Developmental Biology ()

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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line

Daichi Kawaguchi 1,n, Setsuko Sahara 2, Andreas Zembrzycki, Dennis D.M. OLeary
Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

art ic l e i nf o

a b s t r a c t

Article history:
Received 30 September 2015
Received in revised form
10 February 2016
Accepted 10 February 2016

Foxg1 expression is highly restricted to the telencephalon and other head structures in the early embryo.
This expression pattern has been exploited to generate conditional knockout mice, based on a widely
used Foxg1-Cre knock-in line (Foxg1tm1(cre)Skm), in which the Foxg1 coding region was replaced by the Cre
gene. The utility of this line, however, is severely hampered for two reasons: (1) Foxg1-Cre mice display
ectopic and unpredictable Cre activity, and (2) Foxg1 haploinsufciency can produce neurodevelopmental
phenotypes. To overcome these issues, we have generated a new Foxg1-IRES-Cre knock-in mouse line, in
which an IRES-Cre cassette was inserted in the 3UTR of Foxg1 locus, thus preserving the endogenous
Foxg1 coding region and un-translated gene regulatory sequences in the 3UTR, including recently discovered microRNA target sites. We further demonstrate that the new Foxg1-IRES-Cre line displays consistent Cre activity patterns that recapitulated the endogenous Foxg1 expression at embryonic and
postnatal stages without causing defects in cortical development. We conclude that the new Foxg1-IRESCre mouse line is a unique and advanced tool for studying genes involved in the development of the
telencephalon and other Foxg1-expressing regions starting from early embryonic stages.
& 2016 Elsevier Inc. All rights reserved.

Keywords:
Foxg1
Telencephalon
Cre recombinase
Knock-in mouse
Haploinsufciency
MicroRNA

1. Introduction
The Cre/loxP system is a powerful and widely utilized technology for the conditional gene manipulation and cell lineage
tracing (Branda and Dymecki, 2004; Nagy, 2000). A key element of
this approach is the creation of a suitable driver line that is phenotypically normal, but expresses Cre robustly, and reproducibly in
a specic pattern. Even commonly used Cre driver lines, however,
can have unexpected Cre activity patterns or show complicating
phenotypes (Harno et al., 2013; Heffner et al., 2012; Schmidtsupprian and Rajewsky, 2007). For example, a Nestin-Cre mouse
line (Tg(Nes-cre)1Kln), is one of the most commonly used Cre
driver lines in the neuroscience eld, but reports have revealed
unexpected Cre expression in many tissues outside the central
nervous system and a signicant metabolic and behavioral phenotype in Nestin-Cre mice (Declercq et al., 2015; Giusti et al., 2014;
Harno et al., 2013). These factors greatly complicate the interpretation of the derived phenotypes, and reduce the utility of this
approach.
The transcription factor Foxg1 (formerly called BF-1), a member
of the forkhead box family proteins, is one of the earliest genes
n

Corresponding author.
E-mail address: dkawaguchi@mol.f.u-tokyo.ac.jp (D. Kawaguchi).
1
Present address: Graduate School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo 113
0033, Japan.
2
Present address: Department of Developmental Neurobiology, Kings College
London, New Hunts House, Guys Campus, London SE1 1UL, UK.

expressed in the emerging telencephalic region starting around

E8.5E9.0 (Hbert and McConnell, 2000; Shimamura et al., 1995;
Xuan et al., 1995). Because of its very early and robust expression
in the telencephalic progenitors and other embryonic regions, a
knock-in mouse line in which the Foxg1 coding sequences was
replaced with Cre gene (Foxg1tm1(cre)Skm) has been widely used to
manipulate function of genes expressed in the telencephalon, inner ear, olfactory epithelium, anterior retina, pharyngeal endoderm, Rathke's pouch, and foregut at early embryonic stages
(Hbert and McConnell, 2000). However in the Foxg1-Cre mice,
ectopic and unpredictable Cre activities have been observed.
Analysis of the Foxg1-Cre line further revealed that Cre activity
pattern varies signicantly in each mouse, and the extent of variation is different depending on genetic background. The least
variability has been found in the 129SvJ strain but even still, about
36% of Foxg1-Cre mice in the 129SvJ background show unintended
and ectopic Cre recombination patterns that differed from the
endogenous Foxg1 expression (Hbert and McConnell, 2000). Due
to its abnormal behavioral responses and congenital hypoplasia in
the corpus callosum (Crawley et al., 1997; Wahlsten, 1982), the
129SvJ strain is not a preferred background strain in the neuroscience eld. In a more widely used genetic background like
C57BL/6, the Foxg1-Cre driver has been reported to generate
widespread ectopic recombination, in some cases in the entire
central nervous system and in others, in almost all tissues both in
head and body regions (Hbert and McConnell, 2000). The dysregulation of expression in the Foxg1-Cre line precludes its use as a

http://dx.doi.org/10.1016/j.ydbio.2016.02.011
0012-1606/& 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

