OSVZ progenitors of human and ferret neocortex are epithelial-like and

OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling Simone A Fietz, Iva Kelava, Johannes Vogt, Michaela Wilsch-Bräuninger, Denise Stenzel, Jennifer L Fish, Denis Corbeil, Axel Riehn, Wolfgang Distler, Robert Nitsch and Wieland B Huttner I. Additional quantitative information to Figures Fig. 1 (a c) Human cortical wall corresponding in total to 3.5-8 mm of ventricular surface was quantified. The number of Pax6+ mitoses/total mitoses and Tbr2+/total mitoses analyzed were: Pax6, ventricular zone (VZ), 10-wpc 305/318, 12-wpc 180/184, 14-wpc 326/329, 16- wpc 204/210; Pax6, SVZ, 10-wpc 114/127, 12-wpc 75/89, 14-wpc 218/242; Pax6, ISVZ, 16- wpc 29/54; Pax6, OSVZ, 16-wpc 215/244; Tbr2, VZ, 10-wpc 8/396, 12-wpc 4/179, 14-wpc 16/413, 16-wpc 11/358; Tbr2, SVZ, 10-wpc 148/178, 12-wpc 88/111, 14-wpc 242/351; Tbr2, ISVZ, 16-wpc 97/105; Tbr2, OSVZ, 152/347. (l, m) The number of Pax6+ mitoses/total mitoses and Tbr2+/total mitoses analyzed in the developing ferret neocortex were: Pax6, VZ, E32 117/118, E39 89/90, P10 41/41; Pax6, SVZ, E32 141/152, E39 136/143, P10 21/22; Tbr2, VZ, E32 15/187, E39 16/131, P10 3/26; Tbr2, SVZ, E32 129/153, E39 78/120, P10 19/46. Fig. 3 (d) Human cortical wall corresponding to a total ventricular surface of 1.3 mm (z-depth 11-13 µm) was analyzed. VZ, 136 mitotic cells; SVZ, 95 mitotic cells. (g) A total of 27 (Pax6+) and 1

54 (Tbr2+) mitotic cells, respectively, were analyzed in 13-wpc human neocortex. The percentage of the total mitotic SVZ-progenitors showing Tbr2 expression (irrespective of the absence or presence of a basal process) in the 13-wpc human fetal neocortex was 57%. This value is slightly lower than that found for the 14-wpc human fetal neocortex (Fig. 1b, black column), which may reflect inter-fetus variation or the differences in the experimental conditions used. (l) Ferret cortical wall corresponding to a total ventricular surface of 1.4 mm (z-depth 10-15 µm) was analyzed. VZ, 73 mitotic cells; SVZ, 87 mitotic cells. (o) A total of 34 (Pax6+) and 93 (Tbr2+) mitotic cells, respectively, were analyzed in E39 ferret neocortex. Fig. 5 (b) A total of 104 (VZ), 160 (ISVZ) and 106 (OSVZ) mitotic figures in 14-16 wpc human neocortex were scored. (c) A total of 82 (Pax6-positive) and 72 (Tbr2-positive) mitotic figures in 14-16 wpc human SVZ were scored. (h) A total of 30 (VZ) and 78 (SVZ) mitotic figures in E39 ferret neocortex were scored. (i) A total of 30 (Pax6-positive) and 34 (Tbr2-positive) mitotic figures in E39 ferret SVZ were scored. Fig. 6 (e n) Data are the mean of 3-10 (Ki67/Tbr2 double immunofluorescence, control for echistatin), 4-9 (Ki67/Tbr2 double immunofluorescence, echistatin), 3-7 (Ki67/Pax6 double immunofluorescence, control for echistatin), 3-4 (Ki67/Pax6 double immunofluorescence, echistatin), and 6-8 (Ki67/Pax6 double immunofluorescence or Ki67/Tbr2 double immunofluorescence, control and αvβ3 antibody) image quantifications. 2

II. Supplementary Figures Supplementary Fig. 1. Human and ferret SVZ progenitors lack apical polarity proteins. (a e) 13-wpc human fetal neocortex (PFA). Immunofluorescence (red) on cryosections for prominin-1 combined with DAPI staining (blue) (a, b), for Par3 (c), for apkc (d) and for ZO1 (e). Upper and lower boxes in (a) indicate SVZ and VZ areas shown at higher magnification in (b), or are representative of the SVZ and VZ areas shown in (c e). (f k) E39 ferret neocortex (PFA). (f i) Immunofluorescence (red) on cryosections for Par3 combined with DAPI staining (blue) (f, g), for apkc (h) and ZO-1 (i). Upper and lower boxes in (f) indicate SVZ and VZ areas shown at higher magnification in (g), or are representative of the SVZ and 3

