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First published online 14 March 2007
doi: 10.1242/dev.02829
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1 Neural Development Unit, UCL-Institute of Child Health, 30 Guilford Street,
WC1N 1EH, London, UK
2 Biochemistry, Endocrinology and Metabolism Unit, UCL-Institute of Child
Health, 30 Guilford Street, WC1N 1EH, London, UK.
3 National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA,
London, UK.
* Author for correspondence (e-mail: j.martinez-barbera{at}ich.ucl.ac.uk)
Accepted 5 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hesx1, Anterior forebrain, Wnt, ß-catenin, Cre, Rosa26
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hermesz et al., 1996;
Dasen et al., 2001).
Hesx1 and its orthologues in other vertebrates (Anf genes) are
expressed in the rostral region of the embryo during gastrulation and
neurulation in all species analysed
(Hermesz et al., 1996;
Thomas and Beddington, 1996;
Kazanskaya et al., 1997;
Knoetgen et al., 1999). In
mouse, Hesx1 expression is very dynamic, and Hesx1
transcripts are detected in anterior regions of the visceral endoderm,
definitive endoderm and neural ectoderm during early development. Later,
Hesx1 expression is restricted to the Rathke's pouch.
Hesx1-deficient embryos show a reduction in anterior forebrain
(AFB) tissue rostral to the zona limitans intrathalamica (ZLI) at 8.5 dpc. The
presumptive AFB is initially induced, but this territory is reduced at a later
stage of development in Hesx1-/- mutants. Chimeric
analysis has shown that this forebrain phenotype is a consequence of the
requirement of Hesx1 within the anterior neural ectoderm, and not due
to disturbances in the anterior visceral endoderm or anterior definitive
endoderm (Martinez-Barbera et al.,
2000). Hesx1 mutants also show defects in dorsal
forebrain commissural structures, eye abnormalities and pituitary dysplasia
(Dattani et al., 1998). A
comparable phenotype is observed in a human congenital disorder called
septo-optic dysplasia (SOD). Indeed, mutations in HESX1 are
associated with familial cases of SOD and other forms of hypopituitarism in
humans (Dattani et al., 1998;
Dattani, 2004;
Sobrier et al., 2006). In
Xenopus, overexpression of the Hesx1 orthologue,
Xanf1, results in enlargement of the neural plate (NP) at the expense
of epidermis and neural crest (NC)
(Ermakova et al., 1999). Thus,
it is now established that Hesx1 is essential for normal AFB
development in vertebrates. However, little is known about how Hesx1
performs its function.
Experiments in Xenopus, zebrafish, chick and mammals have provided
compelling evidence indicating that suppression of posteriorising signals is
an essential requisite for the formation of anterior neural tissue
(Kimura et al., 2000;
Perea-Gomez et al., 2001;
Kudoh et al., 2002;
Wilson and Houart, 2004;
Stern, 2005). Among them, the
Wnt/ß-catenin pathway plays a key role in anterior-posterior patterning
of the NP [(Yamaguchi, 2001;
Wilson and Houart, 2004;
Marikawa, 2006) and references
therein]. It is thought that the action of Wnt signalling inhibitors and
activators create a gradient along the neural ectoderm (high caudally and low
rostrally) that is required for normal anterior-posterior regionalisation of
the NP in distinct subdivisions (Leyns et
al., 1997; Glinka et al.,
1998; Hsieh et al.,
1999; Kiecker and Niehrs,
2001; Nordstrom et al.,
2002). Normal AFB development is particularly sensitive to
Wnt/ß-catenin signalling (Fredieu et
al., 1997; van de Water et
al., 2001; Houart et al.,
2002). For instance, the zebrafish masterblind mutants, which
carry a mutation in Axin1 that abolishes binding to Gsk3ß causing
Wnt/ß-catenin signalling overactivation, show a phenotype in which
telencephalon and eyes are reduced or absent, and dorsal diencephalic fates
are expanded rostrally (van de Water et
al., 2001; Heisenberg et al.,
2001). Similarly, lack of Tcf3/Headless repressor
function leads to the loss of anterior neural tissue
(Kim et al., 2000). In chick,
it has been shown that posterior forebrain character can be induced in
explants, which would normally acquire an AFB fate, through the direct
caudalising activity of Wnt proteins
(Nordstrom et al., 2002). In
mouse, both overexpression of Wnt8 and lack of Wnt/ß-catenin
antagonists lead to suppression of anterior neural fates
(Popperl et al., 1997;
Mukhopadhyay et al., 2001;
Satoh et al., 2004).
Therefore, it is now established that Wnt/ß-catenin signalling must be
modulated during vertebrate development to allow normal anterior-posterior
patterning of the NP; in particular, Wnt/ß-catenin signalling needs to be
suppressed for the AFB to develop.
