Fig. 2. gata2::GFP-positive axons pioneer the POC and also mark the majority of later POC axons. Gata2:GFP cells and their axons are depicted in green and the primary neuronal scaffold in red. Rostral is towards the left and dorsal is towards the top. Images are maximum intensity projection (MIP) views of confocal z stacks. (A-D) Lateral images at 21, 23, 25 and 27 hpf showing the relative position of gata2:GFP cells (green) with respect to the neuronal scaffold revealed with acetylated alpha tubulin (red). (E-H) Corresponding frontal views of the POC show gata2::GFP-expressing axons pioneer the POC. At 21 hpf, only early neurons differentiating away from the neuroepithelium can be seen, but no axons are present where the POC will form (broken white line) (E). At 23 hpf, a small number of axons can be seen across the POC; these early axons express GFP although because of fixation some of the GFP signal is too weak to be visible in the overlay image (F). At 25 and 27 hpf, respectively, the POC thickens. GFP-positive axons can be seen spanning the entire width of the commissure (G,H). The small number of axons that appear to not express GFP reflects axons from other brain regions that project their axons along this tract. Scale bar: 20 µm.