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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00857

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Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures

Nadege Bondurand^1,*,, Dipa Natarajan^1,, Nikhil Thapar^1,, Chris Atkins² and Vassilis Pachnis^1,

¹ Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Division of Immunoregulation, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (77K): [in a new window]   Fig. 1. Formation of NLBs in cultures of dissociated foetal and postnatal gut. Whole gut from E11.5 mouse embryos (A-E) or the outer muscle layers from P2-14 gut (F-J) were dissociated and plated in NCSC medium. 2-3 days later characteristic colonies appeared in both foetal and postnatal gut cultures (A and F, respectively) which were composed of flat cells with small processes. Higher magnification of individual cells is shown in the insets. Over the next 3-4 days colonies grew in size (B,G) and eventually formed NLBs which detached and floated in the medium by day 10 (C,H). Immunostaining with antibodies against TuJ1 and GFAP indicated that NLBs from both foetal (D,E) and postnatal (I,J) cultures contained large numbers of neurons and glia. E and J are enlargements of parts of the NLBs shown in D and I, respectively. Note that postnatal NLBs were reproducibly smaller relative to their foetal counterparts.

View larger version (124K): [in a new window]   Fig. 2. NLBs do not form in gut cultures from Retk- homozygous embryos. Cultures were established from individual guts dissected from E11.5 embryos from Ret+/k- heterozygous parents. Neuron- and glia-containing NLBs formed readily in cultures of wild-type (A,B) and Ret+/k- (not shown) embryos but not in cultures from Retk- homozygotes (C,D).

View larger version (60K): [in a new window]   Fig. 3. Retrovirally transduced NLBs yield undifferentiated cells which proliferate and differentiate into neurons and glia. (A,F) 24 hours after infection, GFP-expressing cells were isolated by fluorescence activated cell sorting and replated. (B,G) Bright-field images of GFP+ cells 12 hours after plating from foetal and postnatal NLBs, respectively. (C,H) Fluorescent images of the same cells immunostained for TuJ1 and GFAP. Note that the only fluorescent signal detected is that of nuclear localised GFP. (D,I) Bright-field images of GFP+ cells isolated from foetal and postnatal NLBs and cultured for 10 days. (E,J) The corresponding fluorescent images after immunostaining for TuJ1 and GFAP. Note the presence of large numbers of neurons and glia at this stage.

View larger version (48K): [in a new window]   Fig. 4. Foetal and postnatal NLBs contain bipotential progenitors of enteric neurons and glia. (A-C,G-I) Typical colonies generated from single GFP+ cells isolated from foetal (A-C) and postnatal (G-I) NLBs on day 1 (A,G), day 5 (B,H) and day 10 (C,I). (D-F,J-L) Equivalent colonies immunostained with TO-PRO-3 (blue; to reveal the cell nucleus), TuJ1 (red; to identify neurons) and GFAP (green; to identify glia). The cell shown in D and J (day 1) had weak GFP signal but lacked the characteristic TuJ1 and GFAP staining. To avoid confusion with the GFP-specific signal, E and K (day 5) and F and L (day 10) show sectors of colonies in which the retroviral transgene had been extinguished. TuJ1+ cells were detected in day 5 and day 10 colonies but GFAP+ cells were detected only in day 10 colonies. Arrows in F and L point to two neighbouring cells that express TuJ1 or GFAP. (M) Cells in foetal colonies have higher proliferative capacity. Five-dayold colonies from foetal and postnatal EPCs were stained with the mitotic marker H3p. The fraction of H3p+ cells in foetal colonies was higher relative to that of postnatal colonies.

View larger version (119K): [in a new window]   Fig. 5. Cell commitment and differentiation in colonies from foetal EPCs. Twenty-five percent of GFP+ cells isolated from foetal NLBs expressed Sox10 (A1,A2) but were negative for MASH1 (B1,B2) or RET (C1,C2). On day 3, Sox10 expression was detected only in colony-associated cells (D1,D2), suggesting strongly that only the GFP+/SOX10+ EPCs have clonogenic potential. At this stage, only a subset of cells expressed Mash1 (E1,E2) or Ret (F1,F2) but we detected no TuJ1+ cells (G1,G2). On day 10, Sox10 was expressed in a subset of cells in the colonies (H2,I2). At this stage, all colonies contained PGP9.5+ cells (H1) which were negative for SOX10 (H3; arrow points to a PGP9.5+/SOX10-cell and arrowhead points to a neighbouring PGP9.5-/SOX10+ cell). Also, at this stage 87% of the colonies contained GFAP+ cells (I1) which maintained expression of Sox10 (I3; arrows point to GFAP+/SOX10+ cells). Cells expressing high levels of PGP9.5 were negative for MASH1 (J1-J3). Also, GFAP and MASH1 are present on mutually exclusive cells (K1-K3). Finally, the highest levels of RET were detected in TuJ1+ cells (L1-L3; arrows in L3) but GFAP+ cells did not express Ret (M1-M3).

