The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures
Paula Alexandre and
Marion Wassef*
Régionalisation Nerveuse CNRS/ENS UMR 8542, Département de
Biologie Ecole normale supérieure, 46 rue d'Ulm, 75005 Paris,
France
*
Author for correspondence (e-mail:
wassef{at}wotan.ens.fr)
Accepted 15 July 2003
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SUMMARY
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During vertebrate development, an organizing signaling center, the isthmic
organizer, forms at the boundary between the midbrain and hindbrain. This
organizer locally controls growth and patterning along the anteroposterior
axis of the neural tube. On the basis of transplantation and ablation
experiments in avian embryos, we show here that, in the caudal midbrain, a
restricted dorsal domain of the isthmic organizer, that we call the isthmic
node, is both necessary and sufficient for the formation and positioning of
the roof plate, a signaling structure that marks the dorsal midline of the
neural tube and that is involved in its dorsoventral patterning. This is
unexpected because in other regions of the neural tube, the roof plate has
been shown to form at the site of neural fold fusion, which is under the
influence of epidermal ectoderm derived signals. In addition, the isthmic node
contributes cells to both the midbrain and hindbrain roof plates, which are
separated by a boundary that limits cell movements. We also provide evidence
that mid/hindbrain roof plate formation involves homeogenetic mechanisms. Our
observations indicate that the isthmic organizer orchestrates patterning along
the anteroposterior and the dorsoventral axis.
Key words: Mid/hindbrain junction, Isthmic organizer, Mid/hindbrain organizer, Roof plate, Homeogenetic mechanisms, Hensen's node, Dorsoventral patterning, Chick, Quail
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Introduction
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The isthmic organizer (IO), located at the constriction (or isthmus) that
marks the mid/hindbrain (MHB) junction of the developing neural tube, is
essential for the growth and patterning of the midbrain and anterior hindbrain
(Joyner et al., 2000
; Rhin and
Brand, 2001; Simeone, 2000
;
Wurst and Bally-Cuif, 2001
).
When transplanted ectopically into competent territories, the IO induces the
adjacent neuroepithelium to form supernumerary MHB structures
(Martinez et al., 1991
;
Martinez et al., 1995
), an
activity mediated by FGF8 that is expressed at the MHB boundary
(Crossley et al., 1996
). The
isthmic region is also the site of extensive cell rearrangements
(Alvarez Otero et al., 1993
;
Louvi et al., 2003
;
Millet et al., 1996
). Although
much attention has been devoted to the understanding of the genetic
interactions that position and maintain the IO
(Joyner et al., 2000
; Rhin and
Brand, 2001; Simeone, 2000
;
Wurst and Bally-Cuif, 2001
),
less is known about the morphogenetic mechanisms that it induces. Ectopic IO
grafts induce morphological distortions of the host and graft neuroepithelium
(Martinez et al., 1999
) that
contrast with the perfect morphological and genetic integration of other
transplanted regions of the midbrain neuroepithelium
(Alvarado-Mallart et al., 1990
;
Martinez and Alvarado-Mallart,
1990
; Nakamura et al.,
1986
). Our previous transplantation experiments
(Louvi et al., 2003
) indicated
that the midline regions of both the midbrain and hindbrain are populated by
divergent migrations of cells generated from a restricted dorsal midline
region in the caudal midbrain. The roof plate (RP) is a specialized structure
that marks the dorsal midline of the neural tube and is involved in
dorsoventral patterning (Lee et al.,
1998
; Liem et al.,
1995
). At early stages, the expression of two RP markers,
Gdf7 and Wnt1, displays singularities at the level of the
MHB (Louvi et al., 2003
).
Gdf7 expression is delayed in the MHB domain compared to adjacent
regions of the neural tube that express the transcript at the 19 somite stage
(ss) whereas Wnt1 is expressed in a wide dorsal MHB domain at the
10-13ss that encircles the neural tube at the level of MHB junction
(Bally-Cuif and Wassef, 1994
).
Experimental manipulations involving rotation or partial ablation of the avian
midbrain, although not previously connected to IO activity, were found to
affect or to completely prevent the formation or positioning of the roof plate
(RP) (Cowan and Finger, 1982
;
Marin and Puelles, 1994
).
Finally, the expression of Wnt1 is deflected in the direction of
ectopically transplanted (Bally-Cuif and
Wassef, 1994
) or FGF8-induced
(Crossley et al., 1996
) IOs.
