A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors

Article (PDF Available)inBMC Developmental Biology 7(1):122 · February 2007with59 Reads
DOI: 10.1186/1471-213X-7-122 · Source: PubMed
Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing. We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type. The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.
BioMed Central
Page 1 of 11
(page number not for citation purposes)
BMC Developmental Biology
Open Access
Methodology article
A new GFP-tagged line reveals unexpected Otx2 protein
localization in retinal photoreceptors
Nicolas Fossat
, Coralie Le Greneur
, Francis Béby
, Stéphane Vincent
Pierre Godement
, Gilles Chatelain
and Thomas Lamonerie*
IGFL, UMR CNRS 5242-INRA 1237-ENS, IFR128 Lyon-Gerland, 46 allée d'Italie, 69364 Lyon Cedex 07, France,
5239 -ENS, IFR128 Lyon-Gerland, 46 allée d'Italie, 69364 Lyon Cedex 07, France,
Institut Pasteur, CNRS URA 2578, 28 rue du Docteur Roux,
75724 Paris cedex 15, France and
Embryology Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW 2145, Australia
Email: Nicolas Fossat - nfossat@cmri.com.au; Coralie Le Greneur - coralie.le.greneur@ens-lyon.fr; Francis Béby - francis.beby@ens-lyon.fr;
Stéphane Vincent - svincent@pasteur.fr; Pierre Godement - pierre.godement@ens-lyon.fr; Gilles Chatelain - gchateli@ens-lyon.fr;
Thomas Lamonerie* - thomas.lamonerie@ens-lyon.fr
* Corresponding author
Background: Dynamic monitoring of protein expression and localization is fundamental to the
understanding of biological processes. The paired-class homeodomain-containing transcription
factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional
knockout studies have pointed to multiple roles of this protein during late development and post-
natal life. Yet, later expression and functions remain poorly characterized as specific reagents to
detect the protein at any stage of development are still missing.
Results: We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2
coding sequence to monitor the gene activity while preserving most of its functions. Our results
demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene
expression from early embryonic stages to adulthood. In addition, we could visualize the
intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein
distribution and show a further level of regulation of intranuclear protein localization, which
depends on the cell type.
Conclusion: The GFP-tagged Otx2 mouse line fully recapitulates previously known expression
patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens
up the way to live imaging of a highly dynamic actor of brain development and can be adapted to
any mutant background to probe for genetic interaction between Otx2 and the mutated gene.
Studying the expression and intracellular localization of
transcription factors is a difficult task because both may be
highly dynamic. This is precisely the case for Otx2. Mouse
Otx2 is a paired-class homeobox gene that belongs to a
gene family also containing Otx1 and the more divergent
Crx [1]. It plays critical roles in early brain induction and
development [2]. It is expressed in a very dynamic fashion
in areas of the central nervous system (CNS) that rapidly
change as development proceeds [3-5]. Several germinal
and conditional knock-out studies have emphasized its
involvement in multiple functions such as head forma-
Published: 2 November 2007
BMC Developmental Biology 2007, 7:122 doi:10.1186/1471-213X-7-122
Received: 28 June 2007
Accepted: 2 November 2007
This article is available from: http://www.biomedcentral.com/1471-213X/7/122
© 2007 Fossat et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 2 of 11
(page number not for citation purposes)
tion [6-8], photoreceptor fate determination [9] or post-
natal survival and growth [10].
Despite the great number of genetic models generated to
address its activities, there is still a need for tools to study
the complex dynamics of expression of this gene in the
CNS. Indeed, one major problem is the lack of specific
Otx2 antibody. The strong sequence similarity between
Otx1 and Otx2 has made it difficult to raise specific sera,
the use of which is therefore particularly delicate [4,11].
The distribution of Otx2 protein has been first investi-
gated during mouse development [4] with a polyclonal
serum that was later shown to cross react with Otx1 pro-
tein [12] and then in newborn rats [11]. In adults, aside
from few studies such as in the retina [13], no general
study has been carried out. As a result, unambiguous Otx2
expression data mostly rely on mRNA detection [5,14].
Otx2 in situ hybridization (ISH) is widely used to charac-
terize the patterning and development of anterior neur-
oectoderm. However, several studies have raised the
possibility of translational regulation at gastrula stage
[15,16], and recent work suggested that Otx2 specific
miRNA decay might time the generation of retinal neu-
rons [17]. Therefore, true Otx2 expression analysis should
rely on protein rather than mRNA detection. Several LacZ
reporter lines have been created [6,18] but due to proba-
ble deletion of splicing and regulatory sequences [19] or
mRNA nonsense mediated decay, none of them allowed
the complete monitoring of Otx2 gene expression. In
addition, these models do not allow the precise determi-
nation of Otx2 protein intracellular localization. Yet, this
appears to be tightly regulated in the developing retina
[13,20]. Moreover it would be interesting to be able to
examine OTX2 expression and detailed localization, and
to identify directly Otx2-expressing cells, in live tissues.
To overcome these difficulties, we have generated a new
mouse line harbouring a GFP tag within Otx2 natural
genome context. Genetic modifications were made to
ensure as a normal expression as possible. This line
allowed to visualize the full Otx2 development pattern
and to discover an unexpected control of Otx2 protein
subcellular localization.
