1 Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstraße 108, 01307 Dresden, Germany
2 Department of Genetics, University of Technology, Dresden, Germany
* Author for correspondence (e-mail: brand{at}mpi-cbg.de)
Accepted 18 September 2003
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SUMMARY |
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Key words: ace, acerebellar, Fgf8, Midbrain, Hindbrain, Cerebellum, isthmus rhombencephali, MHB, Rhombomere 1, Rostralization, Transformation, Patterning, Lineage analysis, Bead implantation, Plasticity, Modularity, Zebrafish, D. rerio
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Introduction |
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During gastrulation, the boundary between the prospective midbrain and
hindbrain can be defined as the interface of a rostral Otx2 and a
caudal Gbx2 (gbx1 in zebrafish) expression domain in the
neural plate of both amniotes and zebrafish
(Broccoli et al., 1999;
Millet et al., 1999
;
Rhinn et al., 2003
) (reviewed
by Simeone, 2000
). Later on at
this interface, activation of a genetic network composed of various
transcription factors triggers localized expression of a secreted organizer
signal (Fgf8), which in turn determines the development of the surrounding
tissue (reviewed by Liu and Joyner,
2001
; Rhinn and Brand,
2001
; Wurst and Bally-Cuif,
2001
). Molecularly, expression of fgf8 is controlled by
distinct regulators, a combinatorial interaction between inductive and
modulatory factors (Reifers et al.,
1998
; Lun and Brand,
1998
; Ye et al.,
2001
). Analysis of noi/pax2a mutants in zebrafish
demonstrates that induction of fgf8 is independent of pax2a
(Lun and Brand, 1998
). In
spite of the fact that the isthmic organizer develops at the interface of the
otx2 and Gbx expression domains
(Broccoli et al., 1999
;
Millet et al., 1999
;
Rhinn et al., 2003
) (reviewed
by Simeone, 2000
), these
factors are only involved in the maintenance and refinement of fgf8
expression and not in its induction (Ye et
al., 2001
; Martinez-Barbera et
al., 2001
; Li and Joyner,
2001
). Beside positive autoregulatory circuits, Fgf8 triggers
expression of the Fgf target gene sprouty, a negative feedback
modulator of Fgf signaling at the MHB
(Fürthauer et al., 2001
).
Many of the MHB cascade genes are initially induced independently of Fgf8, and
become dependent on Fgf8 activity only around the mid-somitogenesis stages
(Reifers et al., 1998
). By
contrast, expression of the ETS transcription factors erm, pea3 and
gbx2 are tightly dependent on Fgf8, and may mediate Fgf8 responses
during early MHB development (Raible and
Brand, 2001
; Roehl and
Nusslein-Volhard, 2001
; Rhinn
et al., 2003
). Fgf8 thus fulfills multiple functions during
development. Emitted from a perpendicular narrow stripe in the rostral
hindbrain gbx expression domain, Fgf8 is required for
self-maintenance of the MHB domain
(Reifers et al., 1998
;
Lun and Brand, 1998
). Fgf8
also controls the morphogenetic events leading to the formation of the
anatomical isthmic constriction (isthmus rhombencephali) that
separates the midbrain and hindbrain domains macroscopically
(Brand et al., 1996
;
Reifers et al., 1998
). Fgf8
has a crucial role in polarizing the midbrain tectum and defining ordered
ingrowth of retinotectal axons (Lee et
al., 1997
; Picker et al.,
1999
). It also strongly influences the patterning of the dorsal
metencephalon, a part of which eventually gives rise to the cerebellum
(Reifers et al., 1998
).
