Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, UMR 7104, BP 10142, CU de Strasbourg, 67404 Illkirch Cedex, France
Author for correspondence (e-mail:
rijli{at}igbmc.u-strasbg.fr)
Accepted 8 September 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hox, Cranial neural crest plasticity, Craniofacial development, Mouse, CreERT2, Middle ear, Gonial bone, Skeletal derivatives, Conditional knockout
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The way each subpopulation of cranial NCCs acquires the competence to
generate distinctive cartilaginous structures and bones represents a
fascinating matter of investigation that has inspired seminal experiments and
a fervent discussion over the past few decades. By means of chick-to-quail
transplants of pre-migratory NCCs, Noden observed that grafting of presumptive
first branchial arch NCCs to replace presumptive second arch NCCs resulted in
the mirror duplication of first arch skeletal elements in the place of second
arch structures (Noden, 1983).
This result suggested that NCCs were intrinsically endowed with the
potentiality to form specific structures, or pre-patterned, according to their
rostrocaudal origin in the neural tube. However, recent work has shown that
the surrounding environment provides instructive signals to NCCs in order to
trigger their differentiation process, as well as to achieve the correct
shaping, positioning and orientation of the developing skeletal structures
(Couly et al., 2002
;
Crump et al., 2004
;
Ruhin et al., 2003
;
Trainor et al., 2002
). In
turn, the NCCs initiate morphogenesis following an intrinsic,
species-specific, patterning program that may involve feedback signalling to
epithelia (Eames and Schneider,
2005
; Ferguson et al.,
2000
; Schneider and Helms,
2003
; Shigetani et al.,
2002
; Tucker and Lumsden,
2004
). Thus, craniofacial morphogenesis appears to be the result
of reciprocal signalling between neural crest mesenchyme and the surrounding
environment, where timing is an essential component (reviewed by
Le Douarin et al., 2004
;
Santagati and Rijli, 2003
).
Although a significant advance has been provided by the identification of
molecules influencing cranial NCC patterning ability (reviewed by
Helms et al., 2005
), little is
known about the temporal span during which cranial NCCs can generate an
appropriate pattern in response to extrinsic stimuli. Another aspect that is
poorly understood is how positional information along the anteroposterior axis
of distinct subpopulations of NCCs impinges on the patterning programs to give
rise to specific subsets of skeletal elements. These issues are related to the
spatiotemporal plasticity of the NCCs during craniofacial development.
An important molecular distinction of cranial NCCs populations along the
anteroposterior axis concerns their patterns of Hox gene expression. The NCCs
contributing to skull bones, frontonasal and first arch-derived jaw structures
do not express Hox genes (Couly et al.,
1998) (reviewed by Shigetani
et al., 2005
). By contrast, NCCs contributing to hyoid and throat
skeletal elements derived from the second and more posterior branchial arches,
respectively, do express various combinations of Hox genes
(Hunt et al., 1991
). The
involvement of Hox genes in providing rostrocaudal patterning information to
branchial arch derivatives first became evident with the targeted inactivation
of Hoxa2 in the mouse
(Gendron-Maguire et al., 1993
;
Rijli et al., 1993
). As a
result of Hoxa2 functional depletion, the second arch skeletal
elements were homeotically transformed in a duplicated set of first arch-like
elements with reverse polarity
(Gendron-Maguire et al., 1993
;
Rijli et al., 1993
),
strikingly phenocopying the outcome of Noden's heterotopic grafts in the
chick. This result indicated that Hoxa2 acts as a key genetic switch,
enabling the patterning of second arch instead of first arch derivatives.
Hoxa2 gain-of-function experiments in the first branchial arch of
chick, Xenopus and zebrafish embryos yielded a reverse outcome, i.e.
second arch-like structures developed in the place of first arch elements,
demonstrating a conserved role of Hoxa2 as a selector of second arch
patterning information in vertebrates
(Grammatopoulos et al., 2000
;
Hunter and Prince, 2002
;
Pasqualetti et al., 2000
).
Given the unique role of Hoxa2, its mutagenesis in the mouse represents a suitable mammalian genetic model with which to address unsolved questions about the spatiotemporal control of cranial NCC patterning. Here, we set up a conditional mutagenesis system in the mouse, and observed the effects of Hoxa2 deletion in a tissue- and time-dependent manner. Our analysis yielded several novel insights. First, we found that Hoxa2 is selectively required in second arch NCCs. Removal of Hoxa2 in NCCs phenocopied the full knockout transformation of second into first arch morphology. Second, by using a Cre/loxP-based system, we temporally induced Hoxa2 deletion and showed that Hoxa2 function at pre-migratory stages does not provide NCCs with irreversible information for patterning second arch derivatives. Rather, instruction about shape, size and orientation of second arch skeletal elements is provided after NCC migration. However, the execution of the NCC patterning program is strictly Hoxa2 dependent. In fact, homeotic changes can still be obtained upon Hoxa2 inactivation well after NCCs reached their final destination in the second arch. These data illustrate for the first time that second arch NCCs not only retain a degree of plasticity over a remarkably long period, but also that they require the expression of Hoxa2 as an irreplaceable and integral component of their intrinsic patterning program. Our data also suggest that Hoxa2 may directly regulate the spatial expression of a number of target genes involved in the morphogenetic process. Finally, we found that Hoxa2 function is required at separate time points to pattern distinct second arch derivatives. Altogether, this study provides the first temporal analysis of Hox gene function in a vertebrate embryo and illustrates a Hox requirement during late morphogenetic processes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Founder animals were identified by PCR amplification with Cre-specific
primers and bred onto a C57BL/6 genetic background. Offspring carrying the
Cre-ERT2 transgene were genotyped by PCR. Nine distinct founder
transgenic lines were characterized for integration of the transgene by
Southern blot hybridization and for ubiquitous expression of the
Cre-ERT2 mRNA by in situ hybridization. The functionality of the
Cre-ERT2 fusion protein upon tamoxifen induction was ascertained by crossing
it with a ROSA 26R (Soriano,
1999) reporter line. Embryos collected as early as 24 hours after
treatment carrying both the ROSA 26R reporter allele and the Cre-ERT2
transgene stained ubiquitously upon X-gal exposure (data not shown).
