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Fig. 6. Dhh expression by progenitor populations in the developing nerve. We isolated p75⁺P₀^– NCSCs (A; 83% of these cells formed multilineage colonies, and 6% formed colonies that contained myofibroblasts and glia in a clonal analysis), p75⁺P₀⁺ NCSCs (A; 74% of these cells formed multilineage colonies, and 12% formed myofibroblasts and glia), p75^–P₀⁺ myofibroblast progenitors (A; 97% of these cells formed myofibroblast-only colonies) and p75^–P₀^– myofibroblast progenitors (A; 99% of these cells formed myofibroblast-only colonies) from E14.5 rat sciatic nerve (Morrison et al., 1999). Only small numbers of restricted glial progenitors (that form glial-only colonies in culture) are present in the E14.5 sciatic nerve, but the frequency of these cells increases from E15.5 to E17.5, as NCSCs undergo glial restriction (Morrison et al., 1999). As negative controls, we also isolated p75⁺₄⁺ gut NCSCs (B; 76% of these cells formed multilineage colonies) and p75^–₄^– gut epithelial progenitors (B; none of these cells gave rise to colonies that contained neural cells, although they did give rise to fibroblasts in culture). By quantitative (real-time) RT-PCR, we found that nerve p75⁺P₀^– NCSCs expressed the highest levels of Dhh, followed by slightly lower levels in p75⁺P₀⁺ NCSCs (1.45-fold lower), gut epithelial cells (15-fold lower), gut NCSCs (16-fold lower), and nerve myofibroblasts (20 to 40-fold lower) (C). As no reporter gene expression was observed within the intestines of Dhh-Cre⁺loxpRosa⁺ mice, the low level of Dhh expression in nerve myofibroblasts appears to be inconsistent with reporter gene activation within these cells. D shows a representative experiment in which cDNA was random primed and normalized based on ß-actin content, and then Dhh expression levels were compared between samples. We confirmed that each reaction product was homogeneous based on the melting curve, and that a single product was generated of the expected size (data not shown). Sequencing of the Dhh band confirmed that the correct sequence had been amplified.

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