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Genetic Mosaic Dissection of Lis1and Ndel1 in Neuronal MigrationSimonHippenmeyer,1,* YongHa Youn,2 HyangMiMoon,2,3 Kazunari Miyamichi,1 Hui Zong,1,5 AnthonyWynshaw-Boris,2,4

and Liqun Luo1,*1Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA2Department of Pediatrics and Institute for Human Genetics3Biomedical Sciences Graduate Program4Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchUniversity of California, San Francisco School of Medicine, San Francisco, CA 94143, USA5Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA*Correspondence: [email protected] (S.H.), [email protected] (L.L.)DOI 10.1016/j.neuron.2010.09.027

SUMMARY

Coordinatedmigration of newly born neurons to theirprospective target laminae is a prerequisite for neuralcircuit assembly in the developing brain. The evolu-tionarily conserved LIS1/NDEL1 complex is essentialfor neuronal migration in the mammalian cerebralcortex. The cytoplasmic nature of LIS1 and NDEL1proteins suggest that they regulate neuronal migra-tion cell autonomously. Here, we extendmosaic anal-ysis with double markers (MADM) to mouse chromo-some 11 where Lis1, Ndel1, and 14-3-33 (encodinga LIS1/NDEL1 signaling partner) are located. Anal-yses of sparse and uniquely labeled mutant cells inmosaic animals reveal distinct cell-autonomous func-tions for these three genes. Lis1 regulates neuronalmigration efficiency in a dose-dependent manner,while Ndel1 is essential for a specific, previously un-characterized, late step of neuronal migration: entryinto the target lamina. Comparisons with previousgenetic perturbations of Lis1 and Ndel1 also suggesta surprising degree of cell-nonautonomous functionfor these proteins in regulating neuronal migration.

INTRODUCTION

The assembly of functional neural circuits requires the segrega-tion and interconnection of distinct classes of neurons. In thevertebrate central nervous system, a prevalent motif in neuronalorganization is the coalescence of neuronal types into stratifiedlayers or laminae (Ramon y Cajal, 1911). Coordinated migrationof newly born neurons from their birthplace to their final positionrepresents a fundamental mechanism to achieve laminationwithin all structures of the brain. In the past decades, distinctneuronal migration modes as well as a rich catalog of moleculescontrolling neuronal migration have been identified (Heng et al.,2010; Marin et al., 2010).Neuronal migration and the laminar positioning of projection

neurons within the mammalian neocortex has been intensely

studied. Cortical layering occurs in an ‘‘inside-out’’ fashionwhereby earlier born neurons occupy deep layers and succes-sively later born neurons settle in progressively upper layers (An-gevine and Sidman, 1961; Rakic, 1974). Upon radial glia progen-itor cell (RGPC)-mediated neurogenesis, newborn migratingcortical projection neurons are bipolar-shaped in the ventricularzone (VZ) but then convert to a multipolar morphology within thesubventricular zone (SVZ) andmigrate into the intermediate zone(IZ). A switch from the multipolar state back to a bipolarmorphology precedes radial glia-guided locomotion of projec-tion neurons toward the cortical plate (CP), with the trailingprocess concomitantly developing into the axon. Once theneuron arrives in the CP, the leading process attaches to thepial surface and the neuron undergoes terminal somal transloca-tion to reach its final location (Nadarajah et al., 2001; Noctoret al., 2004; Rakic, 1972; Tsai et al., 2005).The importance of neuronal migration for cortical lamination

is highlighted in patients that suffer from isolated lissencephalysequence (ILS) or Miller-Diecker syndrome (MDS). Lissence-phaly is characterized by a smooth brain surface with anabsence or severe reduction of gyri, abnormal lamination, andthickening of the cerebral cortex. About 40% of ILS and virtually100% of MDS cases occur due to the loss of one copy of theLissencephaly-1 (LIS1, also known as PAFAH1B1) gene onhuman chromosome 17 (Reiner et al., 1993; Wynshaw-Boris,2007). LIS1 is a central component of a protein complex, evolu-tionarily conserved from fungus to human, that regulatesnuclear migration through the cytoplasmic microtubule motordynein (Morris, 2000). In mice, reduced LIS1 activity results insevere defects in the radial migration of multiple types ofneurons including neocortical projection neurons (Cahanaet al., 2001; Gambello et al., 2003; Hirotsune et al., 1998; Tsaiet al., 2005). NDEL1 (nuclear distribution gene E-like homolog1) binds to both LIS1 and cytoplasmic dynein heavy chain (Niet-hammer et al., 2000; Sasaki et al., 2000). Ablation or knock-down of cortical NDEL1 function also results in impaired migra-tion of neocortical projection neurons (Sasaki et al., 2005; Shuet al., 2004; Youn et al., 2009). NDEL1 is a substrate for theserine/threonine protein kinase CDK5 (Niethammer et al.,2000; Sasaki et al., 2000), which is also essential for corticalneuronal migration (Gilmore et al., 1998). The adaptor protein14-3-33 binds NDEL1 in a phosphorylation-dependent manner

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to maintain NDEL1 phosphorylation, which is important forbinding to LIS1 and the dynein motor (Toyo-oka et al., 2003).Loss of one copy of 14-3-33 (also known as YWHAE), whichresides within the MDS deletion in human, enhances clinicalsymptoms of LIS1 heterozygosity in humans and neuronalmigration defects in Lis1 heterozygous mice (Toyo-oka et al.,2003). Thus, the tripartite LIS1/NDEL1/14-3-33-complex isa key regulator of cortical neuronal migration (Wynshaw-Boris,2007).

The coupling of the nucleus and centrosome mediated by theLIS1-complex is a key cell biological mechanism for neuronalmigration (Marin et al., 2010; Vallee et al., 2009). The cytoplasmicnature of these proteins suggests that they function cell autono-mously to regulate neuronal migration, but this has not beendirectly tested in vivo. Mice carrying homozygous null allelesdie either at implantation (Lis1, Ndel1) or neonatal (14-3-33)stages (Hirotsune et al., 1998; Sasaki et al., 2005; Toyo-okaet al., 2003). Thus, previous studies addressing the in vivo func-tions of the LIS1-complex relied on the analysis of heterozygousor compound heterozygous animals where all cells were mutant,on conditional mutants or on RNAi knockdown approacheswhere large groups of neurons were affected. To assess thecell-autonomous in vivo function of Lis1, Ndel1, and 14-3-33,we applied the MADM (mosaic analysis with double markers)strategy (Zong et al., 2005) to knock out these genes in sparsesubpopulations of neurons.

RESULTS

Extension of MADM to Chromosome 11The Lis1, Ndel1, and 14-3-33 genes are located on Chr. 11 in themouse. In order to perform mosaic analyses of these genesusing the MADM strategy, we cloned the Hipp11 locus nearthe centromere of Chr. 11 to insert the ‘‘MADM cassettes’’(Figures 1A, 1B, and 2A and Experimental Procedures). We re-placed Dsred2 in the original chimeric MADM cassettes (Zonget al., 2005) with tandem dimer Tomato (tdT) (Shaner et al.,2004), inserted an FRT site 50 to the LoxP site, and targeted thesecassettes to the Hipp11 locus using homologous recombinationin ES cells to produceMADM-11GT (GT:GFPN-term–tdTC-term) andMADM-11TG (TG: tdTN-term–GFPC-term) mice (Figure 1B).

In the absence of recombinase, we did not find any fluorescentcells in trans-heterozygous MADM-11GT/TG mice (data notshown). As predicted by the MADM scheme (Figure S1), intro-duction of Emx1Cre/+ (Gorski et al., 2002) produced fluorescentlyMADM-labeled (GFP only, tdT only, or GFP+/tdT+) cellsrestricted to the forebrain (Figures 1D–1G). MADM-11 labelingin isolated single cells (Figure 1H) can be induced using a specificNestin-CreERT2 transgenic line (referred to as Nestin-spCrehereafter) where CRE recombinase is active in sparse, randomsubsets of neuronal progenitors without tamoxifen (TM) induc-tion (line 1 in Imayoshi et al., 2006).

MADM-11 offers several advantages as a general tool for line-age tracing and labelingof single neuronsover theoriginalMADMatRosa26 (Zong et al., 2005). First, like GFP, tdT allows visualiza-tion of the red fluorescence marker in live animals. Thus,genotypes of distinctly labeled cells in mosaic animals can beunequivocally determined before fixation and immunostaining.

This property greatly improves physiological and imaging studiesof live tissues in MADM animals. Second, tdT+ and GFP+ axonalprojections (e.g., hippocampal mossy fibers; Figures 1D–1G) canbe clearly traced without signal amplification by antibodystaining. Third, the interchromosomal recombination rate inMADM-11 is markedly increased compared with the originalMADM (data not shown). This allows for temporal control of cloneinduction in many areas of the brain, including the cerebralcortex, using TM-inducible Nestin-CreER (e.g., lines 4 and 5 inImayoshi et al., 2006). As an example, we show a single G2-Xclone (seeFigureS1available online for details) ofMADM-labeledprojection neurons in the neocortex, which was induced atembryonic day 10 (E10) by TM and analyzed at E16 (Figures 1I–1M). Fourth, FLPe recombinase (Farley et al., 2000) can also beused to drive interchromosomal recombination with MADM-11(Figure 1C). Lastly, the location of the MADM cassettes allows> 99% of genes located on mouse Chr. 11 to be subjected toMADM-based mosaic analyses.

Lis1 Is Cell Autonomously Required for Cortical Neuronand Astrocyte ProductionTo genetically dissect the cell-autonomous functions of the Lis1,Ndel1, and 14-3-33 genes, we generated separate recombinantMADMGT andMADMTG strains for null mutants of Lis1 (Hirotsuneet al., 1998), Ndel1 (Sasaki et al., 2005) and 14-3-33 (Toyo-okaet al., 2003), respectively (Figure S3A). These recombinants werecrossed to mice that carried the reciprocal MADM cassette anda Cre recombinase transgene to generate experimental MADManimals (Figure S3A). We examined individual Lis1!/!, Ndel1!/!,and 14-3-33!/! projection neurons during neocortical develop-ment using Emx1-Cre expressed in cortical progenitors (Figure 2).First, we analyzed the role of Lis1, Ndel1, and 14-3-33 in

cortical neuron production. We compared the number of homo-zygousmutant (green) and homozygous wild-type (WT; red) cellsin the somatosensory cortex at postnatal day 21 (P21) (Figures2B–2F). G2-X events allow the visualization of two progeny ofthe same mitosis with two distinct colors (Figure S1). If cell divi-sion were symmetric, the number of red and green cells within anisolated clone would be identical (see Figures 1I–1M). Even if thecell division were asymmetric such that red and green progenynumbers were different in individual clones, the random distribu-tion of colors in asymmetric cloneswould ensure that the numberof red and green progeny were equal overall, given a largeenough number of independent G2-X events.Indeed, the green/red ratio in control-MADM (MADM-11GT/TG;

Emx1Cre/+) was not significantly different from 1 (Figures 2B and2F). By contrast, in Lis1-MADM (MADM-11GT/TG,Lis1;Emx1Cre/+),the mutant/WT (green/red) ratio is drastically reduced to "0.1(Figures 2C and 2F). These results indicate that LIS1 is requiredcell autonomously for production or survival of cortical neurons.A similarly low mutant/WT ratio in the cortex was also observedat P1 and P7 (Figures S4A and S4C; data not shown). These dataindicate that LIS1 is not required for survival of neurons duringthe postnatal period, although we cannot rule out the possibilitythat LIS1 is required for survival of prenatal neurons (Gambelloet al., 2003). In addition, no Lis1!/! astrocytes were observed(Figures 2C and 2F). The most likely interpretation for our resultsis that LIS1 is essential for cortical neural progenitor cell divisions

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Mosaic Analysis of Lis1 and Ndel1

696 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.