View larger version (112K): [in a new window]   Fig. 6. Cell commitment and differentiation in colonies from postnatal EPCs. 25% of GFP+ cells isolated from postnatal NLBs expressed Sox10 (A1,A2) but were negative for MASH1 (B1,B2) or RET (C1,C2). As in the case of foetal EPCs, on day 3 Sox10 expression was detected only in colony-associated cells (D1,D2). At this stage, a subset of cells expressed Mash1 (E1,E2) or Ret (F1,F2) but we detected no TuJ1+ cells (G1,G2). On day 10, Sox10 was expressed in a subset of cells (H2,I2) and all colonies contained PGP9.5+ cells (H1) which were negative for SOX10 (H3; arrow points to a PGP9.5+/SOX10-cell and arrowhead points to a PGP9.5-/SOX10+ cell). Also, at this stage, 89% of the colonies contained GFAP+ cells (I1) which maintained expression of Sox10 (I3; arrows point to GFAP+/SOX10+ cells). Cells expressing high levels of PGP9.5 were negative for MASH1 (J1-J3). Also, GFAP and MASH1 were present on mutually exclusive cells (K1-K3; arrow points to a GFAP+/MASH1-cell). Finally, as for foetal EPC colonies, the highest levels of RET were detected in TuJ1+ cells (L1-L3; arrows in L3) while Ret was not expressed in GFAP+ cells (M1-M3).

View larger version (111K): [in a new window]   Fig. 7. EPCs differentiate into ENS-appropriate neuronal subtypes. 15-day-old colonies established from foetal (A-C) and postnatal (D-F) EPCs were fixed and immunostained with antibodies for GFP (green; all panels) and TH (red; A,D), VIP (red; B,E) and NPY (red; C,F). Subsets of neurons expressing these markers (arrows in all panels) were generated with similar efficiencies in both foetal and postnatal colonies. Arrowhead in A points to a neurite-bearing TH+ cell.

View larger version (41K): [in a new window]   Fig. 8. Effects of GDNF on the progeny of foetal and postnatal EPCs. (A) Day-5 clonal cultures of EPCs fixed and stained for H3p or TuJ1. In the upper part of the graph, bars represent the percentage increase in the number of H3p+ cells in the presence of GDNF relative to those present in control (absence of GDNF) culture conditions. In the lower part of the graph, bars represent the percentage decrease in the number of TuJ1+ cells in the presence of GDNF relative to those present in control cultures. (B-E) GDNF also promotes the morphological differentiation of EPC progeny maintained in defined medium. Foetal (B,C) and postnatal (D,E) EPCs were cultured for 4 days in complete NCSC medium. Subsequently, they were cultured for a further 4 days in defined medium (NCSC medium lacking CEE, FGF and EGF) in the absence (B,D) or presence (C,E) of GDNF. GDNF promotes axonal growth and branching of neurons in both foetal and postnatal EPC colonies.

View larger version (65K): [in a new window]   Fig. 9. EPCs colonise foetal gut and have the potential to generate neurons and glia. (A,B) DiI-labelled pieces of foetal and postnatal NLBs were grafted into the wall of gut dissected from Retk-/k- E11.5 mouse embryos and maintained in organotypic culture. A large number of neurons and glial cells were detected in the grafted gut (B) but were absent from control mutant gut (A). Arrow in B points to the site of NLB grafting in the stomach (s). Processing of guts for immunostaining resulted in loss of DiI fluorescence. (C-I) 20-30 foetal EPCs were grafted into the wall of wild-type (D-F) or Retk/k- (H-I) guts which were subsequently cultured for 14 days. C and G are non-grafted control guts. At the end of the culture period, explant sections were immunostained for GFP (C-I) and TuJ1 (C-E,G-I) or GFAP (F). Large numbers of GFP+ cells were detected in the grafted guts (arrows in D and H) but were absent from control guts (C,G). GFP+ cells could be detected at relatively large distances from the site of injection suggesting a considerable migratory ability of grafted cells. Neuronal progeny of EPCs (GFP+/TuJ1+ cells) were detected in grafted wild-type and RET-deficient guts (arrows in E and I). However, GFP+/GFAP+ cells were detected only in wild-type guts (arrows in F). Note the virtual absence of endogenous TuJ1 signal from sections of Retk-/k- guts (G). (J-N) Postnatal EPCs were also introduced into wild-type (J-L) and Retk- homozygous (M-N) guts. Analysis of sections from grafted guts at the end of the culture period indicated that GFP+ cells increased in numbers and colonised relatively large segments of the grafted guts (arrows in J and M point to GFP+ cells). GFP+/TuJ1+ cells were detected in wild-type and RET-deficient gut (arrows in K and N). However, GFP+/GFAP+ cells were only detected in wild-type guts (arrow in L). Arrowheads in D, H and J show the site of EPC grafting. s, stomach; i, intestine.

View larger version (54K): [in a new window]   Fig. 10. Formation of NLBs from miRet51 homozygous mice. Postnatal gut was isolated from individual P2-8 mice born to miRet51 heterozygous parents, dissociated and cultured as described. (A,B) NLBs containing large numbers of TuJ1+ and GFAP+ cells formed in gut cultures from miRet51 animals. (C) In addition, retroviral transduction of NLBs resulted in the efficient isolation of EPCs which generated neuron- and glia-containing colonies (a sector of which is shown here).

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