ablation of Ctip2/Bcl11b in epidermal keratinocytes triggers atopic dermatitis-like skin inflammatory responses in adult mice.

PLoS One 2012 20;7(12):e51262. Epub 2012 Dec 20.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.

Background: Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice.

Methodology/principal Findings: To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2(ep-/-) mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes.

Conclusions/significance: Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in barrier functions. Our results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism enforced by Ctip2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051262PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527437PMC
June 2013

Population-specific regulation of Chmp2b by Lbx1 during onset of synaptogenesis in lateral association interneurons.

PLoS One 2012 21;7(12):e48573. Epub 2012 Dec 21.

Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States of America.

Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene Lbx1 plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of Lbx1 in the embryonic mouse neural tube. Chmp2b was expressed and depended on Lbx1 in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048573PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528757PMC
June 2013

Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

CNS Neurol Disord Drug Targets 2012 Nov;11(7):884-92

Department of Neuroscience, Karolinska Institutet, Retzius väg 8, Stockholm, Sweden.

Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.
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http://dx.doi.org/10.2174/1871527311201070884DOI Listing
November 2012

Pharyngeal mesoderm regulatory network controls cardiac and head muscle morphogenesis.

Proc Natl Acad Sci U S A 2012 Nov 29;109(46):18839-44. Epub 2012 Oct 29.

Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.

The search for developmental mechanisms driving vertebrate organogenesis has paved the way toward a deeper understanding of birth defects. During embryogenesis, parts of the heart and craniofacial muscles arise from pharyngeal mesoderm (PM) progenitors. Here, we reveal a hierarchical regulatory network of a set of transcription factors expressed in the PM that initiates heart and craniofacial organogenesis. Genetic perturbation of this network in mice resulted in heart and craniofacial muscle defects, revealing robust cross-regulation between its members. We identified Lhx2 as a previously undescribed player during cardiac and pharyngeal muscle development. Lhx2 and Tcf21 genetically interact with Tbx1, the major determinant in the etiology of DiGeorge/velo-cardio-facial/22q11.2 deletion syndrome. Furthermore, knockout of these genes in the mouse recapitulates specific cardiac features of this syndrome. We suggest that PM-derived cardiogenesis and myogenesis are network properties rather than properties specific to individual PM members. These findings shed new light on the developmental underpinnings of congenital defects.
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http://dx.doi.org/10.1073/pnas.1208690109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503185PMC
November 2012

Prediction of gene network models in limb muscle precursors.

Gene 2012 Nov 20;509(1):16-23. Epub 2012 Aug 20.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331-3507, USA.

The ventrolateral dermomyotome gives rise to all muscles of the limbs through the delamination and migration of cells into the limb buds. These cells proliferate and form myoblasts, withdraw from the cell cycle and become terminally differentiated. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Gene expression arrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1(EGFP), which resulted from the loss of Pitx2. Target genes of Pitx2 were clustered using the "David Bioinformatics Functional Annotation Tool" to bin genes according to enrichment of gene ontology keywords. This provided a way to both narrow the target genes and identify potential gene families regulated by Pitx2. Representative target genes in the most enriched bins were analyzed for the presence and evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the -20kb, intronic, and coding regions of the genes. Fifteen Pitx2 target genes were selected based on the above analysis and were identified as having functions involving cytoskeleton organization, tissue specification, and transcription factors. Data from these studies suggest that Pitx2 acts to regulate cell motility and expression of muscle specific genes in the muscle precursors during forelimb muscle development. This work provides a framework to develop the gene network leading to skeletal muscle development, growth and regeneration.
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http://dx.doi.org/10.1016/j.gene.2012.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506249PMC
November 2012

Loss of abdominal muscle in Pitx2 mutants associated with altered axial specification of lateral plate mesoderm.

PLoS One 2012 31;7(7):e42228. Epub 2012 Jul 31.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.

Sequence specific transcription factors (SSTFs) combinatorially define cell types during development by forming recursively linked network kernels. Pitx2 expression begins during gastrulation, together with Hox genes, and becomes localized to the abdominal lateral plate mesoderm (LPM) before the onset of myogenesis in somites. The somatopleure of Pitx2 null embryos begins to grow abnormally outward before muscle regulatory factors (MRFs) or Pitx2 begin expression in the dermomyotome/myotome. Abdominal somites become deformed and stunted as they elongate into the mutant body wall, but maintain normal MRF expression domains. Subsequent loss of abdominal muscles is therefore not due to defects in specification, determination, or commitment of the myogenic lineage. Microarray analysis was used to identify SSTF families whose expression levels change in E10.5 interlimb body wall biopsies. All Hox9-11 paralogs had lower RNA levels in mutants, whereas genes expressed selectively in the hypaxial dermomyotome/myotome and sclerotome had higher RNA levels in mutants. In situ hybridization analyses indicate that Hox gene expression was reduced in parts of the LPM and intermediate mesoderm of mutants. Chromatin occupancy studies conducted on E10.5 interlimb body wall biopsies showed that Pitx2 protein occupied chromatin sites containing conserved bicoid core motifs in the vicinity of Hox 9-11 and MRF genes. Taken together, the data indicate that Pitx2 protein in LPM cells acts, presumably in combination with other SSTFs, to repress gene expression, that are normally expressed in physically adjoining cell types. Pitx2 thereby prevents cells in the interlimb LPM from adopting the stable network kernels that define sclerotomal, dermomyotomal, or myotomal mesenchymal cell types. This mechanism may be viewed either as lineage restriction or specification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042228PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409154PMC
January 2013

