Department of Genetics, University of Dresden (TU), Pfotenhauer Strasse
108, 01307 Dresden, Germany
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG),
Dresden, Germany
* Author for correspondence (e-mail: brand{at}mpi-cbg.de)
Accepted 23 June 2003
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SUMMARY |
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Key words: CNS, Engrailed, Fgf8, Forebrain, Midbrain, Isthmus, Zebrafish, Danio rerio
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Introduction |
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The paired-domain proteins Pax6 in the diencephalon and Pax2/5/8 in the
midbrain and MHB organizer serve an important function during the maintenance
phase and subdivide the anterior neural plate into forebrain, midbrain and
hindbrain domains [Pax6 (Walther and
Gruss, 1991; Stoykova et al.,
1996
; Grindley et al.,
1997
) and Pax2/5/8 (Urbanek et
al., 1994
; Favor et al.,
1996
; Schwarz et al.,
1997
; Schwarz et al.,
1999
; Lun and Brand,
1998
; Pfeffer et al.,
1998
; Scholpp and Brand,
2003
)]. Loss-of Pax6 function in mice and in chicken cause a fate
change of the caudal diencephalon into mesencephalic tissue
(Stoykova et al., 1996
;
Matsunaga et al., 2000
).
Conversely, ectopic Pax6 expression in the chick midbrain causes a
downregulation of Pax2 and Engrailed gene expression and posterior
enlargement of the diencephalon (Matsunaga
et al., 2000
). Similarly, posterior forebrain expression of
Pax6 expands into the presumptive midbrain in Pax2 and
Pax5 double deficient mutant mice (Pax2/5)
(Schwarz et al., 1999
) or
pax2.1 mutant zebrafish (Scholpp
and Brand, 2003
) with concomitant enlargement of the posterior
commissure as the anatomical landmark of the diencephalic-mesencephalic
boundary (DMB) (Macdonald et al.,
1994
; Mastick et al.,
1997
). These experiments show that Pax6 plays an essential role in
determining forebrain fate, whereas Pax2/5/8 is essential for development of
the midbrain and MHB territory.
In addition to Pax genes, the Engrailed homeodomain transcription factors
are necessary to maintain midbrain fate in chicken, mice and zebrafish
(Wurst et al., 1994;
Araki and Nakamura, 1999
;
Scholpp and Brand, 2001
).
Mis-expression of En1 in the anterior neural tube represses
Pax6 expression, resulting in a rostral shift of the DMB
(Araki and Nakamura, 1999
).
Similarly, mis-expression of the Medaka eng2 gene can repress
forebrain fate, including optic vesicle formation
(Ristoratore et al., 1999
).
The phenotype of En1 knockout mice demonstrates its importance for
proper development of the mesencephalon
(Wurst et al., 1994
;
Liu and Joyner, 2001
).
Zebrafish eng2 and eng3 (eng2a and eng2b
Zebrafish Information Network) are the functional orthologues of the
murine En1 gene and are expressed in similar spatial domains during
late stages of gastrulation and early somitogenesis
(Force et al., 1999
). A
knock-down of these genes causes a loss of pax2.1 expression and
severe defects in midbrain development, similar to those observed in mice
(Scholpp and Brand, 2001
).
Furthermore, in Pax2/5 deficient mice and fish, Engrailed expression
is strongly reduced or eliminated (Favor
et al., 1996
; Lun and Brand,
1998
; Pfeffer et al.,
1998
; Schwarz et al.,
1999
).
Little is known about intercellular signals regulating the formation of the
DMB. In addition to Pax6 and En, the secreted factor Fgf8 is involved during
establishment and maintenance of the anterior CNS. Fgf8 knockout
studies in mice and zebrafish showed that Fgf8 is essential to maintain the
MHB, to induce the cerebellum and to pattern the midbrain
(Crossley et al., 1996;
Brand et al., 1996
;
Reifers et al., 1998
;
Picker et al., 1999
;
Chi et al., 2003
). These
observations raised the possibility that Engrailed expression is maintained in
the midbrain through Fgf signaling, but so far evidence for direct action by
Fgf is lacking (Reifers et al.,
1998
; Liu and Joyner,
2001
).
Although the mechanisms involved in establishing AP patterning in the anterior neural tube are well studied, it is unclear how the AP subdivisions are maintained, and how this relates to organizer function in the anterior neural plate. We study the formation and maintenance of the zebrafish diencephalic-mesencephalic boundary to understand such maintenance mechanisms. Based on expression studies and functional analysis during DMB formation, we find that eng2 and eng3 genes play a crucial role in maintenance of the DMB. In addition, we find that Fgf signalling molecules, in particular Fgf8, act synergistically with eng2 and eng3 as non-autonomous signals to maintain midbrain identity and hence position the DMB.
