Article
Mosaic Analysis with Doub
le Markers RevealsDistinct Sequential Functions of Lgl1 in NeuralStem CellsHighlights
d MADM-based genetic dissection of intrinsic gene function
and community effects
d Sparse and complete Lgl1 ablation distinctly affects RGP-
mediated neurogenesis
d Lgl1 cell-autonomously controls astrocyte generation in an
EGFR-dependent manner
d Critical role for Lgl1 in postnatal NSC lineage progression and
neurogenesis
Beattie et al., 2017, Neuron 94, 517–533May 3, 2017 ª 2017 Elsevier Inc.http://dx.doi.org/10.1016/j.neuron.2017.04.012
Authors
Robert Beattie, Maria Pia Postiglione,
Laura E. Burnett, ..., Valeri Vasioukhin,
Troy H. Ghashghaei,
Simon Hippenmeyer
In Brief
Beattie et al. determined the relative
contribution of novel intrinsic Lgl1 gene
functions and non-cell-autonomous
community effects in neural stem cell
proliferation behavior. They found
distinct but sequential Lgl1 functions
controlling embryonic neurogenesis and
postnatal astrocyte and olfactory bulb
interneuron generation.
Neuron
Article
Mosaic Analysis with Double MarkersReveals Distinct Sequential Functionsof Lgl1 in Neural Stem CellsRobert Beattie,1 Maria Pia Postiglione,1,4 Laura E. Burnett,1 Susanne Laukoter,1 Carmen Streicher,1 Florian M. Pauler,1
Guanxi Xiao,2 Olga Klezovitch,3 Valeri Vasioukhin,3 Troy H. Ghashghaei,2 and Simon Hippenmeyer1,5,*1Institute of Science and Technology Austria, Am Campus 1, 3400 Klosterneuburg, Austria2Department of Molecular Biomedical Sciences, Program in Genetics, W.M. Keck Center for Behavioral Biology, College of VeterinaryMedicine, North Carolina State University, Raleigh, NC 27607, USA3Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA4Present address: Discovery Sciences, AstraZeneca, 43183 Molndal, Sweden5Lead Contact*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.neuron.2017.04.012
SUMMARY
The concerted production of neurons and glia byneural stem cells (NSCs) is essential for neural circuitassembly. In the developing cerebral cortex, radialglia progenitors (RGPs) generate nearly all neocor-tical neurons and certain glia lineages. RGP prolifer-ation behavior shows a high degree of non-stochas-ticity, thus a deterministic characteristic of neuronand glia production. However, the cellular and mo-lecular mechanisms controlling RGP behavior andproliferation dynamics in neurogenesis and glia gen-eration remain unknown. By using mosaic analysiswith double markers (MADM)-based genetic para-digms enabling the sparse and global knockoutwith unprecedented single-cell resolution, we identi-fied Lgl1 as a critical regulatory component. Weuncover Lgl1-dependent tissue-wide community ef-fects required for embryonic cortical neurogenesisand novel cell-autonomous Lgl1 functions control-ling RGP-mediated glia genesis and postnatal NSCbehavior. These results suggest that NSC-mediatedneuron and glia production is tightly regulatedthrough the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.
INTRODUCTION
The human cerebral cortex is the seat of our cognitive abilities
and is composed of an extraordinary number of neurons and
glia cells. The developmental programs regulating the accurate
generation of postmitotic cells, by neural stem cells (NSCs),
need to be precisely implemented and regulated. At the end of
neurulation, the early neuroepithelium is composed of neuroepi-
thelial stem cells (NESCs) from which all subsequent neural
progenitor cells and their lineages derive. NESCs initially divide
symmetrically but then transform into radial glia progenitor
(RGP) cells. RGPs have been demonstrated to be the major neu-
ral progenitors in the developing cortex responsible for produc-
ing the vast majority of cortical excitatory neurons (Lui et al.,
2011; Taverna et al., 2014).
The RGP division patterns and dynamics determine the num-
ber of neurons in the mature cortex. The mitotic RGP cell divi-
sion at the surface of the embryonic ventricular zone (VZ) can
be either symmetric or asymmetric, which is defined by the
fate of the two daughter cells (Homem et al., 2015; Lui et al.,
2011; Taverna et al., 2014). Symmetric divisions can generate
two RGPs to amplify the progenitor pool (symmetric prolifer-
ative division). In contrast, asymmetric neurogenic divisions
produce a renewing RGP and a neuron, or an intermediate
progenitor (IP) that can further divide in the subventricular
zone (SVZ) to produce neurons (Noctor et al., 2004). RGPs
may also generate SNPs (Stancik et al., 2010) and outer SVZ
radial glia progenitors (oRGs, a.k.a. basal RGs or bRGs) (Fietz
et al., 2010; Hansen et al., 2010; Wang et al., 2011). RGPs
can also produce glia cells, including astrocytes and oligoden-
drocytes, which have critical roles in the development, main-
tenance, and function of neuronal circuits (Chung et al.,
2015; Freeman and Rowitch, 2013). Although gliogenesis is
generally known to follow neurogenesis in the developing brain
(Costa et al., 2009; Magavi et al., 2012; Schmechel and Rakic,
1979; Voigt, 1989), the principles regulating glia generation,
especially at the individual RGP and successive glia progenitor
level(s), are not clear (Bayraktar et al., 2014; Molofsky and
Deneen, 2015). Shortly after birth, the embryonic neuroepithe-
lium transforms into the postnatal NSC niche in the ventricu-
lar-subventricular zone (V-SVZ) within the lateral ventricle (LV)
(Lim and Alvarez-Buylla, 2016). While certain subpopulations
of RGPs give rise to ependymal (E1) cells (Spassky et al.,
2005) postnatally, other RGPs transform into V-SVZ type B1
cells (Merkle et al., 2004). Type B1 cells function as the main
progenitors in adult neurogenesis (Doetsch et al., 1999) and
generate type C cells that in turn differentiate into type A neuro-
blasts migrating to the olfactory bulb (OB) (Lim and Alvarez-
Buylla, 2016).
Neuron 94, 517–533, May 3, 2017 ª 2017 Elsevier Inc. 517
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Figure 1. Sparse and Whole Neuroepithelium Ablation of Lgl1 in RGPs Differentially Affects Cortical Neurogenesis
(A–C) Schematic illustration of experimental paradigm in control-MADM (A, wild-type), Lgl1-MADM (B, genetic mosaic), and cKO-Lgl1-MADM (C, conditional/full
knockout). In control-MADM, GFP+ (green), tdT+ (red), and GFP+/tdT+ (yellow) and unlabeled (vast majority) cells are all WT. In Lgl1-MADM, GFP+ (green) cells are
Lgl1�/�, tdT+ (red) cells are Lgl1+/+, and GFP+/tdT+ and unlabeled cells are Lgl1+/�. In cKO-Lgl1-MADM, GFP+ (green), tdT+ (red), and GFP+/tdT+ (yellow) and the
vast majority of unlabeled cortical projection neurons are all Lgl1�/�.
(legend continued on next page)
518 Neuron 94, 517–533, May 3, 2017
While previous studies provided a rough framework of cortical
neurogenesis and glia production, the cellular and molecular
mechanisms dictating the quantitative neuron and glia output
at the individual NSC level remain to be resolved. Recent mosaic
analysis with double markers (MADM)-based lineage tracing,
however, indicates that the proliferation behavior of RGPs is
remarkably coherent and predictable (Gao et al., 2014). RGPs
initially undergo symmetric amplification division with a defined
proliferation potential before transiting to asymmetric neuro-
genic division. Because MADM affords single-cell resolution,
and thus a quantitative assessment of the neurogenic potential
(Hippenmeyer, 2013; Postiglione and Hippenmeyer, 2014;
Zong et al., 2005), RGPs in their neurogenic phase were shown
to follow a defined nonrandom program of cell-cycle exit, result-
ing in a unitary output of approximately eight to nine neurons per
individual RGP. Upon completion of neurogenesis approxi-
mately one in six neurogenic RGPs proceed to produce glia
(Gao et al., 2014). Although the above MADM-based clonal anal-
ysis provided a quantitative model of NSC proliferative behavior,
the cellular and molecular mechanisms controlling the size of
pre-programmed RGP output through neurogenesis and glio-
genesis are currently unknown.
Key regulators orchestrating the RGP mode of cell division
include the signaling protein LGL1 (a.k.a. Llgl1, lethal giant larvae
homolog 1 [Drosophila]), which regulates intracellular polarity in a
variety of cellular contexts (Betschinger et al., 2003; Klezovitch
et al., 2004; Yamanaka et al., 2003). Although Lgl1 is predicted
to regulate embryonic RGPproliferation behavior, the cell-auton-
omous function of Lgl1 in vivo is not clear. The possible require-
ment of Lgl1 at postnatal stages during NSC lineage progression
is unknown due to lethality of Lgl1 knockout mice at birth. The
analysis of RGP-mediated neurogenesis in Lgl1 mutant mice is
further compromised due to the progressive disruption of the
VZ resulting in disorganization and tumor-like growth of RGPs
in the form of rosettes (Klezovitch et al., 2004). This raises the
possibility that substantial aspects of the phenotype in whole
tissue Lgl1 knockout could be the result of a combination of
both cell-autonomous and non-cell-autonomous and/or com-
munity effects. In this study, we address the above questions
and determine the relative contribution of cell-autonomous
Lgl1 signaling and non-cell-autonomous mechanisms in RGP
proliferation behavior in neurogenesis and glia production. By
capitalizing on the MADM system, we established genetic para-
digms to either ablate Lgl1 in very sparse mosaic and single RGP
clones or neuroepithelium-wide in all RGPs, both coupled with
single-cell labeling enabling high resolution quantitative pheno-
typic analysis. Our MADM-based functional analysis led to the
identification of previously unknown cell-autonomous Lgl1 func-
tions in RGPs and suggests that a concerted interplay of cell-
(D–I) Overview of MADM-labeling pattern in somatosensory cortex in control-MA
magnification of boxed areas in (D), (F), and (H) illustrating the CPwith emerging la
‘‘Double Cortex’’). Scale bars, 500 (D, F, and H) and 60 mm (E, G, and I).
(J–R) Analysis of cortical marker expression and laminar distribution. Expression
MADM+ cells, J00–L00), CTIP2 (white, M–O and M0–O0; % CTIP2+, MADM+/total MA
MADM+ cells, P00–R00) in MADM-labeled cells in control-MADM, Lgl1-MADM, and
Nuclei were stained using DAPI (blue). Cortical layers are indicated (roman nume
See also Figures S1 and S2 and Table S1.
intrinsic mechanisms coupled with stem cell niche interactions
are essential for faithful NSC proliferation behavior.