(Tsai et al., 2005; Yingling et al., 2008), which give rise to bothcortical projection neurons and astrocytes (Costa et al., 2009).A similar reduction in the number of Lis1!/! cells was evident

in all brain areas analyzed, including the hippocampus, the olfac-tory bulb and the cerebellum (data not shown). Together, thesefindings suggest that LIS1 is cell autonomously required for

A B C

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Figure 1. Extension of MADM to Mouse Chromosome 11(A) The Hipp11 genomic locus in cytoband A1 ("3 cM) between Eif4enif1 and Drg1 genes.

(B) Targeting of Hipp11 with GT and TG cassettes to generate MADM-11GT and MADM-11TG. Top panel shows the organization of the Hipp11 genomic locus.

Grey boxes indicate exons 1 and 19 of the flanking gene Eif4enif1, and exons 1 and 9 of Drg1. Middle and bottom panels show the Hipp11 genomic locus with

integrated GT and TG cassettes. LoxP (black triangles), FRT (rectangle), and the direction of transcription (green arrow) are indicated. Details about recombina-

tion products and reconstituted marker genes upon CRE/FLPe-mediated interchromosomal recombination can be found in Figures S1–S3.

(C–H) GFP (green in C–E and H; white in F) and tdT (red in C–E and H; white in G) expression in cortex and hippocampus in P21MADM-11mice. (C and H) Sparse

MADM labeling of cortical pyramidal cells inMADM-11GT/TG;Rosa26FLPe/+ (C) orMADM-11GT/TG;Nestin-spCre+/! (H). (D) Overview of labeling pattern inMADM-

11GT/TG;Emx1Cre/+. (E–G) Higher magnification of D (boxed area) illustrating CA3 pyramidal cells and mossy fiber projections from dentate gyrus granule cells.

(I) Schematic depicts TM-mediated MADM-clone induction at E10 inMADM-11GT/TG;Nestin-CreER+/! in symmetrically dividing neuroepithelial stem cell (NESC).

A G2-X event (see Figure S1 for a description of the MADM principle) results in two columns of green and red labeled neurons migrating along the processes of

radial glia progenitor cells (RGPCs).

(J–M) AG2-XMADMclone in the cortex with neurons expressing GFP (green) and tdT (red), andmigrating along RGPCs at E16 (J). Inset in (J) marks area shown in

(K)–(M) and depicts red and green apical endfeet of MADM-labeled radial glia with migrating neurons in the VZ. Nuclei (D, J, and K) were labeled using DAPI (blue).

CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone; TM, Tamoxifen.

Scale bar, 100 mm (C); 1 mm (D); 30 mm (E–G and K–M); 70 mm (H and J). See also Figures S1 and S2.

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production of most if not all neuronal classes. Indeed, LIS1 isalso cell autonomously required for the production of neuronsin the Drosophila brain (Liu et al., 2000), indicating that this isan evolutionarily conserved function.

In contrast to Lis1, the mutant/wt (green/red) ratio for Ndel1and 14-3-33 neurons and astrocytes was not significantlydifferent from 1 (Figures 2D–2F) under identical experimentalconditions. These results indicate that NDEL1 and 14-3-33 arenot cell autonomously required for proliferation of neural progen-itors or survival of postmitotic neurons and astrocytes. Onepossible explanation for these divergent phenotypes could begenetic redundancy. For example, the function of NDEL1 incell proliferation may be compensated for by NDE1, which

shares 55% sequence homology with NDEL1; deletion of Nde1results in mice with microcephaly (small brain), likely caused bya reduction in progenitor cell division (Feng et al., 2000; Fengand Walsh, 2004). Similarly, multiple isoforms of 14-3-3 are ex-pressed in the brain (Takahashi, 2003) and distinct 14-3-3 iso-forms may compensate for loss of 14-3-33 function. Alterna-tively, LIS1 may act independently of NDEL1 or 14-3-33 toregulate cell proliferation.

Lis1 and Ndel1 Display Distinct Cell-AutonomousPhenotypes in Cortical Neuron MigrationNext, we analyzed the role of Lis1, Ndel1, and 14-3-33 in radialmigration of cortical projection neurons. Since the vast majority

A

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B C D E

Figure 2. MADM Analysis of Lis1, Ndel1, and 14-3-33 in Somatosensory Cortex(A) Genomic location of MADM-11 and Lis1, Ndel1 and 14-3-33 genes on Chr. 11. Physical and genetic distances to the centromere are indicated.

(B–E) MADM-labeled cells in P21 somatosensory barrel cortex in control-MADM (B; MADM-11GT/TG;Emx1Cre/+), Lis1-MADM (C; MADM-11GT/TG,Lis1;Emx1Cre/+),

Ndel1-MADM (D; MADM-11GT/TG,Ndel1;Emx1Cre/+) and 14-3-33-MADM (E; MADM-11GT/TG,14-3-33;Emx1Cre/+). In control-MADM (B), GFP+ (green), tdT+ (red), and

GFP+/tdT+ (yellow) cells are all WT. In Lis1-,Ndel1-, and 14-3-33MADM (C–E), homozygousmutants are GFP+ (green), heterozygous cells are GFP+/tdT+ (yellow)

or unlabeled (vast majority), and homozygous WT cells are tdT+ (red). Nuclei were stained using DAPI (blue). White star in (B) marks tdT+ cortical astrocytes.

Arrows in (C) indicate sparse green Lis1!/! mutant neurons. Arrows in (D) point to Ndel1!/! astrocytes. Cortical layers are numbered in roman digits. WM: white

matter. Scale bar, 150 mm.

(F) Quantification of green/red ratio of neurons (upper panel) and cortical astrocytes (lower panel) corresponding to respective genotypes in (B–E). No Lis1!/!

mutant astrocytes were observed in any sample analyzed.

(G–J) Quantification of the relative distribution (%) of mutant green, heterozygote yellow and WT red neurons (upper panels) and astrocytes (lower panels) for

genotypes corresponding to (B)–(E). Values represent mean ± SEM ns: nonsignificant, *p < 0.05, **p < 0.01, and ***p < 0.001.

See also Figures S2–S4.

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of cells in Lis1-, Ndel1-, and 14-3-33-MADM mice are heterozy-gous for the respective gene to be analyzed, we can study theeffect of gene dosage in addition to homozygous loss of eachgene by comparing the layer distribution of homozygous mutant(green), heterozygous (yellow), and homozygousWT (red) cells inthe same animals (Figures 2C–2E), and to control-MADManimals (Figure 2B). We quantified such distributions in the P21somatosensory cortex (Figures 2G–2J).Despite their stark reduction in number, we observed Lis1!/!

neurons in all cortical layers, similar to corresponding Lis1+/!

and Lis1+/+ in the same animals (Figure 2C), or WT controls inseparate animals (Figure 2B). However, the numbers of bothLis1!/! and Lis1+/! neurons were significantly decreased inlayers II/III but increased in layer VI compared to Lis1+/+ neurons(Figures 2C and 2H). Reduced numbers of Lis1!/! and Lis1+/!

neurons in the uppermost layers of the cortex were evidentfrom the earliest postnatal stages on (Figure S4). These data indi-cate that Lis1+/+ cells exhibit a cell migration advantagecompared to Lis1+/! or Lis1!/! cells. Moreover, since Lis1+/+

cells reside in a largely heterozygous environment, these dataindicate that the migration advantage of Lis1+/+ cells is cellautonomous.Perhaps surprisingly, Lis1!/! neurons do not exhibit obvious

defects when compared to Lis1+/! neurons. However, whenexamined at postnatal day 1 (P1), we found a significant accu-mulation of Lis1!/! cells in the white matter compared toLis1+/! and Lis1+/+ cells, and a concomitant reduction of Lis1!/!

cells in upper cortical layers (Figures S4A and S4B). The differ-ence between Lis1!/! and Lis1+/! cells became non-significantat P7 (Figures S4C and S4D). These findings suggest that homo-zygous loss of Lis1 delays migration of cortical neurons, but thatthis defect is rescued later in development.The distribution of Ndel1+/! neurons is similar to Ndel1+/+

neurons (Figures 2D and 2I) in the same mosaic or in controlanimals (Figures 2B and 2G), indicating that cortical neuronalmigration is not sensitive to Ndel1 dosage. However, the vastmajority (>85%) of Ndel1!/! neurons accumulated in the whitematter below layer VI at P21 (Figures 2D and 2I), indicatinga profound migration deficit in neurons lacking NDEL1 function.We observed similar migration deficits of cortical Ndel1!/!

neurons in all major cortical areas (data not shown). Identicaldefects were seen when Ndel1!/! neurons were labeled withtdT instead of GFP (Figure S3D). The accumulation of Ndel1!/!

neurons below layer VI was not caused by a developmental delayas this defect persisted in an 8 month old animal (data notshown). Because Ndel1!/! neurons are sparsely intercalatedamong the vast majority of unlabeled cells that are Ndel1+/! inthese mosaic Ndel1 animals, this defect reflects a cell-autono-mous function of NDEL1 in regulating cortical neuronalmigration.Interestingly, despite the severe neuronal migration defect, the

layer distribution of Ndel1!/! cortical astrocytes was not signifi-cantly altered when compared to heterozygous or WT cells(Figures 2D and 2I). Thus, NDEL1 is selectively required cellautonomously for migration of cortical neurons but notastrocytes.Contrary to the phenotypes observed with Lis1 and Ndel1

mutant neurons, 14-3-33!/!, 14-3-33+/!, and 14-3-33+/+ cells

are distributed across layers II–VI similar to cells labeled incontrol-MADM (Figures 2E, 2G, and 2J). These data suggestthat neither reduced dosage nor homozygous loss of 14-3-33in isolated cells causes any defects in cortical neuron migration.It is possible that distinct 14-3-3 isoforms may compensate forloss of 14-3-33 function in neuronal migration.

