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Trans-mesenteric neural crest cells are the principal source of the colonic enteric nervous system

Abstract

Cell migration is fundamental to organogenesis. During development, the enteric neural crest cells (ENCCs) that give rise to the enteric nervous system (ENS) migrate and colonize the entire length of the gut, which undergoes substantial growth and morphological rearrangement. How ENCCs adapt to such changes during migration, however, is not fully understood. Using time-lapse imaging analyses of mouse ENCCs, we show that a population of ENCCs crosses from the midgut to the hindgut via the mesentery during a developmental time period in which these gut regions are transiently juxtaposed, and that such 'trans-mesenteric' ENCCs constitute a large part of the hindgut ENS. This migratory process requires GDNF signaling, and evidence suggests that impaired trans-mesenteric migration of ENCCs may underlie the pathogenesis of Hirschsprung disease (intestinal aganglionosis). The discovery of this trans-mesenteric ENCC population provides a basis for improving our understanding of ENS development and pathogenesis.

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Figure 1: Generation of Ednrb-hKikGR mice.
Figure 2: Developing ENCC chains colonize the hindgut by expanding the wavefront region.
Figure 3: Mesenteric ENCCs are the principal source for the ENS in the hindgut.
Figure 4: ENCCs shortcut from the midgut to hindgut via the mesentery.
Figure 5: Biological significance of tmENCCs in hindgut colonization by ENCCs.
Figure 6: Spatiotemporal distribution of tmENCCs is abnormal in a mouse model of Hirschsprung's disease.

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Acknowledgements

We are indebted to T. Attie-Bitach (Hôpital Necker-Enfants Malades) and J. Milbrandt (Washington University in St. Louis) for supplying human embryos and Ret-EGFP knock-in mice, respectively. We also thank H. Niwa, members of the Enomoto laboratory and the Laboratory for Animal Resources and Genetic Engineering for their excellent technical assistance. We are also grateful to D. Sipp for editing this manuscript. This work was supported by RIKEN and by a Grant-in-Aid for Scientific Research (B, 21390122, H.E.) and Scientific Research on Innovative Areas “Cellular and Molecular Basis for Neuro-Vascular Wiring” (22122005) from the Ministry of Education, Science, Sports and Culture, Japan.

Author information

Authors and Affiliations

Authors

Contributions

H.E. conceived the project and generated Ednrb-hKikGR mice. Techniques for gut organ culture and imaging of ENCCs were instructed by H.M.Y. C.N., T.M. and T.U. performed time-lapse imaging analysis for the gut preparations and conducted quantitative and morphometric analyses. T.N. provided genetically engineered mice. H.E., C.N., Y.Y. and T.U. performed immunohistochemical analyses. H.E. wrote the manuscript with D.F.N. and H.M.Y.

Corresponding author

Correspondence to Hideki Enomoto.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 1650 kb)

Supplementary Video 1

ENCCs migrate caudally by forming chains in mouse hindgut. Time-lapse observation of ENCCs in the hindgut from an E12.5 Ret+/EGFP embryo. (MOV 3056 kb)

Supplementary Video 2

Time-lapse observation of individual ENCCs by photoconversion. Time-lapse observation of E13.5 gut explant in which two distinct regions near the wavefront of ENCC chain were photoconverted. Multipolar cells, yellow arrows; monopolar cells, white arrows. (MOV 4479 kb)

Supplementary Video 3

ENCCs derived from the wavefront region migrate caudally at variable distances. Portions of the wavefront region of E12.5 hindgut ENCCs were photoconverted and subjected to time-lapse observation. Note that almost all ENCCs labeled with red chromophores moved in the caudal direction. (MOV 3799 kb)

Supplementary Video 4

Invasion of the hindgut by tmENCCs precedes that by cfENCCs. White dotted lines in the first image delineate the margin of the hindgut (E11.5). Red cells represent photoconverted ENCCs originating from the mesenteric border (tmENCCs) of the hindgut. Note that tmENCCs invade the hindgut first, followed by ENCCs from the cecum (circumflex ENCCs, or cfENCCs). The wavefront of cfENCCs is indicated by yellow arrows. (MOV 4624 kb)

