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A Genomic Atlas of Human Adrenal and Gonad Development [Version 2; Referees: 4 Approved] Ignacio Del Valle1, Federica Buonocore1, Andrew J

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A Genomic Atlas of Human Adrenal and Gonad Development [Version 2; Referees: 4 Approved] Ignacio Del Valle1, Federica Buonocore1, Andrew J Wellcome Open Research 2017, 2:25 Last updated: 08 NOV 2017 RESEARCH ARTICLE A genomic atlas of human adrenal and gonad development [version 2; referees: 4 approved] Ignacio del Valle1, Federica Buonocore1, Andrew J. Duncan1, Lin Lin1, Martino Barenco2, Rahul Parnaik1, Sonia Shah3,4, Mike Hubank5, Dianne Gerrelli2, John C. Achermann 1 1Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, London, UK 2Developmental Biology and Cancer, UCL Great Ormond Street Institute of Child Health, London, UK 3Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia 4Institute of Cardiovascular Science, University College London, London, UK 5The Centre for Molecular Pathology, Royal Marsden Hospital, Sutton, UK v2 First published: 07 Apr 2017, 2:25 (doi: 10.12688/wellcomeopenres.11253.1) Open Peer Review Latest published: 23 Oct 2017, 2:25 (doi: 10.12688/wellcomeopenres.11253.2) Referee Status: Abstract Background: In humans, the adrenal glands and gonads undergo distinct biological events between 6-10 weeks post conception (wpc), such as testis Invited Referees determination, the onset of steroidogenesis and primordial germ cell 1 2 3 4 development. However, relatively little is currently known about the genetic mechanisms underlying these processes. We therefore aimed to generate a detailed genomic atlas of adrenal and gonad development across these critical version 2 report report stages of human embryonic and fetal development. published Methods: RNA was extracted from 53 tissue samples between 6-10 wpc 23 Oct 2017 (adrenal, testis, ovary and control). Affymetrix array analysis was performed and differential gene expression was analysed using Bioconductor. A version 1 mathematical model was constructed to investigate time-series changes across published report report report report 07 Apr 2017 the dataset. Pathway analysis was performed using ClueGo and cellular localisation of novel factors confirmed using immunohistochemistry. Results: Using this approach, we have identified novel components of adrenal 1 Olle Söder, Karolinska Institutet, Sweden development (e.g. ASB4, NPR3) and confirmed the role of SRY as the main human testis-determining gene. By mathematical modelling time-series data 2 Andrew H. Sinclair , Murdoch we have found new genes up-regulated with SOX9 in the testis (e.g. CITED1), Children's Research Institute, Australia which may represent components of the testis development pathway. We have Katie Ayers, Murdoch Children's shown that testicular steroidogenesis has a distinct onset at around 8 wpc and identified potential novel components in adrenal and testicular steroidogenesis Research Institute, Australia (e.g. MGARP, FOXO4, MAP3K15, GRAMD1B, RMND2), as well as testis 3 Rodolfo A. Rey , Hospital de Niños R. biomarkers (e.g. SCUBE1). We have also shown that the developing human Gutiérrez, Argentina ovary expresses distinct subsets of genes (e.g. OR10G9, OR4D5), but enrichment for established biological pathways is limited. Universidad de Buenos Aires, Argentina Conclusion: This genomic atlas is revealing important novel aspects of human Romina P. Grinspon , Hospital de development and new candidate genes for adrenal and reproductive disorders. Niños R. Gutiérrez, Argentina Page 1 of 42 Wellcome Open Research 2017, 2:25 Last updated: 08 NOV 2017 4 Jorma Toppari , University of Turku, Finland Juho-Antti Mäkelä , University of Turku, Finland Discuss this article Comments (0) Corresponding author: John C. Achermann ([email protected]) Author roles: del Valle I: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Buonocore F: Data Curation, Formal Analysis, Investigation, Resources, Validation, Visualization, Writing – Review & Editing; Duncan AJ: Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Review & Editing; Lin L: Conceptualization, Investigation, Methodology, Validation, Writing – Review & Editing; Barenco M: Data Curation, Formal Analysis, Methodology, Software, Writing – Original Draft Preparation; Parnaik R: Investigation, Methodology, Validation, Visualization, Writing – Review & Editing; Shah S: Data Curation, Formal Analysis, Validation; Hubank M: Conceptualization, Methodology, Resources, Supervision, Writing – Review & Editing; Gerrelli