Zebrafish acerebellar (ace) mutants have a point mutation
in the fgf8 gene (Brand et al.,
1996; Reifers et al.,
1998
; Araki and Brand, 2002). In ace mutants a number of
important regulatory genes prefiguring the position of the future anatomical
isthmic constriction are initially present, but their expression is later
abrogated. Consequently, proper patterning of the midbrain/hindbrain along the
rostrocaudal axis is disturbed in ace mutants. The isthmic
constriction fails to form between the midbrain and rhombomere 1 (r1), and a
separate cerebellar anlage is not recognizable in the mutants. Interestingly,
the mutants have no obvious truncation along the rostrocaudal extent of the
mesencephalic/hindbrain alar plate. Rather, the mutants appear to have a
caudally enlarged tectum in place of the cerebellum. This raises the question
of whether the cells in ace located at equivalent rostrocaudal
positions along the neuraxis as the isthmic and cerebellar primordia in wild
types, retain their original fate, or adopt a new one in both domains in
mutant embryos. In the present study we investigated whether the special
morphological features of the ace mutants are due to a simple
dysmorphology or whether they are associated with fate alteration. To
distinguish between these scenarios we analyzed the molecular and positional
identities of the morphologically reorganized tectal compartment of mutant
embryos by comparing tectal/cerebellar specific gene expression patterns,
neuronal subtypes, and cell proliferation, cell death and cell lineage
characteristics of both wild-type and mutant embryos. We provide evidence that
the primordial cells of the rhombencephalic isthmus undergo marked cell fate
changes, demonstrated by their rostralized gene expression pattern and by
cell-lineage analysis. Implantation of Fgf8-protein coated beads suggests that
the observed cell fate transformation and lack of cerebellar development is
due to the missing polarizing and patterning activity of the organizer signal
Fgf8.
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Materials and methods |
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Whole-mount in situ hybridization and whole-mount detection of Eph
receptor tyrosine kinase ligands with Epha3-AP fusion protein
Embryos were fixed in 4% paraformaldehyde (PFA) at the required stage of
development. After dechorionation, embryos were dehydrated in 100% methanol
and stored at -20°C until use. Whole-mount in situ hybdridization was
performed as described (Reifers et al.,
1998).
Membrane-bound ephrin A molecules were detected with an Epha3-AP fusion
protein as described (Picker et al.,
1999).
In situ hybridization and immunohistochemistry on sections
Zebrafish larvae were anaesthetized and, after fixation in 4% PFA, were
cryoprotected in 30% sucrose and embedded in OCT compound (Sakura). Sections
were then cut at 15 µm thickness on a Microm cryostat. Sections were
mounted onto positively charged microscope slides (Superfrost Plus). In situ
hybridization was performed according to a protocol established by Henrique
(Henrique et al., 1995).
For zebrin II/aldolase C immunolocalization, sections were incubated with a 1:1000 dilution of the primary antibody (anti-zebrin II mouse monoclonal; kindly provided by M. Mione, UCL, London) for 48 hours at 4°C. The imunoreactivity was detected with a biotinylated horse anti-mouse antibody (Vector), avidin-biotin-peroxidase complex (ABC Elite Vectastain kit, Vector) and DAB chromogen.
Cell proliferation and cell death detection
Cell proliferation was detected by whole-mount immunohistochemical
detection of phosphorylated histone H3, using a rabbit polyclonal (IgG)
antibody, according to the manufacturer's instructions (Upstate
Biotechnology). To detect apoptotic cells an in situ nick-end labeling
procedure was performed using a commercial kit (Roche).
SU5402 inhibitor treatment
To inhibit Fgf signaling a pharmacological inhibitor, SU5402 (Calbiochem),
was used at a final concentration of 24 µM as described previously
(Reifers et al., 2000a).
Bead implantation and cell lineage tracing
Bead implantation was carried out as described by Reifers et al.
(Reifers et al., 2000a). Cell
lineage analysis was as follows: ace mutant embryos were
distinguished from their siblings by morphology at the 5-somite stage
(Brand et al., 1996
) and
manually dechorionated. Mutant and wild-type embryos were fixed in the desired
position by attaching them with one side to a stripe of 3% methyl cellulose on
a microscope cover slip in Ringer's embryo solution. A glass capillary was
pulled, covered with crystalline DiI (Molecular Probes), and inserted into the
embryo at the level of the midbrain-hindbrain boundary. The capillary was left
inside for not more than 5 seconds. The first pictures were taken at the age
of 10 somites, after the embryos had completely recovered from the labeling
procedure. Images were captured on an Olympus BX61 microscope equipped with a
Spot RT camera and Metamorph imaging software. Measurements were made using
Metamorph. The system was calibrated using a micrometer calibration slide.
Images were taken at 10x or 20x magnification. Fluorescent images
were captured using a standard rhodamine filter set.