Generation of an Otx2
reporter line
In order to clearly identify Otx2 expressing cells during
mouse development and throughout life, we created a
new allele bearing the MuGFP coding sequence [21] in
frame with Otx2 coding sequence (Figure 1). We had pre-
viously shown that fusing the GFP polypeptide both at N-
and C-terminus of Otx2 does not modify its in vitro local-
ization, DNA binding, and transcription properties [22].
We chose a C-terminal GFP fusion, and put an excisable
neo selection cassette immediately downstream the GFP
stop codon. Since subtle modifications of the Otx2 3'UTR
coding region result in impaired mRNA translation in
early embryos [16], we took particular care to keep this
part of the gene as intact as possible. After homologous
recombination of the targeting molecule in ES cells, neo
selection cassette was removed by flp recombinase medi-
ated excision, leaving behind a single FRT site between
GFP stop codon and Otx2 3'UTR (Figure 1a). All steps of
homologous recombination and neo excision were mon-
itored by appropriate PCR and southern blot analyses
(Figure 1b–d).
Recombinant ES cells were injected into recipient blasto-
cysts to generate the Otx2
transgenic line. Chi-
maeric males were obtained who transmitted the Otx2
allele (thereafter referred to as Otx2
allele) to their
Hypomorphic phenotypes in Otx2
Unexpectedly, the breeding of Otx2
mice yielded only
4% living homozygous and fertile Otx2
(Table 1), meaning the Otx2
allele is hypomorphic. This
was surprising for two reasons. First, Otx2 and Otx2-GFP
protein display identical activities in vitro [22]. Second,
× Otx2
crosses yielded 50% heterozygous ani-
mals (Table 1), whereas in the 129/Sv background, only
30% hemizygous Otx2
newborn survive [10]. To check
for defects in homozygous embryos, mice were sacrificed
at various gestational stages. Homozygous embryos were
found in mendelian proportions up to birth (see Table 1),
but showed variable facial abnormalities (Figure 2b). This
evoked a problem of gene dosage or protein activity. West-
ern blot analysis of E9.5 heterozygous embryos showed
comparable amount of Otx2 and Otx2-GFP proteins, indi-
cating a similar expression level of both alleles (Figure 2c).
Thus, activity rather than quantity of Otx2-GFP protein
must be rate limiting, though subtly since it is sufficient to
achieve complete mouse development in a small propor-
tion of animals.
To understand in which manner and when this activity
could be limiting, we explored the major early functions
of Otx2 in homozygous Otx2
embryos. It is known
that this gene controls anterior visceral endoderm (AVE)
movement and subsequent gastrulation [18], but mor-
phological examination showed no obvious difference
between Otx2
and Otx2
embryos up to E8.5 (Fig-
ure 2a). This indicates that AVE migration and gastrula-
tion proceeded normally. By contrast, we could observe
variable brain reduction from E9.5 on (Figure 2b). Otx2 is
required in the anterior neuroectoderm to properly
receive signals from the anterior neural ridge (ANR) and
to maintain forebrain development [23]. Six3 is a good
indicator of forebrain response to ANR induction. In E9.5
embryos of mild or strong phenotype, Six3 was
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 3 of 11
(page number not for citation purposes)
expressed, confirming normal forebrain induction and
maintenance (Figure 2b). As Otx2 itself monitors fore-
brain maintenance, Otx2-GFP fluorescence present in
every phenotype confirmed an established forebrain iden-
tity. It is well known that Otx2 controls the compartmen-
talization and maturation of the forebrain and midbrain
[24]. In particular, it is necessary to set the midbrain-hind-
brain boundary (MHB). Although a slight anterior shift of
Fgf8 expression could not be completely excluded, Fgf8
and Gbx2 pattern showed no obvious alteration (Figure
2b), indicating that the MHB was correctly established.
On the contrary, the En2 domain, which normally labels
mesencephalon, appeared shifted close to the telen-
cephalon and was reduced in strong phenotypes, showing
Generation of the Otx2
alleleFigure 1
Generation of the Otx2
allele. a. The structure of targeting vector (first line), Otx2 wild type locus (second line), tar-
geted OGN allele after homologous recombination (third line) and OG allele after removal of the Neo cassette by FLP recombi-
nase (fourth line) is presented. Gray boxes are Otx2 coding regions, whites boxes are Otx2 5' and 3' UTR regions, yellow box
is PGK-Neo selection cassette, green box is muGFP cDNA. Red triangles are FRT sites. Bent arrows symbolize the three main
transcription starts sites known for Otx2 gene [19, 32]. Dotted lines show BamH I fragments detected by southern blot analysis
using probe represented by a thick line. PCR primers used are indicated by arrows. Product obtained with A and B primers is
shown. Scale bar and sizes of fragments are indicated. b. PCR analysis of Neo
ES clones using primers A and B showing two
non-homologous (N) and one homologous (H) recombinants. c. Southern blot analysis using BamH I digested genomic DNA
and probe indicated in a of wild type and homologous recombinant clones. Genotypes are indicated. d. PCR genotyping of
mice produced from Otx2
ES cells. Analyse was done using C, D and E primers. Sizes, schematic representations of ampli-
fied fragments (see part a for legend) and deduced genotypes are indicated.
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 4 of 11
(page number not for citation purposes)
partial or complete disappearance of diencephalon and
mesencephalon respectively – in all cases, however, Six3
expression showed that telencephalon developed nor-
mally (Figure 2b). Altogether, these data demonstrate that
Otx2-GFP protein activity possesses most essential activi-
ties of native Otx2, but that it may in some instances be
rate limiting for diencephalon and mesencephalon matu-
ration. Interestingly, analysis of surviving homozygous
adults showed no obvious difference with het-
erozygous animals. For instance photoreceptor cells of the
retina, which are highly sensitive to Otx2 expression [9],
appeared normal in Otx2
adults (Figure 2d). To assay
for photoreceptor cells health status, we performed
immunostaining of the glial fibrillary acidic protein
(GFAP) in glial Müller cells. This protein is up regulated in
Müller cells in a variety of degenerative conditions [25].