Cell-Autonomous Function of Lis1, Ndel1, and 14-3-33in Hippocampal Neuronal MigrationWe extended the MADM analysis of Lis1, Ndel1, and 14-3-33to the migration of hippocampal pyramidal neurons (Figure 3).CA1 pyramidal neurons are born in the hippocampal neuroepi-thelium and migrate radially toward the developing target layerwhere they settle in an inside-out fashion similar to the projec-tion neurons in the neocortex (Altman and Bayer, 1990; Fig-ure 3G). The positions of labeled CA1 pyramidal neurons incontrol-MADM are consistent with the normal migrationpattern described above (Figures 3A and 3E). Likewise, thesoma of Ndel1+/+ and 14-3-33+/+ or Ndel1+/! and 14-3-33+/!

pyramidal cells were all located within the CA1 layer (Figures3C and 3D, red and yellow). These data indicate that CA1pyramidal neuron migration is not sensitive to the partialreduction of the Ndel1 or 14-3-33 gene dosage, similar toour findings in cortical neurons. By contrast, the vast majorityof Ndel1!/! CA1 (Figures 3C, 3E, and 3G) and CA3 (data notshown) pyramidal neurons accumulated at the base of the CAsubfields. A significant fraction of 14-3-33!/! CA1 pyramidalneurons also failed to reach the CA1 layer but were scatteredbetween the base and the CA1 cell layer (Figures 3D and 3E).These data indicate that both Ndel1 and 14-3-33 are cellautonomously required for hippocampal pyramidal neuronmigration, although the defect in 14-3-33!/! is milder than inNdel1!/! neurons.LIS1 affects neuronal migration in a dose-dependent manner

both in human and mice (Wynshaw-Boris, 2007). In Lis1-MADM animals, we observed significant heterotopia (ectopic‘‘islands’’ of cells as revealed by DAPI staining; see also Hirot-sune et al. [1998]) containing MADM-labeled CA1 pyramidalneurons (Figure 3B). Given these heterozygous effects, we couldnot clearly define the CA1 layer and determine the amount ofectopically located pyramidal cells in Lis1-MADM. We thereforequantified migration phenotypes in Lis1-MADM based on thedistance of the pyramidal cell soma from the base of CA1 (Fig-ure 3F). Interestingly, the average distance of Lis1+/! andresidual Lis1!/! cells appeared equal (Figures 3B and 3F).However, Lis1+/+ cells were most often found at the ‘‘leadingedge’’ of the CA1 pyramidal layer, with a fraction of Lis1+/+ cellsmigrating beyond the dense CA1 field (Figures 3B and 3F).Consequently, the average distance of Lis1+/+ soma from thebase of CA1 was almost doubled when compared to Lis1+/! orLis1!/! (Figure 3F). These data reinforced our findings in corticalneurons (Figure 2H), that two copies of normal Lis1 gene providemigrating Lis1+/+ cells with a competitive advantage cell autono-mously over Lis1!/! and Lis1+/! cells.The migration phenotypes of MADM-labeled dentate gyrus

granule cells (dGCs) in control-, Lis1-, Ndel1-, and 14-3-33-MADM appeared strikingly similar to pyramidal neurons in theCA1 field and cortical projection neurons (Figure S5): Ndel1 is

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cell autonomously required for dGCmigration; Lis1+/+ dGCs dis-played an advantage over Lis1+/! and Lis1!/! cells, which ex-hibited indistinguishable migration behaviors; 14-3-33!/! dGCshad no detectable migration defects, similar to cortical neurons(Figure 2; see also Figure S6).

Ndel1 Is Essential for the Migration of Many DistinctTypes of NeuronsPrior to this study, Ndel1 function in vivo in neuronal migrationwas analyzed mostly in cortical projection neurons and hippo-campal neurons (Shu et al., 2004). The severe Ndel1!/! migra-tion phenotype in MADM mice in both the cortex (Figures 2Dand 2I) and hippocampus (Figure 3G) prompted us to expandthe mosaic analysis of Ndel1 to other neuronal types. Wefocused on the olfactory bulb (OB; Figure 4) and the cerebellum

A B DC

E

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Figure 3. MADM Analysis of Lis1, Ndel1,and 14-3-33 in the Hippocampus(A–D) MADM-labeled cells in P21 CA1 hippo-

campus in control- (A), Lis1- (B), Ndel1- (C), and

14-3-33- (D) MADM. Genotypes and fluorescent

labeling are as depicted in Figure 2. The CA1 layer

is indicated in (A) and double arrow indicates the

distance of the CA1 field from the base of the

hippocampus. White arrows in (B) mark ectopic

CA1 cell masses. Green stars mark ectopic cells

in (C) and (D). Scale bar, 50 mm.

(E) Relative distribution (%) of pyramidal cells

within the CA1 layer (CA1) or at ectopic locations

(ect. CA1) in control- (top), Ndel1- (middle), and

14-3-33- (bottom) MADM.

(F) Distance (mm) of CA1 pyramidal cell soma from

the base of the hippocampus in CA1 in control-,

Lis1-, Ndel1-, and 14-3-33-MADM. Values repre-

sent mean ± SEM ns: nonsignificant; **p < 0.01

and ***p < 0.001.

(G) Schematic summary of migration of WT (red)

and Ndel1!/! (green) MADM-labeled hippo-

campal CA1 and CA3 pyramidal neurons (CA)

and dGCs. Red WT CA1 and CA3 pyramidal

neurons migrate radially and exit the ventricular

zone to form the pyramidal cell layers (gray

circles). Green Ndel1!/! CA1 and CA3 pyramidal

neurons remain at the base of the CA1/CA3

subfields. Red WT dGCs migrate radially to

different sublayers of the dentate gyrus granule

layer (gray circles). Green Ndel1!/! dGCs accu-

mulate at the hilus and at the base of the dentate

granule cell layer but do not properly invade the

target layer.

See also Figure S5.

(Figure 5) to test whether NDEL1 regu-lates a common step during themigrationof distinct neuronal types.OB interneurons (oINs) are mostly

GABAergic and migrate tangentiallyfrom the SVZ of the lateral ventricle alongthe rostral migratory stream (RMS) to theOB (Lledo et al., 2008; Lois and Alvarez-Buylla, 1994). After reaching the OB,

oINs exit the RMS and migrate centrifugally to occupy differentlayers within the OB (Figure 4H). We found Ndel1!/! oINs alongthe entire RMS and within the OB. However, the distribution ofNdel1!/! cells differed significantly from Ndel1+/+ cells (Figures4A–4F). The ratio of the total number of green/red (mutant/WT)cells within the central segment of the RMS ("600–800 mm)was increased "3-fold compared to the ratio of green/red (WT/WT) cells in control-MADM, indicating that Ndel1!/! oINs accu-mulate in the RMS. Correspondingly, the ratio of green/redneurons inNdel1-MADMwas reduced"3-fold in the OB granulecell layers compared to control-MADM (Figure 4G). Furthermore,Ndel1!/! oINs that have exited the RMS migrated significantlyshorter distances within the OB when compared to Ndel1+/+ inthe same mosaic animal (Figure 4G). Similar results were ob-tained when mosaic brains were examined at P6/7 (data not

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shown). Thus, Ndel1!/! oINs accumulate along the RMS and inthe most central granule cell layers within the OB, indicatinga general migration defect along the entire path (Figure 4H).Cerebellar Purkinje cells normally occupy a single layer in the

cerebellar cortex (Figures 5A and 5F) as a consequence of radialmigration from the ventricular zone outward around the time ofbirth (Altman and Bayer, 1997; Miale and Sidman, 1961). WeusedNestin-spCre to analyzeMADM-labeled cerebellar Purkinjecells, which were readily identifiable on the basis of their largecell bodies and characteristic dendritic trees. We found that>80% of Ndel1!/! Purkinje cells were located within the cere-bellar whitematter at P21 (Figures 5B and 5E), indicating a severemigration defect.Cerebellar granule cells (cGCs) exhibit a unique migration

path. In the embryo, cGC progenitors migrate from the rhombiclip to occupy the surface of the developing cerebellar cortex.During the first 3 postnatal weeks, cGC progenitors proliferatein the outer external granule layer (EGL) at the surface of thecerebellar cortex. Postmitotic cGCs thenmigrate radially inward,

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D

B E

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Figure 4. Ndel1 Function is Essential forMigration of Olfactory Interneurons(A and B) Distribution of olfactory bulb interneu-

rons (oINs) across the granule cell layer (GCL) in

the P21 OB. Genotypes are indicated, Ndel1!/!

cells labeled with GFP (green) and WT cells with

tdT (red). Note the reduction of green Ndel1!/!

cells in (B).

(C–F) Distribution of migrating oINs in cross

sections of the RMS. Nuclei were stained using

DAPI to outline the cytoarchitecture of the OB

(A and B) and RMS (C and E). G, glomerular layer;

M, mitral cell layer. Scale bar, 100 mm (A and B);

150 mm (C–F).

(G) Quantification of the green/red ratio in the RMS

and the OB (upper panels) and the relative distri-

bution (%) of oINs across three equal sectors in

the GCL (lower panels). Values represent mean ±

SEM ns: nonsignificant; *p < 0.05 and ***p < 0.001.

(H) Schematic summary of migration of WT (red)

and Ndel1!/! (green) MADM-labeled oINs. Red

WT oINs originate from the subventricular zone

of the lateral ventricle (SVZ/LV), migrate along

the RMS to the OB, where oINs exit the RMS to

migrate centrifugally to occupy different layers of

the GCL. Green Ndel1!/! oINs accumulate along

the RMS as indicated by the higher number of

green circles in the RMS, and accumulate signifi-

cantly closer to the ependymal layer (E) (extension

of RMS) within the OB than red WT cells, suggest-

ing a defect in migration in the target lamina (gray

circles in the high magnification scheme on the

right). G, glomerular layer; M, mitral cell layer;

GCL, granule cell layer.

See also Figure S6.

passing the already-formed Purkinje celllayer and eventually settle within theinternal granule layer (IGL) (Figure 5F; Alt-man and Bayer, 1997; Miale and Sidman,

1961). At P21, all labeled cGCs in control-MADM as well as redNdel1+/+ and yellow Ndel1+/! cells in Ndel1-MADM hadcompleted neurogenesis and migration, and all Ndel1+/+ andNdel1+/! cGCs were located within and throughout the IGL(Figures 5C and 5D). By contrast, a significant fraction ofNdel1!/! cells was observed within the deep molecular layer(derivative of the EGL). Ndel1!/! cGCs that did migrate pastthe Purkinje cell layer accumulated within the most superficiallayer of the IGL next to the Purkinje cells (Figures 5D and 5E).The marked accumulation of Ndel1!/! cGCs at the borderbetween the Purkinje cell layer and the IGL indicates that themajority of Ndel1!/! cGCs can complete the initial migrationfrom their site of birth and past the molecular and even the Pur-kinje cell layer, but accumulate at the border of their final desti-nation, the IGL. In conclusion, we infer from the analyses of sixdistinct neuron types that NDEL1 is generally required cell auton-omously for neuronal migration. However, it appears that not allphases of the migration process are equally blocked in Ndel1!/!

neurons.

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Ndel1 Regulates Cortical Neuron Entry intothe Developing Cortical PlateTo identify the critical step in neuronal migration controlled byNDEL1, we traced the developmental origin of the migrationdefects in Ndel1!/! neurons. We focused mosaic analyses onthe development of the neocortex because the cortical lamina-tion process with sequential inside-out layering has been wellcharacterized and can be traced unambiguously by costainingwith a nuclear marker.

We used Emx1-Cre for time course MADM analyses. Startingat E12, the distribution of migrating Ndel1!/! neurons acrossthe developing cortex in the VZ and developing preplate (PP)did not differ from WT neurons (Figures 6A and 6B). This findingindicates that somal translocation, the predominant migrationmode of early born neurons (Nadarajah et al., 2001), is notaffected by loss ofNdel1. By E14, a thin CP has formed. The frac-tion of Ndel1!/! neurons located within the nascent CP wassignificantly decreased when compared to the fraction of controlneurons. Instead,Ndel1!/! cells showed a tendency to accumu-

A C

B D

E

F

Figure 5. Ndel1 Is Required for Migration ofCerebellum Purkinje and Granule Cells(A–D) Distribution of Purkinje cells and cGCs in

P21 cerebellum. Genotypes are indicated,

Ndel1!/! cells labeled with GFP (green) and WT

cells with tdT (red). (A and B) Central part of the

cerebellum with the white matter (WM), Purkinje

cell (PC) layer (white dotted line), internal granule

cell layer (IGL) and molecular layer (ML) labeled.

Ndel1!/! Purkinje cells are mostly localized in

the white matter (B, white arrow). See Figure 8F

for high-resolution image of Ndel1!/! Purkinje

cell. (C and D) Distribution of cGCs in control- (C)

and Ndel1-MADM (D). Scale bar, 150 mm (A and

B); 60 mm (C and D).