telencephalon specic Cre driver line, as ectopic Cre activity is

invariably observed in regions around the mid- and hindbrain
(Achim et al., 2012; Fuccillo et al., 2004; Hbert and McConnell,
2000; Kasberg et al., 2013; Li et al., 2008, 2012; Ma et al., 2002;
Zembrzycki et al., 2007). The reasons behind these inconsistent
Cre activities are not completely known but could be due in part
by deletion of functional endogenous microRNA (miRNA) target
sites in the Foxg1 3UTR from the Cre coding messenger RNA
(mRNA) (Miyoshi and Fishell, 2012) that would normally repress
Foxg1 expression (Choi et al., 2008; Garaffo et al., 2015; Shibata
et al., 2011, 2008). In addition, the Foxg1-Cre allele contains a PGKneo cassette, which in some case has been shown to result in
unpredictable Cre activity (Iwasato et al., 2004; Pham et al., 1996).
The other notable caveat of using the Foxg1-Cre line is that
intrinsic phenotypes due to the haploinsufciency of Foxg1 gene
have been observed. Although earlier reports have claimed that
heterozygous Foxg1 mice appear normal (Dou et al., 1999; Hanashima et al., 2004, 2002; Hbert and McConnell, 2000; Xuan et al.,
1995), recent papers have reported that the heterozygous Foxg1Cre mice show a variety of signicant neurodevelopmental defects
including microcephaly, aberrant cortical area patterning, and
impaired neurogenesis in the telencephalon (Eagleson et al., 2007;
Frullanti et al., 2015; Shen et al., 2006; Siegenthaler et al., 2008).
Therefore, phenotypes observed in the Foxg1-Cre-mediated conditional knockout mice could at least in part be caused by Foxg1haploinsufciency.
To overcome these limitations, we have generated a new Foxg1IRES-Cre mouse line. We show here that this new driver produces
consistent Cre recombination patterns that faithfully recapitulate
the endogenous Foxg1 expression at embryonic and postnatal
stages without causing Foxg1-haploinsufciency. We conclude that
the Foxg1-IRES-Cre line is a new tool for manipulating gene expression involved in the development and function of telencephalon and other Foxg1-expressing tissues from early embryonic stages.

Foxg1 gene. A PGK-Diphtheria toxin (DTA) was included to select

against random insertion events. Targeted embryonic stem cell clones
were screened by Southern blot with 5 and 3 probes to identify
Foxg1-IRES-Cre/ clones. These identied clones were injected into
C57BL/6J blastocysts at the Salk Transgenic Core Facility and the resulting chimeras were mated to C57BL/6J females to obtain germ-line
transmission. Heterozygous mice were mated with FLPe mice
(Rodrguez et al., 2000) to remove the PGK-neo cassette. Genotyping
PCR was performed using three primers for detecting wild type allele
and Foxg1-IRES-Cre allele (Foxg1 forward (WT-F): 5-GGGGGACCAGACTGTAAG-3, Foxg1 reverse (WT-R): 5-CTCCCACATTGCACCTCG-3,
Cre forward (Cre-F): 5-GTGTTGCCGCGCCATCTGC-3).
2.3. Western blot analysis
Western blot analysis was performed as described previously
(Lim et al., 2008). Brain lysates were prepared from P0 neocortex
using RIPA buffer, and the concentration of protein in the lysates
was measured using DC protein assay (Bio-Rad). The following
antibodies were used: rabbit anti-Foxg1 (1:5000, Abcam) and
mouse anti-Gapdh (1:5000, GeneTex).
2.4. X-gal staining, Nissl staining, immunohistochemistry, in situ
hybridization

2. Materials and methods

X-Gal staining was performed on whole mount embryo or

14 m cryosections. -galactosidase activity was developed in
staining solution (PBS containing 1 mg/ml X-Gal, 2 mM MgCl2,
0.01% SDS, 0.02% NP40, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6) for
several hours or overnight at 32 C or 37 C. Specimens were then
washed in PBS and postxed in 4% PFA. Sections were counterstained with nuclear fast red (Vector lab). Nissl staining and immunostaining were carried out as described (Zembrzycki et al.,
2015). The following primary antibodies were used: rabbit antiCux1 (1:1000, Santa Cruz), rabbit anti-Foxp2 (1:3000, Abcam), and
rabbit anti-serotonin (1:50,000; Immunostar). In situ hybridization was done using digoxigenin (DIG)-labeled riboprobe for Cad8
on whole brains as described previously (Sahara et al., 2007).

2.1. Mice

2.5. Statistical analysis

All animal experiments were approved and conducted following the guidelines of the Institutional Animal Care and Use Committee at the Salk Institute and were in full compliance with the
guidelines of the National Institutes of Health for the care and use
of laboratory animals. The day of insemination and the day of birth
are designated as embryonic day 0.5 (E0.5) and postnatal day 0
(P0), respectively. Foxg1-IRES-Cre mice were backcrossed to C57BL/
6 background mice (Harlan Laboratories) for at least six times
except for Fig. 6B in which we used mice backcrossed for four
times. To assess specicity of Cre recombination, Rosa26-LacZ mice
(Soriano, 1999) were crossed to Foxg1-IRES-Cre mice.

Quantitative data in Figs. 1, 5 and 6 are means 7s.e.m for the

numbers of brains (n) indicated in the corresponding gures. Data
were compared between groups with one-way ANOVA (Fig. 1) or
unpaired Student's t test (Figs. 5 and 6). A P value of o0.05 was
considered statistically signicant.

2.2. Generation of the Foxg1-IRES-Cre knock-in mouse line

For generation of Foxg1-IRES-Cre mice, Foxg1 gene targeting was
carried out using homologous recombination in embryonic stem cells
(derived from 129 Sv/ter mouse strain) at the Salk Genome Manipulation Core Facility. The 9154 bp genomic region of the mouse Foxg1
gene (anked by EcoRI sites) was used for the targeting vector. The 5
fragment (anked by EcoRI and AclI sites) and the 3 fragment
(anked by AclI and EcoRI sites) were isolated from BamHI anked
genomic region (13634 bp) of the mouse Foxg1 gene. A DNA cassette
containing an internal ribosormal entry site (IRES) preceding a Cre
recombinase gene followed by an frt-anked PGK-neo was inserted at
the AclI site, 27 bp downstream of the stop codon in the 3UTR of