The absence of marked GFAP induction in Otx2
ina confirmed that photoreceptors were healthy thus
demonstrating the functionality of Otx2-GFP protein in
these neurons. For the sake of animal care simplicity, het-
erozygous animals were used for the continuation of this
Expression pattern in Otx2
To test whether the Otx2
mouse line is an accurate
reporter of Otx2 gene activity, we analyzed Otx2-GFP
expression pattern. First, early expression of the transgenic
allele was studied by direct fluorescence observation of
Characterization of Otx2
homozygous embryosFigure 2
Characterization of Otx2
homozygous embryos. a. Morphology of Otx2
and control Otx2
animals at E8.5.
b. Analysis of Otx2-GFP and brain markers in Otx2
and control Otx2
E9.5 embryos. Otx2-GFP is seen by direct GFP
fluorescence visualisation and Six3, En2, Gbx2 and Fgf8 mRNAs are detected by whole-mount in situ hybridization. For E9.5
animals, upper and lower panels show embryos with weak and strong abnormalities respectively. Anterior is left-
ward. Scale bar: 500 µm. c. Detection of Otx2, Otx2-GFP and Otx1 proteins by western blot analysis of nuclear extracts from
tails and heads of Otx2
and Otx2
E9.5 embryos using an anti-Otx antibody. d. Normal structure of the retina in surviving
adults. Comparison of bright field (BF left panels), GFP fluorescence (middle panels) and GFAP expression (right pan-
els) in adult retinas of the indicated genotype. Scale bars : 50 µm.
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 5 of 11
(page number not for citation purposes)
Table 1: Genotypes of offspring obtained from Otx2
× Otx2
and Otx2
× Otx2
intercrosses at various stages of
Genotype +/+ +/OG OG/OG Total
Intercrosses Stage
+/OG × +/+ Post-natal 149 146 295
% 50,5 49,5
+/OG × +/OG E7.5 0 2 5
E13.5 3 4 3
E15.5 1 6 1
E17.5 2 5 4
E18.5 4 2 1
Ante natal 44 98 47 189
% 23,3 51,9 24,9
Post-natal (P10–P15) 28 47 3 78
% 35,9 60,3 3,8
Early Otx2-GFP expression in epiblast and AVEFigure 3
Early Otx2-GFP expression in epiblast and AVE. a. GFP fluorescence and transmission observations of Otx2
and con-
trol Otx2
embryos at E6.5 and E7.5. Anterior is leftward. Scale bars: 100 µm. b. Direct visualisation of GFP fluorescence
(green) and immunofluorescent staining of F-actin (Phalloidin – red) and DNA (Hoechst – blue) on 10 µm thin uterus sections
containing E6.5 Otx2
embryo. Green staining in and around the extraembryonic part of the conceptus is not due to GFP flu-
orescence but to autofluorescence of decidual and blood cells that are present in the tissue. Images on the right correspond to
magnification of white box. Arrow indicates the boundary between embryonic and extra-embryonic part of the conceptus.
Dotted line delimits the frontier between anterior visceral endoderm (ave) and epiblast (ep). Anterior is leftward. Scale bars:
50 µm.
E6.5 and E7.5 embryos. As expected, strong GFP fluores-
cence was detected in the embryonic part of E6.5 Otx2
embryos and became regionalized toward the anterior
pole in E7.5 embryos (Figure 3a). As Otx2 translation in
epiblast has been shown to be very sensitive to locus mod-
ification [16], we wondered whether Otx2-GFP was cor-
rectly expressed both in epiblast and anterior visceral
endoderm (AVE). To verify this, we observed sections of
E6.5 embryos. Indeed, nuclear Otx2-GFP was found in
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 6 of 11
(page number not for citation purposes)
both layers (Figure 3b). This expression pattern matches
exactly the one described for endogenous Otx2 gene [6].
Otx2-GFP protein was then analyzed in developing heter-
ozygous embryos between E8.5 and E12.5 (Figure 4) and
compared to Otx2 mRNA pattern. Again, the dynamics of
Otx2-GFP protein expression paralleled that of Otx2
mRNA at all stages. In line with this, we noticed at E10.5
and E11.5 a rapid decrease of Otx2-GFP protein in telen-
cephalic areas where mRNA was down-regulated. Trans-
verse sections across developing sensory organs of E12.5
embryos show GFP signals in olfactory epithelium, eye
and inner ear (Figure 4i–k) that are characteristic of Otx2
pattern, indicating that Otx2-GFP products recapitulate
endogenous Otx2 expression.
We then addressed Otx2-GFP expression at later stages.
This was of particular importance because a previous
reporter line failed to express ß-galactosidase beyond
E12.5 [6]. In E16.5 sections, as expected again, expression
was found in olfactory epithelium, diencephalon, roof of
mesencephalon, and choroid plexuses (Figure 5a–d). In
P2 brain sections, this expression was maintained. We
also identified a diffuse group of Otx2-GFP positive cells
in the basal telencephalon (Figure 5f). Furthermore, our
line allowed to easily visualize gradients of Otx2 expres-
sion: such was the case in superior colliculus (SC), where
superficial optic layers showed stronger labelling than
deep layers, and in cerebellum with high posterior to low
anterior labelling of the external granular layer (Figure 5g,
h). In adults, very strong labelling could still be seen,
among other locations, in choroids plexuses, thalamus as
well as SC (Figure 5i–m). Thus, the Otx2
allele appears
to be functional from early development till adulthood.