Conversely, no staining was observed on control untreated embryos from the
same intercross (not shown). Three transgenic lines (F34, F85 and F105a) were
selected after this screening. The data presented in this work were primarily
obtained using the F34 line, although the F85 line was used for confirmation
purposes when needed. No noticeable differences in the outcomes were observed
using the two lines.
Mating scheme
The Cre-ERT2 and the Wnt1-Cre
(Danielian et al., 1998)
transgenic lines were crossed with either the
Hoxa2EGFPfloxNeo knock-in
(Pasqualetti et al., 2002
) or
the Hoxa2flox conditional alleles
(Ren et al., 2002
) for the
different experimental purposes. To obtain Hoxa2 conditional
homozygous mutant mice, a first breeding round was accomplished to generate
compound Hoxa2flox/+;Cre-ERT2 or
Hoxa2flox/+;Wnt1-Cre mice, respectively. These animals
were in turn mated to Hoxa2flox/flox mice. Homozygous
mutant newborns were obtained with an expected Mendelian ratio. All genotypes
were determined by PCR.
Tamoxifen treatment
Tamoxifen (Sigma) was dissolved in pre-warmed corn oil (Sigma) to make a 20
mg/ml solution and stored at 4°C. Tamoxifen was administered orally to
pregnant females by means of a gavage syringe. Optimal doses for the different
developmental stages were experimentally determined as described in the
Results section. In particular, the amount of administered tamoxifen per
pregnant female (about 35-40 g of body weight) was estimated as follows: 5 mg
at 7.0 dpc, 6 mg at 8.0 dpc, 7 mg at 9.5 dpc, 8 mg at 10.0 dpc, 9 mg at 10.5
dpc and 10 mg at 11.0 dpc. To obtain maximal Cre-ERT2 recombination efficiency
at later embryonic stages, double (10 mg at 11.5 dpc and 12.0 dpc) and triple
(10 mg at 12.5 dpc, 13.0 dpc and 13.5 dpc) successive administrations (chronic
treatment) were provided with 12-hour intervals. For precise embryonic stage
estimation at the time of treatment, mated females were checked for vaginal
plugs every 2 hours. The time of a detected plug was considered as embryonic
day 0. Specimens were collected at the desired time after the last
treatment.
Skeletal preparation
Neonatal mice were skinned and eviscerated. Skeletons were fixed overnight
in 95% ethanol and then stained in Alcian Blue (750 µg/ml in 80 ml of 95%
ethanol, 20 ml of glacial acetic acid) for at least 24 hours. Skeletons were
cleared in 2% KOH for 8-10 hours, then 1% KOH overnight and then stained in
Alizarin Red (100 µg/ml in 1% KOH) overnight. Further clearance was
performed in 20% glycerol/1% KOH. Skeletons were stocked in 50% glycerol/50%
ethanol.
Immunohistochemistry
Immunostaining for EGFP on cryosections was performed using a polyclonal
rabbit anti-GFP antibody (Molecular Probes Europe BV) and a
peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Beckman Coulter
France SA). Detection was performed with DAB chromogen (DAKO SA).
In situ hybridisation
In situ hybridisation on whole-mount embryos and cryosections was performed
as previously described (Samad et al.,
2004). The following RNA probes were used: Hoxa2
(Ren et al., 2002
),
Alx4 (Qu et al.,
1997
), Bapx1 (Nkx3.2)
(Lettice et al., 2001
),
Msx1 (Robert et al.,
1989
), Six2 (Oliver
et al., 1995
).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thus, Hoxa2 is selectively required in second arch NCCs. Hoxa2 inactivation in NCCs is sufficient to yield the defects associated with middle and external ear development, while expression of Hoxa2 in adjacent tissues does not alleviate the overall mutant phenotype.
