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S. Key, . Table, R. Or, . Source, and . Identifier, Antibodies Rabbit polyclonal anti-mCherry 1/300 Clontech Cat#632496, p.10013483

, Chicken polyclonal anti-GFP 1/1000 Aves lab Cat#GFP-1020, p.10000240

, Mouse monoclonal (IgG2a) anti-PCNA Santa Cruz Cat# sc-56 AB-16.0387; RRID: AB_628110 Rabbit polyclonal (IgG2a) anti-PCNA 1/500 GeneTex Cat#GTX124496 AB-16, p.11161916

, Mouse monoclonal (IgG1) anti-Flag 1/500 Sigma Cat#F1804 AB-16.0331, p.262044

, Mouse monoclonal (IgG2a) anti-GS 1, pp.1000-1006

, Millipore Cat#MAB302 AB-16, p.342

, Human polyclonal anti-Hu 1/10000 Gift from B. Zalk AB-16, p.356

. Anti-parvalbumine, , 2000.

, Chemicals, Peptides, and Recombinant Proteins 4-Hydroxytamoxifen Sigma-Aldrich T176, pp.68392-68427

, 9-tert-ButylDoxycycline, hydrocloride salt Echelon Biosciences B-0801, pp.233585-94

D. Sigma-aldrich and D. , , pp.28718-90

, Critical Commercial Assays Taq DNA polymerase EmeraldAmp Takara Cat# 330A Experimental Models: Organisms/Strains Zebrafish: Tg(T2Kbactin:lox-stop-lox-hmgb1-mcherry) jh15, pp.110221-110225

, Tg(her4.1:EGFP) y83 [25] ZFIN ID: ZDB-FISH, Zebrafish, 14830.

, Zebrafish: Tg(À3her4, vol.3, pp.298-331

, Tg(-3.5ubi:loxP-GFP-loxP-mCherry), Zebrafish, vol.72

, Tg(ubb:LOX2272-LOXP-RFP-LOX2272-CFP- LOXP-YFP) a131 , ubi:Zebrabow-M [73, Zebrafish

, Tg(her4:rtTA, cmlc2:GFP) [71] ZFIN ID: ZDB-PUB, Zebrafish, pp.101209-101235

, Tg(GFP:biTRE:H2amCherry,crist:Venus), Zebrafish, vol.36, pp.130816-130818

. Oligonucleotides-rtta-probe-fw:-atgtctagactggacaagagcaaagtc and . N. Gga-tccattaaccctcactaaagggactaactgtcgaccttgtc, GGGGACAACTTTGTATAGAAAAGTTGTTTTGCAT TATTTCCCTAATTTTAAATGTC (À999-1028bp from AY691485.1) her4AttB1-Rv: GGGGACTGCTTTTTTGTACAAACTTGGTCA GGATCAGATCTGAGCTG (À3389-3409bp from AY691485.1) N/A N/A 4OHT and 9TB treatments 4OHT treatments were performed as described [35] (10so.: 5mM, 4 hr; 1 or 2 dpf: 5mM, 6 hr; 5 dpf: 5mM, 30 hr; 1.5mpf: 5mM, 100 hr). Fish were then washed, transferred into fresh water, and grown as usual. For clonal analyses, zebrabow) embryos were recombined for 1 hr with 5mM 4OHT and 5dpf Tg, pp.1-4

, 6 hr; 5 dpf ? 15 dpf: 10mg/mL, 6 hr; 1.5 mpf: 10mg/mL, 96 hr) 9TB treatments were performed in the dark at 28 CGFP:biTRE:H2amCherry, crist:Venus) line, a small GFP cluster was visible in the parenchyma at posterior levels even in the absence of treatment (not shown); it did not interfere with our analysis. For polyclones analysis, 9TB was applied at 0.5mg/mL for 10min onto 1dpf embryos. The embryos were then rinsed, grown and analyzed at 5dpf. Control experiments to assess clonality were analyzed on embryos treated with 0.5mM 4OHT for 10min at 5dpf, followed by a one-to two-day chase (for mCherry to be visible) Nearest neighbor distances were calculated per hemisphere (Imaris spot to spot closest distance on 20 larval hemispheres ) and their cumulative probability distribution was plotted. We chose induction conductions such that in more than 85% of cases, induced cells were distant from each other of more than 15mm (equivalent to 4 cells diameters) Our brainbow analysis indicates very few cases, if any, of clone fragmentations. We therefore estimated that an initial distance of 4-cell diameters in the majority of cases should be sufficient to avoid this issue. To assess the occurrence of clone fusion, we also compared the estimated numbers of clones at time t+2days and at the analysis time point, 1.5mpf, and found that both were equivalent (Figure S5) Three hemispheres with ambiguous cases were discarded, and isolated mCherry cells, larvae for 10min with 0.5mM 4OHT. 9TB was dissolved the Tg