Altogether these observations suggest a specific behavior of the developing RP
at the level of the MHB.
We show that the dorsal IO is arranged around a restricted central node
that contributes cells to the midbrain and cerebellar RPs and is both
necessary and sufficient for the formation of the midbrain RP. Interfering
locally with IO node function and plasticity prevents RP formation in the
caudal midbrain. We also present evidence that the induction or maintenance of
the expression of RP markers Gdf7 and Wnt1 involve
homeogenetic mechanisms. Finally, we show that a stream of cells extends from
ectopic, FGF8-induced, IOs to populate the locally induced ectopic RP. These
findings demonstrate that the IO is responsible for RP induction and
positioning in the caudal midbrain. They indicate that the IO is involved in
dorsoventral patterning and in the selection of the symmetry axis of the optic
tectum. This process resembles that organized in the ventral spinal cord by
the regressing Hensen's node (Catala et
al., 1996
; Charrier et al.,
1999
), the avian counterpart of the Spemann organizer. Thus, our
observations emphasize that axis organization and contribution to axial
structures may represent a common function of secondary organizers in the
vertebrate brain.
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Materials and methods
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White Leghorn chick and Japanese quail embryos, were operated on between
somite stages (ss) 9 and 13 and fixed 2 days later between stages 19 and 21
(HH19-21) of Hamburger and Hamilton
(Hamburger and Hamilton,
1951
). The methods for performing small ablations, homotopic
isochronic grafts (Alvarez Otero et al.,
1993
; Bally-Cuif and Wassef,
1994
), bead implantation
(Martinez et al., 1999
)
mesencephalic vesicle rotation including the notochord [type 1A in Marin and
Puelles (Marin and Puelles,
1994
)] in situ hybridization
(Bally-Cuif and Wassef, 1994
)
and cell death assay using Nile Blue Sulfate
(Louvi and Wassef, 2000
) were
as described with minor modifications. The DiI crystals were inserted into a
small slit made in the neuroepithelium using tungsten needles. Heparin acrylic
beads were soaked sequentially in FGF8 (R&D, 0.2 mg/ml) and DiI emulsion
(Selleck and Stern, 1991
) or
only in DiI emulsion.
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Results
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A discontinuity in early cell behavior at the presumptive midbrain
beak
Previous quail to chick transplantation experiments have indicated that
there is a global convergence of the caudal midbrain neuroepithelium towards
the midline (Millet et al.,
1996
; Louvi et al.,
2003
). In addition, some medial grafts were observed to elongate
markedly more along the dorsal midline [figure 2B,C in Louvi et al.
(Louvi et al., 2003
)]. This
pattern could have been a mere consequence of the presence of a midline
boundary deflecting the flow of converging cells in the orthogonal directions,
on both sides. Alternatively, and more in line with the interpretation of
Millet et al. (Millet et al.,
1996
), a restricted midline region at the caudal limit of the
midbrain could generate the midline structures in an extended anteroposterior
domain around the MHB junction. A first series of experiments was thus
performed that aimed at better characterizing the origin of the MHB midline.
We first inserted small homotopic and isochronic quail-to-chick transplants
(Fig. 1A) or DiI crystals
(Fig. 1B) laterally in the
caudal mesencephalic vesicle of 10-13ss embryos. The operated embryos were
analyzed 2 days later, at stage HH 19-21, when the isthmic constriction
coincides with the limit between the midbrain and anterior hindbrain (Haleness
et al., 1990; Martinez and
Alvarado-Mallart, 1989
; Millet
et al., 1996
). The lateral grafts or DiI-labeled cells ended
medially without extending much along the anteroposterior axis and both were
excluded from a small midline domain (purple arrows in
Fig. 1A,B). These observations
suggested that a restricted medial region in the dorsal IO differed either in
growth or in cell movements from the adjacent neuroepithelium. Quail
transplants inserted into this region were rarely recovered 2 days later (see
however Fig. 1C). Extensive
cell death affecting the MHB midline region at the 14-16ss
(Lumsden et al., 1991
) (see
also Fig. 3F-I) and, possibly,
a lower proliferation rate could explain why small quail transplants grafted
into the dorsal IO failed to survive. Small medial grafts inserted slightly
more caudally often contributed scattered cells to the cerebellar RP [figures
2B and 3 in Louvi et al. (Louvi et al.,
2003
)]. DiI crystals inserted in the dorsal IO marked a small
compact cell population in HH19-21 embryos
(Fig. 1D). At this stage, the
DiI-labeled cells ended into a protrusion (arrowheads in
Fig. 1E,F) that marks the
posterior midbrain midline and has been described as a wedge in mouse
(Bally-Cuif et al., 1995
) or a
beak in chick (Millet et al.,
1996
). Subsequent experiments described below indicated that cells
destined to populate the RPs of the midbrain and anterior hindbrain were
produced at the periphery of the prospective beak.

