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First published online 1 February 2006
doi: 10.1242/dev.02248
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1 MRC Centre for Developmental Neurobiology, King's College London, Guy's
Campus, London SE1 1UL, UK.
2 Biotechnology Center, University of Technology, c/o Max Planck Institute of
Molecular Cell Biology and Genetics, Dresden, Germany.
* Author for correspondence (e-mail: andrew.lumsden{at}kcl.ac.uk)
Accepted 14 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Regionalization, Forebrain, Shh, Zebrafish, ZLI
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wilson and Houart,
2004; Kiecker and Lumsden,
2005). This may involve specialized groups of cells in `signalling
centres' (reviewed by Rhinn and Brand,
2001), the best-characterized of which are the anterior neural
border (ANB) (Houart et al.,
2002) (reviewed by Wilson and
Houart, 2004), the roof plate (reviewed in
Chizhikov and Millen, 2004),
the floor plate (reviewed in Strähle
et al., 2004) and the midbrain-hindbrain boundary (MHB). The cells
that release signal molecules from such centres are often located at
boundaries between distinct territories, e.g. the MHB forms at the interface
between the mesencephalon and anterior rhombencephalon, whereas the ANB lies
at the boundary between the telencephalon and anterior epidermal ectoderm. The
zona limitans intrathalamica (ZLI), a narrow transverse region between the
prethalamus (also known as the ventral thalamus) and the thalamus (also known
as the dorsal thalamus) (Kuhlenbeck,
1937; Shimamura et al.,
1995) also bears the hallmarks of a signalling centre. Fate
mapping experiments in chick have shown that the ZLI is cell lineage
restricted at its boundaries and is thus a true developmental compartment
(Zeltser et al., 2001;
Garcia-Lopez et al., 2004).
Furthermore, the ZLI is the only structure in the alar plate that expresses
signal molecules of the Hedgehog family (Hh)
(Figdor and Stern, 1993;
Puelles and Rubenstein, 2003).
Well-described functions of Hh signalling from basal and floor plates are
ventralization of the neural tube (reviewed by
Briscoe and Ericson, 1999;
Jessell, 2000), promotion of
growth and proliferation (Britto et al.,
2002; Ruiz i Altaba et al.,
2002), and formation of the hypothalamus
(Chiang et al., 1996;
Mathieu et al., 2002). The
function of Hh signalling at the ZLI has not been addressed directly in mouse
Shh mutants owing to loss of the diencephalon
(Ishibashi and McMahon, 2002),
but studies in chick have shown that Shh is both necessary and sufficient for
thalamic gene induction in vitro (Hashimoto-Torri et al., 2003) and in vivo
(Kiecker and Lumsden,
2004).
In zebrafish, two Hh genes are expressed in the ZLI: sonic
hedgehog (shh) (Krauss et
al., 1993) and tiggy-winkle hedgehog (twhh)
(Ekker et al., 1995). Normal
development of the ZLI has not been described in zebrafish nor have the
mechanisms of its formation been investigated. In addition, it is important to
examine whether recent observations in chick
(Kiecker and Lumsden, 2004)
could be ascribed to an evolutionary conserved mechanism for diencephalic
patterning.
Here, we describe how the mid-diencephalic territory (MDT, composed of prethalamus, the ZLI and thalamus) develops in zebrafish. We show that the expression of shh and twhh mark the ZLI, and that ZLI development is accompanied by expression of dlx2a, a marker of the prethalamus, and of dbx1a, a marker of the thalamus. Furthermore, we show that Hh signalling is sufficient for molecular differentiation of both the prethalamus and the thalamus, but is not required for their maintenance. Interestingly, shh and twhh function similarly during prethalamic induction, whereas thalamic induction appears to require Shh signalling exclusively. Finally, we show that the ZLI forms independently of the basal plate and that Hh signalling from the ZLI is sufficient for maturation of prethalamic and thalamic territories while ventral Hh signals are dispensable.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Our data derive from analysis of wild-type (wt) fish and
the following homozygous mutant embryos and transgenic fish: sonic you
tbx392 (referred to as syu)
(Schauerte et al., 1998),
slow muscle omittedb641 (referred to as smu)
(Barresi et al., 2000),
one-eyed-pinheadtz57 (referred to as oep)
(Hammerschmidt et al., 1996;
Schier et al., 1997) and
2.2shh:gfp:ABC#15 (referred to as shh:GFP)
(Shkumatava et al., 2004).
