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First published online 1 August 2007
doi: 10.1242/dev.001461
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MRC Centre for Developmental Neurobiology, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, UK.
Author for correspondence (e-mail:
steffen.scholpp{at}kcl.ac.uk)
Accepted 13 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Forebrain patterning, Thalamus development, Zona limitans intrathalamica (ZLI), Hedgehog, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The thalamic complex differentiates from the
mid-diencephalic territory (MDT) and consists of three parts: the anteriorly
located prethalamus, the posteriorly located thalamus and an intervening
ventricular ridge - the zona limitans intrathalamica (ZLI). An essential step
towards the establishment of a functional thalamic complex is the release of
Sonic hedgehog (Shh) from the ZLI; this protein is necessary and sufficient to
induce the characteristic set of pro-neural transcription factors within the
MDT (Hashimoto-Torii et al.,
2003; Kiecker and Lumsden,
2004; Vieira et al.,
2005; Scholpp et al.,
2006). shh expression within the ZLI is induced in a
characteristic ventral-to-dorsal progression from the basal plate into the
alar plate, suggesting a dependency on the basal plate; however, studies in
zebrafish have shown that neither the shh-positive basal plate nor
the underlying axial mesoderm per se are required for ZLI formation
(Scholpp et al., 2006).
Positioning this signalling centre along the anteroposterior axis is less well
understood. It is believed that, in mouse, the ZLI forms directly above the
anterior tip of the notochord, and thus at the interface between the
prechordal neuraxis and the epichordal neuraxis
(Shimamura et al., 1995).
Grafting experiments in chick have suggested that alar Shh expression
similar to that observed in the ZLI is induced by an ectopic interface between
prechordal and epichordal neuroepithelia
(Vieira et al., 2005).
Fate-mapping studies in chick have suggested that the pre-ZLI is a
wedge-shaped region of neuroepithelium - characterised by the absence of
lunatic fringe (L-fng) expression - that gives rise to the ZLI
compartment by presumed morphological changes in the diencephalon
(Larsen et al., 2001;
Zeltser et al., 2001). A
further model suggests that mutual repression between Six3 anteriorly and Irx3
posteriorly is involved in positioning the ZLI in chick
(Kobayashi et al., 2002;
Braun et al., 2003). So far,
absence of Six3 expression in the presumptive ZLI of zebrafish and
chick, and the presence of a ZLI in Six3 knock-out mouse embryos, has
raised doubts about the direct involvement of a Six3-Irx3 interaction
(reviewed in Wilson and Houart,
2004; Kiecker and Lumsden,
2005). Recently, it has been shown that mice deficient for both
forebrain-embryonic zinc finger gene (Fezf1, formerly known as
Fez) and Fezf2 (formerly known as Fezl) gene
functions, and fish deficient for fezf2 only, lack the prethalamus
and show a mis-specification of the ZLI, suggesting that either the ZLI
precursors require Fezf1/Fezf2 cell-autonomously or that the ZLI is induced by
interaction between the prethalamus and thalamus via an unknown posterior
molecular component (Hirata et al.,
2006; Jeong et al.,
2007).
Members of the Otx family, which comprises orthologues of the
Drosophila orthodenticle (also known as ocelliless -
FlyBase) gene, play an important role in the regionalisation of the vertebrate
rostral neural tube (Simeone et al.,
2002). During neural plate specification, the Otx genes - in
zebrafish, otx1l and otx2 (collectively referred to as
otx1l/2) - are expressed in a highly regulated
spatiotemporal pattern (Mercier et al.,
1995). During gastrulation, Otx responds to the Wnt gradient
within the neural plate, and becomes downregulated posteriorly by a
combination of high Wnt signalling (Wnt8 from the marginal zone) and
activation of the Gbx genes in the prospective hindbrain
(Kiecker and Niehrs, 2001;
Rhinn et al., 2005). At the
end of the neural plate stage, Otx gene expression marks the forebrain and
midbrain primordia, forming a distinctive border at the midbrain-hindbrain
boundary (reviewed in Rhinn and Brand,
2001; Wurst and Bally-Cuif,
2001), and is required to maintain midbrain identity
(Acampora et al., 1997;
Foucher et al., 2006). A
previous study involving Otx2+/-; Otx1-/-
double-mutant mice suggested that the Otx genes might contribute to the
specification of the diencephalon, because the mutant embryos lack expression
of Shh and diencephalic markers by embryonic day 9.5
(Acampora et al., 1997).
Recently, it has been shown that, when clones of Otx2-/-;
Otx1-/- cells develop in a thalamic location in mosaic mice,
they show posterior diencephalic, most probably pretectal, characteristics,
such as expression of GABAergic markers, suggesting that Otx genes drive
glutamatergic thalamic fate (Puelles et
al., 2006).