Temporally controlled deletion of the Hoxa2 locus
In order to carry out a time-course mutagenesis of the Hoxa2 gene,
we generated the transgenic mouse line
CMV-ßactin-Cre-ERT2 (Cre-ERT2) bearing a
tamoxifen (TM)-inducible form of Cre recombinase, Cre-ERT2
(Feil et al., 1997), driven by
the ubiquitous chicken ß-actin promoter. Transgenic lines expressing
Cre-ERT2 can be efficiently used to perform time-specific excision of
a target gene by a Cre/loxP-based conditional knockout system
(reviewed by Metzger et al.,
2005
). We tested the efficiency of the Cre-ERT2 recombinase in
mediating excision at the Hoxa2 locus by directly monitoring the
levels of residual Hoxa2 mRNA in TM-induced
Hoxa2flox/flox;Cre-ERT2 embryos. The doses and conditions
of TM treatment (see Materials and methods) were optimized for different
developmental time points on the basis of the maximal degree of
Cre-ERT2-induced loss of expression of Hoxa2, as detected by
whole-mount or tissue section in situ hybridisation. Tamoxifen was
administered to pregnant females at 9.5, 10.5 and 11.0 dpc (single gavage), as
well as at 11.5 or 12.5 dpc (chronic administration; see Materials and
methods), and embryos were collected 24 hours after the last TM
administration. We detected no or very little residual Hoxa2
expression as a result of Cre-ERT2 induction at any of the selected time
points (Fig. 2B,D,F; and data
not shown).
|
Distinct temporal requirements of Hoxa2 in the patterning of second arch structures
In the mouse, Hoxa2 is expressed in second arch NCCs from the
onset of their migration (Maconochie et
al., 1999) through the stage of prechondrogenic condensation
(Kanzler et al., 1998
). When
chondrogenesis begins, Hoxa2 is downregulated in the cells that
undergo differentiation, whereas it remains expressed in the NC-derived
surrounding mesenchyme (Kanzler et al.,
1998
) until at least 14.5 dpc
(Fig. 2E), a stage at which
chondrogenesis is well underway.
To address the temporal requirement of Hoxa2 in NC patterning, pregnant females were given tamoxifen at different doses and gestational stages following the scheme described above (see also Materials and methods), and Hoxa2flox/flox;Cre-ERT2 fetuses or newborns were collected and analysed for mutant phenotypes.
We started by inducing Hoxa2 deletion after TM treatment at 7.0
dpc. TM induces Cre-ERT2 translocation to the nucleus within 6 hours, it
reaches its maximal accumulation at about 24 hours and is still present after
about 36 hours (Hayashi and McMahon,
2002; Zervas et al.,
2004
). Considering that skeletogenic NC migration in the mouse
begins at around 8.25 dpc (Serbedzija et
al., 1992
), TM treatment at 7.0 dpc was expected to induce
efficient Hoxa2 inactivation before the onset of NCC migration, and
therefore to reproduce the phenotypes of conventional and conditional
Wnt1-Cre-induced Hoxa2 knockouts. As anticipated,
the TM-induced Hoxa2flox/flox;Cre-ERT2 mutant newborns
died perinatally, and displayed mirror image duplication of first arch-like
structures and absence of external ear pinna
(Fig. 3C; and data not shown).
This outcome confirmed that the TM-inducible targeting system could be
efficiently employed to induce a full Hoxa2 knockout phenotype.
Conversely, no morphological abnormalities were detected in
Hoxa2flox/flox;Cre-ERT2 newborn mice that did not undergo
TM treatment (Fig. 3B).
Next, we tested the effect of Hoxa2 inactivation at NC
post-migratory stages. We started with TM treatment at 9.5 dpc. By this stage,
skeletogenic NCCs have already completed their migration
(Serbedzija et al., 1992) and
filled the branchial arches. Notably, deletion of Hoxa2 at this stage
also yielded a lack of external ear pinna and homeotic replacement of second
with first arch-like structures (Fig.
3E,J). Interestingly, the only distinct feature in the middle ear
skeleton of `post-migratory' Hoxa2flox/flox;Cre-ERT2
mutants was a rather normal looking gonial bone
(Fig. 3E). In the conventional
and `pre-migratory' Hoxa2 mutants, this membranous bone appears, on
the contrary, transformed, as it is abnormally enlarged and stretches across,
bridging over the two halves of the mirror duplication
(Rijli et al., 1993
)
(Fig. 1E,F,
Fig. 3A). Notably, deletion of
Hoxa2 by TM treatment at 8.0 dpc resulted in a variable gonial bone
phenotype, ranging from a malformed gonial bone to a complete transformation
of the element (Fig. 3D; data
not shown). These results indirectly identify a time window for gonial bone
specification and indicate that the first arch NCCs contributing to this
membranous element may be irreversibly committed at an early migratory stage,
before the NCCs give rise to the other elements of the middle ear region.
|
The external ear was systematically affected in all TM-induced Hoxa2 mutants. In particular, Hoxa2 mutagenesis induced by TM treatment up to 11.5 dpc always resulted in the loss of the pinna (Fig. 4D,E). In addition, mutant pups obtained by chronic tamoxifen treatments between 12.5 and 13.5 dpc displayed a smaller external ear (Fig. 4F). This indicated a late role of Hoxa2 in the morphogenesis of the external ear pinna, temporally distinct from its involvement in middle ear patterning.
Altogether, these results have revealed an unprecedented plasticity of second arch NCCs, even after they completed their migration. They have also shown that the execution of their morphogenetic program is absolutely dependent on Hoxa2 function at post-migratory stages. Moreover, our data indicate that Hoxa2 exerts temporally distinct functions on specific subpopulations of NCCs within the second arch, each giving rise to individual skeletal elements or external ear structures.