The transcriptional repressors Hesx1, Six3 and Rax
(Rx) show overlapping expression patterns in the anterior neural
ectoderm during early mouse development
(Oliver et al., 1995;
Thomas and Beddington, 1996;
Mathers et al., 1997).
Moreover, Six3 and Rax mutants show defects in the
forebrain, such as reduced telencephalon and eyes, which are very similar to
those observed in Hesx1-/- mutants
(Mathers et al., 1997;
Martinez-Barbera et al., 2000;
Lagutin et al., 2003). A
mechanism has been postulated in which Six3 is thought to directly
repress the Wnt1 promoter and thereby reduce Wnt/ß-catenin
signalling in the AFB. Whether or not Hesx1 and Rax perform
a similar function during forebrain formation is an important question that
remains unanswered.
To further understand the function of Hesx1 in forebrain development, we have analysed in detail the neural patterning of Hesx1 mutant embryos and performed a genetic fate map of Hesx1-expressing cells in normal embryos and Hesx1 homozygous mutants. Our data indicate that in the absence of Hesx1 there is a posterior transformation of the AFB. Contrary to the observations in the Six3 mutants, HESX1 appears not to directly repress the Wnt1 locus, although absence of Hesx1 leads to an anteriorisation of Wnt1 expression and ectopic activation of the Wnt/ß-catenin targets Sp5 and Axin2 in the AFB. Gain-of-function experiments suggest that Hesx1 alone cannot anteriorise posterior neural tissue, but can rescue the forebrain defects of the Hesx1-deficient mutants.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Two correctly targeted clones were isolated and
injected into blastocysts from C57BL/6J mice. The PGK-Neo cassette
was excised by crossing heterozygous mice with the ACTB:FLPe strain
(Rodriguez et al., 2000).
Hesx1Cre/+ mice were maintained on the C57BL/6J background
and to date have been backcrossed a total of eight times.
Hesx1Cre/Cre embryos showed the forebrain and pituitary
defects observed in Hesx1-/- mutants. It is worth noting
that the severity of the phenotype of the Hesx1-deficient embryos
(Hesx1Cre/Cre and Hesx1-/-) has
increased compared with the initial analysis
(Dattani et al., 1998). No
viable homozygous mice are now identified at weaning and eye defects are fully
penetrant with variable expressivity. We think this is a consequence of
maintaining the colony on the C57BL/6J, whereas it was previously kept on a
C57BL/6 inbred colony maintained at the National Institute for Medical
Research, UK.
Generation of the R26Cond-Hesx1/+ mouse line
The components of the Rosa26 (R26) targeting vectors were
a gift (Soriano, 1999;
Srinivas et al., 2001). To
avoid possible interference with the translation of Hesx1 owing to
the presence of an ATG within the loxP sites, we generated a
pBigT-invloxP by inverting the orientation of both loxP
sites (Ivanova et al., 2005).
After electroporation, a total of 300 clones were picked, expanded and frozen
(see Fig. S6 in the supplementary material). Five correctly targeted clones
were identified and two were used for blastocyst injection (C57BL/6J
background). R26Cond-Hesx1/+ heterozygous mice were
backcrossed three times with C57BL/6J mice and then intercrossed to generate
R26Cond-Hesx1/Cond-Hesx1 homozygous mice, all of which
were viable and fertile.
Genetic crosses
Hesx1Cre/+mice were crossed with
R26Cond-lacZ/Cond-lacZ reporter animals to generate the
Hesx1Cre/+;R26Cond-lacZ/+ compound mice, then
backcrossed to R26Cond-lacZ/Cond-lacZ mice to obtain
Hesx1Cre/+;R26Cond-lacZ/Cond-lacZ mice and
embryos. Hesx1Cre/+;R26Cond-lacZ/+ and
Hesx1Cre/-;R26Cond-lacZ/+ embryos were
generated from crosses between
Hesx1Cre/+;R26Cond-lacZ/Cond-lacZ and
Hesx1+/- mice. Hesx1+/- mice carry a
null allele and have been described previously
(Dattani et al., 1998).
Genotyping of mice and embryos
Embryos and neonates were genotyped by PCR on DNA samples prepared from
tail tips, yolk sacs or whole embryos. Primer sequences and PCR protocols are
available on request.
Histology, X-Gal staining, in situ hybridisation (ISH), TUNEL staining and cell proliferation
Histology, X-Gal staining, TUNEL staining and ISH were performed as
described (Martinez-Barbera et al.,
2002). Proliferation was assessed using the anti-phospho-histone
H3 antibody (mitosis marker; Upstate Biotechnology). Three embryos of each
genotype at similar developmental stages were embedded in wax and sectioned.
Immunostaining was performed following standard procedures and sections were
counterstained with DAPI. For analysis, a total of over 5,000 nuclei were
counted from matching sections containing forebrain tissue. The mitotic index
was expressed as a percentage of mitotic nuclei from the total number of
nuclei.
