Genetic Mosaic Dissection of Lis1
and Ndel1 in Neuronal Migration
and Liqun Luo1,*
1Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA
2Department of Pediatrics and Institute for Human Genetics
3Biomedical Sciences Graduate Program
4Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research
University of California, San Francisco School of Medicine, San Francisco, CA 94143, USA
5Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
*Correspondence: firstname.lastname@example.org (S.H.), email@example.com (L.L.)
Coordinated migration of newly born neurons to their
circuit assembly in the developing brain. The evolu-
tionarily conserved LIS1/NDEL1 complex is essential
for neuronal migration in the mammalian cerebral
cortex. The cytoplasmic nature of LIS1 and NDEL1
proteins suggest that they regulate neuronal migra-
ysis with double markers (MADM) to mouse chromo-
some 11 where Lis1, Ndel1, and 14-3-33 (encoding
a LIS1/NDEL1 signaling partner) are located. Anal-
yses of sparse and uniquely labeled mutant cells in
tions for these three genes. Lis1 regulates neuronal
migration efficiency in a dose-dependent manner,
while Ndel1 is essential for a specific, previously un-
characterized, late step of neuronal migration: entry
into the target lamina. Comparisons with previous
genetic perturbations of Lis1 and Ndel1 also suggest
a surprising degree of cell-nonautonomous function
for these proteins in regulating neuronal migration.
The assembly of functional neural circuits requires the segrega-
tion and interconnection of distinct classes of neurons. In the
vertebrate central nervous system, a prevalent motif in neuronal
organization is the coalescence of neuronal types into stratified
layers or laminae (Ramon y Cajal, 1911). Coordinated migration
of newly born neurons from their birthplace to their final position
represents a fundamental mechanism to achieve lamination
within all structures of the brain. In the past decades, distinct
neuronal migration modes as well as a rich catalog of molecules
controlling 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’’ fashion
whereby 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 migrating
cortical projection neurons are bipolar-shaped in the ventricular
zone (VZ) but then convert to a multipolar morphology within the
subventricular zone(SVZ)andmigrate intothe intermediate zone
(IZ). A switch from the multipolar state back to a bipolar
morphology precedes radial glia-guided locomotion of projec-
tion neurons toward the cortical plate (CP), with the trailing
process concomitantly developing into the axon. Once the
neuron arrives in the CP, the leading process attaches to the
pial surface and the neuron undergoes terminal somal transloca-
tion to reach its final location (Nadarajah et al., 2001; Noctor
et al., 2004; Rakic, 1972; Tsai et al., 2005).
The importance of neuronal migration for cortical lamination
is highlighted in patients that suffer from isolated lissencephaly
sequence (ILS) or Miller-Diecker syndrome (MDS). Lissence-
phaly is characterized by a smooth brain surface with an
absence or severe reduction of gyri, abnormal lamination, and
thickening of the cerebral cortex. About 40% of ILS and virtually
100% of MDS cases occur due to the loss of one copy of the
Lissencephaly-1 (LIS1, also known as PAFAH1B1) gene on
human 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 regulates
nuclear migration through the cytoplasmic microtubule motor
dynein (Morris, 2000). In mice, reduced LIS1 activity results in
severe defects in the radial migration of multiple types of
neurons including neocortical projection neurons (Cahana
et al., 2001; Gambello et al., 2003; Hirotsune et al., 1998; Tsai
et al., 2005). NDEL1 (nuclear distribution gene E-like homolog
1) 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; Shu
et al., 2004; Youn et al., 2009). NDEL1 is a substrate for the
serine/threonine protein kinase CDK5 (Niethammer et al.,
2000; Sasaki et al., 2000), which is also essential for cortical
neuronal migration (Gilmore et al., 1998). The adaptor protein
14-3-33 binds NDEL1 in a phosphorylation-dependent manner
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 695
to maintain NDEL1 phosphorylation, which is important for
binding to LIS1 and the dynein motor (Toyo-oka et al., 2003).
Loss of one copy of 14-3-33 (also known as YWHAE), which
resides within the MDS deletion in human, enhances clinical
symptoms of LIS1 heterozygosity in humans and neuronal
migration defects in Lis1 heterozygous mice (Toyo-oka et al.,
2003). Thus, the tripartite LIS1/NDEL1/14-3-33-complex is
a key regulator of cortical neuronal migration (Wynshaw-Boris,
The coupling of the nucleus and centrosome mediated by the
LIS1-complex is a key cell biological mechanism for neuronal
nature of these proteins suggests that they function cell autono-
mously to regulate neuronal migration, but this has not been
directly tested in vivo. Mice carrying homozygous null alleles
die either at implantation (Lis1, Ndel1) or neonatal (14-3-33)
stages (Hirotsune et al., 1998; Sasaki et al., 2005; Toyo-oka
et al., 2003). Thus, previous studies addressing the in vivo func-
tions of the LIS1-complex relied on the analysis of heterozygous
or compound heterozygous animals where all cells were mutant,
on conditional mutants or on RNAi knockdown approaches
where large groups of neurons were affected. To assess the
cell-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 sparse
subpopulations of neurons.
Extension of MADM to Chromosome 11
The Lis1, Ndel1, and 14-3-33 genes are located on Chr. 11 in the
mouse. In order to perform mosaic analyses of these genes
using the MADM strategy, we cloned the Hipp11 locus near
the 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 (Zong
et al., 2005) with tandem dimer Tomato (tdT) (Shaner et al.,
cassettes to the Hipp11 locus using homologous recombination
MADM-11TG(TG: tdTN-term–GFPC-term) mice (Figure 1B).
cells in trans-heterozygous MADM-11GT/TGmice (data not
shown). As predicted by the MADM scheme (Figure S1), intro-
duction of Emx1Cre/+(Gorski et al., 2002) produced fluorescently
MADM-labeled (GFP only, tdT only, or GFP+/tdT+) cells
restricted to the forebrain (Figures 1D–1G). MADM-11 labeling
Nestin-CreERT2transgenic line (referred to as Nestin-spCre
hereafter) where CRE recombinase is active in sparse, random
subsets 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-
tion of the red fluorescence marker in live animals. Thus,
genotypes of distinctly labeled cells in mosaic animals can be
unequivocally determined before fixation and immunostaining.
of live tissues in MADM animals. Second, tdT+and GFP+axonal
be clearly traced without signal amplification by antibody
staining. Third, the interchromosomal recombination rate in
MADM-11 is markedly increased compared with the original
induction in many areas of the brain, including the cerebral
cortex, using TM-inducible Nestin-CreER (e.g., lines 4 and 5 in
Imayoshi et al., 2006). As an example, we show a single G2-X
projection neurons in the neocortex, which was induced at
embryonic day 10 (E10) by TM and analyzed at E16 (Figures 1I–
1M). Fourth, FLPe recombinase (Farley et al., 2000) can also be
used 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 to
MADM-based mosaic analyses.
