Article

A network of genetic repression and derepression specifies projection fates in the developing neocortex

Department of Biology, Stanford University, Stanford, CA 94305.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2012; 109(47). DOI: 10.1073/pnas.1216793109
Source: PubMed

ABSTRACT

Neurons within each layer in the mammalian cortex have stereotypic projections. Four genes-Fezf2, Ctip2, Tbr1, and Satb2-regulate these projection identities. These genes also interact with each other, and it is unclear how these interactions shape the final projection identity. Here we show, by generating double mutants of Fezf2, Ctip2, and Satb2, that cortical neurons deploy a complex genetic switch that uses mutual repression to produce subcortical or callosal projections. We discovered that Tbr1, EphA4, and Unc5H3 are critical downstream targets of Satb2 in callosal fate specification. This represents a unique role for Tbr1, implicated previously in specifying corticothalamic projections. We further show that Tbr1 expression is dually regulated by Satb2 and Ctip2 in layers 2-5. Finally, we show that Satb2 and Fezf2 regulate two disease-related genes, Auts2 (Autistic Susceptibility Gene2) and Bhlhb5 (mutated in Hereditary Spastic Paraplegia), providing a molecular handle to investigate circuit disorders in neurodevelopmental diseases.

Full-text

Available from: Susan K McConnell, Jan 14, 2015
A network of genetic repression and derepression
species projection fates in the developing neocortex
Karpagam Srinivasan
a,1
, Dino P. Leone
a
, Rosalie K. Bateson
a
, Gergana Dobreva
b
, Yoshinori Kohwi
c
,
Terumi Kohwi-Shigematsu
c
, Rudolf Grosschedl
b
, and Susan K. McConnell
a,2
a
Department of Biology, Stanford University, Stanford, CA 94305;
b
Max-Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany;
and
c
Lawrence Berkeley National Laboratory, Berkeley, CA 94720
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2011.
Contributed by Susan K. McConnell, September 28, 2012 (sent for review August 24, 2012)
Neurons within each layer in the mammalian cortex have stereotypic
projections. Four genesFezf2, Ctip2, Tbr1, and Satb2regulate
these projection identities. These genes also interact with each
other, and it is unclear how these interactions shape the nal pro-
jection identity. Here we show, by generating double mutants of
Fezf2, Ctip2, and Satb2, that cortical neurons deploy a complex
genetic switch that uses mutual repression to produce subcortical
or callosal projections. We discovered that Tbr1, EphA4, and Unc5H3
are critical downstream targets of Satb2 in callosal fate specica-
tion. This represents a unique role for Tbr1, implicated previously in
specifying corticothalamic projections. We further show that Tbr1
expression is dually regulated by Satb2 and Ctip2 in layers 25.
Finally, we show that Satb2 and Fezf2 regulate two disease-related
genes, Auts2 (Autistic Susceptibility Gene2) and Bhlhb5 (mutated in
Hereditary Spastic Paraplegia), providing a molecular handle to in-
vestigate circuit disorders in neurodevelopmental diseases.
cell fate
|
cerebral cortex
|
axon guidance
|
transcription factor
C
ortical neurons project to specic targets located locally
within the cortex or in distant subcortical regions, depending
on the neurons birthdate and laminar position. Neurons in layer
5 project subcortically to targets that include the spinal cord,
whereas neurons in layers 2/3 typically project corticocortically,
including across the corpus callosum (CC) (13). Four key
genesFezf2, Ctip2, Tbr1, and Satb2are involved in specifying
projection neuron identity (3 7). Fezf2 and Ctip2 regulate the
identities of layer 5 subcerebral projection neurons (SCPNs),
whereas Satb2 regulates the fate of callosally projecting neurons
(Fig. 1O). Without Fezf2, SCPNs up-regulate Satb2, switch fate,
and extend axons across the CC (8), suggesting that Fezf2
represses Satb2 and the acquisition of a callosal identity. Con-
versely, without Satb2, callosal neurons up-regulate Ctip2 (but
not Fezf2) and project subcortically (9, 10). Biochemical experi-
ments revealed that Satb2 binds to and represses the Ctip2 locus
(9) but have not ascertained whether the fate switch in Satb2
mutant neurons is a direct consequence of Ctip2 upregulation.
Layer 6 corticothalamic neurons and layer 5 SCPNs show
a similar mutual repression between Tbr1 (layer 6) and Fezf2
(layer 5) (37). Deep layer neurons in Tbr1
/
mice up-regulate
Fezf2 and project inappropriately to the pons, while Fezf2
/
neurons up-regulate Tbr1 and form thalamic projections (Fig.
1O). Thus, Fezf2 represses both Tbr1 and Satb2, which repress
corticothalamic and callosal fates in SCPNs. The nature of these
repressive interactions, however, is unknown. Here we analyze
mice bearing mutations in Fezf2, Ctip2, and Satb2 to examine the
genetic relationships between these pathways. Using in vivo
electroporation to rescue callosal axons in Satb2 mutants, we
identied downstream targets of Satb2 that play key roles in the
formation of callosal projections.
In this study, data obtained from double knockouts of Satb2,
Fezf2, and Ctip2 (together with previously published data) pro-
vide evidence for a genetic model of cortical projection neuron
fate determination during early corticogenesis (Fig. S1A). In Fezf2-
expressing neurons, expression of Tbr1 and Satb2 is repressed,
which results in a suppression of corticothalamic and callosal
fates, respectively, and axon extension along the corticospinal
tract (CST). Fezf2 mutant neurons fail to repress Satb2 and Tbr1,
thus their axons cross the CC and/or innervate the thalamus
inappropriately.
In callosal projection neurons, Satb2 represses the expression
of Ctip2 and Bhlhb5, leading to a repression of subcerebral fates.
Interestingly, we found that Satb2 promotes Tbr1 expression in
upper layer callosal neurons, and Tbr1 expression in these neu-
rons is required for callosal specication (not corticothalamic
specication). Our data suggest that the projection fates of deep
layer and upper layer cortical pyramidal neurons are specied by
complex and mutually inhibitory genetic interactions between
postmitotic determinants during late embryonic development.
Results
Visualization of Callosal and Subcortical Axonal Connections in Satb2
and Fezf2 Mutants.
We visualized callosal axons using mice in
which LacZ was targeted to the Satb2 locus (9). In embryonic day
(E) 18.5 control Satb2
LacZ/+
mice, β-galactosidase positive
(β-gal
+
) axons cross the CC and form corticocortical connections
(Fig. S2 B and O) but do not extend subcortically (Fig. S2 A and
O). As shown previously, β-gal
+
axons in E18.5 Satb2
LacZ/LacZ
mutants fail to cross the CC and instead descend subcortically
through the striatum and along the cerebral peduncle and to the
thalamus (Fig. S2 C, D, and P). Thus, without Satb2, callosal
neurons seem to be respecied as subcortical projection neurons
(9, 10). Satb2
LacZ/LacZ
mice die within a few hours after birth,
which precludes the analysis of axonal connectivity at postnatal
stages. Here we also use mice bearing a conditional allele of Satb2
(Satb2
ox/ox
, generated in the laboratory of R.G.) bred with mice
that express Cre recombinase under the Emx1 promoter (Emx1-
Cre) to produce viable, cortex-specic Satb2 knockouts. Mice
carrying Emx1-Cre also carried the Satb2
LacZ
allele, enabling us to
generate conditional Satb2
LacZ/ox
;Emx1-Cre knockout mice in
which the axons of neurons that normally express Satb2 neurons
are marked by β-gal (Fig. 1 B, DG). A detailed description of
Satb2 conditional knockout mice is in preparation.
The insertion of a placental alkaline phosphatase (PLAP) cas-
sette into the Fezf2 locus (11) enabled us to visualize the projections
of deep layer cells that normally express Fezf2.PLAP
+
axons in
Fezf2
PLAP/+
controls are observed in the pyramidal tract (Fig. 2 A
and E) and thalamus (Fig. S4I), whereas labeled axons are absent
from the CC, as previously described (8, 11). At P0, PLAP
+
axons
Author contributions: K.S. and S.K.M. designed research; K.S., D.P.L., and R.K.B. per-
formed research; K.S., D.P.L., and S.K.M. analyzed data; G.D., Y.K., T.K.-S., and R.G. con-
tributed new reagents/analytic tools; K.S., D.P.L., and S.K.M. wrote the paper; Y.K. and
T.K.-S. provided technical advice with ChIP experiments.