3. Results
3.1. Generation of a Foxg1-IRES-Cre knock-in allele
To generate a mouse line that expresses the Cre recombinase in
Foxg1-expressing cells without disturbing its endogenous Foxg1
gene expression, we targeted an IRES-Cre cassette into the 3UTR
of the Foxg1 gene (Fig. 1A). This targeting strategy was designed to
generate a bicistronic messenger RNA (mRNAs), encoding both
endogenous Foxg1 and the transgenic Cre. Moreover, the targeting
strategy was designed to insert the IRES-Cre and frt-anked
PGKneo cassettes 27 bp after the stop codon of the full length
Foxg1 gene, thus maintaining the previously described miRNA
target sites in 3UTR of Foxg1 gene (e.g., miR-9 and miR-200) (Choi
et al., 2008; Garaffo et al., 2015; Shibata et al., 2011, 2008). Importantly, this strategy did not incorporate an exogenous polyadenylation (polyA) sequence, which causes abnormal miRNA
regulation in the Foxg1-Cre line due to the deletion of Foxg1 3UTR

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

Fig. 1. Generating a Foxg1-IRES-Cre knock-in allele by gene targeting. (A) Diagram showing the targeting vector, wild type Foxg1 allele, Foxg1-IRES-Cre-neo knock-in allele,
and Foxg1-IRES-Cre knock-in allele. Targeted insertion of an IRES-Cre was positioned at AclI site in the 3UTR region of Foxg1 locus. Blue block indicates the entire Foxg1
coding region. The positions of 5 and 3 probes for southern blot analysis, and primers for genotyping PCR (WT-F, Cre-F, and WT-R) are indicated. pA, endogenous
polyadenylation sequence of Foxg1 gene; RI, EcoRI; N, NcoI; B, BamHI. (B, C) Southern blot analysis with 5 and 3 probes in ES cells (clone #1#6) and mice (wile type (WT),
Foxg1-IRES-Cre-neo heterozygous (Het) and homozygous (Hom)) after digestion of the genomic DNA with BamHI and NcoI. Both 5 and 3 probes detected  10.3 kb band in
WT allele. The 5 probe detected  9.0 kb, and the 3 probe detected  3.1 kb band in Knock-in (KI) allele. (D) Genotyping PCR products for WT, Foxg1-IRES-Cre heterozygous
and homozygous mice. WT-F/WT-R primer set produced 180 bp band in WT allele, and Cre-F/WT-R primer set produced 532 bp band in Foxg1-IRES-Cre allele. (E) Western
blot analysis of telencephalic protein at P0 from WT, Foxg1-IRES-Cre Het and Hom mice, using anti-Foxg1 and anti-Gapdh antibodies. Quantitative comparisons of Foxg1 level
(relative to Gapdh) among WT, Het and Hom were shown in the lower panel. n, the number of brains; n.s., not signicant.

from the Cre coding mRNA (Miyoshi and Fishell, 2012). Instead, the
endogenous polyA sequence of the Foxg1 gene was used to
terminate the transcription of the Foxg1-IRES-Cre bicistronic
sequence.
The targeting construct was correctly targeted in ES cells using
homologous recombination, conrmed by southern blot analysis
(Fig. 1B), and then transferred into blastocysts and into the mouse
germline (clone #6) (Fig. 1C). The PGK-neo cassette, which might
cause unpredictable Cre activity (Iwasato et al., 2004; Pham et al.,
1996), was subsequently removed by crossing founder mice to
mice expressing the Flp recombinase under the control of human
actin promoter (Rodrguez et al., 2000) (Fig. 1D). We then examined Foxg1 protein levels in P0 telencephalic lysates. We found
no signicant difference in Foxg1 protein levels among wild type,
Foxg1-IRES-Cre heterozygous and homozygous mice (Fig. 1E), indicating normal expression of Foxg1 proteins in the new Foxg1IRES-Cre mouse line.

3.2. Characterization of Cre activity in the Foxg1-IRES-Cre mouse

line
The Foxg1-IRES-Cre allele was backcrossed into C57BL/6 since it
is the most commonly used background strain in the neuroscience
eld. Cre activity produced by this allele was assessed by examining -galactosidase expression in embryos obtained by mating the Foxg1-IRES-Cre mouse to a Rosa26-LacZ reporter mouse
(Soriano, 1999). Endogenous Foxg1 expression starts around E8.5
E9.0 in the telencephalon, and Foxg1 expression is also observed in
other head regions such as the otic vesicle, olfactory placode,
anterior half of the optic vesicle, pharyngeal endoderm, and also
foregut by E10.5 (Hatini et al., 1999, 1994; Hbert and McConnell,
2000; Shimamura et al., 1995; Tao and Lai, 1992; Xuan et al., 1995).
-galactosidase activity in Foxg1-IRES-Cre; Rosa26-LacZ double
heterozygous embryos at different stages shows a spatial and
temporal pattern that matched closely to the endogenous Foxg1
expression (Fig. 2AI). Reporter gene expression occurred

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

Fig. 2. Detection of Cre-mediated recombination activity in Foxg1-IRES-Cre; Rosa26-LacZ double heterozygous mice at embryonic stages. Cre activity produced by the Foxg1IRES-Cre driver was detected using a Rosa26-LacZ reporter based on X-gal staining (blue). Nuclear Fast Red was used for counterstaining in sections (red). (A) Whole mount
embryos stained at the indicated stage to reveal tissues with Cre activity. (B) Cre activity in the telencephalon at the indicated stages. (CI) Cre activity in the telencephalon
and other embryonic structures at E11.5. anr, anterior neural ridge; tel, telencephalon; ov, otic vesicle; opv, optic vesicle; fg, foregut; oe, olfactory epithelium; rp, Rathke's
pouch; aret, anterior retina; th, thymus rudiment. Scale bars, 300 m.

uniformly in telencephalic progenitors starting around E9.0-E9.5

(Fig. 2B), but was undetectable in the cortical hem at E11.5 (Fig. 2C
and D), where Foxg1 is not expressed (Hanashima et al., 2007;