EMSA, cell transfections, luciferase assays and western blots
EMSA was performed as previously described
(Dattani et al., 1998). DNA
transfections were carried out in CHO and 293 cells as described
(Brickman et al., 2001) using
reporter plasmids encompassing specific regions of the Wnt1 locus
containing putative Hesx1-binding sites. The western blot for HESX1
used a specific rabbit serum we have generated.
qRT-PCR and RT-PCR analysis
Total RNA was isolated from embryos using the RNeasy Micro Kit (Qiagen).
First-strand cDNA synthesis was performed using the Omniscript RT Kit (Qiagen)
according to the manufacturer's recommendations. Amplification of Hesx1,
Cre and Gapdh (endogenous control) with specific primers was
performed using SYBRGreenER (Invitrogen) on an ABI 7500 Real-Time PCR machine
(Applied Biosystems) and analysed using the ABI system software. For RT-PCR
analysis, specific primers for Hesx1, Cre and Gapdh were
used to amplify cDNA templates from RNA extracted from whole embryos or
specific regions of the neural tube using Trizol (Invitrogen). Primer
sequences and PCR conditions are available on request.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To find out the cellular basis of the early forebrain
defects, we analysed neural patterning, proliferation and apoptosis of
Hesx1-/- in comparison with Hesx1+/-
and wild-type embryos from 7.5 to 9.5 dpc.
At presomitic stages, the expression domains of Six3 (AFB marker),
Atx (Dmbx1) and Pax2 (posterior forebrain and
midbrain markers at these stages), and Hoxb1 (hindbrain marker) were
identical in all genotypes (Fig.
1A,B and data not shown)
(Oliver et al., 1995;
Nornes et al., 1990;
Hunt and Krumlauf, 1991;
Gogoi et al., 2002). At the 0-
to 1-somite stage, Pax6 is expressed in the prospective forebrain
region of the developing mouse embryo
(Walther and Gruss, 1991).
Pax6 expression in the anterior NP was significantly reduced in
Hesx1-/- mutants when compared with
Hesx1+/- or wild-type littermates at this stage, thus
confirming that the prospective forebrain region is reduced only at the early
somite stage (Fig. 1C)
(Martinez-Barbera et al.,
2000). The possibility that prospective forebrain was being
respecified was analysed using the neural crest (NC) markers Pax3 and
Foxd3. These markers are expressed on the margin of the anterior NP
at the 2- to 3-somite stage in wild-type embryos, but the most rostral region
is free of Pax3 and Foxd3 transcripts
(Fig. 1D,F,H,J)
(Goulding et al., 1991;
Labosky and Kaestner, 1998).
By contrast, their expression domains were anteriorised in the 2- to 3-somite
stage Hesx1-/- mutants, and the region devoid of
Pax3 and Foxd3 expression was clearly reduced when compared
with Hesx1+/- or wild-type embryos
(Fig. 1E,G,I,K). It is
important to note that these changes were observed prior to a gross lack of
neural tissue, although there was a slight change in the shape of the anterior
NP (Figs 1 and
2; see below). NC cells are
posterior NP derivatives that delaminate from the boundaries of the NP along
the entire axis, with the exception of the most anterior neural ectoderm fated
to be AFB (Couly and Le Douarin,
1987; Sechrist et al.,
1995; Muhr et al.,
1997). Therefore, our data suggest that the AFB loses its identity
in Hesx1-/- mutants in favour of NC.
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;
Echelard et al., 1994;
Meyers et al., 1998;
Storm et al., 2006). In the 3-
to 4-somite wild-type embryo, there was a segment of the NP that expressed
Wnt1 (prospective midbrain), but the most anterior part of the NP was
devoid of Wnt1 transcripts (Fig.
2A). By contrast, in Hesx1-/- mutants, the
Wnt1 expression domain was expanded rostrally throughout the edge of
the NP until its most anterior tip (Fig.
2B). This anteriorisation was not observed at earlier stages, but
Wnt1 expression is barely detectable at the 2-somite stage
(Echelard et al., 1994). The
onset of Fgf8 expression in the anterior neural ridge (ANR) occurs at
the 4- to 5-somite stage in wild-type embryos
(Crossley and Martin, 1995;
Shimamura and Rubenstein,
1997). At this stage, the Fgf8 expression domain in the
ANR was either reduced or absent in Hesx1-null mutants
(Fig. 2C-F).
To check whether Wnt/ß-catenin signalling was ectopically activated
within the prospective AFB, we analysed the expression of the
Wnt/ß-catenin targets Axin2 and Sp5
(Jho et al., 2002;
Takahashi et al., 2005;
Weidinger et al., 2005).