The authors declare no conict of interest.
1
Present address: Neurodegeneration Laboratories, Department of Neuroscience, Genen-
tech, South San Francisco, CA 94080.
2
To whom correspondence should be addressed. E-mail: suemcc@stanford.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1216793109/-/DCSupplemental.
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NEUROSCIENCE INAUGURAL ARTICLE
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descend subcerebrally and branch toward the thalamus (Fig. S3B)
andcerebralpeduncle(Fig. S3B) but do not extend past the pons.
At postnatal day (P) 4, Fezf2
PLAP/PLAP
mutants show a striking re-
duction in the number of PLAP
+
axons that extend into the pyra-
midal tract at the level of the hindbrain (Fig. 2 B and F). In addition,
asubsetofPLAP
+
axons cross the CC (Fig. 2 I and J), consistent
with the interpretation (Fig. S1B) that at least some neurons that
normally express Fezf2 acquire a callosal identity in the absence of
Fezf2, possibly owing to acquisition of Satb2 expression (8).
Interactions Between Fezf2 and Satb2 in the Regulation of Subcortical
Identity.
We tested the role of Satb2 in the respecication of deep
layer fates by generating Fezf2;Satb2 double mutants, predicting
that PLAP
+
axons should fail to extend across the CC. Indeed,
analysis of Fezf2
PLAP/PLAP
;Satb2
LacZ/LacZ
mice revealed a loss of
PLAP
+
axons in the CC at P0 (Fig. 2K), providing strong evi-
dence that the acquisition of a callosal fate of Fezf2 mutant
neurons is due to up-regulation of Satb2. Visualization of PLAP
+
axons in the sagittal plane showed axons projecting subcortically
through the striatum and exiting through two branches, one di-
rected toward the thalamus (Fig. S3 B and C) and the other into
the cerebral peduncle (Fig. S3 B and C), although the latter axons
fail to reach the hindbrain (Fig. S3B, asterisk). The β-gal
+
axons
of Satb2-expressing neurons in the same section follow a similar
tract, although they extend past the peduncle toward the pons
(Fig. S3 A and C) but fail to reach it.
This difference in the extent of subcortical innervation by
PLAP
+
vs. β-gal
+
axons suggested that loss of Satb2 might affect
the formation of subcerebral projections by layer 5 neurons,
which normally express Fezf2 but not Satb2. To ascertain
whether mice lacking both Fezf2 and Satb2 simply show a de-
velopmental delay in the growth of subcerebral projections, we
analyzed PLAP staining at P4 in Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;
Emx1-Cre double mutants. Whole mounts revealed a complete
loss of PLAP
+
axons in the pyramidal tract (Fig. 2D), whereas
littermates lacking only Fezf2 retain some PLAP staining of py-
ramidal tract axons (Fig. 2B).Thesedatasuggestthesurprising
possibility that Satb2 plays a role in the normal targeting of SCPNs.
If this hypothesis is correct, subcerebral axons in Satb2 mu-
tants s hould display defects in their projection pattern s. We
therefore examined the pattern of PLAP labeling in mice lacking
Satb2 and carrying one copy of the Fezf2
PLAP
allele. In Fezf2
PLAP/+
;
Satb2
LacZ/LacZ
mutants at E18, a thin tract of PLAP
+
axons extends
subcortically toward the cerebral peduncle but does not reach
LacZ
Ctip2
-/-
; Satb2
LacZ/Flox
;
Emx1-Cre
Ctip2
+/+
; Satb2
LacZ/Flox
;
Emx1-Cre
Ctip2
+/+
; Satb2
LacZ/Flox
CC
A B C
D
cp
Th
F
CC
I
H
Ctip2
-/-
;
Satb2
LacZ/+
CC
Ctip2
+/+
; Satb2
LacZ/Flox
; Emx1-Cre
Ctip2
-/-
; Satb2
LacZ/Flox
; Emx1-Cre
cp
K
PyT
G
pons
PyT
PyT
PyT
J
St
E
CP
Ctip2
-/-
;
Satb2
LacZ/LacZ
St
Th
N
L
pons
PyT
M
cp
O
Fezf2
Satb2 Tbr1
Ctip2
callosal subcerebral corticothalamic
Layer 5 Layer 6
wildtype
Layers 2-5
?
?
?
?
Fig.3
Fig.1
Fig.2
Fig.5,6
Fig.5,6
Fig. 1. Loss of Ctip2 partially rescues callosal projec-
tions in Satb2 mutant neurons. (AC) LacZ staining of
P0 whole-mount brains (ventral view) reveals aberrant
β-gal
+
axons (blue) in the pyramidal tract (arrow) in
Satb2
LacZ/ox
;Emx1-Cre mutants (B) but not in control
mice (A)orCtip2
/
;Satb2
LacZ/ox
;Emx1-Cre double
knockouts (C). (D)Themajorityofβ-gal
+
callosal axons
in Satb2 conditional mutants fail to project across the
CC at P0. (E and F) Instead, these axons project sub-
cerebrally into the cerebral peduncle (cp) and thala-
mus (Th). (G) A small number of β-gal
+
axons are seen
in the pyramidal tract (PyT). (H) Coronal sections of
Ctip2
/
;Satb2
LacZ/+
mice reveal β-gal
+
axons crossing
normally at the CC. (I and N) β-gal
+
callosal axons in
Satb2;Ctip2 double mutants are seen at the CC (arrow)
at P0. Defasciculated β-gal
+
axons also descend sub-
cortically into the striatum (J), along the cerebral pe-
duncle (K and L) but not to the level of the pyramidal
tract (M). (O) Model outlining genetic interactions
between Fezf2, Ctip2, Satb2,andTbr1 described pre-
viously during cortical projection neuron fate speci-
cation. Question marks denote interactions that are
not clearly understood. (Scale bar in D,200μm.)
CC
PLAP
Fezf2
PLAP/+
;
Satb2
LacZ/Flox
Fezf2
PLAP/PLAP
;
Satb2
LacZ/Flox
Fezf2
PLAP/PLAP
;
Satb2
LacZ/Flox
; Emx1-Cre
Fezf2
PLAP/+
;
Satb2
LacZ/Flox
; Emx1-Cre
Fezf2
PLAP/PLAP
AB CD
I
Fezf2
PLAP/PLAP
;
Satb2
LacZ/LacZ
CC
K
pd
PyT
E
PyT
F
pd
PyT
G
PyT
PyT
PyT PyT
J
CC
LacZ PLAP
PyT
H
Fig. 2. Loss of Satb2 does not rescue PLAP
+
axons
in Fezf2
PLAP/PLAP
mutants. (AD) Whole-mount PLAP
staining of Fezf2
PLAP/+
heterozygous mice (A)and
Satb2 conditional mutants (C) reveals that PLAP
+
axons extend into the pyramidal tract (PyT). (B)
Fezf2
PLAP/PLAP
mutants show a dramatic decrease
in PLAP staining in the pyramidal tract. (D)In
Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre double mu-
tants, there is a complete loss of PLAP staining in
the pyramidal tract. (EH) PLAP staining of sagittal
sections from the brains shown in AD. PLAP
+
axons
in heterozygous Fezf2
PLAP/+
controls (E)andSat-
b2
LacZ/ox
;Emx1-Cre mutants (G) project subcorti-
cally through the pyramidal tract (PyT) and
pyramidal decussation (pd). (H) PLAP
+
axons in
Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre double mu-
tants fail to enter the pyramidal tract. (Scale bar,
200 μm.) (I and J)InFezf2 mutants, PLAP
+
axons are
present in the CC in both sagittal (I) and coronal (J)
sections. (K) Both PLAP
+
(red) and β-gal
+
(green) axons
fail to cross the CC in Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-
Cre double mutants. (Scale bars, 200 μm.)
