Content uploaded by Andreas Zembrzycki
Author content
All content in this area was uploaded by Andreas Zembrzycki on Jun 21, 2019
Content may be subject to copyright.
Generation and analysis of an improved Foxg1-IRES-Cre driver
mouse line
Daichi Kawaguchia,1,*, Setsuko Saharaa,2, Andreas Zembrzyckia, and Dennis D. M. O’Learya
aMolecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037,
USA
Abstract
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
haploinsufficiency 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 3′UTR of
Foxg1
locus, thus preserving the endogenous
Foxg1
coding region and
un-translated gene regulatory sequences in the 3′UTR, including recently discovered microRNA
targeting 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-IRES-
Cre
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.
Keywords
Foxg1; Telencephalon; Cre recombinase; Knock-in mouse; Haploinsufficiency; MicroRNA
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 specific pattern. Even commonly
used Cre driver lines, however, can have unexpected Cre activity patterns or show
*Corresponding Author: Tel: 858-453-4100 x1449, Fax: 858-558-6207, dkawaguchi@mol.f.u-tokyo.ac.jp.
1Present address: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
2Present address: MRC Centre for Developmental Neurobiology, Kings College London, New Hunts House, Guys Campus, London
SE1 1UL, UK.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of
the resulting proof before it is published in its final citable form. Please note that during the production process errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HHS Public Access
Author manuscript
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Published in final edited form as:
Dev Biol
. 2016 April 01; 412(1): 139–147. doi:10.1016/j.ydbio.2016.02.011.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
complicating phenotypes (Harno et al., 2013; Heffner et al., 2012; Schmidt-supprian 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 field, but reports have revealed
unexpected Cre expression in many tissues outside the central nervous system and a
significant 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 expressed in the emerging telencephalic region starting
around E8.5–E9.0 (Hébert 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 (Hébert 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 significantly 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 (Hébert 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 field. 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 (Hébert and McConnell, 2000). The
dysregulation of expression in the
Foxg1-Cre
line precludes its use as a telencephalon
specific 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; Hébert 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 microRNAs (miRNAs) sites in the
Foxg1
3′UTR 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 PGK-neo 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
haploinsufficiency 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; Hébert and McConnell, 2000; Xuan et al., 1995), recent papers have reported that the
heterozygous
Foxg1-Cre
mice show a variety of significant neurodevelopmental defects
including microcephaly, aberrant cortical area patterning, and impaired neurogenesis in the
Kawaguchi et al. Page 2
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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
Foxg1
-haploinsufficiency.
To overcome these limitations, we have generated a new
Foxg1-IRES-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
-haploinsufficiency. 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 regions from early embryonic stages.
Materials and methods
Mice
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 specificity of Cre
recombination,
Rosa26-LacZ
mice (Soriano, 1999) were crossed to
Foxg1-IRES-Cre
mice.
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 129Sv/ter mouse strain)
at the Salk Genome Manipulation Core Facility. The 9154 bp genomic region of the mouse
Foxg1
gene (flanked by EcoRI sites) was used for the targeting vector. The 5′ fragment
(flanked by EcoRI and AclI sites) and the 3′ fragment (flanked by AclI and EcoRI sites)
were isolated from BamHI flanked 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-flanked PGK-neo was inserted at the AclI site, 27bp
downstream of the stop codon in the 3′UTR of
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 identified 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 (Rodríguez
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′).
Kawaguchi et al. Page 3
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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).
X-gal staining, Nissl staining, immunohistochemistry, in situ hybridization
X-Gal staining was performed on whole mount embryo or 14 μm cryosections. β-
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 postfixed 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 anti-Cux1 (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).
Statistical analysis
Quantitative data in Fig. 1, 5 and 6 are means ± s.e.m for the numbers of brains (n) indicated
in the corresponding figures. Data were compared between groups with one-way ANOVA
(Fig. 1) or unpaired Student’s t test (Fig. 5, 6). A P value of <0.05 was considered
statistically significant.
