We and others previously demonstrated that self-renewing and multipotent cells with properties similar to those of NCSCs persist throughout adult life in the mammalian ENS (33
). These cells proliferate extensively and give rise to PNS neurons, glia, and myofibroblasts in culture, forming colonies similar to those formed by fetal NCSCs (32
). These cells also express the NCSC marker p75 and upon transplantation into chick embryos migrate through the neural crest migration pathway to form neurons and glia (33
). Although our prior studies suggested that these cells reside in or near the myenteric plexus, the identity, localization, and physiological function of these cells remained uncertain, partly because p75 alone was not sufficient for the unambiguous identification of these cells (33
In the current study, we discovered that these cells could be isolated to a high degree of purity using CD49b (integrin α2) (Figure C). Nearly all CD49b+
cells expressed nestin, SOX10, S100B, GFAP, and p75, markers of NCSCs and/or enteric glia, but not the neuronal marker HuD (Figure , H and I). Staining of whole mount preparations of the myenteric plexus revealed that most staining of CD49b overlapped with S100B, p75, GFAP, and nestin within myenteric ganglia in a pattern consistent with enteric glia (Figure J and Supplemental Figure 2, C–E). Many enteric glia, therefore, have the potential to form multilineage colonies in culture, and the cells we previously identified as adult gut NCSCs (33
) could also be considered enteric glia.
The observation that about 60% of Lin–S100B+ or Lin–GFAP+ enteric glia stained positive for CD49b (Supplemental Figure 2B) and that 44% of CD49b+Lin– cells formed multipotent neurospheres in culture (Figure C) raises the possibility that adult enteric glia may be heterogeneous. It is possible that a minority of enteric glia are capable of forming multilineage colonies in culture and that these cells represent a distinct subpopulation. Nonetheless, our data indicate that this would represent a major subpopulation of enteric glia (at least 60% × 44% = 26%).
Using BrdU incorporation assays, we readily detected neurogenesis in the CNS dentate gyrus (Supplemental Figure 3) but generally failed to detect neurogenesis in the adult ENS of the same rats (Table ). We administered BrdU to many mice and rats in a variety of different circumstances including normal young adults, old adults, pregnant rats, hyperglycemic rats, rats undergoing dietary changes, mice undergoing voluntary exercise, mice with gut inflammation due to bacterial infection or chemical treatment, rodents with focal ablation of the myenteric plexus due to topical BAC treatment, Gfap-tk mice after ablation of enteric glia, and rats with osmotic minipumps that released growth factors into their peritoneum. We administered BrdU to these rodents for variable periods of time, from 10 days to 19 weeks. Yet in no case did we consistently observe new neurons. The only exception was a single rat with a focal ablation of the myenteric plexus from topical BAC treatment, which did have convincingly BrdU+HuC/D+ neurons (Supplemental Figure 7). However, we were never able to replicate that observation, despite examining 58 additional rats and mice that had been treated with BAC. These results suggest that cells capable of forming new neurons persist within the adult gut but that little neurogenesis occurs under the conditions we studied.
Given that neurogenesis occurs in the adult gut after administration of exogenous 5-HT4
), it remains possible that there are some physiological circumstances in which endogenous 5-HT4
agonists and gut neurogenesis are induced. However, Liu et al. (46
) also did not observe the formation of new neurons upon BrdU administration in the normal adult gut in the absence of exogenous 5-HT4
agonist. It remains unknown what physiological circumstances might induce higher levels of 5-HT4
agonists in the adult gut.
We also performed lineage-tracing studies using GFAP-Cre and GFAP-CreERT2 mice to test whether GFAP+ enteric glia could give rise to neurons in the adult gut, with or without cell division. The frequency of EYFP+ myenteric neurons in the guts of adult GFAP-Cre;Rosa-loxpEYFP mice did not increase during aging or after BAC treatment, despite efficient labeling of 90% ± 3% of GFAP+ glia in these mice (Figure ). This suggested that few new neurons are generated by GFAP+ cells during adulthood. We obtained similar results in GFAP-CreERT2;Rosa-loxpEYFP mice, in which only rare neurons were EYFP+ under steady-state conditions or after BAC treatment (Figure and Table ). These results therefore complement the results of the BrdU incorporation studies in suggesting that there is little neurogenesis in the adult ENS under the physiological conditions that we studied. On the other hand, our data leave open the possibility that GFAP– progenitors might be able to engage in neurogenesis without cell division or that neurogenesis occurs under circumstances we did not study.