Lis1 Is Cell Autonomously Required for Cortical Neuron
and Astrocyte Production
To genetically dissect the cell-autonomous functions of the Lis1,
Ndel1, and 14-3-33 genes, we generated separate recombinant
MADMGTand MADMTGstrains for null mutants of Lis1 (Hirotsune
et al., 1998), Ndel1 (Sasaki et al., 2005) and 14-3-33 (Toyo-oka
et al., 2003), respectively (Figure S3A). These recombinants were
crossed to mice that carried the reciprocal MADM cassette and
a Cre recombinase transgene to generate experimental MADM
animals (Figure S3A). We examined individual Lis1?/?, Ndel1?/?,
and 14-3-33?/?projection neurons during neocortical develop-
mentusing Emx1-Creexpressed incorticalprogenitors (Figure2).
First, we analyzed the role of Lis1, Ndel1, and 14-3-33 in
cortical neuron production. We compared the number of homo-
in the somatosensory cortex at postnatal day 21 (P21) (Figures
2B–2F). G2-X events allow the visualization of two progeny of
the same mitosis with two distinct colors (Figure S1). If cell divi-
sionwere symmetric, thenumberofred andgreencellswithin an
isolated clone would be identical (see Figures 1I–1M). Even if the
cell division were asymmetric such that red and green progeny
numbers were different in individual clones, the random distribu-
of red and green progeny were equal overall, given a large
enough 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 and
2F). 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 required
cell autonomously for production or survival of cortical neurons.
A similarly low mutant/WT ratio in the cortex was also observed
atP1 and P7 (Figures S4Aand S4C; data not shown).These data
indicate that LIS1 is not required for survival of neurons during
the postnatal period, although we cannot rule out the possibility
that LIS1 is required for survival of prenatal neurons (Gambello
et al., 2003). In addition, no Lis1?/?astrocytes were observed
(Figures 2C and 2F). The most likely interpretation for our results
isthatLIS1isessential forcorticalneuralprogenitor celldivisions
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 both
cortical projection neurons and astrocytes (Costa et al., 2009).
A similar reduction in the number of Lis1?/?cells was evident
inall brainareas analyzed,includingthe hippocampus,theolfac-
tory bulb and the cerebellum (data not shown). Together, these
findings suggest that LIS1 is cell autonomously required for
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-11GTand 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 P21 MADM-11 mice. (C and H) Sparse
MADM labeling of cortical pyramidal cells in MADM-11GT/TG;Rosa26FLPe/+(C) or MADM-11GT/TG;Nestin-spCre+/?(H). (D) Overview of labeling pattern in MADM-
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 in MADM-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-XMADMcloneinthecortex withneuronsexpressing GFP (green)and tdT(red),andmigrating alongRGPCs atE16 (J).Inset in(J)marksareashown in
(K)–(M)and depicts redand green apical endfeetof MADM-labeledradialgliawithmigrating neuronsintheVZ.Nuclei (D,J,and K)werelabeled usingDAPI(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.
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 697
production of most if not all neuronal classes. Indeed, LIS1 is
also cell autonomously required for the production of neurons
in the Drosophila brain (Liu et al., 2000), indicating that this is
an evolutionarily conserved function.
In contrast to Lis1, the mutant/wt (green/red) ratio for Ndel1
and 14-3-33 neurons and astrocytes was not significantly
different from 1 (Figures 2D–2F) under identical experimental
conditions. These results indicate that NDEL1 and 14-3-33 are
not cell autonomously required for proliferation of neural progen-
itors or survival of postmitotic neurons and astrocytes. One
possible explanation for these divergent phenotypes could be
genetic redundancy. For example, the function of NDEL1 in
cell proliferation may be compensated for by NDE1, which
shares 55% sequence homology with NDEL1; deletion of Nde1
results in mice with microcephaly (small brain), likely caused by
a reduction in progenitor cell division (Feng et al., 2000; Feng
and 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 to
regulate cell proliferation.
Lis1 and Ndel1 Display Distinct Cell-Autonomous
Phenotypes in Cortical Neuron Migration
Next, we analyzed the role of Lis1, Ndel1, and 14-3-33 in radial
migration of cortical projection neurons. Since the vast majority
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 14-3-33 MADM (C–E), homozygous mutants 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.
