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Supplementary Figure 1. a-i, Genes that control cortical neuron migration and have been proposed to act downstream of Neurog2 remain expressed in Neurog2 mutant telencephalon. Expression of p35, RhoA and Dcx was analyzed by RNA in situ hybridization on frontal sections of E14.5 telencephalon. p35 expression in the cortical plate of a wild-type embryo (a) was reduced but not abolished in Neurog2-/- (b) and Neurog1-/-, Neurog2-/- (c) embryos. Expression of RhoAin wild-type cortical progenitors (d) was not significantly affected in Neurog2-/- (e) and Neurog1-/-, Neurog2-/- (f) embryos. Expression of Dcx in the wild-type cortical plate (g) was not extinguished in Neurog2-/- (h) and Neurog1-/-, Neurog2-/- (i) embryos. j, Strategy used to search for Neurog2 targets in the embryonic cerebral cortex. The dorsal telencephalon of wild-type, Neurog2-/- and Neurog1-/-, Neurog2-/- E13.5 embryos were analyzed by Affymetrix expression microarrays to identify genes regulated by loss-of-function (LOF) of Neurog2. Neurog1 and Neurog2 have partially redundant roles in cortical development, although Neurog1 has no overt function on its own . The dorsal telencephalon of E10.5 wild-type embryos was electroporated with a Neurog2 expression construct and the electroporated tissue harvested after 18hrs and analyzed with control electroporated tissue to identify genes regulated by gain-of-function (GOF) of Neurog2. Genes that showed reciprocal changes to their expression in LOF and GOF experiments were selected as good candidate targets of Neurog2 and their Gene Ontology annotation was examined. k, The strategy depicted in (j) identified Rnd2 as a potential target of Neurog2involved in signal transduction, based on its down-regulation and up-regulation in LOF and GOF experiments, respectively, and its Gene Ontology annotation. Scale bars, 500 μm. (a-i).

doi: 10.1038/nature07198 SUPPLEMENTARY INFORMATION

www.nature.com/nature 1

Neurog2-/- cortexpCIG pCIG-Neurog2

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Supplementary Figure 2. Overexpression of Neurog2 induces Rnd2 expression in the embryonic cerebral cortex. a, Electroporation of Neurog2 in the dorsal telencephalon of E10.5 wild-type embryos induced Rnd2 expression after 2 days of whole embryo culture. The development of the hybridisation signal was stopped as soon as the induction of Rnd2could be detected. b, Electroporation of Neurog2 in the dorsal telencephalon of a E14.5 Neurog2 null mutant embryos followed by 1.5 days of slice culture resulted in induction of Rnd2 expression. Scale bars, 500 μm, (a), 100 μm, (b).



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Supplementary Figure 3. A mutant form of Rnd2 that is unable to bind GTP (Rnd2T21N) lacks a dominant negative activity. a-d, Ex vivo slice culture experiments of wild-type E14.5 embryonic brains electroporated with GFP (a), a Rnd2siRNA (b) or an expression construct encoding a mutant form of Rnd2 protein that is unable to bind GTP due to mutation of threonine-21 to asparagine (Rnd2T21N), and has also been proposed to act as a dominant negative molecule by analogy with the activity of similar mutant forms of small Rho GTPases (c). (d) Quantification of neuronal migration in the experiments illustrated in (a-c) (n=3, >1400 cells per condition). Electroporation of Rnd2T21N does not perturb cortical neuron migration in the brain slice assay, in contrast with the Rnd2siRNA, suggesting that Rnd2T21N does not interfere with activity of endogenous Rnd2. (e) We also examined the capacity of Rnd2T21N to suppress the activity of exogenous wild-type Rnd2. Overexpression of wild type Rnd2 (using pCIG-Rnd2 construct) in E14.5 cortex results in a block of migration of cortical neurons (see also Suppl. Fig. 11). Co-electroporation of increasing molar concentrations of Rnd2T21N with wild-type Rnd2(from a 1:1 to a 10:1 ratio) did not interfere with Rnd2 activity and did not revert the migration phenotype (e). These experiments rule out a dominant negative activity for the GTP-binding mutant protein Rnd2T21N and are consistent with the observations of Nakamura et al and with their interpretations . The graph plots mean + s.e.m. Scale bars, 100 µm.



