Ascl1 Participates in Cajal--Retzius Cell Development in the

Cerebral Cortex November 2011;21:2599--2611

doi:10.1093/cercor/bhr046

Advance Access publication April 5, 2011

Ascl1 Participates in Cajal--Retzius Cell Development in the Neocortex

Rajiv Dixit1, Celine Zimmer2,3, Ronald R. Waclaw4,5, Pierre Mattar1,6, Tarek Shaker1, Christopher Kovach1, Cairine Logan1,

Kenneth Campbell4, Francxois Guillemot2 and Carol Schuurmans1

1Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada T2N

4N12Division of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

3Current address: Institut de biologie du developpement de Marseille Luminy/Unite Mixte de Recherche 6216, 13009 Marseille,

France4Current address: Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati

College of Medicine, Cincinnati, OH 45229, USA5Current address: Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center,

University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA6Current address: Cellular Neurobiology Research Unit, Institut de recherches cliniques de Montreal, Montreal, Quebec, Canada

H2W 1R7

Rajiv Dixit, Celine Zimmer, and Ronald R. Waclaw have contributed equally to this work

Address correspondence to Carol Schuurmans, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.

Email: [email protected].

Cajal--Retzius cells are essential pioneer neurons that guideneuronal migration in the developing neocortex. During develop-ment, Cajal--Retzius cells arise from distinct progenitor domainsthat line the margins of the dorsal telencephalon, or pallium. Here,we show that the proneural gene Ascl1 is expressed in Cajal--Retzius cell progenitors in the pallial septum, ventral pallium, andcortical hem. Using a short-term lineage trace, we demonstrate thatit is primarily the Ascl1-expressing progenitors in the pallial septumand ventral pallium that differentiate into Cajal--Retzius cells.Accordingly, we found a small, albeit significant reduction in thenumber of Reelin1 and Trp731 Cajal--Retzius cells in the Ascl12/2

neocortex. Conversely, using a gain-of-function approach, we foundthat Ascl1 induces the expression of both Reelin, a Cajal--Retziusmarker, and Tbr1, a marker of pallial-derived neurons, in a subset ofearly-stage pallial progenitors, an activity that declines overdevelopmental time. Taken together, our data indicate that theproneural gene Ascl1 is required and sufficient to promote thedifferentiation of a subset of Cajal--Retzius neurons during earlyneocortical development. Notably, this is the first study that reportsa function for Ascl1 in the pallium, as this gene is best known for itsrole in specifying subpallial neuronal identities.

Keywords: basic-helix-loop-helix, dorsal telencephalon, pallium, proneuralgene, transcription factor

Introduction

The neocortex is comprised of 6 neuronal layers that are

generated in a sequential inside-out manner during develop-

ment. The laminar pattern of the neocortex is established in

part by Cajal--Retzius cells, which are essential pioneer neurons

that guide neuronal migration during development. One of the

critical molecules secreted by Cajal--Retzius cells is the

extracellular protein Reelin, the mutation of which results in

a disruption of neuronal layering (Caviness 1982; Morante-Oria

et al. 2003; Hammond et al. 2006; Pla et al. 2006; Yabut et al.

2007; Wagener et al. 2010). Notably, Reelin has also been

implicated in regulating the tangential and subsequent radial

migration of late-born cortical interneurons (Morante-Oria

et al. 2003; Hammond et al. 2006; Yabut et al. 2007), dendrite

formation (Yabut et al. 2007; Chameau et al. 2009), synapto-

genesis (Borrell et al. 1999), and cortical column formation

(Nishikawa et al. 2002; Janusonis et al. 2004), although some of

these roles remain controversial (Pla et al. 2006; Wagener et al.

2010).

Lineage tracing and birthdating experiments have demon-

strated that Cajal--Retzius cells differentiate between embry-

onic day (E) 10 and E13.5 in mouse, originating at focal sites in

the margins of the dorsal telencephalon (i.e., pallium) (Wood

et al. 1992; Hevner et al. 2003; Takiguchi-Hayashi et al. 2004;

Muzio and Mallamaci 2005). The sites of Cajal--Retzius cell

genesis include the ventral pallium, pallial septum, cortical

hem, presumptive choroid plexus, and rostral telencephalon

(Takiguchi-Hayashi et al. 2004; Bielle et al. 2005; Yoshida et al.

2006; Imayoshi et al. 2008; Zimmer et al. 2010). According to

their pallial sites of origin, Cajal--Retzius cells express pan-

cortical-specific markers (e.g., the T-box transcription factor

Tbr1) and have a glutamatergic neurotransmitter phenotype

(del Rio et al. 1995; Gorski et al. 2002; Hevner et al. 2003).

However, Cajal--Retzius cells are also molecularly heteroge-

neous, as highlighted by their regional-specific marker expres-

sion. For example, Trp73 is expressed in Cajal--Retzius cells

derived from the pallial septum and cortical hem, while Er81/

Etv1 is specific to rostral telencephalic derivatives (Meyer et al.

2002; Takiguchi-Hayashi et al. 2004; Bielle et al. 2005; Zimmer

et al. 2010). The subtype specificity of Cajal--Retzius cells is

thought to be functionally significant, allowing these cells to

participate in patterning the embryonic neocortex through

their regional-specific secretion of distinct morphogens

(Borello and Pierani 2010; Griveau et al. 2010).

The heterogeneity of Cajal--Retzius cells arises in part due to

the involvement of distinct signaling molecules patterning each

progenitor domain. For instance, Fgf8 signaling is implicated in

specifying an Er81/Etv1+Cajal--Retzius cell identity in the

rostral pallium (Zimmer et al. 2010), while Tgfb signaling is

involved in the cortical hem (Siegenthaler and Miller 2008).

Several transcription factors that control Cajal--Retzius cell

development have also been identified. Negative regulators

include the homeodomain transcription factors Pax6

(Stoykova et al. 2003) and Lhx2 (Bulchand et al. 2001; Monuki

et al. 2001), the winged helix factor Foxg1 (Hanashima et al.

2004; Muzio and Mallamaci 2005; Hanashima et al. 2007), the

basic-helix-loop-helix (bHLH) proteins Hes1, Hes3, and Hes5

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(Imayoshi et al. 2008), and the nuclear receptor CoupTFI

(Studer et al. 2005). While some of these factors appear to

directly suppress Cajal--Retzius cell generation (e.g., CoupTFI),

others may act indirectly as they are required to pattern the

pallial signaling domains from which Cajal--Retzius cells arise.

Examples of indirect factors are Foxg1 (Hanashima et al. 2004;

Muzio and Mallamaci 2005; Hanashima et al. 2007) and Lhx2

(Bulchand et al. 2001; Monuki et al. 2001), both of which are

required to prevent expansion of the Wnt-rich cortical hem

into neocortical domains. In contrast, Hes1/3/5 are required

to promote a non-neural choroid plexus fate at the expense

of a neural (i.e., Cajal--Retzius cell generating) cortical hem

identity (Imayoshi et al. 2008).