(E) Quantification of Purkinje cell distribution (%) in

the PC layer or in ectopic locations (ect. PC), and

cGCs across the molecular (ML) and internal

granule layer (IGL), in control- (upper panel) and

Ndel1-MADM (lower panel). The IGL was divided

into three equal sectors for quantification of the

relative distribution of cGCs. Values represent

mean ± SEM ns: nonsignificant; *p < 0.05 and

***p < 0.001.

(F) Schematic summary of migration of WT (red)

and Ndel1!/! (green) cerebellar Purkinje cells

and cGCs. cGCs are born at the most superficial

sublayer of the external granule layer (EGL) during

the first 3 postnatal weeks (left panel). NascentWT

cGCs migrate inward across the ML, pass the PC

layer, and settle throughout the IGL (small gray

circles representing the final positions of cGCs).

Most Ndel1!/! cGCs also migrate across the

ML, but a fraction fails to pass the PC layer.

Ndel1!/! cGCs that pass the PC layer accumulate

at the most superficial sublayer of the IGL.

See also Figure S6.

late at the upper edge of the IZ (Figures6C, 6D, and 6R). This phenotype becamemore pronounced at E16, when Ndel1!/!

neurons accumulated specifically below the developingCP in theupper IZ (Figures 6E, 6F, and 6S). This migration deficit persistedat P1 when Ndel1!/! neurons were still found to accumulatebelow layer VI, in the region that becomes thewhitematter at laterstages (Figures 6G, 6H, and 6T; see also Figures 2D, S3C, andS3D). These results suggest that Ndel1!/! neurons can migratethrough the VZ and the IZ but not within the developing CP.Because the MADM analysis described so far was performed

with constitutive Emx1-Cre or Nestin-spCre (not shown), theexact timing of CRE-mediated mitotic recombination (and hencethe removal of the Ndel1 gene) for any given labeled neuron wasunknown. Accordingly, we next carried out temporally controlledclonal MADM analyses using TM-inducible Nestin-CreER.Cortical clones were induced at E10, when the vast majority ofRGPCs divide symmetrically (Gotz and Huttner, 2005), andanalyzed at E16. Individual G2-X MADM-clones appeared asdifferentially labeled radial columns with an equal number ofred WT and green Ndel1!/! migrating neurons (Figures 6I–6Land 6Q; see also Figures 1J–1M for an example of WT G2-X

Neuron



clone). These results directly confirmed our earlier finding thatloss of Ndel1 does not affect neuron production. In all MADMclones, the developing CP was populated by "50% of redcontrol cells. By contrast, the fraction of Ndel1!/! cells in theCP was reduced to "10% of all cells; instead, the number ofNdel1!/! neurons in the upper IZ was significantly increased(Figure 6U). Notably, the number of Ndel1+/+ and Ndel1!/!

neurons in the VZ/SVZ was not significantly different (Figure 6U),indicating that both Ndel1!/! and Ndel1+/+ neurons migrateequally across the VZ/SVZ. Further examples whereby cloneswere induced at E8 or E10 and examined at E14, E16, and E18(Figures 6M–6P) also demonstrated that Ndel1!/! neuronsmigrate normally within the VZ/SVZ and across the IZ, but failto enter or migrate within the developing CP.Taken together, the developmental studies and clonal anal-

yses indicate that Ndel1 is not cell autonomously required formigration of cortical neurons within the VZ/SVZ and IZ. Theprimary defect in Ndel1!/! neurons appears to be an inabilityto enter or migrate within the CP, which represents the targetlamina for cortical projection neurons.

A

B D F H

L

Q

R

S

T

U

KJ

NM O P

I

C E G

Figure 6. Ndel1 Cell Autonomously Regu-lates Cortical Neuron Migration into theCortical Plate(A–H) Time course analysis of migration pattern of

MADM-labeled cortical projection neurons using

in control- (A, C, E, and G) and Ndel1-MADM (B,

D, F, and H) at E12 (A and B), E14 (C and D), E16

(E and F) and P1 (G and H). Genotypes are indi-

cated, Ndel1!/! cells labeled with GFP (green)

and WT cells with tdT (red).

(I–P) Clonal analysis in MADM-11GT/TG,Ndel1;Nes-

tin-CreER+/!

embryonic cortex. (I–L) TM was

applied at E10 (TM/E10) and sample analyzed at

E16 (A/E16). G2-X clone is illustrated with

Ndel1+/+ cells (red in I and L; white in J) and

Ndel1!/! cells (green in I and L; white in K). White

star marks the border of the cortical plate where

Ndel1!/! cells accumulate. (M–P) Time course of

clonal analysis in MADM-11GT/TG,Ndel1;Nestin-

CreER+/! with TM applied at E8 (M) or E10 (N–P)

and samples analyzed at E14 (M and N), E16 (O)

and E18 (P). Scale bar, 30 mm (A and B); 60 mm

(C and D); 100 mm (E, F, I–L, O, and P); 150 mm

(G and H); 50 mm (M and N).

(Q–U) Quantification of ratio of green/red cells (Q)

and relative distribution (%) of red and green cells

in the VZ/SVZ, IZ, and CP (R–U). Genotypes (top)

indicate control-MADM (left column) and Ndel1-

MADM (right column) using Emx1Cre/+ at E14 (R),

E16 (S), and P1 (T) or Nestin-CreER+/! (TM/E10;

A/E16) (U). Values represent mean ± SEM ns,

nonsignificant; *p < 0.05 and ***p < 0.001.

Live Imaging Reveals a SpecificBlock of Ndel1–/– ProjectionNeurons in Cortical Plate EntryOur results from sparse mosaic knockoutaremarkedly different from the findings in

conditional Ndel1mutants: upon conditional ablation of Ndel1 inmost or all cells in the cortex, migration of Ndel1!/! neurons iscompletely abolished (Youn et al., 2009). To compare these twoconditions (most cells versus sparse single cells mutant forNdel1), we performed live imaging experiments of MADM-labeled neurons in organotypic slice preparations similar to thoseused previously in Ndel1 cortical conditional knockout experi-ments (Youn et al., 2009). We traced embryonic cortical slicesfrom control- and Ndel1-MADM E14.5 brains over the course ofup to 15 hr (Figures 7 and S7). We found that the average migra-tion speed ofNdel1!/! neurons within the IZ was not significantlydifferent fromNdel1+/+neurons in the sameslices (Figures 7A–7Hand 7Q) or in slices fromcontrol-MADM (Figure S7A–S7H). Thesedata are consistent with the developmental time course analysis(Figure 6), indicating that NDEL1 is not cell autonomouslyrequired for migration of cortical projection neurons within the IZ.We also identified slices where the border between the IZ and

CP was readily identifiable, quantified the behavior of migratingcells when they encountered this IZ-CP border (Figures 7I–7P;Movie S2) and compared to similar experiments performed with

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control-MADM (Figures S7I–S7P; Movie S1). Whereas 94% ofmigrating Ndel1+/+ neurons readily crossed the border, 87% ofmigrating Ndel1!/! neurons failed to traverse the IZ-CP border(Figure 7R). Interestingly,Ndel1!/! neurons displayed significantlyincreased neurite numbers and elevated neurite length (Figures 7Sand 7T). The propensity to formmultiple neurites inNdel1!/! cellsis likely to interfere with neuronal migration (Youn et al., 2009) andthereforemightcontribute to the failureofNdel1!/! toenter theCP.

These results directly confirm that Ndel1!/! neurons in sparsegenetic mosaics in a mostly heterozygous environment canmigrate through the VZ/SVZ and the IZ. They also suggest thatthe inability of Ndel1!/! mutant neurons to migrate in the devel-oping cortex in the Ndel1 cortical conditional knockout mice isa consequence of cell-nonautonomous effects.

Dendrite Morphogenesis and Axonal Projectionsof Ndel1–/– NeuronsThe MADM-labeling strategy afforded high-resolution examina-tion of the morphological differentiation of Ndel1!/! neurons at

ectopic sites and provided an opportunity to determine theextent to which dendrite morphogenesis and axon projectionrelies on neurons entering their appropriate target layers. In prin-ciple, we cannot distinguish whether abnormal morphogenesisis a secondary consequence of aberrant neuronal positioningor reflects independent NDEL1 functions. However, we candeduce that any normal aspects of development must be inde-pendent of appropriate neuronal position.We first analyzed the dendritic arbors of ectopically localized

neurons in the cortex, hippocampus, and cerebellum. Remark-ably, mislocalized projection neurons established dendritic treesthat resembled cell type-specific patterns. For cortical andhippocampal pyramidal neurons, except for some dilation atthe base of apical dendrites and at axon initial segments (Figures8B and 8D), the apical dendrites of Ndel1!/! cortical and hippo-campal projection neurons appeared indistinguishable fromthose of control neurons, despite ectopic cell body location(Figures 3A, 3C, 8A–8D, and S8B). Ndel1!/! CA1 pyramidalneurons extend apical dendrites across the entire CA1 field.

T

RQ

I J K L M N O P

A B C D E F G H

S

Figure 7. Live Imaging of MADM-Labeled Ndel1–/– Cortical Projection Neurons(A–P) Time-lapse images of migrating cortical projection neurons in the IZ (A–H) and at the border to the CP (I–P) in organotypic cortical slices derived fromNdel1-

MADM (MADM-11GT/TG,Ndel1;Emx1Cre/+) mice at E14.5. Open arrowheads mark Ndel1!/! cells (GFP, green) and stars mark Ndel1+/+ control cells (tdT, red). The

border between the IZ and CP is indicated as dotted line in magenta (I–P). Frames are every 900 (A–H) and 300 (I–P). Scale bar, 50 mm (A–H); 40 mm (I–P).

(Q–T) Quantification of (Q) migration speed in IZ (n = 33 each for Ndel1+/+ and Ndel1!/! cells each); (R) fraction of labeled cells crossing the IZ-CP border (n = 19,

24 for Ndel1+/+ and Ndel1!/! cells, respectively); (S) number of neurite branches (n = 58 each for Ndel1+/+ and Ndel1!/! cells); (T) neurite length of migrating cells

in IZ (n = 51, 65 for Ndel1+/+ and Ndel1!/! cells, respectively). Values in (Q), (S), and (T) represent mean ± SEM; ns, nonsignificant; ***p < 0.001.

See also Figure S7 and Movies S1 and S2.

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Given their origin at a more basal level, these primary apicaldendrites are therefore longer than those of control neurons (Fig-ure S8). These data indicate that growth of the apical dendrite inpyramidal neurons is not affected by abnormal locations of theircell bodies. Similar results were observed in 14-3-33!/! andLis1!/! neurons (Figure S8).In the cerebellum, Purkinje cells were readily identified by their

characteristic dendritic tree despite their ectopic cell body loca-

tions in the white matter (Figure 5B; data not shown). This obser-vation indicates a certain degree of cell-autonomous dendritedevelopment despite a lack of contact with their major presyn-aptic partners, the cGC axons. However, Purkinje cells displayeda drastic reduction in dendritic branching. Occasionally, ectopicNdel1!/! Purkinje cells were located at the base of the IGL (Fig-ure 8F). In such cases, Purkinje cells projected their dendritesoutward toward the molecular layers and axons in the opposite

A B C D E F

G I K M O Q

H J L N P R

Figure 8. Dendrite Morphogenesis and Axonal Projections of Ndel1–/– Neurons(A–F) Morphology and dendrite pattern of pyramidal cells in the cortex (A and B), in the CA3 layer of the hippocampus (C and D), and in Purkinje cells of the cere-

bellum (E and F) in control- (A, C, and E) and Ndel1-MADM (B, D, and F). Genotypes of green and red cells are shown below each panel. All images are from P21

animals with the following genotypes: MADM-11GT/TG;Emx1Cre/+ (A and C); MADM-11GT/TG,Ndel1;Emx1Cre/+ (B and D); MADM-11GT/TG;Nestin-spCre+/! (E);

MADM-11GT/TG,Ndel1;Nestin-spCre+/! (F). Arrows in (A and B) point to basal segments of pyramidal cells in the cortex; white stars (A, B, and D) mark apical

dendrites in pyramidal cells in the cortex (A and B) and CA3 hippocampus (D). ML, molecular layer; PC, Purkinje cell layer; IGL, internal granule layer.

