044 and find more P = 0.023, respectively), while KCC2-C568A embryos (n = 3) did not differ from their wild-type littermates (n = 3 per group; Fig. 4 O). In addition, KCC2-FL

and KCC2-ΔNTD embryos displayed a larger proportion of PSA-NCAM-positive cells in the ventricular and intermediate zones relative to the marginal zone than did wild-type littermates (30 and 26% more than wild-type; P = 0.012 and P = 0.0496, respectively; Fig. 4P). These findings suggest that radial migration of neuronal cells may be delayed in KCC2-FL and KCC2-ΔNTD embryos. The phenotypes of the KCC2-FL and KCC2-ΔNTD embryos indicate disturbances in neural crest cell migration. Neural crest contributes to both the facial bone structures and the bone marrow that produces blood cells (Inoue et al., 2004; Nagoshi et al., 2008). To investigate the distribution of migrating neural crest cells, E9.5 embryos were labelled with the neural crest

cell markers AP-2α and SOX-10 (Inoue et al., 2004). In wild-type embryos (n = 3 per group), several transverse sections in the hindbrain area showed a large amount of labelled neural crest cells outside the neural tube (Fig. 5A). SOX-10-positive cells were found both inside the neural tube, in a migrating learn more stream projecting from the tube, and in areas further away from the tube. AP-2α-positive cells were mainly located in the areas with longer distances from the neural tube, and co-localized with SOX-10-positive cells, indicating that AP-2α expression turns on at later migratory stages. KCC2-FL (n = 4) and KCC2-ΔNTD (n = 3) embryos had a lower proportion of transverse sections with detectable neural crest Tyrosine-protein kinase BLK (63 and 70% of wild-type; P = 0.019 and P = 0.011, respectively) and often displayed a diffuse pattern of these cells (Fig. 5B and C). In contrast, KCC2-C568A embryos (n = 4) did not

differ from wild-type embryos in the proportion of sections with neural crest (95% of wild-type; P = 0.846) nor the neural crest cell pattern (Fig. 5D). Connexins mediate early, direct and rapid communication between cells (Jaderstad et al., 2010) and play a key role in radial neuronal migration (Elias et al., 2007). Wild-type staining of connexin-43 showed a focused expression in cell processes of neural tube and neural crest cells (Fig. 6A). However, KCC2-FL and KCC2-ΔNTD embryos displayed numerous cells with a loss of this polarized expression pattern and with a more circumferential distribution of connexin-43 (Fig. 6B and C). This indicates that cell polarization, an essential feature of developing and migrating cells, might be disturbed in KCC2-FL and KCC2-ΔNTD embryos. KCC2 has been shown to interact with the actin cytoskeleton in an ion transport-independent manner (Li et al., 2007). We therefore labelled the actin cytoskeleton in the E9.5 embryos using phalloidin. Wild-type embryos displayed an enriched actin labelling at the adherens junctions lining the neural tube (Fig. 7A and E).