Development 143: doi:10.1242/dev.129783: Supplementary information
Development • Supplementary information
Fig. S1. LGN antibody characterization. Immunostaining for LGN on COS cells transfected with
plasmids expressing GFP alone (CAG-GFP), EGFP::LGN, or AGS3 and GFP. Cultured cells were fixed
48hr after transfection and stained with the anti-LGN antibody (Sigma, red). While 100% of the cells
transfected with EGFP::LGN are stained with the LGN antibody, as expected, 73% of the cells transfected
with AGS3 are also stained with the anti-LGN antibody (arrowheads), showing cross-reactivity. Scale bar:
20 m.
Fig. S2. LGN inactivation does not affect proliferation in the retina at P0. Immunostaining for BrdU
(A, B) and pH3 (C, D) on retinal sections at P0 in Lgn +/- (A,C) and Lgn -/- (B,D). Scale bar : 20 m. (E, F)
Quantification of total BrdU+ (E) and pH3+ (F) cells in 4 Lgn +/- and 4 Lgn -/- retinas at P0 from 2
experiments. Data presented as mean ± s.e.m.; two-tailed t-test. Yellow dashed line identifies the apical
side.
Development • Supplementary information
Development 143: doi:10.1242/dev.129783: Supplementary information
Development 143: doi:10.1242/dev.129783: Supplementary information
Development • Supplementary information
Fig. S3. NUMB localization is not affected in LGN -/- retinas. Confocal images of mitotic cells in the
mouse retina at P0 in Lgn +/- and Lgn -/- . Sections were stained for NUMB and Hoechst to visualize
DNA. NUMB staining is concentrated apically (arrowheads) in both conditions. Scale bar: 10m and 1m.
4 Lgn +/- and 5 Lgn -/- were analyzed.
Fig. S4. LGN inactivation does not significantly affect overall retinal cell type proportions in the
adult retina and PTXa delocalizes LGN from the cell cortex. (A) Predicted retinal cell proportions in
Lgn -/- when 13% of terminal divisions are changed from an asymmetric to a symmetric photoreceptorphotoreceptor daughter cell pair (boxed), as observed in the lineage study presented in Figure 4C. Raw
lineage data from Gomes et al. 2011 was used as a reference for wildtype values. (B) Percentage of cell
type in clonal analysis shown in Figure 4B-D. (C, D) Hematoxylin/Eosin (H/E) coloration on Lgn +/- and
Lgn -/- retinal sections at P14. (E-H) Immunostainning for CHX10, a bipolar cell marker, and PAX6, an
amacrine cell marker, on retinal sections at P14 in Lgn +/- and Lgn -/-, as indicated. GCL : ganglion cell
layer, INL : inner nuclear layer, ONL : outer nuclear layer. Scale bar : 20m. (I-K) Quantification of
photoreceptor nuclei column height (I), CHX10-positive (J) and PAX6-positive (K) cells in the inner nuclear
layer in 4 Lgn +/- and 4 Lgn -/- retinas from 4 independent experiments, mean ± s.e.m., two-tailed t-test.
(L) Immunostaining for LGN on retinal explants 5 days after electroporation at E17.5 with EGFP or EGFPPTXa. LGN labelling at the membrane (arrowhead) is severely reduced in cells expressing EGFP-PTXa
compared to EGFP or untransfected controls (asterisk). Scale bar: 1 m.
Development • Supplementary information
Development 143: doi:10.1242/dev.129783: Supplementary information
Development 143: doi:10.1242/dev.129783: Supplementary information
Supplementary Material and Methods
Mitotic Spindle Orientation
Centrosomes were stained with anti-γtubulin antibody and DNA labeled with Hoechst. The orientation of
the spindle axis was measured from the x, y, and z coordinates of the centrosome pairs relative to the
plane of the neuroepithelium corresponding to the interface between the RPE and the neural retina using
a MATLAB program (The Mathworks Inc., Natick, MA). We analysed the centrosome pairs in cells in either
metaphase or anaphase-telophase. Statistical comparisons were done using Student's t test.
Histology, Immunofluorescence and Image acquisition
Development • Supplementary information
Primary antibodies used were: rabbit anti-LGN (GPSM2) (1:200 Sigma cat# HPA007327 lot# A41537) and
from F. Matsuzaki (Konno et al., 2008), rat anti-ZO-1 (1:200 DSHB cat# R26.4C), rabbit anti-GFP (1:1000
Life Technology cat# A11122, lot#1691382), mouse anti-BrdU (1:400 Dako cat# M074401-8, clone
BU20A, lot# 20006290), rabbit anti-phospho-histone 3 (1:400 Millipore cat # 06-570, lot# 2370127), goat
anti-γ-tubuline (1:400 Santa Cruz Biotechnology cat# SC-7396, lot# J3013), sheep anti-Chx10 (1:400
Exalpha cat# X1180P, lot# 12969), rabbit anti-Pax6 (1:400 Chemicon Millipore AB2237, lot# 2455611),
sheep anti-GFP (1:400 Serotec cat# 4745-1051, lot# 0607), rabbit anti-Numb (1:200 Abcam cat#
AB14140, lot# GR160743-1).