Injections
Expression constructs for shh mRNA
(Krauss et al., 1993) and for
twhh mRNA (Hammond et al.,
2003) were generated in vitro (Message Machine Kit, Amersham).
mRNA was dissolved in 0.25 M KCl including 0.2% of fluorescein-labelled
dextran (Mini Emerald, Molecular Probes) as a lineage tracer. During
injection, 
150 pg mRNA was deposited into one cell of a 32-cell stage
embryo. For transient knock-down of gene expression, Morpholino-antisense
oligomers (MO) were used at a concentration of 0.5 mM as described previously
(Nasevicius and Ekker, 2000;
Scholpp et al., 2003).
twhh morpholinos (twhh-MO: 5'-GCT TCA GAT GCA GCC TTA
CGT CCA T-3') (Lewis and Eisen,
2001) were injected into the yolk cell close to the blastomeres at
one- to eight-cell stages at a concentration of 0.5 mM. A non-binding
morpholino (morpholino-sense twhh oligomer; con-MO) showed no effect
on embryos when injected at 0.5 mM.
Transplantation
At the one-cell stage, wild-type embryos were injected with 0.25%
rhodamine- or biotin-dextran (Molecular Probes). Thirty to 40 cells from the
animal region were grafted into a host embryo at the sphere stage (3.5 hpf) to
generate a random distribution of labelled cells. At 32 hpf, embryos were
identified by morphology or GFP expression.
Inhibitor treatment
3-Keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine
(KAAD-cyclopamine; Toronto Research Chemicals, Canada) was dissolved in
ethanol at 70 µM to block Hh signalling and to treat embryos at different
intervals up to 32 hpf. Control siblings were vehicle treated.
Staining procedures and imaging techniques
Whole-mount mRNA in situ hybridization was carried out as described
previously (Scholpp et al.,
2003), and stained by NBT/BCIP and Fast Red (Roche). Embryos were
dissected and mounted in 70% (v/v) glycerol/PBS or further processed for
antibody staining. Expression patterns have been described for shh
(Krauss et al., 1993),
twhh (Ekker et al.,
1995), dlx2a (originally described as dlx2)
(Akimenko et al., 1994),
dbx1a (originally described as hlx1)
(Fjose et al., 1994),
neurog1 (Blader et al.,
1997), lhx5 (originally described as lim5)
(Toyama et al., 1995),
ptc1 (Concordet et al.,
1996), emx1 and emx2
(Morita et al., 1995).
Antibody staining was performed as described by Scholpp and Brand
(Scholpp and Brand, 2003).
Live transgenic embryos were mounted dorsal upwards in 1% LMP-agarose, and
imaged using a Nikon C1 confocal microscope. For the fate mapping experiment
(Fig. 5), the following
parameters were chosen: pinhole, 30 µm; z-step,10 µm. Images
were acquired by single scan combining red and green channels. Data sets were
deconvolved by AutoDeblur X CF (AutoQuant) and further processed using Imaris
4.1.3 (Bitplane AG).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To follow its development, we mapped expression of the
Hedgehog genes shh and twhh
(Krauss et al., 1993;
Ekker et al., 1995) between 12
somites and 48 hpf. These ZLI markers were mapped relative to the expression
of the prethalamic marker dlx2a (the homologue of Dlx2 in mouse)
(Akimenko et al., 1994) and the
thalamic marker dbx1a (the homologue of Dbx1 in mouse)
(Fjose et al., 1994) by double
in situ hybridization.