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the pyramidal tract (Fig. S4F). In conditional Satb2 mutants
(Fezf2
PLAP/+
;Satb2
ox/ox
;Emx1-Cre) at P4, PLAP
+
axons reach
past the pons to the pyramidal decussation (Fig. 2 C and G), but
the axons fail to enter the spinal cord (Fig. S4 G and H). To
determine whether this reects a failure of corticospinal in-
nervation or a developmental delay, we immunostained condi-
tional Satb2 knockouts at P21 (when PLAP can no longer
be detected in our animals) using antibodies against protein
kinase C γ (PKCγ), which labels the CST (12). These studies
revealed a complete loss of PKCγ staining in the spinal cord,
consistent with a loss of corticospinal connections.
These analyses of PLAP
+
axons in Satb2 mutants yield the
unexpected conclusion that Satb2 is required for the normal
growth of subcerebral axons to their targets in the spinal cord.
How might Satb2 exert this effect? One possibility is that Satb2
plays a non-cell autonomous role in subcerebral neurons de-
velopment. However, our prior studies have shown that Satb2
and Ctip2 are normally coexpressed in layer 5 neurons during
a brief period early in corticogenesis (E13.5E14) (9). Thus,
although Satb2 may be expressed only transiently in subcerebral
neurons, this window of expression may play a critical role in
regulating the expression of key genes such as axon guidance
molecules. For example, Satb2 mutants show reduced expression
of Unc5H3, a well-characterized axonal guidance receptor (9). In
Unc5H3 mutants, subcerebral axons fail to descend past the
decussation into the spinal cord (13), raising the possibility that
Satb2 knockouts display a similar phenotype due to reduced
expression of Unc5H3. An alternative possibility is that Satb2
may play a non-cell autonomous role in guiding subcerebral
axons to the spinal cord. For example, Satb2 is expressed in
interneurons and in the spinal cord, and it is possible that this
expression is required for corticospinal axon guidance. However,
in our analyses of conditional Satb2 mutants (in which expression
in interneurons is maintained), we observe the same phenotype
as the straight Satb2 knockouts. Therefore, it is unlikely that
Satb2 expression in interneurons is essential for guiding sub-
cerebral axons to the spinal cord.
Genetic Interactions Between Satb2 and Ctip2 in the Formation of
Subcerebral Projections.
Previous studies using Ctip2 knockout
mice suggest that Ctip2 is required for the normal formation of
subcerebral projections, which are defasciculated and terminate
prematurely in these mutants (1). Ctip2
/
mice do not carry
a reporter allele (1), therefore analysis of axonal projections in
these animals has been limited to using dye tracers. Because
Fezf2 expression is not altered in Ctip2
/
mutants (Fig. S4A)
and because Fezf2 and Ctip2 seem to be coexpressed in deep
layer neurons (8, 14), we used Fezf2
PLAP
as a marker for sub-
cerebral projections. As expected, PLAP
+
axons in Ctip2
+/
;
Fezf2
PLAP/+
heterozygous brains extend subcortically through the
striatum; some extend into the thalamus, whereas others are
present in the cerebral peduncle (Fig. S4I) and pyramidal tract
(Fig. S4J). In Ctip2
/
mutants carrying a Fezf2
PLAP
allele,
defasciculated PLAP
+
axons pass through the striatum and ex-
tend into the thalamus or cerebral peduncle (Fig. S4K) but do
not reach the pons (Fig. S4K, Inset). This pattern is similar to
that seen by dye-labeling subcerebral axons in Ctip2 mutants (1).
Our model of the genetic interactions that confer projection
neuron fates predicts that in the absence of both Ctip2 and Satb2
(Fig. S1F), PLAP
+
SCPNs should form projections similar to
those seen in Ctip2 single mutants (Fig. S1C). We used PLAP
staining to trace the projections of these neurons in Ctip2;Satb2
double mutants (Ctip2
/
;Fezf2
PLAP/+
;Satb2
LacZ/ox
;Emx1-Cre) (Fig.
S3 DF). PLAP
+
axons (in red) are absent from the CC and can
be seen subcortically at the level of cerebral peduncle (Fig. S3E).
PLAP
+
axons are absent from the thalamus in sagittal sections
(Fig. S3D, asterisk), consistent with the prediction (Fig. S1F) that
layer 5 neurons that lose both Ctip2 and Satb2 expression will
express only low levels of Tbr1. These neurons, however, con-
tinue to express Fezf2 and Bhlhb5, both of which impart a sub-
cortical identity to the neurons, as evidenced by PLAP
+
axons in
the cerebral peduncle. However, without Ctip2, these neurons
are unable project past the pons.
Loss of Satb2 Rescues Ctip2 Expression in Fezf2
PLAP/PLAP
Neurons.
Ctip2 expression is down-regulated in layer 5 of Fezf2 mutants
(11) (Fig. 3B), suggesting that Ctip2 may be a downstream tar-
get of Fezf2. However, because Fezf2 normally represses
Satb2 genetically (8), and Satb2, in turn, represses Ctip2 (9)
(Fig. 3C), it is possible that the loss of Ctip2 expression in
Fezf2
PLAP/PLAP
mutant neurons is an indirect consequence of the
up-regulation of Satb2, rather than the result of direct tran-
scriptional control of Ctip2 by Fezf2. If the regulation is indirect
via Satb2, we predict that Ctip2 expression will be restored in
layer 5 neurons in Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre double
mutants (Fig. S1E). Indeed, we do observe strong Ctip2 expression
in layer 5 neurons in double mutants (Fig. 3D), suggesting that
Fezf2 regulates Ctip2 indirectly by repressing Satb2.
We counted the number of Ctip2-expressing cells across the
cortical layers, rst in single knockouts for Fezf2 or Satb2.In
layer 6 (Fig. 3G), the number of Ctip2
+
cells increased from
29% ± 1.5% in control mice to 39% ± 1.8% (P < 0.006) in Fezf2
mutants and 47% ± 5.4% (P < 0.01) in Satb2 conditional
mutants. In layer 5, the total number of Ctip2
+
cells decreased in
Fezf2 mutants (57% ± 7% in controls vs. 10% ± 3% in mutants,
P < 0.001) but increased to 83% ± 4% in Satb2 mutants (P <
0.01) (Fig. 3F). This suggests that the loss of Ctip2 in layer 5 of
Fezf2 mutants might be due to the acquisition of Satb2 expression
Ctip2
A
D
Fezf2
PLAP/PLAP
;
Emx1-Cre
Satb2
LacZ/Flox
;
Control
Satb2
LacZ/Flox
;
Emx1-Cre
C
Fezf2
PLAP/PLAP
B
2-4
5
6
2-4
5
6
2-4
5
6
2-4
5
6
2-4
5
6
Fezf2
PLAP/PLAP
;
Satb2 ;
Emx1-Cre
control
Satb2 ;
Emx1-Cre
Fezf2
PLAP/PLAP
E
% layer 6 neurons
that are Ctip2+
% layer 5 neurons
that are Ctip2+
% upper layer neurons
that are Ctip2+
F
G
***
***
*
***
***
***
***
Fig. 3. Ctip2 expression is restored in layer 5 neu-
rons of Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre double
mutants. (A) In WT mice, the expression of Ctip2
protein is highest in layer 5 neurons, moderate in
layer 6, and largely absent from the upper layers
(24). (B) Fezf2 mutants show a severe reduction of
Ctip2 expression in layer 5, whereas expression in
layer 6 is increased. (C) Satb2 mutant mice show
increased Ctip2 expression in layer 5, 6, and the
upper layers. (D) Ctip2 expression is rescued in layer
5ofFezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre double
mutants. (AD) Each panel is a composite of tiled
confocal images to form the full gure. (Scale bar,
50 μm.) (EG) Graphs depict the percentages of
Ctip2
+
neurons in the upper layers (E), layer 5 (F),
and layer 6 (G) in various genotypes. Error bars are
average ± SEM (n = 3 different animals). Condence
levels were calculated using a t test (*P < 0.5; **P <
0.05; ***P < 0.005, relative to control s).