Hoxa2 inactivation at post-migratory stages results in rapid molecular changes in downstream target genes
The finding that Hoxa2 acts at late developmental stages, in
concomitance with the onset of the morphogenetic process, prompted us to
investigate whether Hoxa2 may be an integral component of the NCC
molecular patterning program. In particular, we asked whether Hoxa2
could be directly involved in the generation of the second arch-specific
molecular pattern in post-migratory NCCs by regulating the spatial expression
of genes involved in branchial arch patterning.
|
Next, we asked whether the expression pattern changes were the consequence of post-migratory Hoxa2 lack of function. We, therefore, induced the Hoxa2 mutation in 9.5 dpc embryos, collected them 24 hours later, and performed whole-mount in situ hybridisation for the selected genes. Postmigratory Hoxa2 inactivation was sufficient to induce similar expression pattern changes of Alx4, Bapx1, Six2 and Msx1 to those detected in conventional Hoxa2 knockout embryos (Fig. 5C,F,I,L), providing molecular support to the morphological transformation of skeletal structures observed in newborns following a similar TM treatment (Fig. 3). Most interestingly, we obtained similar molecular results by treating embryos at 10.0 dpc and collecting them as early as 12 hours after TM administration (shown for Six2 in Fig. 5L, inset; and data not shown). Such a rapid molecular reorganisation of second arch mesenchyme as a result of Hoxa2 inactivation in post-migratory NCCs suggests that these genes may be direct targets of Hoxa2.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue specific mutagenesis will still be required to investigate what is
the contribution of Hoxa2 expression in other tissues, if any, to NCC
patterning. Also, given that Hoxa2 is necessary in mouse NCCs to
impart a second arch program, it would be interesting to ascertain whether it
might also be sufficient. In this respect, it should be noted that ectopic
Hoxa2 overexpression in Xenopus, chick and zebrafish embryos
resulted in first-to-second arch transformation, although only when all first
arch tissues were targeted (Couly et al.,
1998; Creuzet et al.,
2002
; Grammatopoulos et al.,
2000
; Hunter and Prince,
2002
; Pasqualetti et al.,
2000
). Targeted Hoxa2 overexpression selectively in first
arch NCCs will be required in order to assess such a model in the mouse.
Temporal analysis of Hoxa2 function reveals plasticity of second arch neural crest cells even after their migration
We applied an inducible Cre-ERT2-based approach to achieve time-dependent
inactivation of a conditional allele of Hoxa2. By this means, we were
able to carry out for the first time a functional analysis of the temporal
requirement of a Hox factor in a vertebrate embryo. Hox genes are known to
play a fundamental role in providing segmental identity at early developmental
stages in the vertebrate embryo (Krumlauf,
1994), but little information is available about their potential
involvement in late aspects of morphogenetic processes. The importance of this
type of study has been recently addressed in a work reporting the conditional
inactivation of Hoxb1 in neurogenic NCCs
(Arenkiel et al., 2004
). The
authors performed their conditional mutagenesis by employing pre- and
post-migratory NCC Cre transgenic lines under the control of two distinct
promoters from the Wnt1 and Ap2 genes, respectively.
However, in this type of approach a precise stage-specific dissection of the
role of the gene was precluded. On the one hand, the Cre-ERT2-based
recombination approach is an efficient and reliable system for temporally
controlled, inducible Cre activity both in adult mouse tissues and developing
embryos (Ahn and Joyner, 2004
;
Danielian et al., 1998
;
Feil et al., 1997
;
Hayashi and McMahon, 2002
;
Imai et al., 2001
;
Kimmel et al., 2000
;
Metzger et al., 2005
;
Sgaier et al., 2005
). On the
other hand, the use of Cre-ERT2 recombinase regulated by a ubiquitous promoter
does not consent, on its own, for a tissue-restricted analysis. In this
respect, the finding that the conditional inactivation of Hoxa2 with
a Wnt1-Cre line resulted in a full knockout phenotype strongly
indicates that the abnormalities observed in our time-dependent mutagenesis
are mainly due to the disruption of Hoxa2 activity in the NCC
population.
In the mouse, Hoxa2 expression in second arch NCCs is present from
the time of their emergence from the neural folds, at around 8.25 dpc
(Maconochie et al., 1999;
Nonchev et al., 1996
). We show
that such an early expression does not provide NCCs with irreversible
patterning information. In fact, induction of the Hoxa2 deletion at
post-migratory stages up to at least 11.0 dpc is still sufficient to generate
morphological changes substantially similar to those obtained in embryos in
which Hoxa2 is knocked out from NCC pre-migratory stages. Thus, mouse
Hoxa2 is mainly required after the process of NCC migration, in
keeping with the maintenance of its expression through at least 14.5 dpc
(Fig. 2) (see also
Kanzler et al., 1998
), a stage
at which cartilage differentiation and morphogenesis are underway. This
finding also supports the conclusions from the complementary gain-of-function
experiments performed on Xenopus embryos, in which it was shown that
ectopic overexpression of Hoxa2 mRNA in the Hox-negative first
branchial arch could still cause a homeotic first-to-second arch
transformation, even at stages subsequent to NCC migration
(Pasqualetti et al.,
2000
).