Results and discussion
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 3′UTR 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-
flanked PGKneo cassettes 27bp after the stop codon of the full length
Foxg1
gene, thus
maintaining the previously described miRNAs targeting sites in 3′UTR 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
3′UTR 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,
confirmed 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
Kawaguchi et al. Page 4
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
removed by crossing founder mice to mice expressing the Flp recombinase under the control
of human actin promoter (Rodríguez et al., 2000) (Fig. 1D). We then examined Foxg1
protein levels in P0 telencephalic lysates. We found no significant 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
Foxg1-IRES-Cre
mouse
line.
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 field. Cre activity produced by this allele was
assessed by examining β-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; Hébert and McConnell, 2000; Shimamura et al., 1995; Tao and Lai, 1992; Xuan et al.,
1995). β-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. 2A–I). Reporter gene expression occurred uniformly in
telencephalic progenitors starting around E9.0–E9.5 (Fig. 2B), but was undetectable in the
cortical hem at E11.5 (Fig. 2C, 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
-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, I).
Although the previous
Foxg1-Cre
line is often used as a telencephalon-specific 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; Hébert 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 targeting sites in the
Foxg1-IRES-Cre
3′UTR, we did
not detect ectopic Cre activity in regions around the mid- and hindbrain at embryonic and
postnatal stages (Fig. 2A–C, Fig. 3A, B), demonstrating that the new
Foxg1-IRES-Cre
line
is an early-active and telencephalon-specific Cre driver line in the brain.
We further examined the β-galactosidase activity patterns at postnatal stages (P7, 1–2 month
old) to confirm the specificity of Cre activity in
Foxg1-IRES-Cre
mice at later stages. We
found that the reporter gene activities were specifically 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,
Kawaguchi et al. Page 5
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
medulla in the thymus, trachea, and esophagus (Fig. 3A–I). We did not observe reporter
gene activities in other organs such as the spinal cord, lung, heart, liver, stomach, pancreas,
spleen, intestine, and kidney (Fig. 3J–R). These results suggest that the new
Foxg1-IRES-
Cre
line produces reliable Cre recombination in
Foxg1
-expressing embryonic tissues and its
derivatives at postnatal stages (1–2 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 influence 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
Foxg1-
IRES-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. 4A–C) 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 specific 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.
Cortical development is not affected in the Foxg1-IRES-Cre mouse line
Heterozygous
Foxg1-Cre
mice, due to
Foxg1
-haploinsufficiency, have significant defects in
cortical development, including microcephaly, aberrant cortical area patterning, reduced
neocortical thickness especially in upper layers, and impaired neurogenesis in 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
Foxg1-IRES-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
Foxg1-IRES-Cre
heterozygous mice did not show
differences in the thickness of total neocortical layers (layer 1 to 6), upper layers (layer 2 to
4), 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)
Kawaguchi et al. Page 6
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
(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 flattened 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
-haploinsufficiency and that overall cortical development
is not affected in
Foxg1-IRES-Cre
heterozygous mice.
Conclusions
We have generated and characterized a new
Foxg1-IRES-Cre
knock-in mouse line to prevent
Foxg1
-haploinsufficiency 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 field. 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-IRES-Cre
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 (Hébert and McConnell, 2000). We also
have demonstrated that use of the
Foxg1-IRES-Cre
mice circumvent defects in the cortical
development caused by
Foxg1
-haploinsufficiency. 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 3′UTR 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 the
same strategy as the
Cre
knock-in line, resulting the removal of the 3′UTR of
Foxg1
gene
from
LacZ
or
tTA
encoding mRNAs (Hanashima et al., 2002; Hatini et al., 1999; Xuan et al.,
1995). As these endogenous 3′UTRs often include gene regulatory sequences like miRNA
targeting 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 specific Cre activity in the brain and early timing of robust Cre activation
Kawaguchi et al. Page 7
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
(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 specifically 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 significantly
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 E8.5–9.5,
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-IRES-Cre
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.