RESULTS
MADM-Based Experimental Paradigm for Sparse andComplete Lgl1 AblationIn order to determine the cell-autonomous function of Lgl1 and to
assess the relative contribution of non-cell-autonomous mecha-
nisms in RGP lineage progression, we developed a quantitative
MADM-based genetic strategy (Figures1A–1C and S1; Table
S1). The main assay consists of subtractive RGP phenotypic
analysis at single cell resolution of (1) genetic Lgl1 mosaic with
only sparse Lgl1 deletion in just a few RGPs, within heterozygous
and/or wild-type (WT) genetic background; and (2) conditional
Lgl1 knockout with global Lgl1 deletion in all RGPswithin Lgl1�/�
mutant background. In other words, individual Lgl1�/�RGP cells
are either surrounded by a local microenvironment with ‘‘normal’’
heterozygous and WT cells (abbreviated Lgl1-MADM), or by
Lgl1�/� mutant cells (abbreviated cKO-Lgl1-MADM). In both
Lgl1-MADM and cKO-Lgl1-MADM, Cre recombinase expressed
from the Emx1 locus (Gorski et al., 2002) is used to induce sparse
MADM labeling and/or geneticmanipulation specifically in dorsal
telencephalic RGPs (Figures 1 and S1). In order to generate Lgl1-
MADM animals, we genetically linked the Lgl1-flox allele (Klezo-
vitch et al., 2004) via meiotic recombination to the MADM-TG
cassette on chromosome (chr) 11 (MADM-11) (Hippenmeyer
et al., 2010); in parallel, the MADM-GT cassette is linked to the
corresponding WT allele (Figure S1A). By using a conditional
Lgl1-flox allele, Emx1-Cre mediates interchromosomal trans-
recombination between MADM cassettes, as well as cis-recom-
bination between the LoxP sites of the Lgl1-flox allele (rendering
the Lgl1-flox allele into Lgl1-D mutant allele). Thus, MADM-
labeled green cells are homozygous Lgl1�/� while red cells are
homozygous Lgl1+/+ (Figures 1B and S1A). Yellow cells are
also generated by certain MADM events (for details, see Fig-
ure S1A) (Hippenmeyer et al., 2010, 2013) and are Lgl1+/�. Forthe generation of cKO-Lgl1-MADM, we genetically linked the
Lgl1-flox allele to both TG and GT cassettes on chr11, respec-
tively (Figure S1B). In these experimental cKO-Lgl1-MADM
mice, Emx1-Cre-mediated cis-recombination renders both
Lgl1-flox alleles into Lgl1-D alleles globally in all Emx1 expressing
dorsal telencephalic RGPs (i.e., conditional Lgl1 knockout). In
addition Emx1-Cre-mediated trans-recombination leads to
sparse MADM labeling with green, red, yellow, and unlabeled
cells in the background, all carrying the Lgl1�/� homozygous
mutation (Figures 1C and S1B). Because individual cells can
be traced at high resolution, these Lgl1-MADM and cKO-Lgl1-
MADM paradigms offer an unprecedented quantitative platform
DM (D), Lgl1-MADM (F), and cKO-Lgl1-MADM (H) at P0. (E, G, and I) Higher
yers. Cyan broken line (H) outlines the subcortical band heterotopia (SBH, a.k.a.
and quantification of SATB2 (white, J–L and J0–L0; % SATB2+, MADM+/total
DM+ cells, M00–O00), and TBR1 (white, P–R and P0–R0; % TBR1+, MADM+/total
cKO-Lgl1-MADM at P0. Scale bars, 60 mm (J–R0).rals). NCX, neocortex; ns, nonsignificant. Values represent mean ± SEM.
Neuron 94, 517–533, May 3, 2017 519
to assay for the influence of the local microenvironment on
Lgl1�/� cells. In Lgl1-MADM, the surrounding cells are WT or
Lgl1+/� and thus could potentially exert positive influence on
sparse Lgl1�/� cells (green cells in Figures 1 and S1A).
In contrast, in cKO-Lgl1-MADM, all cells are Lgl1�/� and there-
fore sparsely labeled green Lgl1�/� mutant cells are encom-
passed by a community of mutant cells that could exert
influence by collective effects with the potential to possibly rem-
edy or worsen the individual cell-autonomous loss-of-function
phenotype.
Analysis of Corticogenesis upon Sparse and CompleteLgl1 Deletion in RGPsWe first analyzed cortical neuron production and overall cortical
morphogenesis in control-MADM, Lgl1-MADM, and cKO-Lgl1-
MADM at postnatal day (P) 0 (Figures 1D–1I). In our genetic
paradigms, we employ constitutive Emx1-Cre, and thus
MADM events occur stochastically at any given time in a
random subset of dividing Emx1+ RGPs. Still, if progenitor
cell division were symmetric, the number of red and green cells
within an individual clone would be close to identical (Gao
et al., 2014; Hippenmeyer et al., 2010). Even if a large number
of RGP divisions were asymmetric, such that red and green
progeny numbers were different in individual clones, the by-
chance distribution of colors in asymmetric clones would
ensure that the number of red and green progeny were equal
overall. The green/red (g/r) ratio of all MADM-labeled cells in
the somatosensory area in control-MADM, Lgl1-MADM, and
cKO-Lgl1-MADM was �1 when analyzed at P0 (Figures 1D–
1I; Table S2). Previous studies assessing ventral telencephalic
RGPs in Lgl1 full knockout suggest that Lgl1�/� RGPs show
a lack of differentiation (Klezovitch et al., 2004). We thus
assessed the cell fate specification of Lgl1�/� cells in control-
MADM, Lgl1-MADM, and cKO-Lgl1-MADM. We evaluated the
expression of TBR1 (layer VI), CTIP2 (layer V), and SATB2
(layer IV-II) at P0 (Figures 1J–1R). The coarse laminar architec-
ture appeared similar in all three experimental paradigms, and
there was no significant difference in the relative numbers
of TBR1+, CTIP2+, and SATB2+ neurons in the neocortex in
control-MADM, Lgl1-MADM, and cKO-Lgl1-MADM mice (Fig-
ures 1J–1R).
Lgl1 Is Not Cell-Autonomously Required inRGP-Mediated NeurogenesisA g/r ratio of 1 at P0 in control-MADM and cKO-Lgl1-MADM
was expected due to the identical genotypes of red and green
cells, WT in control-MADM and Lgl1�/� in cKO-Lgl1-MADM,
respectively. In contrast a g/r ratio of 1 in Lgl1-MADM was
against our expectations because LGL1 has been suggested
to control the switch from symmetric to asymmetric RGP divi-
sion mode, and loss of Lgl1 in global Lgl1 knockout leads to
exuberant RGP proliferation (Klezovitch et al., 2004). While the
full Lgl1 knockout is lethal at birth (Klezovitch et al., 2004),
Lgl1-MADM mice survive beyond 1 year of age. We therefore
determined the g/r ratio of mutant to WT neurons at postnatal
stages P21, 3 months, and 12 months. We found that the g/r
ratio was consistently �1 at all postnatal stages analyzed (Table
S2). To more precisely assess the consequences of Lgl1 loss-
520 Neuron 94, 517–533, May 3, 2017
of-function at the individual progenitor level, we conducted
MADM-based clonal analysis (Gao et al., 2014; Hippenmeyer
et al., 2010; Zong et al., 2005). To this end, we utilized tamoxifen
(TM)-inducible Emx1-CreER (Kessaris et al., 2006) to induce
RGP clones at E11 in control-MADM and Lgl1-MADM. We
analyzed the size and composition of these MADM clones at
E13 and E16 (Figures S2A–S2H). We observed no significant
difference in the total clone size and zonal distribution of clonally
related WT and Lgl1�/� cells indicating that RGP-mediated neu-
rogenesis is not dependent on cell-autonomous Lgl1 function.
Next, we analyzed the unitary neuronal output of RGPs (Gao
et al., 2014) in the absence of Lgl1. TM was injected in Lgl1-
MADM at E12 when RGPs transit from symmetric proliferative
division to asymmetric neurogenic division. As expected, we
observed asymmetric clones with a majority population in
one color and a minority population in the other color (Figures
S2I–S2K), whereby the two colors correspond to different geno-
types (i.e., WT and Lgl1�/�). We assessed the size of the major-
ity and minority populations within individual clones but
observed no significant difference, and the overall neuronal
unit size was unchanged regardless of the color and thus Lgl1
genotype (Figures S2L, S2M, and S2P). We next assessed the
relative number of upper versus lower layer neurons, and similar
to the total unit size, there was no significant difference (Figures
S2N and S2O). We conclude from these clonal analyses that
Lgl1 is not cell-autonomously required for RGP-mediated
neurogenesis.
Increased RGPProliferation Results in Subcortical BandHeterotopia in cKO-Lgl1-MADMAlthough the overall g/r ratio in cKO-Lgl1-MADMwas�1, a large
heterotopic cell mass was present beneath the neocortex (Fig-
ures 1H and S3). The ectopic cell mass resembled subcortical
band heterotopia (SBH) or ‘‘double cortex syndrome’’ in human
(Gleeson et al., 1998). While green Lgl1�/� RGPs have an iden-
tical genotype in both Lgl1-MADM and cKO-Lgl1-MADM, their
surrounding cells have distinct genotypes and thus properties
that could differentially influence the proliferation dynamics of
individual Lgl1�/� RGPs. To test whether distinct RGP prolifera-
tion could be the origin of SBH in cKO-Lgl1-MADM, we injected
bromodeoxyuridine (BrdU) at E13 and analyzed its incorporation
after 1 hr. We noticed a significant increase in the number of
BrdU+ cells in cKO-Lgl1-MADM when compared to control-
MADM (Figure S3). Rosette-like structures that stained positively
for PAX6 and included BrdU+ proliferating RGPs were also
frequently observed throughout the VZ and cortical plate (CP)
in cKO-Lgl1-MADM but not control-MADM (Figure S3). Although
we detected a significant increase in apoptosis, the number of
proliferating cells remained significantly higher at P0 in cKO-
Lgl1-MADM when compared to control-MADM (Figure S3). We
did not detect any signs of tumors, and the size of the SBH
was stable throughout adult stages from P21 onward and up
to 12 months of age (Figure S3). Because the sparse ablation
of Lgl1 in Lgl1-MADM did not lead to the formation of an SBH,
we conclude that in cKO-Lgl1-MADM, community effects
emerging in an environment where all RGPs lack Lgl1 expression
influence their mutual RGP proliferation behavior and/or dy-
namics resulting in SBH.