VZ areas shown in (h, i). (j, k) Double immunofluorescence on cryosections for phosphovimentin (pvim, red) and Par3 (green). Stacks of 5-6 single optical sections are shown. Note the presence of Par3 immunoreactivity at the apical cell cortex of the mitotic VZ progenitor, but its absence from the mitotic SVZ progenitor. Scale bars, 50 µm (a, f), 10 µm (b e, g i), 5 µm (j, k). Supplementary Fig. 2. Human VZ, but not SVZ, progenitors express the apical membrane protein prominin-1. In situ hybridization for prominin-1 of cryosections of 10-wpc human fetal neocortex (PFA), combined with DAPI staining (blue) (a, b) or Ki67 immunofluorescence (green) (d i). (a c) Overview of VZ and SVZ; (a, b) antisense probe, (c) sense probe, both used under identical conditions of detection. Bright-field images are shown in (a c). Note that prominin1 mrna can be detected in the human VZ but not SVZ (b versus c). Scale bars, 30 µm. (d i) Higher magnification of the SVZ (d f) and VZ (g i), with each VZ/SVZ pair of panels cropped from the same image; (d, g) Ki67 immunofluorescence, (e, h) in situ hybridization with antisense probe, (f, i) merge. Note the lack of prominin-1 mrna in a cycling human SVZ progenitor but its presence in a cycling human VZ progenitor. Scale bars, 5 µm. 4

Supplementary Fig. 3. Ferret SVZ progenitors express markers of radial glia. Immunofluorescence (red) for nestin combined with DAPI staining (blue) (a, b), for GLAST (c) and for BLBP (d) on cryosections of E39 ferret neocortex (PFA). Upper, middle and lower boxes in (a) indicate cortical plate (CP), SVZ and VZ areas shown at higher magnification in (b), or are representative of the CP, SVZ and VZ areas shown in (c, d), respectively; arrowheads indicate cell body-associated immunoreactivity in the SVZ and VZ. Scale bars, 50 µm (a), 10 µm (b d). 5

Supplementary Fig. 4. Proposed implications of basal process retention for cell polarity, self-renewal, and symmetric versus asymmetric division of SVZ progenitors, and the consequences for neurogenesis. Note that, contrary to Figs. 1-7, the apical-basal polarity of the cortical wall is depicted with the basal lamina down in order to emphasize the present results. 6

(a) Divisions of progenitors in the VZ, the rodent SVZ, and the OSVZ found in ferret and human. (Top) In both, the developing lissencephalic (rodent) and gyrencephalic (ferret, human) cortex, neuroepithelial cells and subsequently RGCs (apical progenitors, APs) expand by symmetric proliferative divisions in the VZ (top row). APs exhibit apical-basal polarity, extend from the basal lamina (red) to the ventricular (apical) surface (blue), possess apical adherens junctions (black squares), their primary cilium protrudes from the apical membrane into the ventricular lumen (grey), and their mitosis occurs at the ventricular surface. (Middle and bottom rows) Note that in the middle row, only asymmetric (rather than symmetric) divisions generating basal progenitors (BPs) (left) or OSVZ progenitors (right) are illustrated. In the developing lissencephalic cortex (left), the switch of APs to repeated asymmetric differentiative divisions leads to their self-renewal and yields one BP with every division (middle row left). BPs delaminate from the VZ to form the subventricular zone (SVZ), retract their processes, lack apical-basal polarity at M-phase, and typically divide once symmetrically to generate two neurons (N), which leads to their consumption (bottom row left). In the developing gyrencephalic cortex (right), the switch of APs to repeated asymmetric differentiative divisions leads to their self-renewal and yields, with every division, either one inner SVZ (ISVZ) progenitor (not illustrated) that resembles the rodent BP, or one progenitor destined for the OSVZ (OSVZ-P) (middle row right). OSVZ progenitors lose apical contact and apical adherens junctions but, importantly, retain their basal process and basal lamina contact and hence basal polarity, and are proposed to undergo repeated asymmetric cell division, resulting in self-renewal and the generation of one neuron (N) with every division (bottom row right). (b) Consequences for neurogenesis. 7

Blue lines, self-renewal of one AP by repeated asymmetric differentiative divisions (left and right). Orange line (left), constant level of one BP generated from the repeated asymmetric differentiative divisions of one AP and consumed by symmetric neurogenic division, each generating two neurons. Red line (right), linear accumulation of OSVZ progenitors generated from the repeated asymmetric differentiative divisions of one AP and self-renewing by asymmetric neurogenic division, each generating one neuron. Green lines, accumulation of neurons in a linear (left) and progressive (right) fashion. Note the increase in neuron number resulting from two consecutive asymmetric self-renewing progenitor divisions, proposed to be made possible by basal process retention (right). 8