At 12 somites, shh expression is confined to the ventral midline
of the neural tube (Fig. 1A)
(Krauss et al., 1993). The
future anteroposterior position of the ZLI is already visible at this stage by
a kinking of the head (white arrow). At 15-somites, dlx2a is
expressed in the anterior forebrain in a `salt-and-pepper' pattern dorsal to
the ventral midline shh expression domain (blue arrow)
(Fig. 1B). At 20 somites, the
dorsal extension of shh in the future ZLI becomes more pronounced
(Fig. 1C, white arrow). We find
that spatial progression of dlx2a expression evolves concomitantly
with shh expression (Fig.
1D, white and blue arrows), in adjacent but non-overlapping
domains (Fig. 1D'). At 42
hpf, shh expression extends transversely in a very narrow domain
across the alar plate, prefiguring the ZLI
(Fig. 1E, white arrow)
(Puelles and Rubenstein,
2003). Notably, the ZLI is the only place in the embryo in which
shh is expressed in such a dorsal domain. Owing to the posterior
invagination of the dorsal forebrain, the shh expression domain
translates mediolaterally, resulting in the typical forked shape, the two
prongs of which reflect the position of the ZLI
(Fig. 1E', white arrow).
A further consequence of invagination is that the prethalamus becomes located
lateral to the ZLI (Fig.
1E,E', blue arrow).
We then analysed the expression of twhh relative to dbx1a. At 12 somites, twhh has an expression profile similar to that of shh (Fig. 1F) and dbx1a is expressed in the anterior neural ectoderm in a ventral domain overlapping with twhh (Fig. 1F, black arrow). By 15 somites, twhh expression is downregulated ventrally and maintained only in a patch of cells in the ventral telencephalon until at least 42 hpf (Fig. 1G-J). dbx1a expression is first detectable at 15 somites ventrally adjacent to the ZLI (Fig. 1G) as well as posterior to the ZLI (Fig. 1G, yellow arrow). Expression of twhh is first observed in the presumptive ZLI at 20 somites, (Fig. 1H, white arrow) and is accompanied by an extended dorsal domain of the dbx1a expression (Fig. 1I,J, marked by white arrows). The thalamic domain of dbx1a increases in both size and intensity over time (Fig. 1G-J). A horizontal section (Fig. 1I') reveals a stripe-like pattern in which we observed characteristic expression subdomains: an anterior region, where twhh and dbx1a are co-expressed (1); a more posterior twhh-positive stripe (2); a region of low dbx1a expression (3); and a region with strong dbx1a expression (4). At 42 hpf, dbx1a is expressed in the fork-shaped ZLI territory (Fig. 1J,J'). In a posteromedial position, dbx1a marks the thalamus, as shown in the section (yellow arrow).
To map gene expression domains onto emergent neuroanatomy at 48 hpf, we visualized the expression domains of shh, dlx2a and dbx1a by fluorescence in situ hybridization, followed by counterstaining with an anti-acetylated tubulin antibody to mark axons.
We analysed these combined patterns in the entire head
(Fig. 1K-M) using confocal
microscopy and three-dimensional reconstruction software. shh is
expressed in a medioventral domain stretching continuously from the anterior
hypothalamus through the tegmentum into the hindbrain
(Fig. 1K). Interestingly, the
shh expression domain in the alar plate appears small in lateral view
(K, white arrow), but reveals it full size in a vertical rotation
(Fig. 1K'). The
fork-shaped ZLI shows its ventral limit by a thin connection to the basal
expression domain (Fig.
1K'). In situ hybridization for dlx2a reveals the
location of the massive cup-shaped expression domain of the prethalamus
lateral to the ZLI on either side (Fig.
1L, blue arrows), from which there is but a very thin connection
to the more ventral expression domains of the preoptic region
(Fig. 1L') (reviewed in
Puelles and Rubenstein, 2003).
In the lateral view, the thalamic expression domain of dbx1a is
located posterior to the ZLI and medial in the neural tube. 3D rotation movies
can be provided on request that show how the original anteroposterior layout
of the MDT becomes translated lateromedially by 48 hpf.
Overexpression of shh increases the size of the MDT
To study local activity of Shh at the ZLI, we generated small
Shh-expressing clones by injecting 150 pg shh mRNA into one
blastomere of a 32-cell embryo (Fig.