To evaluate the sensitivity of detection of the Otx2-GFP
protein based on the direct fluorescence emitted by GFP,
we compared it with that based on immunostaining using
an anti-GFP antibody and a secondary fluorescent anti-
body. As shown in Figure 5k–m for neurons in the thala-
mus (ventral lateral geniculate nucleus), both methods
Dynamics of Otx2-GFP expression at mid-gestationFigure 4
Dynamics of Otx2-GFP expression at mid-gestation. Direct visualisation of GFP fluorescence of Otx2
embryos and
one control Otx2
E11.5 embryo (a-d) versus Otx2 mRNA localisation detected by whole-mount in situ hybridization on
animals (e-h) at indicated stages. Anterior is leftward. The arrow indicates the position of the midbrain-hindbrain
boundary. Lower right panels: direct detection of GFP fluorescence of transverse sections of E12.5 Otx2
embryo at the
level of nasal cavities (i), eyecup (j) and inner ear (k). di, diencephalon; mes, mesencephalon; oe, olfactory epithelium; ov, optic
vesicle; tel, telencephalon. Scale bars: 500 µm in a-d, 100 µm in i-l.
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 7 of 11
(page number not for citation purposes)
detect the same number of Otx2-GFP positive neurons.
Similar observations were made in the retina and superior
colliculus (not shown). We conclude that virtually all sites
of Otx2 protein expression can be recorded by direct
observation of the GFP fluorescence in the Otx2
mouse line. Use of amplification reactions might of
course further reveal sites of Otx2 expression, particularly
at the subcellular level.
Otx2 is located at the inner face of nuclear envelope in
retinal photoreceptors
We then wondered whether Otx2-GFP intracellular local-
ization exhibits a regulation similar to the one of native
Otx2 protein. In adult mouse retina, a previous study
using immunofluorescence with an Otx2 antibody
described the protein as nuclear in retinal pigment (RPE)
and bipolar cells, but cytoplasmic in photoreceptor (PR)
cells [13]. In embryonic chick retina, the protein was
found in the nuclei of photoreceptor cells [26]. We there-
fore used Otx2-GFP to carefully examine cellular localiza-
tion of Otx2 protein in the retina of adult mice using
confocal microscopy. As expected, Otx2-GFP was distrib-
uted throughout the nuclear space in RPE cells and in cells
located at the outermost part of the internal nuclear layer
(INL) (Figure 6a, b), which correspond to bipolar cells
[13]. In photoreceptor cells, which have very little cyto-
plasm around their soma, Otx2-GFP appeared to sur-
round the PR nuclei, but we could not determine whether
it was excluded or not from the nucleus. To resolve this
issue, we labelled the nuclear envelope with lamin-B and
lamin A/C antibodies (Figure 6 and data not shown). This
showed diffuse strictly nuclear localization for Otx2-GFP
in RPE cells and bipolar cells (Figure 6c, d), but also in PRs
– in these cells, the protein concentrated at the periphery
of the nuclei, facing the inner nuclear envelope (Figure
6e–e"). To rule out the possibility of an artefact due to
GFP as the cause of this unexpected protein distribution in
PR cells, we analyzed native Otx2 localization in PRs of
Efficient Otx2-GFP detection during late-development and in adultsFigure 5
Efficient Otx2-GFP detection during late-development and in adults. a-j. Direct visualisation of GFP fluorescence on
10µm thin section of E16.5 (a-d), P2 (e-h) and adult (i-j) Otx2
animals. b-d. Magnifications of part delimitated by the corre-
sponding red boxes in image a. f-h. Magnifications of part delimitated by the corresponding red boxes in image e. Insets in a
and e are Hoechst staining of the same sections (left) or control fluorescence of 10 µm thin sections of Otx2
animals (right).
Anterior is leftward. k-m. Comparison of Otx2-GFP detection by anti-GFP antibody (k) or direct GFP fluorescence (l) in
the ventral lateral geniculate nucleus of adult Otx2
animals. cb, cerebellum; cp, choroid plexus; cx, cortex; di, diencephalon;
ic, inferior colliculus; mes, mesencephalon; oe, olfactory epithelium; sc, superior colliculus. Scale bars: 500 µm (a, e, i, j) and 10
µm (k-m).
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 8 of 11
(page number not for citation purposes)
wild type mice by immunofluorescence using an anti-Otx
antibody (which reacts against both Otx1 and Otx2)
together with an anti-lamin-B antibody. As there is no
reported Otx1 gene expression in PRs, we interpret the
anti-Otx signal as the detection of Otx2 protein. We
obtained the same result as with Otx2-GFP (Figure 6f–f"),
demonstrating that Otx2 protein definitely adopts a peri-
nuclear location in photoreceptors.
