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First published online 3 July 2006
doi: 10.1242/dev.02460
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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 25 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Isthmic organizer, Mid/hindbrain organizer, Roof plate, FGF8, Follistatin, Activin, BMP7, GDF7, Chick, Quail
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It
constitutes a signaling center that influences dorsoventral patterning of the
neural tube, specification of dorsal neuronal types, and axonal guidance
across the dorsal midline (Liem et al.,
1997; Lee et al.,
1998). RP development has been best studied in the spinal cord,
where its specification relies on interactions between inductive signals of
the TGFß family produced by the adjacent epidermal ectoderm and intrinsic
homeodomain transcription factors (Liem et
al., 2000; Chizhikov and
Millen, 2004b; Chizhikov and
Millen, 2004c). Less is known about RP development in the anterior
brain, where modulations of intrinsic or extrinsic factors involved in neural
tube patterning along the AP axis could influence local RP differentiation
(Bach et al., 2003). In chick
embryos, the RP of the midbrain is highly plastic. Experimental perturbations
of the midbrain neuroepithelium that do not directly involve the dorsal
midline result in the disappearance of the RP at later stages or in its
reorientation or bifurcation (Marin and
Puelles, 1994; Bally-Cuif and
Wassef, 1994; Crossley et al.,
1996; Alexandre and Wassef,
2003).
Growth and patterning of the midbrain-hindbrain (MH) domain of the neural
tube depend on the activity of a signaling center called the isthmic organizer
(IsO), located at the constriction or isthmus that links the midbrain and
hindbrain. Fibroblast growth factor 8 (FGF8), a diffusible molecule secreted
by the IsO, is one of the major mediators of its organizing and
growth-inducing properties (Crossley et
al., 1996; Martinez et al.,
1999). Insertion of a FGF8-soaked bead in the MH domain or the
posterior forebrain induces the formation of a supernumerary IsO, the
development of an ectopic MH junction and also of an ectopic RP. The influence
of the IsO, whether endogenous or FGF8 bead induced, on the midbrain RP is
puzzling. It seems to promote RP elongation and maturation when an ectopic
source of FGF8 is inserted into the midbrain
(Bally-Cuif and Wassef, 1994;
Crossley et al., 1996) or to
impair its differentiation when rotation of the midbrain vesicle brings the
anterior RP closer to the IsO or when the isthmic node is ablated
(Marin and Puelles, 1994;
Alexandre and Wassef,
2003).
In the spinal cord, BMP signaling controls RP formation through the
induction of the competence factors Lmx1a and Lmx1b
(Chizhikov and Millen, 2004a;
Chizhikov and Millen, 2004b;
Chizhikov and Millen, 2004c).
Wnt1 has also been implicated in later aspects of RP differentiation
(Shimamura et al., 1994;
Amoyel et al., 2005).
Lmx1b and Wnt1 are also targets of FGF8 signaling widely
expressed in the caudal midbrain. At later stages of development,
Lmx1b and Wnt1 expression becomes confined to the RP and
floor plate and to a ring of cells marking the MH boundary. A cue to the
unexplained behavior of the midbrain RP may therefore reside in
crossregulatory interactions between the BMP and FGF pathways on common
downstream genes essential for RP development. The aim of the present study
was to understand better these interactions. We find that FGF8 orchestrates
midbrain RP differentiation by finely adjusting BMP, GDF and activin signaling
in time and space.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Some embryos were also examined after long survival, mainly
4.5, 7 and 12 days after surgery. The methods for performing small ablations,
homotopic and heterotopic isochronic grafts
(Bally-Cuif and Wassef, 1994;
Alexandre and Wassef, 2003),
bead implantation (Martinez et al.,
1999), in situ hybridization, immunocytochemistry and cryostat
sections (Bally-Cuif and Wassef,
1994) were as previously described with minor modifications.
Rabbit anti-active caspase 3 (Promega) was diluted 1/250, mAb QCPN
(Developmental studies hybridoma bank) was diluted 1/10 and mouse anti-AFP
(Q-biogene) was diluted 1/2000.