2A-A''). To check efficiency, we analysed a bona fide
target gene of Hh signalling – the Hh receptor patched1
(ptc1) (Concordet et al.,
1996). After 28 hpf of normal development, ptc1 is
expressed in regions of normal Hh activity, e.g. the hypothalamus, ZLI and
floor plate (Fig. 2B,B').
In embryos injected with shh, we detected an increased expression of
ptc1 at the sites of endogenous expression, e.g. the ZLI (bracket),
and also ectopic sites of ptc1 expression that co-localised with
shh-positive clones, e.g. in the forebrain
(Fig. 2C,C', arrows).
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; Toyama et al.,
1995). Ectopic expression of shh mRNA resulted in the
expression of dlx2a (Fig.
2E,E') as well as of lhx5
(Fig. 2G,G') being
extended anteroposteriorly, with a higher intensity compared with non-injected
siblings (Fig.
2D,D',F,F'; bracket). In addition, dlx2a
expression was induced in ectopic locations
(Fig. 2E,E', marked by
arrow). Ectopic shh resulted in similar changes to expression of the
thalamic markers dbx1a, emx2 and neurog1
(Fig. 2H-K'; see Fig. S1
in the supplementary material) posterior to the ZLI. Here, mosaic
overexpression of Shh led to the anteroposterior expansion of both
dbx1a (Fig.
2F,F', bracket) and emx2
(Fig. 2G,G', yellow
arrow). The observed phenotypes cannot be explained by ventralisation of the neural tube in response to increased ventral Shh signalling, rather they suggest a function for shh in anteroposterior regionalization of the neural tube. Although our experimental approach produces Shh-overexpressing cells all over the embryo, the ectopic expression of prethalamic markers was observed only anterior to the ZLI, whereas increased expression of thalamic markers was observed only posterior to the ZLI. This suggests that competence fields anterior and posterior to the ZLI are established independently of Shh. Furthermore, we find that shh influences the acquisition of both prethalamic and thalamic fate, and specifies the size of these territories within these fields.
Hh signalling is required for specification of the MDT
To complement our gain-of-function approach, we analysed embryos treated
with the Hh signalling inhibitor cyclopamine
(Incardona et al., 1998), and
those carrying a mutation in the Hh co-receptor smoothened (slow
muscle omitted, smu), in which all Hh signalling is blocked
(Varga et al., 2001).
To elucidate the timing of the Hh requirement, we blocked Hh signalling with cyclopamine for different durations up to 30 hpf (Fig. 3A-D'). Blocking Hh signalling from 10 somites leads to a phenotype similar to that observed in smu mutant embryos with respect to diencephalic development: a strong downregulation of dlx2a and dbx1a (Fig. 3B). To verify our experimental procedure, we studied ptc1 expression in embryos of the same batch and found a severe downregulation, consistent with a blockade of Hh signalling (Fig. 3B'). Inhibition from 20 somites onwards results in a partial downregulation of dlx2a (Fig. 3A,C; blue brackets) and dbx1a (Fig. 3A,C; yellow brackets) compared with control siblings, while ptc1 expression was downregulated, as for the earlier treatments (Fig. 3C'). Cyclopamine treatment at 30 hpf produced only very subtle defects in the MDT by comparison with controls (Fig. 3D, blue and yellow brackets). Thus, we find that Hh signalling is required to induce prethalamic and thalamic markers during the normal induction phase between 10 somites and 24 hpf, and thereafter becomes dispensable for maintenance of marker expression.
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Distinct roles of Shh and Twhh in mid-diencephalic development
Because two Hh genes are expressed in the forming ZLI
(Fig. 1), we analysed the
individual contribution of Shh and Twhh to diencephalic development in a
series of loss-of-function experiments: analysis of the phenotype of
sonic-you embryos, carrying a mutation in the shh gene
(syu) (Schauerte et al.,
1998) and/or morphant embryos created by an antisense morpholino
targetting twhh mRNA (Fig.
