1 Department of Developmental Biology, Max-Planck Institute of Immunobiology,
Freiburg, Germany
2 Instituto Gulbenkian de Ciência, Oeiras, Portugal
3 Research Institute of Molecular Pathology, Vienna, Austria
4 Laboratoire de Génetique Moléculaire, Institut de Recherches
Cliniques de Montréal, Montréal, Canada
* Author for correspondence (e-mail: mallo{at}igc.gulbenkian.pt)
Accepted 16 April 2003
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SUMMARY |
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Key words: Hoxa2, Ptx1, Hox genes, Branchial arches, Mesenchymal patterning, Fgf signaling
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INTRODUCTION |
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Hoxa2 has unique features that make it a good model to address
this issue. First, it the only Hox gene involved in segmental specification of
the second branchial arch (Gendron-Maguire
et al., 1993; Rijli et al.,
1993
; Barrow and Capecchi,
1999
). Second, the area phenotypically affected is mostly well
defined and coincides with one of the major expression domains of the gene
(Prince and Lumsden, 1994
;
Nonchev et al., 1996
;
Mallo, 1997
). Therefore, the
analysis of the role of Hoxa2 in second arch skeletogenesis is
technically more feasible and the knowledge gained can provide insights into
how other Hox genes control development in other body areas.
Hoxa2 is required for proper skeletal development in the
craniofacial area (Gendron-Maguire et al.,
1993; Rijli et al.,
1993
; Barrow and Capecchi,
1999
). In vertebrates, this area develops in a quasi-segmental
fashion. Development of the facial region can be considered to start with the
production of cranial neural crest cells from the dorsal aspect of the
developing brain (Le Douarin and Kalcheim,
1999
). Crest cells originating at particular levels along the
rostrocaudal axis migrate to populate specific areas of the frontonasal mass
and the branchial arches, the prospective face and neck
(Serbedzija et al., 1992
;
Köntges and Lumsden,
1996
). Increasing evidence indicates that signals from the
pharyngeal endoderm provide patterning information to postmigratory crest
cells (Couly et al., 2002
) and
that Hox genes negatively affect the ability of neural crest cells to
interpret these signals to form skeletal elements
(Kanzler et al., 1998
;
Couly et al., 2002
). However, a
primary role for neural crest cells in patterning processes has also been
suggested on the basis of interspecies grafting experiments
(Schneider and Helms, 2003
).
Whatever the precise mechanisms might be, it is clear that precise
coordination of these processes results in the formation of specific
structures from each of the prospective craniofacial areas. For example,
neural crest cells from the caudal midbrain and the first two rhombomeres (r)
populate the first branchial arch
(Serbedzija et al., 1992
;
Köntges and Lumsden,
1996
) to give rise to the mandible and part of the middle ear, in
particular the malleus, incus and tympanic ring
(Mallo, 1998
). Likewise, cells
migrating from r4 populate the second branchial arch to form the third middle
ear ossicle, the stapes, along with the styloid process and the lesser horn of
the hyoid bone (Mallo, 1998
).
Hoxa2 exerts its function in the latter region, this also being the
rostral limit of its expression in the developing face
(Prince and Lumsden, 1994
;
Nonchev et al., 1996
;
Mallo, 1997
).
In the absence of this gene, the second branchial arch develops abnormally,
giving rise to skeletal structures resembling those normally developing from
the first arch, but in a mirror image disposition with respect to their first
arch orthologs (Gendron-Maguire et al.,
1993; Rijli et al.,
1993
; Barrow and Capecchi,
1999
). Previous work from our laboratory indicated that
Hoxa2 defines skeletal second arch identity by negatively restricting
chondrogenic areas and blocking dermal ossification
(Kanzler et al., 1998
). By
contrast, other investigators suggest an active role for Hoxa2 as a
selector gene able to initiate a second arch specific program
(Grammatopoulos et al., 2000
;
Pasqualetti et al., 2000
). It
is then clear that, to be able to understand definitively which processes are
under Hoxa2 control and how this gene performs its job, it is
essential to identify the downstream targets of the Hoxa2 transcription factor
and to elucidate how they are regulated.