Another regionally expressed group of genes in the early neural plate is
the Irx family of transcriptional control genes - homologues of the
Drosophila Iroquois gene complex
(Dambly-Chaudiere and Leyns,
1992; Gomez-Skarmeta et al.,
1996). In zebrafish, expression of two members of this family,
irx1b and irx7, mark the posterior diencephalon and the
midbrain from the neural plate stage onwards
(Wang et al., 2001;
Itoh et al., 2002). In
contrast to the Otx genes, expression of the Irx genes is upregulated by Wnt
signalling (Itoh et al.,
2002). Although it is known that both Otx gene-family expression
and irx1b expression persist within the diencephalon, especially in
the thalamus, their functions in this area have yet to be elucidated.
In the present study, we mapped Otx gene-family expression during
segmentation and pharyngula periods, confirming that this expression is
maintained in the presumptive ZLI and thalamus during the segmentation period.
We show that this persisting expression is a prerequisite for the formation of
the ZLI, because lack of Otx1l/2 function led to the absence of ZLI markers.
In addition, thalamic fate is not maintained in these Otx1l/2 hypomorphic
(OtxH) (Foucher et al., 2006)
embryos. The mis-specified area is transformed mainly into pretectum and, to a
lesser extent, into prethalamus, indicating that Otx1l/2 are required for the
formation of both the ZLI and thalamus. Rescue of the MDT in OtxH morphants by
conditional spatiotemporal induction of Otx2 expression shows that the Otx
proteins are necessary to specify ZLI identity as well as thalamic identity,
depending upon the position along the anteroposterior axis. Furthermore,
loss-of-function experiments demonstrate that expression of irx1b in
the thalamus is necessary to restrict shh expression and thus to
establish the posterior boundary of the ZLI. Therefore, we propose a novel
model of ZLI formation: Otx1l/2 are required for shh expression in
the ZLI and set the anterior boundary of the ZLI, whereas Irx1b, which is
maintained by Otx1l/2 function, represses the expression of shh and
therefore determines the posterior boundary of the ZLI. By discovering this
novel role for Otx1l/2 during regionalisation of the neural tube, we have
directly linked the early Wnt-dependent patterning events with the formation
of the presumptive ZLI and thus with the positioning of the thalamus during
early neural development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos were staged according to Kimmel et al.
(Kimmel et al., 1995). To
prevent pigment formation, some embryos were raised in 0.2 mM
1-phenyl-2-thiourea (PTU, Sigma). The data we present in this study were
obtained from analysis of King's College wild-type (KWT) fish and the Shh-GFP
transgenic line was generated by Carl Neumann
(Neumann and Nuesslein-Volhard,
2000).
Injections
For transient knock-down of gene expression, morpholino-antisense oligomers
(morpholinos; MO) were dissolved in 5 mM Hepes buffer with 0.2% phenol red
(Nasevicius and Ekker, 2000).
otx1l- and otx2-MOs (see
Foucher et al., 2006) and
Irx1b-MO [originally described as iro1-MO
(Itoh et al., 2002)] were
back-loaded into borosilicate capillaries prepared on a Sutter micropipette
puller. The MO-antisense oligomers were injected at a concentration of 0.5 mM
between the one- and eight-cell stage into the yolk cell close to the
blastomeres, and the injected amount was estimated from the concentration and
volume measured by injection of a sphere of MO solution into oil at the same
pressure settings. As a control, a non-priming morpholino
(Scholpp et al., 2006) was
used, which showed no effect on embryos injected at 0.5 mM but caused
unspecific effects at a concentration of 1.5 mM.
For mis-expression experiments, full-length otx2 was cloned into a
pCS2+ vector (Rupp et al.,
1994) and, from this template, mRNA was synthesised in vitro
(Message Machine kit, Amersham). Together with rhodamine dextran as the
lineage tracer, 120 pg mRNA was injected into 1 out of 64 cells (Miniruby,
Invitrogen).
The heat-shock (hs)-inducible otx2-GFP construct was generated by
using the XbaI and BamHI cloning sites in the hs-GFP-vector
described in Lewis et al. (Lewis et al.,
2004). The generated otx2 gene lacks its 5' UTR,
preventing binding to the otx2-MO. The DNA construct (60 ng) was
injected into the first blastomere. At tailbud stage, embryos were
heat-shocked for 1 hour at 37°C. Injected embryos were fixed in 4% PFA
overnight at 4°C before in situ hybridisation or antibody staining.
