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Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ

View ORCID ProfileAaron A. May-Zhang, View ORCID ProfileEric Tycksen, View ORCID ProfileAustin N. Southard-Smith, Karen K. Deal, View ORCID ProfileJoseph T. Benthal, Dennis P. Buehler, View ORCID ProfileMike Adam, View ORCID ProfileAlan J. Simmons, View ORCID ProfileJames R. Monaghan, Brittany K. Matlock, David K. Flaherty, View ORCID ProfileS. Steven Potter, View ORCID ProfileKen S. Lau, View ORCID ProfileE. Michelle Southard-Smith
doi: https://doi.org/10.1101/2020.07.03.187211
Aaron A. May-Zhang
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
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Eric Tycksen
2Genome Technology Access Center, McDonnell Genome Institute, St. Louis, MO, USA
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Austin N. Southard-Smith
3Epithelial Biology Center and the Department of Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
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Karen K. Deal
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
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Joseph T. Benthal
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
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Dennis P. Buehler
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
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Mike Adam
4University of Cincinnati Children’s Medical Hospital Research Center, Cincinnati, OH, USA
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Alan J. Simmons
3Epithelial Biology Center and the Department of Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
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James R. Monaghan
5Northeastern University, Department of Biology, Boston, MA, USA
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Brittany K. Matlock
6Office of Shared Resources, Vanderbilt University School of Medicine, Nashville, TN, USA
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David K. Flaherty
6Office of Shared Resources, Vanderbilt University School of Medicine, Nashville, TN, USA
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S. Steven Potter
4University of Cincinnati Children’s Medical Hospital Research Center, Cincinnati, OH, USA
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Ken S. Lau
3Epithelial Biology Center and the Department of Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
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E. Michelle Southard-Smith
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
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Abstract

BACKGROUND & AIMS The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions.

METHODS Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction (HCR).

RESULTS Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of one or more marker genes.

CONCLUSIONS Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human GI studies.

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Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Grant support: The laser-capture microdissection instrument was funded by NIH grants P30-CA068485-14 and U24-DK059637-13 to the Tissue Pathology Shared Resource at VUMC. Flow sorting was performed in the VUMC Flow Core, supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (P30 DK058404). RNA sequencing and computing support was provided by the Genome Technology Access Center at Washington University, supported in part by NCI Award P30 CA91842 to the Siteman Cancer Center and ICTS/CTSA UL1TR002345 from the NCRR. This work was funded by NIH awards OT2-OD23850 to EMS2, RO1-DK103831 to KSL, OT2-OD024909 to JRM, with support for AAM on T32-DK007673.

  • Disclosures: Authors do not have any relevant conflicts of interest.

  • Abbreviations

    CALB1
    Calbindin 1
    CALB2
    Calbindin 2
    CCKAR
    CCK Receptor Type A
    CDH9
    Cadherin 2
    CHAT
    Choline acetyltransferase
    EN
    Enteric neuron
    ENS
    Enteric nervous system
    FACS
    fluorescence-activated cell sorting
    FISH
    fluorescence in situ hybridization
    GO
    Gene Ontology
    HCR
    Hybridization chain reaction
    IPAN
    Intrinsic Primary Afferent Neuron
    LCM
    Laser-Capture Microdissection
    NEFL
    Neurofilament
    NMU
    Neuromedin U
    NOS1
    Nitric oxide synthase
    NXPH2
    Neurexophilin 2
    PCA
    Principal Components Analysis
    SNAP25
    Synaptosome Associated Protein 25
    snRNA-Seq
    single-nucleus RNA-Sequencing
    Sox10
    SRY (sex determining region Y)-box transcription factor 10
    SST
    Somatostatin
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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    Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ
    Aaron A. May-Zhang, Eric Tycksen, Austin N. Southard-Smith, Karen K. Deal, Joseph T. Benthal, Dennis P. Buehler, Mike Adam, Alan J. Simmons, James R. Monaghan, Brittany K. Matlock, David K. Flaherty, S. Steven Potter, Ken S. Lau, E. Michelle Southard-Smith
    bioRxiv 2020.07.03.187211; doi: https://doi.org/10.1101/2020.07.03.187211
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    Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ
    Aaron A. May-Zhang, Eric Tycksen, Austin N. Southard-Smith, Karen K. Deal, Joseph T. Benthal, Dennis P. Buehler, Mike Adam, Alan J. Simmons, James R. Monaghan, Brittany K. Matlock, David K. Flaherty, S. Steven Potter, Ken S. Lau, E. Michelle Southard-Smith
    bioRxiv 2020.07.03.187211; doi: https://doi.org/10.1101/2020.07.03.187211

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