Acknowledgments
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-flanked
PGK-neo and PGK-DTA; Samuel Pfaff (Salk Institute) for providing the plasmid encoding IRES-Cre; and members
of the O’Leary lab for discussions. This work was supported by NIH grants R01 NS031558 and R01 MH086147,
and the Vincent J. Coates Chair of Molecular Neurobiology (D.D.M.O’L.). 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. Distinct developmental origins and
regulatory mechanisms for GABAergic neurons associated with dopaminergic nuclei in the ventral
mesodiencephalic region. Development. 2012; 139:2360–70. [PubMed: 22627282]
Armentano M, Chou SJ, Tomassy GS, Leingärtner A, O’Leary DDM, Studer M. COUP-TFI regulates
the balance of cortical patterning between frontal/motor and sensory areas. Nat Neurosci. 2007;
10:1277–86. [PubMed: 17828260]
Branda CS, Dymecki SM. Talking about a revolution: The impact of site-specific recombinases on
genetic analyses in mice. Dev Cell. 2004; 6:7–28. [PubMed: 14723844]
Choi PS, Zakhary L, Choi WY, Caron S, Alvarez-Saavedra E, Miska EA, McManus M, Harfe B,
Giraldez AJ, Horvitz HR, Schier AF, Dulac C. Members of the miRNA-200 family regulate
olfactory neurogenesis. Neuron. 2008; 57:41–55. [PubMed: 18184563]
Chou SJ, Perez-Garcia CG, Kroll TT, O’Leary DDM. Lhx2 specifies regional fate in Emx1 lineage of
telencephalic progenitors generating cerebral cortex. Nat Neurosci. 2009; 12:1381–89. [PubMed:
19820705]
Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ, Maxson SC,
Miner LL, Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R. Behavioral phenotypes of inbred
mouse strains: Implications and recommendations for molecular studies. Psychopharmacology
(Berl). 1997
Declercq J, Brouwers B, Pruniau VPEG, Stijnen P, de Faudeur G, Tuand K, Meulemans S, Serneels L,
Schraenen A, Schuit F, Creemers JWM. Metabolic and Behavioural Phenotypes in Nestin-Cre Mice
Are Caused by Hypothalamic Expression of Human Growth Hormone. PLoS One. 2015;
10:e0135502. [PubMed: 26275221]
Dou CL, Li S, Lai E. Dual role of brain factor-1 in regulating growth and patterning of the cerebral
hemispheres. Cereb Cortex. 1999; 9:543–50. [PubMed: 10498272]
Kawaguchi et al. Page 8
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Eagleson KL, Schlueter McFadyen-Ketchum LJ, Ahrens ET, Mills PH, Does MD, Nickols J, Levitt P.
Disruption of Foxg1 expression by knock-in of cre recombinase: effects on the development of the
mouse telencephalon. Neuroscience. 2007; 148:385–99. [PubMed: 17640820]
Frullanti E, Amabile S, Lolli MG, Bartolini A, Livide G, Landucci E, Mari F, Vaccarino FM, Ariani F,
Massimino L, Renieri A, Meloni I. Altered expression of neuropeptides in FoxG1-null
heterozygous mutant mice. Eur J Hum Genet. 2015:1–6.