Loss of RGPCell Polarity Correlateswith theEmergenceof SBH in cKO-Lgl1-MADMIn order to evaluate whether the emergence of SBH in cKO-Lgl1-
MADM results from a disruption of the junctional adhesion belt,
we analyzed cKO-Lgl1-MADM at early embryonic stages (Fig-
ure 2). Indeed, already at E12, small ectopic cell formations
were apparent in cKO-Lgl1-MADM, but not in control-MADM
or Lgl1-MADM, respectively (Figures 2A–2C). We next assessed
the expression of a set of proteins that localize at basolateral
(CDH2 and b-catenin) and apical (CD133, Pals1, and g-tubulin)
sites in RGPs by immunohistochemistry at E13 when the ectopic
cell masses reached sizes of hundreds of cells. While in control-
MADM and Lgl1-MADM, the ventricular adhesion belt was uni-
formly stained for all basolateral and apical proteins indicated
above, all the components were either not expressed or mislo-
calized at sites of ventricular heterotopia (Figure 2). These results
indicate that excessive proliferation of Lgl1�/� RGPs (Figure S3)
in cKO-Lgl1-MADM is accompanied by the disruption of the
ventricular zone and mislocalization or loss of expression of
basolateral and apical components of the junctional adhesion
belt in RGPs.
Sparse and Complete Lgl1 Deletion DifferentiallyAffects Postnatal NeurogenesisShortly after birth, the embryonic neuroepithelium transforms
into the postnatal NSC niche in the V-SVZ within the LV (Lim
and Alvarez-Buylla, 2016). Because RGPs are lineally related
to progenitors in the adult stem cell niche, we assessed the
constitution and neurogenic properties of the V-SVZ in Lgl1-
MADM, cKO-Lgl1-MADM, and control-MADM. Because we
used Emx1-Cre, MADM-based labeling and Lgl1 ablation only
occurred in the dorsal wall (DW) of the V-SVZ but not in the
lateral and medial walls (LW andMW) of the LV. Given the strong
phenotype in the early embryonic neuroepithelium and VZ in
cKO-Lgl1-MADM, we first analyzed the ependymal cell layer
integrity in the DW and LW in cKO-Lgl1-MADM at P21. To this
end, we stained for CD133, which is strongly expressed in the
highly ciliated E1 ependymal cells. While the ependymal layers
in the DW in control-MADM and Lgl1-MADM appeared indistin-
guishable, the DW in cKO-Lgl1-MADM was completely devoid
of CD133 expression (Figures 3A–3C). The LW was not affected
as expected due to absence of Cre recombinase expression
(Figures 3D–3F). Previous studies have demonstrated that an
intact ependymal cell layer is critical for postnatal neurogenesis
and thus olfactory neuron production (Jacquet et al., 2011;
Paez-Gonzalez et al., 2011). We therefore quantified MADM-
labeled cells in the OB in cKO-Lgl1-MADM, Lgl1-MADM, and
control-MADM. Typically, we detected�80–100 MADM-labeled
red and green cells per mm2 on a representative mid-rostrocau-
dal OB section in control-MADM, and the g/r ratio was �1 (Fig-
ures 3M, 3P, and 3S) (Hippenmeyer et al., 2010). In contrast,
we detected less than five MADM-labeled cells in cKO-Lgl1-
MADM OB per mm2 indicating a dramatic defect in postnatal
neurogenesis (Figures 3O, 3R, and 3U). We did not detect any
MADM-labeled cells along the entire rostral migratory stream
(RMS) reflecting a true lack of DW postnatal neurogenesis rather
than a migration deficit in cKO-Lgl1-MADM (Figures 3I and
3L). These results indicate that global embryonic loss of Lgl1
function in all RGPs in cKO-Lgl1-MADM not only disrupts the
embryonic neuroepithelium, resulting in SBH, but subsequently
impedes the establishment of the DW ependymal cell layer and
thus postnatal neurogenesis. The Lgl1-MADM DW ependymal
cell layer integrity appeared indistinguishable when compared
to control-MADM (Figures 3A and 3B). However, we observed
a significant reduction of Lgl1�/� migrating neuroblasts along
the RMS (Figures 3H and 3K) and a decrease of OB neurons
in Lgl1-MADM. The g/r ratio of Lgl1�/� to WT olfactory granule
and periglomerular cells was �0.5 in Lgl1-MADM (Figures 3N,
3Q, and 3T). These data suggest an important cell-autonomous
function for Lgl1 in the V-SVZ stem cell niche and postnatal
neurogenesis.
Lgl1 Cell-Autonomously Controls Postnatal NSCLineage Progression in V-SVZTo identify the crucial Lgl1-dependent step in the establishment
of the adult stem cell niche and/or the role of Lgl1 in postnatal
neurogenesis, we traced the development of the dorsal V-SVZ
in Lgl1-MADM in a time course (Figures 4A–4J). While we
observed no gross abnormalities at P0, a large fraction of
Lgl1�/� mutant cells appeared in multicellular clusters in the
V-SVZ from P7 onward. We determined the frequency at which
multicellular clusters of distinct sizes (number of cells/cluster)
occurred in the V-SVZ. In control-MADM, the vast majority
(�90%) of both green and red MADM-labeled WT cells ap-
peared as single cells (Figure 4K), two cell clusters were
detected at a low rate, and clusters containing more than two
cells were only very rarely detected. In contrast, in Lgl1-
MADM the majority (>50%) of green Lgl1�/� cells were found
in clusters with more than two cells and larger clusters with 15
or more cells were detected frequently (Figure 4L). Next, we
quantified the g/r ratio of Lgl1�/� to WT cells and detected a
slight but significant �2-fold increase of Lgl1�/� at P0 indicating
that Lgl1 is cell-autonomously required in the emerging V-SVZ
immediately after birth (Figure 4M). The g/r ratio increased
to �8 at P21 and remained stable up until at least 12 months.
These results raise the question whether cluster formation
would include and/or affect type B1 cells in their lineage
progression. To this end, we performed high resolution morpho-
logical analysis of the postnatal V-SVZ stem cell niche in con-
trol-MADM and Lgl1-MADM. While at P0, RGPs (transforming
into type B1 cells) with their cell bodies located in the VZ could
be readily detected by virtue of their basal process (Figures 4N
and 4O), no morphological abnormalities could be observed. In
contrast, at P7 when many large clusters of green Lgl1�/� cells
were apparent (Figure 4D), high resolution morphological anal-
ysis revealed unusually long cellular extensions in Lgl1�/� cells
(Figures 4P and 4Q). Morphologically abnormal and clustered
Lgl1�/� cells expressed high levels of glial fibrillary acidic protein
(GFAP) indicating that these cells represent type B1 cells (Fig-
ure S4). At later postnatal stages, Lgl1�/� cells display even
more dramatic morphological abnormalities including syncy-
tium-like cell assemblies with long cytoplasmic extensions be-
tween individual cells (Figures 4R and 4S). These data indicate
that Lgl1 is required for the maintenance of type B1 cellular
morphology and their integrity within the postnatal V-SVZ
stem cell niche.
Neuron 94, 517–533, May 3, 2017 521
A CB
E FD
G H I
J K L
M N O
P Q R
Figure 2. Subcortical Band Heterotopia in
cKO-Lgl1-MADM IsAssociatedwithDisrup-
tion of Ventricular Apical-Basal Polarity
(A–C) Analysis of MADM-labeling pattern and
ventricular neuroepithelium integrity in control-
MADM (A), Lgl1-MADM (B), and cKO-Lgl1-MADM
(C) at E12. White arrows in (C) mark ectopic apical
cells accumulating into the ventricle. Scale
bars, 20 mm.
(D–R) Analysis of CDH2 (white, D–F), b-catenin
(white, G–I), Pals1 (white, J–L), g-tubulin (white,
M–O), and CD133 (white, P–R) expression pattern
in control-MADM (D, G, J, M, and P), Lgl1-MADM
(E, H, K, N, and Q), and cKO-Lgl1-MADM (F, I,
L, O, and R) at E13. White arrows mark the mis-
expression of apical and basolateral components
in RGPs in ectopic cell mass accumulating into the
ventricle in cKO-Lgl1-MADM. Scale bars, 45 (D–F)
and 30 mm (G–R).
See also Figure S3.
522 Neuron 94, 517–533, May 3, 2017
Lgl1 Cell-Autonomously Controls Cortical AstrocyteGenerationWhile Lgl1 appears not to be required cell-autonomously
for RGP-mediated cortical neurogenesis, we observed an
exuberant large number of Lgl1�/� cells with apparent astrocyte
morphology in Lgl1-MADM at P21 (Figures 5A–5D). To confirm
the cell fate of these MADM-labeled cells, we performed immu-
nohistochemistry for the mature astrocyte markers (Figure S5).
Next, we assessed the size and morphology of MADM-labeled
astrocytes in Lgl1-MADM and control-MADM. We quantified
the size of the cell body, total cell volume, and branching pattern
using Sholl analysis (Sholl, 1953) but could not detect any signif-
icant difference when Lgl1�/� astrocytes were compared to WT
(Figure S5). We quantified the g/r ratio of green Lgl1�/� to redWT
cortical astrocytes and noticed an increase of Lgl1�/� astrocytes
by a factor of �10 at P21 and throughout adulthood (Figure 5G).
In order to assess the cell-autonomous Lgl1 function in astrocyte
production at the single RGP level, we carried out MADM-based
clonal analysis using Emx1-CreER (Kessaris et al., 2006). MADM
clones were induced at E11 and analyzed at P21 (Figures 5E and
5F). Because astrocytes are not always present in both red and
green subclones (Gao et al., 2014), we quantified the number of
clonally related astrocytes in distinctly colored (and thus pre-
senting with different genotype) subclones independently (Fig-
ure 5H). We found that the total number of astrocytes per red
subclone, WT in control-MADM, and WT in Lgl1-MADM was
not significantly different. In contrast, the number of astrocytes
per green Lgl1�/� subclone in Lgl1-MADM was significantly
increased when compared to the green WT subclone in con-
trol-MADM (Figure 5H). The clonal analysis corroborates our re-
sults obtained from the population analysis in Lgl1-MADM.