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Fig. 1. An immobile pivotal domain in the caudal midbrain. (A,B) Posterior, (C)
posterolateral, and (D) lateral views of the dissected (A-C) or whole-mount
(D) neural tubes of HH19-21 chick embryos that received a graft or DiI
crystals as schematized on the left. Small lateral grafts (A) and lateral DiI
crystals (B) all converged towards the midline. They avoided a small medial
circular area in the caudal midbrain (arrowheads in A,B). Small grafts (C) or
DiI crystals (D) inserted into the center of the caudal mesencephalic vesicle
apparently remained still (arrowheads in C and D) and ended in a protrusion or
`beak' that marks the caudal midline of the mesencephalon. This is best seen
on lateral views of the dissected neural tube (E,F arrowheads).
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Fig. 3. Expression of midbrain RP markers depends on IO-derived signals. (A-E)
Posterior views of the MHB of HH19-21 embryos that were operated on at the
10-15ss, as schematized on the left, and treated for the detection of
Gdf7 transcripts (purple). Corresponding transverse cryostat sections
counterstained with nuclear Fast Red are illustrated in A'-C'. (A)
In control HH19-21 chick (or quail) embryos Gdf7 expression marks the
RP. (B) Gdf7 expression is completely (left, 4/6) or partially
(right, 2/6) regenerated after ablations of the isthmic dorsal midline. (C-E)
Homotopic quail to chick IO node grafts performed at different stages
interfere with midbrain RP formation. Graft insertion into the IO node
resulted in RP duplication in 14-15ss embryos (C, double arrowhead in
C'; 5/5) and in the formation of a gap in Gdf7 expression in
younger embryos (between arrowheads in D; 2/3). A medial bulge that expresses
Gdf7 is observed in 10-12ss embryos (arrowhead in E; 3/4). (D')
Transverse section of an E16 embryo that received a slightly larger graft
centered on the IO node at the 12ss. The section was immunostained with QCPN
to detect the grafted quail cells and counterstained with Cresyl Violet. Quail
cells are detected at more caudal levels but the illustrated section is
anterior to the graft limit. Note the absence of RP structure on the midline
(arrowhead). Very few quail cells were detected in embryos that received
transplants limited to the presumptive IO node (C-E) either because the low
proliferation rate interfered with graft integration, or because of the high
level of cell death detected by Nile blue sulfate staining on the midline of
the isthmic region between the 14ss and 22ss (F-I) (see also
Lumsden et al., 1991 ).
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Restricted origin of the midbrain and rostral hindbrain roof plates
at the isthmic node
In contrast to midline grafts, calibrated paramedial transplants extended
long distances anteriorly or posteriorly along the midline
(Louvi et al., 2003
). The
organization of the dorsal midline region of the MHB was therefore analyzed by
insertion of small DiI crystals around the central IO region identified
earlier. The dye-labeled cells were observed to disperse widely along the
midline. Depending on the posterior (Fig.
2A) or anterior (Fig.
2D) position of the DiI crystal, the labeled cells were confined
either to the RP of the anterior hindbrain
(Fig. 2A-C) or of the midbrain
(Fig. 2D-F). DiI labeling of
the anterior hindbrain RP was stereotyped: the dye-labeled cells populated its
entire length abutting rostrally the beak protrusion
(Fig. 2B,C) and reaching the
edge of the choroid plexus caudally, with a small bifurcation at the end
(Fig. 2B). Although the
rostrocaudal extension of midbrain RP labeling was less stereotyped, the
DiI-labeled cells in the anterior hindbrain and the midbrain RPs respected the
same limit located beneath the beak protrusion
(Fig. 2B,C,E,F). Thus, we
identified on the midline a sharp limit to cell movements beneath the midbrain
beak. We could not detect, however, any indication of a transverse restriction
to cell movements (Millet et al.,
1996
) more laterally, in the form of a discontinuity in the shape
of the grafts or the distribution of dye-labeled cells
(Fraser et al., 1990
). A small
pattern difference was noticed between the origin of the midbrain RP, clearly
a paired domain interrupted on the midline, and that of the anterior hindbrain
RP that straddled the midline.

