A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System

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1 Cell Reports, Volume 9 Supplemental Information A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System Morgane Belle, David Godefroy, Chloé Dominici, Céline Heitz-Marchaland, Pavol Zelina, Farida Hellal, Frank Bradke, and Alain Chédotal

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4 Supplementary Figure Legends Figure S1 (related to Figure 1) 3DISCO clearing of immunolabeled embryos. (A) Dorsal view of an E11.5 Wnt1:Cre;Tau mgfp embryo labeled with anti-gfp. (B) Optical coronal section through the hindbrain of an E11 mouse embryo stained with anti-robo3 antibodies (shown in Figure1I). (C) measurement of FR length in E16 embryos labeled withanti-robo3. (D) confocal image of the FR of an E16 embryo labeled with anti-robo3 and cleared with 3DISCO. (E) shows the same embryo than in C, but re-imaged after being stored 6 month. (F) IPN view of the FR projection in an E16 embryo labeled with anti-tag-1 antibodies. Scale bars represent 50µm (F), 100µm (A), 150µm (B) and 200µm (C, D, E) Figure S2 (related to Figure 3) Dorsal views of the medial habenula in knockout embryos. E16 embryos immunolabeled with anti-robo3 antibodies. It illustrates the abnormal and variability of dorsal midline crossing defects of habenular axons in Netrin-1 -/- and DCC -/- knockouts. Scale bars represent 150 µm (A), 200 µm (B, C, D, E, F). 3

5 Epitope Species Dilution Source Catalog Number Barlh1 Rabbit polyclonal 1:300 Sigma HPA ß-Gal Rabbit IgG 1:1000 Cappel ChAT Goat polyclonal 1:200 Millipore ab144p DCC Goat polyclonal 1:150 Santa Cruz sc-6535 DsRed Rabbit polyclonal 1:500 Clontech FoxP2 Rabbit polyclonal 1:1000 Abcam ab16046 GFP Chicken polyclonal GFP Rabbit IgG 1:800 1:800 Abcam ab13970 Life Technologies A11122 Robo1 Goat polyclonal 1:300 R&D System AF1749 Robo2 Rabbit polyclonal 1 :300 Abcam ab75014 Robo3 Goat polyclonal 1:300 R&D System AF3076 Tag1 Rabbit 1 :10000 Domna Karagogeos Tag1 Goat polyclonal 1:1000 R&D System AF4439 Supplementary table S1 (related to experimental procedures) 4

6 Epitope Fluorophore Origin Dilution Source Catalog # Goat Alexa 488 Donkey 1:600 Life technologies A11055 Goat Alexa 488 Bovine 1:500 Jackson Mouse Cy3 Donkey 1:400 Jackson Mouse Alexa 488 Donkey 1:600 Rabbit Alexa 488 Donkey 1:600 Life technologies Life technologies A21202 A21206 Rabbit Cy3 Donkey 1:400 Jackson Chicken Cy3 Donkey 1:300 Jackson Rabbit Alexa 647 Donkey 1:400 Jackson Supplementary table S2 (related to experimental procedures) 5

7 Supplementary Table 1 (related to experimental procedures) List of the primary antibodies use for whole-mount staining Supplementary Table 2 (related to experimental procedures) List of the secondary antibodies use for whole-mount staining 6

8 Supplementary movies legends Movie S1, related to Figure 1 3D movie of an E13 embryo labeled with anti-chat antibodies. Movie S2, related to Figure 1 3D Movie of oculomotor nerves in an E13 embryo labeled with anti-chat antibodies. Movie S3, related to Figure 1 3D movie of an E13 embryo double labeled with anti-chat and anti-tag-1 antibodies. Movie S4, related to Figure 1 3D movie of the forelimb motor innervation in an E13 embryo labeled with anti-chat antibodies. Movie S5, related to Figure 1 3D movie of a spinal cord segment from an E11.5 Wnt1:Cre;Tau GFP embryo labeled with anti-gfp. Movie S6, related to Figure 1 7

9 Scan, using the orthoslicer tool, through the longitudinal axis of the spinal cord of an E11.5 Wnt1:Cre;Tau GFP embryo labeled with anti-gfp. Movie S7, related to Figure 1 3D movie of the E11 spinal cord and caudal hindbrain of a wild-type embryo after whole-mount Robo3 immunostaining showing the array of commissural axons crossing the floor plate. Movie S8, related to Figure 1 Scan, using the orthoslicer tool, through the longitudinal axis of the spinal cord and hindbrain of an E11 wild-type embryo labeled with anti-robo3 (see also movie 8) Movie S9, related to Figure 2 3D movie of an E16 mouse brain labeled with anti-robo3 Movie S10, related to Figure 2 3D movie of the fasciculus retroflexus (FR) projection from the medial habenula (mhb) to the interpeduncular nucleus (IPN), followed by a scan through the IPN region. E16 mouse brain labeled with anti-robo3 Movie S11, related to Figure 3 3D movie showing FR guidance defects in a DCC -/- E16 embryo 8

10 Movie S12, related to Figure 3 3D movie showing FR guidance defects in a Netrin-1 -/- E16 embryo Movie S13, related to Figure 4 3D movie showing FR guidance defects in a Robo1 -/- ;Robo2 -/- E16 embryo Movie S14, related to Figure 4 3D movie showing FR guidance defects in a Robo3 -/- E16 embryo Movie S15, related to Figure 4 3D movie showing FR guidance defects in a Shh :Cre ;Slit1 -/- ;Slit2 lox/lox E16 embryo Movie S16, related to Figure 4 3D movie showing FR guidance defects in a Shh :Cre ;Slit1 -/- ;Slit2 lox/lox ;Slit3 -/- E16 embryo. 9