Whole-mount in situ hybridisation
Whole-mount mRNA in situ hybridisations (ISHs) were performed as previously
described (Scholpp et al.,
2003). Digoxygenin- and fluorescein-labelled probes were prepared
from linearised templates using an RNA labelling kit (Roche). Blue staining
was achieved using 1 mg/ml NBT/BCIP in NTMT (pH 9.5), whereas red staining was
performed using Fast Red (Roche) dissolved in 0.1 M Tris-HCl (pH 8.0). Stained
embryos were dissected and mounted in 70% (v/v) glycerol/PBS. Expression
patterns have been described for otx1l (originally described as
otx3) and otx2 (Mercier
et al., 1995), shha [originally described as shh
(Krauss et al., 1993)],
shhb [originally described as twhh
(Ekker et al., 1995)],
dlx2a (Akimenko et al.,
1994), dbx1a [originally described as hlx1
(Fjose et al., 1994)],
lhx5 [originally described as lim5
(Toyama et al., 1995)],
ptc1 (Concordet et al.,
1996), dmbx1a
(Kawahara et al., 2002),
epha4a [originally described as rtk1
(Xu et al., 1994)],
fezf2 (originally described as fezl)
(Hashimoto et al., 2000),
pax6a (Macdonald et al.,
1994), gsh1 (Cheesman
and Eisen, 2004), and pax7
(Seo et al., 1998).
Embryos were photographed on a Zeiss Axiocam/Axioskop or were imaged using a Nikon C1 confocal microscope. The data sets were `deconvolved' by AutoDeblur X Gold-Edition (AutoQuant) and further processed using Imaris 4.1.3 (Bitplane AG). Composites were assembled in Adobe Photoshop 7.0.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
follow changes in expression of the Otx genes during the subsequent period of
forebrain subdivision in zebrafish, we examined the expression of
otx2 and of regionally expressed markers of the MDT
(Krauss et al., 1993;
Mercier et al., 1995) from
early segmentation to the pharyngula period. During this time period, we found
a very similar expression pattern for otx1l (formerly described as
otx3) and otx2, as previously described
(Mercier et al., 1995). We
therefore only show the expression of otx2, described below
(Fig. 1).
At the 10-somite stage, otx2 was downregulated in the most
anterior part of the neural tube and formed a defined anterior boundary, which
was maintained through the pharyngula stages. The anterior otx2
expression co-localised with most of the wnt8b expression domain, a
marker of the presumptive ZLI (pZLI), as described in chick
(Fig. 1A). At the same stage,
irx1b is expressed in the posterior diencephalon, marking the
thalamic anlage (Lecaudey et al.,
2004), whereas the otx2-positive pZLI was negative for
irx1b (Fig. 1B). At
the 15-somite stage, shha expression is detectable in the basal root
of the pZLI - precisely between the prethalamic and thalamic anlage
(Fig. 1C)
(Barth and Wilson, 1995;
Scholpp et al., 2006).
otx2 expression was additionally downregulated in an area of the
posterior-most forebrain, encompassing the pretectal anlage. Interestingly,
the anterior boundary of the remaining otx2 expression domain later
coincided precisely with the anterior boundary of the shha-positive
ZLI domain at pharyngula stage [prim-15; 30 hours post-fertilisation (hpf);
Fig. 1D,D']. Posterior to
the MDT, otx2 was very weakly expressed in the pretectum and strongly
maintained in the midbrain (Fig.
1D,D'). To further examine the molecular profile of the MDT,
we analysed the expression of well-characterised markers of the forebrain
primordium at prim-15. We found that otx2 expression abuts the
prethalamic expression domain of dlx2a
(Fig. 1E,E')
(Akimenko et al., 1994;
Scholpp et al., 2006).
Similarly to shha, the anterior border of dbx1a [formerly
described as hlx-1
(Fjose et al., 1994;
Scholpp et al., 2006)]
expression - marking the anterobasal part of the ZLI - coincided with the
otx2 expression domain (Fig.
1F,F'). Thus, otx2 expression splits the MDT at the
position of the presumptive prethalamus-ZLI border. In addition,
dbx1a expression also marked the thalamus. This thalamic expression
domain overlapped with that of otx2
(Fig. 1F,F').
Interestingly, the posterior boundary of the thalamic domain of dbx1a
expression did not fully resemble the otx2 expression border,
suggesting that otx2 expression might not entirely cover the thalamic
anlage (Fig. 1F'). At
this stage, irx1b expression still marked the thalamic territory but
not the ZLI anlage, which remained otx2-positive
(Fig. 1G,G')
(Lecaudey et al., 2004).
Therefore, we found the following gene expression code within the diencephalon
from anterior to posterior: the prethalamus is dlx2a-positive,
otx2-negative; the ZLI primordium is otx2- and
shh-positive; the anterobasal part of the ZLI is additionally
dbx1a-positive; the thalamus is otx2-, irx1b- and
dbx1a-positive; the pretectum shows weak expression of otx2.