Mosaic Analysis of Lis1 and Ndel1
698 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.
of cells in Lis1-, Ndel1-, and 14-3-33-MADM mice are heterozy-
gous for the respective gene to be analyzed, we can study the
effect of gene dosage in addition to homozygous loss of each
gene by comparing the layer distribution of homozygous mutant
the same animals (Figures 2C–2E), and to control-MADM
animals (Figure 2B). We quantified such distributions in the P21
somatosensory 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 in
separate animals (Figure 2B). However, the numbers of both
Lis1?/?and Lis1+/?neurons were significantly decreased in
layers 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 evident
cate that Lis1+/+cells exhibit a cell migration advantage
compared to Lis1+/?or Lis1?/?cells. Moreover, since Lis1+/+
cells reside in a largely heterozygous environment, these data
indicate that the migration advantage of Lis1+/+cells is cell
Perhaps surprisingly, Lis1?/?neurons do not exhibit obvious
defects when compared to Lis1+/?neurons. However, when
examined at postnatal day 1 (P1), we found a significant accu-
mulation of Lis1?/?cells in the white matter compared to
Lis1+/?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-significant
at P7 (Figures S4C and S4D). These findings suggest that homo-
zygous loss of Lis1 delays migration of cortical neurons, but that
this 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 control
animals (Figures 2B and 2G), indicating that cortical neuronal
migration is not sensitive to Ndel1 dosage. However, the vast
majority (>85%) of Ndel1?/?neurons accumulated in the white
matter below layer VI at P21 (Figures 2D and 2I), indicating
a 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). Identical
defects were seen when Ndel1?/?neurons were labeled with
tdT instead of GFP (Figure S3D). The accumulation of Ndel1?/?
as this defect persisted in an 8 month old animal (data not
shown). Because Ndel1?/?neurons are sparsely intercalated
among the vast majority of unlabeled cells that are Ndel1+/?in
these mosaic Ndel1 animals, this defect reflects a cell-autono-
mous function of NDEL1 in regulating cortical neuronal
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 cell
autonomously for migration of cortical neurons but not
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 in
control-MADM (Figures 2E, 2G, and 2J). These data suggest
that neither reduced dosage nor homozygous loss of 14-3-33
in isolated cells causes any defects in cortical neuron migration.
It is possible that distinct 14-3-3 isoforms may compensate for
loss of 14-3-33 function in neuronal migration.
Cell-Autonomous Function of Lis1, Ndel1, and 14-3-33
in Hippocampal Neuronal Migration
We extended the MADM analysis of Lis1, Ndel1, and 14-3-33
to 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 layer
where 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 in
control-MADM are consistent with the normal migration
pattern described above (Figures 3A and 3E). Likewise, the
soma of Ndel1+/+and 14-3-33+/+or Ndel1+/?and 14-3-33+/?
pyramidal cells were all located within the CA1 layer (Figures
3C and 3D, red and yellow). These data indicate that CA1
pyramidal neuron migration is not sensitive to the partial
reduction of the Ndel1 or 14-3-33 gene dosage, similar to
our findings in cortical neurons. By contrast, the vast majority
of Ndel1?/?CA1 (Figures 3C, 3E, and 3G) and CA3 (data not
shown) pyramidal neurons accumulated at the base of the CA
subfields. A significant fraction of 14-3-33?/?CA1 pyramidal
neurons also failed to reach the CA1 layer but were scattered
between the base and the CA1 cell layer (Figures 3D and 3E).
These data indicate that both Ndel1 and 14-3-33 are cell
autonomously required for hippocampal pyramidal neuron
migration, although the defect in 14-3-33?/?is milder than in
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. ) containing MADM-labeled CA1 pyramidal
neurons (Figure 3B). Given these heterozygous effects, we could
not clearly define the CA1 layer and determine the amount of
ectopically located pyramidal cells in Lis1-MADM. We therefore
quantified migration phenotypes in Lis1-MADM based on the
distance of the pyramidal cell soma from the base of CA1 (Fig-
ure 3F). Interestingly, the average distance of Lis1+/?and
residual Lis1?/?cells appeared equal (Figures 3B and 3F).
However, Lis1+/+cells were most often found at the ‘‘leading
edge’’ of the CA1 pyramidal layer, with a fraction of Lis1+/+cells
migrating beyond the dense CA1 field (Figures 3B and 3F).
Consequently, the average distance of Lis1+/+soma from the
base of CA1 was almost doubled when compared to Lis1+/?or
Lis1?/?(Figure 3F). These data reinforced our findings in cortical
neurons (Figure 2H), that two copies of normal Lis1 gene provide
migrating 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 the
CA1 field and cortical projection neurons (Figure S5): Ndel1 is
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 699
cell autonomously required for dGC migration; Lis1+/+dGCs dis-
played an advantage over Lis1+/?and Lis1?/?cells, which ex-
hibited indistinguishable migration behaviors; 14-3-33?/?dGCs
had no detectable migration defects, similar to cortical neurons
(Figure 2; see also Figure S6).
Ndel1 Is Essential for the Migration of Many Distinct
Types of Neurons
Prior to this study, Ndel1 function in vivo in neuronal migration
was 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 2D
and 2I) and hippocampus (Figure 3G) prompted us to expand
the mosaic analysis of Ndel1 to other neuronal types. We
focused on the olfactory bulb (OB; Figure 4) and the cerebellum
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)
(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
See also Figure S5.
(Figure 5) to test whether NDEL1 regu-
of distinct neuronal types.
OB interneurons (oINs) are mostly
from the SVZ of the lateral ventricle along
the rostral migratory stream (RMS) to the
OB (Lledo et al., 2008; Lois and Alvarez-
Buylla, 1994). After reaching the OB,
oINs exit the RMS and migrate centrifugally to occupy different
layers within the OB (Figure 4H). We found Ndel1?/?oINs along
the entire RMS and within the OB. However, the distribution of
Ndel1?/?cells differed significantly from Ndel1+/+cells (Figures
4A–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/red
neurons in Ndel1-MADM was reduced ?3-fold in the OB granule
Ndel1?/?oINs that have exited the RMS migrated significantly
shorter distances within the OB when compared to Ndel1+/+in
the same mosaic animal (Figure 4G). Similar results were ob-
tained when mosaic brains were examined at P6/7 (data not
Mosaic Analysis of Lis1 and Ndel1
700 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.
shown). Thus, Ndel1?/?oINs accumulate along the RMS and in
the most central granule cell layers within the OB, indicating
a 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 radial
migration from the ventricular zone outward around the time of
birth (Altman and Bayer, 1997; Miale and Sidman, 1961). We
usedNestin-spCre toanalyze MADM-labeled cerebellar Purkinje
cells, which were readily identifiable on the basis of their large
cell bodies and characteristic dendritic trees. We found that
>80% of Ndel1?/?Purkinje cells were located within the cere-
Cerebellar granule cells (cGCs) exhibit a unique migration
path. In the embryo, cGC progenitors migrate from the rhombic
lip to occupy the surface of the developing cerebellar cortex.