Excessive astrocyte production was also observed in Lgl1-
MADM where we recombined the Lgl1-D allele (generated with
Hprt-Cre germline deleter [Tang et al., 2002]) instead of the
Lgl1-flox allele via meiotic recombination to the MADM-TG
cassette on chr11 (Figure S6). No difference in the astrocyte
phenotype was observed when Lgl1�/� cells exhibit either
maternal or paternal uniparental chromosome disomy, respec-
tively (Figure S6), and identical defects in astrocyte production
were seen when Lgl1�/� cells were labeled with tdT (red) instead
of GFP (green) (Figure S6). Increased astrocyte production was
also observed in the hippocampus in Lgl1-MADM (Figures 5B,
S6, and S7) and in the striatum in Lgl1-MADM induced by
Nestin-Cre (data not shown). Altogether, these data indicate an
important general cell-autonomous function of Lgl1 in astrocyte
production in the neocortex and diverse forebrain structures.
Increased Numbers of Early Postnatal AstrocyteProgenitors Precede Excess Astrocyte ProductionIn order to identify the critical Lgl1-dependent step in RGP line-
age progression and/or the transition to astrocyte production,
we traced the developmental origin of increased cortical astro-
cyte production. Once neurogenesis is completed, RGPs adopt
a gliogenic potential and can either proliferate locally to produce
intermediate astrocyte progenitor cells (aIPCs) or directly trans-
form into aIPCs and/or astrocytes (Kriegstein and Alvarez-
Buylla, 2009). We thus reasoned that astrocytes in Lgl1-MADM
could be emerging precociously or as a result of increased pro-
liferation capacity in aIPCs. To address these questions, we first
evaluated late embryonic Lgl1-MADM and early postnatal
stages for the emergence of astrocytes that could be unambig-
uously defined on the basis of their characteristic morphology.
We did not detect any precocious astrocytes at late embryonic
stages throughout the CP (Figure 6). From P7 onward, nascent
(and mature) astrocytes could be clearly discerned and the g/r
rate of Lgl1�/� to WT astrocytes was significantly increased (Fig-
ure 6M). To increase the temporal resolution and pinpoint the
critical Lgl1-dependent stages in RGPs during aIPC and/or
astrocyte production, we stained Lgl1-MADM in a time course
for brain lipid-binding protein (BLBP) (Feng et al., 1994), which
labels RGPs during embryonic development, early postnatal
aIPC, and mature astrocytes. To quantify aIPCs in the devel-
oping CP, we have only included BLBP+ cells that do not contain
apical and/or basal processes that would be indicative for RGPs
integrated either in the ventricular zone or translocating toward
the pia, rather than aIPCs. We quantified the percentage of
BLBP+/GFP+ double-positive Lgl1�/� cells and BLBP+/tdT+ WT
cells in comparison to all MADM-labeled cells. While at E16,
virtually no BLBP+/GFP+ and BLBP+/tdT+ cells could be de-
tected, BLPB+ Lgl1�/� cells are significantly overrepresented
at E17, E18, and P0 when compared to BLBP+ WT MADM-
labeled cells (Figures 6A–6K). Next, we determined the fraction
of proliferating cells that co-labeled for the cell-cycle marker
Ki67. At E17, when neurogenesis has largely ceased, the fraction
of Ki67+ Lgl1�/� cells in the CP was significantly elevated when
compared to Ki67+ WT MADM-labeled cells. Increased prolifer-
ation was not observed before E17 but through E18, P0, and P7
(Figure 6L). Together, these results suggest that proliferating
Lgl1�/� aIPCs are increased in late embryonic Lgl1-MADM, re-
sulting in a larger overall number of mature astrocytes at post-
natal stages.
We next quantified the relative fraction of astrocytes within the
total of MADM-labeled cells (neurons plus astrocytes) (Fig-
ure 6M). At P7, the fraction of mutant astrocytes (�25%) in
Lgl1-MADM was increased already by 2-fold when compared
to the fraction (�13%) of WT astrocytes in control-MADM. While
the astrocyte fraction in control-MADM reached a plateau of
�20% at P21, the relative fraction of Lgl1�/� mutant astrocytes
in Lgl1-MADM was nearly 50% of all MADM-labeled cells. The
relative number of mutant astrocytes remained stable in more
mature mice and no signs of astrocytoma were observed
(Figure S7). These data indicate that the overproduction of
astrocytes upon loss of Lgl1 still obeys tissue homeostasis
mechanisms. Interestingly, the relative fraction of astrocytes in
cKO-Lgl1-MADM was also increased and reached �50% (Fig-
ure 6M). Thus, the loss of Lgl1�/� in aIPCs results in dramatic
increase in mature astrocytes regardless of the state of the envi-
ronment, i.e., normal in Lgl1-MADM or Lgl1�/� in cKO-Lgl1-
MADM. Altogether, these results suggest that the control of
astrocyte production at normal numbers represents a critical
cell-autonomous Lgl1 function.
Genetic Interaction of Lgl1with Egfr Reveals FunctionalRelationship in Cortical Astrocyte GenerationOn the cellular level, loss of Lgl1 function leads to an exuberant
number of aIPCs and mature cortical astrocytes. To obtain
Neuron 94, 517–533, May 3, 2017 523
CD133 / / / GFP tdT DAPI
V
DW LW
cont
rol-M
AD
MLg
l1-M
AD
McK
O-L
gl1-
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DM
A
C
E
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Olfactory Bulb
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io o
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cells
/ m
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P S
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GCL
G
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ns
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neur
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GCL
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NCXDW
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GCL
ERMS
Rat
io R
MS
neur
ons
wt / wt
-/-Lgl1 / wt
Rat
io o
lf. g
ranu
le c
ells
Figure 3. Sparse and Whole Neuroepithelium Ablation of Lgl1 Differentially Affects Postnatal Neurogenesis
(A–F) Analysis of CD133 (white) expression and integrity of postnatal V-SVZ in dorsal (DW, A–C) and lateral (LW, D–F) walls of lateral ventricle (LV) in control-
MADM (A and D), Lgl1-MADM (B and E), and cKO-Lgl1-MADM (C and F) at P21. Note the absence of CD133 expression and irregular arrangement of V-SVZ cells
in DW in cKO-Lgl1-MADM (C). Scale bars, 10 mm.
(legend continued on next page)
524 Neuron 94, 517–533, May 3, 2017
mechanistic insights at the molecular level, we pursued a
candidate gene approach and conceived genetic interaction ex-
periments in a MADM context. We focused on Egfr because pre-
vious studies have suggested a crucial and dose-dependent
regulatory function of Egfr signaling in gliogenesis (Burrows
et al., 1997; Sibilia et al., 1998). We first tested the hypothesis
that exuberant astrocyte generation upon loss of Lgl1 could be
dependent on functional Egfr. Because Egfr is located on
chr11, it was possible to genetically link an Egfr-flox allele (Lee
and Threadgill, 2009) with the MADM cassette and Lgl1-D
allele to generate Egfr�/�,Lgl1�/� double mutant cells in a
mosaic context (MADM-11GT/TG,Egfr-flox,Lgl1-D;Emx1Cre/+ [Egfr-
Lgl1-MADM; red cells, Egfr+/+,Lgl1+/+; green cells, Egfr�/�,Lgl1�/�; background, Egfr+/�,Lgl1+/�]). Whereas in control-
MADM, the relative fractions of redWT and greenWT astrocytes
were each �20% (Figures 7A–7C), the fraction of green
Egfr�/�,Lgl1�/� double mutant astrocytes in Egfr-Lgl1-MADM
was reduced close to zero, and the fraction of red Egfr+/+,Lgl1+/+
astrocytes was increased to �40% (Figures 7J–7L). These data
show that the emergence of the cell-autonomous Lgl1 loss
of function (LOF) phenotype (i.e., excessive astrocyte produc-
tion) is dependent on functional Egfr. Moreover, Egfr+/+
astrocytes in an Egfr+/� background seem to exhibit a competi-
tive dose-sensitive advantage when compared to control-
MADM (all cells Egfr+/+). In order to scrutinize such concepts,
we conditionally ablated Egfr from all Emx1+ progenitor
lineages on top of Lgl1-MADM mosaic (Figures 7P–7R;
MADM-11GT,Egfr-flox/TG,Egfr-flox,Lgl1-D;Emx1Cre/+ [cKO-Egfr-Lgl1-
MADM; red cells, Egfr�/�,Lgl1+/+; green cells, Egfr�/�,Lgl1�/�;background, Egfr�/�,Lgl1+/�]). Strikingly, in such experimental
conditions, we detected almost no green and red astrocytes at
all, indicating not only a loss of dosage sensitivity (all cells are
Egfr�/�) but also confirming the requirement of Egfr signaling
for astrocyte genesis regardless of Lgl1 genotype (green cells
are Egfr�/�,Lgl1�/�). Next, we tested the possibility that loss of
Lgl1 in combination with Egfr dosage-sensitive competitive
advantagemight exhibit synergistic effects on astrocyte produc-
tion. We thus generated MADM-11GT,Lgl1-D/TG,Egfr-flox;Emx1Cre/+
(Figures 7M–7O; Egfr-Lgl1-MADM; red cells, Egfr+/+,Lgl1�/�;green cells, Egfr�/�,Lgl1+/+; background, Egfr+/�,Lgl1+/�) andquantified the relative levels of red and green astrocytes. While
green Egfr�/�,Lgl1+/+ cells were almost completely absent
(similar as in the above conditions), the relative fraction of red
Egfr+/+,Lgl1�/� astrocytes was �70% (Figure 7O). This number
(G–I) Analysis of MADM-labeling pattern in RMS in control-MADM (G), Lgl1-MAD
(J–L) Quantification of green/red ratio in control-MADM (J) and Lgl1-MADM (
neurons/mm2) in cKO-Lgl1-MADM (L) in RMS at P21.
(M–R) Analysis of MADM-labeling pattern in olfactory bulb (OB) in control-MADM
boxed areas in (M–O) illustrating the distribution of OB interneurons (oINs) acros
the drastic decrease of red and green Lgl1�/� MADM-labeled cells in cKO-Lgl1
40 mm (P–R).
(S–U) Quantification of green/red ratio in control-MADM (S) and Lgl1-MADM (T
granule cells/mm2) in cKO-Lgl1-MADM (U) in GCL at P21.
(V) Schematic illustrating the V-SVZ adult stem cell niches in DW (orange) and LW
migrate along the RMS to the OB. NCX, neocortex; RMS, rostral migratory stream
layer (extension of RMS) within the OB.