In summary, these data show that shha expression in the ZLI is
detected exclusively at the rostral part of the Otx-positive diencephalic
domain.
|
). Due to
a strong maternal contribution, Otx1l and Otx2 protein can be detected by
western blot analysis until mid-somitogenesis in embryos injected with Otx1l-
and Otx2-MO; these are thus hypomorphs and are referred to as OtxH
(Foucher et al., 2006). This
approach provides a tool to address the late functions of the Otx gene family
without interfering with those functions occurring earlier, which have been
studied in mouse (reviewed in Simeone et
al., 2002). To address whether the forebrain territory per se is
initiated and formed correctly in OtxH embryos during the segmentation period,
we studied the expression of well-described regional markers - pax6a,
epha4a and barhl2 (Macdonald
et al., 1994; Xu et al.,
1994; Colombo et al.,
2006) - at the 12-somite and prim-15 stages. Although the
hindbrain expression domains of pax6a and epha4a have
already started to expand anteriorly at 12-somites
(Foucher et al., 2006), we
found no significant change in the diencephalic expression domains of these
markers in OtxH embryos (Fig.
2A-F). At prim-15, the forebrain territory still expressed
pax6a and epha4a, but their expression domains were shorter
along the anteroposterior axis (Fig.
2G-J). Interestingly, the downregulation of these two markers
within the ZLI territory at prim-15 that occurred in control embryos was not
observed in OtxH embryos (Fig.
2H,J). Furthermore, we found a dramatic reduction in the
expression of the mid-diencephalic marker barhl2
(Fig. 2K,L) while its
expression persisted unaltered in the telencephalon and in a small patch of
cells in the posterior hypothalamus.
To further investigate the OtxH phenotype at the axial level of the ZLI, we
analysed GFP expression in shha-GFP transgenic embryos
(Fig. 3A-H)
(Neumann and Nuesslein-Volhard,
2000). At the 15-somite stage, the phenotype of OtxH embryos was
indistinguishable from control-injected transgenic siblings
(Fig. 3A,B). From the 20-somite
stage to prim-18, Shha-GFP expression in the ZLI progressed from ventral to
dorsal in control siblings (Fig.
3C,E,G), whereas, in OtxH embryos, GFP expression was not
initiated in the ZLI, although the basal plate expression domain persisted
(Fig. 3D,F,H). As described
previously, the posterior commissure (PC) does not form in OtxH embryos
(Foucher et al., 2006).
However, we found that the nucleus of the medial longitudinal fascicle (nMLF)
was detectable (Fig. 3G,H). To
investigate this phenotype further, we performed an in situ hybridisation
(ISH) analysis for markers of the ZLI: shha, the Shh-dependent target
gene ptc1, and wnt8b. Expression of both shha and
ptc1 was absent from the pZLI in OtxH fish, whereas their expression
was unchanged in their other expression domains, such as the hypothalamus,
basal plate and floor plate, at prim-10
(Fig. 3I-L). In addition,
diencephalic expression of wnt8b, a marker for the pZLI, was
unchanged at the 18-somite stage in OtxH embryos [although the
midbrain-hindbrain boundary (MHB) expression domain shifted anteriorly;
Fig. 3M,N]. However, by
prim-10, wnt8b ZLI expression was missing, while wnt8b
expression was maintained in the dorsal telencephalon and posterior
hypothalamus - presumably the equivalent to the retromamillary region in chick
(Garda et al., 2002) - in
these embryos (Fig. 3O,P).
|
|
). The persistence of these expression domains demonstrates
the maintenance of a prethalamic territory. However, we found that
dlx2a was absent from the prethalamus, as predicted by the known
requirement for Shh signalling in the expression of this gene (see
Fig. 5E,G)
(Scholpp et al., 2006).
Interestingly, the preoptic expression domain of dlx2a was expanded
similarly to the prethalamic markers described above
(Fig. 5G, arrow).
|
). The
irx1b expression domain abuts the ZLI rostrally
(Fig. 4K), and the pretectal
domain caudally marked by gsh1
(Fig. 4M). We found that
irx1b expression was absent in OtxH embryos during the early
segmentation stages, before ZLI formation, suggesting that Otx function is
independently required to maintain irx1b expression in the thalamus
(Fig. 4I,J). At prim-10, we
found that this Shh-independent irx1b expression domain was still
downregulated in OtxH embryos (Fig.
4L,N). Moreover, expression of the Shh-dependent thalamic marker
genes emx2 and dbx1a was absent
(Fig. 4G,H and data not shown)
due to the lack of the ZLI (Scholpp et
al., 2006) or due to an independent requirement for Otx function
in the thalamus.
