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Fig. 7. Midline crossing of early-born PGN/RTN neurons. (A,B) EYFP-expressing PGN/RTN neurons (left panels) labelled with BrdU (middle panels). Right panels show merged views. BrdU was injected at E12.5 (A) or 14.5 (B). Transverse sections of the metencephalon from E18.5 mice. (C,D) High-power views of the area outlined by rectangles in A,B. Neurons labelled by EYFP-electroporation at E12.5 were also labelled by BrdU (white, arrowheads in the right panel of C) when BrdU had been injected at E12.5 (A,C, purple), but not at E14.5 (B,D, purple). E14.5 BrdU injection labelled EYFP-expressing cells occupying the ventral region of the ipsilateral PGN (right panel of B). (E) Quantification of BrdU labelling. Ordinate represents the number of BrdU-positive and EYFP-expressing cells (N_BrdU) divided by the number of EYFP-expressing cells (N_total) in the contralateral pontine region. The day of BrdU injection is shown along the y-axis. Vertical bar represents s.e.m. Each column represents pooled data from five different embryos. (F) Diagram showing the quantification method. The mediolateral extent of the PGN (l) was defined as the distance from the midline to the lateral end of the PGN, as visualized by EYFP labelling on the ipsilateral side. All EYFP-labelled cells located within this distance from the midline on the contralateral side (i.e. the region shown by the red contour) were counted. The labelled side is towards the left. Both heavily and lightly labelled cells, in the region described in F, were counted for the analysis. Broken vertical lines in A and B show the midline. Scale bar: 200 µm for A,B; 50 µm for C,D.

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