4A-E,H-L) (Lewis and Eisen,
2001).
First, we studied the endogenous efficiency of Shh and Twhh by analysis of the expression width and strength of the bona fide target genes ptc1 and nkx2.2 in various loss-of-function combinations (Fig. 4A-D; data not shown) at the ZLI flanking region at 28 hours. The cells anterior and posterior the ZLI, which receive Hh signalling above the threshold required for ptc1 induction, can be used as an indirect readout of the relative efficiency of the Hh signals. ptc1 is expressed in a total width of 16 cells in wild-type embryos (n=42; Fig. 4A, bracket). In a knock-down analysis for twhh, we detect ptc1 expression all along the ZLI, but find the range of ptc1 expression is moderately reduced to 14 cells (7/15; Fig. 4B, bracket). Interestingly, in syu mutants, ptc1 expression resembles the expression pattern from the remaining twhh gene: positive in the ZLI, but missing in the adjacent ventral part (Fig. 4C, compare with Fig. 1I). The width of the expression domain of ptc1 decreases to five cells (n=12; bracket). To generate a combined shh/twhh mutant/knock-down, we injected twhh-MO into syu mutant embryos (Fig. 4H). In the shh/twhh mutant/knock-down, we find that ptc1 expression is strongly reduced and absent at the ZLI (Fig. 4D), arguing that twhh and shh are the only Hh genes acting in the ZLI. We conclude that at the ZLI, Twhh signalling is less effective than Shh signalling.
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). As
expected, the smu phenotype resembles the combinatorial loss of
Shh/Twhh function phenotype: absence of the prethalamic expression of
dlx2a and a strong reduction in hypothalamic expression (arrowhead),
consistent with the complete blockade of Twhh translation in a syu
mutant background, and with twhh and shh being the only Hh
genes acting in the ZLI territory.
To support our loss-of function analysis, we performed a mosaic
twhh overexpression experiment, as with shh
(Fig. 2)
(Hammond et al., 2003). We
analysed the embryos at 28 hpf by in situ hybridization for dlx2a
(Fig. 4J,K). Injection of
twhh mRNA (150 pg) led to a moderate expansion of dlx2a in
the prethalamus (n=14/42; Fig.
4E,J). Misexpression of shh mRNA (150 pg) led to a strong
anteroposterior expansion (20/43; Fig.
2D,D'). Similarly, a combination of shh and
twhh (150 pg each) also led to expansion of the prethalamic domain
(48/70; Fig. 4K). Thus, both Hh
genes are able to influence the formation of the prethalamus by upregulation
of prethalamic gene expression.
twhh morphant embryos display a phenotype similar to controls, allowing but a minor role for Twhh in dbx1a induction (Fig. 4L,M; square brackets). Analysis of syu mutant embryos, however, revealed a strong reduction of dbx1a (Fig. 4N), suggesting that, unlike the situation for the prethalamus, twhh is not able to compensate for the lack of shh posterior to the ZLI. Interestingly, the presumptive ZLI appeared to be slightly broadened (Fig. 4N; arrows). Knock down of twhh in the syu mutant background led to the complete loss of dbx1a expression in the thalamus (Fig. 4O) similar to the smu mutant phenotype (Fig. 4P), lending further support to Shh and Twhh being the only Hh proteins acting at the ZLI. Similar results were observed in the analysis of neurog1, another marker of thalamic differentiation (data not shown).
In a further gain-of-function analysis, we examined the embryos at 28 hpf by in situ hybridization using the thalamic marker dbx1a (Fig. 4Q,R). Misexpression of twhh led to a slight increase of the dbx1a-expression domain (10/34; Fig. 4L,Q), whereas shh was able considerably to expand the expression domain of dbx1a posteriorly (14/18; Fig. 2H,H'). Misexpression of both mRNAs resulted in a phenotype indistinguishable from that of shh overexpression alone (24/70; Fig. 4R), suggesting that shh is required for the induction of the dbx1a expression domain, whereas twhh does not augment the effect of shh under these conditions.