Normally, Axin2 and Sp5 are not expressed in the most
anterior region of the NP of the 2- to 3-somite stage wild-type embryo
(Fig. 2K,M and data not shown).
Strikingly, Axin2 expression was anteriorised in the mutants as
compared with wild-type littermates at the 3- to 4-somite stage
(Fig. 2G,H). Likewise,
Sp5 transcripts were detected all over the anterior NP of
Hesx1-/- mutants at the 2- to 3-somite and 5- to 6-somite
stages (Fig. 2I-N). Taken
together, this marker analysis suggests that the lack of Hesx1 leads
to the ectopic activation of Wnt/ß-catenin signalling in the prospective
AFB prior to the onset of Fgf8 expression in the ANR.
Rostral expansion of dorsal diencephalic markers in Hesx1-/- mutants
The anterior expansion of posterior neural markers was also evident in
Hesx1 mutant embryos at the 8-to 10-somite stage, although at these
stages there was a marked reduction of AFB tissue. Pax3 and
Foxd3 expression domains did not reach the most anterior region of
the developing forebrain in the wild type and heterozygous Hesx1
mutants, but did so in Hesx1-/- mutants
(Fig. 3A-D)
(Goulding et al., 1991;
Labosky and Kaestner, 1998).
Wnt1 is normally expressed in the midbrain and posterior
diencephalon, whereas Wnt3a is expressed only in the dorsal
diencephalon (Parr et al.,
1993). At these stages, Wnt1 and Wnt3a were also
clearly anteriorised and their expression domains were enlarged
(Fig. 3I-P). Therefore, the
Wnt1 and Wnt3a anteriorisation might reflect the
transformation of AFB into posterior diencephalic fates observed at earlier
stages. The expression domains of Pax2, Fgf8, Pax6 and Six3
in the AFB region were significantly reduced in Hesx1-/-
mutants at 8.5 dpc (Fig. 3G,H
and data not shown) (Nornes et al.,
1990; Crossley and Martin,
1995; Walther and Gruss,
1991; Oliver et al.,
1995). However, moreposterior markers, such as En1 (a
midbrain-hindbrain boundary marker) and Hoxb1 (hindbrain marker) were
unperturbed in Hesx1-deficient embryos
(Fig. 3E,F and data not shown)
(Wurst et al., 1994;
Hunt and Krumlauf, 1991). At
9.5 dpc, rostral expansion of the Atx and Pax6 expression
domains in the posterior diencephalic region was evident in
Hesx1-/- mutants (see Fig. S1A,B in the supplementary
material).
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Altogether, these data indicate that the reduction of AFB tissue in Hesx1 mutants from 8.5 dpc onwards is caused by the posteriorisation of AFB precursors at early somite stages.
Cell fate analysis of Hesx1-expressing cells in normal and Hesx1-deficient embryos
We carried out genetic fate mapping of Hesx1-expressing cells
using a novel Hesx1-Cre mouse line
(Fig. 4) and the
R26-floxstop-lacZ reporter line (hereby called
R26-Cond-lacZ) (Soriano,
1999). X-Gal staining of
Hesx1Cre/+;R26Cond-lacZ/Cond-lacZ embryos,
which are phenotypically normal, revealed lacZ expression in all cell
types where Hesx1 is normally expressed and their descendants (see
Fig. S2 in the supplementary material). This analysis revealed that the normal
fate of Hesx1-expressing cells within the NP is to colonise the
AFB.
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First, we analysed the Cre expression pattern in Hesx1Cre/+, Hesx1Cre/- and Hesx1Cre/Cre embryos. ISH on Hesx1Cre/Cre embryos at 8.5 dpc revealed a pattern of Cre expression that was very similar to the Hesx1 expression domain in wild-type embryos (see Fig. S3AC in the supplementary material). No differences in Cre expression were observed between Hesx1Cre/+ and Hesx1Cre/- embryos, although the intensity of the expression domain was weaker in embryos carrying just one copy of Cre (see Fig. S3D,E in the supplementary material). RT-PCR on Hesx1Cre/+ and Hesx1Cre/- embryos from 7.5-9.5 dpc detected Cre expression at all stages analysed, but no expression was detected in the brain of 11.5-dpc embryos (data not shown). qRT-PCR revealed no differences in Cre expression between Hesx1Cre/+ and Hesx1Cre/- embryos at the 2-somite stage, but a reduction in Cre expression was observed in Hesx1Cre/- embryos at the 5-somite stage, possibly reflecting the ongoing posterior transformation of the AFB (see Fig. S3F in the supplementary material). These data suggest that there is no ectopic expression of Cre within the NP of Hesx1-deficient embryos.