Shibata et al., 2008). Most cells in the anterior region of the optic
vesicle (Fig. 2E), otic vesicle (Fig. 2F), olfactory epithelium (Fig. 2G),
and Rathke's pouch (Fig. 2H) were labeled by E11.5. Foxg1-

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
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D. Kawaguchi et al. / Developmental Biology ()

expressing regions in the pharyngeal pouch and the foregut were

also labeled, including ventral third pharyngeal pouch endoderm/
thymus rudiment (Wei and Condie, 2011) (Fig. 2C, D and I).
Although the previous Foxg1-Cre line is often used as a telencephalon-specic Cre driver line, ectopic Cre expression has been
observed consistently in mid- and hindbrain regions starting from
around E10.5 (Achim et al., 2012; Fuccillo et al., 2004; Hbert and
McConnell, 2000; Kasberg et al., 2013; Li et al., 2008, 2012; Ma
et al., 2002; Zembrzycki et al., 2007). This ectopic Cre expression
domain could potentially result from affected miRNA regulation,
since for example miR-9, which suppresses Foxg1 expression
(Garaffo et al., 2015; Shibata et al., 2011, 2008), is strongly expressed in mid- and hindbrain regions at around E9.5-E10.5 in
wild type brains (Kloosterman et al., 2006; Shibata et al., 2008).
Consistent with the preserved miRNA target sites in the

Foxg1-IRES-Cre 3UTR, we did not detect ectopic Cre activity in

regions around the mid- and hindbrain at embryonic and postnatal stages (Fig. 2AC, Fig. 3 A and B), demonstrating that the new
Foxg1-IRES-Cre line is an early-active and telencephalon-specic
Cre driver line in the brain.
We further examined the -galactosidase activity patterns at
postnatal stages (P7, 12 month old) to conrm the specicity of
Cre activity in Foxg1-IRES-Cre mice at later stages. We found that
the reporter gene activities were specically maintained in tissues
that derived from regions that showed Cre activity at embryonic
stages, such as the telencephalon, olfactory bulb, anterior retina,
olfactory epithelium, anterior pituitary gland, inner ear, medulla in
the thymus, trachea, and esophagus (Fig. 3AI). We did not observe reporter gene activities in other organs such as the spinal
cord, lung, heart, liver, stomach, pancreas, spleen, intestine, and

Fig. 3. Detection of Cre-mediated recombination activity in Foxg1-IRES-Cre; Rosa26-LacZ double heterozygous mice at postnatal stages. Cre activity produced by the Foxg1IRES-Cre driver was detected using a Rosa26-LacZ reporter based on X-gal staining (blue) at postnatal stages (P7 in A, C and 12 months old in B, DR). Nuclear Fast Red was
used for counterstaining (red). (AI) Cre activity in derivative tissues from reporter-positive tissues at embryonic stages. (JR) Cre activities are not observed in (J) spinal cord,
(K) lung, (L) heart, (M) liver, (N) stomach, (O) pancreas, (P) spleen, (Q) intestine, and (R) kidney. (R) Peripheral regions in kidney display higher background staining, which
can be detected in mice without Rosa26-LacZ allele (asterisk). tel, telencephalon; ob, olfactory bulb; oe, olfactory epithelium; pit, pituitary gland; aret, anterior retina; ppit,
posterior pituitary; apit, anterior pituitary; co, cochlea; th, thymus; c, cortex; m, medulla; tr, trachea; es, esophagus. Scale bars, 1 mm in AE, GR; 100 m in F.

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

kidney (Fig. 3JR). These results suggest that the new Foxg1-IRESCre line produces reliable Cre recombination in Foxg1-expressing
embryonic tissues and its derivatives at postnatal stages (12
month old), therefore overcomes a major issue in the previous
Foxg1-Cre line, which displays ectopic and unpredictable Cre activities in various tissues.
Recent studies have revealed that certain breeding strategies
can inuence Cre activity patterns (Gil-Sanz et al., 2015; Hayashi
et al., 2003; Heffner et al., 2012; Kobayashi and Hensch, 2013;
Rempe et al., 2006; Schmidt-supprian and Rajewsky, 2007). Since
maternal versus paternal inheritance of the Cre gene has been
shown to potentially affect Cre activity, we aimed to examine this
issue in our Foxg1-IRES-Cre line. We found that some of Foxg1IRES-Cre; Rosa26-LacZ double heterozygous mice displayed overall
weaker and mosaic recombination patterns, but only when the
Foxg1-IRES-Cre allele was inherited paternally (Fig. 4). The weak/
mosaic phenotype was observed similarly in various reporter positive tissues including telencephalon, pituitary gland, thymus
(Fig. 4AC) and other organs (data not shown). We observed obvious weak/mosaic recombination patterns at a rate of 10 out of 24
paternally inherited Foxg1-IRES-Cre mice, while the other 14 mice
displayed robust level of Cre activity (Fig. 4D). Although we detected overall weak/mosaic Cre activity in some of the paternally
inherited Foxg1-IRES-Cre mice, the Cre expression patterns still
matched to the tissue specic patterns of the endogenous Foxg1
expression. Maternally inherited Foxg1-IRES-Cre mice consistently
displayed the same patterns and robust levels of Cre activity. These
results suggest that maternally inherited Foxg1-IRES-Cre allele
should be used for consistent and robust gene manipulation in the
Foxg1-expressing tissues.
3.3. Cortical development is not affected in the Foxg1-IRES-Cre
mouse line
Heterozygous Foxg1-Cre mice, due to Foxg1-haploinsufciency,
have signicant defects in cortical development, including microcephaly, aberrant cortical area patterning, reduced neocortical
thickness especially in upper layers, and impaired neurogenesis in