Fuccillo M, Rallu M, McMahon AP, Fishell G. Temporal requirement for hedgehog signaling in
ventral telencephalic patterning. Development. 2004; 131:5031–40. [PubMed: 15371303]
Fujimiya M, Kimura H, Maeda T. Postnatal development of serotonin nerve fibers in the
somatosensory cortex of mice studied by immunohistochemistry. J Comp Neurol. 1986; 246:191–
201. [PubMed: 3082945]
Garaffo G, Conte D, Provero P, Tomaiuolo D, Luo Z, Pinciroli P, Peano C, D’Atri I, Gitton Y, Etzion T,
Gothilf Y, Gays D, Santoro MM, Merlo GR. 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. 2015; 68:103–119. [PubMed: 25937343]
Gerfen CR, Paletzki R, Heintz N. GENSAT BAC Cre-Recombinase Driver Lines to Study the
Functional Organization of Cerebral Cortical and Basal Ganglia Circuits. Neuron. 2013; 80:1368–
83. [PubMed: 24360541]
Gil-Sanz C, Espinosa A, Fregoso SP, Bluske KK, Cunningham CL, Martinez-Garay I, Zeng H, Franco
SJ, Müller U. Lineage Tracing Using Cux2-Cre and Cux2-CreERT2 Mice. Neuron. 2015;
86:1091–1099. [PubMed: 25996136]
Giusti SA, Vercelli CA, Vogl AM, Kolarz AW, Pino NS, Deussing JM, Refojo D. Behavioral
phenotyping of Nestin-Cre mice: implications for genetic mouse models of psychiatric disorders. J
Psychiatr Res. 2014; 55:87–95. [PubMed: 24768109]
Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc
G, Hatten ME, Heintz N. A gene expression atlas of the central nervous system based on bacterial
artificial chromosomes. Nature. 2003; 425:917–25. [PubMed: 14586460]
Hanashima C, Fernandes M, Hebert JM, Fishell G. The role of Foxg1 and dorsal midline signaling in
the generation of Cajal-Retzius subtypes. J Neurosci. 2007; 27:11103–11. [PubMed: 17928452]
Hanashima C, Li SC, Shen L, Lai E, Fishell G. Foxg1 suppresses early cortical cell fate. Science.
2004; 303:56–9. [PubMed: 14704420]
Hanashima C, Shen L, Li SC, Lai E. Brain factor-1 controls the proliferation and differentiation of
neocortical progenitor cells through independent mechanisms. J Neurosci. 2002; 22:6526–36.
[PubMed: 12151532]
Harno E, Cottrell EC, White A. Metabolic pitfalls of CNS Cre-based technology. Cell Metab. 2013;
18:21–8. [PubMed: 23823475]
Hatini V, Tao W, Lai E. Expression of winged helix genes, BF-1 and BF-2, define adjacent domains
within the developing forebrain and retina. J Neurobiol. 1994; 25:1293–309. [PubMed: 7815060]
Hatini V, Ye X, Balas G, Lai E. Dynamics of placodal lineage development revealed by targeted
transgene expression. Dev Dyn. 1999; 215:332–43. [PubMed: 10417822]
Hayashi S, Tenzen T, McMahon AP. Maternal inheritance of Cre activity in a Sox2Cre deleter strain.
Genesis. 2003; 37:51–3. [PubMed: 14595839]
Hébert JM, McConnell SK. Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in
the telencephalon and other developing head structures. Dev Biol. 2000; 222:296–306. [PubMed:
10837119]
Heffner CS, Herbert Pratt C, Babiuk RP, Sharma Y, Rockwood SF, Donahue LR, Eppig JT, Murray
SA. Supporting conditional mouse mutagenesis with a comprehensive cre characterization
resource. Nat Commun. 2012; 3:1218. [PubMed: 23169059]
Iwasato T, Nomura R, Ando R, Ikeda T, Tanaka M, Itohara S. Dorsal telencephalon-specific expression
of Cre recombinase in PAC transgenic mice. Genesis. 2004; 38:130–8. [PubMed: 15048810]
Kasberg AD, Brunskill EW, Steven Potter S. SP8 regulates signaling centers during craniofacial
development. Dev Biol. 2013; 381:312–23. [PubMed: 23872235]
Kawaguchi et al. Page 9
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RHA. In situ detection of miRNAs
in animal embryos using LNA-modified oligonucleotide probes. Nat Methods. 2006; 3:27–9.