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(Fig. S4C) (8). Satb2 also represses Ctip2 expression in the upper
layers, as evidenced by the dramatic increase in Ctip2
+
cells from
11% ± 1.7% in controls to 77% ± 9.5% (P < 0.05) in Satb2
mutants (Fig. 3E).
To ascertain whether Ctip2 is regulated directly by Fezf2 or
indirectly by Satb2, we counted the numbers of Ctip2
+
cells in
Satb2
LacZ/ox
;Fezf2
PLAP/PLAP
;Emx1-Cre double mutants. The loss
of Ctip2 expression in layer 5 neurons of Fezf2
PLAP/PLAP
mutants
(10% ± 3%) is rescued in double mutants (65% ± 1.8%; P <
0.0005) (Fig. 3F), providing strong evidence that at this stage in
development, Fezf2 regulates Ctip2 indirectly by repressing Satb2.
Satb2 Regulates Bhlhb5 Expression. Knowing that Fezf2 regulates
Ctip2 indirectly by repressing Satb2, we asked whether other
genes important for SCPN identity, such as Bhlhb5, are regu-
lated similarly. Bhlhb5 is expressed by Ctip2
+
neurons in layer 5
as well as in neurons in layers 2/3 (12). Bhlhb5 functions as an
area-specic transcription factor that regulates the postmitotic
acquisition of area-specic identities. In caudal motor cortex,
Bhlhb5 null mice exhibit anomalous differentiation of cortico-
spinal motor neurons, accompanied by failure of CST formation
(12). The axons stop at the base of the pons and fail to enter the
pyramidal tract (similar to Ctip2
/
mutant axons at P0).
To ascertain whether Fezf2 regulates the expression of
Bhlhb5, we immunostained Fezf2
PLAP/PLAP
brains for Bhlhb5
protein. The number of Bhlhb5
+
cells is signicantly reduced in
layer 5 of Fezf2 mutants (Fig. 4C) relative to controls (Fig. 4A)
(64% ± 3% in controls to 12% ± 4.5% in mutants; P < 0.005). If
this is due to the expansion of Satb2 expression in Fezf2 mutants
(Fig. S4C), Bhlhb5 expression should be rescued in layer 5
neurons that lack Satb2. Indeed, the number of Bhlhb5
+
cells in
layer 5 in P4 mice is restored to 49% ± 3.9% in double mutants
(Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre) (Fig. 4 D and H; P <
0.001 compared with Fezf2 single mutants). These data suggest
that Fezf2 regulates Bhlhb5 expression in layer 5 indirectly by
repressing Satb2, and that Satb2 functions as a repressor of
Bhlhb5. Consistent with the latter interpretation, upper layer
neurons of Satb2 mutant mice exhibited a robust increase in both
the number of Bhlhb5
+
neurons in layers 24 (Fig. 4G; control:
28% ± 7.8%; Satb2
LacZ/ox
;Emx1-Cre conditional mutants, 63% ±
2.4%, P < 0.005) (Fig. 4B). Because Bhlhb5 is normally expressed
by upper layer neurons, this regulation by Satb2 is unlikely to
represent an on/off switch, but rather a mechanism that regulates
the timing and levels of Bhlhb5 expression. Indeed, Bhlhb5 ex-
pression seems unchanged in the upper layers of Satb2 mutants
at E18; the change is evident only by P4. This suggests that Satb2
likely regulates the timing of down-regulation of Bhlhb5 in upper
layer neurons rather than an on/off switch for Bhlhb5 expression
itself. It is also possible that Bhlhb5 is regulated by other factors
expressed by upper layer neurons and that the nal expression
pattern reects the action of multiple regulatory pathways.
Finally, we found that normal Bhlhb5 expression in some deep
layer neurons also requires Ctip2. The percentage of Bhlhb5
+
neurons in layer 6 is increased signicantly in Ctip2
/
mice (Fig.
4 E and I; control: 9% ± 4%, mutants: 70% ± 4.5%; P < 0.0005).
However, in contrast to the severe reduction of Bhlhb5
+
neurons
in layer 5 of Fezf2 mutants, there is no signicant change in the
percentage of layer 5 neurons that express Bhlhb5 in Ctip2
mutants. Because Satb2 expression seems to be unaltered in the
absence of Ctip2, these data are consistent with the interpre-
tation that Satb2 is a primary regulator of Bhlhb5 in layer 5.
To determine whether high levels of Bhlhb5 expression are
sufcient to direct axons toward subcortical targets, we coelec-
troporated expression constructs encoding Bhlhb5 and GFP into
WT embyros at E15.5, a time when upper layer neurons are
being generated. As expected, GFP labeled neurons are present
in the upper layers in control (GFP only) (Fig. 4J) and Bhlhb5
plus GFP (Fig. 4M) electroporated brains. At P4, in control
brains, GFP-labeled axons were conned to the ipsilateral and
contralateral cerebral cortices (Fig. 4J), and no GFP
+
axons can
be observed subcerebrally in the striatum (Fig. 4K) or at the level
of the cerebral peduncle. In these brains, GFP
+
axons can be
seen at the CC (Fig. 4L), indicating that as expected, electro-
porated neurons from upper layers may be sending the axons
across the midline to the contralateral hemisphere. In contrast,
the GFP
+
axons of Bhlhb5-electroporated neurons extend be-
yond the striatal boundary into the internal capsule (Fig. 4N) and
toward the cerebral peduncle (Fig. 4P, arrow). We do observe
some GFP
+
axons in the CC (Fig. 4O), which is probably be-
cause not all GFP
+
cells express bhlhb5. These results suggest
G
Fezf2
PLAP/PLAP
Fezf2
PLAP/PLAP
;
Satb2
LacZ/Flox
;
Emx1-Cre
Control
Ctip2
-/-
;
Satb2
LacZ/+
Satb2
LacZ/Flox
;
Emx1-Cre
D
I
H
Bhlhb5
% upper layer neurons
that are Bhlhb5+
Fezf2
PLAP/PLAP
;
Satb2
;
Emx1-Cre
control
Ctip2
-/-
Satb2 ;
Emx1-Cre
Fezf2
PLAP/PLAP
% layer 5 neurons
that are Bhlhb5+
% layer 6 neurons
that are Bhlhb5+
Ctip2
-/-
;
Satb2
LacZ/LacZ
***
***
***
*
*
*
*
***
AB C E F
*
*
*
pCA-GFP pCA-GFP + pCA-Bhlhb5
GFP
Str
Str
cortex
cortex
cc
cc
J
K
L
M
N
O
P
cp
Str
2-4
2-4
2-4
2-4
2-4
2-4
5
5
5
5
5
5
6
6
66
6
6
Fig. 4. Satb2 regulates Bhlhb5 expression in a layer-
specicmanner.(A) In controls, Bhlhb5 protein is
expressed at high levels in layer 5 neurons and at low to
moderate levels in the upper layers. (B)InSatb2
LacZ/ox
;
Emx1-Cre mutants, Bhlhb5 expression is sign icantly
reduced in layer 5. (C)Expressioninbothlayers5and
6 is decreased in the Fezf2 mutant cortex. (D) Bhlhb5
expression in layer 5 is rescued in Fezf2
PLAP/PLAP
;
Satb2
LacZ/ox
;Emx1-Cre double mutants. (E and F)In
Ctip2
/
mutants (E)andCtip 2
/
;Satb2
LacZ/LacZ
double
mutants (F), expression is increased in both layer 5 and
the upper layers. (AF) Each panel is a composite of tiled
confocal images to form the full gure. (Scale bar, 50
μm.) (GI) Graphs depict the percentages of Bhlhb5
+
neurons in different genotypes in different cortical
layers. Error bars depict average ± SEM (n = 3 different
animals). Condence levels were calculated using a t test
(*P < 0.5; **P < 0.05; ***P < 0.005, relative to controls).
