Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury

Abstract

The enteric nervous system (ENS) in mammals forms from neural crest cells during embryogenesis and early postnatal life. Nevertheless, multipotent progenitors of the ENS can be identified in the adult intestine using clonal cultures and in vivo transplantation assays. The identity of these neurogenic precursors in the adult gut and their relationship to the embryonic progenitors of the ENS are currently unknown. Using genetic fate mapping, we here demonstrate that mouse neural crest cells marked by SRY box-containing gene 10 (Sox10) generate the neuronal and glial lineages of enteric ganglia. Most neurons originated from progenitors residing in the gut during mid-gestation. Afterward, enteric neurogenesis was reduced, and it ceased between 1 and 3 months of postnatal life. Sox10-expressing cells present in the myenteric plexus of adult mice expressed glial markers, and we found no evidence that these cells participated in neurogenesis under steady-state conditions. However, they retained neurogenic potential, as they were capable of generating neurons with characteristics of enteric neurons in culture. Furthermore, enteric glia gave rise to neurons in vivo in response to chemical injury to the enteric ganglia. Our results indicate that despite the absence of constitutive neurogenesis in the adult gut, enteric glia maintain limited neurogenic potential, which can be activated by tissue dissociation or injury.

Figure 1. Enteric neurons and glia are derived from a common pool of Sox10-expressing progenitors.

(A and B) Short-term cultures of dissociated gut from E16.5 Sox10::Cre;R26ReYFP transgenic embryos immunostained for YFP (green) and (in red) either the glial marker BFABP (A) or the neuronal marker TuJ1 (B). (C and D) Whole mount preparations of MS-MPs from P30 animals of the same genotype immunostained for YFP (green) and (in red) either the glial marker S100β (C) or the neuronal marker HuC/D (D). Arrows indicate double-positive cells. (E) Quantification of the fraction of YFP⁺ cells that belong to the neuronal of glial lineage of the ENS. Error bars indicate sem. Scale bars: 30 μm (A and B, insets of A and B), 60 μm (C and D, insets of C and D).

Figure 2. Tamoxifen-dependent induction of YFP in Sox10::iCreER^T2;R26ReYFP embryos recapitulates Sox10 expression.

(A) Targeting strategy for generating the Sox10::iCreER^T2 transgene. Top: Schematic representation of the 170-kb Sox10 PAC. White and blue boxes represent noncoding and coding exons, respectively, and solid lines represent introns and flanking DNA sequences. The start of the coding sequence is shown (ATG). Relative positions of BglII and NotI restriction enzyme sites are shown. Bottom: Targeting cassette, which includes 5′ and 3′ homology regions (white boxes), the CreER^T2 coding sequence (gray box), and the chloramphenicol resistance (Cmr) gene (green box) flanked by FRT sites (black arrows). Relative positions of AscI, BglII, SalI, SpeI, and PacI restriction sites and the location of the 3′ UTR probe used for Southern blot analysis of BglII-digested DNA are depicted. (B) Pregnant females were administered 4-OHT (0.2 mg/g) at E9.5, and Sox10::iCreER^T2;R26ReYFP embryos were harvested and analyzed at E12.5. (C–H) Serial sections from Sox10::iCreER^T2;R26ReYFP embryos were hybridized with Sox10- or Cre-specific riboprobes (C, D, F, and G) or immunostained for YFP (E and H). Sections in C–E represent DRG, while sections in F–H represent the gut. (I and J) YFP immunostaining of whole mount preparations of gut from control (I) or tamoxifen-treated E12.5 embryos (J). Note the dramatic increase in the number of YFP-expressing cells in the gut of embryos exposed to tamoxifen. Oe, esophagus; St, stomach; Mg, midgut; Ce, cecum; Hg, hindgut. Scale bars: 50 μm (C–H) and 600 μm (I and J).

Figure 3. Temporal changes in the neurogenic potential of Sox10-expressing eNCSCs.

(A) 4-OHT (0.2 mg/g) was administered to pregnant females or postnatal animals, and the gut of SER26 transgenic mice was analyzed at P84 (for injections at E7.5, E8.5, E12.5, P0, P7, and P30) or P140 (for injections at P84). (B–E) Laser confocal microscopy images of representative whole mount MS-MP preparations from SER26 animals immunostained for YFP (green) and HuC/D (red) and counterstained with TOTO-3 (blue nuclear marker). MS-MP preparations were from P84 (B–D) or P140 (E) mice that were either not treated (B) or treated with 4-OHT at the indicated time points (C–E). Arrowheads indicate double-positive (YFP⁺HuC/D⁺) cells, which correspond to neurons generated after 4-OHT administration. Arrows indicate YFP^–HuC/D⁺ cells. (F) Quantification of the fraction of HuC/D⁺ neurons coexpressing YFP in the gut of P84 or P140 animals. Shown on the x axis are the time points of 4-OHT administration. (G) Laser confocal microscopy images of MS-MP preparations from P140 SER26 animals (tamoxifen administration at P84) immunostained for YFP (green), HuC/D (blue), and S100β (red). Insets are magnification of the boxed area showing YFP⁺HuC/D^–S100β⁺ cells (arrowheads). Arrows indicate YFP^–HuC/D⁺ neurons. (H) Quantification of the fraction of HuC/D⁺ and S100β⁺ cells within the YFP⁺ cell population. Error bars indicate sem. Scale bars: 60 μm (B–E and G) and 10 μm (insets of B–E and G).

Figure 4. YFP⁺ glia from the ENS of adult SER26 mice generate neurons in culture.