We have addressed this issue by using a subtraction approach and have identified Ptx1 (Pitx1 Mouse Genome Informatics) as one of the mediators of Hoxa2 functional activity. We further show that this gene is upregulated in the second branchial arch mesenchyme of Hoxa2 mutants. Moreover, in Hoxa2-/-;Ptx1-/- embryos, part of the Hoxa2 mutant phenotype is reverted to wild type, demonstrating that upregulation of Ptx1 is essential for the genesis of part of the Hoxa2 mutant phenotype. As Ptx1 expression is repressed by Hoxa2, the latter must interfere, directly or indirectly, with the Ptx1 activation process. Our results show that this activation depends on Fgf signaling and suggest that Hoxa2 interferes with this activity. We further find that Lhx6, another known Fgf8 target in the first branchial arch, is also upregulated in the second arch in the absence of Hoxa2, providing more evidence for a role for Hoxa2 in the modulation of Fgf signaling. The implications of these findings toward understanding patterning processes in the branchial arches and Hox gene activity in general are discussed.
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MATERIALS AND METHODS |
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Mutant and transgenic animals and embryos
The Hoxa2 (Gendron-Maguire et
al., 1993) and Ptx1
(Lanctôt et al., 1999
)
mutant strains have been described previously. The Fgf8;Foxg1-cre
mutant embryos were created by intercrosses of
Fgf8flox/flox females and
Fgf8+/
2,3;Foxg1cre/+
males (Meyers et al., 1998
;
Hebert and McConnell, 2000
).
Msx2::Hoxa2 transgenics were generated by pronuclear injection as
described (Kanzler et al.,
1998
).
In vitro culture of branchial arch explants
First and second branchial arches were dissected out from early E9.5
embryos and incubated on top of isopore filters soaked on DMEM without sodium
bicarbonate, containing 20 mM HEPES, pH 7.2, 15% FCS, 50 units/ml penicillin
and 50 µg/ml streptomycin. The epithelia were removed from the mesenchymes
by controlled enzymatic treatment as previously described
(Mallo et al., 2000). The Fgfr
inhibitor SU5402 was applied at 7 mM or 13.5 mM (in DMSO) on AG 1-X2 beads
(BioRad) previously soaked in the inhibitor solution for 2 hours. Fgf8 was
applied at 1 mg/ml in heparin beads. When explants were made for
Hoxa2-/- arches, embryos were obtained from
Hoxa2+/- intercrosses, the branchial arches dissected out,
and the genotype of each embryo tested on the yolk sac as previously described
(Gendron-Maguire et al.,
1993
). One side of the embryo was incubated with the inhibitor and
the other was used as a control. All explants were incubated for 24 hours at
37°C in an atmosphere of 5% CO2/95% air. Then they were fixed
in 4% PFA and processed for in situ hybridization.
Molecular and phenotypic analyses
Whole-mount in situ hybridization was performed as previously described
(Kanzler et al., 1998), using
Hoxa2 (Mallo, 1997
),
Ptx1 (Lanctôt et al.,
1997
), Cbfa1 (Kanzler
et al., 1998
), Fgf8
(Crossley and Martin, 1995
),
Dlx2 (Bulfone et al.,
1993
) and Lhx6
(Tucker et al., 1999
). When
Msx2::Hoxa2 transgenic embryos were analyzed, E10.5 embryos
(transgenics and controls) were cut in half and each half hybridized with a
different probe (Hoxa2 or Ptx1) to allow direct comparison.
To section the specimens hybridized as whole mounts, the embryos were embedded
in gelatin/albumin and sectioned with a vibratome at 30 µm. Skeletal
phenotypes were analyzed by Alcian Blue/Alizarin Red staining as described
previously (Mallo and Brändlin,
1997
). Apoptosis was analyzed by TUNEL using the procedure
described in Kanzler et al. (Kanzler et
al., 2000
). For histological analyses, embryos were fixed in
Bouin's, dehydrated and embedded in paraffin. Sections (10 µm) were then
stained with Hematoxylin and Eosin.