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Materials and methods |
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Injections
For preparation of mRNA the complete ORF for eng3 was amplified.
The following primer were used: forward, 5'-TTC CCG TTC GTT TCT TTT
TG-3'; and reverse, 5'-TCT TTG GAC TTC AGC ATG GA-3'. We
subcloned the cDNA into the vector pCS2+
(Rupp et al., 1994) and used
the SP6 message machine kit (Ambion) for transcription. The amount of injected
mRNA was estimated from the concentration and volume of a sphere of mRNA
injected into oil at the same pressure settings. mRNA was dissolved in 0.25 M
KCl with 0.2% of Phenol Red and back-loaded into borosilicate capillaries
prepared on a Sutter puller. During injection, mRNA was deposited into the
cytoplasm of one- to two-cell stage embryos. Typically, 500 pg eng3
RNA was injected. The embryos were fixed at appropriate stages prior to in
situ hybridization and antibody staining.
For transient knock-down of gene expression, Morpholino-antisense oligomers
(Morpholinos; MO; by GeneTools) were prepared targeting eng2 and
eng3 (Scholpp and Brand,
2001), fgf3 (Raible
and Brand, 2001
), fgf4 and fgf8
(Araki and Brand, 2001
) by
dissolving in 5 mM HEPES-buffer with 0.2% Phenol Red. Morpholinos were
injected into the yolk cell close to the blastomeres between the one- and
eight-cell stages at a concentration of 4 ng/nl. For a control, randomized
mis-priming morpholinos (con-MO) were used, which showed no effect on embryos
injected at 15 ng/nl but cause unspecific effects at a concentration of 30
ng/nl. Morpholino-injected embryos (morphants) were fixed at given stages
prior to in situ hybridization or antibody staining.
Sequences were as follows: eng2-MO, 5'-CGC TCT GCT CAT TCT CAT CCA TGC T-3'; eng3-MO, 5'-CTA TGA TCA TTT TCT TCC ATA GTG A-3'; fgf3-MO, 5'-CAG TAA CAA CAA GAG CAG AAT TAT A-3'; fgf3-4bp-mismatch, 5'-CAC TAA CAA GAA GAC CAC AAT TAT A-3'; fgf4-MO, 5'-GCC GAC TGG ACA CTC ATC CTT CTA A-3'; fgf8-MO, 5'-GAG TCT CAT GTT TAT AGC CTC AGT A-3'; and con-MO, 5'-CCT CTT ACC TCA GTT ACA ATT TAT A-3'.
Inhibition of Fgf signalling
At 90% epiboly, an inhibitor of Fgf signalling, SU5402 (Calbiochem) was
added to the medium at a concentration of 8 µM as described previously
(Reifers et al., 2000). Embryos were incubated for different time periods and
fixed directly after treatment.
Implantation of FGF soaked beads
Heparin-coated acrylic beads (Sigma) were prepared as described previously
(Reifers et al., 2000). The beads were implanted unilaterally into the region
of the presumptive anterior midbrain of wild-type and no isthmus
mutant embryos at the 10 somite stage (10 ss). Embryos were incubated for 2
hours at 28°C and fixed at the 15 ss for further examination.
Transplantation
Embryos were injected with 400 pg mRNA of a truncated FGF receptor (XFD) as
described previously (Launay et al.,
1996). Rhodamine-dextran (Mini Ruby, Molecular Probes) was
co-injected for tracing cells after injection. At shield stage, cells from the
region of the presumptive midbrain region were taken from a donor embryo and
implanted into a non-labeled host embryo. At 10 ss, embryos were fixed prior
to in situ hybridization and antibody staining.
Labeling of cell clones via laser-based activation of caged
fluorescein
Non-fluorescent, photoactivatable (caged) fluorescein as a cell tracer for
fate mapping in the zebrafish embryo was described by Kozlowski et al.
(Kozlowski et al., 1997). We
used a UV laser (Phototronic Instruments) to uncage the dye more locally. A
solution of 5% anionic DMNB-caged fluorescein (2 nl) (Molecular Probes,
D-3310), 0.25 M KCl, 0.25% Phenol Red and 40 mM HEPES-NaOH (pH 7.5) was
injected in embryos at the one-cell stage and for development the embryos were
kept in a dark humid chamber at 28°C. At the 6 ss, embryos were oriented
in a viewing chamber dorsal up and a laser with 365 nm focused through a
40x water-immersion objective was used to activate the dye 2-4
seconds/cell in the presumptive anterior midbrain area. The embryos were fixed
at 26 hpf prior to in situ hybridization.
