DOCSLIB.ORG

Transcription Factor Wilms' Tumor 1 Regulates Developmental Rnas Through 3 ′ UTR Interaction

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Transcription Factor Wilms' Tumor 1 Regulates Developmental Rnas Through 3 ′ UTR Interaction Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION ’ Furthermore, WT1 is a key regulator of the balance be- Transcription factor Wilms tween mesenchymal and epithelial states in these tissues, tumor 1 regulates being required for the mesenchyme-to-epithelial transi- tion (MET), a key step in nephron formation, and the epi- developmental RNAs through thelial-to-mesenchyme transition (EMT) that produces ′ coronary vascular progenitors from the epicardium. WT1 3 UTR interaction also plays essential roles in adult tissue homeostasis. Ruthrothaselvi Bharathavikru,1,5 Hence, ubiquitous deletion of Wt1 in adult mice leads to Tatiana Dudnakova,2,5 Stuart Aitken,1 Joan Slight,1 acute kidney glomerulosclerosis, atrophy of the exocrine pancreas and spleen, and widespread reduction in bone Mara Artibani,1,4 Peter Hohenstein,1,3 2 1 and fat (Chau et al. 2011). A recent study showed that David Tollervey, and Nick Hastie WT1 is reactivated during tissue repair and adult tumori- genesis, where it is required in the mesenchymal compo- 1Medical Research Council Institute of Genetics and Molecular nentand vasculaturefortumor growth(Wagneret al. 2014). Medicine, University of Edinburgh, Western General Hospital, The molecular mechanisms by which WT1 fulfils these Edinburgh EH4 2XU, United Kingdom; 2Wellcome Trust Centre apparently diverse roles have been attributed to its tran- for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, scriptional function. Accordingly, there is substantial ev- United Kingdom; 3Roslin Institute, The University of Edinburgh, idence that WT1 binds genomic DNA and regulates Easter Bush, Midlothian EH25 9RG, United Kingdom transcription, acting as either an activator or a repressor Wilms’ tumor 1 (WT1) is essential for the development (Essafi et al. 2011; Toska and Roberts 2014). A growing and homeostasis of multiple mesodermal tissues. Despite number of physiological WT1 transcriptional targets evidence for post-transcriptional roles, no endogenous have been identified in development, homeostasis, and WT1 target RNAs exist. Using RNA immunopreci- disease (Motamedi et al. 2014; Dong et al. 2015; Kann et al. 2015). However, the evidence suggests that tran- pitation and UV cross-linking, we show that WT1 binds ′ scriptional regulation is not the only WT1 function. preferentially to 3 untranslated regions (UTRs) of devel- The two major essential isoforms (Barbaux et al. 1997; opmental targets. These target mRNAs are down-regulat- Hammes et al. 2001) conserved throughout vertebrate evo- ed upon WT1 depletion in cell culture and developing lution are created by an alternative splice that inserts three kidney mesenchyme. Wt1 deletion leads to rapid turnover amino acids (lysine–threonine–serine [KTS]) between the of specific mRNAs. WT1 regulates reporter gene expres- third and fourth zinc finger (Hastie 2001). The first sugges- sion through interaction with 3′ UTR-binding sites. Com- tion that WT1 may function post-transcriptionally came bining experimental and computational analyses, we from Larsson et al. (1995), who showed that the +KTS iso- propose that WT1 influences key developmental and dis- forms associatewith splicing factors. WT1 wasthen shown ease processes in part through regulating mRNA turnover. to integrate into active splice complexes by interacting with the splicing factor U2AF65 (Davies et al. 1998). More- Supplemental material is available for this article. over, WT1 binds both RNA and DNA with similar binding efficiencies, as demonstrated by structural and kinetic Received October 4, 2016; revised version accepted studies (Caricasole et al. 1996; Bardeesy and Pelletier February 15, 2017. 