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RESULTS |
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To determine whether Hoxa2 could also act dominantly in the first
arch mesenchyme to block Ptx1 expression, we expressed Hoxa2
in this mesenchyme in transgenic embryos, using the Msx2 promoter
(Kanzler et al., 1998). When
compared with wild-type littermates, Ptx1 expression was considerably
reduced in the first arches of transgenic embryos in the areas corresponding
to the ectopic domain of Hoxa2 expression
(Fig. 1G-J). This result shows
that as in the second arch, Hoxa2 is sufficient to downregulate
mesenchymal Ptx1 expression in the first arch, indicating that these
two arches are equally competent to express or downregulate Ptx1 in
the absence or presence of Hoxa2, respectively.
Hoxa2 blocks Fgf8-dependent Ptx1 induction
Hoxa2 blocks mesenchymal expression of Ptx1 in the
branchial arches, physiologically in the second arch but also in the first
when ectopically expressed there. This implies that Hoxa2 interferes
directly or indirectly with some activating mechanism. In the branchial area,
mesenchymal gene expression is often induced by interactions with the
epithelia (Thesleff et al.,
1995). If this is also the case for Ptx1, Hoxa2 could be
interfering with this activation mechanism.
To understand if mesenchymal Ptx1 induction requires interactions with the epithelia, we dissected out branchial arches before Ptx1 is expressed in the mesenchyme (E9.25 to E9.5) and incubated them in vitro with or without their epithelia. After 1 day, Ptx1 was detected in the first branchial arches that had been incubated with ectoderm (n=8) (Fig. 2B) but not in the first arches whose ectoderms were removed before culture (n=6) (Fig. 2A). These results indicate that initiation of Ptx1 expression in the branchial arch mesenchyme is dependent on epithelial signals. Understanding the nature of this inducing process could shed light into the mechanism of Hoxa2 action. Interestingly, the spatial Ptx1 expression in the intact explants resembled that observed in E10.5 wild-type embryos, being restricted to the central part of the first arch and with no detectable expression in the second arch. Hence, the control mechanisms for Ptx1 expression seem to be largely conserved under our culture conditions, suggesting that the in vitro system could be used to address specific aspects of the induction process.
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When we explanted and incubated E9.5 Hoxa2-/- first and second branchial arches, we found that Ptx1 was upregulated in both the first and second arches, thus reproducing the in vivo findings (n=3) (Fig. 2I). When these explants were incubated in the presence of SU5402, Ptx1 expression was completely abolished from the second arch and reduced in the first (n=3) (Fig. 2J). These results indicate that inhibition of Fgf signaling in the second arch of Hoxa2-/- mutants reverts Ptx1 expression to the wild-type (Hoxa2-expressing) situation. These findings, together with those obtained in the first branchial arch, assign Ptx1 under the control of Fgf signaling and suggest that modulation of this signaling pathway could be the mechanism of Ptx1 inhibition by Hoxa2.
Lhx6 and Fgf8 expression in second branchial arches of Hoxa2-/- embryos
The above results clearly show that Hoxa2 blocks Ptx1
expression in the branchial arch mesenchyme. This could be via a direct effect
on the Ptx1 promoter (blocking activity of a transcriptional
activator) or via an indirect effect, most likely by interfering with some
step of the Fgf signaling pathway. Despite extensive studies on the
Ptx1 promoter, we have so far been unable to obtain any evidence for
direct Hoxa2 control. Therefore, the indirect hypothesis seems to be
favored at the moment. If, indeed, Hoxa2 controls Ptx1
expression by modulation of Fgf signaling, the inhibitory effects of
Hoxa2 in the second arch should not be restricted to Ptx1
alone but might also be extended to other Fgf targets. Previous analysis on
Fgf8;Nes-cre and Fgf8;Foxg1-cre mutant embryos revealed that
Lhx6 expression in the first arch mesenchyme requires Fgf8
(Trumpp et al., 1999) (A.L.