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Supplementary Figure 4. Efficiency and specificity of Rnd2 siRNAs. a, Western blot analysis of Rnd2 expression in P19 cells co-transfected with a pCAGGS- Flag-Rnd2 expression construct and with a control siRNA or with siRNAsagainst Rnd2 (Rnd2 siRNA#1 and #2), as indicated. Rnd2 siRNA#1 is named Rnd2 siRNA in the main text and figures. The two Rnd2 siRNAs efficiently silenced Rnd2 expression. Expression of actin was used as a loading control. b, Co-transfection of siRNA#1 with a mutated form of Rnd2 which is mismatched with siRNA#1 but encodes a wild-type Rnd2 protein (Rnd2*, see Full Methods) showed that Rnd2* is resistant to silencing by siRNA#1. c-f, Electroporation of Rnd2 siRNA#1 and Rnd2 siRNA#2 with GFP in E14.5 cortices resulted in similar migration defects after 4 days of slice cultures (n=6, >3000 cells per condition). g-j, Expression of the mutant form Rnd2* fully rescued the neuronal migration defect resulting from Rnd2 silencing in slice cultures, ruling out that this phenotype is due to an off target effect of the siRNA (n=3, >1500 cells per condition). k-n, Electroporation of Rnd2 siRNA#1 and Rnd2 siRNA#2 with GFP in E14.5 cortices resulted in similar cell morphology defects after 3 days of dissociated cell culture (n=3, >500 cells per condition). o-r, Expression of the mutant form Rnd2* fully rescued the cell morphology defect resulting from Rnd2silencing in dissociated cell cultures (n=3, >950 cells per condition). All graphs plot mean + s.e.m. Scale bars (c-e,g-i), 100μm, (k-m,g-i), 50μm.



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Supplementary Figure 5. Different methods of Rnd2 silencing produce similar cortical neuron migration defects. a,b, E14.5 embryos were electroporated in utero with a GFP expression construct (pCIG) together with a control siRNA or Rnd2siRNA, and the brains were harvested 3 days later (E17.5). The cortical neuron migration defect resulting from Rnd2siRNA electroporation is similar to that obtained by electroporating Rnd2siRNA ex vivo followed by 4 days of brain slice culture (Fig. 2b). c, Western blot analysis of Rnd2 expression in P19 cells transfected with pCAGGS-Flag-Rnd2 construct together with a control shRNA vector or with a construct encoding an Rnd2shRNA sequence identical to that of Rnd2siRNA#1 (see Full Methods). The Rnd2shRNA efficiently silenced Rnd2 expression. d,e, cells electroporated with the Rnd2shRNA construct show a migration defect in the slice culture assay after 4 days similar to that obtained by electroporating a Rnd2siRNA (Fig. 2b). A similar migration defect is also observed after in uteroelectroporation of the Rnd2shRNA construct in E14.5 embryonic brains harvested 3 days later (data not shown). shRNA-treated cells could be monitored by EGFP epifluorescence since the vector comprises an EGFP expression cassette together with an shRNA expression cassette. Scale bars, 100 µm.



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Supplementary Figure 6. Rnd2 knockdown affects all stages of cortical neuron migration. a, Distribution of cells electroporated with a control siRNA (white bars), Rnd2 siRNA#1 and Rnd2 siRNA#2 (red and yellow bars) in cortical slices divided in 8 bins after 4 days in culture (n=6, >3000 cells per condition). b-d, Rnd2 knock-down significantly reduced the migration of cells from the VZ and SVZ to the IZ (b), from the IZ to the CP (c) and from the lower and median CP to the upper CP (d). All graphs plot mean + s.e.m.



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Supplementary Figure 8. Rnd2 knockdown does not affect the proliferation or neuronal specification of cortical progenitors or the morphology of radial glial processes. a-f, Rnd2 knockdown does not interfere with the proliferation of cortical progenitors, measured by the fraction of electroporated cells having incorporated BrdU during a 2 hr exposure (a-c) or expressing the mitosis marker phosphohistone H3 (d-f) after 48 hrs of slice culture. The migration defect of Rnd2 siRNA electroporated cells is therefore not due to these cells remaining in a mitotic progenitor stage. g-l, Rnd2 knockdown does not interfere with the correct specification of electroporated cells to a cortical neuron phenotype, as shown by normal expression of Cre in mice where Cre is inserted in the locus of the cortical neuron-specific gene Nex/Math2 (g-i), and by normal expression of the neuronal marker Map2 in dissociated culture of electroporated cortex, 3 days after electroporation (j-l). The radial migration defect of Rnd2 siRNA electroporated cells is therefore not due to these cells switching to a cortical interneuron or astrocytic fate, respectively. m,n, Rnd2 knockdown does not affect the morphology of the radial glial processes marked by nestin, thus ruling out that the migration phenotype of Rnd2 deficient neurons is an indirect consequence of defects in the radial glial processes, which serve as substrate for the radial migration of cortical neurons. o, Rnd2 knockdown does not promote apoptotic cell death, as shown by the similar fraction of electroporated cells labelled with an antibody against activated caspase 3 in control and Rnd2 siRNA electroporatedslices. n=3-4 in each experiment, >600 cells counted per condition. All graphs plot mean + s.e.m; ns denotes p>0.05 from Student’s t-tests. Scale bars, 100 µm.



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