1998; Zhai et al. 2001). Structural modeling studies also identified an RNA recognition motif (RRM) in WT1 (Ken- nedy et al. 1996). Further support for post-transcriptional The different steps in gene expression, from transcription roles for WT1 was provided by Niksic et al. (2004), who through a series of post-transcriptional events, are closely showed that all isoforms shuttle between the nucleus and interconnected, and some multifunctional proteins regu- cytoplasm, where they are located on actively translating late several points in this pathway. One potential example ribosomes. In particular, the +KTS isoforms were shown is the Wilms’ tumor gene WT1. Mice lacking Wt1 die at to recruit transcripts containing a viral RNA sequence to mid-gestation through defective coronary vasculature, suf- polysomes, regulating their translation (Bor et al. 2006). fer from congenital diaphragmatic hernia, and have no kid- A considerable amount of data therefore support roles neys, gonads, spleen, or adrenals. All of these defects can be for WT1 from splicing through to translation. However, traced to a key role for WT1 in the development of tissues causal evidence for direct WT1 functions in specific steps derived from the intermediate and lateral plate mesoderm. and understanding of the mechanisms involved have been In humans, germline WT1 mutations lead to the epony- limited by the absence of characterized endogenous WT1 mous pediatric cancer, genitourinary anomalies, and, in RNA targets. To address this, we identified WT1-in- some cases, congenital diaphragmatic hernia and heart dis- teracting RNAs in mouse embryonic stem (ES) cells and ease (Chau and Hastie 2012). a mesonephric cell line by a combination of RNA immu- noprecipitation (RIP) and sequencing (RIP-seq) and a mod- [Keywords: WT1; FLASH; 3′ UTR; hybrids; RNA secondary structures; ification of the CLASH (cross-linking ligation and developmental pathways] sequencing of hybrids) UV cross-linking technique (Hel- 4Present address: Weatherall Institute of Molecular Medicine, University wak et al. 2013) termed FLASH (formaldehyde-assisted of Oxford, Oxford OX3 9DS, United Kingdom. 5These authors contributed equally to this work. Corresponding author: [email protected] Article published online ahead of print. Article and publication date are on- © 2017 Bharathavikru et al. This article, published in Genes & Develop- line at http://www.genesdev.org/cgi/doi/10.1101/gad.291500.116. Freely ment, is available under a Creative Commons License (Attribution 4.0 In- available online through the Genes & Development Open Access option. ternational), as described at http://creativecommons.org/licenses/by/4.0/. GENES & DEVELOPMENT 31:347–352 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org 347 Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Bharathavikru et al. cross-linking and sequencing of hybrids). The latter identi- RNAs (snoRNAs). Closer examination of the position of fies RNAs that interact directly with WT1 as well as RNA– significant clusters on protein-coding genes revealed high- RNA duplexes formed by these target RNAs. The interac- er recovery of sequences at the 5′ and 3′ ends of transcripts tion of intramolecular RNA hybrids is increased in the and the UTRs (Fig. 1B). A number of transcripts had nota- presence of WT1. This study provides strong evidence bly high read coverage, including those for Igfbp5, shown that the tumor suppressor protein WT1 regulates physio- as an example in Figure 1C. logically important RNAs and their stability through in- To confirm the sequence data, RIP reactions were per- teractions with 3′ untranslated regions (UTRs). formed followed