If NCSCs give rise to plexiform neurofibromas and MPNSTs, then Nf1
-deficient NCSCs would be expected to persist in expanded numbers throughout late gestation and into the postnatal period. However, we did not detect the postnatal persistence of NCSCs in DRG, sympathetic chain, trigeminal ganglion, brachial plexus, or peripheral nerve of conditional Nf1
-deficient mice (), even in mice that went on to develop plexiform neurofibromas (). The expansion of Nf1
-deficient NCSCs occurred only transiently during mid-gestation. Like wild-type NCSCs, Nf1
-deficient NCSCs became increasingly rare in late gestation and failed to persist postnatally, precluding them from participating in tumorigenesis (Suppl. Fig. 3
). NCSCs also did not persist postnatally in Nf1+/−Ink4aArf−/−
mice (; Suppl. Fig. 4
) despite the fact that they went on to develop MPNSTs as adults (; Suppl. Fig. 4
). Since NCSCs did not persist postnatally in regions of the PNS that formed plexiform neurofibromas or MPNSTs during adulthood, NCSCs could not have given rise to these tumors.
Our inability to detect Nf1
-deficient NCSCs postnatally in regions of the PNS that developed tumors did not simply reflect altered differentiation or survival of these cells in culture. In all experiments we were able to detect the postnatal persistence of both wild-type and Nf1
-deficient NCSCs from the adult gut. This positive control demonstrated that we were able to culture adult NCSCs from each of the genetic backgrounds we studied. Moreover, Nf1
deficiency increased the survival and proliferation of NCSCs in culture, making them easier, not more difficult to grow (). Furthermore, Nf1
deficiency, with or without p53
deficiency, did not alter the ability of NCSCs to undergo multilineage differentiation (; ; Suppl. Fig. 1
; Suppl. Fig. 5
-deficient NCSCs appeared to differentiate normally in peripheral nerves during late gestation (). We did not detect any gross alterations in nerve development in P20 Nf1
mutant mice by electron microscopy (). Rather hyperplasia and increased frequencies of p75+ cells were not observed in P0a-Cre+Nf1fl/−
mice until around 3 months of age (Zheng et al., 2008
). We also did not detect any gross alterations in PNS development or a significant increase in the frequency of p75+ cells in PNS tissues from Nf1+/−Ink4aArf−/−
mice during early adulthood prior to the formation of tumors (Suppl. Fig. 6
). When combined with the observation that Nf1
-deficient NCSCs were lost from the late gestation PNS according to a similar time course as wild-type NCSCs (Suppl. Fig. 3
), and the observation that Nf1
-deficient NCSCs failed to form tumors after transplantation into adult Nf1+/−
peripheral nerves (), our data suggest that Nf1
-deficient NCSCs terminally differentiate during late gestation and are long gone by the time tumors arise in the adult PNS.
Our data instead suggest that infrequent differentiated glia, such as non-myelinating Schwann cells within peripheral nerves, begin proliferating inappropriately in the postnatal period and give rise to plexiform neurofibromas. The dividing cells within plexiform neurofibromas were almost exclusively p75+ (). Many of these cells had a phenotype similar to non-myelinating Schwann cells (and dissimilar to fetal NCSCs) as they were also GFAP+ and BFABP- (). Proliferating cells within MPNSTs also expressed some differentiated glial markers as well as having a glial morphology ().
Why would deletion of Nf1
in fetal nerve progenitors lead to the formation of tumors that do not become evident until adulthood in mice (Zhu et al., 2002
)? A co-submitted manuscript by Zheng and colleagues concludes that abnormal differentiation of some non-myelinating Schwann cells in the absence of Nf1
leads to their association with unusually large numbers of axons (Zheng et al., 2008
). These bundles degenerate postnatally, leading to inflammation that precedes Schwann cell hyperproliferation and the formation of plexiform neurofibromas. These observations suggest that Nf1
-deficient Schwann cells differentiate perinatally and do not become hyperproliferative until early adulthood when their behavior is modified by epigenetic (i.e. inflammation, hormones, nerve damage) or genetic (i.e. secondary mutations) triggers.
Our results do not address the origin of dermal neurofibromas. Typical benign dermal neurofibromas did not arise in any of the mice we studied. Neural progenitors have been cultured from adult dermis and at least some of these are neural crest-derived (Fernandes et al., 2004
; Wong et al., 2006
). While these cells express markers similar to NCSCs, the progenitors cultured from trunk skin have little capacity to make neurons (Wong et al., 2006
) in contrast to the NCSC populations we have characterized (Bixby et al., 2002
; Kruger et al., 2002
; Morrison et al., 1999
). More work will be required to identify the in vivo cells that give rise to the dermally-derived neural progenitors. Nonetheless, this is a different neural progenitor population, in a different location, than the NCSC populations that we characterized in this study.
It is interesting that NF1 negatively regulates the frequency, self-renewal, growth factor sensitivity, and gliogenesis of NCSCs in most regions of the PNS but not in the gut. The basis for this regional difference in NF1 function is not clear. Nonetheless, these results are consistent with the observation that neurofibromatosis patients seem more likely to develop tumors from peripheral nerves, DRGs, and sympathetic ganglia than from the gut (Fuller and Williams, 1991
). The failure of gut neural crest progenitors to exhibit increased proliferation or gliogenesis after Nf1
deletion may partly explain this clinical observation.
While many cancers may arise from the transformation of stem cells, our results indicate that NCSCs are not rendered tumorigenic by mutations in Nf1. Benign tumors and cancers of the PNS can arise from differentiated glia.