During the first 3 postnatal weeks, cGC progenitors proliferate
in the outer external granule layer (EGL) at the surface of the
Figure 4. Ndel1 Function is Essential for
Migration 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).
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 cell
layer and eventually settle within the
internal 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 red
Ndel1+/+and yellow Ndel1+/?
completed neurogenesis and migration, and all Ndel1+/+and
Ndel1+/?cGCs were located within and throughout the IGL
(Figures 5C and 5D). By contrast, a significant fraction of
Ndel1?/?cells was observed within the deep molecular layer
(derivative of the EGL). Ndel1?/?cGCs that did migrate past
the Purkinje cell layer accumulated within the most superficial
layer of the IGL next to the Purkinje cells (Figures 5D and 5E).
The marked accumulation of Ndel1?/?cGCs at the border
between the Purkinje cell layer and the IGL indicates that the
majority of Ndel1?/?cGCs can complete the initial migration
from 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 six
omously for neuronal migration. However, it appears that not all
phases of the migration process are equally blocked in Ndel1?/?
cells in Ndel1-MADM had
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 701
Ndel1 Regulates Cortical Neuron Entry into
the Developing Cortical Plate
To identify the critical step in neuronal migration controlled by
NDEL1, we traced the developmental origin of the migration
defects in Ndel1?/?neurons. We focused mosaic analyses on
the development of the neocortex because the cortical lamina-
tion process with sequential inside-out layering has been well
characterized and can be traced unambiguously by costaining
with a nuclear marker.
We used Emx1-Cre for time course MADM analyses. Starting
at E12, the distribution of migrating Ndel1?/?neurons across
the developing cortex in the VZ and developing preplate (PP)
did not differ from WT neurons (Figures 6A and 6B). This finding
indicates that somal translocation, the predominant migration
mode of early born neurons (Nadarajah et al., 2001), is not
tion of Ndel1?/?neurons located within the nascent CP was
significantly decreased when compared to the fraction of control
neurons. Instead, Ndel1?/?cells showed a tendency to accumu-
Figure 5. Ndel1 Is Required for Migration of
Cerebellum Purkinje and Granule Cells
(A–D) Distribution of Purkinje cells and cGCs in
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
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 (Figures
6C, 6D, and 6R). This phenotype became
more pronounced at E16, when Ndel1?/?
upper IZ (Figures 6E, 6F, and 6S). This migration deficit persisted
at P1 when Ndel1?/?neurons were still found to accumulate
stages (Figures 6G, 6H, and 6T; see also Figures 2D, S3C, and
S3D). These results suggest that Ndel1?/?neurons can migrate
through 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), the
exact timing of CRE-mediated mitotic recombination (and hence
the removal of the Ndel1 gene) for any given labeled neuron was
unknown. Accordingly, wenext carried outtemporally controlled
clonal MADM analyses using TM-inducible Nestin-CreER.
Cortical clones were induced at E10, when the vast majority of
RGPCs divide symmetrically (Go ¨tz and Huttner, 2005), and
analyzed at E16. Individual G2-X MADM-clones appeared as
differentially labeled radial columns with an equal number of
red WT and green Ndel1?/?migrating neurons (Figures 6I–6L
and 6Q; see also Figures 1J–1M for an example of WT G2-X
Mosaic Analysis of Lis1 and Ndel1
702 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.
clone). These results directly confirmed our earlier finding that
loss of Ndel1 does not affect neuron production. In all MADM
clones, the developing CP was populated by ?50% of red
control cells. By contrast, the fraction of Ndel1?/?cells in the
CP was reduced to ?10% of all cells; instead, the number of
Ndel1?/?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 migrate
equally across the VZ/SVZ. Further examples whereby clones
were induced at E8 or E10 and examined at E14, E16, and E18
(Figures 6M–6P) also demonstrated that Ndel1?/?neurons
migrate normally within the VZ/SVZ and across the IZ, but fail
to enter or migrate within the developing CP.
Taken together, the developmental studies and clonal anal-
yses indicate that Ndel1 is not cell autonomously required for
migration of cortical neurons within the VZ/SVZ and IZ. The
primary defect in Ndel1?/?neurons appears to be an inability
to enter or migrate within the CP, which represents the target
lamina for cortical projection neurons.
Figure 6. Ndel1 Cell Autonomously Regu-
lates Cortical Neuron Migration into the
(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-
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 Specific
Block of Ndel1–/–Projection
Neurons in Cortical Plate Entry
Our results from sparse mosaic knockout
conditional Ndel1 mutants: upon conditional ablation of Ndel1 in
most or all cells in the cortex, migration of Ndel1?/?neurons is
completely abolished (Youn et al., 2009). To compare these two
conditions (most cells versus sparse single cells mutant for
Ndel1), we performed live imaging experiments of MADM-
used previously in Ndel1 cortical conditional knockout experi-
ments (Youn et al., 2009). We traced embryonic cortical slices
from control- and Ndel1-MADM E14.5 brains over the course of
up to 15 hr (Figures 7 and S7). We found that the average migra-
data are consistent with the developmental time course analysis
(Figure 6), indicating that NDEL1 is not cell autonomously
requiredfor migrationofcortical projection neurons within theIZ.
We also identified slices where the border between the IZ and
CP was readily identifiable, quantified the behavior of migrating
cells when they encountered this IZ-CP border (Figures 7I–7P;
Movie S2) and compared to similar experiments performed with
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 703
control-MADM (Figures S7I–S7P; Movie S1). Whereas 94% of
migrating Ndel1+/+neurons readily crossed the border, 87% of
migrating Ndel1?/?neurons failed to traverse the IZ-CP border
and 7T). The propensity to form multiple neurites in Ndel1?/?cells
is likely to interfere with neuronal migration (Youn et al., 2009) and
These results directly confirm that Ndel1?/?neurons in sparse
genetic mosaics in a mostly heterozygous environment can
migrate through the VZ/SVZ and the IZ. They also suggest that
the inability of Ndel1?/?mutant neurons to migrate in the devel-
oping cortex in the Ndel1 cortical conditional knockout mice is
a consequence of cell-nonautonomous effects.