Most interestingly, the current work reveals for the first time that second arch NCCs maintain plasticity over a remarkably long period, until they undergo skeletal differentiation in the branchial arch, and that they do so irrespective of any previous expression of Hoxa2. As we demonstrate that Hoxa2 is required after NCC migration, our results strongly suggest that Hoxa2 expression in NCCs is necessary for the molecular interpretation of the information provided by the surrounding environment to yield a second arch-specific morphogenetic program. This model integrates the concept of NCC plasticity with the existence of a NC-specific inherent genetic program that responds to extrinsic stimuli taking into account the developmental history and positional origin of distinct NCC subpopulations.
Distinct temporal and qualitative requirements of Hoxa2 for the morphogenesis of second arch structures
Vertebrate Hox genes are known to confer anteroposterior positional values
to developing tissues, but little is known about how their function is
implemented (Knosp et al.,
2004; Samad et al.,
2004
; Stadler et al.,
2001
; Wellik et al.,
2002
). We show that this function can be accomplished by a
localized morphogenetic activity, which, in the case of Hoxa2, is
integral to the control of the shape, size and location of the skeletal
elements of the second branchial arch.
An important aspect of our study is that we unveiled a differential
temporal effect of Hoxa2 inactivation on cartilage and membranous
bone development. Indeed, conventional knockout and gain-of-function studies
suggested that Hoxa2 is involved in patterning cartilaginous elements
(Grammatopoulos et al., 2000;
Hunter and Prince, 2002
;
Pasqualetti et al., 2000
;
Rijli et al., 1993
), while at
the same time mediating a general inhibitory activity on membranous bone
formation (Creuzet et al.,
2002
; Kanzler et al.,
1998
). We now show that these two roles can be temporally
dissected. In particular, intramembranous bone formation can be ectopically
induced in the second arch by Hoxa2 inactivation up to 11.0 dpc,
although not beyond this stage, whereas it is still possible to affect
cartilage patterning by TM treatment until at least 11.5 dpc
(Fig. 3). This observation
indicates that intramembranous bone NC-derived precursors may be specified
earlier then cartilage precursors. This is in keeping with the timing of
appearance of the early osteoblast differentiation marker Cbfa1
(Runx2), whose expression in the maxillary and mandibular components
of the first branchial arch can be detected as early as 11.5 dpc
(Otto et al., 1997
). In the
Hoxa2/ mutant, Cbfa1 is ectopically
expressed in the second branchial arch, implying an inhibitory role of
Hoxa2 on the activation of the osteogenic marker
(Kanzler et al., 1998
). Thus,
the fact that ectopic membranous osteogenesis can be triggered in the second
arch by TM-treatment not later than 11.0 dpc
(Fig. 3G) strongly suggests
that commitment of NCCs to an osteogenic fate occurs close to or at the time
when Cbfa1 expression is induced, that is around 11.5 dpc, even
though the first osteogenic differentiation markers will not appear before
13.0 dpc (Bialek et al., 2004
).
Beyond 11.5 dpc, second arch NCC precursors devoid of Hoxa2
expression are apparently not able to engage into an osteogenic program
(Fig. 3H), either because the
inducing signal is not available or possibly because the NCCs have lost their
responsiveness.
The inhibition of gonial bone formation represents the sole indication of
an early function for Hoxa2, as TM-treatment from 9.5 dpc on did not
result in gonial bone transformation (Fig.
3E). It is not clear how the NCCs that will originate the gonial
condensation are specified during their migratory phase or entry in the arch.
In fact Bapx1, a gene whose expression in the mouse is associated
with incudo-mallear articulation and gonial bone development
(Tucker et al., 2004), is not
detected in the arches until 10.5 dpc, and it is still ectopically activated
in the second arch subsequent to post-migratory Hoxa2 inactivation
(Fig. 5; see below). It is
therefore conceivable that Bapx1 is necessary but not sufficient to
promote gonial bone development, and that it requires some other early factor,
whose expression can be irreversibly inhibited by Hoxa2 in the
presumptive second arch NCCs during or soon after their migration.
Finally, we have shown that Hoxa2 inactivation up to 11.5 dpc can still result in a loss of the pinna, similar to in conventional Hoxa2/ mice, although homeosis of middle ear elements is no longer observed. Moreover, external ear development can still be affected by Hoxa2 inactivation, even at later stages, resulting in a hypomorphic pinna, whereas middle ear ossicles are normally generated (Fig. 4; and data not shown). Thus, Hoxa2 might be involved in the morphogenesis of the second arch ectomesenchyme, contributing to the pinna through advanced developmental stages, later than its requirement for middle ear patterning. Future studies will be required to assess the molecular pathways regulated by Hoxa2 in external ear morphogenesis.
