Fig 2: Melanoblast development coincided with the second phase cell emigration. (A) Pigment containing cells (black) differentiated from the migratory cells derived from G16 trunk neural tube during a 14 day cell culture. (B,C) Anti-Mitf antibody stained melanoblasts in the dorsal neural tube, the CSA, and the surface ectoderm at G15+ and G16. (D) c-Kit positive melanocytes were visible in the CSA above the neural tube and in the surface ectoderm at G21. Scale bars approx. 30 μm (A), 100 μm (B–D).

Fig 3: Mafb is a functional target of miR‐152‐3p. (a) VENN analysis showed that four target‐predicting algorithms were used to identify the potential downstream targets of miR‐152‐3p, Mafb was overlapped in all databases. (b) Construction of Mafb 3′ UTR wild type (wt) and mutant (mut) luciferase reporter vectors. (c) Relative luciferase activity of reporter containing the 3′ UTR of Mafb upon transfection with miR‐152‐3p mimics and mimics negative control in BMMs and RAW264.7 cells, n = 3. (d) Western blot analysis showed the protein levels of MAFB, NFATc1, c‐Fos, MITF and β‐actin in BMMs transfected miR‐152‐3p mimics, inhibitors or cotransfected with miR‐152‐3p mimics and Mafb. (e) Densitometric analysis of MAFB, NFATc1, c‐Fos, and MITF protein levels in indicated groups, n = 3. The data in the figures represent the averages ± SD. Statistically significant differences between the treatment and control groups are indicated as * (P < 0.05) or ** (P < 0.01). N.S. means no significant differences between two groups

Fig 4: Potential migratory pathways for late neural crest cells and melanoblasts. (A) Anti-HNK1 antibody indicated reappearance of migratory neural crest cells in the dorsal neural tube (*) at G16. Paired round areas of HNK-1 positive cells were visible dorsolateral to the neural tube and adjacent to the surface ectoderm (black arrows). (B) At G17, HNK-1 positive cells appeared to follow two migratory pathways: a dorsolateral pathway underneath the surface ectoderm for some of the migratory cells and a pathway where cells travel from the CSA over the dermomyotome laterally (red arrow) or ventrally between the dermomyotome and the sclerotome (orange arrow). (C) PNA was not seen in the dorsal surface ectoderm, carapacial mesenchyme or in or around the dermomyotome, thus allowing entry of migratory neural crest cells to both dorsolateral and medial pathways. (D) Mitf-positive melanoblasts were visible on top of the neural tube, in the CSA, adjacent to the surface ectoderm and in the dermomyotome (boxed area shown in E) and in at G17. (E) The mediodorsal tip of dermomyotome had Mitf-positive cells. (F) Mitf-positive cells were detectable between the dermomyotome and the sclerotome. (G) At G21, c-Kit-positive melanocytes were observed in both the dorsolateral and medial pathways. Some Mitf- and c-Kit-positive cells are indicated by arrows. Scale bar approx. 1 mm (A,B), 100 µm (C,D) and 200 µm (E). c, vertebral cartilage; d, dermomyotome; drg, dorsal root ganglion; l, lung; nt, neural tube; r, rib.