(JP) Ectopic overexpression of Bhlhb5 in upper layer
neurons of WT animals can redirect their axons sub-
cerebrally. (JL) Sagittal sections of P4 control brains
electroporated with GFP at E15 (when upper layer
neurons are born) show (J)GFP
+
neurons in the upper
layers; (K)noGFP
+
axons in the ipsilateral striatum (Str);
and (L)GFP
+
axons in the CC. (MP)Sagittalsectionsof
P4 brains electroporated with Bhlhb5 and GFP at E15
show (M)GFP
+
neurons in the upper layers; and (N)ro-
bust GFP
+
tracts extending through the striatum (Str)
and (P) toward the cerebral peduncle (cp, arrows). (O)
GFP
+
axons are also observed in the CC. (Scale bar, 50 μm.)
19074
|
www.pnas.org/cgi/doi/10.1073/pnas.1216793109 Srinivasan et al.
Page 4
that Bhlhb5 by itself has a limited but real potential to direct axons
to subcortical targets, less robust than that of Ctip2 (8) and similar
to that observed after Sox5 electroporation at E15.5 (15, 16).
Fezf2, Ctip2, and Satb2 Regulate Tbr1 Expression in a Layer-Specic
Manner.
Fezf2 and Tbr1 play important antagonistic roles in the
regulation of layer 6 axons to the thalamus. Tbr1 mutants show
a loss of corticothalamic projections, whereas Fezf2 mutants
show a striking increase in thalamic innervation, most likely due
to increased expression of Tbr1 (5, 7). In controls (Fig. 5A), the
highest level of Tbr1 expression is observed in layer 6, with lower
levels in layer 5 and scattered cells labeled in the upper layers.
Consistent with previously published reports (5, 7), we found that
the number of Tbr1
+
neurons increases signicantly in layer 5
and upper layer neurons of Fezf2 mutants (Fig. 5 E, G, and H)
(control layer 5: 22% ± 4.7%, mutant layer 5: 54% ± 8.9% P <
0.05; control upper layers: 23% ± 7%, mutant upper layers: 43%
± 3%; P < 0.05) but is not signicantly changed in layer 6 (Fig.
5I). Fezf2 mutants also show a robust band of PLAP bers en-
tering (Fig. 6F) and innervating the anterior thalamus (Fig. 6M),
which might be a consequence of increased Tbr1 expression in
layer 5. Interestingly, Ctip2
/
mutants also show a signicant
increase in the number of Tbr1
+
layer 5 neurons (most obviously
in layer 5b) (Fig. 5 C and H; mutants: 54% ± 1.79%, P < 0.002),
along with increased PLAP labeling in the thalamus (Fig. 6 J and
O), suggesting that Ctip2 represses Tbr1. Because Ctip2 is ge-
netically downstream of Fezf2 and because the increase in Tbr1
expression is similar in Fezf2 and Ctip2 mutants, it is possible that
Fezf2 regulates Tbr1 expression indirectly via Ctip2.
The genetic relationship between Satb2 and Ctip2 led us to
predict that Tbr1 expression should decrease in Satb2 mutants,
owing to the up-regulation of Ctip2. The number of Tbr1
+
neurons in layer 5 of Satb2 mutants is signicantly decreased
(Fig. 5 B and H;3%± 1.2%, P < 0.004) relative to controls, and
PLAP innervation of the thalamus is reduced in Satb2 mutants
(Fig. 6 D and L). Interestingly, in Fezf2
PLAP/PLAP
;Satb2
LacZ/LacZ
double knockouts, we observe PLAP
+
axons in the thalamus,
although the extent of innervation qualitatively seems to be less
than in Fezf2 mutants but more than in Sabt2 mutants (Fig. 6 G,
H, and N). Indeed, Tbr1 expression in layer 5 neurons of double
knockouts is signicantly increased compared with Satb2 mutants
(Satb2
LacZ/LacZ
:3%± 1.2%; Fezf2
PLAP/PLAP
;Satb2
LacZ/LacZ
double mutants: 12% ± 1.2%; P < 0.02). Satb2 mutants also
show a nearly complete loss of Tbr1
+
neurons in the upper layers
(Fig. 5G; control: 23% ± 7.2%, mutant: 4% ± 0.3%; P < 0.01).
There is a small but not signicant change in layer 6, where Satb2
expression is relatively low (Fig. 5I; control: 63% ± 7.8%, mu-
tant: 49% ± 5.4%; P < 0.1). Taken together, these data provide
evidence that Tbr1 is regulated negatively by Ctip2 (and Fezf2).
Counting the number of Tbr1
+
neurons in layer 5 of Ctip2
/
;
Satb2
LacZ/ox
;Emx1-Cre double knockouts yielded a surprising
result. Although there are more Tbr1
+
cells in layer 5 of the
double knockouts compared with Satb2 single mutants (Fig. 5H;
8% ± 1.8% in double mutants and 3% ± 1.2% in Satb2 mutants,
P < 0.005), the numbers are neither restored to WT levels
(22% ± 4.8%) nor to that of Ctip2 single mutants (54% ± 1.8%),
suggesting that Ctip2 is not the sole regulator of Tbr1 expression
and that Satb2 might positively regulate Tbr1.Moreover,Ctip2
/
;
Satb2
LacZ/ox
;Emx1-Cre double knockouts fail to show a restora-
tion of Tbr1 expression in the upper layers (Fig. 5 D and G),
further providing evidence that Satb2 is required for the expres-
sion of Tbr1 in these neurons (independent of Ctip2). These
studies suggest the possibility that Satb2 might bind directly to the
Tbr1 genomic locus and promote its expression.
Satb2 Binds Directly to the Tbr1 Genomic Locus. To determine
whether Satb2 can bind directly to the Tbr1 locus, we identied
three so-called MAR sites (Matrix Attachment Sequences) in the
Tbr1 locus that have the potential to serve as Satb2 binding sites
(Fig. 6P). To ascertain whether Satb2 can bind to any of these
sites, we performed ChIP using an antibody against Satb2 (raised
in the laboratory of Y.K. and T.K-S.) and chromatin from the
cortices of WT mice at P0. As a positive control, we tested
previously published sequences in the Ctip2 genomic locus for
binding (Fig. 6Q) (9). As a negative control, we tested genomic
sequences in a known gene desert region, which should show
minimal or no interaction with Satb2. As expected, the negative
control gene desert region showed minimal or no enrichment for
Satb2 binding, whereas MAR sequences from the Ctip2 locus
showed greater than sevenfold enrichment (when normalized to
values observed in the gene desert region) (Fig. 6Q). We then
used three primers specic to each MAR region in the Tbr1
genomic locus and asked whether these showed enrichment for
Satb2 binding. All three sequences resulted in binding levels
similar to that for the published Ctip2 sites. These data suggest
that Satb2 can directly bind to the Tbr1 locus, thereby positively
regulating the expression of Tbr1.
Regulation of Callosal Projections in Satb2 Mutant Neurons. Until
this point we have focused on the genetic interactions that reg-
ulate the formation of subcerebral and subcortical connections
by deep layer neurons. Here we consider the interactions that
Tbr1 Ctip2
DAPI
AB
C
D
E
F
Tbr1
Bhlhb5
D
API
Ctip2
+/+
; Satb2
LacZ/Flox
Satb2
LacZ/LacZ
; Emx1-Cre
Ctip2
-/-
; Satb2
LacZ/Flox
; Emx1-CreCtip2
-/-
Fezf2
PLAP/PLAP
Fezf2
PLAP/PLAP
; Satb2
LacZ/Flox
; Emx1-Cre
I
G
H
% layer 6 neurons
that are Tbr1+
% layer 5 neurons
that are Tbr1+
% upper layer neuron
s
that are Tbr1+
Ctip2
-/-
;
Satb2
;
Emx1-Cre
Fezf2
PLAP/PLAP
;
Satb2
;
Emx1-Cre
control
Ctip2
-/-
Satb2 ;
Emx1-Cre
Fezf2
PLAP/PLAP
**
**
***
*** ***
**
***
***
***
***
**
*
*
**
Fig. 5. Satb2 and Ctip2 regulate Tbr1 expression in
a layer specic manner. (AD) Immunostaining for
Tbr1 (green) and Bhlhb5 (red) in P4 control cortex
(A) reveals heavy staining in layer 6 and moderate
staining in layer 5. A small fraction of cells in the
upper layers are also Tbr1
+
.(B) Satb2 conditional
knockouts show reduced Tbr1 in layers 6 and 5, and
a loss of Tbr1 in the upper layers. Bhlhb5 expression
is increased in the upper layers. (C)InCtip2 mutants,
Tbr1 staining increases in layers 6 and 5. (D) Ctip2
/
;
Satb2
LacZ/LacZ
double knockouts at P0 show reduced
Tbr1 expression in layer 5 and increased Bhlhb5 in
the upper layers. (E and F) Immunostaining for Tbr1
(green) and Ctip2 (red) reveals an increase in Tbr1
and a decrease in Ctip2 expression in layer 5 of
Fezf2
PLAP/PLAP
mutants. Ctip2 (red) but not Tbr1
(green) expression is restored to control levels in
layer 5 of Fezf2
PLAP/PLAP
;Satb2
LacZ/ox
;Emx1-Cre dou-
ble knockouts (F). (AF) Each panel is a composite of
tiled confocal images to form the full gure. (Scale
bar, 100 μm.) (GI) Graphs depicting the percentage
of Tbr1+ neurons in different layers of the geno-
types under study. Error bars are average ± SEM (n = 3 different animals). Condence levels were calculated using a t test (*P < 0.5; **P < 0.05; ***P < 0.005,
relative to controls).