Dendrite Morphogenesis and Axonal Projections
The MADM-labeling strategy afforded high-resolution examina-
tion of the morphological differentiation of Ndel1?/?neurons at
ectopic sites and provided an opportunity to determine the
extent to which dendrite morphogenesis and axon projection
relies on neurons entering their appropriate target layers. In prin-
ciple, we cannot distinguish whether abnormal morphogenesis
is a secondary consequence of aberrant neuronal positioning
or reflects independent NDEL1 functions. However, we can
deduce 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 trees
that resembled cell type-specific patterns. For cortical and
hippocampal pyramidal neurons, except for some dilation at
the base of apical dendrites and at axoninitial segments (Figures
8B and 8D), the apical dendrites of Ndel1?/?cortical and hippo-
campal projection neurons appeared indistinguishable from
those of control neurons, despite ectopic cell body location
(Figures 3A, 3C, 8A–8D, and S8B). Ndel1?/?CA1 pyramidal
neurons extend apical dendrites across the entire CA1 field.
Figure 7. Live Imaging of MADM-Labeled Ndel1–/–Cortical Projection Neurons
(A–P)Time-lapseimagesof migrating cortical projection neuronsinthe IZ(A–H)and at theborder totheCP(I–P) inorganotypiccorticalslicesderived 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.
Mosaic Analysis of Lis1 and Ndel1
704 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.
Given their origin at a more basal level, these primary apical
dendrites aretherefore longer thanthose of control neurons (Fig-
ure S8). These data indicate that growth of the apical dendrite in
pyramidal neurons is not affected by abnormal locations of their
cell bodies. Similar results were observed in 14-3-33?/?and
Lis1?/?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 dendrite
development despite a lack of contact with their major presyn-
a drastic reduction in dendritic branching. Occasionally, ectopic
Ndel1?/?Purkinje cells were located at the base of the IGL (Fig-
ure 8F). In such cases, Purkinje cells projected their dendrites
outward toward the molecular layers and axons in the opposite
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)Axonalprojections incontrol-and Ndel1-MADM usingEmx1Cre/+.(G–J) Corticothalamicprojections inthalamusatP21(GandH) andP1(Iand J).Insetin(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) Timecourse of axonal projections in the internal capsule in control- (K,M and O)and Ndel1-MADM (L, N and P) atP1 (K and L), P7 (M and N) and P21 (Oand
P).Notethe progressive increase innumber and sizeof axonal varicositiesasmarked by white arrows (Nand P) ingreenNdel1?/?mutantsubcorticalprojections.
(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
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.
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 705
Purkinje cells showed disrupted dendritic branching patterns
within the granule cell layer. Interestingly, the dendritic tree
became more elaborate once it extended into the molecular
layer, 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 regulate
dendrite morphogenesis (Scott and Luo, 2001).
Next, we examined axonal projections in ectopic Ndel1?/?
neurons, focusing on efferent axons of cortical and hippocampal
projection neurons. We found that Ndel1?/?axons reached the
thalamus (Figures 8G and 8H) and other subcortical areas (data
not shown), similar to Ndel1+/?and Ndel1+/+axons in the same
mosaic animals. Tracing of nascent axons within the thalamus
at P1 revealed no differences in the patterning or projection of
developing WT or Ndel1?/?axons (Figures 8I and 8J). Since the
vast majority of Ndel1?/?cortical neurons failed to migrate into
cortical layers and were located ectopically in the white matter
into the correct layer is not a prerequisite for establishing the
correct long-range axonal projections, at least at a gross level.
neurons became apparent. At P21, we found axonal swellings
specific to Ndel1?/?axons in the thalamus (green varicosities in
spinal projections (data not shown) but were most prominent
within the internal capsule (IC), a narrow path between the telen-
cephalon and the midbrain containing axons projecting both to
and from the cortex. A developmental time course indicated that
the appearance of axonal swellings in Ndel1?/?neurons is age
dependent within the IC (Figures 8K–8P). We also traced hippo-
campal efferents in the fornix, a major axonal output pathway of
hippocampal projection neurons. Similar to cortical projection
neurons, axons of Ndel1?/?hippocampal projection neurons
reached the fornix but exhibited aberrant swellings (Figure 8R;
data not shown). Importantly, these axonal swellings were never
observed in control-MADM animals (Figures 8G, 8I, 8K, 8M, 8O,
and 8Q), and were entirely axonal in nature (Figure S9). These
role of NDEL1 in maintaining axonal integrity, consistent with
previous studies showing that NDEL1 regulates neurofilament
organization (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 axonal
projections of Ndel1?/?neurons indicates that certain aspects of
growth and patterning, and long-range axonal projections, are not
strictly dependent on neurons occupying their correct target loca-
tion. However, ectopically located neurons exhibit significant
defects in dendrite morphogenesis and axonal integrity. Whereas
some aspects may be a secondary consequence of migration
defects (e.g., Purkinje cell dendrites not able to reach their target
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 also
been used extensively in neurobiology, for example, to identify
clock (Low-Zeddies and Takahashi, 2001) or to elucidate the
cell-autonomous function of the NMDA receptor in dendritic
patterning (Espinosa et al., 2009). Here, we extended the
MADM genetic mosaic tool to mouse chromosome 11 and
analyzed the functions of LIS1 and NDEL1 in neuronal migration.