We have generated a new mouse line which is unique for
direct visualisation of Otx2 gene activity. Until now, no
other reporter line existed that sustains Otx2 driven
expression from early embryonic stages to adulthood. As
a result of the knock-in fusion strategy in the mouse
line, the Otx2-GFP protein is expressed at physi-
ological levels and appears to display most activities and
Regulated Otx2-GFP nuclear localization in adult retinaFigure 6
Regulated Otx2-GFP nuclear localization in adult retina. Confocal microscopy analysis. a, b. Direct visualisation
of GFP fluorescence (green) and propidium iodide staining of DNA (red) on 10 µm thin section of Otx2
(a) and control
(b) adult retina. c-f". Localisation of Otx2-GFP (direct visualisation of GFP fluorescence – green, c,d,e',e"), immun-
ofluorescent staining of nuclear envelope (lamin B – red, c,d,e,e", f, f") and immunofluorescent staining of Otx2 (green – f',
f") on 10 µm thin section of Otx2
and wild type adult retina showing retinal pigment epithelium cells (RPE, c), outer cells of
the inner nuclear layer (INL, d) and photoreceptor cells of the outer nuclear layer (ONL, e-f"). Image e" and f" are overlays of
images e and e' and of images f and f' respectively. Scale bars: 10 µm.
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 9 of 11
(page number not for citation purposes)
subcellular localization as normal Otx2 protein. In addi-
tion, it provides a straightforward mean to discriminate
Otx2- from Otx1-expressing cells throughout the mouse
brain. Homozygous Otx2
animals may develop nor-
mally but often show mild diencephalic and mesen-
cephalic maturation defects. Interestingly, this reveals that
these structures are the most demanding ones for Otx2
activity, in line with the frequent ocular and brain defects
found in heterozygous mice and human mutants [8,27].
The strong signal to noise ratio of the GFP provides a con-
venient readout of the Otx2 gene dynamics of expression
even in living animals. Furthermore, using the Otx2
allele in combination with modified versions of genes
controlling anterior neuroectoderm patterning and devel-
opment will provide a simple way to monitor normal and
perturbed cellular movements.
There is increasing evidence that beyond elaborated con-
trol of expression through regulation of transcription by
complex enhancers, developmental genes may see their
function further modulated by other posttranscriptional
mechanisms. Here, the expression of GFP-tagged Otx2
from its endogenous locus helped us to unravel cell-spe-
cific control of protein localization within the retina.
Using the mouse Otx2
line, we disclosed that in adult
PR cells, Otx2 protein is not cytoplasmic as previously
thought, but restricted to a small volume at the inner
periphery of nuclei. Interestingly, similar tight regulation
of localization was found for the rod photoreceptor spe-
cific nuclear receptor Nr2e3 [28]. The significance of such
an unusual location for a transcription factor is presently
mysterious. The inner face of nuclear envelope gathers
silent heterochromatin but also highly expressed DNA
domains at the vicinity of nuclear pores [29]. The protein
could be sequestrated at the periphery of the PR nucleus
in order to regulate the amount of free active Otx2 in the
nucleoplasm. Alternatively, all Otx2 proteins at the
periphery of the nucleus could be transcriptionally active,
bringing loops of DNA containing Otx2-regulated genes
to the vicinity of nuclear pores to facilitate mRNA export.
Indeed, the chromatin appears to be least condensed at
the nuclear periphery of photoreceptor cells [30]. Fish
analysis of Otx2 target gene should test this hypothesis.
Another possibility is that a specific fraction of chromatin
resides at the edge of the nucleus, to which Otx2 and other
transcription factors, such as Nr2e3 would be associated.
Among the questions raised by this study are the follow-
ing: when does this pattern take place during PR differen-
tiation and does it have a specific role in PR genetic
expression and function ? Conditional ablation of Otx2
function in mature PR will certainly bring interesting
answers to these questions.
The methodology presented here describes the GFP tag-
ging of an important transcription factor from its own
gene locus. This leads to the creation of transgenic animals
where both tissue and cell specific gene expression as well
as precise intracellular protein localization can be easily
followed. The established line allows to monitor the com-
plete expression pattern of Otx2 gene during mouse life
without any interference from Otx1 gene products. Its sen-
sitivity leads us to identify peculiar aspects of Otx2 protein
expression and cellular localization. This last aspect
reveals a new level of regulation for developmental gene
products – cell-type dependent control of intra-nuclear
distribution – which should be taken into account in
future studies.
Generation of the targeting vector, mouse lines production
and genotyping
For Otx2
targeting molecule, a 2.5 kb genomic fragment
encompassing the full 3'UTR of Otx2 gene and 1.5 kb
downstream sequence was amplified using primers 5'-
ACGTACTAGTagacctgtagaagctat and 5'-ACGTACTAG-
Taagtcttgactaggagt and inserted after Spe I-Nhe I restric-
tion into the Spe I site of pOtx2-GFP plasmid [22],
immediately downstream the BamHI site at the 3' end of
GFP sequence. A BamH I-Bgl II PGK-neo cassette flanked
by FRT sites [10] was inserted into the above BamH I site,
resulting in pOtx-GFP-neo-3' plasmid. A 3.3 kb EcoR I –
Aat II Otx2 genomic fragment encompassing Otx2 exon 2
and part of exon 3 was then substituted to the 5' Otx2
cDNA EcoR I-Aat II fragment of pOtx-GFP-neo-3' plasmid.