The loss of ZLI and thalamus markers in OtxH fish, with only the minor loss
of neural tissue, raised the issue of fate identity of this tissue in OtxH
morphant embryos. With the exception of the basal area, we showed that these
cells are mostly not prethalamic, and therefore considered whether the
mis-specified thalamus might possess posterior properties by examining the
expression of two pretectal markers - pax7 and gsh1
(Seo et al., 1998;
Cheesman and Eisen, 2004).
Interestingly, pax7 expression was already expanded anteriorly at the
20-somite stage (Fig. 4I,J).
Similarly, we observed a significant rostral shift in the expression domains
of these genes, suggesting a transformation of the ZLI/thalamus anlage into
pretectum, at the prim-10 stage (Fig.
4C,D,M,N). This anterior shift of the pretectum was also
detectable in the basal plate: the primordium of the nMLF, marked by
lhx5 expression, was also shifted rostrally
(Fig. 4C,D). Our previous
analysis (Foucher et al.,
2006) excludes the possibility of restricted cell death in the
diencephalon of OtxH embryos. We thus conclude that Otx1l/2 function is
required to specify the fate of the ZLI and the thalamus: absence of these
proteins led to transformation of the ZLI/thalamus into pretectum and, to a
lesser extent, into basal prethalamus (Fig.
4O,P). Finally, loss of expression of the early Shh-independent
thalamic marker irx1b in OtxH embryos indicates a direct Otx
requirement for proper specification of the thalamus, possibly via the
maintenance of Irx function.
Ectopic Otx2 can cell-autonomously rescue shha expression in the ZLI in OtxH morphant embryos
To find whether Otx1l/2 function is sufficient to induce shha
expression in the diencephalic alar plate, we generated a heat shock-inducible
construct driving expression of a GFP-tagged Otx2 protein (HS-Otx2-GFP), which
could not be repressed by the otx2 MO (see Materials and methods).
After injection of this construct and the Otx1l- and Otx2-MO mix, we
heat-shocked the embryos at the early neural plate stage (tail bud) and
performed our analysis at prim-10 (Fig.
1A). To reliably detect co-localisation between cell clones
positive for Shha and the Otx2-GFP fusion protein, we performed a single ISH
for shha in combination with 
-GFP-antibody staining
(Fig. 5B-C'').
Furthermore, to evaluate the position of the ectopically induced shha
expression, a set of treated embryos was assessed for shha and for
endogenous otx2 transcripts (Fig.
5D,F,H). Finally, we tested whether expression of dlx2a,
a Shh-dependent prethalamic marker gene, was rescued non-cell-autonomously by
the diencephalic cells expressing otx2-GFP that were also positive
for shha expression (Fig.
5C,G,I).
In OtxH embryos, shha expression was absent from the ZLI territory
and the otx2-positive midbrain territory was lacking
(Fig. 5B,F). After heat-shock,
otx2-positive clones, marked by GFP, were randomly induced throughout
the embryo (Fig. 5B,C) because
of the random distribution of the injected DNA
(Gilmour et al., 2002). In
cases in which embryos were not treated by heat-shock, or when
Otx2-GFP-positive clones were not located within the MDT primordium, we
observed the phenotype described for OtxH embryos
(Fig. 5B,G): the
shha-positive ZLI was missing, the posterior diencephalon had shrunk
and dlx2a expression was not induced in the prethalamic anlage due to
the lack of ZLI signalling. Whenever Otx2-GFP-positive cell clones were
located within the pZLI area, shha expression was induced in these
clones cell-autonomously (Fig.
5C-C''). When the heat-shock was performed during
mid-somitogenesis (15-somite stage), we found no rescue of shha
expression within the ZLI territory (data not shown). To locate the position
of the induced clones in the diencephalic territory, we performed another set
of experiments using otx2 expression itself as a marker to visualise
the prethalamic-thalamic boundary (Fig.
5D,F,H). In control MO-injected embryos, the ZLI formed within the
otx2-positive territory abutting the prethalamic domain
(Fig. 1D and
Fig. 5D). In OtxH embryos, the
ZLI was missing, whereas the endogenous otx2 transcript could still
be detected and used as a landmark for the prethalamus-thalamus border
(Fig. 5F). In OtxH/HS-Otx2GFP
embryos, shha expression could only be detected at the anterior Otx2
expression border (Fig. 5H). A
cross-section at the level of the ZLI anlage of the same embryo showed that
the cell patch rescued for shha expression was not connected to the
shha expression of the basal plate
(Fig. 5H'). We never
found ectopic induction of shha expression in the prethalamus or in
the presumptive posterior thalamus or pretectum.
|
) and, in
a cross-section, the lateral position of the prethalamus could be visualised
in relation to the ZLI (Fig.