In vivo imaging of the development of the DMB
At shield stage, dechorionated wild-type host embryos containing
transplanted donor cells (see above) were stained with 100 µm
Bodipy-FL-ceramide C5 (Molecular Probes) in Ringer medium for 30 minutes. At
26 hpf embryos were scanned by confocal microscopy and fixed prior to in situ
hybridization and antibody staining.
Whole-mount in situ hybridization
Whole-mount mRNA in situ hybridization were carried out as described by
Reifers et al. (Reifers et al.,
1998). Digoxygenin- and fluorescein-labeled probes were prepared
from linearized templates using an RNA labelling and detection kit (Roche).
Stained embryos were dissected and mounted in glycerol. Embryos were
photographed on a Zeiss Axioskop and assembled using Adobe Photoshop.
Expression patterns have been described previously: efna4
(Xu and Brulet, 1984
),
gbx1 (Rhinn et al.,
2003
), pax6.1 (original clone cZK3)
(Krauss et al., 1991b
;
Krauss et al., 1991a
),
eng2 and eng3 (Ekker et
al., 1992
), otx2 (Mori et al., 1994), pax2.2
(Pfeffer et al., 1998
), and
isl1 (Okamoto et al., 2000).
Antibody staining
We visualized the Engrailed proteins with a monoclonal antibody 4D9
(Patel et al., 1989) using the
protocol described by Holder and Hill
(Holder and Hill, 1991
). A
monoclonal antibody against acetylated tubulin (Sigma, T-6793) was used at
1:20 dilution to reveal neurons that have started axogenesis
(Wilson et al., 1990
).
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Results |
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The early pax2.1 expression marks the future midbrain and isthmus
(Krauss et al., 1991a;
Lun and Brand, 1998
). The
expression of pax2.1 starts in two wings at midgastrula stage, 80% of
epiboly, which fuse into one band at the tailbud stage
(Fig. 1F)
(Lun and Brand, 1998
).
Surprisingly, double in situ hybridization experiments with pax6.1
and pax2.1 revealed a gap between these two expression domains
(Fig. 1D-I); that is 4-5 cells
wide on cross-sections at tailbud stage (11 sections examined,
Fig. 1G). At mid-somitogenesis
stage (6 ss) the gap between pax2.1 and pax6.1 widens,
particularly on the dorsal side (Fig.
1H). With progressive restriction of pax2.1 to the
isthmic zone, the midbrain emerges free of expression of either
pax6.1 or pax2.1. At 24 hpf, expression of pax2.1
is restricted to the area of the isthmus proper
(Fig. 1I).
In contrast to pax2.1, mapping of the expression of pax6.1 relative to the Engrailed genes does not reveal a gap (Fig. 1J-M). Expression of eng2 starts at 90% of epiboly, at the same position as the pax2.1 expression domain (Fig. 1J) and generally follows the pattern of pax2.1. However, eng2 expression is detectable more anteriorly than pax2.1 expression at 100% of epiboly (Fig. 1J). From this stage onwards, the posterior boundary of pax6.1 and the anterior boundary of eng2 are immediately adjacent to each other (Fig. 1K,L), with a slight overlap in one or two cell rows. eng3 expression starts around tailbud stage as a transverse band in the forming midbrain. Neither eng2 nor eng3 is detectable elsewhere in the presumptive forebrain. At 24 hpf, all Engrailed genes, including eng1 (eng1a Zebrafish Information Network) and eng4 (eng1b Zebrafish Information Network) are strongly expressed at the isthmus, and anterior and posterior to the pax2.1 expression domain in the midbrain (Fig. 1N). Thus, in contrast to pax2.1, the eng2 and eng3 genes are expressed in the future midbrain territory and are posteriorly adjacent to the pax6.1 domain starting at late gastrulation until mid-somitogenesis stages (Fig. 1M). This close proximity of Engrailed gene and pax6.1 expression suggests a regulative interaction during formation of the DMB.
At the onset of pax6.1 expression, fgf8 is expressed in a
more posterior and wider stripe than pax2.1
(Fig. 1O). The
fgf8-expressing region covers the prospective MHB and continues into
the fourth rhombomere, but leave the midbrain primordium free
(Reifers et al., 1998). At
tailbud stage, a gap of
15 cell rows is visible between the posterior
limit of pax6.1 expression and the anterior limit of fgf8
expression. The distance between these two expression domains remains similar
until mid-somitogenesis (Fig.