and A.N., unpublished). Analysis of the gene chip data revealed that
Lhx6 was moderately upregulated (2.6-fold) in the second arches of
Hoxa2 mutants. Consistent with this finding, in situ analysis in
Hoxa2-/- embryos revealed that Lhx6 is indeed
upregulated in the rostroproximal mesenchyme of the mutant second arches
(Fig. 4A,B). Interestingly,
this domain is located just beneath an area of Fgf8 expression in the
rostral second arch epithelium (Fig.
4C) and corresponds to one of the areas of strong Hoxa2
expression in the second arch mesenchyme
(Fig. 4E). These results
clearly indicate that other genes under Fgf8 control are also upregulated in
the absence of Hoxa2 and further substantiate the role of
Hoxa2 in modulating Fgf signaling.
Hoxa2 repression of Fgf8 targets in the mesenchyme could occur by
modulating the Fgf signaling pathway in the Fgf target cells (i.e. the
mesenchyme) or by modulation of epithelial Fgf8 expression. To test
for the latter possibility, we compared Fgf8 expression in wild-type
and Hoxa2 mutant embryos. As mentioned above, Fgf8
expression can be detected in the second arch epithelium, in particular in
areas corresponding to the caudal second arch ectoderm and in the proximal
part of the first pharyngeal cleft (Fig.
4C). This expression pattern seemed unaffected by the presence or
absence of Hoxa2 (Fig.
4D). We cannot rule out the possibility that Hoxa2 might
affect expression of some other member of the large Fgf family. However, as
Fgf8 seems to be the physiological activator of Lhx6 and
Ptx1 (Trumpp et al.,
1999) (Fig. 6),
these results suggest that Hoxa2 blocks Fgf signaling by interfering
with the signaling pathway in mesenchymal (i.e. target) cells rather than
affecting Fgf expression itself.
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Partial rescue of the Hoxa2-/- phenotype in
Hoxa2-/- Ptx1-/- embryos
The above results indicate that in normal embryos Hoxa2 prevents
Ptx1 expression in the second arch mesenchyme. As the absence of
Hoxa2 leads to a skeletal phenotype in the second arch
(Gendron-Maguire et al., 1993;
Rijli et al., 1993
;
Barrow and Capecchi, 1999
) and
Ptx1 is involved in skeletal development
(Lanctôt et al., 1999
;
Szeto et al., 1999
), it is
possible that the Hoxa2 mutant phenotype is associated, totally or
partially, with the Ptx1 upregulation. To test if this is indeed the
case, we generated Hoxa2-/- mice in which Ptx1
upregulation could not occur (Hoxa2;Ptx1 double mutants).
Skeletal analysis of newborn double mutants revealed the presence of a single
tympanic ring (Fig. 5D,F)
instead of the two observed in Hoxa2-/- mice
(Fig. 5B). The gonial bone (a
part of the malleus that develops by dermal ossification), which is abnormally
extended in Hoxa2-/- embryos
(Fig. 5B), seemed to be
connected in Hoxa2-/-;Ptx1-/- embryos
to the extra dermal element associated with Meckel's cartilage observed in
both the Ptx1-/- and
Hoxa2-/-;Ptx1-/- embryos
(Fig. 5C,D). The rest of the
skeletal phenotype of these double mutants was an additive mix of
Hoxa2-/- and Ptx1-/- characteristics
(Figs 5,
6). For example, similar to
Hoxa2 single mutants
(Gendron-Maguire et al., 1993
;
Rijli et al., 1993
;
Barrow and Capecchi, 1999
), the
incus, malleus and squamous bone were clearly duplicated in the double mutant
(Fig. 5;
Fig. 6K,L), and the
basisphenoid presented the typical laterodorsal extension that connects with
the duplicated incus (Fig.