Transcription factors that act in a positive fashion to

promote Cajal--Retzius cell development include the T-box

factor Tbr1 (Hevner et al. 2001) and the LIM homeodomain

transcription factor Lhx5 (Miquelajauregui et al. 2010), as well

as Trp73 (transformation related protein 73), all of which are

expressed postmitotically and act as differentiation factors. In

addition, the homeodomain genes Emx1 and Emx2 are

required to establish the Wnt-rich cortical hem domain and

thus likely act indirectly to promote cortical hem--derived

Cajal--Retzius cell differentiation (Mallamaci et al. 2000;

Shinozaki et al. 2002; Muzio and Mallamaci 2005; von Frowein

et al. 2006). The proneural bHLH gene Neurogenin (Neurog) 2

is also required and partially sufficient to specify a Cajal--Retzius

cell fate (Imayoshi et al. 2008). Nevertheless, some Cajal--

Retzius cells are generated in the absence of Neurog2 function

(Imayoshi et al. 2008), suggesting that additional proneural

genes may be involved in specifying the identity of these

pioneer neurons.

The proneural bHLH gene achaete-scute-complex-homolog-

like-1 (Ascl1) has a well-established role in ventral telence-

phalic (i.e., subpallial) progenitors, where it is robustly

expressed and is required and sufficient to specify a ventral,

GABAergic neuronal identity (Casarosa et al. 1999; Horton et al.

1999; Britz et al. 2006; Wang et al. 2009). However, Ascl1 is also

expressed at lower levels in pallial progenitors (Britz et al.

2006), where its function(s) remain poorly defined. Here, we

show that Ascl1 is expressed in a subset of Cajal--Retzius cell

progenitors in the cortical hem, pallial septum, and ventral

pallium. Furthermore, using loss-of-function analyses, we show

that Ascl1 is indeed required for the differentiation of a subset

of Cajal--Retzius cells. Finally, using a gain-of-function approach,

we demonstrate that at early stages of Cajal--Retzius cell

development (E10.5), Ascl1 can promote the expression of

Tbr1, a pallial-specific marker (Hevner et al. 2001), and Reelin,

a Cajal--Retzius cell marker, in a subset of pallial progenitors, an

activity that declines over developmental time.

Materials and Methods

Animals and GenotypingTo generate staged embryos, timed pregnant matings were set up, with

the morning of the vaginal plug considered E0.5. The Ascl1 and

Neurog2GFPKI mutant alleles were maintained on a CD1 outbred

background (Charles River). Genotyping of Ascl1 mutant and wild-type

alleles (Casarosa et al. 1999), the Ascl1:GFP transgene (Gong et al.

2003), and Neurog2 wild-type and Neurog2GFPKI mutant alleles (Britz

et al. 2006) were performed using polymerase chain reaction as

previously described. Timed pregnant CD1 females were also used for

all electroporation experiments.

Tissue Processing and RNA In Situ HybridizationEmbryos were dissected at the specified embryonic stages in ice-cold

phosphate-buffered saline (PBS, pH 7.5). Embryos were transferred to

4% paraformaldehyde (PFA)/13 PBS and fixed overnight at 4 �C.Embryos were then washed 3 3 10 min in 13 PBS and transferred to

20% sucrose/13 PBS overnight at 4 �C. Fixed embryos were then

embedded and frozen in Tissue Tek OCT (Sakura Finetek USA, Inc.)

prior to cryosectioning at 10 lm and mounting on Superfrost plus

slides (Fisher Scientific). RNA in situ hybridization was performed as

previously described (Alam et al. 2005), using digoxygenin (dig)-

labeled riboprobes that were generated with a 10X dig-labeling mix

according to the manufacturer’s instructions (Roche Diagnostics

GmbH). Linearized templates and corresponding riboprobes were

generated with the following restriction enzyme/polymerase combi-

nations: Ascl1 (XbaI/Sp6), Reln (EcoR I/T3), Trp73 (Sal I/T3), and

Wnt3a (SacI/Sp6).

ImmunostainingEmbryos were processed as described above. Immunostaining was

performed on 10 lm cryostat sections. Cryosections were blocked

either in 10% normal goat or donkey serum/13 PBS/0.1% Triton X-100

(PBT) or in 3% bovine serum albumin/13 TBST (Tris-buffered saline:

25 mM Tris--HCl, pH 7.4/0.14 M NaCl/0.1% Triton X-100). Primary

antibodies were incubated on sections for 2 h at room temperature or

overnight at 4 �C in blocking solution. Sections were washed 3 3 10

min in PBT or TBST and then incubated with secondary antibodies

for 1 h at room temperature. Sections were then washed 3 3 10 min

in PBT or TBST and counterstained 5 min with 4,6-diamidino-2-

phenylindole (DAPI, 1/10 000, Sigma). Finally, sections were washed

3 3 10 min in PBS or TBST and mounted in AquaPolymount

(Polysciences, Inc.). Some immunolabeling was processed using

secondary antibodies coupled to horseradish peroxidase and a Dia-

minobenzidine substrate, according to the manufacturer’s instruc-

tions (VectaStain ABC kit, Vector Laboratories). Primary antibodies

included: rabbit anti-calretinin (1/2000; Swant), mouse anti-Ascl1 (1/

200; BD Biosciences), rabbit anti-Neurog2 (1/20) (gift of David

Anderson), rabbit anti-Neurog2 (1/500; gift of Masato Nakafuku),

rabbit anti-GFP (1/500; Chemicon), sheep anti-GFP (1/750; Bio-

genesis), rabbit anti-Tbr1 (1/3000; Chemicon), mouse anti-Reelin (1/

500; Chemicon), rabbit anti-pan-Dlx antibody (1/500; gift from Grace

Panganiban), rabbit anti-pan-Otx antibody (1/500; Baas et al. 2000),

rabbit anti-calbindin D-28k (1:1000; Swant), and mouse-anti-Trp73 (1/

200; Lab Vision). Secondary antibodies included goat or donkey anti-

mouse or anti-rabbit antibodies conjugated to Alexa488 (1/500;

Invitrogen Canada Inc.), Cy3 or Cy5 (1/500; Jackson Immunore-

search).

In Utero Electroporation and Embryo CultureIn utero electroporation was performed as previously described (Saito

and Nakatsuji 2001; Langevin et al. 2007). Expression vectors were

generated by subcloning Ascl1 into pCIG2, which contains a chicken

b-actin/Cytomegalovirus promoter/enhancer and an IRES2-EGFP

cassette for visualization of transfected cells (Britz et al. 2006; Mattar

et al. 2008). DNA was purified using a Qiagen Endotoxin free kit

(Qiagen). An electric current (7 pulses of 40--50 mV) was applied

across the uterine wall surrounding injected embryos with 5 mm

platinum tweezer-style electrodes (Protech) and a BTX square wave

electroporator (VWR, CanLab). The uterus was then returned to the

abdominal cavity and embryos were allowed to develop to appropriate

developmental stages. Electroporation and culture of E10.5 embryos

were performed as previously described (Hand et al. 2005; Friocourt

et al. 2008; Gohlke et al. 2008).