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Results |
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|
Lack of cerebellar development in ace
Having seen that dmbx1, otx2 and wnt4 appear at ectopic
caudal locations, we analyzed the expression of genes that mark the upper
rhombic lip where the cerebellar anlage forms in wild-type embryos.
zath1 (zebrafish atonal homologue 1; atoh1 -
Zebrafish Information Network) is a marker of the upper rhombic lips
(Koster and Fraser, 2001). In
ace mutants, we consistently find that zath1 is missing from
the upper rhombic lip region, when compared with wild-type siblings
(Fig. 3A-D).
|
Likewise, staining for the bHLH transcription factor neurod, a
marker expressed by cerebellar granule cells
(Fig. 3I,K)
(Miyata et al., 1999;
Lee et al., 2000
;
Mueller and Wullimann, 2002
),
fails to detect a cerebellar compartment in mutant embryos
(Fig. 3J,L). In addition,
Purkinje cells of the cerebellum cannot be detected by anti-zebrin II/aldolase
C (Brochu et al., 1990
)
immunostaining of mutant embryos (Fig.
3M,N). Thus, complementary to the caudal expansion of gene
expression domains normally excluded from the dorsal metencephalon, specific
markers for the developing cerebellar primordium, or for later granule and
Purkinje cells, are not expressed in ace embryos.
Rhombomere 1 behaves as a bipartite structure in ace
embryos
Rhombomeres are transiently appearing segmental structures during hindbrain
development. Of the 7 (8 in amniotes) rhombomeres, the rostral rhombomere 1
(r1) gives rise to the cerebellum and other important structures of the
vertebrate central nervous system, such as the Locus coeruleus (LC)
(Morin et al., 1997). As our
results revealed that the cerebellar plate derived from rostral r1 lost its
identity, we investigated the expression of further specific marker genes
characteristic of r1 in wild-type embryos. Expression of epha4a
(formerly known as rtk1) marks caudal r1
(Fig. 4A,C), and its expression
is not diminished in ace embryos
(Fig. 4B,D; small arrow).
However, the orientation of its expression domain is altered: instead of being
perpendicular to the rostrocaudal axis of the hindbrain, it becomes slanted
(Fig. 4B). Moreover, double
detection of otx2 and epha4a
(Fig. 4E,F) reveals that their
expression domains now abut, forming a new interface in ace embryos
(Fig. 4F).
|
The presence of epha4a in caudal r1, and the abutting of the
epha4a and otx2 domains in conjunction with the lack of
cerebellar and LC development, suggest that the lack of Fgf8 in ace
mutants affects the rostral and caudal part of r1 unequally. The effect of the
loss of organizer-derived Fgf8 function is restricted to rostral r1. Our
analysis supports the notion raised by C. Moens' and I. Mason's laboratories
that r1 is a bipartite structure, where the rostral part of this rhombomere
should be considered as a separate entity designated as rhombomere 0
(Waskiewicz et al., 2002;
Walshe et al., 2002
). In
ace mutants, rhombomeres rostral or caudal to r4 appear to be often
reduced in width along the rostrocaudal axis of the hindbrain, as judged by
rhombomere marker analysis (Maves et al.,
2002
) (Fig. 4A-H).
This is most probably due to the impaired r4 signaling activity in
ace (Maves et al.,
2002
), rather than to the loss of isthmic organizer function.
However, beyond the smaller rostrocaudal extent of certain rombomeres in
ace, the generation of rhombomere-specific branchiomotor neuron
patterns, including r2 and r3 trigeminal motoneurons, is largely normal
(Maves et al., 2002
).
Rostralization can also be detected at the ventral part of the MHB
primordium
As previously shown, the narrow perpendicular stripe of the isthmic
organizer region emits rostrocaudal patterning signals along the whole extent
of the MHB region. The lack of functional Fgf8 signaling dramatically alters
the morphology of the isthmic and dorsal metencephalic alar plate region in
ace embryos. We also examined how the basal plate (tegmental) region
is affected in ace mutants. To address this, we analyzed expression
of zash1b (ashb - Zebrafish Information Network), gap43,
tag1/cntn2 and epha4a. These genes are expressed at
ventral aspects of the diencephalon and hindbrain leaving a gap at the
tegmental part of the MHB and midbrain. In ace embryos we observe a
fusion of the diencephalic and hindbrain expression domains of zash1b,
gap43 and tag1/cntn2, filling the above mentioned gap
(Fig. 4I,J,
Fig. 3E,F and
Fig. 3G,H, respectively). In
the case of epha4a, a caudal expansion of the diencephalic domain can
be observed (Fig. 4B,D). These
findings are consistent with the recently reported caudal expansion of
fgfr3 expression along the basal plate of the midbrain, and with the
consequent fusion of the diencephalic and hindbrain expression domains in
ace embryos (Sleptsova-Friedrich et al., 2002). Expression of
twhh, instead of being expanded, shows a dramatic reduction in the
ventral mesencephalon in ace mutants
(Fig. 4K,L). The alteration in
expression of twhh may reflect the expansion of more rostral, caudal
diencephalic or rostral mesencephalic fates toward caudal co-ordinates of the
mesencephalon. Taken together, these changes in gene expression are compatible
with the observed rostralization taking place at the dorsal metencephalic alar
plate, and are indicative of a caudal-to-rostral transformation of the basal
plate of the MHB. However, the degree of rostralization along the
mesencephalic/metencephalic basal and alar plates may be different.