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Basal plate is dispensable for the formation of the MDT
From gastrulation stages, the ventral forebrain continuously expresses
hh transcripts (Fig.
1) (Krauss et al.,
1993), allowing the possibility that ventral Shh-expressing cells
could contribute to the formation of the ZLI by dorsalward cell migration or
by `bucket-brigade' Hh signalling.
To test these possibilities, we traced randomly distributed alar cells in a transgenic shh:GFP background (Shkutmatava et al., 2004) and followed their movement from 15 somites until 42 hpf. We found that ventral-to-dorsal cell movement in the alar plate is rather minor (Fig. 5A) and that cells keep their dorsoventral position for at least 24 hours until 42 hpf. In addition, alar plate cells are able to switch on Shh expression if they are located at the correct anteroposterior position of the presumptive ZLI (Fig. 5B,C; yellow arrow). These observations suggest that active cell movement is unimportant.
Another possibility is that Hh signalling from ventral regions is required
for induction of the ZLI, as Hh signalling is needed for dorsoventral
patterning in other regions of the CNS (reviewed by
Jacob and Briscoe, 2003). In
chick, this early ventral expression has been held responsible for Shh
expression within the ZLI itself (Zeltser,
2005). We asked, therefore, whether hh expression in the
ventral neural plate plays a role in the formation of the ZLI and in directly
regulating development of the prethalamus and thalamus. By studying embryos
carrying a mutated form of the EGF-CFC nodal co-receptor one-eyed
pinhead (oep) (Hammerschmidt
et al., 1996; Schier et al.,
1997; Gritsman et al.,
1999), which lack the anterior basal plate, we were able to study
mid-diencephalic development in the complete absence of the ventral source of
Hh signalling.
First, we analysed shh and twhh expression in these
mutant embryos at the 20-somite stage and 30 hpf. As expected, the ventral
expression domain in the diencephalon is absent in these mutants
(Fig. 5D,D'; see Fig. S1
in the supplementary material). At 20 somites, we saw the first expression of
shh in the dorsalmost part of the normal shh expression
domain in the alar plate (by definition, the ZLI) in oep mutants (see
Fig. S1 in the supplementary material). In all mutant embryos analysed, the
presumptive ZLI is similar in size to that of wild-type siblings
(n=63; Fig. 5D',
red arrows) (Schier et al.,
1997). This shows that formation of the ZLI and its Shh expression
domain are independent of the basal plate, and specifically of ventral Hh. To
compare the effective range of Hh signalling, we analysed the expression of
ptc1. Owing to the lack of Hh expression ventrally,
ptc1 becomes reduced in the majority of ventral regions. By contrast,
expression of ptc1 astride the ZLI shows the same anteroposterior and
dorsoventral extent as in wild-type embryos, suggesting that Hh signalling
from the ZLI is unaffected in oep mutant embryos
(Fig. 5E,E').
In further experiments, we analysed the formation of the MDT in oep mutants. Prethalamic dlx2a expression and thalamic neurog1 expression in oep mutants is similar to that in wild-type embryos (Fig. 5F-G', red arrows) indicating that Hh signalling from the ZLI alone is sufficient to induce the characteristic expression of markers in the MDT with no requirement for Hh signalling from the ventral region of the forebrain.
Wt cells are able to receive a Hh signal and display correct expression profile in smu mutant embryos
To address the issue of whether Hh signalling is necessary and sufficient
to induce prethalamic and thalamic fate at a distance, we grafted wild-type
cells into a smu mutant background either anterolateral or posterior
to the presumptive ZLI at sphere stage. At 32 hpf, we performed double in situ
hybridization with shh, as a marker for the ZLI, and a marker for
either the prethalamus (dlx2a) or thalamus (dbx1a). In
addition, we stained the embryos with FITC-conjugated streptavidin to detect
biotin-containing transplanted wild-type cells.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Formation of the MDT
We find that from the 12 somites onwards, shh and twhh
expression in the ZLI extends dorsally into the alar plate from a ventral
origin. Cell tracing experiments (Fig.