Imaging, Cell Counts, and StatisticsImaging was carried out on a Leica TCS 4D confocal microscope or

a Leica DMRXA2 microscope using OpenLab software (Improvision).

For lineage analyses, 2 confocal photographs (212 3 212 lm; stacks of

3 lm) per area per embryo (n = 2) were analyzed. For the

electroporation data: 2--3 confocal photographs (212 3 212 lm;

stacks of 3 lm) per area per embryo (n = 4) were analyzed. For

counting of RNA in situ hybridization signal, 3 digital images were

2600 Ascl1 Function in Cajal--Retzius Cells d Dixit et al.Downloaded from https://academic.oup.com/cercor/article-abstract/21/11/2599/277473by gueston 29 March 2018

captured for each embryo (n = 3 per genotype). Error bars reflect

standard error of the mean. Student’s 2-tailed t-tests were performed

with P values denoted as follows: <0.05*, <0.01**, <0.005***.

Results

Ascl1 Is Expressed in Cajal--Retzius Cell ProgenitorDomains in the Pallium

Ascl1 is a proneural gene that is expressed at high levels in

subpallial progenitors, where it is required and sufficient to

specify a GABAergic interneuron identity (Casarosa et al. 1999;

Horton et al. 1999; Britz et al. 2006; Wang et al. 2009).

However, Ascl1 is also expressed at lower levels in pallial

progenitors (Britz et al. 2006), where its role has yet to be

elucidated. To determine if Ascl1 is expressed in pallial

territories that give rise to Cajal--Retzius cells, we first

performed RNA in situ hybridization between E10.5 and

E12.5, when the bulk of Cajal--Retzius cells differentiate (Wood

et al. 1992; Hevner et al. 2003; Takiguchi-Hayashi et al. 2004).

Cajal--Retzius cells are derived from progenitor domains that

line the pallial margins, including the pallial septum, ventral

pallium, and cortical hem (schematic; Fig. 1A,B). In E10.5

coronal sections through the rostral telencephalon, Ascl1

transcripts were detected at high levels in the lateral ganglionic

eminence (lge) of the subpallium and at lower levels in

scattered pallial progenitors, including in the pallial septum and

ventral pallium (Fig. 1C,C#). In more caudal sections, Ascl1

transcripts were detected in the lge, as well as the ventral

pallium and cortical hem, which serve as lateral and caudome-

dial signaling centers, respectively, and are major sources of

Cajal--Retzius cells (Fig. 1D--D$). Similar expression profiles

were apparent at E11.5 (Fig. 1E,E#,F--F$) and E12.5 (Fig.

1G,G#,H--H$), with high Ascl1 transcript levels detected in

subpallial progenitors and lower Ascl1 expression levels

throughout the pallial ventricular zone (vz), including in the

pallial septum, ventral pallium, and cortical hem. To confirm

that Ascl1 transcripts were translated into protein in pallial

progenitors, we also performed immunolabeling at E10.5 (Fig.

2A--L and Supplementary Fig. S1B--B#), E11.5 (Supplementary

Fig. S1C--C#), and E12.5 (Fig. 1I,I#,J--J$ J$ and Supplementary

Fig. S1D--D#). At all stages, Ascl1 protein was detected at low

levels in pallial progenitors compared with its robust expres-

sion in the vz of the subpallium. Within the pallium, Ascl1

protein was detected at higher levels in the cortical hem and at

lower levels in the ventral pallium and pallial septum (Fig. 1I,J,J$).To confirm that Ascl1 was indeed expressed in progenitors

that had a pallial identity, rather than in subpallial progenitors

that had migrated tangentially into the dorsal telencephalon,

we performed coimmunolabeling with Ascl1 and a series of

pallial markers. Focusing on the E10.5 ventral pallium, we

observed that the vast majority of Ascl1+progenitors coex-

pressed Pax6, a paired homeodomain transcription factor that

shows a bias toward higher expression levels in progenitors on

the pallial side of the pallial--subpallial border (Fig. 2A--D).

Similarly, we found that in the E10.5 pallium, 38.2 ± 4.1% Ascl1+

progenitors coexpressed Neurog2 (n = 2; Fig. 2E--H), a proneu-

ral bHLH transcription factor that is exclusively expressed

on the pallial side of the boundary and which is required for

the generation of a subset of Cajal--Retzius neurons (Imayoshi

et al. 2008). Notably, proneural proteins are expressed in a ‘‘salt-

and-pepper’’ fashion, and only 10.2 ± 1.2% of E10.5 pallial

progenitors (labeled by DAPI) expressed Ascl1 (n = 4) while

6.4 ± 0.8% expressed Neurog2 (n = 2; Fig. 2E--H). We also noted

temporal and spatial differences in Ascl1 and Neurog2 ex-

pression in pallial progenitors. Ascl1 was detected at higher

levels than Neurog2 within the E10.5 pallial septum, while at

later stages (E11.5 and E12.5), Ascl1 was expressed at lower

levels than Neurog2 in the rostromedial pallium (Supplemen-

tary Fig. S1).

Ascl1 and Neurog2 are therefore expressed in distinct tem-

poral and spatial patterns in Cajal--Retzius cell progenitor do-

mains, suggesting they may have nonoverlapping roles in

regulating the differentiation of these pioneer neurons.

Ascl1 Is Expressed in Cajal--Retzius Cell Lineages ofPallial Origin

To better assess the extent to which Ascl1 is expressed in

Neurog2+pallial lineages, we performed short-term lineage

tracing, taking advantage of the perdurance of the GFP reporter

in Neurog2GFPKI/+ embryos (Fig. 2I--L). An analysis of GFP and

Ascl1 coexpression revealed that 41.2% (n = 2 embryos, 438

progenitors) of GFP+pallial progenitors coexpressed Ascl1 in

E10.5 Neurog2GFPKI/+ cortices. Conversely, 66.2% of Mash1

+

progenitors and 58.8% of GFP+progenitors were single positive

in E10.5 Neurog2GFPKI/+ cortices (Fig. 2I--L). These results

suggest either that: 1) Ascl1 is expressed in both Neurog2-

expressing and non-expressing pallial lineages (Nieto et al.

2001) and/or 2) Ascl1 precedes the onset of Neurog2

expression, or persists after GFP is degraded, in a common,

Neurog2-expressing pallial lineage. Regardless of the interpre-

tation, we can conclude that pallial lineages are spatially and/or

temporally heterogeneous with respect to Ascl1 and Neurog2

expression.

