Fig. 2. The boundary cap contains NCSCs. (A) In situ hybridisation for Krox20 on a coronal section of E10.75 mouse embryo showing the dorsal part of the DRG and the boundary cap. (B) Immunocytochemistry for Maob (red) on adjacent section. (C) In situ for Krox20 on a coronal section of E11.75 mouse embryo. (D) Immunocytochemistry for Maob (red) on adjacent section. (E,F) Maob expression in dorsal root ganglion cells 2 hours (E) and 12 hours (F) after plating. (G) Enrichment of stem cells at 12 hours correlated with a several-fold increase of Maob⁺ cells. The enrichment is not due to transcriptional upregulation or proliferation of the Maob-positive population as it persists in the presence of actinomycin D and cytosine arabinoside (G). Notably, no change was seen in the absolute numbers of Maob-positive cells (data not shown). (H) A coronal section through an E11.5 thoracic dorsal root ganglion stained with Cresyl Violet with BC and dorsal root ganglion indicated. The dorsal root ganglion is outlined. The black lines indicate the parts isolated by micro-dissection of the dorsal root ganglia into a BC part (b) and a central part (c). (I) RT-PCR for Krox20 on dissected tissue with HPRT as internal reference for starting material. Krox20 mRNA is present only in the BC but not in the central part. (J) When cells from the BC and central parts were cultured in propagation medium separately, there was a significant difference in the number of stem cell clones produced from the different samples (^***P<0.001, Mann-Whitney), showing that the stem cells are located in the BC. Abbreviations: Actino D, Actinomycin D; Ara-C, cytosine arabinoside; BC, boundary cap; c, central part; b, boundary cap part; DRG, dorsal root ganglia; SC, spinal cord. Scale bars: 50 µm.