5) suggest that a progressive activation of genes is required for
acquisition of ZLI identity rather than it being formed by a stream of cells
migrating from the floor plate. However, we cannot exclude that migration
takes place at an earlier stage (before 15-somites) or that single cells from
the floor plate move dorsally. A 5 hour lag between the detection of GFP in
the ZLI and detection of shh mRNA by in situ hybridization provides
further evidence for progressive maturation
(Shkumatava et al., 2004).
Studies in chick have suggested that Shh is required for the formation of
the ZLI (Kiecker and Lumsden,
2004; Zeltser,
2005). However, we find that Hh expression in the zebrafish ZLI is
independent of Hh, or indeed any ventral signals. Thus, absence of the ventral
midline region of the neural tube, as in the oep mutant, does not
interfere with establishment of the ZLI. This has been observed previously in
other nodal mutants such as cyclops
(Barth and Wilson, 1995).
Furthermore, we can conclude that Hh signalling is dispensable for the
formation of the ZLI, as shown in the smu mutant embryos
(Fig. 3)
(Varga et al., 2001). Although
the ZLI appears narrower in smu mutants when compared with wild-type
siblings, the grafting assay shows that Hh from the ZLI is still able to
regionalize the territory appropriately
(Fig. 6). One possible
explanation for the difference between chick and zebrafish is that the
experimentally induced reduction of ventral Hh signalling in chick causes the
ZLI to mature more slowly. Alternatively, the positive feedback autoregulatory
mechanism for Hh expression, a plausible mechanism in chick
(Kiecker and Lumsden, 2004),
is less evident in fish. The persistence of shh expression in the ZLI
of smu embryos argues that positive feedback autoregulation is indeed
of little importance in zebrafish.
In the absence of dorsalward cell migration from the basal plate, we
propose that the ZLI is formed by process of progressive maturation of alar
plate cells, reflected by the activation of Hh from ventral to dorsal. This
observation could be explained by existence (and decay) of an inhibitory
signal from the roof plate. (Zeltser,
2005) (F. Guinazu, C. Kiecker and A. Lumsden, unpublished). The
ventral limit of twhh expression in the ZLI coincides with the
ventral border of shh expression in oep mutant embryos,
where the basal plate is genetically depleted. Based on this observation, we
can define the dorsoventral extent of the ZLI in zebrafish.
In vitro studies have claimed that thalamic development is partially
dependent on signals from the basal plate, although expression patterns of
prethalamic or thalamic markers have not been investigated directly
(Hashimoto-Torii et al., 2003;
Zeltser, 2005). By contrast,
focal blockade of Hh signal reception has suggested that horizontal Hh
signalling is more important than vertical
(Kiecker and Lumsden, 2004).
Our findings now offer further evidence for this: embryos initially lacking
the ventral Hh signal are able to induce prethalamic and thalamic expression
similar to wild type (Fig. 5).
We conclude, therefore, that the MDT requires direct Hh signalling solely from
the ZLI and that the ventral contribution of Hh signal for induction of
prethalamic and thalamic tissue is dispensible.
Establishment of diencephalic subdivisions
We show that Hh signalling from the ZLI is directly required for induction
or maintenance of dlx2a and lhx5 in the prethalamus and for
induction of dbx1a, emx2 and neurog1 in the thalamus. All of
these are pro-neural genes. It is well known that Shh is needed in the spinal
cord and hindbrain for the induction of specific neuronal progenitor
identities (reviewed by Jessell,
2000). Recently, it was shown that Hh signalling can actively
direct cell-cycle exit and lead cells to differentiation
(Shkumatava and Neumann,
2005); our description of the maturation of the diencephalon
serves as a further example of this. Thus, we suggest that Hh signalling is
important for regionalization of the MDT and the subsequent generation of
various neuronal identities. Interestingly, we find that a regional pattern is
already established in the diencephalon before expression of Hh genes at the
ZLI. In our grafting experiments as well as in our overexpression analysis, we
show that Hh signalling is able to induce expression profiles of various
neuronal subtypes appropriate to their position relative to the ZLI. These
findings confirm by experiments in chick, which have shown that the initial
pattern is set by an interaction of the competence factors Six3 and Irx3 in
the anterior and posterior diencephalon, respectively
(Hashimoto-Torii et al., 2003;
Kiecker and Lumsden,
2004).