Values represent mean ± SEM. ns, nonsignificant.
See also Figure S8.
is much higher than in separate conditions with relative astrocyte
fractions of green Lgl1�/� in Lgl1-MADM (Figures 7D–7F) or red
Egfr+/+ in Egfr-MADM (Figures 7G–7I) reaching�40% each, indi-
cating a synergistic effect in Egfr+/+,Lgl1�/� astrocytes in an
Egfr+/� background. Altogether, our Lgl1/Egfr genetic interaction
experiments suggest a critical functional relationship in cortical
astrocyte generation.
DISCUSSION
In the developing cerebral cortex, NSCs are in charge of gener-
ating cell-type diversity. However, the underlying cellular and
molecular mechanisms controlling NSC proliferation behavior
and lineage progression are poorly defined. In our study, by
using quantitative MADM-based experimental paradigms at sin-
gle cell resolution, we found that Lgl1 is required at distinct
sequential stages in cortical NSCs to control quantitative neuron
and glia output (Figure 8). At early embryonic stages, Lgl1 func-
tion is required at the global neuroepithelium tissue level during
cortical neurogenesis. The loss of Lgl1 in all RGPs triggers a dy-
namic community effect that results in the overproduction of
cortical projection neurons and the formation of an SBH. In
contrast, Lgl1 is cell-autonomously required in aIPCs at later
stages to control astrocyte production and in postnatal V-SVZ
neurogenic niche for lineage progression of NSCs in the LV.
Collectively, our results define distinct sequential non-cell-
autonomous and intrinsic cell-autonomous Lgl1 functions con-
trolling cortical neuron and glia genesis and postnatal stem cell
behavior. We discuss our findings with emphasis on the interplay
of cell-autonomous gene function with non-cell-autonomous
and/or community effects and in the context of the general prin-
ciples of NSC proliferation behavior.
Genetic Dissection of Cell-Autonomous Gene Functionand Environmental Community Effects at Single-CellResolutionWhile genetic loss of function can reveal cell-autonomous gene
functions, the contribution of non-cell-autonomous gene func-
tions and/or community effects often remain poorly defined.
Non-cell-autonomous gene functions may involve directed
cell-to-cell communication either via contact-mediated or
secreted signaling cues (Greenman et al., 2015; Hippenmeyer,
2014). For instance, excess activation of AKT3 in just a small
population of cells is associated with human focal malformations
M (H), and cKO-Lgl1-MADM (I) at P21. Scale bars, 30 mm.
K); and absolute numbers (i.e., almost complete absence; number of RMS
(G), Lgl1-MADM (H), and cKO-Lgl1-MADM (I) at P21. Higher magnification of
s the granule cell layer (GCL) and in periglomerular areas in the P21 OB. Note
-MADM (O and R). Nuclei are stained using DAPI. Scale bars, 200 (M–O) and
), and absolute numbers (i.e., almost complete absence; number of olfactory
(purple), RMS, and OB in the postnatal brain. oINs are generated in the V-SVZ,
; G, glomerular layer; M, mitral cell layer; GCL, granule cell layer; E, ependymal
Neuron 94, 517–533, May 3, 2017 525
GFP
/ /
tdT
DA
PI
P0P7
P21
6Mo
12M
oLgl1-MADM / -/- Lgl1- wtcontrol-MADM / wt- wt
A B
C D
E F
HG
I J
K
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100
1 2-3 4-5 6-10 11-15
50
25
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>15
# cells / cluster
% n
orm
. fre
quen
cy +/+Lgl1
+/+Lgl1
ns
ns
L
# cells / cluster
0
100
1 2-3 4-5 6-10 11-15
50
25
75
>15
+/+Lgl1
-/-Lgl1
% n
orm
. fre
quen
cy
**
***
*** *** *
*
*
*
*
*
* *
**
**
**
Qua
ntifi
catio
n
ratio / cells-/-Lgl1 wt
control-MADM
Lgl1-MADM
0
15
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P0 P7 P21 6Mo 12Mo
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*****
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20
Quantification
M
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/ -
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gl1
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-MA
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L
gl1
-w
t
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rol-M
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/ -
wt
wt
P21P7
N
O
P
Q
R S
Figure 4. Lgl1 Is Cell-Autonomously Required for V-SVZ NSC Lineage Progression
(A–J) Time course analysis of MADM-labeling pattern in V-SVZ in dorsal wall (DW) of the lateral ventricle (LV) in control-MADM (A, C, E, G, and I) and Lgl1-MADM
(B, D, F, H, and J) at P0 (A and B), P7 (C and D), P21 (E and F), 6 months (6Mo; G and H), and 1 year (12Mo; I and J). White asterisks (*) mark aberrant large clusters
of green Lgl1�/� cells in V-SVZ. Nuclei are stained using DAPI. Scale bars, 75 (A and B), 60 (C and D), 30 (E and F), and 50 mm (G–J).
(legend continued on next page)
526 Neuron 94, 517–533, May 3, 2017
in cortical development, which disrupts the architecture of the
entire hemisphere (Baek et al., 2015). These findings suggest
that alteration of the properties of individual neurons collectively
may affect the entire community. Such a phenomenon is also
observed in distinct cellular contexts including collective cell
migration and tissue morphogenesis (Heisenberg and Bellaıche,
2013). The cellular and molecular mechanisms orchestrating
community effects during brain development are mostly un-
known due to the lack of experimental assays enabling the
visualization and quantitative assessment of the non-cell-auton-
omous elements in full or whole tissue conditional loss-of-func-
tion phenotypic analysis (Greenman et al., 2015). To this end,
we have established an unprecedented genetic strategy to visu-
alize and dissect the interplay of relative cell-autonomous gene
function and the contribution of non-cell-autonomous commu-
nity effects to the overall phenotype presentation. Our assay
relies uponMADM-based single-cell phenotypic analysis of indi-
vidual mutant cells in (1) normal environment, and (2) homozy-
gous mutant environment. The approach includes the sparse
mosaic versus global/whole tissue wide ablation of a candidate
gene, resulting in distinct cellular environments permitting the
direct assessment of the non-cell-autonomous influence on the
single-cell phenotype. This assay can be applied in principle to
any candidate gene of interest, provided that knockout or condi-
tional alleles are available, and MADM cassettes have been in-
serted on the particular chromosome where the gene is located.
Here, we utilized such a genetic paradigm to genetically dissect
the function of Lgl1 in controlling NSC proliferation behavior in
neurogenesis and glia production.
Role of Lgl1 in Embryonic Cortical NeurogenesisAblation of Lgl1 from all NESCs results in a severe non-cell-
autonomous community effect. Albeit, the formation of ectopic
clusters of exuberantly proliferating Lgl1�/� progenitors in
cKO-Lgl1-MADM (where all cells lack Lgl1) occurred in a non-
stereotypic fashion but was correlated with absence or misex-
pression of apical and basolateral components. Strikingly, the
phenotype of cKO-Lgl1-MADM is almost congruent to the
phenotype ofNumb/Numbl doublemutants (Li et al., 2003; Rasin
et al., 2007). NUMB has been proposed to control the trafficking
of adherens junction components (Rasin et al., 2007). Because
LGL1 has also been suggested to regulate polarized secretion
and exocytosis (M€usch et al., 2002), it will be interesting in the
future to determine a potential functional relationship of LGL1
and NUMB in the regulation of adherens junctions and possibly
the control of progenitor proliferation. The SBH in cKO-Lgl1-
MADM was composed of neurons with upper and lower cortical
layer identity as well as glia cells, indicating that the generation of
faithful cell fates by embryonic Lgl1�/� mutant RGPs is not
(K and L) Cluster size (number of cells/cluster) distribution of V-SVZ cells at P21 in
green bars in control-MADM and red bar in Lgl1-MADM) are not forming cluster
(M) Quantification of the green/red ratio of MADM-labeled cells in DW V-SVZ at
MADM (Lgl1�/�/WT, gray bars). Scale bar, 7.5 mm.
(N–S) Time course analysis of cellular morphology in V-SVZ in control-MADM (M
P21 (Q and R). Scale bars, 7.5 (N–P) and 9 mm (Q and R).
Values represent mean ± SEM. ns, nonsignificant; *p < 0.05, **p < 0.01, ***p < 0.
See also Figures S4 and S8.
severely disturbed. However, it will be intriguing to determine
the global tissue-wide homeostatic properties and possible
changes associated with the SBH formation and maintenance
in the adult cKO-Lgl1-MADM. In Lgl1-MADM with only sparse
Lgl1 knockout, the individual Lgl1�/� mutant progenitors appear
to proliferate normally, presumably because their integration
within the neuroepithelium is rescued by the surrounding
‘‘normal’’ progenitors maintaining the cell adhesion of mutant
cells in a non-cell-autonomous manner. Future efforts aiming
to determine the genetic fingerprints of Lgl1�/� mutant progeni-
tors in Lgl1-MADM and cKO-Lgl1-MADM by single cell RNA
sequencing (RNA-seq) could help (provided that a large enough
set of progenitors can be faithfully isolated) to identify the
intrinsic signaling pathways associated with either ‘‘rescue’’ of
mutant progenitors in Lgl1-MADM or aberrant proliferation and
SBH formation in cKO-Lgl1-MADM.
Lgl1-Dependent Lineage Progression in PostnatalV-SVZ NSCsThe V-SVZ in the DW of the LV was not properly established in
cKO-Lgl1-MADM, and postnatal OB interneuron (oIN) generation
was virtually absent. It is likely that the strong community effects
observed in the embryonic VZ resulted in a dispersal of the DW
ependymal cells in the SBH. This phenotype reflects the impor-
tance of an intact E1 cell layer that derives from a discrete pool
of RGPs (Jacquet et al., 2011; Paez-Gonzalez et al., 2011) and
that is severely affected in the cKO-Lgl1-MADM. In contrast,
Lgl1-MADM mice show a normal E1 layer. Nevertheless, post-
natal neurogenesis is still compromised in Lgl1-MADM, although
not to the extent like in cKO-Lgl1-MADM, suggesting a distinct
underlying basis and critical cell-autonomous role for Lgl1 in
postnatal neurogenesis. In sparse Lgl1-MADM, mutant Lgl1�/�
type B1 cells display aberrant cellularmorphology and frequently
appear in clusters or syncytia of nuclei with long cytoplasmic
bridges that could represent incomplete cell division. It is
tempting to speculate that Lgl1 could regulate very specific intra-
cellular trafficking events required for correct cytokinesis (Schiel
et al., 2013). In any case, our results indicate a delicate Lgl1-
dependent step in the proliferation pattern of type B1 cells. Lgl1
function could dictate the balance of symmetric versus asym-
metric V-SVZ progenitor division pattern and thereby indirectly
regulate the level of progenitor expansion versus differentiation
and thus quantitative output during postnatal neurogenesis.
