Figure 13

Dextran dye micro-injections (green) into the cerebral artery via the dorsal sinus of embryos at E95% to E100%/POI stage. The S-phase marker BrdU (red) identifies the cap and the immunolocalization of tyrosinated tubulin (white) delineates the protoniche. Panels (A, Ai, Aii, Aiii) show the same specimen. (A) All three channels are shown separately with the composite image at the bottom right: top left, tyrosinated tubulin (white); top right, Brd U-labeled cells (red); bottom left, dextran dye filled blood vessels (green) running through the protoniche (outlined in blue). An example of a lacunae filled with dextran dye (asterisk), a capillary that has an expanded blind ending, is seen in the bottom left of (A), and an asterisk is also placed where it exists in the tyrosinated tubulin channel, top left of (A). (Ai) Leica software for the orthogonal slicer function was applied to this stack of images to obtain coronal (xz-axis) and sagital views (yz-axis) through the pore and the protoniche at the dotted reference lines. The intersection of the reference lines marks the center of the pore, which can be seen in the z-axis at the bottom and right side, showing the vasculature running concommitant with the pore and just underneath the pore and the protoniche. (Aii) A confocal stack (26 μm) from the ventral surface through to the mid-brain in a compressed view, showing that the cerebral artery (asterisk) bifurcates and then pushes ventrally toward the two hemispheres, each branch dividing laterally and then many times over into the neuropile regions as continuous tubes. The solid vertical line demarks the midline of the brain and the arrowhead denotes where the pore of the niche is located. (Aiii) The dextran (green) channel alone, more clearly illustrating the vasculature pathway from the midbrain (via the cerebral artery) to the ventral regions of the brain, and finally to regions around and in the niche. (B) Another example of a protoniche that has blood vessels confluent with the niche structure. The separate confocal channels are the same as in (A), and the arrow points to the central fibrous area of the pore. (Bi) A slightly higher magnification of (B) imaged deeper into the pore, showing the vasculature (arrows) running through the pore itself. All three channels are displayed as described for (A, B). (C) The vasculature can sometimes be observed to be contiguous with the protoniche (outlined in blue). The arrows point to a blood vessel that becomes part of the protoniche structure. Scale bars: 20 μm (A, Aii, Aiii, B, C), 10 μm (Bi).