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Fig. 2. Isthmic origin of MHB roof plate structures. (A,D) Posterior views of the
dissected neural tubes of HH19-21 embryos in which DiI crystals were inserted
at the 10-13ss. The site of insertion is schematized on the left. Midline DiI
crystals inserted at the level of the isthmic constriction (A) label the
entire anterior hindbrain roof plate (RP). Paramedial DiI crystals inserted
into the caudal midbrain vesicle (D) label the midbrain RP. (B,C,E,F)
Midsagittal cryostat sections of two embryos (shown in the insets) similar to
A and D. The DiI-labeled hindbrain (hb) RP ends beneath the beak (B,C). When
it extends to the isthmic region (E,F), DiI labeling in the midbrain (mb) RP
respects a similar sharp limit beneath the beak. (G) Fate map of the dorsal
isthmic region focused on the central IO node. The cells that contribute to
the roof plates of the midbrain (in red) and anterior hindbrain (purple) are
organized around a central domain (gray) whose relative size decreases between
the 10-13ss and HH19-21 stages. They migrate extensively along the midline but
respect a common limit in the caudal midbrain. The 10-13ss and HH19-21 stage
embryos are not drawn at the same scale.
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These observations are schematically illustrated in
Fig. 2G. They indicated that
the cells destined for the midbrain and anterior hindbrain RPs were generated
at the periphery of an immobile midline domain of the IO. We have called this
structure the IO node.
The IO node is required for midbrain RP development
To investigate whether the IO node was necessary for the generation of the
midbrain RP, small midline ablations were performed in the isthmic region of
10-13ss chick embryos. Two days later, expression of Gdf7 (and
Wnt1, not shown), a chick and quail RP marker
(Fig. 3A,A')
(Lee et al., 1998
), was
completely (4/6) or partially (thinner RP, 2/6) regenerated
(Fig. 3A,A',B,B'),
consistent with previous reports on the regulative behavior of the isthmic
region (Alvarado-Mallart et al.,
1990
; Alvarez Otero et al.,
1993
; Marin and Puelles,
1994
; Martinez and
Alvarado-Mallart, 1990
;
Nakamura et al., 1986
). In
contrast, homotopic quail-to-chick grafts inserted into the IO node resulted
in marked alterations of the RP (Fig.
3C,C',D,D',E). Depending on the stage at operation,
Gdf7 midline expression observed 2 days later was either split or
duplicated (Fig. 3C,C'
14-15ss embryos, 5/5), missing (Fig.
3D, between arrowheads; D', 13ss embryos, 2/3) or missing
with a small Gdf7-positive protrusion bulging in the middle of the
Gdf7 gap (Fig. 3E,
arrowhead, 10-12ss, 3/4). Very few or no QCPN-immunoreactive quail cells were
detected in the isthmic region of the grafted embryos. Indeed extensive cell
death was observed on the midline of the mid-hindbrain junction between the
14ss and 22ss (Fig. 3F-I). The
transient presence of the small chunk of grafted quail cells could impair the
regulation of IO node function that we observed after ablation. The bulge
observed in the youngest embryos (Fig.
3E) was interpreted as a tentative regeneration of the IO node. We
also noticed that embryos that received a slightly larger transplant including
the IO node survived better. We interpret this as the result of better healing
of the grafts in these cases. Indeed, it is known that brain growth involves
an increase in ventricular pressure that requires both neural tube closure
anteriorly and a transient obturation of the ventricular lumen posteriorly
(Desmond and Schoenwolf,
1986
). The embryos that received small IO node grafts showed a
marked growth delay and their aspect is suggestive of ventricular leakage
(Fig. 3D,E). Grafts involving
the IO node often resulted in the formation, at E16, of a dome-shaped optic
tectum that lacked a roof plate on the midline rostrally to the graft
(arrowhead in Fig. 3D').
These observations indicated that experimental manipulations that affect a
small MHB region, the IO node, interfere with the formation of the midbrain
RP. They suggest that the IO node is essential for midbrain RP
development.
Homeogenetic mechanisms contribute to midbrain RP formation or
maintenance
Whereas RP markers regenerated after ablation of the midline in the
posterior midbrain, ablations performed more rostrally
(Fig. 4A-F) often resulted in
gaps in the expression of Gdf7 (4/4 in anterior midbrain, not shown,
and 1/6 in intermediate midbrain, Fig.