The LIS1/NDEL1 complex is considered part of the basic
machinery for neuronal migration that couples the nucleus and
centrosome. Given that both LIS1 and NDEL1 are cytoplasmic
proteins, the prediction would be that they should act cell auton-
omously to regulate all steps of migration. However, our mosaic
analysis reveals a far more complex picture and provides novel
insights into the cell-autonomous and nonautonomous functions
of 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 neuronal
migration (Sasaki et al., 2005; Shu et al., 2004), our develop-
mental analyses indicate that NDEL1 is not cell autonomously
required for all aspects of neuronal migration. Using MADM-
mediated sparse knockout and labeling in fixed brains as well
as live imaging, we found that Ndel1?/?neurons migrate nor-
mally within the VZ/SVZ and IZ similar to WT neurons. The first
defect we observed when comparing Ndel1?/?and Ndel1+/+
neurons in the same mosaic animal was the accumulation of
Ndel1?/?neurons at the border of the developing CP (Figures
9A and 9B). This selective defect is unlikely caused by perdur-
mental Experimental Procedures). Since the CP represents the
target lamina for migrating cortical projection neurons, we
propose that a major cell-autonomous function of NDEL1 in
neurons is to regulate the entry into the target laminae (Figures
9A and 9B).
We have extended the analysis of Ndel1 to migration of excit-
atory and inhibitory neurons including hippocampal pyramidal
cells and dentate granule cells (Figure 3G), cerebellar Purkinje
(Figure 4H). NDEL1 is cell autonomously required for the migra-
tionof alltheseneurons,andtheir migrationphenotypes strongly
support our central hypothesis that NDEL1 regulates the entry
into the target lamina. For example, Ndel1?/?cerebellar granule
cells migrate normally across the molecular layer of the cere-
bellar cortex but are accumulated near the Purkinje cell layer
before entering into the granular layer, the target lamina for
granule cells (Figure 5F).
Mechanistically, NDEL1 could exert this cell-autonomous
function of controlling target lamina entry by transducing an
extracellular signal to the intracellular neuronal migration
machinery. Interestingly, NDEL1 is phosphorylated by CDK5/
p35, a protein serine/threonine kinase complex essential for
neuronal 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 phenotype
somewhat similar to the phenotype of Ndel1?/?neurons (Gil-
more et al., 1998; Ohshima et al., 2007). It will be interesting in
the cell-autonomous function of Cdk5 with that of Ndel1, and to
Mosaic Analysis of Lis1 and Ndel1
706 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.
determine the extracellular cues and signaling cascades that
regulate NDEL1 activity to control the entry of migrating neurons
into the target lamina.
LIS1 is well known to regulate neuronal migration in a dosage-
sensitive manner (Wynshaw-Boris, 2007). MADM allowed us to
compare 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 well
as cell autonomy. We find that Lis1+/+cells have a competitive
advantage over cells of all other genotypes in the mosaic animal
in their extent of migration; this can be seen in cortical projection
neurons, CA1 pyramidal neurons and dentate granule cells. In
addition, 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 that
LIS1 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 in
previous reports using other genetic perturbations of the same
genes. 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 complete
block of neuronal migration within the VZ/SVZ and IZ (Youn
etal., 2009). MADM analysis indicates that migration of Ndel1?/?
neurons are specifically blocked at the entry of the cortical plate
but migrate normally in VZ, SVZ, and IZ. For Lis1, analysis of
previous RNAi knockdown, compound heterozygous and whole
cortex conditional knockout indicate that neuronal migration is
severely 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-
mental delay, Lis1?/?and Lis1+/?neurons behave similarly in the
migration of cortical projection neurons (Figure 2H), hippo-
campal CA1 pyramidal neurons (Figure 3F) and dentate granule
cells (Figure S5F) when examined in adult.
a subset of previously described migration functions for Ndel1
and Lis1 can be accounted for by cell-nonautonomous function
of these genes in regulating neuronal migration. Specifically,
migration of Ndel1?/?cells within the IZ can be positively
affected by neighboring Ndel1+/?cells even though entry into
the cortical plate cannot. A cell-nonautonomous function may
also account for the eventual migration of Lis1?/?neurons into
proper cortical and hippocampal layers. We note that small
cell-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 mutually
exclusive. First, a ‘‘community effect’’ may provide support for
individual neurons in the process of migration. In particular,
isolated Ndel1?/?or Lis1?/?neurons, although defective in
the intrinsic essential migration machinery (e.g., centrosome-
nucleus coupling), could nevertheless physically ‘‘piggyback’’
on normally migrating neighboring neurons. However, if most
or all neurons are mutant for Lis1 or Ndel1, community effects
become ineffective. Second, migrating neurons may actively
signal to each other to facilitate the intrinsic migration
program. The generation of such a signal may require the
LIS1/NDEL1 complex, accounting for their cell-nonautono-
Figure 9. Cell-Autonomous and Nonauton-
omous Functions of LIS1 and NDEL1 in
(A) Schematic summary of migration of WT (red)
(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
(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.
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 707
Regardless of the exact mechanism, MADM-based genetic
mosaic analyses revealed extensive cell-cell interactions among
tonomous functions could contribute significantly to the clinical
in LIS1 protein levels exhibit inappropriate community effects or
fail to signal to each other. This may in turn exacerbate the
It will be revealing to investigate the interplay of cell-autonomous
and cell-nonautonomous mechanisms in the control of neuronal
migration. 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 allow
mosaic analysis for ?2000 genes located distal to the HIPP11
locus. The targeted knockin approach presented here also
suggests a general strategy to expand MADM to other mouse
Identification of Hipp11 as a Suitable Genomic Locus for the
Generation of MADM-11 Transgenic Mice
The 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-11GTand
MADM-11TGmice 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 Brains
Postnatal 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-
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 Induction
For MADM clone induction, pregnant MADM-11GT/GTfemales 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,Ndel1allele to label mutant cells in
Live-Imaging Assay for MADM-Labeled Organotypic Slices
Previously established live-imaging protocols (Youn et al., 2009) were used
with slight adaptations (see Supplemental Experimental Procedures).
Supplemental Information includes nine figures, two movies, and Supple-
mental Experimental Procedures and can be found with this article online at
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
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
Accepted: August 30, 2010
Published: November 17, 2010
Altman, J., and Bayer, S.A. (1990). Prolonged sojourn of developing pyramidal
cells in the intermediate zone of the hippocampus and their settling in the
stratum pyramidale. J. Comp. Neurol. 301, 343–364.