1P,Q). After this period, the distance increases owing to the
strongly proliferating midbrain primordium between the pax6.1- and
the fgf8-positive domains (Fig.
1R).
Engrailed-dependent repression of pax6.1 during formation of
the diencephalic-mesencephalic boundary
Our gene expression study of pax6.1 and Engrailed genes suggests
that eng2/eng3, but not pax2.1, might interact with
pax6.1 during formation of the DMB. To investigate this further, we
mis-expressed eng3-mRNA unilaterally by injecting into one blastomere
at the two blastomere stage.
We find that eng3 mis-expression causes a repression of
pax6.1 in the anterior part of the embryo from the onset of its
expression, whereas hindbrain expression is unaffected
(Fig. 2A,B). Otx2,
another gene expressed in the prosencephalon and midbrain, is not affected,
indicating that change of cell fate from forebrain/midbrain to hindbrain fate
does not occur (Fig. 2A,B). In
a later phase at 26 hpf, eng3 mis-expression leads to an alteration
in gene expression and structure of the DMB, visualized by repression of
pax6.1 and efna4 (previously known as ephA4), a
further marker gene respecting the DMB
(Macdonald et al., 1994). In
addition absence of the posterior commissure is observed on the injected side
(Fig. 2D; white arrowheads). We
also found that pax6.1 dependent structures such as the eyes
(Bally-Cuif and Wassef, 1994
;
Halder et al., 1995
) were
reduced or absent in eng3-mis-expressing embryos
(Fig. 2C,D,M) (Ristoratore et al., 1999
).
The injection of different amounts of eng3 mRNA led to a repression
of pax6.1 in a dose-dependent manner specifically in the anterior
part of the embryo (Fig. 2K-M). At the highest injected amount of mRNA (600 pg), development of the
presumptive diencephalons was completely blocked, as seen by total loss of the
pax6.1 expression (Fig.
2M). The same amount of lacZ-mRNA has no effect (not
shown).
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Inactivation of Engrailed gene expression causes forebrain
expansion
To determine whether Engrailed genes are also required to suppress
forebrain identity in the cephalic neural plate, we examined the development
of embryos deficient for eng2 and eng3. To inactivate the
Engrailed genes, we injected morpholino antisense oligomers
(Nasevicius and Ekker, 2000)
to prevent translation of the early expressed genes eng2 and
eng3 (Scholpp and Brand,
2001
). We compared the phenotype of morpholino-injected embryos to
no isthmus (noi) mutant embryos that lack functional Pax2.1
protein. In the noi mutant embryos, the pax5 transcripts are
not detectable (Lun and Brand,
1998
).
With regard to development of the DMB, the repression of either
eng2 or eng3 did not lead to a phenotype different from the
control embryos (Scholpp and Brand,
2001). In contrast to this, double knock-down of both
eng2 and eng3, caused the expression of the diencephalic
marker genes pax6.1 and ephA4 to expand posteriorly, first
detectable at the 7 ss and becomes more prominent at later stages such as the
15 ss (Fig. 3A,B,D,E). This
expansion correlates with a caudal shift of some branches of the posterior
commissure into the presumptive midbrain territory at 32 hpf
(Fig. 3O,P). pax5
expression is not detectable in the MO-eng2 and MO-eng3
injected embryos (Fig. 3I,J).
The observed phenotype resembles that of the pax2.1 mutant
noi (Fig. 3C,F)
(Scholpp and Brand, 2001
). The
lack of a functional pax2.1 protein leads to the absence of
expression of the Engrailed genes, except for a very faint and transient
expression of eng2 at the tailbud stage
(Lun and Brand, 1998
). The
repression of the Engrailed mRNA translation via morpholino injection did not
produce a more pronounced phenotype than the noi mutant, arguing that
pax2.1 largely exerts its function via eng2 and
eng3 (Scholpp and Brand,
2001
). eng2 and eng3 are therefore both
necessary and sufficient to restrict the posterior forebrain boundary in the
cephalic neural plate. In keeping with the transformation of cell type
identity in the neural plate that we observed after eng3
overexpression (Fig. 2), loss
of eng2 and eng3 function does not cause overproliferation
of forebrain cells at the 7 ss, 15 ss and 24 hour stage, as detected in
-Phosphohistone 3 antibody and Pax6 double-stained embryos
(Fig. 3G-L).