5E'). In OtxH embryos, the expression of dlx2a was
missing in the prethalamus (Fig.
5G,G') but was sometimes present posterior to the
prethalamus, close to the shha-positive basal plate
(Fig. 5G,G', arrows),
similarly to the caudo-ventral expansion described for other prethalamic
markers (Fig. 4). Most
importantly, the alar shha expression induced by Otx2-GFP was
sufficient to rescue the expression of dlx2a in the prethalamic
primordium (Fig. 5I). In a
cross-section, the rescue could be seen unilaterally, whereas the control side
still lacked proper dlx2a expression
(Fig. 5I').
|
Thus, we conclude that Otx1l/2 function is necessary cell-autonomously for both the formation of a functional ZLI organiser and the maturation of thalamic identity. Both the pre-ZLI and the prospective thalamus therefore independently require Otx activity for their development. In addition, the highly restricted spatial competence of Otx to induce alar shha expression suggests a mechanism by which shha expression is actively repressed posterior to the pZLI possibly via maintenance of irx1b expression.
Irx1b sets the boundary between the ZLI and the thalamus
From our analysis so far, we can conclude that Otx1l/2 are required for the
induction of the ZLI and the thalamic anlage. However, although Otx1l and Otx2
are expressed in an area comprising both the pZLI and thalamus, our mosaic
analysis showed that they are not able to induce ZLI identity ectopically in
the presumptive thalamus. We therefore set out to address the issue of how ZLI
formation is restricted posteriorly. Like the Irx1 gene expression
domain in mice, the expression domain of the zebrafish orthologue,
irx1b, abuts on the ZLI territory
(Bosse et al., 1997;
Cohen et al., 2000;
Lecaudey et al., 2005). We
analysed the phenotype of embryos with reduced irx1b function by
using the antisense morpholino approach
(Itoh et al., 2002). To
visualise the sequence of events during ZLI formation in irx1b
morphants, we injected the MO into shha-GFP transgenic embryos. Until
the 20-somite stage (19 hpf), shha expression was comparable to
control siblings (compare to Fig.
6A with Fig. 3C).
At prim-5 (26 hpf), we observed some cells posterior to the pZLI domain
switching on GFP expression (Fig.
6B, arrowheads). At prim-18 (32 hpf), we observed a broadening of
the Shha-GFP domain (Fig. 6C).
At prim-10, we found that the ZLI territory marked by shha expression
was massively expanded caudally, whereas the pretectal domain marked by
gsh1 was unaltered (Fig.
6D,E, arrows). Similarly, the expression of shhb
(formerly known as twhh) was also broader, expanding posteriorly,
whereas the pretectal dmbx1a expression domain was not visibly
affected (Fig. 6F,G, arrows).
Consequently, the pax6a expression domain within the thalamic and
pretectal area shrinks upon expansion of the ZLI domain at prim-18
(Fig. 6H,I, arrows). These data
show that, when Irx1b function is perturbed, the ZLI expands into the thalamic
area. In addition, the observation of a normal pretectum and unaltered
formation of the PC in irx1b morphant embryos, implies normal
development of the diencephalic-mesencephalic boundary, in contrast to OtxH
embryos (Scholpp et al., 2003;
Foucher et al., 2006). We
conclude that, in embryos lacking Irx1b function, the ZLI is normally
initiated at the prethalamic-thalamic junction but subsequently expands
posteriorly. Irx1b is therefore required to repress Shh expression and thus
sets the posterior boundary of the ZLI in the diencephalic Otx-expressing
domain.
|
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wilson and Houart, 2004;
Kiecker and Lumsden, 2005). To
date, the mechanisms by which one of these local organisers, the ZLI, is
precisely restricted spatially and temporally, have yet to be unravelled.
Here, we describe a novel mechanism by which the ZLI is induced and positioned
in the presumptive diencephalon. As early as ten somites, the anterior limit
of otx1l/2 expression marked the boundary between the prethalamus and
the thalamus - the precise anteroposterior position of the future ZLI. We show
that lack of otx1l/2 function leads to the absence of the ZLI and
subsequently of ZLI-dependent target genes in the MDT. Moreover, with a lack
of Otx function, the thalamus was mis-specified prior to, and independently
from, ZLI formation. The adjacent territories of the prethalamus and pretectum
expanded into the mis-specified territory and formed a new interface.
Conditional mosaic induction of Otx2 expression during somitogenesis, in
Otx1l/2-deficient embryos, was able to rescue shha expression in the
pZLI area as well as irx1b expression in the thalamic anlage.
