Table 1.
Primary antibodies for immunohistochemistry studies.
Table 2.
Secondary antibodies for immunohistochemistry studies.
Fig 1.
Isolation and culture of YFP+ enteric neural crest cells (ENCC) from postnatal Wnt1-cre;R26RYFP/YFP mouse gut.
A. Flow cytometry profile showing the YFP+ cell population (green) separate from the enteric cell population (pink). B. Selected YFP+ cells form YFP+ neurospheres after 7 days in culture. C. Immunohistochemical analysis confirms that all cells within the neurospheres express YFP (green; Civ, Div). Neurospheres contain cells immunopositive for the neuronal marker TuJ1 (red; C, Ci) and the glial marker GFAP (cyan; C, Cii). DAPI labels nuclei in blue (Ciii). D. Neurospheres (outlined by dotted lines) also contain cells (asterisk and arrowheads) that express Sox10 (red; D, Di and Diii, arrowheads) but are negative for GFAP (cyan; D, Dii) i.e. presumptive ENSSCs. Inset in D shows a cross section through a Sox10+/GFAP- cell (red only; asterisk; presumptive ENSSC) adjacent to a Sox10+/GFAP+ cell (red and cyan; presumptive glial cell). Scale bar in B-D = 20μm.
Fig 2.
ENCCs from transplanted murine neurospheres show appropriate colonisation, localisation and formation of ENS-like networks in recipient wild-type gut.
A. Wholemount gut preparation showing YFP+ transplanted cells (green) projecting along endogenous TuJ1+ (red) ENS nerve fibres. Arrow indicates cell bodies at the presumptive site of transplantation and arrowhead indicates distal extent of projections of transplanted cells. YFP+ transplanted cells also expressing the neuronal marker TuJ1 are seen as yellow. Inset shows a high power image taken from boxed region in S1 Fig panel A revealing interconnections and network formation between YFP+ cell bodies. B. Confocal 3D reconstruction showing YFP+ transplanted cells (green) located at the site of transplantation on the serosal surface (S) and within the myenteric plexus (MYP; arrowheads) between the inner circular (CM) and outer longitudinal (LM) muscle layers. C. YFP+ transplanted cells (green) co-locate with the synaptic marker synaptophysin (red; arrowheads). Scale bar in A = 100μm; C = 25μm. Inset in C shows individual channels.
Fig 3.
Transplanted YFP+ cells show functional integration within host gut.
A. Schematic of experimental protocol demonstrating electrical point stimulation of host enteric nerve fiber at a site distant from YFP+ transplanted cells. B,C. Representative images of YFP+ cells before (B) and after (C) Fluo4-AM loading. Arrows indicate transplanted neurons (TP cell) from which Ca2+ responses are plotted in (D). D. Representative traces showing Ca2+ responses recorded as F/F0 from TP cell in control conditions (solid lines) and after addition of TTX (dotted lines). E. Summary data demonstrating abolition of Ca2+ responses in the presence of TTX (43 cells, n = 6). Also see S1 Movie.
Fig 4.
Transplanted YFP+ mouse ENCCs proliferate and generate enteric neurons and glia but do not spread beyond transplanted gut.
A-C. Z-projections in which transplanted YFP+ cells co-express ENS markers (yellow; arrowheads) including the pan-neuronal marker TuJ1 (A), the inhibitory neuronal marker nNOS (B), and the glial marker S100 (C). Transplanted cells co-expressing ENS markers also demonstrate BrdU incorporation (cyan; arrowheads; A-C and insets). DAPI labels nuclei in blue (A-C and insets). Insets show individual channels. D. Wnt1-cre;R26RYFP/YFP expressing transplanted cells (YFP+) were identified within R26RYFP/YFP recipient mice by the presence of the cre transgene on PCR. Representative cre-PCR to identify cre expressing cells within the major organs from a transplanted mouse. Brain (B), lungs (Lu), heart (H), liver (Li), spleen (S), kidneys (K), adrenal glands (A) and gut mesentery (M) were all negative for cre. Control tissues: Wnt1-cre;R26RYFP/YFP gut tissue (C1), transplanted gut (C2), YFP+ neurospheres (C3) and Wnt1-cre;R26RYFP/YFP ear biopsy (C4) are cre+. Scale bar in A-C = 20μm.
Fig 5.
YFP+ ENCC colonise aganglionic Ednrbtm1Ywa gut in vivo.
A. Neurons in an Ednrbtm1Ywa colon immunolabelled with TuJ1 progressing from proximal ganglionated gut (Ai), through the partially ganglionated transition zone (Aii) to the distal aganglionic gut (Aiii). B. Ednrbtm1Ywa gut 3 days after transplantation with YFP+ ENCCs (green) which could be identified in distal colon. Boxed area is area enlarged in Bi and asterisk denoted presumptive site of transplantation in this region. Bi. Interconnections are visible between transplanted cells that have spread from the site of transplantation. Bii. Z-projections in which endogenous neurons express the neuronal marker Tuj1 (red; arrow) and YFP+ transplanted cells (green) also coexpress Tuj1 (yellow; arrowhead). Projections from YFP+;Tuj1+ transplanted cells closely associate with projections from endogenous Tuj1+ neurons present in the transition zone (insets in Bii; arrowheads and arrows respectively). Scale bar in Bi = 100μm; Bii = 50μm. DAPi is shown in blue.