Vectors
The following vectors were used for in situ hybridization: ChWnt1
and QWnt1 are species specific, although they weakly crossreact
(Bally-Cuif and Wassef, 1994);
Gdf7 (Lee et al.,
1998) (gift from K. Lee), Lmx1b
(Matsunaga et al., 2002) (gift
from N. Nakamura), follistatin
(Graham and Lumsden, 1996)
(gift from F. Giudicelli), noggin
(Hirsinger et al., 1997) (gift
from C. Freitas), Id3 (Kee and
Bronner-Fraser, 2001) (gift from M. Bronner-Fraser), and
ActA and ActB (Merino et
al., 1999) (gift from J. M. Hurle).
Beads
Heparin acrylic (Sigma) or Affigel Blue (BioRad) beads were soaked in a
drop of recombinant protein (R&D, 16-20 beads/µl, unless otherwise
specified) at the following concentrations: FGF8, 0.1, 0.2 and 0.4 mg/ml;
BMP7, 1 mg/ml; GDF7, 0.5 mg/ml; noggin, 1 mg/ml; follistatin, 0.5 and 1 mg/ml;
FGF8/GDF7, 0.2/0.5 mg/ml; activin A, 1 and 2.5 µg/ml). Formatederivatized
AG-1 X2 beads (BioRad) were used to deliver the FGFR1 inhibitor SU5402
(Promega, 4 mg/ml in DMSO); control beads were soaked in PBS or DMSO. The
beads were incubated overnight in a moist chamber at 4°C and rinsed three
times before insertion. When heparin acrylic beads soaked in PBS were inserted
in the caudalmost region of the midbrain, they sometimes induced a relocation
of the MH boundary or widened the Wnt1 expression domain, thus
mimicking the effect of an FGF8 bead. We interpreted this behavior as
resulting from a change in local FGF8 signaling resulting from the diffusion
of heparin, which is known to be a potent co-factor of FGF8, or from a
modification of the shape and time course of the gradient of FGF8, which may
be accumulated and released by the heparin bead. PBS-soaked heparin acrylic
beads had no noticeable effect elsewhere.
Explant culture
The heads of HH14-15 embryos were isolated from the rest of the body at the
level of the otic vesicles then separated into two halves by cutting the
dorsal and ventral midlines. The two halves remained together, one left
unperturbed as control. The caudal part of explants, including the isthmic
region, was ablated to remove the endogenous source of FGF8. FGF8 signaling
was also modulated through implantation of FGF8, PBS, SU5402 or DMSO beads in
the caudal midbrain. The explants were placed ventricular side down on
floating membranes (de Diego et al.,
2002) and cultured for 6 or 18 hours before fixation and
processing for in situ hybridization.
Electroporation
The chick Lmx1b expression vector [pMiw-Lmx1b
(Matsunaga et al., 2002); a
gift from K. Nakamura] was derived from the pMiwIII vector containing
regulatory sequences from the Rous sarcoma virus enhancer and chicken
ß-actin promoter. In ovo electroporation on stage HH10 chick embryos was
performed as described (Funahashi et al.,
1999). A GFP-GPI expression vector
(Keller et al., 2001) (gift of
D. Henrique) derived from pEGFP-N1 (Clontech) was co-electroporated with
pMiw-LMX1B to check for the efficiency of transfection.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Alexandre and Wassef,
2003) that FGF8 beads inserted in the midbrain may induce locally
a short RP segment. The hypothesis that FGF8 derived from the IsO may initiate
development of the endogenous RP is, however, difficult to reconcile with the
observation that expression on the midline of several BMP family members is
transiently downregulated at the level of the IsO
(Louvi et al., 2003). In the
midbrain, BMP signaling is still required after neural tube closure for RP
differentiation. Beads impregnated with the BMP inhibitor noggin inserted in
the dorsal midbrain of HH10 embryos prevent Gdf7 expression on the
midline and RP development (7/16, Fig.
2A-A''). However, even in the most affected embryos
(Fig. 2A',A''), a
short RP segment still develops at both ends of the midbrain. Thus other
factors may compensate for BMP downregulation at the anterior and posterior
poles of the midbrain. Local factors on the midline may also potentiate RP
marker induction by FGF8. In general, the ectopic RP segment induced by FGF8
links the bead to the dorsal midline (Fig.
2B). When the FGF8 induced RP is reduced to a short segment near
the bead (Alexandre and Wassef,
2003) (arrows in Fig.
2C), a small deflection is also induced opposite the bead on the
midline RP (arrowhead in Fig.