BCL11B regulates epithelial proliferation and asymmetric development of the mouse mandibular incisor.

PLoS One 2012 22;7(5):e37670. Epub 2012 May 22.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.

Mouse incisors grow continuously throughout life with enamel deposition uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition is due to the presence of enamel-secreting ameloblasts exclusively within the labial epithelium of the incisor. We have previously shown that mice lacking the transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted ameloblast formation in the developing incisor. We now report that BCL11B is a key factor controlling epithelial proliferation and overall developmental asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the labial epithelium and development of the epithelial stem cell niche, which gives rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and development of stem cells and ameloblasts on the inner, or lingual, side of the incisor. This bidirectional action of BCL11B in the incisor epithelia appears responsible for the asymmetry of ameloblast localization in developing incisor. Underlying these spatio-specific functions of BCL11B in incisor development is the regulation of a large gene network comprised of genes encoding several members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic hedgehog. Our data integrate BCL11B into these pathways during incisor development and reveal the molecular mechanisms that underlie phenotypes of both Bcl11b(-/-) and Sprouty mutant mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037670PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358280PMC
December 2012

Detection of apoptosis by TUNEL assay.

Methods Mol Biol 2012 ;887:41-7

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. The method is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template. This chapter describes an assay for detection of apoptotic cells during mouse odontogenesis using a colorimetric TUNEL system.
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http://dx.doi.org/10.1007/978-1-61779-860-3_5DOI Listing
August 2012

Immunohistochemistry and detection of proliferating cells by BrdU.

Methods Mol Biol 2012 ;887:33-9

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.

Immunohistochemistry is a classic technique used for the localization of antigenic target molecules in -tissue. The method exploits the principle that the target antigen is recognized by specific antibody and is visualized using different detection systems. The subject of this chapter is simultaneous immunohistochemical detection of protein antigens and proliferation marker BrdU in the developing tooth.
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http://dx.doi.org/10.1007/978-1-61779-860-3_4DOI Listing
August 2012

Determination of gene expression patterns by in situ hybridization in sections.

Methods Mol Biol 2012 ;887:23-31

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.

In recent years, in situ RNA hybridization technique has found a widespread application in developmental biology. This method has frequently been used to determine gene expression patterns, which is a first step toward understanding of a gene function. Here, we provide a reliable and sensitive method for in situ RNA hybridization on frozen sections of mouse embryo using digoxigenin-labeled RNA probes. This technique can be used to study gene expression patterns at all stages of odontogenesis.
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http://dx.doi.org/10.1007/978-1-61779-860-3_3DOI Listing
August 2012

Determination of gene expression patterns by whole-mount in situ hybridization.

Methods Mol Biol 2012 ;887:15-22

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.

Whole-mount in situ hybridization (WISH) is a reliable and specific method to study three-dimensional patterns of gene expression. A labeled nucleic acid probe anneals to a complementary target sequence and is visualized and localized in the embryo. This chapter describes a sensitive method for WISH on mouse embryos using digoxigenin-labeled RNA probes. The technique can be used for the analysis of gene expression patterns during early stages of odontogenesis and in tooth explants.
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http://dx.doi.org/10.1007/978-1-61779-860-3_2DOI Listing
August 2012

Regulation of motility of myogenic cells in filling limb muscle anlagen by Pitx2.

PLoS One 2012 27;7(4):e35822. Epub 2012 Apr 27.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.

Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1(EGFP/+), which resulted from the loss of Pitx2. Very few genes showed changes in expression. Many small-fold, yet significant, changes were observed in genes encoding cytoskeletal and adhesion proteins which play a role in cell motility. Myogenic cells from genetically-tagged mice were cultured and subjected to live cell-tracking analysis using time-lapse imaging. Myogenic cells lacking Pitx2 were smaller, more symmetrical, and had more actin bundling. They also migrated about half of the total distance and velocity. Decreased motility may prevent myogenic cells from filling pre-patterned regions of the limb bud in a timely manner. Altered shape may prevent proper assembly of higher-order fibers within anlagen. Pitx2 therefore appears to regulate muscle anlagen development by appropriately balancing expression of cytoskeletal and adhesion molecules.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035822PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338778PMC
September 2012