(G–R) Axonal projections in control- andNdel1-MADM using Emx1Cre/+. (G–J) Corticothalamic projections in thalamus at P21 (G andH) and P1 (I and J). Inset in (G

and H) are higher magnification images highlighting axonal varicosities in Ndel1!/! corticothalamic projections. Inset in (J) highlights red Ndel1+/+, yellow

Ndel1+/!, and green Ndel1!/! nascent growing axons in P1 thalamus.

(K–P) Time course of axonal projections in the internal capsule in control- (K, M andO) andNdel1-MADM (L, N and P) at P1 (K and L), P7 (M and N) and P21 (O and

P). Note the progressive increase in number and size of axonal varicosities asmarked by white arrows (N and P) in greenNdel1!/!mutant subcortical projections.

(Q and R) Hippocampal efferents in the fornix in control- (Q) and Ndel1-MADM (R) at P21. White arrow in (R) marks accumulation of varicosities from green

Ndel1!/! axons.

Scale bar, 30 mm (A–D); 50 mm (E and F); 230 mm (G, H, and M–P); 200 mm (K and L); 110 mm (I, J, Q, and R). See also Figures S8 and S9.

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direction toward thewhitematter. However, thesemispositionedPurkinje cells showed disrupted dendritic branching patternswithin the granule cell layer. Interestingly, the dendritic treebecame more elaborate once it extended into the molecularlayer, where the branches could presumably contact cGC axons(Figure 8F). These results support the general idea that cell-intrinsic programs and external cues act together to regulatedendrite morphogenesis (Scott and Luo, 2001).

Next, we examined axonal projections in ectopic Ndel1!/!

neurons, focusing on efferent axons of cortical and hippocampalprojection neurons. We found that Ndel1!/! axons reached thethalamus (Figures 8G and 8H) and other subcortical areas (datanot shown), similar to Ndel1+/! and Ndel1+/+ axons in the samemosaic animals. Tracing of nascent axons within the thalamusat P1 revealed no differences in the patterning or projection ofdeveloping WT or Ndel1!/! axons (Figures 8I and 8J). Since thevast majority of Ndel1!/! cortical neurons failed to migrate intocortical layers and were located ectopically in the white matter(Figures 2D, 2I, S3C, and S3D), these data suggest thatmigrationinto the correct layer is not a prerequisite for establishing thecorrect long-range axonal projections, at least at a gross level.

Asdevelopmentprogressed, abnormalities inaxonsofNdel1!/!

neurons became apparent. At P21, we found axonal swellingsspecific to Ndel1!/! axons in the thalamus (green varicosities inFigure 8H). Aberrant axonal swellingswere also present in cortico-spinal projections (data not shown) but were most prominentwithin the internal capsule (IC), a narrow path between the telen-cephalon and the midbrain containing axons projecting both toand from the cortex. A developmental time course indicated thatthe appearance of axonal swellings in Ndel1!/! neurons is agedependent within the IC (Figures 8K–8P). We also traced hippo-campal efferents in the fornix, a major axonal output pathway ofhippocampal projection neurons. Similar to cortical projectionneurons, axons of Ndel1!/! hippocampal projection neuronsreached the fornix but exhibited aberrant swellings (Figure 8R;data not shown). Importantly, these axonal swellings were neverobserved in control-MADM animals (Figures 8G, 8I, 8K, 8M, 8O,and 8Q), and were entirely axonal in nature (Figure S9). Theseaxonphenotypes inNdel1!/! neuronsmay reflect an independentrole of NDEL1 in maintaining axonal integrity, consistent withprevious studies showing that NDEL1 regulates neurofilamentorganization (Nguyen et al., 2004), which is essential for the main-tenance of axonal structures (Fuchs and Cleveland, 1998).

In summary, our analysis of dendrite morphogenesis and axonalprojections of Ndel1!/! neurons indicates that certain aspects ofneuronalmorphogenesis, such as neuronal polarity, gross dendriticgrowth and patterning, and long-range axonal projections, are notstrictly dependent on neurons occupying their correct target loca-tion. However, ectopically located neurons exhibit significantdefects in dendrite morphogenesis and axonal integrity. Whereassome aspects may be a secondary consequence of migrationdefects (e.g., Purkinje cell dendrites not able to reach their targetmolecular layers), othersmay reflect additional functions of NDEL1.

DISCUSSION

Genetic mosaic analysis is a powerful tool to tease apart cell-autonomous and nonautonomous functions of genes in regu-

lating developmental processes. Mosaic analysis has alsobeen used extensively in neurobiology, for example, to identifythe contribution of individual neurons to the organismal circadianclock (Low-Zeddies and Takahashi, 2001) or to elucidate thecell-autonomous function of the NMDA receptor in dendriticpatterning (Espinosa et al., 2009). Here, we extended theMADM genetic mosaic tool to mouse chromosome 11 andanalyzed the functions of LIS1 and NDEL1 in neuronal migration.The LIS1/NDEL1 complex is considered part of the basicmachinery for neuronal migration that couples the nucleus andcentrosome. Given that both LIS1 and NDEL1 are cytoplasmicproteins, the prediction would be that they should act cell auton-omously to regulate all steps of migration. However, our mosaicanalysis reveals a far more complex picture and provides novelinsights into the cell-autonomous and nonautonomous functionsof LIS1 and NDEL1 in regulating neuronal migration (Figure 9).Although the adult phenotypes of Ndel1!/! neurons support

the previously established role of NDEL1 in regulating neuronalmigration (Sasaki et al., 2005; Shu et al., 2004), our develop-mental analyses indicate that NDEL1 is not cell autonomouslyrequired for all aspects of neuronal migration. Using MADM-mediated sparse knockout and labeling in fixed brains as wellas live imaging, we found that Ndel1!/! neurons migrate nor-mally within the VZ/SVZ and IZ similar to WT neurons. The firstdefect we observed when comparing Ndel1!/! and Ndel1+/+

neurons in the same mosaic animal was the accumulation ofNdel1!/! neurons at the border of the developing CP (Figures9A and 9B). This selective defect is unlikely caused by perdur-ance ofNdel1mRNAor protein inNdel1!/! neurons (see Supple-mental Experimental Procedures). Since the CP represents thetarget lamina for migrating cortical projection neurons, wepropose that a major cell-autonomous function of NDEL1 inneurons is to regulate the entry into the target laminae (Figures9A and 9B).We have extended the analysis of Ndel1 to migration of excit-

atory and inhibitory neurons including hippocampal pyramidalcells and dentate granule cells (Figure 3G), cerebellar Purkinjecells and granule cells (Figure 5F) and olfactory bulb interneurons(Figure 4H). NDEL1 is cell autonomously required for the migra-tion of all these neurons, and their migration phenotypes stronglysupport our central hypothesis that NDEL1 regulates the entryinto the target lamina. For example, Ndel1!/! cerebellar granulecells migrate normally across the molecular layer of the cere-bellar cortex but are accumulated near the Purkinje cell layerbefore entering into the granular layer, the target lamina forgranule cells (Figure 5F).Mechanistically, NDEL1 could exert this cell-autonomous

function of controlling target lamina entry by transducing anextracellular signal to the intracellular neuronal migrationmachinery. Interestingly, NDEL1 is phosphorylated by CDK5/p35, a protein serine/threonine kinase complex essential forneuronal migration (Chae et al., 1997; Gilmore et al., 1998; Niet-hammer et al., 2000; Sasaki et al., 2000). Indeed, Cdk5!/!

cortical projection neurons exhibit a migration arrest phenotypesomewhat similar to the phenotype of Ndel1!/! neurons (Gil-more et al., 1998; Ohshima et al., 2007). It will be interesting inthe future to performMADManalysis ofCdk5 to directly comparethe cell-autonomous function of Cdk5 with that of Ndel1, and to

Neuron



determine the extracellular cues and signaling cascades thatregulate NDEL1 activity to control the entry of migrating neuronsinto the target lamina.LIS1 is well known to regulate neuronal migration in a dosage-

sensitive manner (Wynshaw-Boris, 2007). MADM allowed us tocompare the behavior of sparse, uniquely labeled Lis1!/!,Lis1+/!, and Lis1+/+ cells in a predominantly Lis1+/! heterozy-gous animal and thus address the questions of dosage as wellas cell autonomy. We find that Lis1+/+ cells have a competitiveadvantage over cells of all other genotypes in the mosaic animalin their extent of migration; this can be seen in cortical projectionneurons, CA1 pyramidal neurons and dentate granule cells. Inaddition, there is a significant delay in the migration of Lis1!/!

compared to Lis1+/! cortical projection neurons to correct layers(Figure S4). Together, these observations support the notion thatLIS1 cell autonomously regulates the migration efficiency (Fig-ure 9C) in a dose-dependent manner.Perhaps the most surprising finding of our study is that the

neuronal migration phenotypes resulting from sparse MADM-based gene knockout are distinct from those observed inprevious reports using other genetic perturbations of the samegenes. For Ndel1, previous RNAi knockdown results in a signifi-cant defect of neurons migrating out of the VZ/SVZ (Shu et al.,2004); conditional whole-cortex knockout causes a completeblock of neuronal migration within the VZ/SVZ and IZ (Younet al., 2009). MADM analysis indicates that migration ofNdel1!/!

neurons are specifically blocked at the entry of the cortical platebut migrate normally in VZ, SVZ, and IZ. For Lis1, analysis ofprevious RNAi knockdown, compound heterozygous and wholecortex conditional knockout indicate that neuronal migration isseverely perturbed (Gambello et al., 2003; Hirotsune et al.,1998; Tsai et al., 2005; Youn et al., 2009). MADM analysis of

Lis1, while revealing an essential role for neural progenitor prolif-eration consistent with previous results, did not show a completearrest ofmigration in Lis1!/! neurons. Indeed, despite a develop-mental delay, Lis1!/! and Lis1+/! neurons behave similarly in themigration of cortical projection neurons (Figure 2H), hippo-campal CA1 pyramidal neurons (Figure 3F) and dentate granulecells (Figure S5F) when examined in adult.Altogether, these phenotypic differences strongly suggest that

a subset of previously described migration functions for Ndel1and Lis1 can be accounted for by cell-nonautonomous functionof these genes in regulating neuronal migration. Specifically,migration of Ndel1!/! cells within the IZ can be positivelyaffected by neighboring Ndel1+/! cells even though entry intothe cortical plate cannot. A cell-nonautonomous function mayalso account for the eventual migration of Lis1!/! neurons intoproper cortical and hippocampal layers. We note that smallcell-nonautonomous effects have been proposed also for nonra-dial migration in Lis1+/! mice (McManus et al., 2004).How can these cytoplasmic proteins exert cell-nonautono-

mous effects? We envision two scenarios that are not mutuallyexclusive. First, a ‘‘community effect’’ may provide support forindividual neurons in the process of migration. In particular,isolated Ndel1!/! or Lis1!/! neurons, although defective inthe intrinsic essential migration machinery (e.g., centrosome-nucleus coupling), could nevertheless physically ‘‘piggyback’’on normally migrating neighboring neurons. However, if mostor all neurons are mutant for Lis1 or Ndel1, community effectsbecome ineffective. Second, migrating neurons may activelysignal to each other to facilitate the intrinsic migrationprogram. The generation of such a signal may require theLIS1/NDEL1 complex, accounting for their cell-nonautono-mous effect.