We next asked if pallial progenitors expressing Ascl1 indeed

give rise to Cajal--Retzius cells by performing short-term lineage

tracing with Ascl1:GFP BAC transgenic embryos (Parras et al.

2007). We quantified the number of GFP+cells that coex-

pressed Reelin, a pan-Cajal--Retzius cell marker (Schiffmann

et al. 1997; Alcantara et al. 1998), and Trp73, which labels

Cajal--Retzius cells derived from the pallial septum and cortical

hem (Meyer et al. 2002, 2004). GFP-labeled Cajal--Retzius cells

were separately quantified in the ventral pallium/piriform

cortex in lateral domains, the pallial septum in the rostral

midline, and the cortical hem in the caudal midline (as shown

in schematic; Fig. 3A). In E11.5 Ascl1:GFP cortices, a significant

proportion of Reelin+and Trp73

+cells coexpressed GFP in the

ventral pallium/piriform cortex (34 ± 1.5% GFP+Reelin

+/Reel-

in+; 10 ± 0.2% GFP

+Trp73

+/Trp73

+; n = 2; Fig. 3B--D) and pal-

lial septum (29 ± 2.6% GFP+Reelin

+/Reelin

+; 38 ± 7.1%

GFP+Trp73

+/Trp73

+; n = 2; Fig. 3B#,C#,D), while many fewer

Cajal--Retzius cells in the cortical hem were GFP+(2 ± 0.6%

GFP+Reelin

+/Reelin

+; 1 ± 1.7% GFP

+Trp73

+/Trp73

+; n = 2; Fig.

3B$,C$,D).Reelin is also expressed in some cortical interneurons,

although it is not thought to be expressed in these cells until

late embryonic stages (Alcantara et al. 1998). However, to

confirm that the GFP+Reelin

+cells in the E11.5 Ascl1

+lineage

trace were indeed Cajal--Retzius neurons and not Reelin+

interneurons, we performed triple immunolabeling with

the cortical-specific neuronal marker Tbr1 (Hevner et al.

2001). The presence of GFP+Reelin

+Tbr1

+cells in the cortical

marginal zone of E11.5 Ascl1:GFP embryos confirmed that

Cerebral Cortex November 2011, V 21 N 11 2601Downloaded from https://academic.oup.com/cercor/article-abstract/21/11/2599/277473by gueston 29 March 2018

Supplementary Fig. S4







Figure 1. Ascl1 is expressed in Cajal--Retzius cell progenitor domains. (A,B) Schematic representation of a coronal section through the E11.5 telencephalon showing the majorCajal--Retzius cell progenitor domains (green; boxed areas), including the ventral pallium (A,B), pallial septum (at rostral levels; A), and cortical hem (at caudal levels; B). (C--H)Distribution of Ascl1 transcripts in coronal sections through the E10.5 (C,C#,D--D#D$), E11.5 (E,E#,F--F#F$), and E12.5 (G,G#,H--H#H$) telencephalon, focusing on rostral(C,C#,E,E#,G,G#) and medial (D--D#D$,F--F#F$,H--H#H$) levels. Black arrowheads mark the pallial--subpallial border, which is demarcated by the transition from low to high Ascl1transcript levels. Boxed areas in C,E,G are in the pallial septum, which are magnified in C#,E#,G#, respectively. Boxed areas in D,F,H are in the ventral pallium and cortical hem,which are magnified in D#,F#,H# and D#D$,F#F$,H#H$, respectively. (I,J) Ascl1 protein expression in coronal sections through the E12.5 telencephalon at rostral (I,I#) and caudal(J-J$) levels. Arrowheads mark the pallial--subpallial border. The boxed area in I is in the pallial septum, which is magnified in I#, and the boxed areas in J are in the ventralpallium and cortical hem, which are magnified in J#,J$, respectively. vp, ventral pallium; ps, pallial septum; ch, cortical hem; mge, medial ganglionic eminence.


Ascl1-expressing progenitors give rise to Cajal--Retzius cells

with a pallial-specific regional identity (Fig. 3E--H).

In E11.5 Ascl1:GFP transgenics, GFP was detected on both

sides of the pallial/subpallial border (Fig. 3B--C), consistent

with the expression of Ascl1 in both pallial and subpallial

progenitors (Fig. 1C--J). This raised the remote possibility that

GFP+Reelin

+and GFP

+Trp73

+cells in E11.5 Ascl1:GFP trans-

genics may have a subpallial origin, an interpretation that

would go against the prevailing view that Cajal--Retzius cells are

derived from pallial progenitors (Wood et al. 1992; Hevner et al.

2003; Takiguchi-Hayashi et al. 2004; Bielle et al. 2005; Muzio

and Mallamaci 2005). Nevertheless, we set out to provide

additional support for the pallial origin of Cajal--Retzius cells,

particularly those in the ventral pallium/piriform cortex, which

overlie the subpallium. Short-term lineage tracing in E11.5

Neurog2GFPKI/+ embryos revealed that the vast majority (if not

all) Reelin+(Fig. 3I--I$,K,L--L$) and Trp73

+(Fig. 3J,J$) Cajal--

Retzius cells were GFP+in the ventral pallium/piriform cortex,

pallial septum, and cortical hem. Furthermore, a significant

proportion of Reelin+cells underlying the basal telencephalon

(Fig. 3K,M--M$), which are thought to be derived in part from

the pallium and thalamic eminence (Abellan et al. 2009; Tissir

et al. 2009), were also GFP+in E11.5 Neurog2

GFPKI/+ embryos.

We can thus conclude that the vast majority of Cajal--Retzius

cells present in the E11.5 neocortex and piriform cortex

(where our Ascl1 lineage tracing was focused) are pallial in

origin, while marginal zone cells underlying the basal telen-

cephalon may have a mixed origin.

Taken together, we conclude that Ascl1-expressing progen-

itors give rise to Cajal--Retzius cells, particularly in the pallial

septum and ventral pallium. However, a long-term lineage trace

(e.g., using an Ascl1-CreKI line crossed with a reporter) would

be required to validate this regional bias.

Ascl1 Is Required for the Generation of Cajal--RetziusNeurons

To test if Ascl1 is required for the generation of Cajal--Retzius

cells, we performed cell counts along the rostral-to-caudal

extent of the neocortical marginal zone in wild-type and

Ascl1–/– embryos sectioned along the sagittal plane (Fig. 4A,B).

