In this study we found that both postnatal (P1-3) and adult loss of Nf1 using the PlpCre driver cause GEM-grade I neurofibroma tumor formation. The models differ in the timing of neurofibroma formation, in the size of the neurofibromas generated, and in prevalence of hematopoietic manifestations. We found that myelinating Schwann cells, p75+ cells, and satellite cells are targeted by the inducible PlpCre driver. Our results support previous studies indicating that loss of Nf1 in subpopulations of nerve Schwann cell lineage cells cause neurofibroma formation, and extend these studies by showing that acute Nf1 loss, after organogenesis and cell differentiation, can be tumorigenic.
We identified EGFP+ cells in order to identify possible tumor cells of origin in the PlpCre
model. Tamoxifen exposure induced peripheral nervous system recombination, as judged by EGFP+ cells, in satellite cells in the DRG (S100β+ or GFAP+) with the characteristic morphology of satellite cells, closely wrapping DRG cell bodies. In DhhCre;Nflfl/fl
mice, satellite cells do not show recombination, yet neurofibromas form (24
). While not definitive, the combination of the two models does not support a role for satellite cells in mouse neurofibroma formation; however, the possibility that satellite cells are important for neurofibroma formation in some settings while not in others cannot be excluded.
Most EGFP+ cells in adult peripheral nerves were S100β+ (myelinating) Schwann cells. This result is expected, as endogenous Plp is a characteristic of myelinating Schwann cells in adult peripheral nerve (33
). At the EM level, Nf1
loss did not dramatically alter myelination.
Remak bundle disruption is a characteristic feature of all neurofibroma models (22
). When the non-myelinated Schwann cell population was examined, no EGFP+;GFAP+ non-myelinating Schwann cells were identified. These data are surprising as electron microscopy shows disruption of the association between axons and non-myelinating Schwann cells in the PlpCre;Nf1fl/fl
model. We conclude that Nf1
loss within GFAP+ cells is not necessary for Remak bundle disruption in the PlpCre;Nf1fl/fl
We identified EGFP+;p75+ cells in peripheral nerve. These may be a sub-population of GFAP-negative non-myelinating cells, and/or an as-yet unidentified population(s) in adult peripheral nerve. Zheng et al. (2008) proposed that the p75+ non-myelinating cell population was the cell of origin for neurofibroma formation (23
). The present study eliminates the GFAP+ non-myelinating Schwann cell as the tumor-initiating cell. It is possible that neurofibroma formation results from the p75+/GFAP-negative cells in peripheral nerve, and/or that loss of Nf1
within mature myelinating Schwann cells have non-cell autonomous effect(s) that promote tumor formation – similar to the non-cell autonomous effect on hematopoietic cells when Nf1
is lost within stromal cells.
Perinatal tamoxifen injection into PlpCre;Nf fl/fl
mice resulted in GEM grade I neurofibroma formation that resulted in morbidity at 15-23 months old. In contrast, adult loss of Nf1
resulted in large neurofibromas which caused morbidity beginning 5 months post tamoxifen introduction. We considered the possibility that recombination occurs in more cells when tamoxifen-induced recombination occurs in adults. However, twice as many cells in sciatic nerve and three times as many in the DRG were EGFP+ in pups as compared to adult mice. Therefore together with previous studies showing that loss of Nf1
in most developing Schwann cells leads to robust neurofibroma formation (22
) we conclude that cells exist in the adult peripheral nervous system that remain susceptible to neurofibroma formation, and that the susceptible population(s) may be enriched in adult and embryonic nervous systems.
Le et al's co-submitted study confirms our observation that neurofibromas can form after perinatal or adult loss of Nf1. However, while they identified small neurofibromas at the thoracic and lumbo-sacral levels after adult loss of Nf1, we generated large neurofibromas throughout the neuroaxis. Some differences between the two studies may account for the slightly different findings. Lu et al. provided 4 mg tamoxifen (2mg twice daily) by oral gavage to adult mice for 5 days, while we dosed 1mg twice day by I.P. injection for 3 days. It is also possible that the different phenotypes result from the different tamoxifen-inducible PlpCre driver lines used in the two studies. This difference may well account for the absence of hematopoietic lesions in their system. Most importantly, our data do not support the idea that there is a critical window for neurofibroma formation.
Mast cells, and other hematopoietic cells, showed <1% recombination in PlpCre;Nf1fl/fl
mice, yet neurofibroma formation was robust. While Krox20Cre;Nf1fl/fl
mice show hyperplasia within the DRG, neurofibroma development required an Nf1+/−
background attributed to Nf1+/−
mast cells (22
animals develop neurofibromas when only Schwann cell lineage cells are Nf1
), and the PlpCre;Nf1fl/fl
model is similar in that a heterozygous background is not necessary for neurofibroma formation.
The peripheral blood of PlpCre;Nf1fl/fl
mice showed a decrease in monocytes with a relative increase in lymphocytes and PMN. In contrast, Nf1
loss driven by Mx1-Cre causes a mouse disorder similar to human juvenile myelomonocytic leukemia (JMML), with hyperproliferation of all hematopoietic cell types and progressive myeloproliferative disorder (36
). Flow cytometry and immunohistochemistry showed that the PlpCre
transgene did not cause Nf1
loss in cells of hematopoietic origin. Rather, EGFP+ cells within these tumors had a stromal/mesenchymal appearance, and some double-labeled with Sca-1. EGFP expression did not co-localize with the expression of hematopoietic lineage markers or c-kit (data not shown), indicating that recombined EGFP+ cells had a non-hematopoietic, mesenchymal origin (30
The idea that Nf1 loss can affect tumorigenesis in a non-cell autonomous fashion (e.g. myelinating cells acting upon other cells in the nerve) is thus supported by analogy to the formation of hematopoietic lesions within the PlpCre;Nf1fl/fl animals, in which mutant stromal cells after either perinatal or adult tamoxifen-induced Nf1 loss, cause lymphoid and myeloid proliferation. However we cannot exclude the possibility that neurofibroma formation requires Nf1 loss of function in a small population of stem/progenitor-like cells that remain unidentified by our analysis, or induces expression of chemoattractants that result in massive tissue infiltration by hematopoietic cells. In either event, neurofibroma formation is not restricted to loss of Nf1 in embryonic life, but can be triggered by Nf1 loss throughout life.