6C,D). Other typical Ptx1 mutant characteristics, such as
the reduced mandible or the proximal extra dermal element associated with
Meckel's cartilage (Lanctôt et al.,
1999
), also seemed unaffected by the Hoxa2 mutation
(Fig. 5C,D;
Fig. 6F,H), and the hindlimb
phenotype of Hoxa2-/-;Ptx1-/- embryos
resembled that of their Ptx1-/- littermates
(Lanctôt et al., 1999
;
Szeto et al., 1999
).
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Further histological analyses of Hoxa2-/-;Ptx1-/- embryos revealed the disappearance of another Hoxa2-/- phenotype in soft tissues. Specifically, the posterior part of the tongue does not show the medial cleft typically present in Hoxa2 single mutants (Fig. 7K,L). In addition, the styloglossus, which shows a medial trajectory in Hoxa2-/- mutants, runs more laterally in Hoxa2-/-;Ptx1-/- embryos, resembling its wild-type trajectory (Fig. 7J-L).
These results, taken together, demonstrate that the upregulation of Ptx1 observed in Hoxa2 single-mutant second branchial arches plays a role in the development of the Hoxa2 mutant phenotype. Thus, one of the functions of Hoxa2 in the second arch must be to block activation of Ptx1.
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DISCUSSION |
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Ptx1 in the Hoxa2-/- phenotype
Our results show that the phenotype of Hoxa2-/- embryos
is modified in the absence of Ptx1. Some of the Hoxa2 mutant
characteristics are lost and reverted to wild-type-like structures. The most
clearly rescued characteristics seem to involve soft tissues, most
particularly the EAM, which is not duplicated anymore, and the dorsal part of
the base of the tongue. This is very surprising, particularly in the case of
the EAM, because this structure derives from the first branchial cleft
ectoderm and upregulation of Ptx1 expression in the second arch is
restricted to the mesenchyme. A variety of embryological and genetic
experiments has previously indicated that invagination of the EAM is
associated to the development of the tympanic ring
(Mallo, 2001). Because in
Hoxa2-/-;Ptx1-/- embryos an ectopic
tympanic ring primordium is formed in the second arch, the absence of EAM
duplication is not simply due to the absence of a duplicated ring. One
possibility is that the interactions between the second arch ring primordium
and the ectoderm covering the second arch side of the cleft occurs differently
in the presence or the absence of Ptx1. In this scenario, considering
that the second arch ring of
Hoxa2-/-;Ptx1-/- embryos seem to be
able to interact with the EAM induced at the first arch side of the cleft, the
ectoderm of the cleft that covers the second arch should have specific
characteristics able to respond differently to Ptx1-expressing and
non-expressing mesenchyme.
Another possible explanation for the phenotypic rescue of EAM in Hoxa2-/-;Ptx1-/- double embryos is that the duplicated tympanic ring is formed in a location that favors interaction with the first arch side-derived EAM rather than with its second arch counterpart. This explanation would imply a role for Ptx1 in the spatial and/or temporal induction of the second arch tympanic ring. Consistent with this hypothesis, the second arch-derived rings seem to be located differently in Hoxa2-/- and Hoxa2-/-;Ptx1-/- embryos, and preliminary data from our laboratory indicate that the timing of tympanic ring induction is different in the presence and absence of Ptx1. A detailed comparison of the spatiotemporal development of the duplicated tympanic ring in Hoxa2-/- and Hoxa2-/-;Ptx1-/- embryos will be required to fully evaluate this possibility.