Srinivasan et al. PNAS
|
November 20, 2012
|
vol. 109
|
no. 47
|
19075
NEUROSCIENCE INAUGURAL ARTICLE
Page 5
mediate corticocortical connectivity as exemplied by the for-
mation of axons that cross the CC, which we visualized using the
Satb2
LacZ
allele (9).
Satb2 mutants exhibit a failure of callosal development and the
extension of β-gal
+
axons to subcerebral targets, accompanied by
a marked up-regulation of Ctip2 staining throughout the cortex
(9, 10), whereas Fezf2 expression is unaltered. The ectopic ex-
pression of either Ctip2 or Fezf2 in WT upper layer neurons can
divert their projections toward subcortical targets (8, 9), sug-
gesting that the redirection of β-gal
+
axons from callosal to
subcerebral targets in Satb2 mutants may be due to the up-reg-
ulation of Ctip2. We therefore set out to explore the genetic
interactions between Satb2 and Ctip2 in the formation of cortical
connectivity. If the upregulation of Ctip2 is responsible for the
alteration in axonal projections, then the loss of Ctip2 in a Satb2
mutant background should result in one of two possible out-
comes: (i) the callosal projections of Satb2 mutant neurons will
be rescued and β-gal
+
axons will again cross the midline, or (ii)
a novel default fate (neither callosal nor subcortical) might
be revealed.
In Fezf2 and Ctip2 single mutants [Fezf2
PLAP/PLAP
;Satb2
LacZ/+
(Fig. S2F)orCtip2
/
;Satb2
LacZ/ox
(Fig. 1H and Fig. S2H)],
β-gal
+
axons from Satb2-expressing neurons cross the CC. As
expected, in Fezf2
PLAP/PLAP
;Satb2
LacZ/LacZ
double knockouts,
β-gal
+
axons are absent from the CC and instead extend sub-
cortically to the thalamus (Fig. S2K) and cerebral peduncle (Fig.
S2L), similar to Satb2 single mutants (Fig. S2 C and D). These
data conrm that Fezf2 is not responsible for the fate change in
Satb2 mutant neurons.
LacZ staining of P0 brains from Satb2 conditional mutants
reveals that β-gal
+
axons enter the pyramidal tract (Fig. 1 B, F,
and G), as expected. In contrast, β-gal
+
axons are absent from
the pyramidal tract of Ctip2
/
;Satb2
LacZ/ox
;Emx1-Cre double
mutants (Fig. 1C), similar to control brains (Fig. 1A). Further,
β-gal
+
axons cross the CC (Fig. 1I) in these double mutants.
These results suggest that the acquisition of Ctip2 expression by
neurons that lack Satb2 is responsible, at least in part, for their
failure to form callosal connections and the extension of β-gal
+
axons to subcerebral targets (Fig. S1D). Interestingly, in Ctip2;
Satb2 double mutants, many β-gal
+
neurons still continue to
project subcortically to the striatum, cerebral peduncle, or thal-
amus (Fig. 1 J, K, and L), although none reach the pyramidal tract
(Fig. 1 L and M). Thus it seems that, without both Satb2 and
Ctip2, neurons that normally express Satb2 may project callosally
(owing to loss of Ctip2) or subcortically to the peduncle.
Overexpression of Tbr1 Can Rescue Callosal Projections in Satb2
Mutants.
Tbr1 plays an important role in specifying the cortico-
thalamic projections of layer 6 neurons (5, 7, 17), but upper layer
neurons that normally express Tbr1 do not project to the thala-
mus. This suggests that Tbr1 might have an unexplored role in
the upper layers. Indeed, in Tbr1
/
mutant cortices, the axons of
upper layer neurons fail to cross the CC and instead terminate in
Probst bundles at the midline (17). Interestingly, restoring ex-
pression of Tbr1 in the upper layers of Tbr1
/
mutants did not
direct those axons to the thalamus (7); instead they projected
across the CC, consistent with the possibility that Tbr1 in up-
per layer neurons plays a role in callosal connectivity. We
therefore asked whether we could rescue the callosal defects in
Satb2
LacZ/LacZ
mutants by reintroducing Tbr1 into the upper
layers. Co-electroporation of expression constructs encoding
Tbr1 and GFP into E15.5 Satb2
LacZ/LacZ
mutants does rescue the
formation of callosal projections: β-gal
+
axons cross the CC in all
10 electroporated mutant embryos (Fig. 7 C and D), suggesting
that expression of Tbr1 can compensate for the loss of Satb2.
This denes a new function for Tbr1 and places Tbr1 genetically
downstream of Satb2 in callosal neuron specication. Thus, in
the developing cortex, Tbr1 has a dual function, rst specifying
layer 6 corticothalamic neurons and later in callosal connectivity.
Loss of Tbr1 Expression in Satb2 Mutants Coincides with a Loss of
Auts2.
Tbr1 binds to and directly activates the expression of
Auts2, a gene expressed in frontal cortex that has been linked to
autism and mental retardation (18). The functional signicance
of this relationship has been puzzling because Tbr1 regulates
corticothalamic identity, and there have been no reports of
defects in corticothalamic tracts in autistic patients. However,
imaging studies in patients with autism have revealed defects in
callosal tracts (2, 1926). A recent publication highlights a decit
in long-range connections (such as callosal connections) and
excess of short-range cortical connections in patients with autism
spectrum disorder (27).