by PCR across different regions of select- ed protein-coding transcripts (Fig. 1D; Supplemental Fig. S1C). For most of the selected targets, including Igfbp5, Results and Discussion the results were in good agreement with the RIP-seq anal- ysis, showing significant enrichment toward the 3′ UTR. WT1 binds to multiple categories of RNA In order to assess the functional relevance of the WT1-in- RIP (Bharathavikru and Dudnakova 2016) was performed teracting RNA, a gene ontology (GO) analysis was per- on ES cells and mesonephric M15 cells along with nega- formed. Several developmental pathways that are tive control RIP to identify RNAs that interact with regulated by WT1 were identified as significant GO cate- endogenous WT1. The specificity of the antibody was gories in this analysis, including coronary vasculature de- verified by immuno-pull-down and Western blotting velopment and the wnt signaling pathway (Supplemental (Supplemental Fig. S1A). Recovered RNAs were identified Fig. S1D,E; Supplemental Tables S1, S2). by RT–PCR and Illumina sequencing. Overall numbers of reads obtained are shown in Supplemental Figure S1B. ′ Reads were mapped to the mouse genome and analyzed UV cross-linking confirms WT1 enrichment over the 3 for different RNA categories (Fig. 1A). RIP data were pro- UTR of mRNAs cessed to identify regions in which the number of uniquely To identify specific protein–RNA interaction sites, we aligned reads passed statistical significance (false discov- adopted a UV cross-linking approach. Since ncRNAs ery rate [FDR] < 0.05; minimum of five reads), and overlap- were identified among WT1-interacting RNA (Fig. 1A), ping regions of aligned reads were clustered (minimum of we applied an experimental strategy that can identify five reads) using pyCRAC software (Webb et al. 2014). RNA–RNA interactions. The CLASH technique can iden- The RNA biotypes of these clusters were determined using tify both RNA–protein and RNA–RNA interactions but Ensembl annotations. Strikingly, WT1 targets were pre- requires the use of epitope-tagged
Load more

Recommended publications
  • Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases
    International Journal of Molecular Sciences Article Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases Yu-Lung Lin y, Yi-Wei Lin y, Jennifer Nhieu y, Xiaoyin Zhang and Li-Na Wei * Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] (Y.-L.L.); [email protected] (Y.-W.L.); [email protected] (J.N.); [email protected] (X.Z.) * Correspondence: [email protected]; Tel.: +1-612-6259402 Contributed equally. y Received: 29 April 2020; Accepted: 7 June 2020; Published: 9 June 2020 Abstract: Cellular retinoic acid-binding protein 1 (CRABP1) is highly expressed in motor neurons. Degenerated motor neuron-like MN1 cells are engineered by introducing SODG93A or AR-65Q to model degenerated amyotrophic lateral sclerosis (ALS) or spinal bulbar muscular atrophy neurons. Retinoic acid (RA)/sonic hedgehog (Shh)-induced embryonic stem cells differentiation into motor neurons are employed to study up-regulation of Crabp1 by Shh. In SODG93A or AR-65Q MN1 neurons, CRABP1 level is reduced, revealing a correlation of motor neuron degeneration with Crabp1 down-regulation. Up-regulation of Crabp1 by Shh is mediated by glioma-associated oncogene homolog 1 (Gli1) that binds the Gli target sequence in Crabp10s neuron-specific regulatory region upstream of minimal promoter. Gli1 binding triggers chromatin juxtaposition with minimal promoter, activating transcription. Motor neuron differentiation and Crabp1 up-regulation are both inhibited by blunting Shh with Gli inhibitor GANT61. Expression data mining of ALS and spinal muscular atrophy (SMA) motor neurons shows reduced CRABP1, coincided with reduction in Shh-Gli1 signaling components.
