Figure 1: Horizontal vRG divisions produce oRG cells.

(a) Time-lapse stills of horizontal vRG division labelled with Adeno-GFP in GW16 human fetal cortical slice that results in generation of an oRG cell. vRG cell (blue arrowhead) starts at a distance from the ventricle, descends during INM, and divides with a horizontal spindle orientation at the ventricular surface (dotted white line at 17:45). Basal daughter (white arrowhead) retains basal fibre (asterisk) and rapidly exits the ventricular zone, adopting an oRG cell morphology. Apical daughter (orange arrowhead) inherits entire apical domain (white arrow) and regrows a basal fibre, becoming a self-renewed vRG cell. Cells of interest are schematized at beginning and end of sequence to more clearly demonstrate morphology and inheritance of basal fibre and apical domain. Scale bar, 10 μm. (b) Fate staining of daughter cells from division in a confirms that the basal daughter (white arrowhead) is Tbr2−/Sox2+, consistent with oRG identity, and the apical daughter (orange arrowhead) is also Tbr2−/Sox2+, consistent with vRG identity. Scale bar, 10 μm. (c) z-plane analysis of basal daughter from division in panel a rotated 90° confirms lack of apical process. Scale bar, 10 μm. (d) Time-lapse stills of vertical vRG division in GW16 human fetal cortical slice after which both daughters inherit half of the AD (white arrows) and neither daughter adopts oRG morphology. Part of the basal fibre is not visible due to its location out of the imaged z-stack. Scale bar, 10 μm. (e) Schematic of vRG cells undergoing mitosis at the ventricular surface (solid red line) with vertical, oblique and horizontal cleavage angles. Mitotic spindle orientation was analysed with respect to the ventricular surface. (f) Images of vRG cells in fixed human fetal cortical slices undergoing mitosis with vertical (left), oblique (center), and horizontal (right) mitotic spindle orientations at the ventricular surface. Scale bars, 5 μm. (g) Quantification of mitotic spindle orientation in fixed slices throughout peak neurogenesis and oRG cell generation, and during time-lapse imaging of GW16/18 human fetal cortical slices. Cells used for calculation in fixed slices were pH3+, pVim+ cells in anaphase or telophase that displayed a basal fibre and were located at the ventricular surface.