OC factors are differentially and dynamically distributed in dI6 population and sub-populations during development. (A–I) Immunodetection of HNF-6, OC-2 or OC-3 (red) in double-labeled Lbx1+ (blue)-Lhx1/5+ (green, A–C) dI6 population or in Bhlhb5+ (green, E–G) dI6 subpopulation on transverse sections of thoracic spinal cord at e10.5. HNF-6 (A,E) and OC-2 (B,F) are present in a majority of Lbx1+Lhx1/5+ dI6 neurons and in a significant number of Bhlhb5+ newly-born dI6 while OC-3 (C–G) is limited to a smaller number of cells. The number (D–H) and percentage (I–M) of OC-positive dI6 neurons per hemisection were quantified accordingly. (J–Q) Immunodetection of HNF-6, OC-2 or OC-3 (red) in double-labeled Lbx1+ (blue)-Lhx1/5+ (green, J–L) dI6 neurons or in Dmrt3+ (green, N,O) dI6 subpopulation on transverse sections of thoracic spinal cord at e12.5. HNF-6 (J,N) and OC-2 (K,O) are mainly detected in dI6 neurons located in ventral spinal cord. OC-3 (L) is also detected in Lbx1+Lhx1/5+ dI6 neurons. Its presence in Dmrt3+ dI6 could not be evaluated due to incompatibility between antibody species. (D,M) Quantifications of ventrally located dI6 neurons containing HNF-6, OC-2 or OC-3 at e12.5 show an important increase in the number of dI6 containing OC factors from stage e10.5 to e12.5 (D) but the percentage of dI6 containing OC decreases (M). (P,Q) The number of Dmrt3+ dI6 containing HNF-6 and OC-2 is small. The pictures show left hemisections as indicated to the left. Dashed lines delineate the spinal cord and the lumen of the ventricle. Insets are magnified views of boxed regions. Arrowheads point to double-labeled cells. Mean values ±SEM, n ≥ 3. Scale bars = 100 μm.