[PubMed: 16369549]
Kobayashi Y, Hensch TK. Germline recombination by conditional gene targeting with Parvalbumin-
Cre lines. Front Neural Circuits. 2013; 7:168. [PubMed: 24137112]
Li Y, Gordon J, Manley NR, Litingtung Y, Chiang C. Bmp4 is required for tracheal formation: a novel
mouse model for tracheal agenesis. Dev Biol. 2008; 322:145–55. [PubMed: 18692041]
Li Y, Yui D, Luikart BW, McKay RM, Li Y, Rubenstein JL, Parada LF. Conditional ablation of brain-
derived neurotrophic factor-TrkB signaling impairs striatal neuron development. Proc Natl Acad
Sci U S A. 2012; 109:15491–6. [PubMed: 22949667]
Lim YS, McLaughlin T, Sung TC, Santiago A, Lee KF, O’Leary DDM. p75(NTR) mediates ephrin-A
reverse signaling required for axon repulsion and mapping. Neuron. 2008; 59:746–58. [PubMed:
18786358]
Ma L, Harada T, Harada C, Romero M, Hebert JM, Mcconnell SK, Parada LF, Cns I. Neurotrophin-3
Is Required for Appropriate Establishment of Thalamocortical Connections. Neuron. 2002;
36:623–634. [PubMed: 12441052]
Miyoshi G, Fishell G. Dynamic FoxG1 Expression Coordinates the Integration of Multipolar
Pyramidal Neuron Precursors into the Cortical Plate. Neuron. 2012; 74:1045–1058. [PubMed:
22726835]
Nagy A. Cre recombinase: the universal reagent for genome tailoring. Genesis. 2000; 26:99–109.
[PubMed: 10686599]
Pham CT, MacIvor DM, Hug BA, Heusel JW, Ley TJ. Long-range disruption of gene expression by a
selectable marker cassette. Proc Natl Acad Sci U S A. 1996; 93:13090–5. [PubMed: 8917549]
Rempe D, Vangeison G, Hamilton J, Li Y, Jepson M, Federoff HJ. Synapsin I Cre transgene expression
in male mice produces germline recombination in progeny. Genesis. 2006; 44:44–9. [PubMed:
16419044]
Rodríguez CI, Buchholz F, Galloway J, Sequerra R, Kasper J, Ayala R, Stewart AF, Dymecki SM.
High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet. 2000;
25:139–40. [PubMed: 10835623]
Sahara S, Kawakami Y, Izpisua Belmonte JC, O’Leary DDM. Sp8 exhibits reciprocal induction with
Fgf8 but has an opposing effect on anterior-posterior cortical area patterning. Neural Dev. 2007;
2:10. [PubMed: 17509151]
Schmidt-supprian M, Rajewsky K. Vagaries of conditional gene targeting. Nat Immunol. 2007; 8:665–
668. [PubMed: 17579640]
Shen L, Nam H, Song P, Moore H, Anderson SA. FoxG1 haploinsufficiency results in impaired
neurogenesis in the postnatal hippocampus and contextual memory deficits. Hippocampus. 2006;
16:875–90. [PubMed: 16941454]
Shibata M, Kurokawa D, Nakao H, Ohmura T, Aizawa S. MicroRNA-9 modulates Cajal-Retzius cell
differentiation by suppressing Foxg1 expression in mouse medial pallium. J Neurosci. 2008;
28:10415–21. [PubMed: 18842901]
Shibata M, Nakao H, Kiyonari H, Abe T, Aizawa S. MicroRNA-9 regulates neurogenesis in mouse
telencephalon by targeting multiple transcription factors. J Neurosci. 2011; 31:3407–22. [PubMed:
21368052]
Shimamura K, Hartigan DJ, Martinez S, Puelles L, Rubenstein JL. Longitudinal organization of the
anterior neural plate and neural tube. Development. 1995; 121:3923–33. [PubMed: 8575293]
Siegenthaler JA, Tremper-Wells BA, Miller MW. Foxg1 haploinsufficiency reduces the population of
cortical intermediate progenitor cells: effect of increased p21 expression. Cereb Cortex. 2008;
18:1865–75. [PubMed: 18065723]
Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999; 21:70–
1. [PubMed: 9916792]
Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Nishikawa S. Neuroepithelial cells
supply an initial transient wave of MSC differentiation. Cell. 2007; 129:1377–1388. [PubMed:
17604725]
Kawaguchi et al. Page 10
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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 SB, Huang ZJ. A Resource of Cre Driver Lines for Genetic Targeting of
GABAergic Neurons in Cerebral Cortex. Neuron. 2011; 71:995–1013. [PubMed: 21943598]
Tao W, Lai E. Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head
gene family, in the developing rat brain. Neuron. 1992; 8:957–66. [PubMed: 1350202]
Wahlsten D. Deficiency of corpus callosum varies with strain and supplier of the mice. Brain Res.