, Whole-mount brain clearing Whole zebrabow dissected adult brains were cleared using the SCALE approach [77] and mounted in glycerol 35% Whole her4 H2a-mCherry,9TB(15dpf) mCherry immunostained brains were cleared using the CUBIC approach [78] with the following incubations: CUBIC-1, 37 C, overnight; primary antibody, 37 C, 48 hr; secondary antibody, Mounting was done in CUBIC-2

, S5, S6, and 7 were taken using an inverted confocal microscope (Zeiss LSM700) and processed with the ZEN 2011 software (Carl Zeiss MicroImaging) Figures 2, 3A?3D, and S4A?S4L images are tile scans followed by maximum intensity projections of 8 squares and around 20 confocal optical sections. Images were then processed with Imaris or Photoshop CS6. Figures 3E?3F 0 , S7M, and S7N were processed with a median filter 3x3x3 followed or not by removal of object bigger than 250mm to remove background in the vessels or surface background, Confocal microscopy, image acquisition and processing All images except Figures 3E?3F 0 situ hybridization pictures (Figure 7; Figure S6) were Current Biology e2 photographed with a Zeiss Axiozoom V6 Macroscope. CUBIC cleared brain was imaged using an upright confocal microscope (Zeiss LSM710) with a Plan-Apochromat 20X/0, pp.3288-3301, 2017.

. Multiphoton-microscopy, Japan) and integrating galvo scanners (VM500+, GSI, USA), a high-index immersion objective with 4mm working distance (XLPN25XSVMP, Olympus) and a motorized sample stage for mosaic acquisition (PS3H122 and ProScan H117, Prior Scientific) Excitation was provided by a Titanium-sapphire oscillator (Chameleon Ultra2, Coherent, USA) and an optical parametric oscillator (compact OPO, Coherent, USA) For simultaneous excitation of CFP, YFP and RFP signals, TiS, OPO and two-color equivalent excitation l 3 = 2/(1/ l TiS + 1/ l OPO ) wavelengths were set to 850 nm, 1100 nm and 959 nm, respectively. Nonlinear signals were selected with appropriate dichroics (Semrock FF520-Di02 FF560-Di01) and filters (Semrock FF01-475/64 FF01-538/40 FF01-607/70), and epidetected on three separate channels by photomultiplier modules (P25PC SensTech UK and H7422P-40 Hamamatsu Japan) and lab-designed photon counting electronics. The pixel dwell time was 12 ms, and the voxel size was 0.8 3 0.8 3 2 mm 3 . For whole pallium imaging, cleared brains were mounted on Scale media between two 150mm-thick glass coverslips separated by a spacer, and a mosaic of 9 3 6 volumes each encompassing 260 3 285 3 1000 mm 3 with a 20% lateral overlap was recorded. Multicolor multiphoton stacks were preprocessed for flat-field correction and stitched with the open-source FIJI Image Stitching plugin using the Max Intensity fusion method [80]. Unlike cells, blood vessels exhibited intense fluorescence in all three channels and appeared ''white.'' These signals were removed using MATLAB by zeroing pixels having this characteristic. This processing step removed the vessels images without affecting cell signals. Semi-automatic cell detection was then performed using the Imaris Spot detection tool separately in the three channels. The automated detection exhibited an error rate The sparsity of labeling led to only a few ambiguous cases, based on cell colors, spatial clustering and cell sizes

, hemispheres or cells processed can be found in the text and legends for figures (Figures S5E and S5F). n = 20 pallial hemispheres at 1dpt and 24 pallial hemispheres at 1.5mpt. The numbers in bracket indicate the number of hemispheres concerned for each number of induced cells/clones (Figure 5F) Data are presented as mean ± SEM, and statistical differences were determined using t test, QUANTIFICATION AND STATISTICAL ANALYSIS All details on the number of brains