Altman, J., and Bayer, S.A. (1997). The Development of the Cerebellar System
in Relation to Its Evolution, Structure, and Functions (Boca Raton, FL: CRC
Angevine, J.B., Jr., and Sidman, R.L. (1961). Autoradiographic study of cell
migration during histogenesis of cerebral cortex in the mouse. Nature 192,
Cahana, A., Escamez, T., Nowakowski, R.S., Hayes, N.L., Giacobini, M., von
Holst, A., Shmueli, O., Sapir, T., McConnell, S.K., Wurst, W., et al. (2001).
Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homo-
dimerization. Proc. Natl. Acad. Sci. USA 98, 6429–6434.
Chae,T.,Kwon,Y.T., Bronson,R.,Dikkes, P.,Li, E.,and Tsai, L.H.(1997).Mice
lacking p35, a neuronal specific activator of Cdk5, display cortical lamination
defects, seizures, and adult lethality. Neuron 18, 29–42.
Costa, M.R., Bucholz, O., Schroeder, T., and Go ¨tz, M. (2009). Late origin of
glia-restricted progenitors in the developing mouse cerebral cortex. Cereb.
Cortex 19 (Suppl 1), i135–i143.
Espinosa, J.S., Wheeler, D.G., Tsien, R.W., and Luo, L. (2009). Uncoupling
dendrite growth and patterning: single-cell knockout analysis of NMDA
receptor 2B. Neuron 62, 205–217.
Farley, F.W., Soriano, P., Steffen, L.S., and Dymecki, S.M. (2000). Widespread
recombinase expression using FLPeR (flipper) mice. Genesis 28, 106–110.
Feng, Y., and Walsh, C.A. (2004). Mitotic spindle regulation by Nde1 controls
cerebral cortical size. Neuron 44, 279–293.
Feng, Y., Olson, E.C., Stukenberg, P.T., Flanagan, L.A., Kirschner, M.W., and
Walsh, C.A. (2000). LIS1 regulates CNS lamination by interacting with mNudE,
a central component of the centrosome. Neuron 28, 665–679.
Mosaic Analysis of Lis1 and Ndel1
708 Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc.
Fuchs, E., and Cleveland, D.W. (1998). A structural scaffolding of intermediate
filaments in health and disease. Science 279, 514–519.
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 cere-
bral cortical development. J. Neurosci. 23, 1719–1729.
Gilmore, E.C., Ohshima, T., Goffinet, A.M., Kulkarni, A.B., and Herrup, K.
(1998). Cyclin-dependent kinase 5-deficient mice demonstrate novel develop-
mental arrest in cerebral cortex. J. Neurosci. 18, 6370–6377.
Gorski, J.A., Talley, T., Qiu, M., Puelles, L., Rubenstein, J.L., and Jones, K.R.
(2002). Cortical excitatory neurons and glia, but not GABAergic neurons, are
produced in the Emx1-expressing lineage. J. Neurosci. 22, 6309–6314.
Go ¨tz, M., and Huttner, W.B. (2005). The cell biology of neurogenesis. Nat. Rev.
Mol. Cell Biol. 6, 777–788.
Heng, J.I., Chariot, A., and Nguyen, L. (2010). Molecular layers underlying
cytoskeletal remodelling during cortical development. Trends Neurosci. 33,
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 reduc-
tion of Pafah1b1 (Lis1) activity results in neuronal migration defects and early
embryonic lethality. Nat. Genet. 19, 333–339.
Imayoshi, I.,Ohtsuka,T.,Metzger, D., Chambon, P., and Kageyama, R.(2006).
Temporal regulation of Cre recombinase activity in neural stem cells. Genesis
Liu, Z., Steward, R., and Luo, L. (2000). Drosophila Lis1 is required for neuro-
blast proliferation, dendritic elaboration and axonal transport. Nat. Cell Biol. 2,
Lledo, P.M., Merkle, F.T., and Alvarez-Buylla, A. (2008). Origin and function of
olfactory bulb interneuron diversity. Trends Neurosci. 31, 392–400.
Lois,C.,andAlvarez-Buylla, A.(1994).Long-distance neuronal migration inthe
adult mammalian brain. Science 264, 1145–1148.
Low-Zeddies, S.S., and Takahashi, J.S. (2001). Chimera analysis of the Clock
mutation in mice shows that complex cellular integration determines circadian
behavior. Cell 105, 25–42.
Marin, O., Valiente, M., Ge, X., and Tsai, L.H. (2010). Guiding neuronal cell
migrations. Cold Spring Harb. Perspect. Biol. 2, a001834–a001834.
McManus, M.F., Nasrallah, I.M., Pancoast, M.M., Wynshaw-Boris, A., and
Golden, J.A. (2004). Lis1 is necessary for normal non-radial migration of inhib-
itory interneurons. Am. J. Pathol. 165, 775–784.
Miale, I.L., and Sidman, R.L. (1961). An autoradiographic analysis of histogen-
esis in the mouse cerebellum. Exp. Neurol. 4, 277–296.
Morris, N.R. (2000). Nuclear migration. From fungi to the mammalian brain.
J. Cell Biol. 148, 1097–1101.
Nadarajah, B., Brunstrom, J.E., Grutzendler, J., Wong, R.O., and Pearlman,
A.L. (2001). Two modes of radial migrationinearly development of thecerebral
cortex. Nat. Neurosci. 4, 143–150.
Nguyen, M.D., Shu, T., Sanada, K., Larivie `re, R.C., Tseng, H.C., Park, S.K.,
Julien, J.P., and Tsai, L.H. (2004). A NUDEL-dependent mechanism of neuro-
filament assembly regulates the integrity of CNS neurons. Nat. Cell Biol. 6,
Niethammer, M., Smith, D.S., Ayala, R., Peng, J., Ko, J., Lee, M.S., Morabito,
M., and Tsai, L.H. (2000). NUDEL is a novel Cdk5 substrate that associates
with LIS1 and cytoplasmic dynein. Neuron 28, 697–711.
