Figure 3.

In vitro myelination by Mtmr13^−/− DRG explant cultures leads to CMT4B2-like myelin outfoldings. (a and b) DRG explant cultures from wild-type and Mtmr13^−/− embryos were analyzed by immunofluorescence after 21 days in culture. Compact myelin and axons were visualized with antibodies to MBP (magenta) and NFH (green), respectively. Elaborate myelin outfoldings are present in Mtmr13^−/− cultures but are not observed in wild-type cultures. See “Materials and Methods” section for operational definition of a myelin outfolding. (a’ and b’) Higher magnification images of the indicated portions of (a) and (b) reveal myelin outfoldings (white arrowheads) in Mtmr13^−/− DRG explant cultures. (c–f) Examples of myelinated segments in Mtmr13^−/− DRG cultures reveal varying morphologies of outfoldings. (c’–f’) Higher magnification images of portions of (c–f), respectively. The location of a Schwann cell nucleus (n) is indicated in (f’). (g–j) Deconvolution microscopy of wild-type and Mtmr13^−/− DRG explant cultures better reveal elaborate, convoluted nature of Mtmr13^−/− myelin outfoldings (white arrowheads), including those at the ends of myelin segments (nodes or heminodes; (h)). A white arrow in (h) indicates a Schmidt–Lanterman incisure. Scale bar: 27 μm (a and b), 17 μm (a’ and b’), 24 μm (c, d, e, and f), 8.5 μm (c’, d’, e’, and f’), 8.2 μm (g–j). (k) Quantification of myelin outfoldings in wild-type and Mtmr13^−/− DRG explant cultures. Percentage of myelin segments containing one or more myelin outfoldings: 7.168 ± 4.784% for wild-type versus 29.42 ± 7.000% for Mtmr13^−/−; mean ± SD; ****p < .0001; n = 7 and 6 explants for wild type and Mtmr13^−/−, respectively. An average of 1,067 myelinated segments per explant were evaluated for the presence of myelin outfoldings (the median value was 806 myelin segments evaluated per explant/coverslip). MTMR = myotubularin-related.