4C,C'). Interestingly, most (4/6) of the intermediate
midbrain ablations resulted in partial gaps containing scattered Gdf7
(Fig. 4B,B') or
Wnt1 (Fig. 4D) cells
at low density that could have migrated from or been induced by the cut ends
of the RP. In addition, local perturbations of the midline often resulted in
moderate alterations in the expression of RP markers that spread on both sides
of the lesion but were more obvious anteriorly
(Fig. 4E,F). Also,
anteroposterior rotations of the midbrain vesicle that maintained dorsoventral
polarity (Marin and Puelles,
1994
) and did not directly involve the dorsal midline resulted in
the disappearance of RP markers at HH19-21
(Fig. 4G) as well as in the
absence of RP structures at later stages
(Bally-Cuif and Wassef, 1994
;
Marin and Puelles, 1994
).
Thus, a temporary interruption or reversal of anteroposterior (AP) signaling
dramatically impaired RP formation. This behavior suggested that homeogenetic
mechanisms are involved in the induction or maintenance of RP identity during
development. To directly demonstrate that a homeogenetic mechanism operates
during RP development, an anteroposterior strip of quail mesencephalon was
rotated 90° and grafted orthogonal to the mesencephalic midline of a chick
host (Fig. 4h). To distinguish
between the host and graft RPs, we took advantage of species-specific chick
and quail Wnt1 probes (Bally-Cuif et al., 1994). The row of ChWnt1 expressing
host RP cells (Fig. 4H1), abutted a stripe of QWnt1-labeled cells within the graft
(Fig. 4H2, H3). The induced
quail RP develops in continuity with the host roof plate, orthogonally to the
presumptive AP axis of the graft. This clearly demonstrates that homeogenetic
mechanisms are involved in roof plate development.

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Fig. 4. Homeogenetic signaling induce the expression of RP markers. (A-D) Dorsal
view of the midbrain region of HH19-21 embryos that received a small midbrain
midline ablation at the 10-12ss as schematized on the left. Corresponding
transverse cryostat sections counterstained with Nuclear Fast Red are
illustrated in B',C'. The embryos were treated for the detection
of Gdf7 (A-C) or Wnt1 (D) transcripts. The ablation resulted
in complete regeneration (A, 1/6), or in the formation of partial (B, 4/6) or
complete (C, 1/6) gaps in the midbrain Gdf7 expression domain.
Scattered cells in the partial gaps expressed Gdf7 (B) or
Wnt1 (D). (E,F) Lateral views (anterior is to the left) of
Gdf7 midline expression in control (E) and ablated (F) embryos.
Gdf7 expression is perturbed (*) on both sides of the ablation
(delimited by the arrowheads). (G) Posterior view of a HH19 embryo
illustrating the lack of Gdf7 expression (between arrowheads) on the
midline of the mesencephalic vesicle after inversion of its anteroposterior
axis as schematized in g. (H) Dorsal view of the midbrain of a 4-day-old
chimera. An anteroposterior strip of quail midbrain neuroepithelium was
transplanted at HH10 perpendicular to the host midline as schematized in h.
H1-H3 illustrate the same chimera. The host roof plate (RP), labeled with
chWnt1 (Fast Red), is seen in red using fluorescent optics (H1); the induced
RP (purple arrowheads in H2 and H3), labeled with QWnt1 (NBT/BCIP), appears
purple under bright-field optics (H2). After dissection of the dorsal
midbrain, a faint QCPN labeling delineates the quail transplant (H3). Anterior
is to the right.