2C). This indicates that, even if dispensable, co-factors on the
midbrain dorsal midline potentiate the inductive activity of FGF8 on RP
differentiation. To identify genetic factors involved in midbrain RP
development, we examined another experimental situation where an ectopic RP is
induced.
Acquisition of RP competence by ectopic transplants
We examined grafts of naive dorsal midbrain neuroepithelium implanted
across the host midline. We have shown previously
(Alexandre and Wassef, 2003)
that ectopic RP segments were induced in these transplants as extensions of
the host RP. The induced RP formed a solid row of cells usually of uniform
width. RP induction could be initiated both anteriorly and posteriorly in the
graft (16/63, Fig.
3A,A',A''). In general, the large dorsal midbrain
transplants grafted perpendicular to the midline did not grow much
(Louvi et al., 2003). Most
transplants did not develop a RP structure. In these transplants,
Wnt1 expression was maintained or induced in a wide domain comprising
the whole graft (9/26, Fig.
3B,B') or a large part of the graft (11/26,
Fig.
3,C,C',D,D',D''). Lmx1b expression was
upregulated in the grafts (10/10). In the transplants, the domain of
Lmx1b expression was always included within the Wnt1 domain
(4/4, Fig. 3B). Gdf7
and Lmx1b expression coincided in most cases (4/6, not shown).
Sometimes (2/6) part of the Lmx1b domain contained only scattered
cells expressing Gdf7 (not shown). Induction of an ectopic RP in the
transplant was accompanied by a downregulation of this widespread expression
of Wnt1 and Lmx1b. Thus, ectopic transplantation stabilizes
the expression of the RP competence factors Lmx1b and Wnt1
which is normally transient in the lateral midbrain. These factors have been
shown essential for RP differentiation in the spinal cord
(Chizhikov and Millen, 2004a;
Chizhikov and Millen, 2004b;
Chizhikov and Millen,
2004c).
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; Chizhikov and Millen,
2004b; Chizhikov and Millen,
2004c). Both are targets of FGF8 and, in addition to their dorsal
(and ventral) midline expression, are expressed in a wide domain of the caudal
midbrain (Fig. 4A,B). We
wondered whether expression of Gdf7 could be induced by
Lmx1b on the anterior midbrain midline. Overexpression of
Lmx1b in the dorsal midbrain was obtained by electroporation.
Confirming the report of Matsunaga et al.
(Matsunaga et al., 2002),
Wnt1 but not Gdf7 was already strongly induced 10 hours
after Lmx1b electroporation (Fig.
4D,D',D''). Gdf7 was induced in a few
scattered cells 24 hours after Lmx1b electroporation in the midbrain
(Fig. 4D). High levels of
Gdf7 expression were not detected before 34 hours after
electroporation, when Gdf7 was expressed in large cell strands or
patches in the lateral midbrain (Fig.
4E,F). Interestingly, Gdf7 induction by Lmx1b
was less efficient in the caudal midbrain (arrowheads in
Fig. 4E,E'). Thus,
initiation of Gdf7 expression on the anterior midbrain midline could
be triggered by Lmx1b and Wnt1, which are both present much
earlier on the midline.
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), noggin
(Fig. 5A) and Id3
(Fig. 5B)] is delayed in the
caudal midbrain. Insertion of a FGF8 bead dorsally in the caudal midbrain
prolonged this transient downregulation of Gdf7 expression
(Fig. 5C-E). This suggests
that, in situ, FGF8 not only promotes RP formation but also inhibits its
maturation. To test if this dual influence could also be detected during the
process of ectopic RP induction by FGF8 beads, we examined the expression of
early (Wnt1) and late (Gdf7) RP markers 24 and 30 hours
after FGF8 bead insertion. One day after bead implantation, a row of cells
prefiguring the ectopic RP expressed Wnt1 along its entire length (in
red in Fig. 5F,F')
bridging the midline to the FGF8 bead. Gdf7 expression was intense
near the midline but became fainter more distally (in purple in
Fig. 5F). In slightly older
embryos (Fig. 5G), both
Wnt1 and Gdf7 expressions in the induced RP reached the
bead. Thus, maturation of the induced and endogenous RP was similarly delayed
near the source of FGF8, suggesting that FGF8, in situ, regulates the caudal
progression of RP differentiation. As mentioned above, Gdf7 induction
by ectopic electroporated Lmx1b was less efficient in the caudal
midbrain than elsewhere, which also suggests the existence of an
isthmus-derived inhibitory influence that prevents or delays Gdf7
induction by Lmx1b.