No significant differences in X-Gal staining were observed from the onset
of gastrulation to the 2- to 3-somite stage between
Hesx1Cre/+;R26Cond-lacZ/+ and
Hesx1Cre/-;R26Cond-lacZ/+ embryos (see Fig. S2B
and Fig. S4F in the supplementary material). However, at the 4- to 5-somite
stage, X-Gal-positive cells reached more posterior regions of the NP in
Hesx1Cre/-;R26Cond-lacZ/+ embryos as compared
with Hesx1Cre/+;R26Cond-lacZ/+ littermates
(Fig. 5A,B). The majority of
lacZ-expressing cells in
Hesx1Cre/+;R26Cond-lacZ/+ compound embryos at
the 8- to 10-somite stage remained anterior to the point of fusion between the
cranial folds at the boundary between anterior and posterior forebrain
(Fig. 5C). By contrast,
abundant lacZ-expressing cells were observed posterior to this
boundary in Hesx1Cre/-;R26Cond-lacZ/+ mutants
(Fig. 5D). These differences
were accentuated at 9.5 dpc. Moreover, lacZ-expressing cells also
colonised the first branchial arch in
Hesx1Cre/-;R26Cond-lacZ/+ embryos from 9.5 dpc
in a manner resembling the pattern of endogenous NC migration
(Fig. 5I-N). This was never
observed in Hesx1Cre/+;R26Cond-lacZ/+ embryos
(n=52). The possibility that lacZ-expressing cells in the
first branchial arch were migratory NC was in agreement with the rostral
expansion of the NC markers Pax3 and Foxd3
(Fig. 1D-K and
Fig. 3A-D). In fact, at 16.5
dpc, lacZ-expressing cells were found in skull bones, dermis, maxilar
and mandibular bones, basisphenoid and vibrissae of
Hesx1Cre/-;R26Cond-lacZ/+ mutants, which are
all tissues normally colonised by cranial NC (see Fig. S4 in the supplementary
material, and data not shown) [Creuzet et al.
(Creuzet et al., 2005) and
references therein]. No differences in X-Gal staining were observed in the
endoderm of the pharyngeal arches and the liver between the two genotypes.
At 12.5 dpc, most of the lacZ-expressing cells colonised the telencephalic vesicles, eyes, hypothalamus and ventral diencephalon in the Hesx1Cre/+;R26Cond-lacZ/+ embryos, and only sporadic stained cells were observed within the frontonasal mass and the posterior forebrain (Fig. 5E,G). By contrast, Hesx1Cre/-;R26Cond-lacZ/+ embryos showed an abundance of lacZ-expressing cells in the posterior forebrain and frontonasal mass, in addition to the ectopic lacZ-expressing cells within the first branchial arch (Fig. 5F,H and data not shown). We noticed that Hesx1-deficient embryos at 11.5-12.5 dpc had more lacZ-expressing cells localised in the AFB (Fig. 5E,F). Since Cre expression is not activated ectopically within the NP, this finding might be due to the fact that in these embryos the AFB derives predominantly from the most rostro-medial (RM) region of the early NP, as more lateral regions change fate to posterior forebrain and NC (Figs 1 and 2). The RM region of the NP contains a higher density of lacZ-expressing cells than more lateral regions (see Fig. S2B and Fig. S4F in the supplementary material). By contrast, the AFB of Hesx1Cre/+;R26Cond-lacZ embryos derives from regions of the early anterior NP, containing both high and low densities of lacZ-expressing cells.
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75%, n=134), in agreement with previous observations
(Dattani et al., 1998). In
these embryos, one side developed better than the other, with the right side
commonly being the most severely affected (95%)
(Fig. 5H). The greater
reduction of telencephalic and eye tissue was concomitant with an increased
colonisation of X-Gal-positive cells in the posterior forebrain of the same
side. This suggests that more cells that would normally colonise the
telencephalon and eyes have reverted their fate to posterior forebrain in the
most-affected side. The reasons underlying the asymmetry defects in
Hesx1-deficient embryos remain unknown at present, as Hesx1
is not expressed asymmetrically within the NP. X-Gal staining on whole brains
followed by ISH with Shh and Pax6 revealed that
lacZ-expressing cells did not cross the forebrain-midbrain boundary,
which is marked by the posterior limit of Pax6 expression in the
forebrain (Inoue et al., 2000)
(see Fig. S5 in the supplementary material). Taken together, these data indicate that a lack of Hesx1 expression causes a transformation of the AFB primordium, which acquires a posterior forebrain identity and generates NC.
Hesx1 might not be sufficient to change posterior neural fates
If a lack of Hesx1 leads to a posteriorisation of the forebrain,
could Hesx1 misexpression alter posterior fates when ectopically
expressed? To address this issue we generated a R26-Cond-Hesx1 mouse
line by gene targeting in ES cells (see Fig. S6 in the supplementary
material). In this mouse, Hesx1 expression is activated upon
Cre-mediated excision of the stop cassette.