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Ectopic IOs contribute cells to a locally induced RP
In vivo, the relative contribution to RP formation of the IO and of
epidermal ectoderm-derived signals cannot be assessed since the position of
the MHB RP corresponds with the location of neural fold fusion. We thus
examined the capacity of the IO to induce RP formation in a different
location. We have previously observed
(Bally-Cuif and Wassef, 1994
)
that ectopic quail-to-chick IO grafts induced the formation of a row of
Wnt1-expressing cells linking the host midline to the graft. Because
Wnt1 is expressed both on the dorsal midline and in an isthmic ring,
the RP identity of the IO-induced cells could not be determined. No directed
migration of quail cells outside the IO graft was observed in these
experiments. Nevertheless, on the basis of the IO node midbrain location
determined in the present study, we would expect midbrain RP-directed
migration to involve host chick cells rather than quail cells. To examine the
behavior of IO cells in an ectopic environment, we induced the formation of an
ectopic IO by inserting heparin acrylic beads soaked sequentially in FGF8 and
in DiI solutions into the lateral mes- or diencephalon
(Crossley et al., 1996
). DiI
served to mark the cells of the FGF8-induced ectopic IO. The embryos were
photographed 2 days after bead implantation under rhodamine fluorescence
optics, then processed for Gdf7 or Wnt1 transcript
detection. Fragments of control beads soaked only in DiI labeled a patch of
cells (Fig. 5A) but did not
modify the morphology of the neural tube or the pattern of Gdf7
(Fig. 5A') or
Wnt1 expression (not shown). In contrast, a dorsally directed row of
DiI-labeled cells was always detected streaming from the FGF8-induced
cerebellum (Fig. 5B,C) that
develops as a bulge around the FGF8/DiI-soaked bead fragments
(Crossley et al., 1996
;
Martinez et al., 1999
). The
row of DiI-labeled cells prefigured an ectopic row of Gdf7
(Fig. 5B') or
Wnt1 (Fig. 5C') expressing cells that in many cases extended further dorsally
(Fig. 5C') to reach the
endogenous RP. Thus, the site of neural fold fusion is not required by the IO
to induce an ectopic RP and to contribute cells to it, suggesting that RP
determination and positioning is a robust property of the IO.

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Fig. 5. Cell migration from the FGF8-induced IO marks the position of the ectopic
roof plate (RP). Lateral views of three HH19-21 undissected embryos, 2 days
after implantation in the lateral midbrain of DiI (A,A'), or FGF8/DiI
(B,B',C,C') -soaked beads as schematized on the left. The same
embryos photographed under rhodamine fluorescence optics in (A-C) were
processed for the detection of Gdf7 (A',B') or
Wnt1 (C') transcripts (purple). (A,A') Control embryo; a
patch of fluorescent cells surrounds the DiI-labeled bead fragment (A).
Midline Gdf7 (or Wnt1, not shown) expression is not
perturbed (A'). In embryos that have received a FGF8/DiI bead fragment,
a row of DiI-labeled cells (B,C) migrated from the FGF8-induced cerebellum in
the direction of the host midline. These cells marked the position of ectopic
RP structures labeled with Gdf7 (B') or Wnt1
(C'). The ectopic RP (arrowheads) either remained confined to the
vicinity of the bead (B') or reached the endogenous midline (C').
The position of the bead is indicated with a red circle.
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Discussion
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The dorsal part of the MHB junction produces two major sensory structures,
the optic tectum and the cerebellum. The development of a smoothly polarized
optic tectum structure is a prerequisite to the establishment of ordered
retinotectal projections and to the accurate representation of the visual
field. It has been shown that signals derived from the IO are necessary in
chick and zebrafish (Nakamura,
2001
; Picker et al.,
1999
) for the development of anteroposterior polarity in the optic
tectum. We show that the IO also governs the development and positioning of
the RP, a structure involved in dorsoventral signaling.
Involvement of the IO in dorsoventral patterning
It is generally believed that when the neural tube forms by fusion of the
dorsal tips of the neural folds, the RP and neural crest are generated at its
dorsal midline under the influence of TGFß-related signals of the BMP
family produced by the epidermal ectoderm
(Liem et al., 1995
). After
neural tube closure, neural crest cells emigrate from the dorsal neural tube.
At the same time, BMP expression is lost from the epidermal ectoderm and
induced in the RP converting the initially lateromedial TGFß signals into
dorsoventral signals. Our observations indicate that, while dorsal signals may
be required to establish the competence to form RP structures, precise
positioning of the MHB RP depends on signaling from the IO. Indeed, we have
observed that expression of members of the BMP/GDF family is downregulated at
the MHB junction (Louvi et al.,
2003
).
Secondary organizers and neural tube axis formation
We describe here an IO-dependent positioning and development of the RP that
resembles, in many respects, the control of floor plate development
(Catala et al., 1996
;
Charrier et al., 1999
;
Placzek et al., 1993
) by the
regressing Hensen's node (Fig.