1982; 239:329–347. [PubMed: 7093694]
Wei Q, Condie BG. A focused in situ hybridization screen identifies candidate transcriptional
regulators of thymic epithelial cell development and function. PLoS One. 2011; 6:e26795.
[PubMed: 22087235]
Xuan S, Baptista CA, Balas G, Tao W, Soares VC, Lai E. Winged helix transcription factor BF-1 is
essential for the development of the cerebral hemispheres. Neuron. 1995; 14:1141–52. [PubMed:
7605629]
Zembrzycki A, Griesel G, Stoykova A, Mansouri A. Genetic interplay between the transcription
factors Sp8 and Emx2 in the patterning of the forebrain. Neural Dev. 2007; 2:8. [PubMed:
17470284]
Zembrzycki A, Perez-Garcia CG, Wang C, Chou S, O’Leary DDM. Postmitotic regulation of sensory
area patterning in the mammalian neocortex by Lhx2. Proc Natl Acad Sci. 2015a; 112:6736–41.
[PubMed: 25971728]
Zembrzycki A, Stocker AM, Leingärtner A, Sahara S, Chou SJ, Kalatsky V, May SR, Stryker MP,
O’Leary DDM. Genetic mechanisms control the linear scaling between related cortical primary
and higher order sensory areas. eLife. 2015b; 4:e11416. [PubMed: 26705332]
Kawaguchi et al. Page 11
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Highlights
-Generation of an improved Foxg1-IRES-Cre driver mouse line.
-Cre activity in this line recapitulates the endogenous Foxg1 expression
pattern.
-This line lacks neurodevelopmental defects associated with Foxg1
haploinsufficiency.
-This driver will facilitate the genetic dissection of forebrain development/
function.
Kawaguchi et al. Page 12
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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 3′UTR 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 significant.
Kawaguchi et al. Page 13
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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
Foxg1-IRES-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. (C–I) 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,
Kawaguchi et al. Page 14
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Rathke’s pouch; aret, anterior retina; th, thymus rudiment; n, number of mice analyzed.
Scale bars, 300 μm.
Kawaguchi et al. Page 15
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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
Foxg1-IRES-Cre
driver was detected using a
Rosa26-LacZ
reporter based on X-gal staining (blue) at postnatal stages (P7 in A, C and 1–2 months old in
B, D–R). Nuclear Fast Red was used for counterstaining (red). (A–I) Cre activity in
derivative tissues from reporter-positive tissues at embryonic stages. (J–R) 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 A–E, G–R; 100 μm in F.
Kawaguchi et al. Page 16
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Fig. 4. Weak/mosaic Cre activity is detected in some of paternally Foxg1-IRES-Cre inherited
mice
(A–C) Weak/mosaic Cre activity patterns in (A) brain at P7, (B) pituitary gland and (C)
thymus at 1–2 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
μm. (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.
Kawaguchi et al. Page 17
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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
Foxg1-IRES-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 1 to 6) (measured by
Nissl staining) and thickness of upper (layer 2 to 4) 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 significant. Scale bars, 2 mm in A; 300 μm in B.
Kawaguchi et al. Page 18
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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 flattened 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 significant; F/M, frontal/motor cortex; V1, primary visual area; S1,
primary somatosensory area; PMBSF, posterior medial barrel subfield; A1, primary auditory
area. Scale bars, 1 mm.
Kawaguchi et al. Page 19
Dev Biol
. Author manuscript; available in PMC 2018 April 11.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