In Hoxa2-/-;Ptx1-/- embryos, the two ring primordia fuse to form a single tympanic ring, a phenomenon never observed in Hoxa2 single mutants. One possible explanation is that the two rings of Hoxa2-/- embryos have an intrinsic inability to fuse, which requires Ptx1 expression. Alternatively, this effect may be secondary to the presence or not of a duplicated EAM. The independence of the two rings in Hoxa2-/- embryos would derive from the independent growth of the two EAMs. In the case of Hoxa2-/-;Ptx1-/- embryos, if each ring primordia is associated with a different part of the same EAM, when the rings and EAMs complete their growth, the tips of the ring primordia, which are directed by the leading edge of the single EAM, will eventually reach each other and fuse. We regard this explanation as more probable because in Hoxa2-/-;Ptx1-/- embryos the two primordia seem to grow toward each other, whereas in Hoxa2-/- embryos they grow in a more parallel fashion.
Finally, the absence of medial cleft in the tongue of
Hoxa2-/-;Ptx1-/- embryos indicates
another role for Ptx1 in patterning of non-skeletal tissues. It has
been suggested that abnormal insertion and trajectory of the hyoglossus might
play a role in the abnormal tongue clefting in Hoxa2-/-
embryos (Ohnemus et al.,
2001). Consistent with this hypothesis, in
Hoxa2-/-;Ptx1-/- double mutants, this
muscle seems to have a more lateral trajectory than in Hoxa2 single
mutants. The hyoglossus is a second arch mesodermal derivative
(Carlson, 1999
), so it is
conceivable that abnormal upregulation of Ptx1 in the second arch
could negatively affect the behavior of the muscle precursors in this area.
Ptx1 expression in the second arch is expected to occur in neural
crest-derived mesenchyme (Hoxa2 is expressed in crest cells). In this
case, the neural crest cells would affect patterning/morphogenetic processes
in adjacent non-neural crest-derived tissues, similar to what has been
described in avian embryos (Köntges
and Lumsden, 1996
; Schneider
and Helms, 2003
). It should be noted, however, that abnormal
Ptx1 expression in the second arch muscle precursors of
Hoxa2-/- embryos cannot be ruled out from our in situ
data.
Hoxa2 and Fgf signaling
A very important finding from this paper is that Hoxa2 acts, at
least in part, by repressing genes that play a role in mesenchymal patterning.
This is clear for Ptx1, and preliminary results from our laboratory
indicate that this might also be the case for other genes involved in the
production of the duplicated endochondral structures (N.B., M.C. and M.M.,
unpublished). The repressive nature of this process implies the existence of a
Hoxa2-independent activating mechanism for those genes susceptible of
a Hoxa2-dependent block. The Hoxa2-dependent block can occur
by direct interaction with the promoter of the gene or by interference with
some upstream step in the inducing process. A combination of both is also
possible, as has been shown for other genes
(Guss et al., 2001). Despite
extensive efforts, we have so far been unable to find any evidence for a
direct interaction of Hoxa2 with the Ptx1 promoter. As these
are negative results, we cannot rule out the existence of such an interaction,
but, they lead us to favor the alternative hypothesis of Hoxa2
controlling Ptx1 (and Lhx6) expression by interference with
the inducing mechanism.
A variety of data indicates that Fgf signaling is a major component of the
inducing mechanism for both genes, making this signaling cascade a prime
candidate for the target of Hoxa2 activity. Both Ptx1 and
Lhx6 are induced beneath an Fgf8-expressing epithelium
(Lanctôt et al., 1997;
Tucker et al., 1999
;
St Amand et al., 2000
). In
addition, not only is the epithelium required for mesenchymal induction of
both genes, but this induction can also be mimicked by addition of Fgf-soaked
beads (Fig. 2D)
(Tucker et al., 1999
;
St Amand et al., 2000
).
Moreover, genetic evidence indicates the absolute requirement of Fgf8 for
mesenchymal induction of these two genes
(Fig. 3) (Trumpp et al., 1999
) (A.L.
and A.N., unpublished). Finally, activation of Ptx1 can be
specifically blocked by an inhibitor of Fgf receptors. As Fgf signaling seems
to be the common feature of Lhx6 and Ptx1 induction, it is
reasonable to hypothesize that Hoxa2 interferes with activation of
these genes by modulating the activity of these Fgf signals. Consistent with
this hypothesis, we have shown that the Fgfr inhibitor is also effective in
blocking the Ptx1 activation observed in Hoxa2 mutant second
arches. Moreover, activation of Lhx6 in the second arch of
Hoxa2-/- embryos occurs in an area of strong
Hoxa2 expression adjacent to a Fgf8 expression domain in the
rostral second arch epithelium.