A

B C

Figure 9. Cell-Autonomous and Nonauton-omous Functions of LIS1 and NDEL1 inNeuronal Migration(A) Schematic summary of migration of WT (red)

and Ndel1!/! (green) MADM-labeled cortical

projection neurons illustrating the cell-autono-

mous function of NDEL1 to control invasion into

the cortical plate, their target lamina. WT neurons

exit the VZ, migrate across the IZ (red arrow), and

into the CP along the RGPC fiber, settling within

distinct layers of the cortex according to their birth

date. Ndel1!/! neurons migrate out of the VZ and

across the IZ (green arrow), but fail to migrate into

the CP (—j), representing the target lamina (gray

circles) for cortical projection neurons. Ndel1!/!

neurons accumulate below the expanding CP

during embryogenesis and eventually remain

ectopically located in the white matter (WM) at

postnatal stages.

(B and C) Models of cell-autonomous (green) and

nonautonomous (blue) in vivo functions of NDEL1

(B) and LIS1 (C) in the developing brain. NDEL1

cell autonomously controls invasion and/or migra-

tion within developing target laminae. LIS1 cell

autonomously regulates the efficiency of neuronal

migration in a dose-dependent manner. In addi-

tion, extensive interactions among migrating

neurons, either mediated by specific cell-nonautonomous effects of LIS1/NDEL1 or through a general community effect, promote migration of Ndel1!/! cells

before reaching the target laminae and Lis1!/! cells along the entire path under sparse knockout conditions.

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Regardless of the exact mechanism, MADM-based geneticmosaic analyses revealed extensive cell-cell interactions amongmigrating neurons (Figures 9B and 9C). Importantly, such nonau-tonomous functions could contribute significantly to the clinicalsymptoms of Lissencephaly, whereby cells with a 50% reductionin LIS1 protein levels exhibit inappropriate community effects orfail to signal to each other. This may in turn exacerbate thealready-impaired, intrinsicmigratory capacity of Lis1+/! neurons.It will be revealing to investigate the interplay of cell-autonomousand cell-nonautonomous mechanisms in the control of neuronalmigration. Our study also reinforced the power of mosaic anal-ysis in dissecting gene function in mammalian brain develop-ment. The successful generation of MADM-11 will now allowmosaic analysis for "2000 genes located distal to the HIPP11locus. The targeted knockin approach presented here alsosuggests a general strategy to expand MADM to other mousechromosomes.

EXPERIMENTAL PROCEDURES

Identification of Hipp11 as a Suitable Genomic Locus for theGeneration of MADM-11 Transgenic MiceThe genomic locus of choice on Chr. 11 for targeted knockin of MADM

cassettes should ideally (1) be located close to the centromere to maximize

the number of genes on Chr. 11 that can be subjected to MADM-based

mosaic analysis; (2) be located within an intergenic region to minimize the

probability of disrupting endogenous gene function; and (3) allow strong

and ubiquitous biallelic expression of marker proteins derived from the

MADM transgenes. We mapped in silico the centromeric-most 10 Mbp of

the acrocentric Chr. 11 and chose the targeting site for generating MADM-

11 in cytoband A1 at "3 cM between the Eif4enif1 and Drg1 genes (Figures

1A and 1B). We named this locus Hipp11. Both Eif4enif1 and Drg1 flanking

Hipp11 displayed broad spatial and temporal EST (expression sequence

tag) expression patterns (http://www.ncbi.nlm.nih.gov/), potentially permit-

ting MADM transgenes to be capable of global expression from a built-in

ubiquitous pCA promoter. Chimeric MADM cassettes were targeted to the

Hipp11 locus by homologous recombination in R1 ES cells. Two independent

ES cell clones for each MADM-11GT/+ and MADM-11TG/+ were expanded and

chimeric founder mice generated by blastocyst injection. Homozygous

MADM-11GT/GT, MADM-11TG/TG, and trans-heterozygous MADM-11GT/TG

mice were born at expected Mendelian ratios, were fully viable and fertile,

and appeared indistinguishable from wild-type mice. MADM-11GT and

MADM-11TG mice are available at Jackson Laboratory Repository (http://

jaxmice.jax.org/query) under JAX Stock No. 013749 (MADM-11-GT) and

JAX Stock No. 013751 (MADM-11-TG).

Analysis of GFP and tdT Markers in MADM-Labeled BrainsPostnatal mice were perfused with 4% PFA and dissected brains postfixed in

4% PFA overnight, cryoprotected in 30% sucrose/PBS and embedded for

cryostat sections. Embryonic brains were immersed in 4% PFA for 4 hr to

o/n for fixation. Fixed brains were sectioned at 20–60 mm using a cryostat (Le-

ica). Cryosections were washed 33 in PBS, stainedwith DAPI andmounted for

imaging using a confocal microscope (Zeiss). Quantification of cell numbers

(Supplemental Experimental Procedures) were derived from R2 animals/

genotype and time points unless indicated otherwise; values in quantification

charts represent mean ± SEM. Student’s t test was used to determine signif-

icance: *p < 0.05, **p < 0.01, and ***p < 0.001.

MADM Clone InductionFor MADM clone induction, pregnant MADM-11GT/GT females that have been

crossed to MADM-11TG/TG;Nestin-CreER+/! males were injected intraperito-

neal with 1–3 mg TM (Sigma) dissolved in corn oil (Sigma) at E8/E10. Embryos

were isolated at E14, E16, and E18 and processed for analysis as described

above. No difference in the clonal pattern was observed when the genotypes

of males and females were switched. For Ndel1-MADM clones, one of the

parent mice contained the MADM-11TG/TG,Ndel1 allele to label mutant cells in

green.

Live-Imaging Assay for MADM-Labeled Organotypic SlicesPreviously established live-imaging protocols (Youn et al., 2009) were used

with slight adaptations (see Supplemental Experimental Procedures).

SUPPLEMENTAL INFORMATION

Supplemental Information includes nine figures, two movies, and Supple-

mental Experimental Procedures and can be found with this article online at

doi:10.1016/j.neuron.2010.09.027.

ACKNOWLEDGMENTS

We thank B. Tasic and L. Li for generating and validating unpublished 3xMyc-

tdT constructs, Y. Chen-Tsai and Stanford Transgenic Facility for help with

generating knockinmice, R. Tsien for providing tdT, R. Kageyama for providing

Nestin-CreER+/!, members of the Luo, Zong, and Wynshaw-Boris labs for

discussion, and S. McConnell, T. Mosca, C. Potter, Y. Chou, C. Liu, L. Swee-

ney, W. Joo, and M. Spletter for comments on the manuscript. This work was

supported by postdoctoral fellowships from the European Molecular Biology

Organization ALTF 851-2005 (S.H.), Human Frontier Science Program

LT00805/2006-L (S.H.) and LT00300/2007-L (K.M.), Swiss National Science

Foundation PA00P3_124160 (S.H.), JSPS Postdoctoral Fellowships for

Research Abroad (K.M.), and NIH grants NS050835 to L.L. and NS041310

and HD047380 to A.W.-B. L.L. is an investigator of the Howard Hughes

Medical Institute.

Accepted: August 30, 2010

Published: November 17, 2010

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Neuron, Volume 68

Supplemental Information

Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration Simon Hippenmeyer, Yong Ha Youn, Hyang Mi Moon, Kazunari Miyamichi, Hui Zong, Anthony Wynshaw-Boris, and Liqun Luo

INVENTORY: Supplemental Figures with Legends Supplemental Figure S1, related to Figure 1.

Supplemental Figure S2, related to Figure 1 and Figure 2.

Supplemental Figure S3, related to Figure 2.



Supplemental Figure S6, related to Figure 4 and Figure 5.




Supplemental Movies Supplemental Movie 1, related to Figure 7.

Supplemental Movie 2, related to Figure 7.

Supplemental Experimental Procedures Supplemental References

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Figure S1. Principle of MADM, Related to Figure 1. MADM employs Cre/LoxP-dependent interchromosomal recombination to generate uniquely labeled homozygous mutant cells in an otherwise heterozygous background in mice. Two reciprocally chimeric marker genes are targeted separately to identical loci on homologous chromosomes (here Hipp11 for MADM-11). The chimeric marker genes (labeled as GT and TG alleles) consist of partial coding sequences for green (eGFP[G]) and red (tdT[T], tandem dimer Tomato) fluorescent proteins separated by an intron containing the LoxP site. MADM-11 contains an additional FRT site that allows the induction of MADM-labeling by the FLP recombinase. Following recombinase-mediated interchromosomal recombination, functional

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green and red fluorescent proteins are reconstituted. If recombination occurred at the G2 phase and the two recombinant chromosomes were segregated into different daughter cells (X-Segregation, left branch; we term these G2-X events), each daughter cell would express a single fluorescent protein. When a mutation of interest is introduced distal to one MADM cassette, then one of the daughter cells would be homozygous mutant (here the green cell) for the gene of interest, whereas its sibling, labeled by a different color (red), would be homozygous wild-type. In addition to G2-X events, recombination in G2 followed by Z-Segregation (G2-Z events, right branch), G1, or postmitotic recombinations (not shown) do not alter the heterozygote genotype, but can produce double-labeled (yellow) cells. Note that the direction of transcription of the transgenes from MADM-11 (proximal to distal with the centromere as reference) is opposite to the direction of transcription from the original MADM at the Rosa26 locus (distal to proximal; Zong et al., 2005).

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Figure S2. Possible Effect of LoxP Sites at Targeted Knockout Loci for MADM Analysis, Related to Figures 1 and 2. MADM-11 provides a genetic mosaic analysis resource for mutations located on Chr. 11 distal to Hipp11. However, given that a significant number of targeted mutations contain LoxP sites (such as the three mutations in Lis1, Ndel1 and 14-3-3 analyzed in this study), there is a possibility that intra-chromosomal cis-recombination with the LoxP site in the MADM cassettes could occur in the absence of, or in combination with, MADM-mediated inter-chromosomal recombination. Such cis-recombination events would cause inversion or deletion depending on the orientation of the LoxP site in target loci. Previous studies in ES cells have provided an estimate for such cis-recombination events (Zheng et al., 2000). If cis-recombination resulted in inversions and therefore unlikely to cause lethality in ES cells (as oppose to deletions which likely cause ES cells to die), the rate of inversion was estimated to be 2.2x10-3, 3.2x10-4, and 8.3x10-5 for distances of 24, 30 and 60 cM (Zheng et al., 2000). Given the large distance between MADM cassette near the centromere and the three target loci we analyzed (>40 cM), we expect a low rate of cis-recombination. Nevertheless, we have modeled the consequence if such events do happen [for simplicity, we assume that the mutant allele is linked with the green GFP marker (TG), as in most of our experiments; see Figure S1 for complete MADM scheme]:

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(A) If the trans-recombination between the MADM cassettes does not occur, cis-recombinations alone that cause deletion (A1) or inversion (A2) would not produce labeled cells and therefore should not affect the outcome of MADM analysis, which focuses on labeled cells. (B) In +/- yellow cells that were generated by G2-Z, G1 and postmitotic recombinations, cis-recombination can only occur on the green chromosome that carries the mutation. This would result in loss of the green marker and the cell would turn from yellow to red. In addition this red cell will carry an additional deletion (B1) or inversion (B2), depending on the loxP orientation. (C-D) In -/- green cells, there are two possibilities. If cis-recombination occurs on the recombinant chromosome (C), the green marker would be disrupted, resulting in the loss of color. The frequency of green cells in MADM labeling is consequently reduced. If cis-recombination occurs on the non-recombinant chromosome (D), the resulting cell would still be green, but will carry an additional deletion (D1) or inversion (D2). Additionally, in +/+ cells (not shown), no cis-recombination occurs because there is no mutant allele carrying the additional loxP site.