Timing and concentration of Hh signalling
Cyclopamine treatment arrests dlx2a and dbx1a expression
at the time of treatment (Fig.
3). Therefore, persistent Hh signalling is necessary between 12
somites and 24 hpf to induce the full extent of adjacent expression domains.
After 24 hpf, Hh signalling is dispensable with respect to marker expression.
Therefore, we conclude that the timing of Hh signalling has to be tightly
controlled. In addition, it has been shown that patterning within the thalamus
reflects dose-dependent Hh signalling for the induction of Sox14
(Hashimoto-Torii et al., 2003;
Kiecker and Lumsden, 2004),
and we find that knocking down one Hh gene reveals another
concentration-dependent mechanism anterior to the ZLI. A further example is
served by the spinal cord, where Shh induces concentration-dependent changes
in ventral genes (Kohtz et al.,
1998).
Differences of activity range anterior and posterior to the ZLI
Prethalamic gene expression is activated in a ventral-to-dorsal direction,
accompanying the dorsal extension the ZLI, whereas the thalamus matures from
anterior to posterior. This could be explained by the topography of the two
territories: the prethalamus forms lateral to the ZLI, such that it remains
close to the source. By contrast, the thalamus lies in a medial position and
stretches far posterior, such that the distance from the Hh source is
comparatively greater. This would lead first to the induction of the anterior
part of the thalamus, followed by progressively more posterior tissues.
Different activity ranges of Hh signalling around the ZLI could be explained
by different propagation mechanisms for Hh anterior and posterior to the
ZLI.
Interestingly, we find that Twhh acts in prethalamic development, whereas it is virtually dispensable for thalamic gene induction, suggesting different concentrations of the Hh signals at the ZLI or different effectivity of the two proteins. A further possibility would be that dlx2a in the prethalamus is induced at a lower threshold compared with dbx1a in the thalamus. Therefore, Shh is able to replace Twhh in most functions, but not vice versa. This would also explain no twhh mutants have been discovered.
In the absence of Hh signalling, the dbx1a expression domain at
the ZLI seems slightly broadened compared with wild type
(Fig. 3). Our data show that
the expression of dbx1a at the ZLI is Hh independent. It has been
suggested that, in addition to functional Hh signalling, Wnts could play a
role during mid-diencephalic regionalization
(Garda et al., 2002;
Braun et al., 2003).
Direct acquisition of diencephalic specification.
To acquire the correct genetic profile, single cells in the MDT have to
integrate Hh signalling directly. Blocking reception of the Hh signal in small
cell clones has a similar effect (Kiecker
and Lumsden, 2004). In addition, the latter experiment suggests
that there is an Hh-independent pre-pattern in the tissue as discussed
previously. We found a single exception where one cell clone located anterior
to the ZLI switched on a thalamic marker. This could be due to a mislocated
tectal cell clone or it could be that mechanical stress of grafting caused the
induction of signals leading to local posterization of these cells
(Storey et al., 1998;
Scholpp et al., 2003)
(reviewed in Chiquet et al.,
2003).
In summary, we have described the ZLI as a new signalling centre in the
zebrafish embryo and have further explored its organising role during
development of the MDT. Although we have elucidated certain aspects of its
formation and function, these findings raise further questions. What are the
consequences of lacking marker gene expression in the prethalamus or thalamus?
How does the absence of dlx2a or dbx1a interfere with (e.g.)
neuronal composition of these territories? In addition, it has been proposed
that other signalling molecules such as Wnts and Fgfs, play roles during the
development of this territory (Braun et
al., 2003; Echevarria et al.,
2003). However, studies that range across different vertebrate
models could reveal the existence of a common basic mechanism leading to the
correct positioning, differentiation and function of the ZLI through
vertebrate evolution.
|
ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/5/855/DC1
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