Auts2, like Tbr1, is expressed in both the deep and upper
layers of the cortex (Fig. 7 K and L). Because Tbr1 regulates
Fezf2
PLAP/+
; Ctip2
-/-
Fezf2
PLAP/+
Satb2
LacZ/LacZ
Fezf2
PLAP/PLAP
Fezf2
PLAP/+
Fezf2
PLAP/PLAP
;
Satb2
LacZ/LacZ
Posterior Anterior
PLAP
EGI
AC
Th
Th
Th
Th
Th
DF HJB
Ex 1
MAR1
61,642,766
61,650,496
61,637,121
61,637,821
61,658,946
61,659,331
61,661,791
61,662,166
MAR2 MAR3
Ex 6
Potential MAR sequences in Tbr1 genomic region
82 46
Ctip2 (A4)
Tbr1_MAR1
Tbr1_MAR3
Tbr1_MAR2
{
{
0
Ratio Enrichment (MAR seq / gene desert)
;
K
L
MN
O
P
Q
PLAP
Th
Th
Th
Th
Fig. 6. Innervation of thalamus by PLAP
+
axons
correlates with Tbr1 expression. (A, B, and K) PLAP
+
control brains revealed robust PLAP staining in the
striatum (st) and thalamus (Th). (C, D, and L) Satb2
mutants show a reduction in PLAP
+
axons traversing
the striatum but almost no expression in the thal-
amus. (E, F, and M) Fezf2 mutants show robust
PLAP staining in the striatum, anterior commissure
(AC), and thalamus. (G, H, and N)InFezf2
PLAP/PLAP
;
Satb2
LacZ/ox
;Emx1-Cre double mutants, PLAP stain-
ing is reduced in the striatum and thalamus but
remains robust in the anterior commissure. (I, J, and
O) Robust PLAP staining is observed in the striatum
and thalamus of Ctip2
/
mutants. (AJ)PLAPin-
nervation using colorimetric subst rate for alkaline
phosphatase. (KO) Immunostaining using an anti-
PLAP antibody to visualize PLAP
+
tracts. (Scale bar,
200 μm.) All images are composites of tiled confocal
images to show the entire section. (P) Genomic map
representation of three MAR (matrix attachment
regions) sequences identied in the Tbr1 genomic
locus. ( Q) ChIP analysis of chromatin from P0 WT
neonatal cortex immunoprecipitated with an anti-
body to Satb2 using multiple primer sets targeting
the Tbr1 MAR regions. These experiments show
comparable levels of enri chment relative to Ctip2 A5 MAR [Ctip2 42/44 primer set (3)] region. Primers were normalized to a gene desert region. Each bar
represents enrichment of the region using a distinct primer set designed to that region. Each primer set was tested at least three times in three inde-
pendent samples.
19076
|
www.pnas.org/cgi/doi/10.1073/pnas.1216793109 Srinivasan et al.
Page 6
Auts2, we investigated whether the loss of Tbr1 expression in
upper layer neurons in Satb2
LacZ/LacZ
mutants coincides with
changes in the expression of Auts2. We observed a striking loss
of Auts2 expression in the upper layers of Satb2 mutants (Fig. 7
M and N), similar to the loss of Tbr1 in Satb2 mutants (Fig. 5B);
there was no change in Auts2 expression in layers 5 or 6 (Fig. 7 M
and N) relative to controls (Fig. 7 K and L). These data are
consistent with the possibility that Satb2 regulates the expression
of Tbr1, which in turn is required for Auts2 expression in callosal
projection neurons. These results may have implications for the
etiology of autism.
Expression of EphA4 and Unc5H3 Restores Callosal Projections in
Satb2 Mutants.
Previously we identi ed several genes that show
altered expression in Satb2
LacZ/LacZ
mutants (9). In particular,
three axonal guidance molecules (EphA4, PlxnA4, and Unc5H3)
are down-regulated in upper layers of mice lacking Satb2. Prior
studies have implicated Ephs and ephrins in callosal develop-
ment (13, 28, 29). EphA4 is normally expressed in upper layer
callosal neurons and the glial wedge (28). In Satb2 mutants,
EphA4 expression is lost in cortical neurons, but expression in
the glial wedge is maintained (9). Unc5H3 mutants have no
reported callosal deciencies (13), but mice lacking Netrin, a li-
gand for Unc5H3, lack both the CC and the anterior commissure
(29, 30). To test the hypothesis that one or more of these genes
is required for the proper guidance of callosal axons to their
destinations, we attempted to rescue the formation of callosal
projections in Satb2 mutants by reintroducing the expression of
individual axon guidance molecules into upper layer neurons. In
utero electroporation of PlxnA4 failed to rescue callosal pro-
jections (Fig. 7 I and J)inSatb2
LacZ/LacZ
mutants, but electro-
poration of EphA4 (Fig. 7 E and F)orUnc5H3 (Fig. 7 G and H)
resulted in the extension of β-gal
+
axons across the CC. These
results suggest that EphA4 and Unc5H3 are critical downstream
targets of Satb2 in callosal fate specication.
Discussion
Our data suggest that cortical projection neurons actively repress
alternate fates to promote appropriate fate choices during de-
velopment (Fig. 7O). When specic repressive interactions are
removed, alternative fates are executed (Fig. S1). Whereas Tbr1,
Ctip2, and Satb2 are expressed in postmitotic neurons, Fezf2 is
expressed in cycling cortical progenitors from very early stages of
corticogenesis (11). Loss of Fezf2 is critical for the specication
of the subcerebral projections of layer 5 neurons, as evidenced by
the loss of corticospinal projections in Fezf2 mutants (11, 14).
During the earliest stages of corticogenesis, Sox5 expression in
subplate and layer 6 neurons represses the expression of Fezf2
(and consequently that of Ctip2) (15, 16). This likely promotes
the expression of Tbr1 in layer 6 neurons. Tbr1 binds to and
represses the Fezf2 genomic locus (5, 7), thereby suppressing a
subcerebral fate and promoting the formation of corticothalamic
projections from layer 6. Sox5 expression is down-regulated early
in layer 5 neurons (15, 16), leading to a derepression of Fezf2,
which consequently leads to a repression of Satb2. The effect of
this is threefold. First, there are no intracellular triggers to
promote a callosal fate. Second, the absence of Satb2-mediated
repression of Ctip2 and Bhlhb5 leads to the continued expression
of these genes and the extension of subcortical axons. (Inter-
estingly, although Bhlhb5 expression in Satb2 mutants at E18
does not differ from that in controls, expression at P4 is signi-
cantly increased in Satb2 mutants. This suggests that Satb2 is
required for the normal down-regulation of Bhlhb5 expression
during early prenatal development, and that the sustained ex-
pression of Bhlhb5 in Satb2 mutants might be important in ex-
ecuting or maintaining the new subcerebral fate of these
neurons.) Third, our data indicate that the absence of Satb2-
mediated activation of Tbr1 suppresses corticothalamic projec-
tions. Thus, Fezf2-expressing layer 5 neurons extend their axons
subcerebrally.
We hypothesize that during production of the upper layers,
the absence of Fezf2 in cortical progenitors allows their daugh-
ters to express Satb2, which in turn promotes a callosal identity
(in part, surprisingly, by activating Tbr1
in upper layer neurons).
Simultaneously, activation of Satb2 results in the repression
of Ctip2 and Bhlhb5, ensuring that executors of subcortical
identity remain inactive in callosal neurons. Thus, each phase of
corticogenesis and neuronal fate specication deploys an active
repression of previous fates and a promotion of the appropriate
layer-specic projection fate (see SI Discussion for further details).
Tbr1 seems to play distinct roles at different stages of cortical
development. At early stages, Tbr1 promotes a frontal identity
while suppressing caudal identity (4). During the formation of
layer 6, Tbr1 plays an essential role in specifying the fates and
projection patterns of corticothalamic neurons (4, 5, 7, 17). In-
terestingly, although corticothalamic projections are decreased
in Tbr1 mutants and increased in Fezf2 mutants (which show an
up-regulation of Tbr1), these alterations in projections are in-
complete: there are still some corticothalamic axons in Tbr1
mutants, and Fezf2 mutant neurons do not completely convert to
LacZ
Satb2
Lacz/LacZ
(GFP) Satb2
Lacz/LacZ
(TBR1) Satb2
Lacz/LacZ
(EphA4) Satb2
Lacz/+
(UNC5H3)
Satb2
Lacz/LacZ
(PlxnA4)
AC E GI
CC
CC
CC
CC CC
BD F HJ
AUTS2 Ctip2
Satb2
LacZ/+
Satb2
LacZ/LacZ
AUTS2 AUTS2
2-4
5
6
1
MNKL
Fezf2
Satb2
Tbr1
Ctip2
callosal subcerebral corticothalamic
Layer 5 Layer 6
Tbr1
wildtype
Layers 2-5
O
Fig. 7. Tbr1, EphA4, and Unc5h3 are downstream
targets of Satb2 and can direct callosal projections.
β-Gal+ axons fail to cross the CC in Satb2
LacZ/LacZ
mutants electroporated with either a GFP control
construct (A and B)oraPlxnA4 expression construct
together with GFP (I and J). β-Gal
+
axons cross the
CC in Satb2 mutants electroporated with TBR1-IRES-
GFP (C and D), EphA4 and GFP (E and F), or Unc5H3
and GFP (G and H). E and F are composit es of tiled
images to form the full gure. (Scale bar, 100 μm.)