Cell counts were not performed in a regional-specific manner

as the loss of one Cajal--Retzius cell population is known to be

compensated for by alterations in the migratory routes of Cajal--

Retzius cells derived from other progenitor domains (Bielle

et al. 2005; Pla et al. 2006; Griveau et al. 2010). At E12.5, 1.35-

fold fewer Reelin+Cajal--Retzius cells were detected in Ascl1

–/–

cortices compared with wild-type controls (wild-type: 126.4 ±4.0; n = 3 vs. Ascl1

–/–: 93.1 ± 3.4 Reelin+cells/field; n = 2; P <

0.0001; Fig. 4C,C#,D,D#,G). Similarly, there was a 2.2-fold

decrease in Trp73+Cajal--Retzius cells in E12.5 Ascl1

–/– cortices

(wild-type: 31.2 ± 1.9; n = 3 vs. Ascl1–/–: 14.2 ± 2.1 Trp73

+cells/

Figure 2. Ascl1 is coexpressed with pallial-specific markers. (A--D) Confocal images of Pax6 (green), Ascl1 (red), and DAPI (nuclear marker, blue) expression in theE10.5 pallium, focusing on the pallial--subpallial border (demarcated by dotted line and white arrowhead). Boxed cell in panels A--D is magnified in D to demonstrate thatsome DAPIþ pallial progenitors in the ventral pallium coexpress Ascl1 and Pax6. (E--H) Confocal images of Neurog2 (green), Ascl1 (red), and DAPI (nuclear marker, blue)expression in the E10.5 pallium, focusing on the pallial--subpallial border (demarcated by dotted line and white arrowhead). Boxed cell in E--H is magnified in H todemonstrate that some DAPIþ pallial progenitors in the ventral pallium coexpress Ascl1 and Neurog2. (I--L) Confocal images of GFP (green), Ascl1 (red), and DAPI(nuclear marker, blue) expression in E10.5 Neurog2GFPKI/þ pallium, focusing on the pallial--subpallial border (demarcated by dotted line and white arrowhead). Boxedcell in I--L is magnified in L to demonstrate that some DAPIþ pallial progenitors in the ventral pallium coexpress Ascl1 and GFP in E10.5 Neurog2GFPKI/þ cortices. vp,ventral pallium.


field; n = 2; P < 0.0001; Fig. 4E,E#,F,F#,G). Notably, the reduction

in Cajal--Retzius cell number, including Trp73+cells, which are

derived from the pallial septum and cortical hem, was not due

to a mispatterning of medial signaling centers. Indeed, Wnt3a

was expressed in the hem of both wild-type and Ascl1–/– E12.5

cortices (Fig. 4H,I).

To determine if the reduction in Cajal--Retzius cell number

observed at E12.5 was due to a delay in the differentiation of

these cells in Ascl1 mutants, we also performed cell counts at

E15.5 and E18.5, analyzing both sagittal and coronal sections

through the neocortex (Fig. 5A--C). At E15.5, the number of

Reelin+(wild-type: 58.6 ± 2.1; n = 3 vs. Ascl1

–/–: 51.8 ± 2.0

Reelin+cells/field; n = 3; P = 0.03; Fig. 5J) and Trp73

+(wild-

type: 50.9 ± 1.4; n = 3 vs. Ascl1–/–:44.0 ± 2.1 Trp73

+cells/field;

n = 3; P = 0.01; Fig. 5K) Cajal--Retzius cells was reduced inAscl1–/–

cortices compared with wild-type controls. The presence of

fewer Reelin+(wild-type: 85.9 ± 1.7; n = 3 vs. Ascl1

–/–: 77.0 ± 1.7

Reelin+cells/field;n = 6; P = 0.006; Fig. 5D--D#,E--E#,J) and Trp73+

(wild-type: 54.4 ± 2.8; n = 5 vs. Ascl1–/–: 42.6 ± 2.5 Trp73

+cells/

field; n = 3; P = 0.01; Fig. 5F,F#,G,G#,H,I,K) Cajal--Retzius cells inthe Ascl1

–/– marginal zone was maintained even at E18.5, when

neocortical neurogenesis is complete (Takahashi et al. 1999).

Ascl1 is therefore required for the generation of a subset of

Cajal--Retzius cells, including Trp73+cells, which are specifi-

cally derived from the cortical hem and pallial septum.

Ascl1 Is Required for the Differentiation of Reelin+

Cortical Interneurons

By E18.5, Reelin is not only expressed in Cajal--Retzius cells in

the upper marginal zone but also in interneurons of subpallial

origin that are interspersed throughout the lower marginal

zone and in neocortical layers II--VI (Fig. 5D--D$) (Alcantara

et al. 1998; Morante-Oria et al. 2003). In E18.5 Ascl1–/– cortices,

the number of Reelin+cells was reduced not only in the

marginal zone but also throughout the cortical plate (Fig. 5E--

E$). The reduction in Reelin+cells in E18.5 Ascl1

–/– cortices is

consistent with the loss of cortical interneurons previously

reported in these mutants (Casarosa et al. 1999; Horton et al.

1999). We verified that fewer cortical interneurons were

generated in E18.5 Ascl1–/– cortices by examining the

expression of several additional interneuron markers. Calreti-

nin, which labels both Cajal--Retzius cells of pallial origin and

cortical plate interneurons, was expressed in fewer cells in

E18.5 Ascl1–/– cortices (Supplementary Fig. S2A,B), as was

Figure 3. Cajal--Retzius cells are derived from Ascl1þ lineages. (A) Schematic representation of coronal section through the E11.5 telencephalon showing sites of analysis in thefollowing figures (red, boxed areas), including the ventral pallium/piriform cortex, pallial septum (at rostral levels), and cortical hem (at medial levels). (B--B$,C--C$) Coexpressionof GFP (green) and Reelin (red; B--B$) or GFP (green) and Trp73 (red; C--C$) in E11.5 Ascl1:GFP cortex at the level of the ventral pallium/piriform cortex (B--C), pallial septum(B#,C#), and cortical hem (B$,C$). Boxed cells in each panel are shown at higher magnification in panels to the right. Yellow dotted line in B,C marks the pallial--subpallial border.Scale bar: 53 lm. (D) Graph showing the percentage of Cajal--Retzius cells labeled with Reelin (red bar) or Trp73 (yellow bar) that derive from the Ascl1 lineage (i.e., GFPþ) in theventral pallium/piriform cortex, pallial septum, and cortical hem. (E--H) Confocal images of GFP (green, E,H), Reelin (red, F,H), and Tbr1 (blue, G,H) expression in E11.5 Ascl1: EGFPpallium, focusing on the pallial--subpallial border (demarcated by dotted line). Boxed cell in E--H is magnified in H to demonstrate that some GFPþ cells in the ventral palliumcoexpress Reelin and Tbr1. (I--M) Coexpression of GFP (green) and Reelin (red; I--I$,K,L--L$,M--M$) or GFP (green) and Trp73 (red; J--J$) in E11.5 Neurog2GFPKIþ/- cortex. Highmagnification images are at the level of the ventral pallium/piriform cortex (I,J,L--L$), pallial septum (I$,J$), cortical hem (I$,J$), or basal telencephalon (M--M$). Boxed cells inI--J$ are shown at higher magnification in panels to the right. Yellow dotted line in I--J marks the pallial--subpallial border. Scale bar in I is 53 lm. ch, cortical hem; mge, medialganglionic eminence; pc, piriform cortex; ps, pallial septum; vp, ventral pallium.



calbindin, which labels interneurons in the lower marginal

zone and infragranular layers (Supplementary Fig. S2C,D). A

reduction in cortical interneurons in E18.5 Ascl1–/– cortices

was also evident using pan-Dlx (Supplementary Fig. S2E,F) and

pan-Otx (Supplementary Fig. S2G,H) antibodies, the former

labeling interneurons of subpallial origin, and the latter labeling

both infragranular neurons in the cortical plate and interneur-

ons in supragranular layers and in the marginal zone.