Therefore, Otx1l/2 is necessary for the specification of both the ZLI and
thalamus. Finally, the fate distinction between the Otx-positive ZLI anlage
and the Otx-positive presumptive thalamus is mediated by Irx1b, as shown by
the broadening of the ZLI at the expense of the thalamus when Irx1b function
is decreased.
|
;
Foucher et al., 2006). In the
diencephalon, we found that ZLI formation, including shha induction,
is dependent on proper Otx1l/2 function. Mice embryos with reduced Otx1/2
transcripts show a similar lack of Shh ZLI expression. In these mice,
the direct or indirect nature of such loss could not be addressed and the lack
of ZLI was proposed to be a consequence of the proximity of the MHB organiser
and its repressive influence on the diencephalon
(Acampora et al., 1997;
Li and Joyner, 2001;
Scholpp et al., 2003). In this
report, we used a mosaic analysis and temporally controlled mis-expression in
zebrafish, and were able to show that the formation of the ZLI is a novel
specific function of Otx1l/2 proteins. By contrast, a recent study suggests
that Otx2 protein is also able to restrict Shh expression in the
floor plate in a dose-dependent manner during mouse midbrain development
(Puelles et al., 2003). The
authors propose a model in which Otx1/2 and Groucho, a family of
transcriptional co-repressors, are needed to mediate a repressive interaction
that limits Shh expression to the basal plate. Although Groucho
activity has been described for the formation of the MHB organiser
(Wurst and Bally-Cuif, 2001),
there are no Groucho genes known to be involved in ZLI induction, neither in
zebrafish nor in other species. This opens the possibility of a dual
regulation of shh expression by Otx proteins: inhibitory in the
presence of Groucho and activating in its absence.
Formation of the ZLI
There is an ongoing debate about the early regionalisation of the neural
tube and the positioning of the ZLI. It is widely believed that, in mouse and
chick, the ZLI forms directly above the anterior tip of the notochord, and
thus at the interface between the prechordal neuraxis, induced by prechordal
plate mesoderm, and the epichordal neuraxis, induced by the chordamesoderm
(Shimamura et al., 1995).
Because Fgf8-coated beads are able to induce Foxg1 in the prechordal
neural plate and En1/2 in the epichordal neural plate in mice, it has
been suggested that these two tissues have different competence mediated by
the Iroquois complex (Shimamura and
Rubenstein, 1997; Kobayashi et
al., 2002). However, direct evidence of mesodermal inductive
influence on ZLI positioning is lacking, and in other model organisms, such as
fish, it has been shown that the influence of the mesoderm on induction and
anteroposterior pattern of the neural tube, and therefore positioning and
induction of the ZLI, is dispensable
(Schier et al., 1997;
Scholpp et al., 2006). It has
been suggested that, in chick, the pre-ZLI territory is a Wnt8b-positive and
L-fng-free wedge-shaped area that collapses during development and forms the
definitive ZLI compartment with lineage restriction at both its rostral and
caudal boundaries (Zeltser et al.,
2001). Furthermore, grafting experiments in chick have shown that
an ectopic border between neural tissue from a prechordal and epichordal
origin is sufficient to induce an ectopic ZLI
(Vieira et al., 2005).
The molecular players involved in ZLI induction have yet to be identified.
Gain-of-function experiments in chick have suggested a model in which the
interaction between Six3 and Irx3 induces the ZLI
(Kobayashi et al., 2002;
Braun et al., 2003). However,
temporal analysis of the expression patterns for these two genes show a gap
between the two domains at the time of ZLI formation, precluding direct
interaction between the two territories at this stage (reviewed in
Kiecker and Lumsden, 2005).
Moreover, the ZLI forms in Six3-deficient mice, excluding an
inductive role for Six3 (Lagutin et al.,
2003). Finally, our data show that irx1b function is also
dispensable for ZLI induction.
Recently, a new border of gene expression has been suggested to be involved
in positioning the ZLI organiser, involving an interaction between the
prechordally expressed Fezf1 and Fezf2 genes with an unknown
epichordal factor (Hirata et al.,
2006; Jeong et al.,
2007). Fezf1/Fezf2-deficient mice lack Shh expression in
the ZLI, due to mis-specification of the prethalamus. Interestingly,
mis-expression of Fezf2 shifts caudally and reduces the Shh-positive
ZLI in fish and mice. We believe that our data allow us now to integrate the
various morphological and genetic data and suggest a new model for the
induction and positioning of the ZLI along the anteroposterior axis of the
neural tube. The spatially restricted stripe-like expression domain of Otx1l/2
patterns the neural tube prior to ZLI formation, setting the anterior border
of the pZLI. We show that the ZLI is formed by the rostral half of this
Otx-expressing domain, which co-localises with the stripe of wnt8b
expression. Thus, this Otx1l/2/Wnt8b domain would appear to correspond
directly with the L-fng-negative/Wnt8b-positive compartment that was
previously described as the pZLI in chick
(Larsen et al., 2001;
Zeltser et al., 2001;
Garcia-Lopez et al., 2004).