According to the LoxP orientations in the three alleles we analyzed, cis-recombination in Lis1 and Ndel1 should result in large deletions, whereas cis-recombination in 14-3-3 should result in an inversion. We can apply the above scenario to our mutants to estimate cis-recombination frequency.

Assuming that inversions cause little effect on the viability of cells (Zheng et al., 1999), these events should reduce the number of green cells (C2) and increase the number of red cells (B2) and therefore reduce green/red ratio. Yet in our 14-3-3 mutant neuron and glia counts, we found green/red ratio to be indistinguishable from 1:1 (Figure 2F, S5D and S6, data not shown). These results suggest that the frequency of such cis-recombination is negligible.

For deletion events, if they result in cell lethality due to partial monosomy of Chr. 11, then green cells would be reduced (C1, D1) but red cells would not increase (red cells die in B1). If such deletions are compatible with cell viability, then the situation is similar to inversion discussed above. In both cases we expect to see a reduction of the green/red ratio if the frequency of such cis-recombination is significant. For Ndel1, cis-recombination events would delete most of Chr. 11, and would cause reduction of green/red ratio if such events happen frequently. However, Figure 2F and 6Q shows that the green/red ratio is not different from 1 in Ndel1-MADM.

Taken together, these conclusions are consistent with previous data in ES cells (Zheng et al., 2000) that the frequency of Cre-LoxP mediated cis-recombination occurs rarely if the genomic distance between the LoxP sequences is sufficiently large, and therefore should not affect the outcome of phenotypic analysis using MADM.

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Figure S3. General Breeding Scheme and Results for Reciprocal Ndel1-MADM Analysis, Related to Figure 2. (A) Breeding strategy to generate recombinant (2 crosses) and experimental (1 cross) MADM-mice. Using this strategy, the mutant cells will be labeled in green as seen throughout most of the

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study, including the cells in panel S3C, as well as in the schematic depicted in Figure S1. First cross: homozygous MADM-11TG/TG is crossed to heterozygous mutant+/-. Second cross: double heterozygote MADM-11TG,+/+,mutant is backcrossed to homozygous MADM-11TG/TG and meiotic recombinants between the mutant allele and the TG allele are selected. Third cross: MADM-11TG,mutant/TG,+ is crossed to MADM-11GT/GT;XCre/Cre (where X represents any locus with Cre recombinase inserted as transgene). Typically, MADM-11GT/TG,mutant;XCre/+ were selected with mutant cells labeled in green and wt cells labeled in red (mutant-MADM), and MADM-11GT/TG;XCre/+ were used as control (control-MADM). Homozygous mutant cells can also be labeled in red and their homozygous wild-type siblings in green by reversing the crossing scheme with respect to GT and TG alleles to create a GT, mutant recombinant in the first cross (see Figure S3D as an example).

(B-D) Examples of MADM-labeled cells in mosaics in the somatosensory cortex using Emx1Cre/+ with control-MADM (B) (MADM-11GT/TG;Emx1Cre/+) and mutant-MADM where mutant cells are green (C) (MADM-11GT/TG,Ndel1;Emx1Cre/+) or red (D) (MADM-11GT,Ndel1/TG;Emx1Cre/+). It is evident that Ndel1-/- neurons (but not astrocytes), whether labeled in green (C) or red (D), exhibit severe migration defects. Cortical layers are indicated in roman digits, WM: white matter. Scale bar, 150 m.

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Figure S4. MADM Analysis of Lis1 in Cortical Development, Related to Figure 2. Time course analysis of the migration pattern of cortical projection neurons in Lis1-MADM (MADM-11GT/TG,Lis1;Emx1Cre/+) at P1 (A and B) and P7 (C and D). Mutant Lis1-/- cells are labeled with GFP (green only), heterozygous Lis1+/- cells are GFP/tdT double positive (yellow) or unlabeled (vast majority), and Lis1+/+ cells are marked with tdT (red only). Nuclei were stained using DAPI (blue). White stars (A and C) indicate sparse green Lis1-/- mutant neurons. Cortical layers are numbered in roman digits. WM: white matter. Quantification charts (B and D) indicate the relative distribution (%) of mutant green, heterozygote yellow and wt red neurons. Values represent mean ± SEM. ns: non-significant, *p<0.05, **p<0.01, and ***p<0.001. Scale bar, 50 m (A); 90 m (C).

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Figure S5. MADM Analysis of Lis1, Ndel1 and 14-3-3 in Hippocampal Dentate Gyrus Granule Cell Layer, Related to Figure 3. Distribution of MADM-labeled dGCs in the hippocampus of control-MADM (A and E; MADM-11GT/TG;Emx1Cre/+), Lis1-MADM (B and F; MADM-11GT/TG,Lis1;Emx1Cre/+), Ndel1-MADM (C and G; MADM-11GT/TG,Ndel1;Emx1Cre/+) and 14-3-3 -MADM (D and H; MADM-11GT/TG,14-3-

3 ;Emx1Cre/+). Nuclei were stained using DAPI (blue). Scale bar, 40 m.

For quantification (E-H) of the relative distribution (%) of dGCs, the dentate gyrus was divided into three equal horizontal sectors as seen in (A). Note the significant fraction of green Ndel1-/- dGCs in the hilus/polymorphic layer below sector 1 (h/pl). Occasionally green Ndel1-/- and Lis1-/- dGCs were found in the stratum moleculare (mol). Values represent mean ± SEM. ns: non-significant, *p<0.05, **p<0.01, and ***p<0.001.

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Figure S6. MADM Analysis of 14-3-3 in Olfactory Bulb and Cerebellum, Related to Figure 4 and Figure 5. (A-C) Distribution of olfactory bulb interneurons (oINs) in the olfactory bulb (A), and Purkinje cells (B) and cGCs (C) in the cerebellum in P21 MADM-11GT/TG,14-3-3 ;Emx1Cre/+ (A) and MADM-11GT/TG,14-3-3 ;Nestin-spCre+/- (B and C). Mutant 14-3-3 -/- cells are labeled with GFP (green only), heterozygous 14-3-3 +/- cells are GFP/tdT double positive (yellow) or unlabeled, and 14-3-3 +/+ cells are marked with tdT (red). Nuclei in (A) were stained using DAPI (blue). Scale bar, 90 m in (A); 40 m in (B and C).

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(D) Quantification of relative distribution (%) of oINs across three equal sectors in the granule cell layer (GCL) in the olfactory bulb in 14-3-3 -MADM. G: glomerular layer; M: mitral cell layer.

(E) Quantification of relative distribution (%) of Purkinje cells in Purkinje cell (PC) layer or ectopic locations (ect. PC); and cGCs across the molecular (ML) and internal granule cell layer (IGL) in 14-3-3 -MADM. The IGL was divided into three equal sectors for quantification of the relative distribution of cGCs. Values represent mean ± SEM. Note that no difference was observed in the distribution of mutant 14-3-3 -/- oINs, Purkinje cells and cGCs when compared to control 14-3-3 +/+ cells.

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Figure S7. Live-Imaging of MADM-labeled Wild-type Cortical Projection Neurons, Related to Figure 7. Time-lapse images of migrating cortical projection neurons in the IZ (A-H) and at the border to the CP (I-P) in cortical slices derived from control-MADM (MADM-11GT/TG;Emx1Cre/+) mice at E14.5. Open arrowheads and stars mark migrating green (GFP), red (tdT) and yellow (GFP+/tdT+) wild-type neurons. The border between the IZ and CP is indicated as dotted line in magenta (I-P). Frames are at 30’ (A-H) and 15’ (I-P). Scale bar, 50 m in (A-H); 40 m in (I-P).

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Figure S8. Analysis of Apical Dendrite Morphology in CA1 Pyramidal Cells, Related to Figure 8. (A-C) Dendrite pattern in pyramidal CA1 cells in the hippocampus in P21 Lis1-MADM (A; MADM-11GT/TG,Lis1;Emx1Cre/+), Ndel1-MADM (B; MADM-11GT/TG,Ndel1;Emx1Cre/+), and 14-3-3 -MADM (C; MADM-11GT/TG,14-3-3 ;Emx1Cre/+). Mutant Lis1-/- (A), Ndel1-/- (B) and 14-3-3 -/- (C)

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cells are labeled with GFP (green); heterozygous Lis1+/- (A), Ndel1+/- (B) and 14-3-3 +/- (C) are GFP/tdT double positive (yellow) or unlabeled; and homozygous wt Lis1+/+ (A), Ndel1+/+ (B) and 14-3-3 +/+ (C) cells are marked with tdT (red). Cyan star in (C) marks mutant 14-3-3 -/- located in CA1 and green star in (C) marks ectopically located 14-3-3 -/- cells. In rare cases, mutant Ndel1-/- were observed in CA1 (see quantification below) but the vast majority of mutant Ndel1-/- pyramidal cells were located at the base of the CA1 field. See also Figure 3 for quantification of relative distribution and distance of cell body from base of hippocampus in control-MADM, Lis1-MADM, Ndel1-MADM and 14-3-3 -MADM. Scale bar, 40 m.

(D) Quantification of the length ( m) of apical CA1 pyramidal cell dendrites in the hippocampus in control-MADM, Lis1-MADM, Ndel1-MADM and 14-3-3 -MADM. Note the increased length of ectopically located Lis1-/-, Lis1+/-, Ndel1-/- (but not Ndel1+/- or rare Ndel1-/- located in CA1), and 14-3-3 -/- (but not 14-3-3 +/- or 14-3-3 -/- located in CA1). Values represent mean ± SEM. ns: non-significant, ***p<0.001.

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Figure S9. Variability in the Morphology of Axonal Varicosities in Ndel1-/- Cortical Projection Neurons, Related to Figure 8. Pattern of axonal varicosities within the internal capsule in P21 Ndel1-MADM (MADM-11GT/TG,Ndel1;Emx1Cre/+) in overview (A, C and E) and at high resolution (B, D and F). Mutant Ndel1-/- subcortical projecting axons are labeled with GFP (white in A and B; green in E and F) and DAPI (white in C and D; blue in E and F) marks nuclei. Note that axonal varicosities are highly variable in size and shape and do not co-localize with DAPI. Scale bar, 40 m in (A, C and E); 10 m in (B, D and F).

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SUPPLEMENTAL MOVIES

Movie S1. Related to Figure 7. Live-imaging of MADM-labeled green, red, and yellow wild-type migrating cortical projection neurons at the IZ-CP border in control-MADM at E14.5 over the course of 7h30min.

Movie S2. Related to Figure 7. Live-imaging of MADM-labeled green Ndel1-/-, yellow Ndel1+/- and red Ndel1+/+ migrating cortical projection neurons at the IZ-CP border in Ndel1-MADM at E14.5 over the course of 11h15min.