Fig. 4. Weak/mosaic Cre activity is detected in some of paternally Foxg1-IRES-Cre

inherited mice. (AC) Weak/mosaic Cre activity patterns in (A) brain at P7,
(B) pituitary gland and (C) thymus at 12 months old in paternally Foxg1-IRES-Cre
inherited mice are shown. Cre activity produced by the Foxg1-IRES-Cre driver was
detected using a Rosa26-LacZ reporter based on X-gal staining (blue) with Nuclear
Fast Red counterstaining (red). Scale bars, 300 m. (D) Proportions of mice showing
weak/mosaic Cre activity are indicated. The data were obtained from maternally or
paternally Foxg1-IRES-Cre inherited mice. Some of paternally Foxg1-IRES-Cre inherited mice displayed weak/mosaic Cre activity, but maternally Foxg1-IRES-Cre
inherited mice did not show any weak/mosaic Cre activity. n, number of mice
analyzed.

the telencephalon (Eagleson et al., 2007; Frullanti et al., 2015;

Shen et al., 2006; Siegenthaler et al., 2008). Although Foxg1 protein expression levels in Foxg1-IRES-Cre heterozygous and homozygous mice are comparable to wild type mice (Fig. 1E), we further
determined if our new Foxg1-IRES-Cre line shows any of the cortical developmental defects observed in the Foxg1-Cre heterozygous mice. First, we compared overall cortical size at P7 and in
adult mice and found no differences between wild type and Foxg1IRES-Cre heterozygous mice (Fig. 5A). We next examined neocortical thickness and cortical layer pattern formation by Nissl
staining and immunostaining for the upper layer marker Cux1, as
well as for the deep layer marker FoxP2 at P7. Staining in Foxg1IRES-Cre heterozygous mice did not show differences in the
thickness of total neocortical layers (layer 16), upper layers (layer
24), and deep layers (layer 5 and 6), and also in the location and
distribution of cortical layers, compared to wild type mice
(Fig. 5B). We also analyzed cortical area patterning by using whole
mount in situ hybridization at P7 to examine the expression of the
cortical sensory area marker gene Cad8. Cad8 expression demarcates several rostral sensory areas, such as frontal/motor cortex (F/M) and caudal areas including the primary visual area (V1)
(Armentano et al., 2007; Zembrzycki et al., 2015b). F/M size and V1
size in Foxg1-IRES-Cre heterozygous mice, measured by the size of
the Cad8 expression domains, were comparable to wild type mice
(Fig. 6A). Finally, when area patterning was examined using Serotonin (5-HT) immunostaining on tangential sections of the attened cortex, which reveals terminations of thalamocortical axons
in cortical layer 4 of the corresponding primary sensory areas
(Fujimiya et al., 1986; Zembrzycki et al., 2015a), we found no differences in overall cortical or area sizes and their patterning between wild type and Foxg1-IRES-Cre heterozygous mice (Fig. 6B).
These results suggest that the Foxg1-IRES-Cre transgene does not
cause Foxg1-haploinsufciency and that overall cortical development is not affected in Foxg1-IRES-Cre heterozygous mice.

4. Discussion
We have generated and characterized a new Foxg1-IRES-Cre
knock-in mouse line to prevent Foxg1-haploinsufciency and ectopic
Cre expression that is typically found in the widely used Foxg1-Cre
knock-in mouse line. We have focused our analysis on Foxg1-IRES-Cre
mice in the C57BL/6 genetic background, since it is the most-commonly used background strain in the neuroscience eld. Our results
revealed that Foxg1-IRES-Cre allele generates a pattern of Cre activity
that matches the endogenous Foxg1 expression both spatially and
temporally. It is to be noted that our results indicate the Foxg1-IRESCre allele should be inherited maternally to ensure inheritance of
strong Cre activity in all animals, as this strategy resulted in the most
consistent pattern and levels of Cre activity. The consistency of Cre
activity in the new Foxg1-IRES-Cre line has considerable advantages
compared to the previous Foxg1-Cre line, in which about 88% of mice
show unintended and ectopic Cre expression patterns in the C57BL/6
background (Hbert and McConnell, 2000). We also have demonstrated that use of the Foxg1-IRES-Cre mice circumvent defects in the
cortical development caused by Foxg1-haploinsufciency. While the
homozygous Foxg1-Cre mice die perinatally due to the full deletion of
Foxg1 gene, our new Foxg1-IRES-Cre mice are viable and fertile even
when carrying the homozygous allele.
Mice carrying engineered driver or reporter genes are often
produced using a knock-in or transgenic approach. In both cases,
this approach often removes the 3UTR of targeted genes from
driver/reporter encoding mRNAs, by adding an exogenous polyA
sequence after the driver/reporter coding sequences (Gerfen et al.,
2013; Gong et al., 2003; Taniguchi et al., 2011). For example in
Foxg1 allele, LacZ and tTA knock-in lines were both generated by

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

Fig. 5. Cortical size, cortical thickness and layer pattern formation are normal in Foxg1-IRES-Cre heterozygous mice. (A) Dorsal views of P7 and adult brains of WT and Foxg1IRES-Cre heterozygous (Het) mice. Quantitative comparison of total cortical size between WT and Het mice were shown in the right panel. (B) Nissl staining and immunostaining of Cux1 and Foxp2 in the neocortex at P7. Quantitative comparison of cortical thickness (layer 16) (measured by Nissl staining) and thickness of upper (layer
24) and deep (layer 5 and 6) layers (measured by Cux1 staining) between WT and Het mice were shown in the lower panel. n, the number of brains; n.s., not signicant.
Scale bars, 2 mm in A; 300 m in B.