    [Show full text]
  • Role of Gas1 Down-Regulation in Mitogenic Stimulation of Quiescent NIH3T3 Cells by V-Src
    Oncogene (1998) 17, 1629 ± 1638 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Role of Gas1 down-regulation in mitogenic stimulation of quiescent NIH3T3 cells by v-Src Milena Grossi1, S Anna La Rocca1, Gloria Pierluigi1, Serena Vannucchi1, Elisabetta M Ruaro2, Claudio Schneider2 and Franco TatoÁ 1 1Istituto Pasteur-Fondazione Cenci-Bolognetti, Dipartimento di Biologia Cellulare e dello Sviluppo, UniversitaÁ di Roma `La Sapienza', Vie degli Apuli, 100185-Roma, Italy and 2Laboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie, 34012-Trieste, Italy Quiescent mammalian ®broblasts can be induced to re- interfering with the early serum response (Del Sal et enter the cell cycle by growth factors and oncoproteins. al., 1992). Gas1-induced growth arrest appears to be We studied the pathway(s) through which v-Src, the mediated by p53, with no requirement for its sequence- oncogenic tyrosine kinase encoded by the v-src oncogene speci®c transactivating function (Del Sal et al., 1995). of Rous sarcoma virus, forces serum-starved NIH3T3 Ample evidence documents that positive signalling cells to enter S-phase. To this purpose, we isolated and from growth factors and oncogene products very often characterized a polyclonal population of NIH3T3 cells involves the activation of a major biochemical pathway transformed by the MR31 retroviral vector, encoding known as the Ras-pathway (Kazlauskas, 1994; Mar- G418 resistance and the v-src temperature-sensitive allele shall, 1994; McCormick, 1994; Schlessinger, 1994). In from the mutant ts LA31 PR-A. NIH(MR31) cells order to activate this pathway, ultimately leading to S- displayed a temperature-conditional transformed pheno- phase entry, transduction of signals generated at the type and could be made quiescent by serum deprivation cell membrane is needed and performed by adaptor at the restrictive temperature.
    [Show full text]
  • Gastrointestinal Defects of the Gas1 Mutant Involve Dysregulated Hedgehog and Ret Signaling
    144 Research Article Gastrointestinal defects of the Gas1 mutant involve dysregulated Hedgehog and Ret signaling Sandrine Biau1,2,*, Shiying Jin1,* and Chen-Ming Fan1,` 1Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, Maryland 21218, USA 22iE Foundation, International Institute for Water and Environmental Engineering, Rue de la Science, 01 BP 594, Ouagadougou 01, Burkina Faso *These authors contributed equally to this work `Author for correspondence ([email protected]) Biology Open 2, 144–155 doi: 10.1242/bio.20123186 Received 24th September 2012 Accepted 2nd October 2012 Summary The gastrointestinal (GI) tract defines the digestive system and in the GI tract. Because Gas1 has been shown to inhibit Ret is composed of the stomach, intestine and colon. Among the signaling elicited by Glial cell line-derived neurotrophic factor major cell types lining radially along the GI tract are the (Gdnf), we explored whether Gas1 mutant enteric neurons epithelium, mucosa, smooth muscles and enteric neurons. displayed any alteration of Ret signaling levels. Indeed, isolated The Hedgehog (Hh) pathway has been implicated in directing mutant enteric progenitors not only showed increased levels of various aspects of the developing GI tract, notably the mucosa phospho-Ret and its downstream effectors, phospho-Akt and and smooth muscle growth, and enteric neuron patterning, phospho-Erk, but also displayed altered responses to Gdnf and while the Ret signaling pathway is selectively required for Shh. We therefore conclude that phenotypes observed in the enteric neuron migration, proliferation, and differentiation. The Gas1 mutant are due to a combination of reduced Hh signaling growth arrest specific gene 1 (Gas1) encodes a GPI-anchored and increased Ret signaling.
    [Show full text]
  • Multiple Roles of Sonic Hedgehog in the Developing Human Cortex Are Suggested by Its Widespread Distribution
    Brain Structure and Function https://doi.org/10.1007/s00429-018-1621-5 ORIGINAL ARTICLE Multiple roles of Sonic Hedgehog in the developing human cortex are suggested by its widespread distribution Fani Memi1,3 · Nada Zecevic1 · Nevena Radonjić2 Received: 29 September 2017 / Accepted: 25 January 2018 © The Author(s) 2018. This article is an open access publication Abstract Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurode- velopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10–40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences inSHH -pathway gene expression between humans and rodents. Keywords Cerebral cortex · Human fetal brain · Morphogen · SHH receptors Introduction generally well conserved in mammals, a number of changes and novelties have emerged during evolution that may Corticogenesis is a developmental process that requires underlie the biological basis for the higher cognitive and coordination of the signaling pathways providing mitogenic motor abilities that are specific to humans (Geschwind and signals and guiding the regulation of symmetric/asymmetric Rakic 2013; Silbereis et al.