Hoxa2 interference with Fgf signaling could explain the defects in
bone development observed in transgenic mice upon Hoxa2 expression in
the first arch and developing skull bones
(Kanzler et al., 1998). The
mandibular hypoplasia can result from either direct or indirect Hoxa2
activity on Ptx1 in the first arch, as this phenotype resembles, to a
large extent, that of Ptx1 mutants
(Lanctôt et al., 1999
;
Szeto et al., 1999
). However,
in the skull bones, where a direct interaction with Ptx1 or a similar
gene is very unlikely, Hoxa2 interference with Fgf signaling
represents a plausible explanation of the phenotype, especially considering
that Fgfs are required for bone development in this area
(Iseki et al., 1999
;
Sarkar et al., 2001
).
The key question is therefore how Hoxa2 interferes with Fgf
signaling. It is unlikely that this is achieved by control of the signal
itself, as Fgf8 expression is unaffected in
Hoxa2-/- embryos. Although other Fgfs could be affected,
it seems likely that, at least for Lhx6 and Ptx1, Fgf8 is
the main player. Other possibilities involve the components of the signal
transduction cascades (Boilly et al.,
2000), or other modulators, such as genes of the Spry or Sef
families (Niehrs and Meinhardt,
2002
). Experiments are currently in progress in our laboratory to
address this issue.
Interestingly, it has been reported that Fgf signaling can affect Hox gene
expression (Cho and De Robertis,
1990; Kolm and Sive,
1995
; Partanen et al.,
1998
; Trainor et al.,
2002
). In the branchial arches, exogenous Fgf sources are able to
downregulate Hoxa2 expression
(Trainor et al., 2002
). This
finding, together with our present and previous data, suggests a feedback
mechanism (Fig. 9) that could
play an important role in establishing the skeletogenic areas in the second
arch, which correspond to those areas of low Hoxa2 activity
(Kanzler et al., 1998
). When
postmigratory second arch crest cells are exposed to Fgf signals, those cells
with high Hoxa2 content will be refractory to these signals, whereas
those expressing Hoxa2 below a given level will be capable of some
response. The initial responses to Fgfs in those cells with low Hoxa2
contents will have a negative effect on Hoxa2 expression, which in
turn will increase their response to the Fgfs. This generates a feedback loop,
eventually resulting in cells with very low (or no) Hoxa2 expression
and high responsiveness to Fgf signals. If Hoxa2-expressing and
non-expressing cells are able to segregate from each other (M.M.,
unpublished), this feedback loop will eventually result in areas without
Hoxa2 and with high Fgf competence. As Fgfs promote skeletogenesis in
the branchial arch mesenchyme (Moore et
al., 2002
), it is reasonable to assume that these areas belong to
the skeletogenic-competent ones, in agreement with our previous results
(Kanzler et al., 1998
).
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This interpretation of Hoxa2 activity is in agreement with
previously published data from other laboratories and our own. Expression
studies have shown that during normal development both in mouse and chicken
embryos, Hoxa2 is excluded from skeletogenic areas
(Kanzler et al., 1998;
Grammatopoulos et al., 2000
).
Conversely, in the Hoxa2 mutant second arches, skeletal elements
develop within the Hoxa2 expression domain
(Kanzler et al., 1998
). These
results are consistent with the Hoxa2-expressing mesenchyme being
unable to generate skeletal elements and with the notion that the mutant
phenotype results from activation of skeletogenesis in normally silent areas.
