Genetics, Development and Cell Biology Genetics, Development and Cell Biology Publications 2017 Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment Sweta Roy-Carson Iowa State University, [email protected] Kevin Natukunda Iowa State University, [email protected] Hsien-chao Chou Iowa State University Narinder Pal Iowa State University Caitlin Farris Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/gdcb_las_pubs See next page for additional authors Part of the Cell and Developmental Biology Commons, and the Molecular Genetics Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ gdcb_las_pubs/202. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Genetics, Development and Cell Biology at Iowa State University Digital Repository. It has been accepted for inclusion in Genetics, Development and Cell Biology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment Abstract Background: Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The og al of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed. Results: Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP)w37]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP- negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development. Conclusion: We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders. Keywords Enteric nervous system, Neural crest, Transcriptome, RNA-sequencing, Zebrafish, Hirschsprungs, phox2b Disciplines Cell and Developmental Biology | Genetics and Genomics | Molecular Genetics Comments This article is published as Roy-Carson, Sweta, Kevin Natukunda, Hsien-chao Chou, Narinder Pal, Caitlin Farris, Stephan Q. Schneider, and Julie A. Kuhlman. "Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment." BMC genomics 18 (2017): 290. doi: 10.1186/ s12864-017-3653-2. This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/gdcb_las_pubs/202 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. Authors Sweta Roy-Carson, Kevin Natukunda, Hsien-chao Chou, Narinder Pal, Caitlin Farris, Stephan Q. Schneider, and Julie A. Kuhlman This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/gdcb_las_pubs/202 Roy-Carson et al. BMC Genomics (2017) 18:290 DOI 10.1186/s12864-017-3653-2 RESEARCH ARTICLE Open Access Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment Sweta Roy-Carson1, Kevin Natukunda1, Hsien-chao Chou1,2, Narinder Pal1,3, Caitlin Farris1,4, Stephan Q. Schneider1 and Julie A. Kuhlman1,5* Abstract Background: Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed. Results: Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP)w37]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development. Conclusion: We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders. Keywords: Enteric nervous system, Neural crest, Transcriptome, RNA-sequencing, Zebrafish, Hirschsprungs, phox2b * Correspondence: [email protected] 1Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA 5642 Science II, Iowa State University, Ames, IA 50011, USA Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roy-Carson et al. BMC Genomics (2017) 18:290 Page 2 of 24 Background Adhesion molecule (L1CAM), Neurotrophin 3 (NTF3), The enteric nervous system (ENS), the largest division of Neuregulin 1 (NRG1), Neuregulin 3 (NRG3), Neurturin the peripheral nervous system (PNS), is composed of a (NRTN), Neurotrophic Receptor Tyrosine Kinase 3 network of neurons and glia that innervate the gastro- (NTRK3), PHOX2B, Prokineticin 2 (PROK2), Prokineticin intestinal tract [1]. The ENS functions within the gastro- Receptor 1 (PROKR1), Prokineticin Receptor 2 (PORKR2), intestinal tract to control motility for mixing and Persephin (PSPN), Semaphorin 3A (SEMA3A), Sema- moving food, absorption of nutrients, epithelial secre- phorin 3D (SEMA3D), SRY-Box 10 (SOX10), Transcription tions, and blood circulation [2]. It arises from a popula- Factor 4 (TCF4),