Because Lgl1 has been associated with polarity-driven pro-
cesses and is involved in controlling the switch from symmetric
to asymmetric RGP divisions, it will be intriguing to assess in
future studies whether the loss of Lgl1 in asymmetrically dividing
type B1 cells results in ‘‘more symmetric’’ division that may be
not fully completed due to possible cell-cycle checkpoints.
control-MADM (K) and Lgl1-MADM (L). Note that�90% of Lgl+/+ cells (red and
s and occur as single cells but <40% Lgl�/� appear as single cells.
P0, P7, P21, 6Mo, and 12Mo in control-MADM (WT/WT, white bars) and Lgl1-
, O, and Q) and Lgl1-MADM (N, P, and R) at P0 (M and N), P7 (O, and P), and
001.
Neuron 94, 517–533, May 3, 2017 527
Clonal Analysis
GFP / / tdT DAPI
TM /
E11
- A
naly
sis
/ P21
Cre
ER/+
Lgl1
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DM
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x1 /
-/-
Lgl
1-
wt
Cre
/+Lg
l1-M
AD
M ;
Emx1
/ -/-
L
gl1
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t
Cre
ER/+
cont
rol-M
AD
M ;
Emx1
/
wt
-w
t
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/+co
ntro
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; Em
x1 /
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t-
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Clonal AnalysisPopulation Analysis
I
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Population Analysis
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******
# astrocytes /red subclone
0
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Lgl1-MADM - w
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# astrocytes /green subclone
0
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Lgl1-MADM -
-/-
Lgl1
10075
50
25
***
Figure 5. Lgl1 Function Is Cell-Autono-
mously Required for Cortical Astrocyte
Generation
(A–D) Population analysis of MADM-labeled
cortical astrocytes in control-MADM (A and C) and
Lgl1-MADM (B and D) at P21. White arrows in (D)
mark groups with increased numbers of Lgl1�/�
astrocytes. For high-resolution analysis of cell size
and arborization, refer to Figure S5. Scale bars,
500 (A and B) and 60 mm (C and D).
(E and F) Clonal analysis ofMADM-labeled cortical
astrocytes in control-MADM (E) and Lgl1-MADM
(F). Tamoxifen (TM) was applied at E11 and brain
samples analyzed at P21. G2-X clones are illus-
trated. Cortical layers are indicated (roman nu-
merals). Scale bars, 70 mm.
(G) Quantification of the green/red ratio of cortical
astrocytes in population analysis at P21, 3 months
(3Mo), and 1 year (12Mo) in control-MADM (WT/
WT) and Lgl1-MADM (Lgl1�/�/WT).
(H) Quantification of the number of astrocytes in
red (left) and green (right) MADM subclones. Note
that red cells within red subclones in both control-
MADM and Lgl1-MADM are WT, and green cells
within green subclones are WT in control-MADM
but Lgl1�/� in Lgl1-MADM.
Values represent mean ± SEM. ns, nonsignificant,
***p < 0.001.
See also Figures S5–S7.
Alternatively, Lgl1 could act as an intracellular sensor and/or
mediator of extracellular signals to regulate the transition from
quiescent to active status in type B1 cells. Importantly, however,
typeB1 cells produce typeCcells that in turn can give rise to neu-
roblasts migrating toward the RMS. Because there is a lineage
relationship from type B1 to type C to neuroblast, the loss of
Lgl1 function in type B1 cells results in a block of lineage progres-
sion and thus affects all downstream progenitor cells and their
output. Type C cells not only produce neuroblasts destined for
528 Neuron 94, 517–533, May 3, 2017
the olfactory granule cell layer but also
give rise to oligodendrocyte lineage
(Kriegstein and Alvarez-Buylla, 2009). It
will thus be important in future lineage
tracing experiments to evaluate whether,
and to what extent, a block in lineage
progression from type B1 to type C cells
may affect the oligodendrocyte lineage
emerging from type C cells. Perhaps
even more important will be the determi-
nation of progenitor cell-type diversity
among type B and type C cells and to
evaluate whether the loss of Lgl1 may
result in changes of progenitor cell fates
and how it affects neuron and glia output.
Cell-Autonomous Lgl1 Function inPostnatal Cortical AstrocyteProductionMADM is a powerful approach to study
cell-autonomous gene function at high
spatiotemporal resolution (Hippenmeyer et al., 2010; Joo et al.,
2014). Here, the analysis of Lgl1-MADM allowed us to identify
as-yet-unknown cell-autonomous Lgl1 functions in NSC prolifer-
ation behavior (Figure 8). Once RGPs cease the production of
projection neurons, they adopt gliogenic potential (Bayraktar
et al., 2014; Kriegstein and Alvarez-Buylla, 2009). However, the
cellular and molecular mechanisms controlling glia production
and quantitative output are not well understood. In our analysis
of Lgl1-MADM, we discovered that the production of astrocytes
BLBP / / / GFP tdT DAPI
control-MADMLgl1-MADMcontrol-MADM Lgl1-MADM
E16 E17
A B C D
F
E
H
I
J
G
+ + +% BLBP ,MADM / total MADM
0
6
4
E16 E17 E18 P0
2
+/+Lgl1
-/-Lgl1***
8
**
***
+ +% MADM astrocytes / total MADM cells
0
50
P7 P14 P21
25
P210
50
25
control-MADM +/+(R+G Lgl1 cells)
Lgl1-MADM -/-(G Lgl1 mutant cells)
cKO-Lgl1-MADM -/-(R+G Lgl1 mutant cells)
M
****** ***
+ + +% Ki67 ,MADM / total MADM
0
6
4
E17 E18 P0 P7
2
+/+Lgl1
-/-Lgl1
8
***
******
***
E16
LK
Figure 6. Increased Numbers of Astrocyte Progenitors Precede Overproduction of Postnatal Cortical Astrocytes
(A–J) Analysis of BLBP (white) expression pattern in CP in control-MADM (A and C–F) and Lgl1-MADM (B and G–J) at E16 (A and B) and E17 (C–J). Higher
magnification of yellow boxed areas in (C and G) illustrate MADM-labeled cells expressing BLBP (marked by cyan arrows) in control-MADM (D–F) and Lgl1-
MADM (H–J). Scale bars, 50 (A–C and G) and 60 mm (D–F and H–J).
(K) Quantification of fraction (%) BLBP+/MADM+ double-positive cells of total number of MADM-labeled cells (red tdT+ or green GFP+) in Lgl1-MADM at E16, E17,
E18, and P0. Red bars represent tdT+/BLBP+ double-positive Lgl1+/+ cells, and green bars represent GFP+/BLBP+ Lgl1�/� cells.
(L) Quantification of fraction (%) Ki67+/MADM+ double-positive cells of total number of MADM-labeled cells (red tdT+ or green GFP+) in Lgl1-MADM at E16, E17,
E18, P0, and P7. Red bars represent tdT+/Ki67+ double-positive Lgl1+/+ cells, and green bars represent GFP+/Ki67+ Lgl1�/� cells.
(M) Quantification of fraction (%) of MADM-labeled astrocytes of total number of MADM-labeled cells (neurons and astrocytes). Population analysis in control-
MADM, Lgl1-MADM, and cKO-Lgl1-MADM at P7 and P14 (control-MADM, Lgl1-MADM) and P21 (control-MADM, Lgl1-MADM, and cKO-Lgl1-MADM). Note that
the fraction of mutant Lgl1�/� astrocytes in cKO-Lgl1-MADM is elevated to similar levels like the mutant Lgl1�/� astrocytes in Lgl1-MADM at P21.
Values represent mean ± SEM. **p < 0.01, ***p < 0.001.
See also Figure S7.
Neuron 94, 517–533, May 3, 2017 529
++
% M
AD
M a
stro
cyte
s / t
otal
MA
DM
cel
ls
0
80
40
20
60
ns++
% M
AD
M a
stro
cyte
s / t
otal
MA
DM
cel
ls
0
80
40
20
60
***
ns
++
% M
AD
M a
stro
cyte
s / t
otal
MA
DM
cel
ls
0
80
40
20
60
++
% M
AD
M a
stro
cyte
s / t
otal
MA
DM
cel
ls
0
80
40
20
60 ***
++
% M
AD
M a
stro
cyte
s / t
otal
MA
DM
cel
ls
0
80
40
20
60 ***
++
% M
AD
M a
stro
cyte
s / t
otal
MA
DM
cel
ls
0
80
40
20
60 ***
GT/TG Cre/+MADM-11 ; Emx1 GT/TG,Lgl1 Cre/+MADM-11 ; Emx1
GT/TG,Egfr,Lgl1 Cre/+MADM-11 ; Emx1GT/TG,Egfr Cre/+MADM-11 ; Emx1
+/+Lgl1 +/- Lgl1-/-Lgl1wt wtwt
GT,Egfr/TG,Egfr,Lgl1 Cre/+MADM-11 ; Emx1GT,Lgl1/TG,Egfr Cre/+MADM-11 ; Emx1
+/+ +/+Lgl1 ; Egfr +/- +/- Lgl1 ; Egfr-/- -/-Lgl1 ; Egfr+/+Egfr +/- Egfr-/-Egfr
+/+ -/-Lgl1 ; Egfr +/- -/- Lgl1 ; Egfr-/- -/-Lgl1 ; Egfr-/- +/+Lgl1 ; Egfr +/- +/- Lgl1 ; Egfr+/+ -/-Lgl1 ; Egfr
GFP / / tdT DAPI
I
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HCNCX
A B C D FE
G H I J LK
M N O P RQ
Figure 7. Genetic Interaction of Lgl1 with Egfr Reveals Functional Relationship in Cortical Astrocyte Generation
Analysis of MADM-labeled cortical astrocytes in MADM-11GT/TG;Emx1Cre/+ (A–C; control-MADM; all cells WT), MADM-11GT/TG,Lgl1-D;Emx1Cre/+ (D–F, Lgl1-
MADM; red cells: Lgl1+/+; green cells: Lgl1�/�; background: Lgl1+/�), MADM-11GT/TG,Egfr-flox;Emx1Cre/+ (G–I, Egfr-MADM; red cells: Egfr+/+, green cells: Egfr�/�,background: Egfr+/�), MADM-11GT/TG,Egfr-flox,Lgl1-D;Emx1Cre/+ (J–L; Egfr-Lgl1-MADM; red cells: Egfr+/+,Lgl1+/+; green cells: Egfr�/�,Lgl1�/�; background:
Egfr+/�,Lgl1+/�), MADM-11GT,Lgl1-D/TG,Egfr-flox;Emx1Cre/+ (M–O; Egfr-Lgl1-MADM; red cells: Egfr+/+,Lgl1�/�; green cells: Egfr�/�,Lgl1+/+; background:
Egfr+/�,Lgl1+/�), and MADM-11GT,Egfr-flox/TG,Egfr-flox,Lgl1-D;Emx1Cre/+ (P–R; cKO-Egfr-Lgl1-MADM; red cells: Egfr�/�,Lgl1+/+; green cells: Egfr�/�,Lgl1�/�; back-ground: Egfr�/�,Lgl1+/�) at P21. Quantification (C, F, I, L, O, and R) of fraction (%) of MADM-labeled cortical astrocytes of total number of MADM-labeled cells
(neurons and astrocytes) is indicated. Note that in Egfr-MADM (G–I) the relative fraction of red Egfr+/+ astrocytes is increased up to �40% (presumably due to
dosage sensitivity and growth advantage in an Egfr+/� background) whereas green Egfr�/� astrocytes are almost completely absent. Introduction of Lgl1�/� into
Egfr�/� green cells (J–L) does not rescue the number of green Egfr�/�,Lgl1�/� double mutant astrocytes. In contrast, the relative fraction of red Egfr+/+ astrocytes
that also lack Lgl1 (i.e., Egfr+/+,Lgl1�/�) is increased up to�70% (M–O). Global ablation of Egfr (P–R) results in near complete loss of cortical astrocytes regardless
of Lgl1 genotype. Cortical layers are indicated (roman numerals). Values represent mean ± SEM. ns, nonsignificant, ***p < 0.001. Scale bars, 500 (A, D, G, J,
N, and P) and 60 mm (B, E, H, K, N, and Q).