6). Both the IO and Hensen's node behave as neural organizers and
induce convergent extension cell movements. They both initiate the formation
of the neural tube midline signaling structures by directly contributing cells
to them. The identity of both ventral and dorsal midline structures is
propagated and reinforced by homeogenetic mechanisms
(Placzek et al., 1993
). A
major difference between the two organizers is that the Hensen's node actively
moves posteriorly whereas the IO seems to remain still. We suggest that the IO
node is immobilized by the opposing forces that result from the progression of
two divergent axial structures. This is consistent with the observation that
in En1Otx2 mutants the midbrain roof plate extends more
caudally in the absence of a cerebellar roof plate
(Louvi et al., 2003
). The
formation of the beak protrusion could also result from these constraints
exerted on the central neuroepithelium of the IO node.

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Fig. 6. The immobile IO node and the regressing Hensen's node. Schematic drawing
comparing the organizing properties of the IO node (left, same colour code as
in Fig. 2G) and the regressing
Hensen's node [right, the dotted area represents the neural plate; modified
from Charrier et al. (Charrier et al.,
1999 )]. The IO and Hensen's node organize similar convergent
extension cell movements and produce cells destined for axial structures. In
both cases, the differentiation of the definitive midline neural structures
depends on homeogenetic mechanisms (curved arrows). The stability of the IO
node contrasts with the active caudalward movement of the regressing Hensen's
node that leads to extension of the neural tube. The opposing centrifugal
migrations arising from the IO node may explain why it remains still. Black
arrows point to the direction of cell movements.
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Positioning the optic tectum symmetry axis
Why rely on the IO rather than on ectoderm-derived signals to position the
MHB RP? It may be argued that maintaining the symmetry in size and structure
of the two optic tecta before the establishment of the retinotectal connection
is of particular physiological importance to prevent asymmetry in the
representation of the visual world. Visual lateralization exists in birds. It
relies on asymmetric visual signals that depend on the prehatch position of
the embryo so that the right eye is exposed to light through the translucent
shell (Vallortigara et al.,
2001
). This asymmetry is part of the functional adaptation of the
brain to the visual environment and does not preclude the basic need for
isotropy in the visual representation. Two kinds of developmental mechanisms,
those involved in symmetry breaking and those controlling midbrain growth
could interfere with the development of left-right symmetric midbrain
structures. Several signals involved in symmetry breaking are operating at
successive developmental stages around the MHB domain. FGF8
(Boettger et al., 1999
) and
FGF18 (Ohuchi et al., 2000
),
two FGF family members, as well as SHH
(Levin et al., 1995
;
Pagan-Westphal and Tabin,
1998
) are expressed asymmetrically in the Hensen's node. FGF8
(Crossley et al., 1996
;
Joyner et al., 2000
;
Martinez et al., 1999
; Rhin
and Brand, 2001; Simeone,
2000
; Wurst and Bally-Cuif,
2001
) and SHH (Zhang et al.,
2000
) are known to affect later stages of MHB development and
their asymmetric expression could lateralize the early MHB. Also, the anterior
midbrain abuts the dorsal diencephalon that expresses clear morphological
asymmetries affecting in particular the habenula and the pineal organ in
zebrafish (Concha et al.,
2000
). The development of these morphological asymmetries is
preceded by the asymmetric expression of several genes in the left and right
dorsal diencephalon (Concha et al.,
2000
). Asymmetric signaling in the prosencephalon could indirectly
affect midbrain development. Finally, compared to the neighboring structures,
the MHB grows enormously. Even a transient increase in FGF8 protein has been
shown to produce long lasting growth stimulation in the mesencephalic
neuroepithelium (Shamim et al.,
1999
). Thus, any difference in FGF8 signaling between the left and
right IO would result in marked asymmetries in the mature optic tecta, had the
two midbrain halves been delimited before the IO is fully active. Postponing
the positioning of the roof plate and linking it to the source of growth
signals may aid to circumvent the consequences on dorsal midbrain development
of the symmetry-breaking processes.
 |
ACKNOWLEDGMENTS
|
---|
We thank R. Goiame for technical assistance, K.J. Lee for the chGdf7 probe,
the DSHB for the QCPN antibody. We acknowledge L. Bally-Cuif, B. Barbour, M.
Cohen-Tannoudji, A. Pierani and F. Rosa for helpful comments on the
manuscript. P.A. was a fellow of the Fundação para a
Ciência e a Tecnologia (Portugal). This work was supported by CNRS/ENS
and ACI-Développement.
 |
Footnotes
|
---|
This paper is dedicated to Cuca Alvarado-Mallart who retired recently
 |
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