R26Cond-Hesx1/+ mice were crossed with the
ß-Actin-Cre transgenic line, in which Cre expression is
driven by the ß-Actin (Actb) promoter
(Meyers et al., 1998).
R26Cond-Hesx1/+;ß-Actin-Cre compound embryos
expressed Hesx1 transcripts ubiquitously (except in the heart where
ß-Actin is not active), whereas
R26Cond-Hesx1/+, ß-Actin-Cre and wild-type
littermates expressed Hesx1 only within the endogenous domain in the
ventral forebrain and/or Rathke's pouch (see Fig. S6D,E in the supplementary
material). Western blot analysis of whole heads demonstrated that
R26Cond-Hesx1/+;ß-Actin-Cre
compound embryos contained more HESX1 protein than
R26Cond-Hesx1/+, ß-Actin-Cre or wild-type
littermates (see Fig. S6F in the supplementary material). Protein levels were
even higher when embryos contained two copies of the
R26-Cond-Hesx1 allele in
R26Cond-Hesx1/Cond-Hesx1;ß-Actin-Cre
compound embryos (data not shown).
Marker analysis revealed no significant differences in the expression of Six3, Pax3, Foxd3, Wnt1 and Fgf8 between R26Cond-Hesx1/+;ß-Actin-Cre, R26Cond-Hesx1/Cond-Hesx1;ß-Actin-Cre, single mutants and wild-type littermates (data not shown). Therefore, it seems likely that Hesx1 is not sufficient to confer anterior identity to posterior neural tissues. However, a proportion of compound embryos overexpressing Hesx1 had exencephaly (10% of R26Cond-Hesx1/+;ß-Actin-Cre, n=65; and 80% of R26Cond-Hesx1/Cond-Hesx1;ß-Actin-Cre, n=35) (see Fig. S6E in the supplementary material). The reasons underlying the exencephaly are at present unknown, but this phenotype is background-dependent and not detected in a mixed C57BL6/J;CD1 background (data not shown).
AFB is likely to exhibit a differential sensitivity to Hesx1 levels
We used the R26-Cond-Hesx1 mouse line in an attempt to rescue the
forebrain defects of the Hesx1 homozygous mutants. Controlling the
Hesx1 dosage from the R26 locus, by adding either one or two
copies on a Hesx1-null background, might reveal distinct requirements
for HESX1 protein levels for normal development of specific AFB
structures.
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). By
contrast,
Hesx1Cre/Cre;R26Cond-Hesx1/+ embryos
showed a significant improvement in AFB development
(Fig. 6C,D). The size and shape
of the telencephalic vesicles in
Hesx1Cre/Cre;R26Cond-Hesx1/+ embryos
was comparable with wild-type littermates in nine out of 12 embryos. However,
there was no major improvement in eye development. Of the embryos recovered,
all had abnormal eyes (n=12), and either showed bilateral
anophthalmia (n=10) or unilateral microphthalmia (n=2). Eye
phenotype rescue was accentuated in
Hesx1Cre/Cre;R26Cond-Hesx1/Cond-Hesx1
compound embryos (Fig. 6E,F).
Bilateral anophthalmia was observed in only three out of ten embryos. From the
remaining seven embryos, three had normal eyes, two had bilateral
microphthalmia and two unilateral anophthalmia. Telencephalic development was
normal in nine out of ten embryos analysed. No phenotypic defects were seen in
embryos of all other genotypes. The results from these experiments allow us to
draw two conclusions. First, that Hesx1 expression from the
R26-Cond-Hesx1 allele can rescue the forebrain defects in
Hesx1-deficient embryos. Second, that the telencephalon and eyes
might exhibit a distinct sensitivity to HESX1 dosage. The rescue of the forebrain defects was analysed at the molecular level in 8.5-dpc embryos. Hesx1 expression was restricted to the AFB in Hesx1Cre/Cre;R26Cond-Hesx1/Cond-Hesx1 and wild-type littermates and levels of expression were comparable (Fig. 7A,B). Foxd3 expression was anteriorised in the Hesx1-deficient embryos (Fig. 3C,D), but the Foxd3 expression domain in Hesx1Cre/Cre;R26Cond-Hesx1/Cond-Hesx1 was similar to that of wild-type embryos (Fig. 7C,D). Finally, the anteriorisation of Axin2 expression in the Hesx1-/- mutants (Fig. 2G,H) was reverted in Hesx1Cre/Cre;R26Cond-Hesx1/Cond-Hesx1 compound embryos, and an Axin2-free region of the anterior NP was evident (Fig. 7E,F). Overall, these experiments suggest that the rescue of the morphological forebrain defects correlates with an improvement of the neural patterning of the anterior NP.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Martinez-Barbera et al.,
2000). In this study we demonstrate that lack of Hesx1
leads to a fate transformation of anterior to posterior forebrain. Cells that
normally colonise the telencephalon, eyes, hypothalamus and ventral
diencephalon are found in more-posterior regions, such as the dorsal thalamus
and pretectum in Hesx1-/- mutants. Gain-of-function
experiments indicate that Hesx1 neither promotes AFB identity when
misexpressed in the posterior NP, nor increases the size of the AFB when
overexpressed in its endogenous expression domain. Moreover, our experiments
suggest that the eyes might require higher levels of HESX1 protein than the
telencephalon for normal development. This study has revealed that one of the
functions of Hesx1 is to prevent the Wnt/ß-catenin signalling
pathway from being activated within the AFB.