See also Figure S8.
530 Neuron 94, 517–533, May 3, 2017
E14E12E8 - E10
Neuroepithelialcell
Neurogenic radial gliaprogenitor cell
Postnatal / Adult
V-SVZ type B1 cell
IV
I
II
III
VI
V
V-SVZ
Neuroblasts
WM
VentricularZone(VZ)
CorticalPlate
SubventricularZone (SVZ)
VZ
E18 / Birth
Gliogenic radial gliaprogenitor cell
II-IV
VI
V
SVZ
VZ
Astrocytes
Lgl1 function required for VZ integrity(control of embryonic neurogenesis)
Lgl1 regulates corticalastrocyte production
Lgl1 controls postnatalneurogenesis in V-SVZ
LGL1
LGL1 LGL1
Figure 8. Lgl1 Controls NSC Lineage Progression in Embryonic and Postnatal Stem Cell Niches
Schematic model of NSC lineage progression and the role of Lgl1 non-cell-autonomous (indicated in green) and cell-autonomous (indicated in blue) functions at
discrete sequential steps.
is strongly increased upon cell-autonomous loss of Lgl1. Inter-
estingly, the relative number of astrocytes was also significantly
higher in cKO-Lgl1-MADM indicating a dominant cell-autono-
mous component, not strongly influenced by non-cell-autono-
mous community effects. What can we learn from the Lgl1
loss-of-function astrocyte phenotype with regard to the general
principles of RGP proliferation behavior? It has been suggested
that RGPs give rise to aIPCs that locally amplify astrocyte pro-
duction in a tightly controlled manner (Ge et al., 2012). It is
currently not clear whether astrocyte production follows a strictly
deterministic program similar to neurogenic RGPs (Gao et al.,
2014) or whether a certain degree of stochasticity contributes
to the proliferation dynamics of aIPCs. Regardless of the precise
mechanism, clonally related astrocytes do not disperse broadly
(Gao et al., 2014; Molofsky et al., 2014), and because astrocytes
exhibit precise tiling (i.e., do not overlap their fine projections), it
has been suggested that astrocyte production is controlled by
homeostatic cues to ensure complete coverage of the local neu-
ropil (Molofsky and Deneen, 2015). In any case, loss of Lgl1 func-
tion results in a scalable overproduction of Lgl1�/� astrocytes.
Given that astrocytes tile the neuropil, do Lgl1�/� aIPCs have a
competitive advantage over WT progenitors? And if yes, which
Lgl1-dependent signaling cascades are misregulated in aIPCs?
The astrocyte overproduction in Lgl1-MADM could reflect the
loss of a specific Lgl1-dependent function in polarized secretion
and/or exocytosis in order to regulate cell-surface abundance of
astrocyte production stimulating and/or inhibiting factors. It is
intriguing to note that the control of polarized secretion, exocy-
tosis (M€usch et al., 2002), and possibly further intracellular traf-
ficking events, could actually represent one unifying function of
Lgl1 in the control of proliferating NSCs. In such a mechanistic
framework, Lgl1 could regulate the cell-surface abundance of
junctional complex components in embryonic RGPs, control traf-
ficking events critical for cytokinesis in type B1 NSC, and tune
growth factor receptor levels at the plasmamembrane in prolifer-
ating aIPCs. In this regard, we could observe genetic interaction
between Lgl1 and Egfr suggesting, indeed, a functional relation-
ship. Although the precise nature of Lgl1/Egfr interaction remains
to be determined, it seems highly specific for cortical astrocyte
generation but not V-SVZ NSC behavior (Figure S8). It will be
revealing toprobewhether LGL1andEGFR interact at the protein
level and to assay EGFR cell surface levels and/or turnover in
Lgl1�/� context during astrocyte generation. We cannot exclude
that Egfr and Lgl1 also play independent functions in astrocyte
generation, and it will be interesting to dissect those putative
functions. Alternatively, but not mutually exclusive, Lgl1 could
regulate the number of symmetric amplification versus asym-
metric differentiation divisions by regulating intracellular polarity
Neuron 94, 517–533, May 3, 2017 531
and/or the symmetry of the division plane in aIPCs. It will be inter-
esting in the future to assess the mechanisms and dependence
on Lgl1 function dictating the total astrocyte unit production in
distinct functional areas in the cortex and beyond in other brain
areas. Lastly, Lgl1 is highly expressed in mature astrocytes
(Zhang et al., 2016), and it will be intriguing to assess the expres-
sion status of Lgl1 in reactive astrocytes during injury and
whether the local response in quiescent astrocyte progenitors
require the downregulation and/or inhibition of Lgl1 function in
order to initiate the astrocyte production at injury sites.
Collectively, by using sparse and whole tissue genetic MADM
approaches to ablate Lgl1 gene function in NSCs, we define
distinct sequential Lgl1 functions in neurogenesis, astrocyte pro-
duction, and postnatal stem cell behavior. Our study emphasizes
the importance of the local stem cell niche environment and thus
non-cell-autonomous contributions in concert with cell-autono-
mous gene function in the control of NSC proliferation behavior.
More generally, single-cell phenotypes in conditional or full
knockout reflect a combination of both cell-autonomous gene
function andenvironment-derivedcues thatmay remedyor exac-
erbate any observed phenotype. It will thus be important in future
genetic loss-of-function paradigms to qualitatively and quantita-
tively determine the relative contributions of the intrinsic and
extrinsic components to the overall loss-of-function phenotype.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Mouse Lines and Maintenance
d METHOD DETAILS
B Preparation of MADM-Labeled Tissue
B Immunostaining of MADM-Labeled Brains
B Imaging and Analysis of Marker Expression in MADM-
Labeled Brains
B Generation of MADM Clones in the Neocortex
B Astrocyte Morphology Filament Tracing
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental Information includes eight figures and two tables and can be
found with this article online at http://dx.doi.org/10.1016/j.neuron.2017.
04.012.
AUTHOR CONTRIBUTIONS
Conceptualization, S.H.; Methodology, R.B., C.S., and S.H.; Investigation,
R.B., M.P.P., L.E.B., C.S., S.L., F.M.P., and S.H.; Resources, G.X., T.H.G.,
O.K., and V.V.; Writing – Original Draft, S.H.; Writing – Review & Editing, all
authors; Funding Acquisition and Supervision, S.H.
ACKNOWLEDGMENTS
We thank Drs. N. Sans andM.Montcouquiol for transferring Lgl1-floxmice and
Dr. Threadgill for providing Egfr-flox mice; A. Heger (Preclinical Facility) and
532 Neuron 94, 517–533, May 3, 2017
E. Papusheva (Bioimaging Facility) for technical support; C. Schwayer,
E. Fisher, P. Hirschfeld, M. Frank, and J. Rodarte for initial experiments and/or
assistance; J. Knoblich, M. Loose, C.P. Heisenberg, and members of the
Hippenmeyer lab for discussion; and A. Kicheva, C. Mieck, N. Amberg, and
C. Duellberg for comments on the manuscript. This work was supported by
IST Austria institutional funds, NO Forschung & Bildung n[f+b] (C13-002), the
European Union (FP7-CIG618444), and a program grant from the Human Fron-
tiers Science Program (RGP0053/2014) to S.H.
