olig3 expression pattern. (a) High magnification of the right side of the rhomboencephalon region showing that olig3 (brown) is expressed medially to the neural crest marker sox10 (blue); the dashed line represents the boundary of expression of the two markers. (b) High magnification of the right side of the trunk region shows that the lateral (l) olig3 (brown) expression domain comprises the region where ngn1-positive interneurons (arrows) differentiate. RB cells (arrowheads) are in a more lateral position and are outside the olig3 expression domain. (c) The anterior border of the olig3 domain (blue) is delimited by krox20 (red) in rhombomere-5 (r5). (d) The olig3 domain has elongated rostrocaudally, and a two-cell-wide gap separates olig3 and dlx3. (e) ngn1:GFP transgenics showing that RB-cell precursors (red) and olig3-expressing cells are in close contact. (f) At the five-somite stage, the lateral domain has further elongated. (f′) Transverse section at the level of the posterior trunk of the embryo in f. The embryos are in dorsal view, anterior to the top.

olig3 controls specification of the neural crest. (a–d) The levels and the expression domains of sox10 and foxd3 are decreased in four-somite embryos injected with olig3 mRNA (b and d) (phenotype in >90% of injected embryos, n = 30). (e and f) The expression of foxd3 is increased in 15-somite embryos treated with olig3MO (f) (morphant phenotype > 90%, n = 20). Similarly, the expression of crestin is increased in 30-hpf embryos treated with olig3MO (h and f) (morphant phenotype > 90%, n = 40). Similar results are obtained in 24-hpf embryos hybridized with sox10 (k and l) (morphant phenotype > 90%, n = 30). Embryos are in dorsal (a–h, k, and l) or lateral (i and j) view, anterior to the top (a–d) or to the left (e–l).

olig3 knockdown affects differentiation of lateral neural plate cells. (a–f) isl1/2-expressing interneurons (a and b; black arrowheads in a) (18, 19) are absent in 20-somite (b and d) and 24-hpf (f) embryos injected with olig3MO. Conversely, RB cells (RB) are increased in number in olig3 morphants (b, d, and f) (morphant isl1-phenotype > 90%, n = 40; morphant isl2-phenotype > 90%, n = 30). mn, motoneurons. (g and h) lim1 is expressed at 24 hpf in D3 interneurons of the dorsal spinal cord (g). D3 interneurons are slightly affected in embryos injected with olig3MO (h) (morphant phenotype > 90%, n = 15). (i and j) The glial fibrillar acidic protein gene (gfap) is expressed at 3 days postfertilization in astrocytes of the dorsal spinal cord (i). gfap expression is absent in embryos injected with olig3MO (j) (morphant phenotype > 90%, n = 20). Embryos are in transverse sections (a and b) or in lateral view with anterior to the left (c–j).

olig3 is controlled by Notch and regulates ngn1 and deltaA expression in the lateral neural plate. (a–d) Notch signaling controls olig3 expression. (a) olig3 expression at the two-somite stage showing the medial (m) and lateral (l) domains. (b) mib embryo at the two-somite stage showing increased and broadened expression of olig3. (c and d) Tail bud-stage embryo injected with Notch-ICD (NIC) showing dramatic reduction of olig3 expression (d) (shown phenotype > 90%, n = 20). (e–g) olig3MO rescues pigmentation in mib mutants. (e) Trunk pigmentation at 30 hpf. (f) mib embryo lacks melanocytes. (g) Rescue of the pigmentation in a mib mutant injected with olig3MO (morphant phenotype 9/10). (h–j, h′, and j′) olig3MO rescues the neural crest in DAPT-treated embryos. (h) crestin expression in a normal embryo at 30 hpf. (h′) Transverse section of the embryo in h. (i) A DAPT-treated embryo lacks most of the neural crest cells. (j) Injection of olig3MO in an embryo treated with DAPT rescues neural crest cells (morphant phenotype 9/10). (j′) Transverse section of the embryo in j. (k and l) olig3 controls the expression of deltaA in interneurons. (k) deltaA expression pattern in a four-somite-stage control embryo injected with olig3^mismMO. (l) The stripe of deltaA-positive cells corresponding to interneurons (in) is missing in embryos injected with olig3MO (morphant phenotype > 90%, n = 40). (m–o) In situ hybridizations at two-somite stage showing that olig3 activates ngn1 expression in the lateral neural plate. (m) Expression of ngn1 in motoneurons (mn), interneurons (in), and RB cells (RB) in control embryos injected with olig3^mismMO. (n) In an olig3MO-injected embryo, ngn1 is expressed only in the medialmost (mn) and lateralmost (RB) neuronal populations (morphant phenotype > 90%, n = 20). (o) Injection of olig3 mRNA up-regulates ngn1 expression (shown phenotype > 90%, n = 20). (p–s) RB and dorsal root ganglion (drg) cells are present in ngn1:GFP-transgenic embryos (13) injected with olig3MO (r and s), as in controls (p and q). Interneurons (in) are reduced or absent (q and s). Embryos are shown in dorsal view with anterior to the top (a–d and k–o), lateral view with anterior to the left (e–j), and dorsal view with anterior to the left (p–s).

olig3 mediates mediolateral specification. isl2 staining of RB cells is shown. RB cells originating in the neural crest domain are increased in number in olig3MO-injected embryos (morphant isl2 phenotype > 90%, n = 20) (b) as well as in DAPT-treated embryos (morphant isl2 phenotype > 90%, n = 40) (c) compared with controls (a). DAPT treatment and olig3MO act synergistically (d) (morphant isl2 phenotype > 90%, n = 20). Embryos are shown in dorsal view with anterior to the left.

BMP-dependent restriction of olig3 expression domains. (a) Normal embryo. (b) swr embryo showing a widened space between the two medial domains (m). (c) Ventral view of a swr embryo showing that the lateral olig3 domains are broadened and merge on the ventral side. (d) Normal embryo. (e) din embryo with reduced lateral (l) domain. (f and g) Normal (f) and swr (g) embryos show a similar olig2 pattern. (h and i) Normal expression of olig3 in the dorsal spinal cord of the smu embryo. Embryos in a–g are in dorsal view with anterior to the top, except in c (ventral view). Embryos in h and i are in lateral view, anterior to the left.

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