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We conclude that Fgfs are involved in maintaining eng3 expression in the midbrain territory independent of functional Pax2.1 protein. Furthermore, Fgfs are able to repress forebrain fate, as monitored by expression of pax6.1.
Fgf-blind cells in the midbrain acquire forebrain fate
Our findings raise the possibility that cells located in the presumptive
midbrain neural plate might require Fgf to keep them from following a
forebrain fate. To test this idea, we blocked reception of Fgf signalling in
small cell clones in the midbrain by expressing the dominant negative
Fgf-receptor (Fgfr) XFD (Amaya et al.,
1991). We co-injected XFD-mRNA with a fluorescent lineage
tracer into the one blastomere stage. At shield stage we transplanted small
cell clones from the injected embryo into the territory of the presumptive
midbrain of a wild-type embryo, i.e. before the onset of pax6.1 and
pax2.1 expression (Fig.
5A-C). The resulting chimaeras were examined for fluorescent cell
clones located in the midbrain and subjected to in situ hybridization at the
10 ss. Transplanted cells autonomously expressed the forebrain markers
pax6.1 and efna4 in the presumptive midbrain
(Fig. 5D,G; n=5),
whereas surrounding midbrain cells express neither pax6.1 nor
efna4.
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Fgf signalling is necessary to maintain the position of the DMB until
10 ss
The above results lead us to determine when Fgf signalling is required to
suppress forebrain fate, and to compare this with the onset of gene expression
in the forming DMB. We treated embryos with the pharmaceutical Fgfr inhibitor
SU5402 (Mohammadi et al.,
1997) at different developmental time points and for varying time
periods. Wild-type embryos treated with SU5402 showed only a weak posterior
expansion of the diencephalic pax6.1 expression, similar to the
phenotype observed in ace mutant embryos (not shown). We then
repeated the SU5402 inhibition with noi mutant embryos, which lack
eng2 and eng3 expression
(Lun and Brand, 1998
) and
therefore provided an opportunity to study the role of Fgf signalling in a
sensitized background. We applied the inhibitor from 80% epiboly, i.e. before
the onset of pax6.1 expression, until 15 ss. This treatment causes a
strong expansion of pax6.1 expression into the territory of the
presumptive midbrain around 15 ss in noi embryos
(Fig. 6A,B). Inhibition between
5 ss to 15 ss led to a weaker expansion of pax6.1 expression
(Fig. 6C), and with inhibition
from the 10 ss onwards, posterior expansion of pax6.1 is no longer
observed (Fig. 6D), even when
the treatment was continued until 24 hpf (not shown). We conclude that Fgf
signalling is necessary in combination with eng2/eng3 for positioning
of the DMB until about the 10 ss.
Fgf8 and Engrailed act in a synergistic fashion to position the
DMB
To determine which Fgf is required for correct positioning of the DMB, we
blocked the translation of fgf3, fgf4 and fgf8, all of which
are expressed at the MHB, via injection of morpholino (MO) antisense oligos,
alone or in combination. (Araki and Brand,
2001; Raible and Brand,
2001
). The embryos were injected with 4 ng of MO and showed
gene-specific defects. MO-fgf8 injection phenocopies the ace
mutant in 79% of the injected embryos (not shown)
(Araki and Brand, 2001
). The
injected embryos showed a weak expansion of the forebrain marker
pax6.1 into the presumptive midbrain, a loss of eng2
expression from the 15 ss onwards, and failure of MHB formation
(Fig. 6G). The same phenotype
is observed in ace mutant embryos
(Reifers et al., 1998
). The
injection of MO-fgf8 into noi embryos caused a stronger
expansion of pax6.1 expression into the presumptive midbrain than
that observed after inactivation of either fgf8 or pax2.1
alone (compare Fig. 6H, 6F and
6G). In the injected embryos, we observe a fusion of the forebrain
and hindbrain expression domains of pax6.1. By contrast, the
injection of MO-fgf3 and MO-fgf4 did not lead to any changes
with regard to the formation of the DMB (not shown). In addition, no single
injection of the other fgf morpholinos, or a combination of them, was
able to increase the pax6.1 expansion in the MO-fgf8
injected morphants (data not shown). We conclude from these experiments that
among the Fgfs tested, Fgf8 is the primary signalling molecule that restricts
pax6.1 expression to the forebrain cephalic neural plate. We next
compared the phenotype of MO-fgf8 injected noi mutant
embryos with that of SU5402-treated noi mutant embryos. In both
situations, the forebrain and hindbrain expression domains of pax6.1
were fused ventrally (Fig.
