All three SoxE genes failed to drive differentiation of the transfected population into any particular lineage. A small proportion of transfected cells differentiated into Schwann cells as detected by the marker Po. The location of transfected cells was in many cases appropriate for Schwann cells or melanocytes, yet only some axon-associated cells expressed the Schwann cell marker Po, and no cells in the dermis expressed the nielanocyte marker MEBL-1. None of the very few SoxE transfected cells observed within ganglia expressed the neuronal marker HuC/HuD. These findings are in accord with and extend the in vitro observation that overexpression of SoxlO in neural crest cells inhibited differentiation into neuronal lineages and preserved multipotency (Kim et al., 2003). These results contrast with previous observations of neural crest differentiation after Sox9 over-expression (Cheung and Briseoe, 2003), possibly resulting from axial level differences.

Our data indicate high-level expression of all SoxE genes can maintain neural tube-derived cells in an undif-ferentiated state in vivo and inhibit transfected cells from localizing in sites of neurogenesis. However, the reason for lack of differentiation remains unclear. High-level expression of Sox9 is associated with and necessary for cartilage formation by both cranial neural crest and mesoder-mally derived cells (Bi et al., 2001; Spokony et al., 2002; Mori-Akiyama et al., 2003), and SoxlO promotes expression of melanocyte and Schwann cell genes (Bondurand et al., 2000, 2001; Peirano et al., 2000; Potterf et al., 2000; Britsch et al., 2001). This finding indicates that, expressed at normal levels, SoxE genes are not in themselves inhibitory to differentiation. Of interest, SoxlO overexpres-sion from the two-cell stage in Xeno-pus greatly increased the number of pigment cells expressing Trp-2 (Aoki et al., 2003).

Sox family members are believed to often pair with a cofactor to activate expression of downstream targets (Kamachi et al., 2000; Wilson and Koopman, 2002). It is possible that overexpression of SoxE genes at an early stage in neural crest development gives an imbalance in relation to the expression of cofactors required for differentiation. SoxlO and Pax3 interact to promote expression of the melanocyte gene Mitf (Bondurand et al., 2000; Potterf et al., 2000); however, we did not observe Pax3 expression in SoxlO transfected cells, so this imbalance may have been sufficient to prevent melanocyte differentiation. Furthermore, SoxlO is expressed only transiently in melanoblasts, whereas SoxlO transfected cells maintained expression until at least E8. Similarly, high-level expression driven by the p-actin promoter used in our expression constructs may provide an excess of SoxE protein, overriding the effects of differentiation-promoting co-factors. Promoters of several SoxE gene targets such as Po contain dimeric and monomeric binding sites (Peirano et al., 2000; Peirano and Wegner, 2000; Schlierf et al., 2002; Bernard et al., 2003). The functional consequences of monomeric and dimeric binding are unclear but expression of high levels of SoxE genes may interfere with the balance between these binding modes, resulting in failure to activate normally SoxE responsive promoters. Alternatively, overexpression of SoxE genes within the neural tube results in extensive migration of neural crest-like cells but at a considerably later stage than the endogenous neural crest. It is possible that a proportion of SoxE induced migratory cells are unable to access the normal differentiation microenviron-ments and signals due to the late stage at which they migrate and, therefore, are inhibited from differentiating.