We thus conclude that Ascl1 is required for the generation

of most cortical interneurons, including those that initiate

Reelin expression in the late embryonic period.

Induction of Cajal--Retzius Cell Markers by Ascl1

To test if Ascl1 is sufficient to promote a Cajal--Retzius cell

identity, we misexpressed this gene in the E10.5 pallium using

a bicistronic vector coexpressing GFP and Ascl1 and compared

the results with a control vector expressing only b-galactosi-dase (b-gal; Fig. 6I). Electroporated brains were cultured for 2

days in vitro (DIV) and then immunolabeled with antibodies to

Reelin, a pan-Cajal--Retzius cell marker, and Tbr1, a marker of

postmitotic neurons of pallial derivation (Hevner et al. 2001).

Strikingly, Ascl1 promoted a 2.1-fold increase in the number

of cells that expressed Tbr1 compared with control electro-

porations (control: 23.2 ± 1.3% b-gal+ cells coexpressed Tbr1+;

n = 4; 1092 electroporated cells; Fig. 6A--D,J vs. 47.6 ± 4.4%

GFP+cells coexpressed Tbr1

+after Ascl1 overexpression; n = 4

733 electroporated cells; Fig. 6E--H,J; P < 0.0001). Moreover,

2.1-fold more Ascl1-electroporated cells expressed both Tbr1

and Reelin (i.e., 14.9 ± 1.8% of GFP+cells wereGFP

+Tbr1

+Reelin

+;

n = 4; 733 electroporated cells; P = 0.0009; Fig. 6E--H,J) compared

with control electroporations (i.e., 7.2 ± 0.9% b-gal+ cells were

b-gal+Tbr1+Reelin+;n = 4; 1092 electroporated cells; Fig. 6A--D,J).Notably, Ascl1 did not induce the expression of other Cajal--

Retzius markers in this assay, including calretinin (Supplemen-

tary Fig. S3A--H) and Trp73 (data not shown).

The cell fate specification properties of the proneural genes,

including Ascl1, are both spatially and temporally restricted

(Britz et al. 2006; Mattar et al. 2008). We therefore tested if

Ascl1 could promote the expression of Cajal--Retzius cell

markers in the E12.5 pallium, at the tail end of their

differentiation period. An advantage of the E12.5 stage was

our ability to efficiently use in utero electroporation to

introduce expression constructs into the pallium, allowing for

normal in vivo development posttransfection (Fig. 7G) (Saito

and Nakatsuji 2001; Langevin et al. 2007; Mattar et al. 2008). In

E12.5 / E15.5 pallial electroporations, Ascl1 promoted a

modest, albeit significant, 1.06-fold increase in the number of

cells that expressed Tbr1 compared with control electro-

porations (Ascl1: 93.09 ± 1.03% GFP+cells coexpressed Tbr1

+;

n = 3; 3990 GFP+electroporated cells vs. control pCIG2: 87.24 ±

1.60% GFP+

cells coexpressed Tbr1+; n = 3; 2826 GFP

+

electroporated cells; Fig. 7A--F,H). However, the minimal ability

Figure 4. Fewer Cajal--Retzius cells are generated in E12.5 Ascl1�/� cortices. (A) Dissected E12.5 brain showing plane of sectioning (marked by line; sagittal plane) used inpanels C--F,H,I. (B) Schematic illustration of sagittal section through E12.5 telencephalon/diencephalon. (C--F) RNA in situ hybridization on sagittal sections of E12.5 wild-type(C,C#,E,E#) and Ascl1�/� (D,D#,F,F#) cortices showing expression of Reelin (C--D#) and Trp73 (E--F#). (E) Reelinþ and Trp73þ cell quantitation in the E12.5 preplate. Error barsrepresent standard error of the mean (s.e.m.). Student’s unpaired t-tests were performed with P values denoted as follows:\0.05*,\0.01**,\0.005***. (H,I) Expression ofWnt3a in E12.5 cortical hem in wild-type (H) and Ascl1�/� (I) cortices. ch, cortical hem; ctx, cortex; dienc, diencephalon; ge, ganglionic eminences; pp, preplate.







of Ascl1 to induce ectopic Tbr1 expression in the E12.5 pallium

was significantly below the >2-fold induction observed at E10.5.

Moreover, Ascl1 was not able to induce detectable increases in

Reelin (Supplementary Fig. S4A--F) or Trp73 (data not shown)

protein expression in the E12.5 pallium.

Taken together, we thus conclude that Ascl1 gradually loses

its ability to promote the differentiation of Tbr1+Reelin

+Cajal--

Retzius cells in pallial progenitors over developmental time.

Our data thus reinforce the idea that there are temporal

changes in Ascl1 function and/or in the competence of pallial

progenitors to respond to the specification activities of Ascl1.

Discussion

Cajal--Retzius cells are a heterogeneous neuronal population,

displaying regional-specific patterns of gene expression accord-

ing to their sites of origin, which include several distinct

progenitor domains that line the pallial margins. Here, we

demonstrate that the proneural gene Ascl1 is expressed in

several domains of Cajal--Retzius cell genesis and that pallial

progenitors expressing this proneural gene give rise to a subset

of these pioneer neurons. Accordingly, we found that Ascl1 is

required for the differentiation of a subset of Reelin+and

Trp73+

Cajal--Retzius cells of pallial origin. Moreover, we

showed that Ascl1 can promote the expression of Reelin,

a Cajal--Retzius cell marker, and Tbr1, which labels neurons of

pallial origin, in early (i.e., E10.5) pallial progenitors. This study

is of particular significance as it identifies a precise role for

Ascl1 in the embryonic pallium, where a role for this gene had

not previously been defined.

Ascl1 Is Expressed in Cajal--Retzius Cell Lineages

Three major focal sources of Cajal--Retzius cell genesis have

been identified in the pallial margins, including the ventral

pallium, cortical hem, and pallial septum (Takiguchi-Hayashi

et al. 2004; Bielle et al. 2005; Yoshida et al. 2006; Imayoshi et al.