6H,I). To distinguish whether the lack of Eng gene expression in
noi mutants, or the lack of some other function controlled by
pax2.1, is responsible for forebrain repression, we injected
MO-eng2/eng3 into the acerebellar (ace) mutant. We
again observe the fusion of the forebrain and hindbrain domains of
pax6.1 expression (Fig.
6J), arguing that the Eng genes are responsible for forebrain
repression in noi mutants.
To determine the neuroanatomical consequences of pax6.1 expression
in embryos lacking both fgf8 and eng2/eng3-dependent
forebrain restriction, we examined the phenotype of 28-hour-old embryos. We
examined in particular the formation of isl1-positive neurons in the
epiphysis (Masai et al., 1997)
and the location of the posterior commissure with an antibody against
acetylated tubulin, in relation to pax6.1 and eng2
expression. Expanded forebrain expression of pax6.1 into the midbrain
was visible in the MO-fgf8 injected embryos, when compared to the
noi mutant embryos (Fig.
7B,C). A significantly enhanced phenotype is shown in embryos
lacking both fgf8 and eng2/eng3
(Fig. 7D). As observed during
mid-somitogenesis stages, the forebrain and hindbrain expression domains fuse
ventrally, and only a dorsal patch of cells with unknown identity remains free
of pax6.1 expression at 28 hpf. Expression of the Engrailed genes was
absent in the MO-fgf8 injected embryos, in the noi mutants
and in MO-fgf8/noi double mutants
(Fig. 7B-D).
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Discussion |
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Positioning of the DMB requires eng2/eng3 function
Previous work in mice had suggested that the DMB may be generated by mutual
repression of Pax6 and Pax2 plus Pax5
(Schwarz et al., 1999). Loss
of Pax2/5 in mice or Pax2.1 in fish leads to expansion of the forebrain, and
loss of Pax6 leads to expansion of the midbrain into forebrain territory
[Pax2/5 (Schwarz et al.,
1999
), Pax2.1 (Scholpp and
Brand, 2003
), Pax6 (Stoykova
et al., 1996
)]. The nature of these interactions is not understood
during the early embryonic period when the DMB division is established. In
particular, because Pax2/5 controls the expression of En1 and
En2 transcription factors, it was not clear whether Pax2/5 act
directly onto Pax6 transcription, as suggested for Pax2 during optic stalk
development (Schwarz et al.,
2000
), or if regulation occurs indirectly via En1 and En2. Our
expression analysis shows that pax2.1 and pax6.1 are
initially not adjacent or overlapping at the DMB, which would be a
prerequisite for mutual repression. pax6.2 is a duplicate
Pax6 gene in teleosts with a potentially similar function as
pax6.1. However, because expression of pax6.2 is restricted
to the mid-diencephalon, it is unlikely to contribute to formation of the DMB
(Nornes et al., 1998
). In
contrast to pax2.1, eng2 and eng3 (the functional zebrafish
orthologues of the mammalian En1 gene)
(Force et al., 1999
;
Scholpp and Brand, 2001
) abut
or overlap with the pax6.1 domain already at the onset of expression,
suggesting that eng2 and eng3 may serve this repressive
function. Evidence from mis-expression of En1 and En2 in
chick and OL-eng2 in Medaka previously suggested that En can suppress
Pax6 expression in the forebrain neural plate
(Araki and Nakamura, 1999
;
Ristoratore et al., 1999
). We
find that mis-expression of eng3 has the same effect in zebrafish,
causing suppression of forebrain development and expansion of midbrain fate.
In loss-of-function conditions, the En genes function redundantly in mice and
zebrafish (Hanks et al., 1995
;
Scholpp and Brand, 2001
), and
although we have not tested eng2 mis-expression, we expect this to
give the identical result as eng3 mis-expression. These findings
suggest that repression of pax6.1 by eng2/eng3 operates
during establishment of the DMB at neural plate stages. Our laser-uncaging
experiments in eng3-injected embryos support the idea that the
crucial interactions take place during early neural plate stages, and thus
argue that transformation of the forebrain neural plate into a midbrain
identity is the likely basis of the observed neuroanatomical alterations at
later stages embryos. An interesting side aspect is that pax6.1
expression is specifically affected in the forebrain, but not the hindbrain
neural plate in such embryos. Expression of pax6.1 in the hindbrain
and spinal cord may therefore be under different genetic control, and indeed
murine Pax6 is thought to act during dorsoventral, but not anteroposterior
patterning processes in the spinal chord
(Goulding et al., 1993
). A
direct action of Eng2/Eng3 onto forebrain pax6.1 expression is also
supported by our loss-of-function studies for eng2/eng3.