Hoxa2 is a positive and negative regulator of gene expression in second arch mesenchyme
The gene expression analysis conducted in this work has provided additional
insights on the molecular program regulated by Hoxa2 in the second
arch. Previous studies reported that first arch specific genes can be
ectopically activated in the second arch territory of Hoxa2 mutant
embryos (Bobola et al., 2003;
Kutejova et al., 2005
). Thus,
it has been proposed that Hoxa2 has a mainly repressive role in the
second arch NCCs, and that in some cases it is directly implicated in
transcriptional inhibition, as with respect to Six2
(Kutejova et al., 2005
). We
found here that additional first arch-specific homeobox containing genes, such
as Alx4 and Bapx1, are similarly repressed by
Hoxa2. However, the expression of Msx1, which is normally
higher and displays a broader distribution in the hyoid than in the mandibular
arch at 10.5 dpc, decreases and acquires a first arch-like pattern in the
absence of Hoxa2 (Fig.
5). Thus, Hoxa2 can regulate gene expression both
positively and negatively, possibly by interacting with specific cofactors, in
order to constitute a second arch specific pattern.
It is interesting to note that the asymmetrical distribution between the
mandibular and hyoid arches of these gene transcripts is not evident in the
NCC mesenchyme of 9.5 dpc embryos (data not shown), despite Hoxa2
expression in the second arch. This is a further indication that
Hoxa2 mainly plays a later role, noticeable from 10.5 dpc onwards.
Interestingly, even at 10.5 dpc, when the arch specific expression pattern has
already been established, Hoxa2 inactivation results in homeotic
transformation of skeletal elements (Fig.
3). At the molecular level, induction of Hoxa2 deletion
by TM treatment of 9.5 or even 10.5 dpc embryos equally results in a
transformation of the second arch gene expression pattern to a first arch-like
pattern (Fig. 5; and data not
shown). This finding provides strong molecular support to the evidence of
long-lasting plasticity of second arch NCCs, as discussed above. In fact, at
least up until 10.5-11 dpc, NCCs are still able to undergo rapid molecular
changes and switch their developmental course. In this respect, it is also
noteworthy that the switch in gene expression upon Hoxa2 inactivation
can occur as rapidly as within 12 hours. Considering that tamoxifen takes
approximately 6 hours from the time of administration to start accumulating in
the nucleus, the time needed to induce a response on the NCC patterning can be
considered to be even shorter than 12 hours. This is an indication that
Hoxa2 is likely to directly regulate some of those molecular targets.
Accordingly, Six2 has recently been proposed to be a direct target of
Hoxa2 (Kutejova et al.,
2005). However, ectopic expression of Six2 in the second
arch NCCs was not sufficient to reproduce the Hoxa2 mutant phenotype
(Kutejova et al., 2005
),
suggesting a requirement for additional players. Indeed, our results suggest
that a number of genes involved in patterning NCC derivatives may be under the
direct transcriptional control of Hoxa2 to generate a second
arch-specific pattern. In this respect, the employment of our inducible system
to compare second arch transcripts at different stages between wild type and
mutant should allow the identification of the Hoxa2 target genes
responsible for the development of distinct structures.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Center for Biotechnology, College of Science and
Technology, Temple University, Philadelphia, PA 19122, USA
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahn, S. and Joyner, A. L. (2004). Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 118,505 -516.[CrossRef][Medline]
Arenkiel, B. R., Tvrdik, P., Gaufo, G. O. and Capecchi, M.
R. (2004). Hoxb1 functions in both motoneurons and
in tissues of the periphery to establish and maintain the proper neuronal
circuitry. Genes Dev.
18,1539
-1552.
Bialek, P., Kern, B., Yang, X., Schrock, M., Sosic, D., Hong, N., Wu, H., Yu, K., Ornitz, D. M., Olson, E. N. et al. (2004). A twist code determines the onset of osteoblast differentiation. Dev. Cell 6, 423-435.[CrossRef][Medline]
Bobola, N., Carapuco, M., Ohnemus, S., Kanzler, B., Leibbrandt,
A., Neubuser, A., Drouin, J. and Mallo, M. (2003).
Mesenchymal patterning by Hoxa2 requires blocking Fgf-dependent
activation of Ptx1. Development
130,3403
-3414.
Couly, G., Grapin-Botton, A., Coltey, P., Ruhin, B. and Le
Douarin, N. M. (1998). Determination of the identity of the
derivatives of the cephalic neural crest: incompatibility between Hox
gene expression and lower jaw development. Development
125,3445
-3459.
Couly, G., Creuzet, S., Bennaceur, S., Vincent, C. and Le Douarin, N. M. (2002). Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development 129,1061 -1073.[Medline]
Creuzet, S., Couly, G., Vincent, C. and Le Douarin, N. M. (2002). Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton. Development 129,4301 -4313.[Medline]
Crump, J. G., Swartz, M. E. and Kimmel, C. B. (2004). An integrin-dependent role of pouch endoderm in hyoid cartilage development. PLoS Biol. 2, E244.[CrossRef][Medline]
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8,1323 -1326.[CrossRef][Medline]
Dupe, V., Davenne, M., Brocard, J., Dolle, P., Mark, M.,
Dierich, A., Chambon, P. and Rijli, F. M. (1997). In
vivo functional analysis of the Hoxa-1 3' retinoic acid
response element (3'RARE). Development
124,399
-410.
Eames, B. F. and Schneider, R. A. (2005).
Quail-duck chimeras reveal spatiotemporal plasticity in molecular and
histogenic programs of cranial feather development.