    [Show full text]
  • Gas1 Is a Receptor for Sonic Hedgehog to Repel Enteric Axons
    Gas1 is a receptor for sonic hedgehog to repel PNAS PLUS enteric axons Shiying Jina, David C. Martinellia,1, Xiaobin Zhenga, Marc Tessier-Lavigneb, and Chen-Ming Fana,2 aDepartment of Embryology, Carnegie Institution of Washington, Baltimore, MD 21218; and bRockefeller University, New York, NY 10065 Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved November 26, 2014 (received for review September 26, 2014) The myenteric plexus of the enteric nervous system controls the neurons (1, 2). Germ-line and endoderm-specific conditional mouse movement of smooth muscles in the gastrointestinal system. They mutants for Shh and/or Ihh display reduced mesenchyme and extend their axons between two peripheral smooth muscle layers smooth muscle mass (6, 8). Shh and Ihh directly act on the gut to form a tubular meshwork arborizing the gut wall. How a tubular mesoderm, as inactivation of smoothened (Smo), the obligatory Hh axonal meshwork becomes established without invading centrally signaling component, in gut mesoderm causes the same defect (8). toward the gut epithelium has not been addressed. We provide Mice mutant for growth arrest specific gene 1 (Gas1), which encodes evidence here that sonic hedgehog (Shh) secreted from the gut an Hh surface binding receptor (9, 10), also share this defect (11). epithelium prevents central projections of enteric axons, thereby The role of Hh signaling in enteric neuron development is less forcing their peripheral tubular distribution. Exclusion of enteric defined. Shh mutant intestines contained more punctuated Tuj1 central projections by Shh requires its binding partner growth (an antibody recognizing neuronal βΙΙΙ-tubulin) staining signals er- arrest specific gene 1 (Gas1) and its signaling component smooth- roneously located near the base of the villi and were interpreted to ened (Smo) in enteric neurons.
    [Show full text]
  • A Genome-Wide Shrna Screen Identifies GAS1 As a Novel Melanoma Metastasis Suppressor Gene
    Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene Stephane Gobeil,1 Xiaochun Zhu,1 Charles J. Doillon,2 and Michael R. Green1,3 1Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA; 2Oncology and Molecular Endocrinology Research Center, CHUL’s Research Center, CHUQ, Laval University, Quebec City, Québec G1V 4G2, Canada Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples.
    [Show full text]
  • Solution Structure and Biophysical Characterization of the Multifaceted
    Rosti et al. BMC Biochemistry (2015) 16:8 DOI 10.1186/s12858-015-0037-6 RESEARCH ARTICLE Open Access Solution structure and biophysical characterization of the multifaceted signalling effector protein growth arrest specific-1 Katja Rosti1, Adrian Goldman2,3 and Tommi Kajander1* Abstract Background: The protein growth arrest specific-1 (GAS1) was discovered based on its ability to stop the cell cycle. During development it is involved in embryonic patterning, inhibits cell proliferation and mediates cell death, and has therefore been considered as a tumor suppressor. GAS1 is known to signal through two different cell membrane receptors: Rearranged during transformation (RET), and the sonic hedgehog receptor Patched-1. Sonic Hedgehog signalling is important in stem cell renewal and RET mediated signalling in neuronal survival. Disorders in both sonic hedgehog and RET signalling are connected to cancer progression. The neuroprotective effect of RET is controlled by glial cell-derived neurotrophic factor family ligands and glial cell-derived neurotrophic factor receptor alphas (GFRαs). Human Growth arrest specific-1 is a distant homolog of the GFRαs. Results: We have produced and purified recombinant human GAS1 protein, and confirmed that GAS1 is a monomer in solution by static light scattering and small angle X-ray scattering analysis. The low resolution solution structure reveals that GAS1 is more elongated and flexible than the GFRαs, and the homology modelling of the individual domains show that they differ from GFRαs by lacking the amino acids for neurotrophic factor binding. In addition, GAS1 hasanextendedloopintheN-terminaldomainthatisconserved in vertebrates after the divergence of fishes and amphibians. Conclusions: We conclude that GAS1 most likely differs from GFRαs functionally, based on comparative structural analysis, while it is able to bind the extracellular part of RET in a neurotrophic factor independent manner, although with low affinity in solution.