Nevertheless, it remained possible that Pax2.1 would confine pax6.1
expression at the DMB both directly and indirectly, via regulating Eng2/Eng3.
We think this is unlikely, because FGF8-soaked beads can suppress
pax6.1 expression and forebrain identity even in a
pax2.1-mutant genetic background, when implanted into noi
mutant embryos (see below). Together, these results argue that
eng2/eng3 expression is both necessary and sufficient as the key
determinant for restricting pax6.1 expression and forebrain identity
at the DMB.
Non-autonomous repression of forebrain fate by Fgf signalling
A key observation of our work is that, as well as eng2/eng3, Fgf
signalling is also necessary for normal formation of the DMB. Morpholino
inactivation suggests that among the Fgfs expressed in the early neural plate,
Fgf8, but not Fgf3 or Fgf4, perform this function, a notion that is further
confirmed by our studies of the ace (fgf8) mutant. The
closest source for Fgf8 during the crucial phase of development is the forming
midbrain-hindbrain organizer, located at the junction between the midbrain and
hindbrain primordia. The temporal requirement for Fgf signalling, as seen by
pharmacological inhibition with the Fgfr inhibitor SU5402, is consistent with
the time of Fgf8 expression in the forming MHB organizer. Beyond 10 ss, Fgf
signalling is no longer required. Wnt signalling during this period is thought
to subdivide the forebrain domain
(Heisenberg et al., 2001;
Kudoh et al., 2002
) (reviewed
by Wilson et al., 2002
).
Interestingly, the likely source for Fgf8 at the MHB does not directly abut
the cells at the DMB, suggesting a possible long-range effect of Fgf8
signalling. We suggest that long-range signalling may occur directly: our
results show that an Fgf signal needs to be directly received by midbrain
cells, as midbrain cells expressing a dominant-negative Fgf receptor construct
lose midbrain identity, and switch to a pax6.1-positive forebrain
fate. This finding predicts that Fgf8 can signal over a considerable distance
through the forming midbrain neural plate, consistent with its role as a
secreted factor. During later stages of midbrain development, it is thought
that Fgf8 polarizes ephrin ligand expression to allow proper
formation of the retinotectal projection of retinal axons
(Lee et al., 1997
;
Picker et al., 1999
;
Yates et al., 2001
), which may
also involve long-distance signalling by Fgf8 in the forming tectum.
Synergistic repression by Eng2/Eng3 and Fgf8
A key finding of our work is that the most extreme disruption of DMB
formation and concomitant reduction of midbrain formation is seen only after
inactivation of both Eng2/Eng3 and Fgf8 function, arguing that both have
parallel, independent functions in maintaining the DMB
(Fig. 8). We observed this in
various genetic situations, e.g. when Eng2/Eng3 function is knocked-down
either through morpholino injection, or in noi mutant embryos, in
combination with SU5402 inhibition of Fgf signalling, after more specific loss
of Fgf8 in ace mutants, or after morpholino-inhibition of Fgf8
(Fig. 6). However, Fgf8 is
apparently also involved in Engrailed maintenance in the midbrain primordium,
because ace mutants fail to maintain eng2/eng3 expression
(Reifers et al., 1998).
Moreover, Fgf8-bead implantation also causes activation of eng3
expression in the forebrain, and Fgf8 may therefore repress pax6.1
expression also indirectly via eng2/eng3 expression. Importantly, the
activation of eng3 expression in response to Fgf8 can occur even in
the absence of pax2.1, and is linked to concomitant reduction of
pax6.1 (Fig. 4).
Similarly, in mice, implantation of an Fgf8 bead causes repression of Pax6
even in an
En1//En2/
background (Liu and Joyner,
2001
). Together, these findings provide evidence that Fgf8 can act
directly on the DMB. In chick, FGF8-soaked beads can activate En1 and Pax2
expression in the forebrain, which was suggested to reflect induction of an
ectopic MHB organizer (Garda et al.,
2001
). Based on our studies of DMB formation, an early step in
these events may the suppression of forebrain and support of midbrain fate by
FGF8-bead implantation, without a need to induce an ectopic MHB organizer.
