Figure S2.

Evidence Supporting Abundant Direct Neurogenesis in OB, but Not NCx, at E12.5, Related to Figure 2

(A) Images from parasagittal sections of mouse OB and NCx at E12.5 stained for PH3 (mitoses), Pax6 (RGCs) and Tbr2 (IPCs). Arrowheads point at double-positive nuclei shown at high magnification in the insets.

(B) Quantification of linear density of apical and basal PH3+ nuclei in OB and NCx positive for Pax6 or Tbr2. Values are mean + SEM; n = 4 embryos per group and 2-3 confocal planes for each embryo; t tests; ^∗p < 0.05; ^∗∗∗p < 0.001.

(C) Single confocal plane images from a sagittal section of the OB at E12.5 stained for Pax6 and Tuj1. Cells 1 and 2 indicated by arrowheads co-express the two markers, as demonstrated by the high-magnification images including orthogonal planes at the levels indicated.

(D and E) Identification of neurons born from apical divisions in vivo. Experimental design used shown in (D). The timing for analysis after BrdU administration was defined following our previous measures of the duration of G2+M phases of the cell cycle. Images in (E) are from a sagittal section of the OB at E12.5 stained with BrdU and Tbr1. The arrowhead indicates the cell magnified to the right, with orthogonal views demonstrating co-localization. This cell is full of BrdU and expresses Tbr1 close to the ventricular border. Similar cells were never observed in NCx.

(F and G) Experimental design and examples of cells born directly or indirectly from VZ mitoses. Upon BrdU injection at E12.5, this is incorporated by RGCs and IPCs in S-phase (F). BrdU+ cells start entering mitosis 3hr later, and then the ventricular side is electroporated, transducing RGC cell bodies next to the apical side but not IPCs in the SVZ, as these are distant from the electroporation surface. After allowing subsequent rounds of cell division, only neurons born by direct neurogenesis (not dividing again) will express GFP and contain 100% BrdU; cells with 50% BrdU or less are born after additional cell cycles, when direct versus indirect neurogenesis are indistinguishable by this method. (G) Examples of neurons in OB and NCx at E18.5 labeled as indicated in (F). High magnifications show examples of GFP+ cells retaining high (100%), medium (50%) or low (25%) levels of BrdU.

Scale bars: 100 μm (A), 25 μm (C, E, and G).