Normal fate of Hesx1-expressing cells
The fate of Hesx1-expressing cells in our study is in agreement
with previous fate mapping studies in the mouse. Soon after the onset of
gastrulation, the anterior visceral endoderm is displaced towards the
extraembryonic region by the definitive endoderm, which is formed at the tip
of the primitive streak (Lawson and
Pedersen, 1987; Thomas and
Beddington, 1996). X-Gal staining of
Hesx1Cre/+;R26Cond-lacZ/Cond-lacZ
embryos detected patches of blue cells in the visceral endoderm of the
extraembryonic region from late streak stages. It was noticeable that
lacZ-expressing cells remained clumped together and, as previously
described, not much intermingling was observed
(Gardner and Cockroft, 1998).
Hesx1 is expressed in the axial levels of the anterior definitive
endoderm, an area containing the precursors of some anterior foregut
derivatives (Lawson and Pedersen,
1987; Thomas et al.,
1998). In fact, lacZ-expressing cells colonised the
anterior foregut in
Hesx1Cre/+;R26Cond-lacZ/+ embryos at
8.5 dpc and were found in its derivatives, including pharyngeal endoderm,
thyroid gland, liver and ventral pancreas.
Strong X-Gal staining was detected in the Rathke's pouch from 9.0 dpc onwards. Interestingly, blue cells mainly colonised the anterior and intermediate lobes, both of which are derived from the oral ectoderm, but blue cells were rarely observed in the posterior pituitary, which has neural origin from a recess in the floor of the hypothalamus (see Fig. S4E in the supplementary material).
Cell fate analysis of lacZ-expressing cells within the NP showed that the majority of the labelled cells colonised the forebrain region anterior to the ZLI, i.e. telencephalon, eyes, hypothalamus and ventral diencephalon. Only scattered cells ended up in the dorsal thalamus and pretectum and, very rarely, lacZ-expressing cells crossed the forebrain-midbrain boundary. Therefore, Hesx1 marks only prospective forebrain, in particular the AFB.
Anterior to posterior transformation of the forebrain in Hesx1-deficient embryos
A main finding of this study is that the absence of Hesx1 brings
about a posterior transformation of the AFB, which is evidenced by rostral
expansion of the expression domains of the NC markers Foxd3 and
Pax3, prior to an overt morphological defect of the anterior NP.
Numerous fate mapping studies have demonstrated that premigratory NC cells are
dorsal cell types that form at the border of the NP, from mid-diencephalon to
spinal cord levels, but not rostral to the mid-diencephalon
(Couly and Le Douarin, 1987;
Sechrist et al., 1995;
Muhr et al., 1997). However,
cells within this NC-free region can be induced to form NC when exposed to
caudalising signals in explant experiments
(Muhr et al., 1997). We
believe that the Hesx1 expression domain in the anterior NP
corresponds to this crest-free area. For instance, X-Gal staining of
Hesx1Cre/+;R26Cond-lacZ/+ embryos,
which are phenotypically normal, showed that the majority of
lacZ-expressing cells colonised the AFB and did not form NC. By
contrast, lacZ-expressing cells populated the posterior forebrain in
Hesx1Cre/-;R26Cond-lacZ/+ mutants,
thus reaching the area of the NP that yields migratory NC of the first
branchial arch and the frontonasal mass
(Inoue et al., 2000;
Creuzet et al., 2005).
The expression domains of Wnt3a, Wnt1, Atx and Pax6 were expanded rostrally and lacZ-expressing cells were found throughout the dorsoventral axis of the dorsal thalamus and pretectum. This suggests that in the absence of Hesx1, cells initially specified to AFB fates colonise the entire segment of the posterior forebrain, not only NC-generating regions. However, the size of the posterior forebrain is not affected in Hesx1-deficient embryos at 12.5-18.5 dpc, possibly because the increased cell death in the posterior forebrain compensates for the excess of cells populating this region.