Importantly, the inability of Hoxa2-expressing mesenchyme to give
rise to skeletal structures has also recently been reported in chicken embryos
after grafting prospective second arch crest into the first branchial arch
(Couly et al., 2002
). These
results argue against the ability of Hoxa2-expressing cells to
trigger an endogenous second arch-specific skeletogenic program and are
consistent with their being unable to respond to skeletogenic signals.
The results of Hoxa2 misexpression experiments in the first arch
of chicken and Xenopus embryos have suggested a dominant role of
Hoxa2 in producing second arch structures, with phenotypes that were
interpreted as homeotic transformations
(Grammatopoulos et al., 2000;
Pasqualetti et al., 2000
).
Conversely, we have shown that activation of Hoxa2 expression in the
first arch of mouse embryos, either in transgenic experiments or by induction
with retinoic acid, resulted in deletion of first arch structures without any
sign of posterior transformation (Mallo
and Brändlin, 1997
;
Kanzler et al., 1998
). The
discrepancies between these interpretations could be due to different criteria
being used to define the identity of skeletal elements. Alternatively, for
Xenopus embryos, the discrepancies might be attributed to differences
among species, because, in contrast to mouse or chicken, the Xenopus
Hoxa2 homolog seems to be expressed in skeletogenic regions
(Pasqualetti et al., 2000
).
For the chicken experiments, a similar explanation is not plausible as
Hoxa2-positive cells do not contribute to the skeleton when
transplanted to the first arch (Couly et
al., 2002
).
Implications for a common mechanism of Hox gene function
Hox genes play essential roles in determining segmental identities in
different parts of the vertebrate embryo, including the skeletal elements of
the paraxial mesoderm and the limbs
(Krumlauf, 1994;
Zakany and Duboule, 1999
;
Burke, 2000
). So far, there is
no clear picture of how Hox genes perform this task, but our findings suggest
an interesting explanation. It has been shown that several signaling pathways,
including those of Bmps, Fgfs and Hhs, are involved in patterning and
morphogenesis in somitic and limb mesoderm
(Pourquie et al., 1996
;
Oh and Li, 1997
;
Partanen et al., 1998
;
Murtaugh et al., 1999
;
Murtaugh et al., 2001
;
Pizette and Niswander, 2000
).
We propose that Hox genes define the competence of these mesenchymal cells to
respond to these signals. If each Hox gene has a specific effect on the
ability of the mesenchyme to respond (to permit or to block) to one or several
of these signaling pathways, and the different Hox genes are able to compete
with each other in such activities, a particular Hox combination would result
in a specific pattern of response to skeletogenic signals, eventually
generating a structure. In this context, the Hox code would be the readout of
the responses to these induction processes. Alterations in Hox gene expression
would result in altered responses, eventually resulting in abnormal
structures, and depending on the particular cases involved, these could be
scored as homeotic transformations.
Interestingly, mutations in Fgfr1 and Acvr2b have
produced skeletal phenotypes in the vertebrae and limbs similar to those
obtained from altered Hox gene expression
(Partanen et al., 1998;
Oh and Li, 1997
). As subtle
changes in the expression of some Hox genes were observed, it was suggested
that Hox gene expression was under the control of Fgf and Bmp signals, and
that the observed phenotypes were secondary to the alterations in Hox gene
expression. Based on our results, another (and not mutually exclusive)
explanation is possible. If Hox genes modulate Fgf and Bmp signaling,
deviations from the normal signaling activities mediated by particular
receptors would interfere with the normal readout of the Hox code, eventually
producing phenotypic changes. As Fgf activity can affect Hox gene expression
(Cho and De Robertis, 1990
;
Kolm and Sive, 1995
;
Partanen et al., 1998
;
Trainor et al., 2002
), the
abnormal Fgf or Bmp signaling could elicit alterations in expression of
specific Hox genes and start a feedback loop, similar to that outlined in
Fig. 9, that would eventually
potentiate and perpetuate the altered mesenchymal competence to the
signals.
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ACKNOWLEDGMENTS |
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