This explanation is supported by the fact that FGF8-soaked beads do not induce
later markers of the MHB such as pax2.2 or pax5 in
noi mutant embryos under these circumstances (not shown).
|
Even with the most extreme loss of Eng2/Eng3 and Fgf8, a small group of
cells located in the dorsal midbrain does not acquire pax6.1
expression. These cells express the dorsal markers wnt1, wnt4 and the
dorsal midbrain marker pax7c (not shown), and are therefore most
likely neural tube cells with a dorsal identity. Why do these cells not
acquire forebrain character? One possibility is that an unknown signal can
suppress pax6.1-positive dorsal diencephalic identity, or promote
dorsal midbrain identity. This signal is unlikely to be Wnt1 or Wnt4 itself,
because these are normally co-expressed in dorsal p1 with pax6.1.
Furthermore, knock-down of wnt1 and the partially redundant
wnt10b do not lead to predominately dorsal neural tube defects
(Lekven et al., 2002). Promising candidates are bone morphogenetic proteins
(Bmps), which can repress Pax6 in the hindbrain and dorsal spinal cord
(Goulding et al., 1993;
Timmer et al., 2002
). Indeed,
in mice, Bmp6 and Bmp7 are expressed in the roof plate of the telencephalon,
the midbrain and in the hindbrain (Furuta
et al., 1997
). Interestingly, pax6.1 expression becomes
excluded in the dorsal part of the telencephalon and hindbrain already during
midsomitogenesis stages (Fig.
1J,K). However, expression of zebrafish bmp7 has not been
detected in the roof plate of the midbrain
(Schmid et al., 2000
),
suggesting that another member of the Bmp family, e.g. Bmp6, might perform
this function.
Pax2 and its transient requirement in midbrain development and
forebrain suppression
pax2.1-deficient zebrafish noi mutant embryos and
Pax2/5 mutant mouse embryos lack Engrailed expression and later the
midbrain territory (Favor et al.,
1996; Lun and Brand,
1998
; Schwarz et al.,
1999
). One possibility is therefore that pax2.1 (Pax2/5
in mice) may itself confer midbrain character to neuroepithelial cells.
Alternatively, Pax2 function might only be needed to ensure spatially
restricted activation of Engrailed-type genes, which in turn repress forebrain
fate and control a midbrain specific program. An important finding of our
study in favour of the latter possibility is that pax2.1 is not
necessary to achieve repression of forebrain fate, because Fgf8-soaked beads
implanted into the forebrain primordium can suppress pax6.1, even in
the absence of functional pax2.1. Likewise, the normally stringent
requirement for pax2.1 for eng2/eng3 activation
(Lun and Brand, 1998
) can be
circumvented if expression is directly activated by implantation of an
Fgf8-soaked beads into the midbrain primordium. We therefore suggest that the
main, and perhaps only, function of pax2.1 in the midbrain may be to
activate eng2/eng3 expression. This finding is consistent with our
observation that morpholino-mediated knock-down of eng2 and
eng3 gives an exact phenocopy of the noi mutant phenotype in
the midbrain (Scholpp and Brand,
2001
).
The DMB and lineage restriction
Neither the mutual repression model of pax6.1 and
eng2/eng3, nor the non-autonomous repression of Pax6 genes by Fgf8
explains cellular behavior at the DMB. Previous studies have shown that cell
mixing across the DMB is restricted (Araki
and Nakamura, 1999; Larsen et
al., 2001
), and classical markers of rhombomere boundaries, like
tenascin and vimetin, are expressed also at the DMB in chick and zebrafish
(Larsen et al., 2001
;
Cerdá et al., 1998
);
however, the mechanism that restricts mixing is not clear. Among the marker
genes we have used to follow forebrain fate was the ephrin receptor
efna4, the expression of which expanded into the midbrain in embryos
with disrupted midbrain development, e.g. after knock-down of
eng2/eng3. Efn receptors and their ligands have been implicated in
restricting cell mixing across segment boundaries in the hindbrain
(Wilkinson, 2000
), and ephrin
A2 and ephrin A5a are two ligands of Efna4 that are expressed in the midbrain,
complementary to the receptor expression. These ligands are not only important
for mediating retinotectal projection in zebrafish, but are also expressed in
earlier somitogenesis stages of midbrain development, prior to ingrowth of
retinotectal axons (Brennan et al.,
1997
), and expression is missing in noi and ace
mutants (Picker et al., 1999
)
(S.S., C.L. and M.B., unpublished) We therefore suggest that Eph receptors and
their ligands may also perform a similar function at the DMB. One implication
of these findings is that segmentation in the hindbrain may be mechanistically
related to a potentially neuromeric organisation of the more rostral neural
plate.
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ACKNOWLEDGMENTS |
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