the same strategy as the Cre knock-in line, resulting the removal of
the 3UTR of Foxg1 gene from LacZ or tTA encoding mRNAs (Hanashima et al., 2002; Hatini et al., 1999; Xuan et al., 1995). As these
endogenous 3UTRs often include gene regulatory sequences like
miRNA target sites, their removal might alter the expression of a
transgene, thus failing to maintain the spatiotemporal characteristics of the endogenous gene expression patterns. Our results
suggest that maintaining these sites when engineering new lines
might help to avoid unintended driver/reporter activities from
introduced transgenes.
The new Foxg1-IRES-Cre mouse line is a unique and advanced tool
for studying genes involved in the development of the telencephalon
from early embryonic stages. The telencephalon specic Cre activity
in the brain and early timing of robust Cre activation (E9.0) in the
telencephalon are remarkable advantages compared to other Cre
lines producing Cre activity in the telencephalon. For example, the

Emx1-Cre line produces Cre activity specically in the dorsal telencephalon, but its robust Cre activation happens around E10.5. The
Nestin-Cre line displays much broader Cre activities in entire central
nervous system (CNS), compared to more restricted recombination
patterns in Foxg1-IRES-Cre and Emx1-Cre mice. Robust Cre activation
in the telencephalon of Nestin-Cre starts signicantly later, around
E11.5 (Chou et al., 2009). Sox1-Cre mice displays robust Cre activation
in the telencephalon at time points comparable to Foxg1-IRES-Cre
mice between E8.59.5, however Sox1-Cre activities are observed
widespread in the CNS regions and in the dorsal root ganglia (Takashima et al., 2007).
In summary, our study demonstrates that the new Foxg1-IRESCre mouse line does not show intrinsic defects that are typically
found in Foxg1-Cre mice providing an alternative tool that allows
for early and consistent gene manipulation in the telencephalon
and other Foxg1-expressing embryonic and postnatal tissues.

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

Fig. 6. Cortical arealization is not affected in Foxg1-IRES-Cre heterozygous mice. (A) Dorsal views of P7 brains of WT and Foxg1-IRES-Cre heterozygous (Het) mice processed
for whole mount in situ hybridization with a DIG-labeled Cad8 riboprobes. Relative F/M area and V1 area to total cortical area were measured based on Cad8 expression
domains. Quantitative comparisons between WT and Het were shown in the lower panel. (B) 5-HT immunostaining on tangential sections of attened P7 cortex in WT and
Het mice. Quantitative comparisons of cortical surface area, F/M area, S1 area, PMBSF area, and V1 area between WT and Het were shown in right and lower panels. n, the
number of brains; n.s., not signicant; F/M, frontal/motor cortex; V1, primary visual area; S1, primary somatosensory area; PMBSF, posterior medial barrel subeld; A1,
primary auditory area. Scale bars, 1 mm.

Acknowledgements
We thank Berta Higgins, Haydee Gutierrez, and Seti Moghadam
for technical assistance; Chris Kintner (Salk Institute) for comments on the manuscript; Goichi Miyoshi (New York University)
for helpful discussion and providing the Foxg1 genomic DNA;
Martyn Goulding (Salk Institute) for providing plasmids encoding
frt-anked PGK-neo and PGK-DTA; Samuel Pfaff (Salk Institute) for
providing the plasmid encoding IRES-Cre; and members of the
OLeary lab for discussions. This work was supported by NIH
Grants R01 NS031558, R01 MH086147, and P30 NS072031, and the
Vincent J. Coates Chair of Molecular Neurobiology (D.D.M.OL.). D.
K. was supported by fellowships from the JSPS Postdoctoral Fellowships for Research Abroad, the Uehara Memorial Foundation,
and the Naito Foundation.

References
Achim, K., Peltopuro, P., Lahti, L., Li, J., Salminen, M., Partanen, J., 2012. Distinct
developmental origins and regulatory mechanisms for GABAergic neurons associated with dopaminergic nuclei in the ventral mesodiencephalic region.
Development 139, 23602370.
Armentano, M., Chou, S.-J., Tomassy, G.S., Leingrtner, A., OLeary, D.D.M., Studer,
M., 2007. COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas. Nat. Neurosci. 10, 12771286.
Branda, C.S., Dymecki, S.M., 2004. Talking about a revolution: The impact of sitespecic recombinases on genetic analyses in mice. Dev. Cell 6, 728.
Choi, P.S., Zakhary, L., Choi, W.-Y., Caron, S., Alvarez-Saavedra, E., Miska, E. a,
McManus, M., Harfe, B., Giraldez, A.J., Horvitz, H.R., Schier, A.F., Dulac, C., 2008.
Members of the miRNA
200 family regulate olfactory neurogenesis. Neuron
57, 4155.
Chou, S.J., Perez-Garcia, C.G., Kroll, T.T., OLeary, D.D.M., 2009. Lhx2 species regional fate in Emx1 lineage of telencephalic progenitors generating cerebral

cortex. Nat. Neurosci. 12, 13811389.