Development 132,1499
-1509.
Feil, R., Wagner, J., Metzger, D. and Chambon, P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237,752 -757.[CrossRef][Medline]
Ferguson, C. A., Tucker, A. S. and Sharpe, P. T.
(2000). Temporospatial cell interactions regulating mandibular
and maxillary arch patterning. Development
127,403
-412.
Gammill, L. S. and Bronner-Fraser, M. (2003). Neural crest specification: migrating into genomics. Nat. Rev. Neurosci. 4,795 -805.[CrossRef][Medline]
Gendron-Maguire, M., Mallo, M., Zhang, M. and Gridley, T. (1993). Hoxa-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75,1317 -1331.[CrossRef][Medline]
Grammatopoulos, G. A., Bell, E., Toole, L., Lumsden, A. and
Tucker, A. S. (2000). Homeotic transformation of
branchial arch identity after Hoxa2 overexpression.
Development 127,5355
-5365.
Hayashi, S. and McMahon, A. P. (2002). Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244,305 -318.[CrossRef][Medline]
Helms, J. A., Cordero, D. and Tapadia, M. D.
(2005). New insights into craniofacial morphogenesis.
Development 132,851
-861.
Hu, D., Marcucio, R. S. and Helms, J. A.
(2003). A zone of frontonasal ectoderm regulates patterning and
growth in the face. Development
130,1749
-1758.
Hunt, P., Wilkinson, D. and Krumlauf, R.
(1991). Patterning the vertebrate head: murine Hox 2
genes mark distinct subpopulations of premigratory and migrating cranial
neural crest. Development
112, 43-50.
Hunter, M. P. and Prince, V. E. (2002). Zebrafish hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch. Dev. Biol. 247,367 -389.[CrossRef][Medline]
Imai, T., Jiang, M., Chambon, P. and Metzger, D.
(2001). Impaired adipogenesis and lipolysis in the mouse upon
selective ablation of the retinoid X receptor alpha mediated by a
tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes.
Proc. Natl. Acad. Sci. USA
98,224
-228.
Kanzler, B., Kuschert, S. J., Liu, Y. H. and Mallo, M.
(1998). Hoxa-2 restricts the chondrogenic domain and
inhibits bone formation during development of the branchial area.
Development 125,2587
-2597.
Kimmel, R. A., Turnbull, D. H., Blanquet, V., Wurst, W., Loomis,
C. A. and Joyner, A. L. (2000). Two lineage boundaries
coordinate vertebrate apical ectodermal ridge formation. Genes
Dev. 14,1377
-1389.
Knosp, W. M., Scott, V., Bachinger, H. P. and Stadler, H. S.
(2004). HOXA13 regulates the expression of bone morphogenetic
proteins 2 and 7 to control distal limb morphogenesis.
Development 131,4581
-4592.
Krumlauf, R. (1994). Hox genes in vertebrate development. Cell 78,191 -201.[CrossRef][Medline]
Kutejova, E., Engist, B., Mallo, M., Kanzler, B. and Bobola,
N. (2005). Hoxa2 downregulates Six2 in the
neural crest-derived mesenchyme. Development
132,469
-478.
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest, 2nd edn. Cambridge: Cambridge University Press.
Le Douarin, N. M., Creuzet, S., Couly, G. and Dupin, E.
(2004). Neural crest cell plasticity and its limits.
Development 131,4637
-4650.
Lettice, L., Hecksher-Sorensen, J. and Hill, R. (2001). The role of Bapx1 (Nkx3.2) in the development and evolution of the axial skeleton. J. Anat. 199,181 -187.[CrossRef][Medline]
Maconochie, M., Krishnamurthy, R., Nonchev, S., Meier, P.,
Manzanares, M., Mitchell, P. J. and Krumlauf, R.
(1999). Regulation of Hoxa2 in cranial neural crest cells
involves members of the AP-2 family. Development
126,1483
-1494.
Metzger, D., Li, M. and Chambon, P. (2005). Targeted somatic mutagenesis in the mouse epidermis. Methods Mol. Biol. 289,329 -340.[Medline]
Noden, D. M. (1983). The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues. Dev. Biol. 96,144 -165.[CrossRef][Medline]
Nonchev, S., Vesque, C., Maconochie, M., Seitanidou, T.,
Ariza-McNaughton, L., Frain, M., Marshall, H., Sham, M. H., Krumlauf, R. and
Charnay, P. (1996). Segmental expression of Hoxa-2
in the hindbrain is directly regulated by Krox-20.
Development 122,543
-554.
Oliver, G., Wehr, R., Jenkins, N. A., Copeland, N. G., Cheyette,
B. N., Hartenstein, V., Zipursky, S. L. and Gruss, P.
(1995). Homeobox genes and connective tissue patterning.
Development 121,693
-705.
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R., Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R. et al. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89,765 -771.[CrossRef][Medline]
Pasqualetti, M. and Rijli, F. M. (2002). Developmental biology: the plastic face. Nature 416,493 -494.[CrossRef][Medline]
Pasqualetti, M., Ori, M., Nardi, I. and Rijli, F. M.
(2000). Ectopic Hoxa2 induction after neural crest migration
results in homeosis of jaw elements in Xenopus.
