title: Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Sweta
authors.initials: S
authors.lastname: Roy-Carson
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Natukunda
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA.|Present Address: National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Hsien-Chao
authors.initials: HC
authors.lastname: Chou
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA.|Present address: North Central Regional Plant Introduction Station, 1305 State Ave, Ames, IA, 50014, USA.
authors.forename: Narinder
authors.initials: N
authors.lastname: Pal
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA.|Present address: Pioneer Hi-Bred International, Johnson, IA, 50131, USA.
authors.forename: Caitlin
authors.initials: C
authors.lastname: Farris
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Stephan Q
authors.initials: SQ
authors.lastname: Schneider
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA. jkuhlman@iastate.edu.|642 Science II, Iowa State University, Ames, IA, 50011, USA. jkuhlman@iastate.edu.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Kuhlman
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002465:Cell Movement; D060833:Cellular Microenvironment; D017615:Enteric Nervous System / Q000254:growth & development*; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D053263:Gene Regulatory Networks; D018398:Homeodomain Proteins / Q000235:genetics*; D055495:Neurogenesis; D017423:Sequence Analysis, RNA / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics*
chemical_list: D018398:Homeodomain Proteins; C109220:NBPhox protein; D014157:Transcription Factors
keywords: Enteric nervous system; Hirschsprungs; Neural crest; RNA-sequencing; Transcriptome; Zebrafish; phox2b