D: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Writing – Review & Editing; Achermann JC: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. How to cite this article: del Valle I, Buonocore F, Duncan AJ et al. A genomic atlas of human adrenal and gonad development [version 2; referees: 4 approved] Wellcome Open Research 2017, 2:25 (doi: 10.12688/wellcomeopenres.11253.2) Copyright: © 2017 del Valle I et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Grant information: JCA is a Wellcome Trust Senior Research Fellow in Clinical Science [grant no. 098513/Z/12/Z], with support from the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust, and University College London. The human embryonic and fetal material was provided by the Joint MRC/Wellcome Trust [grant no. 099175/Z/12/Z] Human Developmental Biology Resource (www.hdbr.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. First published: 07 Apr 2017, 2:25 (doi: 10.12688/wellcomeopenres.11253.1) Page 2 of 42 Wellcome Open Research 2017, 2:25 Last updated: 08 NOV 2017 Development of these structures is closely linked as they arise REVISED Amendments from Version 1 from a shared region of intermediate mesoderm in early embryo- We have undertaken further qRTPCR validation of adrenal gene genesis and subsequently have common biological processes, expression and include these data as a revised Figure 5b. We such as the ability to synthesise steroid hormones (see Figure 1 for also include higher power immunohistochemistry of FOXO4 an overview). and NR5A1 in the nuclei of fetal testis Leydig cells as a revised Figure 12d. We include a dataset of differential gene expression in the testis versus ovary (Dataset 7, OSF). We now include three The primordial adrenal gland arises as a distinct structure at Supplementary Figures of further validation data to show serial around 28 days post-conception (dpc) and undergoes rapid upregulation of novel genes in the testis with time (qRTPCR, growth during late embryonic and early fetal life (Ishimoto & Supplementary Figure 1); upregulation of novel steroidogenic Jaffe, 2011). The adrenal cortex develops into definitive and components with time (qRTPCR and immunohistochemistry, Supplementary Figure 2); higher powered adrenal expression fetal zones that can produce steroid hormones (cortisol, aldoster- data for GRAMD1B and RMDN2 in the adrenal gland as well one) and large amounts of adrenal androgens, such as dehydroe- as additional negative control data (immunohistochemistry, piandrosterone (DHEA) (Miller & Auchus, 2011; Xing et al., Supplementary Figure 3). Finally, we have extended the 2015). Chromaffin cells derived from the sympathetic nervous discussion to include a paragraph outlining several limitations of the study and possible future approaches. system migrate into the developing adrenal gland and later coa- lesce to form the adrenal medulla, which secretes adrenaline and See referee reports noradrenaline (Figure 1). It has been proposed that the entire hypothalamic-pituitary-adrenal (HPA) axis is functionally intact during early fetal life (Goto et al., 2006). Introduction The development of the adrenal gland and gonads (testes, ovaries) In contrast, the developing gonad remains “bipotential” until are two of the most important embryological events, but relatively around 41 dpc as it can differentiate into either a testis or ovary little is known about the exact mechanisms of these processes in (Figure 1) (Bashamboo et al., 2017; Svingen & Koopman, humans. 2013). Figure 1. Current model of human gonad and adrenal development between Carnegie Stage 13 (CS13) and Fetal Stage 3 (F3). The bipotential gonad and adrenal gland both arise from the adrenogonadal primordium around CS13. Sex determination in the bipotential gonad occurs between CS17 and CS23 when transient SRY expression promotes the upregulation of genes, including SOX9, in the developing testis. Sex differentiation starts with the onset of steroidogenesis around CS23, which results in development of the external genitalia and phallic growth (right panel). Ovary-specific genes are thought to suppress male pathway specification. The ovary also supports germ cell expansion and entry into meiosis. The fetal adrenal cortex develops into definitive and fetal zones, producing steroid hormones and adrenal androgens. Chromaffin cells derived from the sympathetic nervous system migrate into the developing adrenal gland, merging later to form the adrenal medulla. Page 3 of 42 Wellcome Open Research 2017, 2:25 Last updated: 08 NOV 2017 In
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