Development 127,5367
-5378.
Pasqualetti, M., Ren, S. Y., Poulet, M., LeMeur, M., Dierich, A. and Rijli, F. M. (2002). A Hoxa2 knockin allele that expresses EGFP upon conditional Cre-mediated recombination. Genesis 32,109 -111.[CrossRef][Medline]
Prince, V. and Lumsden, A. (1994).
Hoxa-2 expression in normal and transposed rhombomeres: independent
regulation in the neural tube and neural crest.
Development 120,911
-923.
Qu, S., Niswender, K. D., Ji, Q., van der Meer, R., Keeney, D.,
Magnuson, M. A. and Wisdom, R. (1997). Polydactyly and
ectopic ZPA formation in Alx-4 mutant mice.
Development 124,3999
-4008.
Ren, S. Y., Pasqualetti, M., Dierich, A., Le Meur, M. and Rijli, F. M. (2002). A Hoxa2 mutant conditional allele generated by Flp- and Cre-mediated recombination. Genesis 32,105 -108.[CrossRef][Medline]
Rijli, F. M., Mark, M., Lakkaraju, S., Dierich, A., Dolle, P. and Chambon, P. (1993). A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene. Cell 75,1333 -1349.[CrossRef][Medline]
Robert, B., Sassoon, D., Jacq, B., Gehring, W. and Buckingham, M. (1989). Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J. 8, 91-100.[Abstract]
Ruhin, B., Creuzet, S., Vincent, C., Benouaiche, L., Le Douarin, N. M. and Couly, G. (2003). Patterning of the hyoid cartilage depends upon signals arising from the ventral foregut endoderm. Dev. Dyn. 228,239 -246.[CrossRef][Medline]
Samad, O. A., Geisen, M. J., Caronia, G., Varlet, I.,
Zappavigna, V., Ericson, J., Goridis, C. and Rijli, F. M.
(2004). Integration of anteroposterior and dorsoventral
regulation of Phox2b transcription in cranial motoneuron progenitors
by homeodomain proteins. Development
131,4071
-4083.
Santagati, F. and Rijli, F. M. (2003). Cranial neural crest and the building of the vertebrate head. Nat. Rev. Neurosci. 4,806 -818.[CrossRef][Medline]
Schneider, R. A. and Helms, J. A. (2003). The
cellular and molecular origins of beak morphology.
Science 299,565
-568.
Serbedzija, G. N., Bronner-Fraser, M. and Fraser, S. E.
(1992). Vital dye analysis of cranial neural crest cell migration
in the mouse embryo. Development
116,297
-307.
Sgaier, S. K., Millet, S., Villanueva, M. P., Berenshteyn, F., Song, C. and Joyner, A. L. (2005). Morphogenetic and cellular movements that shape the mouse cerebellum; insights from genetic fate mapping. Neuron 45,27 -40.[CrossRef][Medline]
Shigetani, Y., Sugahara, F., Kawakami, Y., Murakami, Y., Hirano,
S. and Kuratani, S. (2002). Heterotopic shift of
epithelial-mesenchymal interactions in vertebrate jaw evolution.
Science 296,1316
-1319.
Shigetani, Y., Sugahara, F. and Kuratani, S. (2005). A new evolutionary scenario for the vertebrate jaw. BioEssays 27,331 -338.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Stadler, H. S., Higgins, K. M. and Capecchi, M. R.
(2001). Loss of Eph-receptor expression correlates with loss of
cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs.
Development 128,4177
-4188.
Trainor, P. and Krumlauf, R. (2000). Plasticity in mouse neural crest cells reveals a new patterning role for cranial mesoderm. Nat. Cell Biol. 2, 96-102.[CrossRef][Medline]
Trainor, P. A., Ariza-McNaughton, L. and Krumlauf, R.
(2002). Role of the isthmus and FGFs in resolving the paradox of
neural crest plasticity and prepatterning. Science
295,1288
-1291.
Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and
Martin, G. R. (1999). Cre-mediated gene inactivation
demonstrates that FGF8 is required for cell survival and patterning of the
first branchial arch. Genes Dev.
13,3136
-3148.
Tucker, A. S. and Lumsden, A. (2004). Neural crest cells provide species-specific patterning information in the developing branchial skeleton. Evol. Dev. 6, 32-40.[CrossRef][Medline]
Tucker, A. S., Yamada, G., Grigoriou, M., Pachnis, V. and
Sharpe, P. T. (1999). Fgf-8 determines rostral-caudal
polarity in the first branchial arch. Development
126, 51-61.
Tucker, A. S., Watson, R. P., Lettice, L. A., Yamada, G. and
Hill, R. E. (2004). Bapx1 regulates patterning in
the middle ear: altered regulatory role in the transition from the proximal
jaw during vertebrate evolution. Development
131,1235
-1245.
Wellik, D. M., Hawkes, P. J. and Capecchi, M. R.
(2002). Hox11 paralogous genes are essential for
metanephric kidney induction. Genes Dev.
16,1423
-1432.
Zervas, M., Millet, S., Ahn, S. and Joyner, A. L. (2004). Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1. Neuron 43,345 -357.[CrossRef][Medline]
Related articles in Development:
|