Received: October 3, 2016
Revised: March 2, 2017
Accepted: April 5, 2017
Published: May 3, 2017
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
GFP – Chick Aves Labs Cat#GFP-1020; RRID: AB_10000240
RFP – Rabbit MBL Cat#PM005; RRID: AB_591279
tdTomato – Goat Sicgen Antibodies Cat#AB8181-200
GFAP – Rabbit Dako Cat#Z0334; RRID: AB_10013382
Ki67 – Rabbit Leica Microsystems Cat#NCL-Ki67p; RRID: AB_442102
BLBP – Rabbit Millipore Cat#AB9558; RRID: AB_2314014
S100b – Mouse Sigma-Aldrich Cat#S2532; RRID: AB_477499
Prominin1 – Rat Affymetrix eBioscience Cat#14-1331; RRID: AB_2314136
Satb2 – Mouse Abcam Cat#AB51502; RRID: AB_882455
Ctip2 – Rat Abcam Cat#AB18465; RRID: AB_2064130
Pax6 – Rabbit BioLegend Cat#901301; RRID: AB_2565003
Tbr1 – Rabbit Abcam Cat#AB31940; RRID: AB_2200219
N-cadherin (CDH2) – Mouse Thermo Fisher Scientific Cat#333900; RRID: AB_2313779
BrdU – Mouse BD Bioscience Cat#347580; RRID: AB_400326
Caspase-3 pAb – Rabbit Promega Cat#G7481; RRID: AB_430875
Glast (EAAT1) – Rabbit Abcam Cat#AB416; RRID: AB_304334
g-tubulin – Mouse Sigma-Aldrich Cat#T3559; RRID: AB_477575
b-catenin – Mouse BD Transduction labs Cat#610153; RRID: AB_397554
Pals1 – Rabbit Proteintech Cat#17710-1-AP; RRID: AB_2282012
DyLight 405 Anti-Mouse IgG Jackson ImmunoResearch Labs Cat#715-475-150; RRID: AB_2340839
Alexa Fluor 488 Anti-Chicken IgG Jackson ImmunoResearch Labs Cat#703-545-155; RRID: AB_2340375
Cy3 Anti-Goat IgG Jackson ImmunoResearch Labs Cat#705-165-147; RRID: AB_2307351
Alexa Fluor 647 Anti-Goat IgG Jackson ImmunoResearch Labs Cat#705-605-147; RRID: AB_2340437
Alexa Fluor 647 Anti-Mouse IgG Jackson ImmunoResearch Labs Cat#715-605-151; RRID: AB_2340863
Cy3 Anti-Rabbit IgG Jackson ImmunoResearch Labs Cat#711-165-152; RRID: AB_2307443
Alexa Fluor 647 Anti-Rabbit IgG Jackson ImmunoResearch Labs Cat#711-605-152; RRID: AB_2340624
Alexa Fluor 647 Anti-Rat IgG Jackson ImmunoResearch Labs Cat#712-605-153; RRID: AB_2340694
Experimental Models: Organisms/Strains
Mouse: MADM-11-GT The Jackson Laboratory RRID: IMSR_JAX:013749
Mouse: MADM-11-TG The Jackson Laboratory RRID: IMSR_JAX:013751
Mouse: Lgl1-Flox Klezovitch et al., 2004 MGI: 102682
Mouse: Egfr-Flox Lee and Threadgill, 2009 MGI: 3513096
Mouse: Emx1-Cre The Jackson Laboratory RRID: IMSR_JAX:005628
Mouse: Emx1-CreER The Jackson Laboratory RRID: IMSR_JAX:027784
Mouse: Hprt-Cre The Jackson Laboratory RRID: IMSR_JAX:004302
Software and Algorithms
IMARIS 7.7.1 Bitplane http://www.bitplane.com/imaris/imaris
MATLAB Mathworks https://www.mathworks.com/products/matlab/index.
html?s_tid=gn_loc_drop
ZEN Digital Imaging for Light Microscopy Zeiss https://www.zeiss.com/microscopy/en_us/products/
microscope-software/zen.html#introduction
Other
LSM 800 Confocal Zeiss N/A
LSM 880 with Airy Scan Confocal Zeiss N/A
Cryostat Cryostar NX70 Thermo Fisher Scientific N/A
e1 Neuron 94, 517–533.e1–e3, May 3, 2017
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Simon
Hippenmeyer ([email protected]).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
See Table S1 for complete information regarding genotypes used in this study.
Mouse Lines and MaintenanceMouse protocols were reviewed by institutional preclinical core facility (PCF) at IST Austria and all breeding and experimentation was
performed under a license approved by the Austrian Federal Ministry of Science and Research in accordance with the Austrian and
EU animal laws.Mice weremaintained and housed in animal facilities with a 12 hr day/night cycle and adequate food andwater under
conditions according to IST Austria institutional regulations. Mouse lines with chromosome 11MADM cassettes (Hippenmeyer et al.,
2010), Lgl1-flox allele (Klezovitch et al., 2004), Egfr-flox (Lee and Threadgill, 2009), Emx1-Cre (Gorski et al., 2002), and Emx1-CreER
(Kessaris et al., 2006) have been described previously. For the generation of Lgl1-D allele, Lgl1-flox was crossed toHprt-Cre germline
delete (Tang et al., 2002) and double heterozygous Lgl1-flox; Hprt-Cre backcrossed to wild-type. All analyses were carried out in a
mixed C57/Bl6, CD1 genetic background. Littermates were randomly assigned to experimental groups based on genotype. No sex
specific differences were observed under any experimental conditions or in any genotype.
METHOD DETAILS
Preparation of MADM-Labeled TissueMice were deeply anesthetized through injection of a ketamine/xylazine/acepromazine solution (65 mg, 13 mg and 2 mg/kg body
weight, respectively), and confirmed to be unresponsive through pinching the paw. Perfusion was performed with ice-cold phos-
phate-buffered saline (PBS) followed immediately by 4% PFA prepared in PBS. Tissue was further fixed in 4% PFA over night to
ensure complete fixation. Brains were washed with PBS 4-5 times, and cryopreserved with 30% sucrose solution in PBS for approx-
imately 48 hr. Brains were then embedded in Tissue-Tek O.C.T. (Sakura) and for postnatal and adult time points, 30mm coronal
sections were collected in 24 multi-well dishes (Greiner Bio-one) and stored at�20�C in antifreeze solution (30% v/v ethyleneglycol,
30% v/v glycerol, 10% v/v 0.244M PO4 buffer) until use. For embryonic time points, 20mmcoronal sections were collected directly on
Superfrost glass slides (Thermo Fisher Scientific). For clonal analysis with P21 time points, coronal 45mm sections were collected
sequentially in individual wells of 24 multi-well dishes, and mounted onto glass slides while maintaining the order of sectioning.
Immunostaining of MADM-Labeled BrainsFor sections fixed to glass slides, tissue was first rehydrated by incubating for 15 min in PBS at room temperature. All following steps
apply to both tissue sections fixed to glass slides and floating sections in multi-well plates. Tissue sections were blocked for 45min in
a buffer solution containing 2% normal donkey serum (Life Technologies), 1% Triton X-100 in PBS. Primary antibodies were diluted
(see table) in blocking buffer and incubated overnight at 4�Con either a rocking table (floating sections) or a stationary platform (tissue
on slides). Sections were washed 4 times for 10 min each with PBS and incubated with corresponding secondary antibody diluted in
blocking buffer solution for 1 hr. Sections were washed 4 times with PBS, with the penultimate wash step containing the nuclear stain
DAPI (Invitrogen). For primary antibodies requiring antigen retrieval, tissue was first processed by incubating in sodium citrate buffer
(10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) at 80�C for 10 min. For primary antibodies requiring DNA denaturation, sections
were first treated with 2N HCl at 37�C for 30 min. Once cooled to room temperature, sections were washed with PBS and standard
immunostaining protocol as described above was carried out. Floating sections were mounted on Superfrost glass slides (Thermo
Fisher Scientific), dehydrated and embedded in mounting medium containing 1,4-diazabicyclooctane (DABCO; Roth) and Mowiol
4-88 (Roth).
Imaging and Analysis of Marker Expression in MADM-Labeled BrainsSections were imaged using either an inverted LSM800 or LSM880 with airy scan confocal microscope (Zeiss) and processed using
Zeiss Zen Blue software. Tiled images, encompassing the entire region of interest, were taken for a minimum of three brain sections
per animal. Confocal images were imported into Photoshop software (Adobe) and the boundaries for the region of interest were
traced. MADM-labeled cells were manually counted based on respective marker expression. Quantification of cell clusters lining
the V-SVZ was performed by first measuring the average cell area of control-MADM cells in the V-SVZ. Next, the number of cells
in Lgl1�/� mutant cell clusters was estimated by measuring their total area and dividing by the average cell area. These estimates
were confirmed by manually counting DAPI labeled nuclei. Quantification of BrdU and Caspase-3 labeling was performed using a
40x oil objective.
Neuron 94, 517–533.e1–e3, May 3, 2017 e2
Generation of MADM Clones in the NeocortexMADM clone induction was performed as described previously (Gao et al., 2014; Hippenmeyer et al., 2010). In brief, timed pregnant
females injected intraperitoneally with tamoxifen (TM) (Sigma) dissolved in corn oil (Sigma) at E11 or E12 at a dose of 2-3mg/pregnant
dam. Live embryos were recovered at E18–E19 through caesarean section, fostered, and raised for further analysis at P21. For
embryonic time point analysis, caesarean section and analysis was performed at either E13 or E16. Brains containing MADM clones
were isolated, and tissue sections processed as described above. 3D reconstruction was performed by using a custom MATLAB
script and IMARIS-based imaging analysis platform. Cortical areas were identified by using the Allen Brain Atlas (http://mouse.
brain-map.org/static/atlas).
Astrocyte Morphology Filament TracingThe Sholl method of concentric spheres was used tomeasure astrocyte branching complexity (Sholl, 1953). First, brain sectionswere
stained for GFAP, labeling the main processes of the majority of cortical astrocytes. Then individual GFP+ layer II/III astrocytes from
control-MADM and Lgl1-MADM mice were imaged using a 63x oil objective. 3D reconstruction and analysis was performed using
IMARIS software. Briefly, using the Filament Tracer algorithm, the GFAP labeled processes were 3D reconstructed, concentric
sphere Sholl analysis was performed, and total cell volume and cell body volume was calculated from the 3D structure.
QUANTIFICATION AND STATISTICAL ANALYSIS
See Table S2 for complete information regarding quantifications and statistics used in this study. This table includes all graphed
values, including SEMs, p values, and exact values of n. For all graphs except Figures S5L, S5N, and S8M, statistical analysis
was performed in Excel. Values represent mean ± SEM and significance was determined using the two-tailed unpaired Student’s
t test. n was defined as an individual section green/red ratio quantification. For Figures S5L, S5N, and S8M, statistical analysis
was performed in Graphpad Prism 7.0. For Figures S5L and S5N, multiple t tests were performed and discovery determined using
the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. Each rowwas analyzed individually, without
assuming a consistent SD n was defined as an individual reconstructed astrocyte. For Figure S8M, significance was determined
using One-way Anova and corrected with Tukey’s multiple comparisons test, with n being defined as one single section green/
red ratio quantification. By plotting the raw data we were able to determine that our data met the criteria of normal distribution. Sig-
nificance was established at *p < 0.05, **p < 0.01, ***p < 0.001.
e3 Neuron 94, 517–533.e1–e3, May 3, 2017