    [Show full text]
  • Towards an Understanding of Kidney Diseases Associated with WT1 Mutations Lihua Dong1, Stefan Pietsch1 and Christoph Englert1,2
    mini review http://www.kidney-international.org © 2015 International Society of Nephrology OPEN Towards an understanding of kidney diseases associated with WT1 mutations Lihua Dong1, Stefan Pietsch1 and Christoph Englert1,2 1Molecular Genetics, Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany and 2Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, Jena, Germany Mutations in Wilms’ tumor 1 (WT1)causeawidespectrum Kidney failure is a worldwide public health problem, with of renal manifestations, eventually leading to end-stage increasing incidence and prevalence, high costs, and poor kidney failure. Insufficient understanding of WT1’smolecular outcomes.1 Some of those renal manifestations are caused functions in kidney development has hampered efficient by Wilms’ tumor 1 (WT1) mutations, including Wilms’ therapeutic applications for WT1-associated diseases. tumor, the Denys-Drash syndrome (DDS), the Frasier Recently, the generation and characterization of mouse syndrome (FS), and the steroid-resistant nephrotic syndrome. models and application of multiple state-of-the-art To understand WT1 function in kidney development and approaches have significantly expanded our understanding homeostasis, multiple mouse models have been generated and of the molecular mechanisms of how WT1 mutations lead to characterized. Particularly, the generation of Wt1 conditional kidney failure. Here, we discuss the WT1 binding consensus knockout mice crossed to inducible and cell-specific Cre mice and illustrate the
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • Wilms' Tumour 1 (WT1) in Development, Homeostasis and Disease
    © 2017. Published by The Company of Biologists Ltd | Development (2017) 144, 2862-2872 doi:10.1242/dev.153163 PRIMER Wilms’ tumour 1 (WT1) in development, homeostasis and disease Nicholas D. Hastie* ABSTRACT particularly those involving mesenchyme-to-epithelial transitions The study of genes mutated in human disease often leads to new (MET) and the reverse process, epithelial-to-mesenchyme transition insights into biology as well as disease mechanisms. One such gene (EMT); (3) the cellular origins of mesenchymal progenitors for a is Wilms’ tumour 1 (WT1), which plays multiple roles in development, variety of tissue types, pinpointing the key role of the mesothelium; tissue homeostasis and disease. In this Primer, I summarise how this and (4) fundamental aspects of transcription and epigenetic multifaceted gene functions in various mammalian tissues and regulation. WT1 is also an example of how protein isoforms organs, including the kidney, gonads, heart and nervous system. differing by just a few amino acids may have profoundly different This is followed by a discussion of our current understanding of the functions. Here, I provide an overview of the WT1 gene, molecular mechanisms by which WT1 and its two major isoforms highlighting how it functions in the development and homeostasis regulate these processes at the transcriptional and post- of various organs and tissues, and how its mutation can lead to transcriptional levels. disease. KEY WORDS: WT1, Developmental disorders, Homeostasis, WT1 structure, evolution and isoforms Molecular mechanisms of disease The mammalian WT1 gene is ∼50 kb in length, encoding proteins from as many as ten exons. There are at least 36 potential Introduction mammalian WT1 isoforms, the diversity created through a The Wilms’ tumour 1 (WT1) gene was first identified in 1990 as a combination of alternative transcription start sites, translation start strong candidate predisposition gene for Wilms’ tumour(Calletal., sites, splicing and RNA editing (Fig.