The 5' homology region was further extended by inserting
a neighbouring 2.3 kb EcoRI Otx2 genomic fragment
encompassing Otx2 exon 1C, into the unique EcoR I site
of the above plasmid. The Not I linearized targeting plas-
mid was electroporated into ENS ES cells [31], homolo-
gous recombinant clones were identified by PCR using
primers A and B and confirmed by southern blot analysis
of BamH I digested genomic DNA with the indicated
probe (Figure 1c). One positive clone was transfected with
a plasmid (pFlp-puro) expressing the flp recombinase to
remove the neo cassette. Mice were produced by standard
blastocyst injection procedure. Genotypes were assessed
by PCR using the primers C, D and E (Figure 1d). Animals
were maintained in the 129/Sv background. Day of vagi-
nal plug was taken as E0.5. All animals were handled in
accordance with French regulation. Protocols were
approved by CREEA, the local ethic committee for animal
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 10 of 11
(page number not for citation purposes)
Analyses of embryos, brains and retinas
For whole mount analyses, samples were dissected in PBS
and directly visualised for GFP fluorescence or fixed in 4%
paraformaldehyde (PFA) for in situ analyses. For sections
analyses, samples were dissected in PBS, directly frozen on
dry ice for E6.5 or fixed 1 hour in 4% PFA at 4°C, rinsed
3 times in PBS, protected over-night in 30% sucrose and
frozen in cryomount. 10 µm section were made, fixed 10
minutes in 4% PFA for E6.5, rinsed 3 times in PBS and
mounted in Gel/Mount (Biomeda, CA, USA) or processed
before with Hoechst (Ref. 33258, Sigma-Aldrich, Saint
Louis, USA) staining (1:200, 5 minutes), Phalloidin (Ref.
A34055, Molecular Probes, OR, USA) staining (1:50, 1
hour), Propidium Iodide (Ref. P4170, Sigma-Aldrich,
Saint Louis, USA) staining (1:10000, 15 minutes) or
Immunostaining followed by 3 times rinsed in PBS. In situ
hybridization and Immunofluorescence were done as pre-
viously described [10]. Probes for in situ hybridization are:
0.15 kb Otx2 exon2; Otx2, Six3, En2, Fgf8 and Gbx2 probes
were gifts from S.L. Ang, A.P. McMahon, E.J. Robertson, G.
Martin and A. Joyner, respectively. Primary antibodies
used: 1:100 Goat anti-lamin B (M-20): sc-6217 (Santa
Cruz Biotechnology, Santa Cruz, USA), 1:300 Rat polyclo-
nal anti-Otx (gift from M. Wassef), 1:500 Rabbit anti-GFP
(Invitrogen, Carlsbad CA, USA) and 1:200 Goat anti-
GFAP (Santa Cruz Biotechnology).
Microscope analysis
Images were done with fluorescent stereomicroscope
"Lumar", wide field microscope "Axioplan" or "Axiovert"
and confocal microscope "LSM 510" from Zeiss, Jena,
Western blot analysis
Extraction of nuclear proteins from E9.5 head (Region in
front of MHB) and tail (Region of the most posterior part
of the embryo in volume equivalent of the region of the
head) was done as previously described [22]. Extracts (15
µg) were subjected to electrophoresis on 6 to 18% gradi-
ent gel and transferred to nitrocellulose according to
standard protocol. Rat anti-Otx antibody was used at
1:2000 dilution.
ANR: anterior neural ridge. AVE: anterior visceral endo-
derm. CNS: central nervous system. E: Embryonic day. ES:
Embryonic Stem Cells. GFP: green fluorescent protein.
INL: inner nuclear layer. ISH: in situ hybridization. MHB:
midbrain-hindbrain boundary. ONL: outer nuclear layer.
PR: photoreceptor. RPE: retinal pigment epithelium. SC:
superior colliculus.
Authors' contributions
NF was involved in designing the study, performed micro-
scopy, histology, ISH, western blot analysis and helped to
draft the manuscript. CLG and FB raised the line and per-
formed genotyping, statistics and microscopy. PG did the
vLGN immunostaining and improved the manuscript. SV
performed the homologous recombination of targeting
molecule in ES cells and neo cassette excision. GC started
to raise the line and performed initial fluorescence analy-
ses. TL conceived and supervised the study, generated the
constructs, performed genotyping, statistics and micros-
copy and drafted the manuscript. All authors have read
and approved the final manuscript.
Authors thank the PBES crew for injection of ES cells into blastocysts and
expert handling of mice, A. Ottaviani for anti-lamin antibodies, M. Wassef
and A. Prochiantz for anti-Otx antibody, C. Medina-Palazon and F. Mure for
assistance with the gradient gel system and many other colleagues for plas-
mids and reagents. Claire Lionnet and Fabienne Simian from PLATIM gave
us decisive help for microscopy. Lotty Moon kindly proofread the article.
This work was supported by grants from the CNRS and Retina France to
TL. NF received a postdoctoral fellowship from Retina France.
1. Germot A, Lecointre G, Plouhinec JL, Le Mentec C, Girardot F,
Mazan S: Structural evolution of Otx genes in craniates. Mol
Biol Evol 2001, 18:1668-78.
2. Rhinn M, Dierich A, Shawlot W, Behringer RR, Le Meur M, Ang SL:
Sequential roles for Otx2 in visceral endoderm and neuroec-
toderm for forebrain and midbrain induction and specifica-
tion. Development 1998, 125:845-56.
3. Frantz GD, Weimann JM, Levin ME, McConnell SK: Otx1 and Otx2
define layers and regions in developing cerebral cortex and
cerebellum. J Neurosci 1994, 14:5725-5740.