2008; Zimmer et al. 2010). We show here that Ascl1 is

expressed in all 3 of these progenitor domains from E10.5, at

the onset of Cajal--Retzius cell differentiation. To verify that

Ascl1-expressing progenitors give rise to Cajal--Retzius neu-

rons, we performed short-term lineage tracing using an

Ascl1:GFP BAC transgenic line, which had previously been

Figure 5. Fewer Cajal--Retzius cells and Reelinþ interneurons are detected in Ascl1�/� cortices in late embryogenesis. (A) Dissected E18.5 brain showing sagittal and coronalplanes of sectioning used in panels D--E and F--I, respectively. (B) Schematic illustration of coronal section through E18.5 brain at the level of the cerebral cortex. (C) Schematicillustration of sagittal section through E18.5 cortex. (D--G) Coronal (D--E$) or sagittal (F--G#) sections of E18.5 cortices showing the distribution of Reelin (D--D$,E--E$) and Trp73transcripts (F--F#,G--G#) and Trp73 protein (H,I) expression in wild-type (D--D$,F--F#,H) and Ascl1�/� (E--E$,G,G#,I) embryos. (J,K) Quantification of the number of Relnþ (J) andTrp73þ (K) Cajal--Retzius cells in the marginal zones of E15.5 and E18.5 wild-type and Ascl1�/� cortices. Error bars represent s.e.m. Student’s unpaired t-tests were performedwith P values denoted as follows:\0.05*,\0.01**,\0.005***. cp, cortical plate; dp, dorsal pallium; hp, hippocampus; mz, marginal zone; ncx, neocortex; Reln, Reelin.



reported to faithfully recapitulate the endogenous expression

of Ascl1 (Parras et al. 2007). In Ascl1:GFP BAC transgenics, we

found that GFP was primarily expressed in Cajal--Retzius cells

that were derived from the ventral pallium and pallial septum.

Surprisingly, despite relatively high Ascl1 transcript and protein

expression in the cortical hem, very little GFP expression was

detected in Cajal--Retzius cells lining the caudomedial marginal

zone (i.e., hem derived) in Ascl1:GFP BAC transgenics. We

speculate that the ability of Ascl1 to specify a Cajal--Retzius cell

fate in the cortical hem may be prevented by the formation of

complexes between Ascl1 and inhibitory factors specifically

present in cortical hem progenitor cells. However, we cannot

rule out the possibility that the Ascl1:GFP BAC transgenic line

does not faithfully label all cells derived from the Ascl1 lineage.

Future long-term lineage tracing experiments using an Ascl1-

creKI line would be necessary to image the full complement of

Ascl1-derived pallial neurons.

What are the signals that regulate Ascl1 expression in the

pallium? Previous reports demonstrated that Ascl1 can be

induced by Fgf signaling in the telencephalon (Kuschel et al.

2003; Gutin et al. 2006), the activity of which is high in the

pallial septum (Zimmer et al. 2010), where Ascl1 is expressed

at high levels from E10.5. Conversely, Wnts repress Ascl1

expression (Backman et al. 2005), yet we found high Ascl1

transcript and protein levels in the cortical hem, which is a rich

source of Wnt signaling (Grove et al. 1998; Lee et al. 2000;

Shimogori et al. 2004). However, the hem is also under the

influence of Bmp and Tgfb signaling (Yoshida et al. 2006;

Hanashima et al. 2007; Siegenthaler and Miller 2008), and it may

be that these signals combined are permissive (or instructive)

for Ascl1 expression.

Ascl1 Is Required in Cajal--Retzius Cell Lineages

Previous clonal analyses of Ascl1–/– pallial progenitors in

a Neurog2-negative lineage revealed that Ascl1 is required to

regulate neuronal differentiation in these cells in an in vitro

clonal assay (Nieto et al. 2001). To date, this has been the only

indication that Ascl1 may have a pallial-specific function. Here,

we report the first in vivo evidence supporting a role for Ascl1

in pallial progenitors. Specifically, we found that Ascl1 is

required for the generation of a subset of Cajal--Retzius cells,

with decreased numbers of both Reelin+and Trp73

+Cajal--

Retzius neurons observed in Ascl1–/– cortices. We suggest that

the reduction in Trp73+Cajal--Retzius cells in Ascl1 mutants,

which are derived from both the cortical hem and the pallial

septum (Nieto et al. 2001; Meyer et al. 2002; Takiguchi-Hayashi

et al. 2004; Bielle et al. 2005), mainly corresponds to Cajal--

Retzius cells derived from the pallial septum for several rea-

sons. Firstly, our short-term lineage tracing (albeit with

the caveats outlined above) suggests that not many Ascl1-

expressing progenitors in the cortical hem differentiate into

Cajal--Retzius cells, whereas the pallial septum is a rich source

of Ascl1-derived Cajal--Retzius cells. Secondly, we show here

that the cortical hem expression of Wnt3a is not perturbed in

Ascl1–/– cortices, suggesting that there are no major patterning

defects in this signaling center in the absence of Ascl1 function.

While additional studies would be required to confirm that the

cortical hem is a functional signaling center in Ascl1 mutants,

we suggest that this is the case. Indeed, the cortical hem is

known to pattern adjacent hippocampal domains (Galceran

et al. 2000; Bulchand et al. 2001), which are normal in Ascl1

mutants (data not shown). Finally, we found that in the E10.5

pallial septum, more progenitors express high levels of Ascl1

protein as compared with Neurog2, a proneural transcription

factor that is also required for the generation of a subset of

Cajal--Retzius cells (Imayoshi et al. 2008), suggesting that Ascl1

may have a nonredundant function in this territory at the

earliest stages of Cajal--Retzius cell differentiation. Should Ascl1

indeed function in the early pallial septum, which is Fgf

signaling dependent (Zimmer et al. 2010), it would be

Figure 6. Ascl1 promotes the differentiation of Tbr1þReelinþ Cajal--Retzius cells in the early E10.5 pallium. (A--H) Triple immunostaining on coronal sections of E10.5 þ 2DIVtelencephalon electroporated with nls-b-gal (b-galactosidase) (A--D) or Ascl1-IRES-GFP (E--H) using antibodies directed against b-gal (A--D) or GFP (E--H) and Reelin (red) andTbr1 (blue). (I) Schematic illustration of the pallial area analyzed in A--H. (J) Quantification of the percentage of markerþ (i.e., b-galþ in A--D or GFPþ in E--H) electroporated cellscoexpressing Tbr1 over the total number of electroporated cells. Quantification of the percentage of markerþ (i.e., b-galþ in A--D or GFPþ in E--H) electroporated cellscoexpressing Tbr1 and Reelin over the total number of electroporated cells. Error bars represent s.e.m. Student’s unpaired t-tests were performed with P values denoted asfollows:\0.05*,\0.01**,\0.005***. mz, marginal zone.


interesting in the future to determine if Ascl1 pallial function(s)

in Cajal--Retzius cell generation depends on Fgf signaling.

Given that Cajal--Retzius cells are reduced in number in

Neurog2–/– (Imayoshi et al. 2008) and Ascl1

–/– cortices, both

genes must have nonredundant roles in regulating the

differentiation of these cells. At first glance, the reduction in

Cajal--Retzius cell number in Neurog2 mutants is somewhat

surprising given the pallial-specific upregulation of Ascl1

expression previously reported in these embryos (Fode et al.