Although required for the maintenance of AFB identity, Hesx1
cannot alter posterior fates in caudal neural tissue when misexpressed in the
mouse embryo. To do so, Hesx1 might require specific co-factors only
present in anterior and not posterior neural tissue. (see Fig. S6 in the
supplementary material) (Ermakova et al.,
1999). The partial rescue of the forebrain defects and attenuation
of the posterior transformation observed in
Hesx1Cre/Cre;R26Cond-Hesx1/+ and
Hesx1Cre/Cre;R26Cond-Hesx1/Cond-Hesx1 compound
embryos suggest that only the AFB is competent to respond to HESX1. Overall,
these data indicate that Hesx1 is required for AFB development but,
on its own, it is not sufficient to promote AFB fates.
The genetic rescue experiments have revealed that whereas one copy of
R26-Cond-Hesx1 in a Hesx1-null background is sufficient to
improve telencephalic development, two copies of the R26-Cond-Hesx1
allele are required to rescue both telencephalon and eye development. Although
we cannot rule out the possibility that telencephalic and eye precursors could
express different levels of Hesx1 from the R26 locus, we
think that our experiments point to a differential sensitivity of the eye and
telencephalon to Hesx1 levels. Fate mapping studies in the mouse
embryo have shown that telencephalic precursors reside in the rostrolateral
regions of the anterior NP and that the eye field is located caudal to this
and more medially (Inoue et al.,
2000). It is worth noting that Hesx1 expression is higher
in the area of the NP that is thought to correspond to the eye field
(Thomas and Beddington, 1996).
In this scenario, eye development is expected to be more sensitive than
telencephalic development to HESX1 levels, and indeed,
Hesx1-deficient embryos show a higher penetrance of eye versus
telencephalic abnormalities. In agreement with this concept, mutant mice
bearing a single amino acid change in position 26 that yields a HESX1 protein
with reduced repressing activity (Carvalho
et al., 2003), display eye abnormalities but have normal
telencephalic development (our unpublished results). This suggests that
Hesx1 might have a dual role within the anterior NP: to promote
anterior versus posterior forebrain development and to segregate telencephalic
and eye field identities (Stigloher et
al., 2006; Mathers et al.,
1997).
Hesx1 antagonises caudalising signals within the NP
Our data strongly suggest that the mechanism underlying the posterior
transformation of Hesx1-/- mutants is the ectopic
activation of Wnt/ß-catenin signalling within the prospective AFB at
early somite stages. Experiments in Xenopus and zebrafish have
indicated that the eye is the most sensitive region of the NP to
Wnt/ß-catenin activation (Fredieu et
al., 1997; van de Water et
al., 2001). When zebrafish embryos were exposed for a short time
to LiCl, an inhibitor of Gsk3ß that enhances Wnt/ß-catenin
signalling, only eye development was disrupted and the treated embryos
displayed microphthalmia or anophthalmia. However, when embryos were treated
with LiCl for longer, they developed telencephalic as well as eye defects. An
excess of Wnt/ß-catenin signalling might explain the reduction in the AFB
markers Six3 and Pax6 and the concomitant rostral expansion
of the NC markers Pax3 and Foxd3.
It seems likely that Hesx1 and Six3 might work in parallel in the maintenance of AFB identity, but through distinct mechanisms. Hesx1 can bind to Wnt1 regulatory elements in in-vitro assays, but it cannot repress reporter vectors containing Wnt1 regulatory elements (see Fig. S7 in the supplementary material). Moreover, expansion of the Sp5 expression domain occurs prior to Wnt1 rostralisation in Hesx1-/- mutants. Therefore, in contrast to the Six3-/- mutants, ectopic Wnt1 expression within the prospective AFB might not be the primary reason for the forebrain defects; rather, the excess of Wnt1 might contribute to the final morphological defects later in development.
The possibility exists that Hesx1 may antagonise
Wnt/ß-catenin signalling by modulating the activity of some component(s)
of the pathway causing a cell-autonomous inhibition within
Hesx1-expressing cells. Alternatively, or in addition, Hesx1
might repress one or more Wnt/ß-catenin target genes directly, thus
preventing the acquisition of posterior fates. Chimeric experiments suggest
that Hesx1 is required cell-autonomously within the NP
(Martinez-Barbera et al.,
2000). However, Hesx1 function can be partially
compensated for, as most Hesx1-/- mutants show some AFB
development. Moreover, in chimeric embryos, Hesx1-deficient cells, as
well as wild-type cells, can also colonise the AFB. The possibility exists
that a subset of Hesx1-/- cells might be more susceptible
to respond to Wnt/ß-catenin signalling and acquire a posterior fate, but
this might be difficult to observe in chimeric embryos, where the bulk of
wild-type cells can colonise the same regions of the neural tube
(Martinez-Barbera et al.,
2000). Notably, the forebrain defects were completely rescued only
in embryos with a high degree of chimerism, and the AFB was mainly populated
with wild-type cells (Martinez-Barbera et
al., 2000). Future research will elucidate the molecular function
of Hesx1.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/8/1499/DC1
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ACKNOWLEDGMENTS |
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