4. Mallamaci A, Di Blas E, Briata P, Boncinelli E, Corte G: OTX2 home-
oprotein in the developing central nervous system and
migratory cells of the olfactory area. Mech Dev 1996,
5. Simeone A, Acampora D, Mallamaci A, Stornaiuolo A, D'Apice MR,
Nigro V, Boncinelli E: A vertebrate gene related to orthodenti-
cle contains a homeodomain of the bicoid class and demar-
cates anterior neuroectoderm in the gastrulating mouse
embryo. Embo J 1993, 12:2735-47.
6. Acampora D, Mazan S, Lallemand Y, Avantaggiato V, Maury M, Sime-
one A, Brulet P: Forebrain and midbrain regions are deleted in
Otx2-/- mutants due to a defective anterior neuroectoderm
specification during gastrulation. Development 1995,
7. Ang SL, Jin O, Rhinn M, Daigle N, Stevenson L, Rossant J: A targeted
mouse Otx2 mutation leads to severe defects in gastrulation
and formation of axial mesoderm and to deletion of rostral
brain. Development 1996, 122:243-252.
8. Matsuo I, Kuratani S, Kimura C, Takeda N, Aizawa S: Mouse Otx2
functions in the formation and patterning of rostral head.
Genes Dev 1995, 9:2646-2658.
9. Nishida A, Furukawa A, Koike C, Tano Y, Aizawa S, Matsuo I, Furu-
kawa T: Otx2 homeobox gene controls retinal photoreceptor
cell fate and pineal gland development. Nat Neurosci 2003,
10. Fossat N, Chatelain G, Brun G, Lamonerie T: Temporal and spatial
delineation of mouse Otx2 functions by conditional self-
knockout. EMBO Rep 2006, 7:824-30.
Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
BMC Developmental Biology 2007, 7:122 http://www.biomedcentral.com/1471-213X/7/122
Page 11 of 11
(page number not for citation purposes)
11. Nothias F, Fishell G, Ruiz i Altaba A: Cooperation of intrinsic and
extrinsic signals in the elaboration of regional identity in the
posterior cerebral cortex. Curr Biol 1998, 8:459-62.
12. Acampora D, Avantaggiato V, Tuorto F, Briata P, Corte G, Simeone
A: Visceral endoderm-restricted translation of Otx1 medi-
ates recovery of Otx2 requirements for specification of ante-
rior neural plate and normal gastrulation. Development 1998,
13. Baas D, Bumsted KM, Martinez JA, Vaccarino FM, Wikler KC, Barn-
stable CJ: The subcellular localization of Otx2 is cell-type spe-
cific and developmentally regulated in the mouse retina.
Brain Res Mol Brain Res 2000, 78:26-37.
14. Rath MF, Munoz E, Ganguly S, Morin F, Shi Q, Klein DC, Moller M:
Expression of the Otx2 homeobox gene in the developing
mammalian brain: embryonic and adult expression in the
pineal gland. J Neurochem 2006, 97:556-66.
15. Acampora D, Boyl PP, Signore M, Martinez-Barbera JP, Ilengo C,
Puelles E, Annino A, Reichert H, Corte G, Simeone A: OTD/OTX2
functional equivalence depends on 5' and 3' UTR-mediated
control of Otx2 mRNA for nucleo-cytoplasmic export and
epiblast-restricted translation. Development 2001, 128:4801-13.
16. Boyl PP, Signore M, Acampora D, Martinez-Barbera JP, Ilengo C,
Annino A, Corte G, Simeone A: Forebrain and midbrain devel-
opment requires epiblast-restricted Otx2 translational con-
trol mediated by its 3' UTR. Development 2001, 128:2989-3000.
17. Decembrini S, Andreazzoli M, Vignali R, Barsacchi G, Cremisi F: Tim-
ing the generation of distinct retinal cells by homeobox pro-
teins. PLoS Biol 2006, 4:e272.
18. Kimura C, Yoshinaga K, Tian E, Suzuki M, Aizawa S, Matsuo I: Vis-
ceral endoderm mediates forebrain development by sup-
pressing posteriorizing signals. Dev Biol 2000, 225:304-21.
19. Courtois V, Chatelain G, Han ZY, Le Novere N, Brun G, Lamonerie
T: New Otx2 mRNA isoforms expressed in the mouse brain.
J Neurochem 2003, 84:840-53.
20. Rath MF, Morin F, Shi Q, Klein DC, Moller M: Ontogenetic expres-
sion of the Otx2 and Crx homeobox genes in the retina of
the rat. Exp Eye Res 2007.
21. Zernicka-Goetz M, Pines J, Ryan K, Siemering KR, Haseloff J, Evans MJ,
Gurdon JB: An indelible lineage marker for Xenopus using a
mutated green fluorescent protein. Development 1996,
22. Chatelain G, Fossat N, Brun G, Lamonerie T: Molecular dissection
reveals decreased activity and not dominant negative effect
in human OTX2 mutants. J Mol Med 2006, 84:604-15.
23. Tian E, Kimura C, Takeda N, Aizawa S, Matsuo I: Otx2 is required
to respond to signals from anterior neural ridge for forebrain
specification. Dev Biol 2002, 242:204-23.
24. Kurokawa D, Kiyonari H, Nakayama R, Kimura-Yoshida C, Matsuo I,
Aizawa S: Regulation of Otx2 expression and its functions in
mouse forebrain and midbrain. Development 2004, 131:3319-31.
25. Eisenfeld AJ, Bunt-Milam AH, Sarthy PV: Muller cell expression of
glial fibrillary acidic protein after genetic and experimental
photoreceptor degeneration in the rat retina. Invest Ophthal-
mol Vis Sci 1984, 25:1321-8.