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Regulation of roof plate differentiation by BMP signaling
Because BMP signals are involved at successive stages of RP development, we
wondered if a BMP family member could mediate this RP homeogenetic signal.
Insertion of BMP7-soaked beads at stage HH9-10 affected the medial domain of the RP but did not induce Gdf7 (Fig. 6A) or Wnt1 (Fig. 6B,B') locally, indicating that BMP7 is not directly involved in the homeogenetic behavior of the RP. The thin sheet of cells that develops between the two RP halves resembled the neural component of the choroid plexus but it did not express choroid plexus markers such as transthyretin (TTR, Fig. 6B,B') or BMP5 (not shown). Downregulation of BMP signaling through the insertion of a noggin-soaked bead locally impaired RP differentiation (Fig. 6C,C'), but did not result in the formation of a choroid plexus.
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).
We asked whether FGF8 could widen the competence domain for GDF7
auto-activation. Gdf7 expression was widely induced, though at a low
level, by beads soaked in a mixture of FGF8 and GDF7, and the refinement of RP
differentiation into a compact linear structure was prevented
(Fig. 6G,H). Overexpression of
FGF8 and GDF7 together also induced a large number of activated caspase
3-immunoreactive cells (7/7; Fig.
6I), whereas beads soaked in GDF7 (1/5;
Fig. 6F) or FGF8 (not shown)
did not induce an increase in cell death. Thus, FGF8 signaling, by inducing
cell death, precludes a rapid progression of the front of
Gdf7-expressing cells, therefore stabilizing it [see the pattern of
cell death in the caudal midbrain in Alexandre and Wassef
(Alexandre and Wassef, 2003);
Fig. 3].
Increase in activin signaling interferes with RP patterning and maintenance
Activins belong to the activin/nodal/TGFß subgroup of the TGFß
superfamily. At the difference of BMPs, which signal through SMADs1/5/8, the
members of the activin subgroup signal through SMAD2/SMAD3. Two observations
point to a possible function of activins in dorsal midbrain development.
First, the neural folds of the MH domain express high levels of active SMAD2
in E8.5-E9.5 mouse embryos (de Sousa Lopes
et al., 2003). Second, follistatin a high-affinity activin
inhibitor, is expressed in a dynamic pattern in the MH domain (see below). We
first examined if the pattern of expression of activins between HH9 and HH18
is consistent with a possible function in dorsal midbrain patterning and RP
differentiation. Although activin A expression was not detected above
background levels, we found that activin B is expressed in the
midbrain-forebrain domain with a progressive anterior shift in its domain of
expression. Activin B is first expressed at low level throughout the midbrain
vesicle at HH10 (Fig. 7A). Its
expression increases and becomes restricted to the mid-forebrain junction at
stage HH12-13 (Fig. 7B). By
stage HH18, activin B expression becomes confined to the pretectum
(Fig. 7C). Overexpression of
activin through the insertion of activin A-soaked beads induced a local
increase in Lmx1b expression 7 and 24 hours later
(Fig. 7D,E). Although clear,
the induction of Lmx1b expression by activin does not compare with
its widespread and extremely rapid (less than 3 hours) upregulation by FGF8
(Adams et al., 2000). Two
apparently opposite modifications of the RP were observed 2 days after activin
bead insertion. In about half of the cases (8/18,
Fig. 7F,H,H'), a thin RP
bifurcation was induced between the bead and dorsal midline. In 3/18 embryos,
the expression of RP markers was destabilized in the caudal midbrain
(Fig. 7G). Both phenotypes
could be observed in the same embryo (Fig.
7H). At later stages (E7.5, 6 days after bead insertion), most
embryos lacked a RP in the posterior midbrain and the locally induced RP could
no longer be detected (not shown). Thus, activin could be a major player in
midbrain RP lability, because increasing activin signaling positively
modulates RP extension but also results in its destabilization.
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), and
inhibits their binding to activin receptors. At HH11, a small region at the
midbrain forebrain junction is delineated by two follistatin domains
(Fig. 7I), flanking an intense
midline spot of Gdf7 expression
(Fig. 7J). Follistatin
expression progresses posteriorly in a decreasing gradient between stages HH11
and HH18 (Fig. 7K).
Overexpression of follistatin at HH9-11 did not impair RP differentiation.
