Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Nancy M. Joseph 2 Yoh-suke Mukouyama 1 Jack T. Mosher 2 Martine Jaegle 0 Steven A. Crone 3 Emma-Louise Dormand 1 Kuo-Fen Lee 3 Dies Meijer 0 David J. Anderson 1 4 Sean J. Morrison ) 2 4 0 Department of Cell Biology, Erasmus University Medical Center , 3000DR Rotterdam , The Netherlands 1 Division of Biology 216-76, California Institute of Technology , Pasadena, CA 91125 , USA 2 Departments of Internal Medicine and Cell and Developmental Biology, 1500 East Medical Center Drive, University of Michigan , Ann Arbor, MI 48109-0934 , USA 3 The Salk Institute , 10010 North Torrey Pines Road, La Jolla, CA 92037 , USA 4 Howard Hughes Medical Institute - Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Crerecombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to Current models of peripheral nerve development address the generation of Schwann cells from restricted neural crest progenitors. The undifferentiated neural crest cells in the embryonic day 14 (E14) peripheral nerve have been described as Schwann cell precursors that undergo overt differentiation to Schwann cells over the next few days of development (Jessen et al., 1994). These Schwann cell precursors were assumed to be glial-restricted (Jessen and Mirsky, 1992; Mirsky and Jessen, 1996). We have more recently discovered that multipotent neural crest stem cells (NCSCs) persist in the E14-E17 peripheral nerve (Bixby et al., 2002; Morrison et al., 1999). The persistence of NCSCs raises the question of whether the neural crest gives rise to additional nerve cell types in addition to Schwann cells. If so, nerve development is much more complex than was previously thought, involving NCSC self-renewal (Morrison et al., 1999), lineage commitment and multilineage differentiation. Circumstantial evidence suggests that NCSCs give rise to more than just Schwann cells in developing nerves. Culture of single cells from the developing sciatic nerve yields several different types of colonies, including multilineage NCSC fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation. colonies (containing neurons, Schwann cells and myofibroblasts), colonies that contain only Schwann cells and myofibroblasts, colonies that contain only Schwann cells, and colonies that contain only myofibroblasts (Morrison et al., 1999). This raises the possibility that NCSCs give rise to restricted progenitors within the nerve, which in turn differentiate into Schwann cells and fibroblasts. If single sciatic nerve NCSCs are subcloned after proliferating in culture they generate additional NCSCs, as well as colonies that contain only Schwann cells and myofibroblasts, colonies that contain only Schwann cells, and colonies that contain only myofibroblasts (Morrison et al., 1999). Nerve NCSCs may undergo progressive restrictions in vitro and in vivo to form both Schwann cells and fibroblasts. Recent fate-mapping studies in vivo have raised questions about whether the fate of neural progenitors in vivo can be accurately predicted based on their function in culture (Gabay et al., 2003). Because culture conditions often dysregulate progenitor patterning in an unphysiological way (Anderson, 2001), neural progenitors might readily generate cell types in culture that they would never generate in vivo. In this regard, the ability of nerve (and other trunk) NCSCs to generate myofibroblasts in culture (Morrison et al., 1999) appeared to contrast with the failure to observe a contribution of trunk neural crest cells to fibroblast-type derivatives in vivo (Le Douarin, 1982). This raises the question of whether the potential of nerve NCSCs to make fibroblasts in culture is ever expressed in vivo. More generally, it is important to begin to assess the fate of neural stem cell populations during normal development, in addition to examining the developmental potential of these cells in culture. If NCSCs differentiate into both Schwann cells and fibroblasts within nerves, what is the ultimate fate of the fibroblasts? There are a variety of cell types present within peripheral nerves in addition to Schwann cells, including perineurial cells, pericytes and endoneurial fibroblasts (Fig. 1). The embryonic origins of perineurial cells, pericytes and endoneurial fibroblasts are uncertain and controversial. Based on morphological criteria, some investigators have argued that perineurial cells are neural crest derived (Hirose et al., 1986), while others have argued they are not (Low, 1976). The observation that embryonic fibroblasts from the cranial periosteum could form perineurial cells in culture supported a mesodermal origin for these cells (Bunge et al., 1989). Although pericytes have been presumed to be mesodermally derived, the vascular smooth muscle around the cardiac outflow tracts is neural crest derived (Jiang et al., 2000; Kirby and Waldo, 1995), and so some vascular smooth muscle cells in other locations might also be neural crest derived. Little is known about the properties of endoneurial fibroblasts or their embryonic origin. We have used Cre-recombinase fate mapping (Chai et al., 2000; Jiang et al., 2000; Yamauchi et al., 1999; Zinyk et al., 1998) to examine which cells in peripheral nerve are neural crest derived (see Fig. S1 in supplementary material). Nerve sheath perineurial cells, vascular pericytes and endothelial cells were not neural crest derived. By contrast, endoneurial fibroblasts were neural crest derived, identifying a previously unrecognized, non-glial neural crest-derivative. Both Schwann cells and endoneurial fibroblasts appeared to arise from a common desert hedgehog (Dhh)-expressing progenitor population within the nerve environment, and nerve NCSCs strongly expressed Dhh, suggesting that nerve NCSCs give rise to both Schwann cells and endoneurial fibroblasts. To begin to identify the signals within the nerve environment that promote multilineage dif (...truncated)