26. Bovolenta P, Mallamaci A, Briata P, Corte G, Boncinelli E: Implica-
tion of OTX2 in pigment epithelium determination and neu-
ral retina differentiation. J Neurosci 1997, 17:4243-52.
27. Ragge NK, Brown AG, Poloschek CM, Lorenz B, Henderson RA,
Clarke MP, Russell-Eggitt I, Fielder A, Gerrelli D, Martinez-Barbera JP,
et al.: Heterozygous Mutations of OTX2 Cause Severe Ocular
Malformations. Am J Hum Genet 2005, 76:.
28. Chen J, Rattner A, Nathans J: The rod photoreceptor-specific
nuclear receptor Nr2e3 represses transcription of multiple
cone-specific genes. J Neurosci 2005, 25:118-29.
29. Akhtar A, Gasser SM: The nuclear envelope and transcriptional
control. Nat Rev Genet 2007, 8:507-17.
30. Carter-Dawson LD, LaVail MM: Rods and cones in the mouse
retina. I. Structural analysis using light and electron micros-
copy. J Comp Neurol 1979, 188:245-62.
31. Gauthier K, Chassande O, Plateroti M, Roux JP, Legrand C, Pain B,
Rousset B, Weiss R, Trouillas J, Samarut J: Different functions for
the thyroid hormone receptors TRalpha and TRbeta in the
control of thyroid hormone production and post-natal devel-
opment. Embo J 1999, 18:623-31.
32. Fossat N, Courtois V, Chatelain G, Brun G, Lamonerie T: Alterna-
tive usage of Otx2 promoters during mouse development.
Dev Dyn 2005, 233:154-60.
    • "The Crx lacZ/Dlx1/Dlx2 DKO reporter mice are currently being established in the lab. An Otx2-GFP reporter mouse (Fossat et al., 2007) will also be bred with the Dlx1/2 DKO colony for characterization of reporter gene expression in vivo. Designing loss-of-function knock-in mouse models specific for putative sites regulated by DLX2 would provide us with in vivo determinations of DLX2 function on a candidate target gene's transcriptional regulation, specifically which sites would be necessary and/or sufficient. "
    [Show description] [Hide description] DESCRIPTION: ABSTRACT DLX HOMEOBOX TRANSCRIPTIONAL REGULATION OF CRX AND OTX2 GENE EXPRESSION DURING VERTEBRATE RETINAL DEVELOPMENT Objectives: We are interested in identifying and characterizing DLX transcriptional targets during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 double knockout (mutant) mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Methods: CRX and OTX2 expression in mutants and wild-type littermates was detected at RNA and protein levels. Chromatin immunoprecipitation (ChIP) of embryonic retina was utilized to identify DLX protein-genomic DNA complexes in situ. Quantification of expression was assessed by qRT-PCR and cell counting. In vitro assays such as electrophoretic mobility shift assays (EMSA) and luciferase reporter assays were used to detect the direct binding and activity, respectively, of DLX2 on the Crx and Otx2 promoters in vitro. Results: Qualitative and quantitative assessment of the temporal and spatial expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression, thereby restricting CRX expression to the outer nuclear layer. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. ChIP assays demonstrated that DLX proteins are bound to specific regions of the Crx and Otx2 promoters in situ, supporting a direct role for Dlx genes in repressing Crx and Otx2 expression during retinal development. Conclusion: The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data supports our hypothesis that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.
    File · Research · Apr 2015 · Cell Biology International
    • "At E12.5, photoreceptor and bipolar cell fate is determined by the expression of Otx2 in retinal progenitor cells [7], which controls the subsequent induction of the related Crx transcription factor. Expression of both Otx2 and Crx is then maintained throughout life in photoreceptor and bipolar cells8910. The function of Otx2 has been addressed in the adult eye. "
    [Show abstract] [Hide abstract] ABSTRACT: During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.
    Full-text · Article · Feb 2014
    • "GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment. The doughnut-ring-like staining of nuclei with the anti-NRL antibody was reproducible, and similar staining of Nr2e3 and Otx2 proteins in mouse photoreceptor nuclei was previously reported (Chen et al., 2005; Fossat et al., 2007). "
    [Show abstract] [Hide abstract] ABSTRACT: Although the gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis, it is ubiquitously expressed in all body tissues, including the retina. To study the function of OPTN in retinal ganglion cells as well as the whole retina, we previously isolated OPTN-interacting proteins and identified the gene encoding the bZIP transcription factor neural retina leucine zipper (NRL), which is a causative gene for retinitis pigmentosa. Herein, we investigated the binding between OPTN and NRL proteins in HeLaS3 cells. Co-expression of HA-tagged NRL and FLAG-tagged OPTN in HeLaS3 cells followed by immunoprecipitation and western blotting with anti-tag antibodies demonstrated the binding of these proteins in HeLaS3 cells, which was confirmed by proximity ligation assay. NRL is the first OPTN-binding protein to show eye-specific expression. A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids [aa]) of OPTN was necessary for binding with NRL. Immunostaining showed that Optn (rat homologue of OPTN) was expressed in rat photoreceptors and localised in the cytoplasm of photoreceptor cells. This is a novel demonstration of Optn expression in photoreceptor cells. OPTN was not detected in photoreceptor nuclei under our experimental conditions. Further analyses are necessary to elucidate the function of OPTN and the significance of its possible binding with NRL in photoreceptor cells.
    Article · Jan 2014
Show more