Caudal enlargement of the tectum in ace mutants is not a
consequence of enhanced cell proliferation or decreased cell death
Various mechanisms could account for the successive rostralization and loss
of isthmic indentation in ace mutants, such as regionally distinct
cell proliferation or apoptosis, or transformation of cell fate in the
affected tissue. We therefore analyzed the mitotic behaviour of the dorsal
mesencephalic (tectal) and MHB primordial cells using an antibody raised
against the proliferation marker phospho histone H3 (PH3). H3 phosphorylation
has previously been described to correlate with mitosis in mammalian cells,
Xenopus and Tetrahymena. Before the actual formation of the
anatomical isthmic constriction, marked differences cannot be detected in cell
proliferation between wild-type and mutant embryos
(Fig. 5A-D). However, at later
stages [36, 57 and 72 hours postfertilization (hpf)], the immunoreactivity of
PH3 is markedly reduced at the caudal-most edges of ace tecta, and
the cerebellar proliferative zone typical for wild-type embryos is absent in
ace (Fig. 5E-G; data
not shown). Increased proliferation, therefore, is very likely not the cause
of the enlarged tectum. However, the decreased proliferation observed in young
ace mutant larvae, may account for the overall smaller than wild-type
size of ace mutants at later stages, at day 5, for example
(Fig. 3I,J)
(Picker et al., 1999).
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Cells at the location of the wild-type MHB give rise to caudal tectum
in ace embryos
The rostralized gene expression and the lack of cerebellar cell types in
ace mutants, along with the results of the proliferation and the
cell-death studies, all suggest that the isthmic and cerebellar primordium
might have already adopted a new tectal identity by early- to
mid-somitogenesis stages. To address this issue directly, we performed cell
lineage tracing experiments to reveal the fate of the MHB primordial cells. We
investigated whether MHB primordial cells in ace mutants are retained
in a position that corresponds to the MHB compartment of wild types, or
whether they end up elsewhere, acquiring a new fate. We labeled groups of
cells at the level of the prospective MHB with the lipophilic dye DiI during
early somitogenesis (5-somite stage) in wild-type and ace embryos.
The morphological MHB has not yet formed at this stage, but MHB primordial
cells can be targeted by their position along the rostrocaudal axis relative
to the posterior edge of the optic vesicle. By comparing the position of
DiI-labeled cells at the 10-somite stage
(Fig. 6A,D), and at 24 hpf
(Fig. 6B,C,E,F), in wild-type
and ace mutant embryos, we were able to compare the fate of these
marked cells. Table 1 summarizes the results of our labeling experiments. In all wild-type cases,
the labeled cells ended up in either the posterior tectum or in the
cerebellum/r1, i.e. in the MHB region (Fig.
6B,C). In all ace cases, cells ended up in the posterior
part of the enlarged tectum (Fig.
6E,F). Moreover, we did not observe a loss of the labeled cell
population in ace embryos, when compared with wild-type siblings.
These results suggest that cells that are normally fated to become MHB tissue
in the wild-type are transformed into tectal cells in the ace
mutants.