Affigel Blue beads soaked in follistatin and inserted on the midline slightly
widened the Gdf7 expression domain (8/24;
Fig. 7L). This modest effect of
follistatin overexpression contrasts with that of noggin beads and indicates
that follistatin has little influence on BMP signaling in the midbrain. The
anterior high/posterior low gradient of expression of follistatin is
suggestive of a negative modulation by FGF8. In order to test this, FGF8
signaling in the midbrain was modulated in several ways. When FGF8 beads were
inserted in the midbrain at stage HH10, the caudal progression of follistatin
expression was prevented around the beads
(Fig. 7M). At later survival
times, follistatin expression was, however, normally turned on caudally beyond
the circular range of action of the FGF8 bead (not shown). Downregulation of
FGF8 signaling was obtained through the use of SU5402, a FGF signaling
inhibitor. Follistatin expression was increased on the side of SU5402-soaked
beads inserted in vivo at stage HH10 (Fig.
7N). The downregulation of follistatin expression around the FGF8
beads could reflect the transformation of the midbrain neuroepithelium into
cerebellum and isthmus. In order to manipulate FGF8 signaling in older
embryos, we used MH explants. The MH region of the neural tube of stage
HH14-15 embryos was cut on the dorsal and ventral midlines, the two halves
were cultured side by side on floating membranes, one serving as control.
Follistatin expression was increased compared with the control side 6 hours
after ablation of rhombomere 1, which suppressed the posterior source of FGF8
(Fig. 7O,P). Insertion of beads
soaked in SU5402 or DMSO in the posterior midbrain of explants gave similar
results as in vivo, although less consistent. Thus, follistatin expression
responds rapidly to variations in FGF8 signaling, allowing it to control
activin signaling indirectly.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) a patterning mechanism depending on the IsO and
mimicked by an FGF8 source that locally induces the formation of a short RP
segment. However, a posterior trigger for midbrain RP differentiation was not
easy to reconcile with the initial anterior expression of markers of RP
maturation. In addition, the long-range RP duplications induced by FGF8 beads
or IsO grafts or the failure of RP differentiation observed after 180°
rotation of the midbrain vesicle remained unexplained. The aim of the present
study was to characterize new regulations involved in midbrain RP formation
that could relate to its plasticity. We show here that activin dynamically
expressed at the midbrain-forebrain junction acts as a potent modulator of RP
differentiation.
FGF8 influence on RP differentiation: taking precedence over the BMPs
RP differentiation (Liem et al.,
1995; Liem et al.,
1997; Furuta et al.,
1997; Bach et al.,
2003) (present work noggin bead treatment) and the dorsal midline
expression of Lmx1b (Chizhikov
and Millen, 2004b; Liu et al.,
2004) generally depend on BMP signals. However, FGF8 controls the
expression of genes such as Lmx1b and Wnt1, which act as
competence factors and general markers of the RP. Changes in FGF8 signaling
rapidly up- or downregulate Lmx1b expression in the caudal midbrain.
In chick, we confirmed the observation of Adams et al.
(Adams et al., 2000) that
FGF8-soaked beads broadly increased Lmx1b in less than 3 hours.
Conversely, in zebrafish, a 4 hour treatment with SU5402 completely abolished
expression of Lmx1b in the caudal midbrain including the dorsal
midline (O'Hara et al., 2005).
Midline expression of Lmx1b was not affected in the anterior
midbrain. Thus, on the caudal midbrain midline, FGF8 is the major regulator of
Lmx1b expression which, in addition, is not maintained by BMP
signaling. This is consistent with the observation that the level of
expression of several ligands and targets of the BMP pathway is transiently
downregulated on the caudal midbrain midline
(Louvi et al., 2003) (this
work). Taken together, these observations indicate that expression of
Lmx1b in the caudal midbrain is under the control of FGF8, which
locally abolishes the neural folds competence for RP differentiation. Thus,
similar to the action of ectopic FGF8 beads, endogenous FGF8 may direct the
differentiation of the caudal midbrain RP at the center of the field it
organizes, independently of the site of early neural tube closure. It is
interesting to note that the same behavior, pattern reorganization around the
bead and RP duplication, is also induced by FGF8 beads inserted into the
forebrain (Crossley et al.,
2001). This strengthens the idea that regulatory mechanisms
involved in FGF8 patterning may be co-opted for axis formation.