(KN) Auts2 expression is lost in the upper layers of
Satb2 mutants. (K and L) Auts2 protein (green in K,
white in L) is expressed in both the upper and deep
layers of control Satb2
LacZ/+
brains at P0. Ctip2 ex-
pression is shown in red. (M and N) Auts2 expression
is maintained in layer 6 of Satb2 mutants but is down-
regulated in the upper layers. (Scale bar, 50 μm.) (O)
Model of genetic interactions between Fezf2, Ctip2,
Satb2,andTbr1 in wild type cortex. Fezf2 expression
in layer 5 neurons represses Tbr1 and Satb2, which
repress corticothalamic and callosa l fates, respec-
tively. The repression of Satb2 enables expression of
Ctip2 and Bhlhb5, which are required for the spec-
ication and execution of a subcerebral identity. In
Satb2 expressing neurons, Ctip2 is repressed, leading to a repression of subcerebral identity and acquisition of callosal and corticocortical axonal projections.
Srinivasan et al. PNAS
|
November 20, 2012
|
vol. 109
|
no. 47
|
19077
NEUROSCIENCE INAUGURAL ARTICLE
Page 7
corticothalamic identity, suggesting that Tbr1 cannot be the sole
specier of a corticothalamic fate.
In addition, we found that Satb2 and Ctip2 dynamically regu-
late the expression of Tbr1 in a layer dependent manner. Satb2
seems to promote expression of Tbr1, whereas Ctip2 represses
Tbr1 expression. This complex bidirectional regulation might
explain why we observe a partial rescue of callosal axon targeting
in Ctip2;Satb2 double mutants (Fig. S1F). In these animals, Tbr1
expression in layer 5 neurons is partially rescued (relative to
Satb2 single mutants) and might account for the partial rescue of
callosal projections in these animals. In the upper layers, Tbr1
seems to play a unique role in promoting the formation of cal-
losal projections, which may explain why Tbr1 mutants display
callosal agenesis (17). This role for Tbr1 in callosal development
is also intriguing in the context of autism. Autistic patients seem
to have callosal abnormalities (1926), and in this context it is
relevant that Auts2, a gene associated with autism, is regulated by
Tbr1, which in turn is regulated by Satb2, a callosal fate speci-
cation gene. Recent studies suggest that a mutation in CAv1/2
(an L-type calcium channel) that is associated with Timothy
syndrome, a form of autism, results in lower numbers of Satb2-
expressing cells and an increase in Ctip2-expressing cells (31).
The authors suggest that the reduction in callosal neurons in
Timothy syndrome is consistent with the emerging view that
autism spectrum disorders arise from defects in connectivity
between cortical areas (31).
The role of axon guidance molecules, specically Ephrins and
their receptors, in directing callosal neurons has been studied
extensively using genetic knockouts. Although the loss of single
genes does not result in dramatic callosal abnormalities, double
knockouts of various genotypes do compromise callosal devel-
opment, suggesting that there is redundancy built into the sys-
tem. Indeed, it is interesting to note that although restroring
EphA4 expression in Satb2 mutants rescued callosal projections,
EphA4 knockout mice fail to display callosal defects (28). However,
multiple EphB receptors (B1, B2, and B3) and ligands (eph-
rinB1, B2, and B3) are expressed in callosal bers and midline
guidepost cells, respectively, and it is likely that their functions
are redundant. Restoring expression of Unc5h3 also rescued
callosal projections in Satb2 mutants. Unc5H3 is normally ex-
pressed throughout the cortical plate during development, and
no callosal defects have been observed in Unc5h3
rcm
null mice,
although these mice display subcortical defects in CST devel-
opment (13). Our ndings suggest an unexpected role for this
receptor in callosal development.
Methods
Animals. Satb2
LacZ/+
, Fezf2
PLAP/+
, and Ctip2
+/-
animals were mated to gener-
ate appropriate double knockouts. Details of generatio n of the conditional
Satb2 single and double mutants are described in SI Methods.
Immunohistochemistry. Standard immunohistochemistry techniques were
used. Details of antibodies used in the study are listed in Table S1. PLAP
staining was performed as described previously (11).
In Utero Electroporation: Methods are as described in previous work (8).
ChIP. Experiments were performed as described earlier (9) using a rabbit anti-
Satb2 antibody. MAR sequence predictions were based on www.genomatix.
de. For primer sequences, refer to Table S2.
ACKNOWLEDGMENTS. We thank Bin Chen and William McKenna (University
of California, Santa Cruz) for sharing reagents; Geetu Tuteja, Lee Shoa Long
Clarke, and Bruce Schaar (Stanford University) for help and advice with ChIP
experiments; Jeffrey Macklis (Harvard University) for Ctip2 mutant mice;
Avraham Yaron (Weizmann Institute) for EphA4 and PlxnA4 express ion con-
structs; and Robert Nechanitzky and Thomas Manke (laboratory of R.G.) for
helping with in silico analyses of Satb2 binding sites. This study was funded
by National Institutes of Health Grants EY08411 (to S.K.M.) and K99
MH086720 (to K.S.).
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www.pnas.org/cgi/doi/10.1073/pnas.1216793109 Srinivasan et al.
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    • "TBR1 is highly expressed in deep layers of the cerebral cortex, which have been suggested to participate in ASD pathology (Willsey et al. 2013). TBR1 target genes include multiple other autism risk factors (Chuang et al. 2015), and in particular TBR1 activates AUTS2 (Bedogni et al. 2010a; Srinivasan et al. 2012). Pathogenic de novo truncating and missense mutations disrupted multiple aspects of TBR1 function, including subcellular localization, interactions with co-regulators and transcriptional repression (Deriziotis et al. 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: Autism Spectrum Disorders (ASD) encompass a group of neurodevelopmental diseases that demonstrate strong heritability, however the inheritance is not simple and many genes have been associated with these disorders. ASD is regarded as a neurodevelopmental disorder, and abnormalities at different developmental stages are part of the disease etiology. This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration. This article is protected by copyright. All rights reserved.
    No preview · Article · Oct 2015 · Journal of Neurochemistry
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    • "The molecular logic underlying this DL neuron fate bias of progenitors was again uncovered through Foxg1 downstream transcriptome analysis. Of the layer transcription factors, Tbr1, which is expressed in the majority of early-born neurons (Hevner et al., 2001) and establish the corticothalamic projection neuron identity within the layer-subtype transcriptional network (Han et al., 2011; McKenna et al., 2011; Srinivasan et al., 2012), exhibited a significant downregulated response to Foxg1 induction. A reporter assay revealed that this repression was mediated through a 4-kb Tbr1 promoter region consisting of multiple conserved Foxg1 binding sequences. "
    [Show abstract] [Hide abstract] ABSTRACT: Information processing in the cerebral cortex requires the activation of diverse neurons across layers and columns, which are established through the coordinated production of distinct neuronal subtypes and their placement along the three-dimensional axis. Over recent years, our knowledge of the regulatory mechanisms of the specification and integration of neuronal subtypes in the cerebral cortex has progressed rapidly. In this review, we address how the unique cytoarchitecture of the neocortex is established from a limited number of progenitors featuring neuronal identity transitions during development. We further illuminate the molecular mechanisms of the subtype-specific integration of these neurons into the cerebral cortex along the radial and tangential axis, and we discuss these key features to exemplify how neocortical circuit formation accomplishes economical connectivity while maintaining plasticity and evolvability to adapt to environmental changes.
    Full-text · Article · Aug 2015 · Frontiers in Neuroscience
    • "The increased number of Bcl11b-positive neurons might be caused by compensatory upregulation of this closely related gene. Upregulation of Bcl11b could be responsible for Tbr1 repression in the deep cortical layers of the Bcl11a mutants; such a mechanism was previously observed in Satb2 mutants that show upregulation of Bcl11b as well (Srinivasan et al., 2012). At P2, we did not detect changes in the distribution of deep-layer neurons within the CP. "
    [Show abstract] [Hide abstract] ABSTRACT: During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Jul 2015 · Neuron
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