In a companion to the present article, Laranjeira et al. did report neurogenesis at the border of injured regions of the distal ileum after BAC treatment of adult mice (55
). The authors observed this neurogenesis by lineage tracing SOX10+
gut cells in Sox10-CreER
). Consistent with our results, they never observed neurogenesis in the adult gut after 1–3 months of age in the absence of injury and could not detect the injury-induced neurogenesis by fate mapping with GFAP-CreERT2
). In principle, Sox10-CreER
should fate map the same GFAP+
cells that we studied, as most enteric glia are CD49b+
, and SOX10+
(Figure ). However, if small numbers of neurons are generated without cell division after BAC treatment, it is possible we were unable to detect these cells in the background of EYFP+
neurons generated during development in GFAP-Cre
mice and that the efficiency of recombination was too low in adult GFAP-CreERT2
mice to detect these cells. It is also possible that a rare population of SOX10+
progenitors may exist and may contribute to adult gut neurogenesis without undergoing cell division. Overall, neurogenesis may occur in the adult gut in response to certain injuries, though it is not clear what kinds of injuries might occur under physiological conditions that are mimicked by BAC treatment. Our data suggest that the adult ENS does not have constitutive neurogenesis by dividing progenitors, unlike the CNS.
We readily observed gliogenesis under steady-state conditions and in response to injury (Figure A and Figure ). While most enteric glia were quiescent at any one time (Figure K), at least some of these cells appeared to be recruited into cycle, as 2.8% ± 0.3% of enteric glia were BrdU+
after 6 weeks of BrdU administration (Figure ). This suggests a low level turnover of enteric glia under steady-state conditions as well as considerable capacity to regenerate glia after injury (Figure ). Our data suggest that the primary progenitor function of enteric glia is to form new glia in the ENS throughout adult life. The fact that Gfap-tk
mice die with degeneration of the myenteric plexus after sustained ganciclovir treatment implies that this adult gliogenesis is critical for the maintenance of ENS function, as ganciclovir preferentially kills dividing cells (51
Although we did not generally observe regeneration of new neurons in the injured gut after BAC injury (Figure D), we did observe extensive regeneration of nerve fibers in the injured region, in association with a re-colonization of the injured region by glia (data not shown). It is unclear whether this contributed to a recovery of ENS function, though it does raise the possibility that ENS function may be restored more through a regeneration of axons and a rewiring of existing circuits than by generation of new neurons. Hanani et al. also reported the regeneration of nerve fibers in close association with glia in BAC-injured regions of the gut, though they observed morphological evidence for the regeneration of neurons in the injured region, which we did not detect (43
In stark contrast to our failure to consistently observe neurogenesis in vivo, adult CD49b+
enteric glia readily formed neurons in culture (Figure F). This raises the question of what mechanisms stimulate this latent neurogenic potential in culture and not generally in vivo. One possibility is that the culture medium contains factors that promote the dedifferentiation of enteric glia into multipotent cells; however, freshly isolated, uncultured adult rat gut p75+
cells form neurons after transplantation into chick embryos, albeit in smaller numbers than fetal p75+
). This suggests that even uncultured adult enteric glia (which are p75+
in addition to being CD49b+
, and S100B+
; Figure and Supplemental Figure 2) have neurogenic potential, though the chick embryo may also reprogram these cells. Alternatively, it is possible that CD49b+
adult enteric glia are bona fide multipotent progenitors that possess intrinsic neurogenic potential; however, the adult ENS may lack the signals required to promote neuronal differentiation by these cells. The observation that serotonin receptor agonists can induce neurogenesis in the adult ENS (46
) raises the possibility that serotonin receptor stimulation may be inadequate for neurogenesis in the adult gut under most physiological conditions and that serotonin from chick embryo extract may promote the elevated levels of neurogenesis observed in culture. It is also possible that the adult ENS is not permissive for neurogenesis in vivo due to inhibitory signals.
Our data suggest that many adult enteric glia are capable of generating multipotent, self-renewing colonies in culture, similar to NCSCs. However, we observed little or no adult neurogenesis under physiological conditions in the ENS. Low levels of neurogenesis may occur in response to certain types of injury or under restricted circumstances. However, the primary physiological function of the progenitor activity retained by adult enteric glia is to form new glia under steady-state conditions and to regenerate glia that are lost to injury. The robust gliogenesis that we observed is likely necessary to maintain adult ENS and gut function.