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Discussion |
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The lack of isthmic indentation is preceded by marked changes in
molecular identity
The examination of MHB region specific marker gene expression enabled us to
investigate the processes underlying the special anatomical features of
ace embryos (Brand et al.,
1996; Reifers et al.,
1998
). In ace mutants, expression of specific genes that
prefigure the future anatomical constriction (pax2a, her5, wnt1, eng2,
eng3, erm, pea3, spry4, spa1a and pax8) is perturbed
(Brand et al., 1996
;
Reifers et al., 1998
;
Raible and Brand, 2001
;
Fürthauer et al., 2001
)
(present study). Consequently, in ace embryos a caudal shift of the
expression of forebrain and mesencephalic markers (otx2, dmbx1, wnt4)
is evident in positions where the isthmic primordium and upper rhombic lips
should differentiate. From the expression of wnt4, it can also be
estimated how rostral the molecular identity of the structures in ace
embryos is. This gene normally starts to be expressed at the junction of the
caudal diencephalon and the rostral tectum
(Ungar et al., 1995
). Its
appearance at more caudal positions indicates that ace mutants show
gene expression profiles characteristic for the rostral tectum of wild-type
embryos. Pharmacological inhibition of Fgf signaling in wild-type embryos
faithfully mimics the expansion of otx2 expression seen in
ace mutants. The rostralized gene expression profiles of the mutants,
along with the lack of the rhombic lip/early cerebellar marker zath1
and other markers of the developing cerebellum (gap43, tag1/cntn2, neurod,
zebrin II), suggest a possible fate transformation of the isthmic and
cerebellar regions preceding the agenesis of the isthmic fold and the lack of
a distinct cerebellar domain at later stages. Our present results, combined
with earlier observations (Reifers et al.,
1998
; Raible and Brand,
2001
), show that a molecular MHB is initially formed but is then
lost during the early- to midsomitogenesis period in the mutants. The
significant structural reorganization taking place at the embryonic neuraxis
of the mutant is preceded by a marked alteration in the molecular identity of
the cells of the isthmic and cerebellar primordia. The identity of the MHB
domain is not maintained and, as a consequence, both the isthmic and
cerebellar primordia acquire a more rostral, mesencephalic character in
ace mutants. In fact, in ace mutants the dorsocaudal
compartment (upper rhombic lip/dorsal r1), which normally gives rise to the
cerebellum, develops as an enlargement of the midbrain tectum. Taken together,
these results show that the agenesis of the isthmic constriction in
ace embryos is not only a simple dysmorphology. The scheme in
Fig. 8 summarizes the most
important features of ace mutants in comparison with wild-type
siblings.
|
Our analysis suggests that quantitative processes, such as increased proliferation or a decreased apoptotic rate, are unlikely to play a major role in the restructuring process occuring within the mesencephalic-metencephalic region in ace mutants. By contrast, our cell lineage tracing experiments clearly indicate that the isthmic and cerebellar primordial cells become part of the tectum in ace embryos. Cells at the location of the wild-type MHB give rise to caudal tectum in ace embryos. Therefore, we think that the cells of the putative isthmic primordium in the mutants are qualitatively different to those of the wild type. The marked rostralization in gene expression is connected to the alteration of positional information and caudal-to-rostral transformation in the absence of the functional organizer signal Fgf8. Our results suggest that MHB and cerebellar primordial cells are transformed to tectal ones, or show a default tectal fate in ace.
Possible molecular mechanisms underlying the isthmus-to-midbrain
transformation
In our bead implantation experiments the molecular and morphological
features of the MHB could be restored in ace mutant embryos,
indicating that Fgf8 is either directly or indirectly necessary to execute the
proper morphogenetic program at the mesencephalic and hindbrain alar plate.
Moreover, when Fgf8 is provided ectopically in wild-type embryos it is able to
restructure even the caudal parts of the forebrain, as was known from previous
work in chick (Martinez et al.,
1999; Shamim et al.,
1999
). Fgf8, besides being necessary and sufficient to reprogram
and restructure the surrounding tissues, seems to act in a dose-dependent
manner. Several pieces of evidence support the idea that different doses of
Fgf8 may be responsible for the specification of distinct structures of the
MHB region, and that the activity of Fgf8 is directly coupled to the dose of
Fgf8 protein acting on the target tissue
(Xu et al., 2000
;
Sato et al., 2001
).