Supplementary Video 5

Wholemount immunohistochemical detection of ENCCs at the period of gut hairpin-bending. Confocal imaging of Ret+/EGFP gut (E11.0) stained with anti-GFP and anti-PECAM antibodies. The sample was cleared with benzyl alcohol/benzyl benzoate solution to examine the entire thickness of the gut. Note substantial numbers of ENCCs detected along the mesentery. (MOV 4076 kb)

Supplementary Video 6

ENCCs cross the mesentery during the period of loop-like gut bending. Time-lapse observation of ENCCs in an E10.7 Ednrb-hKikGR gut. In the first image, margin of the gut mesenchyme is indicated by dotted lines. This video was taken from the mesentery side (left side of the object) because observation from this angle facilitates visualization of ENCCs in the mesentery due to the relatively flat configuration of the mesenchyme between the gut and the mesentery (see Fig. 3c). (MOV 3721 kb)

Supplementary Video 7

ENCCs crossing the mesentery contribute to the most advanced region of ENCC chains in the hindgut. Intermittent photoconversion (every 24 min) was performed on the mesentery of E10.7 Ednrb-hKikGR gut, and ENCC migration was examined. Blue rectangle in the first image depicts the area subjected to photoconversion. Margin of the gut is indicated by dotted lines in the first image. Note that photoconverted cells preceded the wavefront of cfENCCs and were located in the most advanced region of ENCC chains. (MOV 3966 kb)

Supplementary Video 8

ENCCs initiate mesentery crossing in a delayed fashion in Ret9/EGFP gut. Time-lapse observation of ENCCs in an E11.7 gut explant from a Ret9/EGFP embryo. Dotted lines in the first image delineate margins of the gut mesenchyme. Note that a substantial numbers of ENCCs cross the mesentery and colonize the hindgut before the cfENCCs pass through the cecum (the wavefront of cfENCCs indicated by white arrows). (MOV 5237 kb)

Supplementary Video 9

Reduced motility of ENCCs in the mesentery of a Ret9/EGFP embryo. ENCCs found in the mesentery of E12.5 Ret9/EGFP embryos (arrows) display reduced cell body movement and are unable to invade the hindgut. (MOV 2301 kb)

Supplementary Video 10

ENCCs in the mesentery of wild type embryos. ENCCs are occasionally found in the mesentery of E11.5 Ret+/EGFP embryos (blue arrows). Note that these ENCCs are highly motile, enter the hindgut and contribute to the ENS. (MOV 2800 kb)

Supplementary Video 11

Trans-mesenteric migration of ENCCs detected by Gfrα1-EGFP reporter. ENCCs of Gfrα1Gfrα1-EGFP/+; CAGGCre-ER embryos became GFP-positive after tamoxifen-induced Cre activation. Note that these GFP-expressing ENCCs retain the wild type Gfrα1 allele. Arrows depict some ENCCs undergoing trans-mesenteric migration. (MOV 2455 kb)

Supplementary Video 12

Impaired trans-mesenteric migration of ENCCs lacking GFRα1. Time-lapse observation of ENCCs of Gfrα1Gfrα1-EGFP/−; CAGGCre-ER embryos after Cre activation. By Cre-mediated excision of floxed Gfrα1, ENCCs become Gfrα1-null and GFP-positive. These ENCCs were unable to exit from the midgut mesenchyme and never entered the mesentery, although many of these cells still retained motility (arrows). (MOV 3575 kb)

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Nishiyama, C., Uesaka, T., Manabe, T. et al. Trans-mesenteric neural crest cells are the principal source of the colonic enteric nervous system. Nat Neurosci 15, 1211–1218 (2012). https://doi.org/10.1038/nn.3184

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