Crawley, J.N., Belknap, J.K., Collins, A., Crabbe, J.C., Frankel, W., Henderson, N., Hitzemann, R.J., Maxson, S.C., Miner, L.L., Silva, A.J., Wehner, J.M., Wynshaw-Boris,
A., Paylor, R., 1997. Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology (Berl).
Declercq, J., Brouwers, B., Pruniau, V.P.E.G., Stijnen, P., de Faudeur, G., Tuand, K.,
Meulemans, S., Serneels, L., Schraenen, A., Schuit, F., Creemers, J.W.M., 2015.
Metabolic and behavioural phenotypes in Nestin-Cre mice are caused by hypothalamic expression of human growth hormone. PLoS One 10, e0135502.
Dou, C.L., Li, S., Lai, E., 1999. Dual role of brain factor
1 in regulating growth and
patterning of the cerebral hemispheres. Cereb. Cortex 9, 543550.
Eagleson, K.L., Schlueter McFadyen-Ketchum, L.J., Ahrens, E.T., Mills, P.H., Does, M.
D., Nickols, J., Levitt, P., 2007. Disruption of Foxg1 expression by knock-in of cre
recombinase: effects on the development of the mouse telencephalon. Neuroscience 148, 385399.
Frullanti, E., Amabile, S., Lolli, M.G., Bartolini, A., Livide, G., Landucci, E., Mari, F.,
Vaccarino, F.M., Ariani, F., Massimino, L., Renieri, A., Meloni, I., 2015. Altered
expression of neuropeptides in FoxG1-null heterozygous mutant mice. Eur. J.
Hum. Genet., 16.
Fuccillo, M., Rallu, M., McMahon, A.P., Fishell, G., 2004. Temporal requirement for
hedgehog signaling in ventral telencephalic patterning. Development 131,
50315040.
Fujimiya, M., Kimura, H., Maeda, T., 1986. Postnatal development of serotonin nerve
bers in the somatosensory cortex of mice studied by immunohistochemistry. J.
Comp. Neurol. 246, 191201.
Garaffo, G., Conte, D., Provero, P., Tomaiuolo, D., Luo, Z., Pinciroli, P., Peano, C., DAtri,
I., Gitton, Y., Etzion, T., Gothilf, Y., Gays, D., Santoro, M.M., Merlo, G.R., 2015. The
Dlx5 and Foxg1 transcription factors, linked via miRNA
9 and
200, are required for the development of the olfactory and GnRH system. Mol. Cell.
Neurosci. 68, 103119.
Gerfen, C.R., Paletzki, R., Heintz, N., 2013. GENSAT BAC Cre-recombinase driver lines
to study the functional organization of cerebral cortical and basal ganglia circuits. Neuron 80, 13681383.
Gil-Sanz, C., Espinosa, A., Fregoso, S.P., Bluske, K.K., Cunningham, C.L., MartinezGaray, I., Zeng, H., Franco, S.J., Mller, U., 2015. Lineage tracing using Cux2-Cre
and Cux2-CreERT2 mice. Neuron 86, 10911099.
Giusti, S. a, Vercelli, C. a, Vogl, A.M., Kolarz, A.W., Pino, N.S., Deussing, J.M., Refojo,
D., 2014. Behavioral phenotyping of Nestin-Cre mice: implications for genetic
mouse models of psychiatric disorders. J. Psychiatry Res. 55, 8795.
Gong, S., Zheng, C., Doughty, M.L., Losos, K., Didkovsky, N., Schambra, U.B., Nowak,

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i

D. Kawaguchi et al. / Developmental Biology ()

N.J., Joyner, A., Leblanc, G., Hatten, M.E., Heintz, N., 2003b. A gene expression
atlas of the central nervous system based on bacterial articial chromosomes.
Nature 425, 917925.
Hanashima, C., Fernandes, M., Hebert, J.M., Fishell, G., 2007. The role of Foxg1 and
dorsal midline signaling in the generation of Cajal-Retzius subtypes. J. Neurosci.
27, 1110311111.
Hanashima, C., Li, S.C., Shen, L., Lai, E., Fishell, G., 2004. Foxg1 suppresses early
cortical cell fate. Science 303, 5659.
Hanashima, C., Shen, L., Li, S.C., Lai, E., 2002. Brain factor
1 controls the proliferation and differentiation of neocortical progenitor cells through independent mechanisms. J. Neurosci. 22, 65266536.
Harno, E., Cottrell, E.C., White, A., 2013. Metabolic pitfalls of CNS Cre-based technology. Cell Metab. 18, 2128.
Hatini, V., Tao, W., Lai, E., 1994. Expression of winged helix genes, BF
1 and BF
2,
dene adjacent domains within the developing forebrain and retina. J. Neurobiol. 25, 12931309.
Hatini, V., Ye, X., Balas, G., Lai, E., 1999. Dynamics of placodal lineage development
revealed by targeted transgene expression. Dev. Dyn. 215, 332343.
Hayashi, S., Tenzen, T., McMahon, A.P., 2003. Maternal inheritance of Cre activity in
a Sox2Cre deleter strain. Genesis 37, 5153.
Hbert, J.M., McConnell, S.K., 2000. Targeting of cre to the Foxg1 (BF
1) locus
mediates loxP recombination in the telencephalon and other developing head
structures. Dev. Biol. 222, 296306.
Heffner, C.S., Herbert Pratt, C., Babiuk, R.P., Sharma, Y., Rockwood, S.F., Donahue, L.
R., Eppig, J.T., Murray, S. a, 2012. Supporting conditional mouse mutagenesis
with a comprehensive cre characterization resource. Nat. Commun. 3, 1218.
Iwasato, T., Nomura, R., Ando, R., Ikeda, T., Tanaka, M., Itohara, S., 2004. Dorsal
telencephalon-specic expression of Cre recombinase in PAC transgenic mice.
Genesis 38, 130138.
Kasberg, A.D., Brunskill, E.W., Steven Potter, S., 2013. SP8 regulates signaling centers
during craniofacial development. Dev. Biol. 381, 312323.
Kloosterman, W.P., Wienholds, E., de Bruijn, E., Kauppinen, S., Plasterk, R.H.A., 2006.
In situ detection of miRNAs in animal embryos using LNA-modied oligonucleotide probes. Nat. Methods 3, 2729.
Kobayashi, Y., Hensch, T.K., 2013. Germline recombination by conditional gene
targeting with Parvalbumin-Cre lines. Front. Neural Circuits 7, 168.
Li, Y., Gordon, J., Manley, N.R., Litingtung, Y., Chiang, C., 2008. Bmp4 is required for
tracheal formation: a novel mouse model for tracheal agenesis. Dev. Biol. 322,
145155.
Li, Y., Yui, D., Luikart, B.W., McKay, R.M., Li, Y., Rubenstein, J.L., Parada, L.F., 2012.
Conditional ablation of brain-derived neurotrophic factor-TrkB signaling impairs striatal neuron development. Proc. Natl. Acad. Sci. USA 109, 1549115496.
Lim, Y.-S., McLaughlin, T., Sung, T.-C., Santiago, A., Lee, K.-F., OLeary, D.D.M., 2008.
p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and
mapping. Neuron 59, 746758.
Ma, L., Harada, T., Harada, C., Romero, M., Hebert, J.M., Mcconnell, S.K., Parada, L.F.,
Cns, I., 2002. Neurotrophin
3 is required for appropriate establishment of
thalamocortical connections. Neuron 36, 623634.
Miyoshi, G., Fishell, G., 2012. Dynamic FoxG1 expression coordinates the integration
of multipolar pyramidal neuron precursors into the cortical plate. Neuron 74,
10451058.
Nagy, A., 2000. Cre recombinase: the universal reagent for genome tailoring.
Genesis 26, 99109.
Pham, C.T., MacIvor, D.M., Hug, B.A., Heusel, J.W., Ley, T.J., 1996. Long-range disruption of gene expression by a selectable marker cassette. Proc. Natl. Acad. Sci.
USA 93, 1309013095.