(A–D) MS-MP cultures from 4-OHT–treated SER26 mice immunostained for YFP (green), TuJ1 (red), and GFAP (blue) at 12 hours (A), 4 days (B), 10 days (C), and 20 days (D) after plating. On DIV1, all YFP⁺ cells coexpressed GFAP (arrowhead in A), and all Tuj1⁺ neurons were YFP^– (arrow in A). On DIV4, the YFP⁺ population includes glial (YFP⁺Tuj1^–GFAP⁺) and non-glial non-neuronal (YFP⁺Tuj1^–GFAP^–) cells (arrowhead and arrow in B, respectively). After 10 DIV, YFP⁺ cells include glia (arrows in C), non-glial non-neuronal cells, and neurons (YFP⁺Tuj1⁺GFAP^–; arrowheads in C). On DIV20, most YFP⁺ cells are found in NLBs. (E) Quantification of the fraction of neurons and glia in the YFP⁺ population of DIV1, DIV4, and DIV10 cultures. Error bars indicate sem. (F–I) DIV4 cultures immunostained for YFP and GFAP and Sox10 (F), GFAP and Sox2 (H), Phox2B (G), and Ascl1 (I). Sox10 and Sox2 were expressed by YFP⁺GFAP⁺ cells as well as by YFP⁺GFAP^– cells (arrows and arrowheads, respectively, in F and H). (J–M) Immunostaining of DIV10 cultures for YFP and Tuj1 (J), nNOS (K), VIP (L), and NPY (M). (N) Immunostaining of 6-month-old cultures for YFP, HuC/D, and GFAP. In these cultures the YFP⁺ population included HuC/D⁺ neurons (arrow), GFAP⁺ glial cells (arrowhead), and HuC/D^–GFAP^– cells (diamond arrow). Scale bars: 60 μm (A–D, N), 10 μm (F–I), 10 μm (J), 10 μm (K–M).

Figure 5. Enteric glial cells generate functional neurons in vitro.

(A) DIC image of enteric glial cell culture established from Sox10::iCreER^T2;R26R^FP635 mice. Numbers 1–4 indicate cells that are studied in detail. (B) Fluorescence image (recorded at 620/50 nm) of the cells shown in A. Note that cells 1 and 3 express the fluorescent protein FP635. (C) Fluo-4 signal (recorded at 525/50 nm) of the culture before stimulation. Note low background levels of signal in cells 1–4. (D–G) AoT images in which only pixels responding to high K⁺ (D), EFS (E), and application of DMPP (F) and ATP (G) are shown. (H) Merge of images in B, D, and G. (I) Merge of images in B, E, and G. (J) Recordings of the responses of neurons 1 (red) and 2 (black) and glia 3 (red) and 4 (black) to high K⁺, EFS, DMPP and ATP. Neuron 1 and glia 3 express FP635. Insets show the fast upstroke in neuronal cells to high K⁺, compared with a slow secondary response in glial cells. Neurons typically show a fast response to high K⁺, EFS and DMPP, while glial cells show a slow response to high K⁺ and ATP. (K) Average response of FP635⁺ (red) and negative neurons to high K⁺. (L) Average response of FP635⁺ (red) and FP635^– neurons to EFS. Scale bars: 20 μm.

Figure 6. Glial cells generate synapse-forming neurons in vitro.

(A–D) Fluorescence images of the same field of view of an ENS culture established from adult (>P84) 4-OHT–treated SER26 animals. Culture was immunostained for YFP (A), nNOS (B), and synaptophysin (SYN; C). A merge of images in A–C is shown in D. Arrows indicate a YFP⁺nNOS⁺ neuron that exhibits punctuate expression of the synaptic protein synaptophysin. (E) DIC image of an ENS culture established from Sox10::iCreER^T2;R26R^FP635 mice. Arrows point to varicosities analyzed in detail in the other panels. (F) Fluo-4 image without stimulation. (G) AoT at the varicosities indicated in E upon high-K⁺ stimulation. (H) Typical Ca²⁺ transients in the indicated neuronal varicosities following high-K⁺ (red) and DMPP (blue) stimulation. (I) AoT of DMPP stimulation. (J) Merge of images in F, G, and I in which the varicose nerve fiber is visible in purple. Scale bars: 20 μm.

Figure 7. YFP⁺ glial cells from the ENS of adult SER26 mice can generate neurons in vivo.

(A) Laser confocal microscopy of MS-MP preparations from the gut of 4-OHT– and BAC-treated SER26 mice (P84 or older). Inset shows absence of YFP⁺, HuC/D⁺, or S100β⁺ cells in the treated area. (B) Schematic representation of enteric plexus that includes an area that as a result of BAC treatment becomes aganglionic (area 3). (C–H) Laser confocal microscope images of MS-MP preparations representing areas of the enteric plexus that are located distant to (area 1; C and F), adjacent to (area 2; D and G), or within the BAC-treated area of the gut (area 3; E and H) and analyzed 1 month (C–E) or 3 months (F–H) after BAC treatment. MS-MP strips were immunostained for YFP (green), HuC/D (red), and S100β (blue). Arrowheads in insets of G indicate YFP⁺HuC/D⁺ double-labeled cell. Arrows indicate YFP⁺HuC/D^–S100β⁺ cells. Diamond arrow in D indicates space within the ganglia suggesting that neuron loss has occurred. (I) Quantification of the fraction of YFP⁺HuC/D⁺ cells in areas 1, 2, and 3. Note that the fraction of YFP⁺ neurons present within enteric ganglia of area 2 is much greater relative to that in area 1. *F(2) = 5.613; P = 0.03; ANOVA. Error bars indicate sem. Scale bars: 600 μm (A), 100 μm (C, E, F, H, and inset of A), 60 μm (D and G), 30 μm (insets of E and H), 10 μm (insets of C, D, and F), 10 μm (insets of G).