Gain-of-function experiments performed with two different Fgf8 isoforms
(MacArthur et al., 1995
;
Liu et al., 1999
;
Sato et al., 2001
) show that
the type-difference in the activity of the a and b isoforms can be attributed
to the signal intensity, as electroporation of 100-fold less Fgf8b expression
vector exerts similar effects to Fgf8a in chick embryos
(Sato et al., 2001
). To
achieve cerebellar differentiation a high Fgf8 concentration is required, as
reflected by the transformation of the tectal structures into cerebellum upon
ectopic overexpression of the stronger Fgf8b isoform. During normal
development, the stronger Fgf8b is the predominant isoform of Fgf8 in the
isthmic region (Sato et al.,
2001
). The region that is exposed to strong Fgf8 signal (e.g. to
the more abundant and physiologically more active Fgf8b), which expresses
Gbx2 and Irx2, may then acquire r1 characteristics, from
which the dorsal rhombic lip and, later, the cerebellum will differentiate
(Sato et al., 2001
). Regions
in which the tectal differentiation program will be executed receive only weak
Fgf8 signaling, as inferred from the results of diencephalic overexpression of
the weakly active Fgf8a isoform (Sato et
al., 2001
). In addition, different doses of Fgf8 might influence
the fine patterning of midbrain regions
(Lee et al., 1997
;
Picker et al., 1999
). Our bead
implantation experiments support dose dependency of Fgf8 action, as reflected
by the graded induction of tectal ephrin A expression at ectopic locations in
wild-type embryos, or in ace embryos at positions where the isthmic
organizer normally forms. Close to the Fgf8 source, this protein is normally
expressed at high levels, and its expression gradually decreases as a function
of the distance from the source. In ace embryos the gradient is
absent, and the mutants show no or only low levels of expression,
characteristic of the anterior tectum in wild types
(Picker et al., 1999
) (present
study).
The next question is how Fgf8 triggers and orchestrates the reorganization
of the isthmic region when applied in the form of coated beads. We have
suggested previously that the implantation of Fgf8 beads into the
mesencephalic alar plate of the mutants may re-activate a set of regulatory
genes that are acting during an earlier phase of the normal MHB development
(Reifers et al., 1998). During
normal development, these genes are initially independent of Fgf8, but at the
time the bead implantation experiments were performed many of these key
regulators (her5, wnt1, pax2a, pax8, spa1a, spry4) are directly or
indirectly dependent on Fgf8 function, as is evident from the fact that they
disappear in the absence of functional Fgf8 in ace mutants. Indeed,
bead implantation experiments done on chick and fish embryos support this
notion. Fgf8- or Fgf4-soaked beads implanted into the posterior forebrain or
hindbrain alar plate are capable of inducing isthmic, cerebellar or midbrain
structures, and the key regulatory genes (Wnt1, En2 and Fgf8
itself) are induced around the bead in the implanted embryos
(Crossley et al., 1996
;
Martinez et al., 1999
;
Shamim et al., 1999
).
Similarly in zebrafish, we found that implanted Fgf8-coated beads in wild-type
embryos are able to induce fgf17, en2, sprouty4 and other targets of
the MAPK pathway (Reifers et al.,
2000b
; Fürthauer et al.,
2001
; Raible and Brand,
2001
). Similar to the results of the above-mentioned in vivo bead
implantation experiments, Fgf8b-coated beads are able to trigger expression of
En1, En2, Pax5, Wnt1 and Gbx2 in mouse embryonic midbrain
and diencephalic explants, and repress Otx2 in mesencephalic explants
(Liu et al., 1999
). This
proposed action of Fgf8 triggering key regulators of MHB development, which
then create ectopic MHB identity, is presumably different from the normal
steps of MHB development because early expression of these markers is
independent of Fgf8. These events can be considered as a shortcut in the
developmental program, leading to recapitulation of the MHB cascade. Upon
re-initiation of key regulators of the MHB cascade, it is likely that the
mutual effects of regulatory genes create secondary genetic events that then
stabilize the identity of the tissue, as is presumed to happen during the
maintenance phase of MHB development.