|
). FGF8
induces a battery of negative regulators (reviewed by
Thisse and Thisse, 2005) that
could slow down the normal process of differentiation in the RP, as it does in
the adjacent neuroepithelium. The complex behavior of the midbrain RP suggests
that midbrain-specific targets of FGF8, such as E1/E2, which are
known to be potent inhibitors, or Pax2, which acts as activator or
inhibitor depending on cellular context, could preferentially affect specific
steps of RP maturation. First expressed independently of FGF8, Pax2
is later regulated by FGF8 signaling
(Crossley et al., 1996) and it
is required for Lmx1b induction by FGF8
(O'Hara et al., 2005). Pax2 is
known to interact directly with LMX1B
(Marini et al., 2005), which
could explain why induction of Gdf7 by Lmx1b is inhibited in
the caudal midbrain.
Extending RP differentiation in the caudal midbrain
In several systems (Sun et al.,
2002; Dubrulle et al.,
2001; Delfini et al.,
2005; Akai et al.,
2005; Mathis et al.,
2001; Diez del Corral et al.,
2002; Delfino-Machin et al.,
2005), a local pool of undifferentiated progenitors is maintained
by high levels of FGF signaling, while the establishment of the polarity of
the neuroepithelium and the progression of cell differentiation are
synchronized by the decreasing gradient in FGF signaling
(Lee et al., 1997). RP
differentiation seems to be linked to the establishment or maintenance of
planar cell polarity in the midbrain. Convergent extension movements induced
by FGF8 are observed at the midline
(Millet et al., 1996;
Louvi et al., 2003;
Alexandre and Wassef, 2003).
They may be important to prevent discontinuities between the Lmx1b
expression domains controlled by FGF8 and BMPs. Convergent extension also
repels the Gdf7-expressing cells beyond the influence of high FGF8
signaling, thus rescuing them from death. It is interesting to note that
discontinuities in midbrain polarity induced by neuroepithelium rotation are
rapidly regulated in the isthmic region
(Martinez and Alvarado-Mallart,
1990), but that elsewhere the transplants tend to adopt or
maintain the signature of the isthmic midbrain [Lmx1b and
Wnt1, this work; Pax2
(Vieira et al., 2006)], which
is considered to be less differentiated, and thus become competent to develop
a RP (see Fig. 8C). However,
signals from the host RP, possibly mediated by GDF7, are essential to
initiating the formation of an ectopic RP.
Follistatin and activin function in the dorsal midbrain
Follistatin is often considered to be an inhibitor of BMP signaling, but it
plays distinct roles, depending on the context, and interacts with members of
several groups of TGFß family ligands, including BMPs, GDFs and activins.
Interestingly, we find that BMPs, GDF and activin ligands all play distinct
roles in midbrain RP development. BMPs are involved as RP competence factors,
GDF7 in auto-activation of RP differentiation, and activin B in RP expansion
and stabilization. Muscle development is controlled by BMP7 and myostatin,
both of which are modulated by follistatin, but in different ways. Follistatin
binds BMP7 reversibly with low affinity. It converts the muscle
growth-inhibiting effect of BMP7 into a strong stimulatory one that is blocked
by noggin. Therefore, as follistatin does not prevent BMP7 binding to its
receptor (Iemura et al.,
1998), it has been suggested that follistatin influences BMP7
binding to its receptors. Follistatin binding to myostatin/GDF8 seems to
completely prevent receptor activation
(Amthor et al., 2002;
Amthor et al., 2004). Because
follistatin binds TGFß ligands with distinct affinities and
differentially affects interaction with their receptors, its progressive
expansion across the midbrain may be important to modulate their function.
Follistatin binds to activin with a much higher affinity than to other
TGFß ligands. The dynamic expression patterns of follistatin and activin
suggest that they tightly regulate each other's availability in the midbrain.
It remains unclear if the destabilization of the posterior midbrain RP that we
observe after insertion of activin beads relates to an increase in activin
signaling or whether sequestration by activin impairs other functions of
follistatin.
Conclusion
Similar mechanisms are involved in RP differentiation in the anterior
midbrain and in the spinal cord. FGF8 signaling prevents their normal
deployment in the caudal midbrain through its modulation of the function of
several members of the TGFß superfamily. The need for a progressive
transition between these two modes of regulation may increase the
vulnerability of RP differentiation to experimental manipulation.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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