Figure 1.
Developmental expression of Olig3 in the embryonic chick spinal cords at the thoracic level.
Cross sections from various embryonic chick spinal cord tissues were subjected to immunofluorescent staining with rat anti-Olig3 antibody. (A) cE3. (B) cE4. (C) cE5. (D) cE6. (E) cE7. (F) cE8. (F) cE9. (G) cE10. (H) cE11. (I) cE12. (J) cE15. (K) cE18. The dorsal part is up. Bars, 100 µm.
Figure 2.
Olig3 expression pattern in the developing mouse spinal cord.
Cross sections from various embryonic mouse spinal cord tissues were subjected to immunofluorescent staining with rat anti-Olig3 antibody. (A) E10.5. (B) E11.5. (C) E12.5. (D) E13.5. (E) E14.5. (F) E18.5. (G) P4. (H) P8. (I) P31. Olig3 was initially expressed in the dorsal-most neuroepithelial cells at E10.5, and transiently expressed in three ventral groups at E12.5 (Shown by arrows). At E13.5, Olig3 was expressed in the lateral marginal zone of the entire spinal cord along dorsal-ventral axis. The expression pattern of Olig3 in the spinal cord is maintained from E14.5 to E18.5. Olig3 expression remained detectable at postnatal day 4 (P4) but not at P8 and later stages. The dorsal part is up. Bars, 100 µm.
Figure 3.
Short-term BrdU birth-dating analysis of Olig3+ cells in the developing mouse spinal cords.
Mouse embryos from E10.5 (A), E12.5 (B), E14.5 (C), and E18.5 (D) were pulse-labeled with BrdU for 2 hours. Transverse spinal cord sections were subjected to double immunolabeling with anti-BrdU (green) and anti-Olig3 (red). Many Olig3/BrdU double-positive cells were observed at the dorsal-most domain of spinal cord at E10.5. In contrast, no Olig3/BrdU double-positive cells could be found at E12.5, E14.5 and E18.5. The dorsal part is up. Bars, 100 µm.
Figure 4.
Long-term BrdU birth-dating analysis of Olig3+ cells in the developing mouse spinal cords.
BrdU was injected into pregnant mice at various embryonic stages and embryos were harvested at E18.5. Transverse spinal cord sections were subjected to double immunolabeling with anti-BrdU (green) and anti-Olig3 (red). Spinal cord section from mice injected at E10.5, E11.5 and E12.5 contained BrdU+/Olig3+ cells. However, injection after E13.5 did not produce any Olig3/BrdU double-positive cells. A’–C’ are the high-magnification images of A–C, respectively. Double positive cells are indicated with arrows. (G) The percentage of Olig3+ cells that co-express BrdU was calculated. The dorsal part is up. Bars: A–F are 100 µm; A’–C’ are 50 µm.
Figure 5.
Olig3 is expressed in the cells derived from p2 and p3 domains.
cE6 and cE9 chicken spinal cord sections were double-immunostained with anti-Olig3 (red), anti-Chx10 (green), anti-Olig2 (green) and anti-Nkx2.2 (green). Olig3 was expressed in the cells derived from p2 and p3 domains of ventricular zone, but not in Olig2+ motor neuron progenitor cells and oligodendrocytes. C, F, I are the high power views of B, E, H, respectively, in the ventrolateral region. Double positive cells are indicated with arrows. The dorsal part is up. Bars, 100 µm.
Figure 6.
Nkx2.2 controls Olig3 expression in V3 interneurons.
Transverse spinal cord sections from wild-type (A) and Nkx2.2−/− (B) embryos were subjected to immunolabeling with anti-Olig3 antibody. In E13.5 Nkx2.2 knockout mice, Olig3 expression was almost completely lost in ventral-most region of spinal cord. Arrows indicate the region in which Olig3 expression is affected. (C) The number of Olig3+ cells in the ventral-most region of E13.5 wild type and Nkx2.2−/− spinal cords. p<0.001. The dorsal part is up. Bars, 100 µm.
Figure 7.
Nkx2.2 overexpression induces ectopic Olig3 expression.
(A–C) The spinal cords of cE2 chick embryos were electroporated in ovo with replication competent RCASBP retroviral vectors harboring Nkx2.2 gene. The embryos were harvested 2 days later (cE4). Over-expression of Nkx2.2 induced ectopic expression of Olig3 and V3 interneuron marker Sim1. Ectopic Olig3+ and Sim1+ neurons are indicated with arrows. (D) The ratio of the positive cells generated in the Nkx2.2 electroporated (EP) and the control side of the spinal cord. p<0.005. (E–G) Over-expression of Olig3 did not affect Nkx2.2 and Sim1 expression. (H) The ratio of neuron numbers generated on Olig3 electroporated side and the control side. p>0.05. The dorsal part is up. Bars, 100 µm.
Figure 8.
Loss of Olig3 function does not obviously affect the development of V2 and V3 interneurons and motor neurons.
Spinal cord sections from E13.5 wild-type (A, C, E) and Olig3−/− (B, D, F) embryos were subjected to immunostaining with anti-Chx10 (A, B), anti-Nkx2.2 (C, D) and anti-Islet1 (E, F). The production and distribution of these interneurons was similar in wild-type and Olig3−/− spinal cords. Loss of Olig3 didn’t affect the generation of motor neurons, but inhibited the production of Islet1+dI3 interneurons. (G) The ratio of positive cells in Olig3−/− spinal cords compared to wild type. p>0.05. The dorsal part is up. Bars, 100 µm.