During normal development, repressive genetic interactions between Otx2 and
Gbx2 (Gbx1 in zebrafish) seem to determine the caudal limit of the tectum and
the position of the molecular MHB (Broccoli
et al., 1999; Millet et al.,
1999
; Katahira et al.,
2000
; Martinez-Barbera et al.,
2001
; Rhinn et al.,
2003
) (M. Rhinn and M.B., unpublished). In vertebrates, both
Fgf8 and Gbx2 have a repressive activity on Otx2
expression in the metencephalic alar plate (Liu and Joyner, 1999;
Martinez et al., 1999
;
Millet et al., 1999
;
Tour et al., 2002
). In
addition, zebrafish gbx2 is fully dependent on Fgf8 during the
maintenance period (Rhinn et al.,
2003
). In ace mutants, one of the key determinants of the
MHB position, otx2, expands at the expense of the fgf8 and
gbx1 (Gbx2 in amniotes) domain. It is therefore conceivable
that upon displaying functional Fgf8 to otx2-expressing ace
mutant tectal cells, the repressive genetic interactions
(Martinez-Barbera et al.,
2001
) playing a role during normal MHB maintenance are
re-established, accounting for the reversal of the tectal morphology and
identity on the operated side in ace embryos. Conversely, in
ace mutants where functional Fgf8 signaling is absent
(Reifers et al., 1998
;
Araki and Brand, 2001
),
otx2 transcripts are not repressed, causing a strong similarity to
rostral mesencephalic regions experiencing putatively low or no Fgf8 signal as
a function of the distance from the emitting source. Consequently, the
mesencephalic/tectal differentiation program is executed in place of the
metencephalic alar plate in ace mutants. Our observations are in good
agreement with the results of Otx2 misexpression studies, where the
ectopic appearance of Otx2 changes the fate of the metencephalic alar
plate to a more rostral, tectal fate
(Broccoli et al., 1999
;
Katahira et al., 2000
). Taken
together, the transformation process that takes place at the expense of the
isthmic and cerebellar primordia can be viewed as an imbalance between mutual
repressive effects of the MHB cascade genes, caused by lack of the
orchestrating Fgf8 signal. As a consequence of the lack of Fgf8 function, the
self-maintenance of isthmic and cerebellar primordial cells is impaired
resulting in a rostralization of gene expression profiles, and transformation
of the isthmic and cerebellar primordium in ace mutants. In agreement
with recent observations (Tallafuß
and Bally-Cuif, 2003
), our results suggest a role for Fgf8 in
protecting the isthmic and metencephalic precursors from acquiring more
rostral identities, and in self-maintaining the MHB identity.
Modularity in the developing upper brainstem structures
The embryonic brain seems to be composed of sequential modules that
represent histogenetic fields specified by position-dependent expression of
patterning genes (reviewed by Redies and
Puelles, 2001). This early, embryonic modularity of the nervous
system is later on translated into functional modularity. Furthermore, the
suggested modular organization of the early brain allows for adaptive
modification during evolution (Redies and
Puelles, 2001
). The modularity of the developing brainstem
structures allows for spatial and temporal changes at early stages of
development, for example, transformation of one particular structure to
another one. During early stages of ontogenesis, such plasticity of the
developing CNS makes escape from an immediate developmental arrest possible in
CNS mutant embryos. Cases of transformation and their mechanisms are well
documented during insect development (Sato
and Denell, 1985
; Klingensmith
et al., 1989
), and during development of the vertebrate caudal
hindbrain, where the modular organization is evident owing to the rhombomeres
(McKay et al., 1994
;
Gavalas et al., 1998
;
Popperl et al., 2000
;
Wiellette and Sive, 2003
)
(reviewed by Lumsden and Krumlauf,
1996
). The rostralization, transformation and reversal of the
abnormalities by bead implantation in ace mutants reveal the
plasticity of the developing upper brainstem. An interesting question is
therefore whether the observed transformation of the isthmic region hints at a
modular organization of this region as well. Anatomically, a modular
organization of the midbrain and the isthmic region has not yet been revealed
(Redies and Puelles, 2001
).
Experiments performed in chick reveal the unique developmental fate of r1,
being the only rhombomere in which no Hox genes are expressed as consequence
of Fgf8 action in the anterior hindbrain. Fgf8 acts to set aside the territory
from which the cerebellum will eventually develop through restriction of
Hoxa2 expression in r1 (Irving
and Mason, 2000
). Visualizing the formation of a new
otx2/epha4a interface in ace mutants reveals a
bipartite organization of r1, where the rostral part of r1 acquires a new,
tectal identity. The modular plasticity also applies to more anterior parts of
the mesencephalon and the diencephalon as seen, supported by bead implantation
and electroporation experiments, both in chick
(Martinez et al., 1999
;
Sato et al., 2001
) and fish
(present study). We demonstrated that functional Fgf8 is able to both revert
the transformation of the isthmic and cerebellar primordia in mutant embryos
and induce the diencephalon and mesencephalon to undergo transformation in
wild-type embryos. Taken together, this reveals an extensive plasticity of the
isthmic and r1 region, suggesting that transformation of cell fates seems to
be the most economic way of restructuring the MHB territory in the absence of
functional organizer activity in ace mutants.
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ACKNOWLEDGMENTS |
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