    [Show full text]
  • Additional File 2
    Additional Table 2 All biological function categories and involved cytokines at 1, 7, 14 and 28 days after sciatic nerve injury List Pop Pop Category Term Count % P -value Genes total hits total Fold enrichment Bonferroni Benjamini False discovery rate Day 1 GOTERM_B GO:0021828~gonadotrophin- 3 0.0666370501999111 0.0000218920775503996 NRP2, NRP1, SEMA3A 31 3 10910 351.935483870967 0.004063691764008 0.004063691764008 0.0271097576750367 P_DIRECT releasing hormone neuronal migration to the hypothalamus GOTERM_B GO:1903375~facioacoustic 3 0.0666370501999111 0.0000218920775503996 NRP2, NRP1, SEMA3A 31 3 10910 351.935483870967 0.004063691764008 0.004063691764008 0.0271097576750367 P_DIRECT ganglion development GOTERM_B GO:0097490~sympathetic neuron 3 0.0666370501999111 0.0000437092389619988 NRP2, NRP1, SEMA3A 31 4 10910 263.951612903225 0.00809713627958896 0.00405679694050276 0.054120022480375 P_DIRECT projection extension GOTERM_B GO:0097491~sympathetic neuron 3 0.0666370501999111 0.0000437092389619988 NRP2, NRP1, SEMA3A 31 4 10910 263.951612903225 0.00809713627958896 0.00405679694050276 0.054120022480375 P_DIRECT projection guidance GOTERM_B GO:0048846~axon extension 3 0.0666370501999111 0.0000437092389619988 NRP2, NRP1, SEMA3A 31 4 10910 263.951612903225 0.00809713627958896 0.00405679694050276 0.054120022480375 P_DIRECT involved in axon guidance GOTERM_B GO:0036486~ventral trunk neural 3 0.0666370501999111 0.0000437092389619988 NRP2, NRP1, SEMA3A 31 4 10910 263.951612903225 0.00809713627958896 0.00405679694050276 0.054120022480375 P_DIRECT
    [Show full text]
  • Gas1 Is a Receptor for Sonic Hedgehog to Repel Enteric Axons
    Gas1 is a receptor for sonic hedgehog to repel PNAS PLUS enteric axons Shiying Jina, David C. Martinellia,1, Xiaobin Zhenga, Marc Tessier-Lavigneb, and Chen-Ming Fana,2 aDepartment of Embryology, Carnegie Institution of Washington, Baltimore, MD 21218; and bRockefeller University, New York, NY 10065 Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved November 26, 2014 (received for review September 26, 2014) The myenteric plexus of the enteric nervous system controls the neurons (1, 2). Germ-line and endoderm-specific conditional mouse movement of smooth muscles in the gastrointestinal system. They mutants for Shh and/or Ihh display reduced mesenchyme and extend their axons between two peripheral smooth muscle layers smooth muscle mass (6, 8). Shh and Ihh directly act on the gut to form a tubular meshwork arborizing the gut wall. How a tubular mesoderm, as inactivation of smoothened (Smo), the obligatory Hh axonal meshwork becomes established without invading centrally signaling component, in gut mesoderm causes the same defect (8). toward the gut epithelium has not been addressed. We provide Mice mutant for growth arrest specific gene 1 (Gas1), which encodes evidence here that sonic hedgehog (Shh) secreted from the gut an Hh surface binding receptor (9, 10), also share this defect (11). epithelium prevents central projections of enteric axons, thereby The role of Hh signaling in enteric neuron development is less forcing their peripheral tubular distribution. Exclusion of enteric defined. Shh mutant intestines contained more punctuated Tuj1 central projections by Shh requires its binding partner growth (an antibody recognizing neuronal βΙΙΙ-tubulin) staining signals er- arrest specific gene 1 (Gas1) and its signaling component smooth- roneously located near the base of the villi and were interpreted to ened (Smo) in enteric neurons.
    [Show full text]