2000; Schuurmans et al. 2004). However, we have found that

Ascl1 is not upregulated in the Neurog2–/– pallium at E10.5,

with increased Ascl1 transcription not evident until ~E12.5(data not shown), when Cajal--Retzius cell genesis is almost

complete. We thus favor the idea that Neurog2 and Ascl1

function in nonoverlapping populations of pallial progenitors

destined to differentiate into distinct types of Cajal--Retzius

cells. Alternatively, we suggest that Neurog2 and Ascl1 may

function in the same lineages but at different times in lineage

progression.

Ascl1 Fate Specification Properties Are TemporallyRegulated in the Pallium

Proneural genes promote both generic aspects of neuronal

differentiation and specify neuronal subtype identities (Talikka

et al. 2002). In the embryonic telencephalon, Ascl1 has

previously been shown to specify a subpallial, GABAergic in-

terneuron identity (Casarosa et al. 1999; Horton et al. 1999;

Britz et al. 2006; Wang et al. 2009). Here, we misexpressed

Ascl1 in the E10.5 pallium, and to our surprise, found that Ascl1

could induce the expression of Tbr1, a pallial-specific neuronal

marker, as well as Reelin, a pan-Cajal--Retzius cell marker. This

suggests that Ascl1 can play an instructive role in specifying

a cortical-specific Cajal--Retzius cell identity in early pallial

progenitors. However, Ascl1 was not sufficient to induce all

Cajal--Retzius cell markers, including calretinin and Trp73. We

suggest that the inability ofAscl1 to induce calretinin expression,

which is normally excluded from pallial septum-derived Cajal--

Retzius cells (Bielle et al. 2005), may reflect a specific role for

Ascl1 in promoting a pallial septum-specific identity. In contrast,

it is not so easy to explain the inability of Ascl1 to induce

Trp73 expression, a marker of Cajal--Retzius cells derived from

the pallial septum and cortical hem (Takiguchi-Hayashi et al.

2004; Bielle et al. 2005). We thus suggest that Ascl1 does not

have the capacity to fully initiate a Cajal--Retzius cell differen-

tiation program, possibly because it is only one component of a

complex of transcription factors that might together be re-

quired. Additional studies examining the combinatorial effects

of different transcription factor complexes on Cajal--Retzius

cell differentiation would be required to further dissect the

transcriptional cascades that are involved in Cajal Retzius cell

fate specification and differentiation.

We have previously shown that the proneural activities of Ascl1

and Neurog2 are temporally and spatially regulated in the

embryonic telencephalon (Schuurmans et al. 2004; Britz et al.

2006; Mattar et al. 2008). Here, we found that the ability of Ascl1

to promote the expression of Cajal--Retzius cell markers is

strikingly reduced by E12.5. Instead, at this later developmental

stage, we previously showed that Asc1l promotes the ectopic

expression of subpallial markers (Britz et al. 2006). Accordingly,

we found here that Ascl1 is required for the differentiation of

a subset of Reelin+Cajal--Retzius cells and almost all Reelin

+

cortical interneurons, reflecting a requirement for Ascl1 in both

Figure 7. Ascl1 has a reduced ability to promote Tbr1 expression in the E12.5 pallium. (A--F) Expression of GFP (green, A,C,D,F) and Tbr1 (red, B,C,E,F) in brains electroporated atE12.5 with pCIG2 (A--C) or pCIG2-Ascl1 (D--F) expression vectors and harvested and analyzed at E15.5. (G) Schematic illustration of coronal section through E15.5 pallium,showing site of analysis (boxed area). (H) Quantification of the percentage of GFPþ cells coexpressing Tbr1. Error bars represent s.e.m. Student’s unpaired t-tests were performedwith P values denoted as follows:\0.05*,\0.01**,\0.005***. ctx, cortex; ig, infragranular layers; iz, intermediate zone; mz, marginal zone; str, striatum; sg, supragranularlayers; svz, subventricular zone.


pallial and subpallial domains, respectively. However, we suggest

that the Reelin+Cajal--Retzius cells and Reelin

+cortical inter-

neurons are derived from distinct progenitor domains, an idea

supported not only by lineage studies but also by genetic analyses.

For example, the homeodomain genes Cux1/2 are required for

the generation of Reelin+interneurons in the cortical plate but

not for the generation of Reelin+Cajal--Retzius neurons (Cubelos

et al. 2008). It would thus be of interest in the future to examine if

there are genetic interactions between Ascl1 and Cux1/2 in

subpallial progenitors.

We showed that Ascl1 is expressed at higher levels in subpallial

versus pallial progenitors. This raises the question of whether the

dose of Ascl1 plays an instructive role in cell fate specification,

with the elevated levels of endogenous Ascl1 expression observed

in subpallial progenitors preferentially promoting an interneuron

fate, while the lower Ascl1 expression levels observed in pallial

progenitors promoting a Cajal--Retzius cell fate. However, if Ascl1

gene dosage were simply instructive, we would not expect that

the high levels of Ascl1 expression obtained with our electro-

poration technique would promote the differentiation of Cajal--

Retzius cells at any embryonic stage. Instead, our data support the

idea that there are either changes in Ascl1 itself (e.g., through

posttranslational modifications) and/or temporal changes in

the competence of pallial progenitors to respond to the fate

specification properties of Ascl1. Notably, precedents exist for

temporal changes in the abilities of different transcription factors

to specify cell fates. For instance, the homeobox gene Gsx2

preferentially specifying a striatal projection neuron identity early

in telencephalic development, while later it promotes an olfactory

bulb interneuron identity (Waclaw et al. 2009).

Supplementary Material

Supplementary material can be found at: http://www.cercor

.oxfordjournals.org/

Funding

Canadian Institutes of Health Research (CIHR) Operating Grant to

C.S. (grant number MOP-44094) and Medical Research Council

Institutional Funds to F.G. C.S. is an Alberta Heritage Foundation

for Medical Research (AHFMR) Senior Scholar. C.K. and P.M. were

supported by a CIHR Training Grant in Genetics, Child De-

velopment and Health. P.M. was also supported by a CIHR Canada

Graduate Scholarship,; Alberta Heritage Foundation for Medical

Research Studentship; and a Heart and Stroke Foundation

Studentship and C.K. received a QEII Scholarship. R.D. was

supported by a CIHR Canada HOPE Fellowship. C.Z. was

supported by a Medical Research Development Fellowship.

Notes

We thank Siew-lan Ang, Masato Nakafuku, David Anderson, Flora

Vaccarino, and Grace Panganiban for generously providing reagents.

We thank Nicole Gruenig and Laidlaw Green for technical assistance

with animal maintenance and genotyping, Dawn Zinyk for help

preparing figures, and Deborah Kurrasch for critical reading of the

manuscript. Conflict of Interest : None declared.

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