Rempe, D., Vangeison, G., Hamilton, J., Li, Y., Jepson, M., Federoff, H.J., 2006. Synapsin I Cre transgene expression in male mice produces germline recombination in progeny. Genesis 44, 4449.
Rodrguez, C.I., Buchholz, F., Galloway, J., Sequerra, R., Kasper, J., Ayala, R., Stewart,
A.F., Dymecki, S.M., 2000. High-efciency deleter mice show that FLPe is an
alternative to Cre-loxP. Nat. Genet. 25, 139140.
Sahara, S., Kawakami, Y., Izpisua Belmonte, J.C., OLeary, D.D.M., 2007. Sp8 exhibits
reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior
cortical area patterning. Neural. Dev. 2, 10.
Schmidt-supprian, M., Rajewsky, K., 2007. Vagaries of conditional gene targeting.
Nat. Immunol. 8, 665668.
Shen, L., Nam, H., Song, P., Moore, H., Anderson, S.A., 2006. FoxG1 haploinsufciency results in impaired neurogenesis in the postnatal hippocampus and
contextual memory decits. Hippocampus 16, 875890.
Shibata, M., Kurokawa, D., Nakao, H., Ohmura, T., Aizawa, S., 2008. MicroRNA
9
modulates Cajal-Retzius cell differentiation by suppressing Foxg1 expression in
mouse medial pallium. J. Neurosci. 28, 1041510421.
Shibata, M., Nakao, H., Kiyonari, H., Abe, T., Aizawa, S., 2011. MicroRNA
9 regulates
neurogenesis in mouse telencephalon by targeting multiple transcription factors. J. Neurosci. 31, 34073422.
Shimamura, K., Hartigan, D.J., Martinez, S., Puelles, L., Rubenstein, J.L., 1995. Longitudinal organization of the anterior neural plate and neural tube. Development
121, 39233933.
Siegenthaler, J. a, Tremper-Wells, B. a, Miller, M.W., 2008. Foxg1 haploinsufciency
reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression. Cereb. Cortex 18, 18651875.
Soriano, P., 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain.
Nat. Genet. 21, 7071.
Takashima, Y., Era, T., Nakao, K., Kondo, S., Kasuga, M., Smith, A.G., Nishikawa, S.,
2007. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129, 13771388.
Taniguchi, H., He, M., Wu, P., Kim, S., Paik, R., Sugino, K., Kvitsani, D., Fu, Y., Lu, J., Lin,
Y., Miyoshi, G., Shima, Y., Fishell, G., Nelson, S.B., Huang, Z.J., 2011. A resource of
Cre driver lines for genetic targeting of GABAergic neurons in Cerebral Cortex.
Neuron 71, 9951013.
Tao, W., Lai, E., 1992. Telencephalon-restricted expression of BF-1, a new member of
the HNF
3/fork head gene family, in the developing rat brain. Neuron 8,
957966.
Wahlsten, D., 1982. Deciency of corpus callosum varies with strain and supplier of
the mice. Brain Res. 239, 329347.
Wei, Q., Condie, B.G., 2011. A focused in situ hybridization screen identies candidate transcriptional regulators of thymic epithelial cell development and
function. PLoS One 6, e26795.
Xuan, S., Baptista, C. a, Balas, G., Tao, W., Soares, V.C., Lai, E., 1995. Winged helix
transcription factor BF
1 is essential for the development of the cerebral
hemispheres. Neuron 14, 11411152.
Zembrzycki, A., Griesel, G., Stoykova, A., Mansouri, A., 2007. Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain. Neural. Dev. 2, 8.
Zembrzycki, A., Perez-Garcia, C.G., Wang, C., Chou, S., OLeary, D.D.M., 2015a.
Postmitotic regulation of sensory area patterning in the mammalian neocortex
by Lhx2. Proc. Natl. Acad. Sci. USA 112, 67366741.
Zembrzycki, A., Stocker, A.M., Leingrtner, A., Sahara, S., Chou, S.J., Kalatsky, V., May,
S.R., Stryker, M.P., OLeary, D.D.M., 2015b. Genetic mechanisms control the
linear scaling between related cortical primary and higher order sensory areas.
eLife 4, e11416.

Please cite this article as: Kawaguchi, D., et al., Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line. Dev. Biol.
(2016), http://dx.doi.org/10.1016/j.ydbio.2016.02.011i