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SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Construction of MADM-11 Transgenes by Targeted Knockin and Mouse Genetic Techniques Modified chimeric GT and TG MADM-cassettes were generated by splitting coding sequences for eGFP (Zong et al., 2005), and tdT (Shaner et al., 2004) tagged at the C-terminus with 3xMyc epitope, with an intron that contains a LoxP-neo-LoxP cassette (Zong et al., 2005) as well as an additional FRT site. GT and TG marker genes: GT: GFPN-term (1-273) - intron - tdTC-term (4-1544) and TG: tdTN-term (1-3) - intron - GFPC-term (274-724).

The genomic Hipp11 locus for targeting the MADM cassettes was cloned by PCR amplification from 129/SvJ genomic DNA. GT and TG MADM-cassettes were inserted at Hipp11 to construct GT and TG targeting vectors, which were linearized and electroporated into R1 ES cells. Two correctly targeted ES clones for each GT and TG were injected into blastocyst embryos to generate chimeric mice. All MADM experiments were carried out in mixed 129/C57Bl6/CD1 genetic background. Details on the generation of recombinant and experimental MADM mice are described Figure S3.

Heterozygote Lis1+/- (Hirotsune et al., 1998), Ndel1+/- (Sasaki et al., 2005), and 14-3-3 +/- (Toyo-oka et al., 2003); Emx1Cre/+ (Gorski et al., 2002); Nestin-CreER+/- (Imayoshi et al., 2006) and Rosa26Flpe/+ (Farley et al., 2000) mice have been described previously. Timed pregnancies were setup to generate embryos and mice at defined developmental stages as indicated throughout the study.

All experimental procedures involving the above listed mice were carried out in accordance with the APLAC (Administrative Panel on Laboratory Animal Care) protocol and the institutional guidelines by the Veterinary Service Center (VSC) at Stanford University.

Generation of Recombinant Mice for MADM Analysis Mouse breeding for generation of recombinant mice was carried out according to the scheme in Figure S3. The scale of the mating for generating recombinants using meiotic recombination was adjusted according to the genetic distance between the MADM cassettes and the gene of interest. In particular, MADM-11 cassettes are ~3 cM (centi-Morgan) distal to the centromere while for example Lis1 is located ~44 cM distal to the centromere, or ~41 cM away from the MADM cassettes. Thus, there is 41% probability that meiotic recombination will occur in the recombinant generating mating (Figure S3). However, since only half of the progeny will be of the desired genotype (TG,mutant), while the other half will be wild-type (+,+), animals with the appropriate genotype will be generated at a rate of ~20% (41% x ½). Upon recombination of the mutant onto the TG-containing MADM chromosome, mutant cells are labeled in green and wild-type control cells in red (see also Figure S3C for Ndel1 mutant). A similar crossing strategy using the reciprocal MADM cassette (GT) in the crosses described above is typically used to generate mice ready for MADM analysis with labeling of mutant and wild-type cells in the ‘opposite’ direction (mutant in red and wild-type control in green, see also Figure S3C and S3D). Generally, performance of MADM analysis in both directions with reciprocal labeling scenarios should eliminate any possible bias.

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Live-Imaging Assay for MADM-Labeled Organotypic Slices

Whole E14.5 MADM-labeled brains (control- and Ndel1-MADM) were isolated and 350 m coronal slices obtained using a vibratome (Leica). Slices were transferred to transparent cell culture filter insets (Millipore) and cultured in DMEM/F12 medium containing 1X N2 supplement (Gibco) for 1-2h in glass-bottom 6-well plates (MatTek) prior to live-imaging sessions lasting typically 10-15h (individual frames at 10-15min.) using an inverted confocal microscope equipped with an environmental chamber (Leica). Movies for analysis of cell migration were generated using Leica TCS SP5 software. Average speed of migration ( m/h) was determined by measurement of the distance travelled during the time period of active locomotion of migrating cortical projection neurons (derived from 3 independent movies). IZ-CP borders were determined by bright field microscopy based on the cell density difference. Quantifications of neurite length and branch numbers were carried out as previously described (Youn et al., 2009). Values represent mean ± SEM. Student’s t-test was used to determine significance with ***p<0.001.

Total Cell Numbers for Quantification of Phenotypes and Statistical Analyses Total cell numbers that were included in the quantification charts throughout the study were:

Figure 2 (control-MADM: 4667; Lis1-MADM: 2750; Ndel1-MADM: 4923; 14-3-3 -MADM: 3242). Figure 3 (control-MADM: 529; Lis1-MADM: 307; Ndel1-MADM: 602; 14-3-3 -MADM: 481). Figure 4 (control-MADM: 475 in RMS, 876 in OB; Ndel1-MADM: 1076 in RMS, 705 in OB). Figure 5 (control-MADM: 94 Purkinje cells, 574 cGCs; Ndel1-MADM: 306 Purkinje cells, 1651 cGCs). Figure 6 (control-MADM: 3604; Ndel1-MADM: 3891; clonal-MADM: 756). Figure 7 (Ndel1-MADM: 341). Figure S4 (Lis1-MADM: 4112). Figure S5 (control-MADM: 387; Lis1-MADM: 236; Ndel1-MADM: 716; 14-3-3 -MADM: 289). Figure S6 (14-3-3 -MADM: 298 in OB; 107 Purkinje cells, 1216 cGCs). Figure S8 (control-MADM: 30; Lis1-MADM: 43; Ndel1-MADM: 63; 14-3-3 -MADM: 36). Values in all graphs and charts generally represent mean ± SEM. Student’s t-test was used to determine significance with *p<0.05, **p<0.01, and ***p<0.001.

Possible Effect of Perdurance in Mosaic Analysis In MADM analysis, it is possible that the perdurance of remnant mRNA and/or protein from parental heterozygous cells may provide residual Ndel1 function, which could in principle support a certain level of neuronal migration in the Ndel1-/- cells. However, the following lines of evidence strongly argue against the possibility that perdurance of Ndel1 contributes significantly to the phenotypic difference between our sparse knockout and genetic perturbations of Ndel1. The Emx1-Cre and Nestin-spCre drivers that we used to generate MADM clones induce recombination well before the hGFAP-Cre (Zhuo et al., 2001) (which is not active before E12.5/E13.5 in cortical RGPC) used for whole cortex Ndel1 knockout using the same mutant allele (Youn et al., 2009). Yet live imaging of hGFAP-Cre induced Ndel1 knockout neurons fail to migrate in E14.5 slices (Youn et al., 2009), whereas live imaging of MADM knockout Ndel1 neurons under similar imaging conditions show an average migration speed indistinguishable

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from control neurons (Figure 7Q). Furthermore, we have generated TM-induced MADM-clones as early as E8 (Figure 6M). These early E8 clones included dozens of MADM-labeled Ndel1-/- neurons - residual Ndel1 mRNA and/or NDEL1 protein would have been serially diluted to a few percent even without degradation. For example, in all MADM experiments, the mitotically active ‘mother’ progenitor cell is heterozygous (starting with ~50%) and after 4 divisions, one could expect maximal RNA/protein perdurance to be less than 5%. Nevertheless, Ndel1-/- neurons from early E8 induced clones exited the VZ/SVZ properly and migrated across the IZ without significant defects when compared to labeled control neurons. However, TM-induced conditional whole cortex knockout of Ndel1 one day prior to live-imaging at E14.5 results in a complete block of neuronal migration (Youn et al., 2009), indicating minimal perdurance of Ndel1 activity even within a day after gene inactivation.

For Lis1 mosaic analysis, it is possible that the 10% or so remaining Lis1-/- neurons observed in Lis1-MADM were direct progeny of Lis1+/- progenitors undergoing the final cell division and therefore could have the highest perdurance. Significant perdurance in a fraction of Lis1-/- neurons may allow those neurons to migrate into and within the cortical plate. However, several lines of evidence argue against this interpretation. First, neuronal migration is extremely sensitive to Lis1 dosage and neurons with ~35% of LIS1 protein show significant deficits in migration (Gambello et al., 2003; Hirotsune et al., 1998). Second, conditional deletion of Lis1 in the cortex resulted in complete block of migration within a day (Youn et al., 2009). Third, Lis1-/- cortical projection neurons exhibited a significant migration delay when examined at P1, and somehow “caught up” with Lis1+/- neurons at P7 and P21 (Figure S3). Perdurance would result in an opposite time-dependence.

For all of the above reasons, we consider that potential perdurance of NDEL1 or LIS1 cannot account for the difference of phenotypes we observed in sparse knockout and previous cortical knockout. Rather, these differences are most likely caused by cell-non-autonomous effect.

SUPPLEMENTAL REFERENCES Farley, F.W., Soriano, P., Steffen, L.S., and Dymecki, S.M. (2000). Widespread recombinase expressionusing FLPeR (flipper) mice. Genesis 28, 106 110.Gambello, M.J., Darling, D.L., Yingling, J., Tanaka, T., Gleeson, J.G., and Wynshaw Boris, A. (2003).Multiple dose dependent effects of Lis1 on cerebral cortical development. J Neurosci 23, 1719 1729.Gorski, J.A., Talley, T., Qiu, M., Puelles, L., Rubenstein, J.L., and Jones, K.R. (2002). Cortical excitatoryneurons and glia, but not GABAergic neurons, are produced in the Emx1 expressing lineage. J Neurosci22, 6309 6314.Hirotsune, S., Fleck, M.W., Gambello, M.J., Bix, G.J., Chen, A., Clark, G.D., Ledbetter, D.H., McBain, C.J.,and Wynshaw Boris, A. (1998). Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migrationdefects and early embryonic lethality. Nat Genet 19, 333 339.Imayoshi, I., Ohtsuka, T., Metzger, D., Chambon, P., and Kageyama, R. (2006). Temporal regulation of Crerecombinase activity in neural stem cells. Genesis 44, 233 238.

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Sasaki, S., Mori, D., Toyo oka, K., Chen, A., Garrett Beal, L., Muramatsu, M., Miyagawa, S., Hiraiwa, N.,Yoshiki, A., Wynshaw Boris, A., et al. (2005). Complete loss of Ndel1 results in neuronal migrationdefects and early embryonic lethality. Mol Cell Biol 25, 7812 7827.Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. (2004).Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. redfluorescent protein. Nat Biotechnol 22, 1567 1572.Toyo oka, K., Shionoya, A., Gambello, M.J., Cardoso, C., Leventer, R., Ward, H.L., Ayala, R., Tsai, L.H.,Dobyns, W., Ledbetter, D., et al. (2003). 14 3 3epsilon is important for neuronal migration by binding toNUDEL: a molecular explanation for Miller Dieker syndrome. Nat Genet 34, 274 285.Youn, Y.H., Pramparo, T., Hirotsune, S., and Wynshaw Boris, A. (2009). Distinct dose dependent corticalneuronal migration and neurite extension defects in Lis1 and Ndel1 mutant mice. J Neurosci 29, 1552015530.Zheng, B., Sage, M., Cai, W.W., Thompson, D.M., Tavsanli, B.C., Cheah, Y.C., and Bradley, A. (1999).Engineering a mouse balancer chromosome. Nat Genet 22, 375 378.Zheng, B., Sage, M., Sheppeard, E.A., Jurecic, V., and Bradley, A. (2000). Engineering mousechromosomes with Cre loxP: range, efficiency, and somatic applications. Mol Cell Biol 20, 648 655.Zhuo, L., Theis, M., Alvarez Maya, I., Brenner, M., Willecke, K., and Messing, A. (2001). hGFAP cretransgenic mice for manipulation of glial and neuronal function in vivo. Genesis 31, 85 94.Zong, H., Espinosa, J.S., Su, H.H., Muzumdar, M.D., and Luo, L. (2005). Mosaic analysis with doublemarkers in mice. Cell 121, 479 492.

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