This expression domain abuts the Irx-expressing territory in fish,
chick and mouse. Finally, in both chick and zebrafish, it collapses into a
narrow stripe of Hh-expressing cells as it matures into the ZLI
(Fig. 7) (Zeltzer et al.,
2001).
Independent positioning of the anterior and posterior ZLI boundaries
A striking similarity between the ZLI and the MHB organisers is their
location at the interface between an Otx-positive and an Otx-negative
territory. Besides the involvement of Otx proteins, the two signalling centres
also each release a combination of signal molecules (ZLI: Wnt8b and Shh; MHB:
Wnt1 and Fgf8). The boundary between the midbrain and hindbrain has
lineage-restriction properties (Zervas et
al., 2004; Langenberg and
Brand, 2005), whereas the ZLI is a narrow stripe of cells defined
by both an anterior and a posterior lineage-restriction boundary
(Larsen et al., 2001).
Therefore, because the ZLI is a zone rather than an interface, we considered
whether the position of its prethalamic border is set independently of its
thalamic border. In addition to the requirement for an
otx1l/2-positive tissue for the formation of the ZLI, and concomitant
establishment of its anterior limit, we uncovered an irx1b-dependent
regulation setting the posterior limit of the ZLI. The striking shape of the
irx1b-expressing domain suggests that it might give the
Shh-expressing cell population its characteristic tapering shape. In chick,
Irx3 has been shown to mediate thalamic competence and, furthermore,
Irx3 has a repressive function on ZLI formation
(Kiecker and Lumsden, 2004);
however, chick loss-of-function data are missing, preventing direct comparison
with our fish results. Neither irx3a nor irx3b, the fish
orthologues of Irx3, are expressed directly adjacent at the future
ZLI boundary, whereas Irx1 in mouse and Irx1b in fish abut the ZLI
(Bosse et al., 1997;
Cohen et al., 2000;
Lecaudey et al., 2005). By
knock-down of Irx1b function, we have been able to show that the expression
domains of the Shh genes - shha and shhb - expand
posteriorly at the expense of thalamic tissue, while the anterior boundary is
unaltered. Complementarily, a recent study has shown that Fezf proteins,
expressed in the presumptive prethalamus and ZLI during early segmentation
stages, are required for ZLI formation. Otx proteins need to interact with
another protein inside the anterior diencephalon, because we showed that Otx
proteins are able to induce the ZLI solely inside the diencephalic territory
anterior to the presumptive thalamus. We therefore propose that Fezf could be
such a partner. The nature of the proposed Otx/Fezf relationship in the
rostral neural tube therefore needs to be addressed. Thus, we conclude that
the establishment of the anterior and posterior boundaries of the ZLI are
independent events and require different mechanisms.
Otx requirement for thalamic identity
In OtxH embryos, we observed that cells located at the anteroposterior
position of the thalamic anlage expressed pretectal markers such as
pax7 (Fig. 4). The
Otx1cre/-; Otx2flox/- mouse shows small patches
of cells deficient for Otx1/2 function in the thalamic anlage
(Puelles et al., 2006). These
patches show characteristics of inhibitory GABAergic neurons in the thalamic
anlage, although the thalamus normally contains mostly excitatory
glutamatergic cells. Due to characteristic pretectal pax3 and
pax7 expression and the fact that the adjacent pretectum generates
mainly GABAergic neurons, the authors claim that the lack of Otx1/2
transcripts leads to induction of pretectal fate. We found that thalamic
tissue, which is marked by neurogenin 1 expression and is a
prerequisite for the generation of glutamatergic neurons, is not specified in
OtxH embryos. Interestingly, a recent mouse study showed that gsh1
expression is required for the formation of GABAergic neurons in the spinal
cord (Mizuguchi et al., 2006).
We found that pretectal gsh1 expression was shifted to the location
of the thalamic anlage in OtxH embryos. Due to the fact that neither cell
proliferation nor cell death is dramatically increased in OtxH embryos
(Foucher et al., 2006), we
suggest a fate change of these cells from a thalamic to a pretectal identity,
in agreement with the finding in mouse
(Puelles et al., 2006).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Inserm UMRS587, Unité de Génétique
des Déficits Sensoriels, Collège de France, Institut Pasteur,
75015 Paris, France
|
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