Noctor, S.C., Martı ´nez-Cerden ˜o, V., Ivic, L., and Kriegstein, A.R. (2004).
Cortical neurons arise in symmetric and asymmetric division zones and
migrate through specific phases. Nat. Neurosci. 7, 136–144.
Ohshima, T., Hirasawa, M., Tabata, H., Mutoh, T., Adachi, T., Suzuki, H.,
Saruta, K., Iwasato, T., Itohara, S., Hashimoto, M., et al. (2007). Cdk5 is
required for multipolar-to-bipolar transition during radial neuronal migration
and proper dendrite development of pyramidal neurons in the cerebral cortex.
Development 134, 2273–2282.
Rakic, P. (1972). Modeof cell migration to thesuperficial layersof fetal monkey
neocortex. J. Comp. Neurol. 145, 61–83.
Rakic, P. (1974). Neurons in rhesus monkey visual cortex: systematic relation
between time of origin and eventual disposition. Science 183, 425–427.
Ramon y Cajal, S. (1911). Histology of the Nervous System of Man and
Vertebrates (Oxford: Oxford University Press).
Reiner, O., Carrozzo, R., Shen, Y., Wehnert, M., Faustinella, F., Dobyns, W.B.,
Caskey, C.T., and Ledbetter, D.H. (1993). Isolation of a Miller-Dieker lissence-
phaly gene containing G protein beta-subunit-like repeats. Nature 364,
Sasaki, S., Shionoya, A., Ishida, M., Gambello, M.J., Yingling, J., Wynshaw-
Boris, A., and Hirotsune, S. (2000). A LIS1/NUDEL/cytoplasmic dynein
heavy chain complex in the developing and adult nervous system. Neuron
Sasaki, S., Mori, D., Toyo-oka, K., Chen, A., Garrett-Beal, L., Muramatsu, M.,
Miyagawa, S., Hiraiwa, N., Yoshiki, A., Wynshaw-Boris, A., and Hirotsune, S.
(2005). Complete loss of Ndel1 results in neuronal migration defects and early
embryonic lethality. Mol. Cell. Biol. 25, 7812–7827.
Scott, E.K., and Luo, L. (2001). How do dendrites take their shape? Nat.
Neurosci. 4, 359–365.
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 fluores-
cent proteins derived from Discosoma sp. red fluorescent protein. Nat.
Biotechnol. 22, 1567–1572.
Shu, T., Ayala, R., Nguyen, M.D., Xie, Z., Gleeson, J.G., and Tsai, L.H. (2004).
Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to
regulate cortical neuronal positioning. Neuron 44, 263–277.
Takahashi, Y. (2003). The 14-3-3 proteins: Gene, gene expression, and func-
tion. Neurochem. Res. 28, 1265–1273.
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). 14-3-33 is
important for neuronal migration by binding to NUDEL: a molecular explana-
tion for Miller-Dieker syndrome. Nat. Genet. 34, 274–285.
Tsai, J.W., Chen, Y., Kriegstein, A.R., and Vallee, R.B. (2005). LIS1 RNA inter-
ference blocks neural stem cell division, morphogenesis, and motility at
multiple stages. J. Cell Biol. 170, 935–945.
Vallee, R.B., Seale, G.E., and Tsai, J.W. (2009). Emerging roles for myosin II
and cytoplasmic dynein in migrating neurons and growth cones. Trends Cell
Biol. 19, 347–355.
Wynshaw-Boris,A.(2007). Lissencephaly and LIS1:Insightsintothe molecular
mechanisms of neuronal migration and development. Clin. Genet. 72,
Yingling,J., Youn, Y.H., Darling, D., Toyo-Oka, K., Pramparo, T., Hirotsune, S.,
and Wynshaw-Boris, A. (2008). Neuroepithelial stem cell proliferation requires
LIS1 for precise spindle orientation and symmetric division. Cell 132, 474–486.
Youn, Y.H., Pramparo, T., Hirotsune, S., and Wynshaw-Boris, A. (2009).
Distinct dose-dependent cortical neuronal migration and neurite extension
defects in Lis1 and Ndel1 mutant mice. J. Neurosci. 29, 15520–15530.
Zong, H., Espinosa, J.S., Su, H.H., Muzumdar, M.D., and Luo, L. (2005).
Mosaic analysis with double markers in mice. Cell 121, 479–492.
Mosaic Analysis of Lis1 and Ndel1
Neuron 68, 695–709, November 18, 2010 ª2010 Elsevier Inc. 709
Neuron, Volume 68
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
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 S4, related to Figure 2.
Supplemental Figure S5, related to Figure 3.
Supplemental Figure S6, related to Figure 4 and Figure 5.
Supplemental Figure S7, related to Figure 7.
Supplemental Figure S8, related to Figure 8.
Supplemental Figure S9, related to Figure 8.
Supplemental Movie 1, related to Figure 7.
Supplemental Movie 2, related to Figure 7.
Supplemental Experimental Procedures
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
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).
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]:
(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-
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-
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.
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
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
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).
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.
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).
(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
(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.
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).
Figure S8. Analysis of Apical Dendrite Morphology in CA1 Pyramidal Cells, Related to
(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)
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.
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).
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.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Construction of MADM-11 Transgenes by Targeted Knockin and Mouse Genetic
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.
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
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
Gambello,? M.J.,? Darling,? D.L.,? Yingling,? J.,? Tanaka,? T.,? Gleeson,? J.G.,? and? Wynshaw?Boris,? A.? (2003).?
Hippenmeyer?et?al.? Download full-text
Yoshiki,? A.,? Wynshaw?Boris,? A.,? et? al.? (2005).? Complete? loss? of? Ndel1? results? in? neuronal? migration?
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.? red?
Zheng,? B.,? Sage,? M.,? Sheppeard,? E.A.,? Jurecic,? V.,? and? Bradley,? A.? (2000).? Engineering? mouse?
Zhuo,? L.,? Theis,? M.,? Alvarez?Maya,? I.,? Brenner,? M.,? Willecke,? K.,? and? Messing,? A.? (2001).? hGFAP?cre?