SEMA6A is expressed in neural crest cells that give rise to boundary cap cells. (a) Neural crest cells that give rise to boundary cap cells express SEMA6A while they are still migrating ventrally (HH19; arrow). Boundary cap cells start to cluster first at the VMEP. (b) Only those cells that have formed clusters at the VMEP express the BCC marker KROX20 (arrowhead). The neural crest markers (c) SOX10 and the (d) 1E8 epitope are expressed by many neural crest-derived cell populations and are not restricted to BCCs at HH19. Note that neither SOX10 (open arrow in (c)), nor 1E8 (open arrow in (d)) are expressed in BCCs while they are still migrating. First clusters of BCCs next to the DREZ marked by (e) SEMA6A expression (open arrowhead) or (f) KROX20 (open arrowhead) are detectable at lumbosacral levels by HH21. At this stage, many SEMA6A-expressing cells are still migrating along the neural tube to reach the ventral BCC cluster (arrow in (e)). (i-l) A similar situation is found at HH24. SEMA6A expression is clearly detectable in dorsal BCCs (open arrowhead in (i); compare with (j)). (n, o) After HH30, SEMA6A expression in dorsal (open arrowhead in (n)) and ventral (arrowhead in (n)) BCCs decreased but was still visible by HH36 (o). In situ hybridizations on adjacent transverse sections of the lumbosacral spinal cord are shown for SEMA6A (a, e, i, m-p), KROX20 (b, f, j), and SOX10 (c, g, k) at HH19 (a-d), HH21 (e-h), HH24 (i-l) as indicated. Sections shown in (d, h, l) were stained for 1E8 (red) and neurofilament (green). Bars are 100 μm in (a-n), 200 μm in (o), and 500 μm in (p).