1 Department of Developmental Biology, Max-Planck Institute of Immunobiology,
Stuebeweg 51, 79108 Freiburg, Germany
2 Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156
Oeiras, Portugal
* Author for correspondence (e-mail: bobola{at}immunbio.mpg.de)
Accepted 16 November 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hoxa2, Six2, Hox genes, Branchial arches, Neural crest, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hox genes are key developmental regulators required to specify segmental
identity in the developing embryo (Carroll,
1995). Genetic analyses in flies and vertebrates have yielded
extensive knowledge about the developmental processes regulated by Hox genes,
but the molecular events directly controlled by these genes are still largely
unknown (for reviews, see Krumlauf,
1994
; Zakany and Duboule,
1999
; Burke, 2000
;
Trainor and Krumlauf, 2001
;
Alonso, 2002
).
Hox-negative and Hox-positive domains can be distinguished in the cranial
neural crest (Prince and Lumsden,
1994; Graphin-Bottom et al., 1995;
Couly et al., 1996
;
Köntges and Lumsden,
1996
). The visceral skeleton of the face derives from the
Hox-negative first branchial arch, with a very limited contribution from the
more posterior Hox-positive branchial arches. Moreover, ectopic expression of
Hox genes in anterior Hox-negative crest cells in chicken
(Creuzet et al., 2002
) results
in the absence of the facial skeleton. A similar effect is observed in mouse,
following Hoxa2 overexpression in the head mesenchyme
(Kanzler et al., 1998
).
Among Hox genes, Hoxa2, together with Hoxb2, shows the
most anterior domain of expression in the cranial neural crest, corresponding
to the population that migrates to the second branchial arch
(Prince and Lumsden, 1994;
Nonchev et al., 1996
;
Mallo, 1997
). In mouse,
disruption of the Hoxa2 gene mainly affects second branchial arch
development. Second arch skeletal elements (stapes, styloid process, lesser
horn of the hyoid bone) are transformed into first arch-specific skeletal
elements (incus, malleus and tympanic ring), arranged in a mirror image
disposition to their first arch counterparts
(Gendron-Maguire et al., 1993
;
Rijli et al., 1993
;
Barrow and Capecchi, 1999
).
This arrangement suggests a common source of information located between the
first and second branchial arch (Rijli et
al., 1993
; Mallo and Brandlin,
1997
), with a different interpretation of this common signal in
neural crest cells expressing Hoxa2. More specifically,
Hoxa2 is thought to interfere negatively with the response of the
neural crest cells to skeletogenic cues
(Couly et al., 2002
;
Bobola et al., 2003
);
accordingly, chondrogenesis in the second arch takes place exclusively in
areas that are free of Hoxa2 expression
(Kanzler et al., 1998
). Thus,
Hoxa2 patterns the second arch skeleton by limiting its formation.
Consistent with a role in controlling the size of the condensations,
overexpression of Hoxa2 in chick and in frog confers a late,
postmigratory, patterning role to Hoxa2 during development of the
branchial arch-derived skeleton
(Grammatopoulos et al., 2000
;
Pasqualetti et al., 2000
).
Because of the negative effect on the formation of the facial skeleton, the
absence of Hox gene expression in the anterior part of the embryo has been
proposed as a crucial factor to allow the evolution of the head and lower jaw
in gnathostomes (Creuzet, 2002; Manzanares
and Nieto, 2003). As with most developmental processes regulated
by Hox genes, the molecular cascade initiated by these genes to culminate in
the inhibition of craniofacial skeletogenesis is unknown.
In a subtraction approach, designed to clarify the molecular cascades
initiated by Hoxa2 to control skeletogenesis in the second branchial arch
(Bobola et al., 2003), we have
found Six2 as a gene regulated by Hoxa2.
The Six family of homeobox transcription factors, characterized by a Six
domain and a homeodomain, counts six members in mammals
(Kawakami et al., 2000).
Members of this family share transcriptional properties and the ability to
interact physically and functionally with Eya proteins, both in
Drosophila and in vertebrates
(Bonini et al., 1997
;
Pignoni et al., 1997
;
Heanue et al., 1999
;
Ohto et al., 1999
;
Ikeda et al., 2002
;
Ozaki et al., 2002
;
Li et al., 2003
;
Ruf et al., 2004
). Six genes
differ largely in their expression pattern during embryogenesis, and gene
inactivation experiments have revealed that these genes control a variety of
developmental processes (Klesert et al.,
2000
; Carl et al.,
2002
; Li et al.,
2002
; Laclef et al.,
2003a
; Laclef et al.,
2003b
; Lagutin et al.,
2003
; Xu et al.,
2003
; Zheng et al.,
2003
; Ozaki et al.,
2004
).
Six2 function has not yet been characterized. Its expression is
restricted to the head mesenchyme, foregut, stomach, kidney and genital
tubercle (Oliver et al.,
1995). Inactivation of Six1, its closest homolog, affects
muscle, kidney, branchial arch derivatives and inner ear development
(Laclef et al., 2003a
;
Laclef et al., 2003b
;
Xu et al., 2003
;
Zheng et al., 2003
;
Ozaki et al., 2004
). The
presence of incomplete and/or smaller cartilages and bones characterizes the
craniofacial phenotype of Six1 null mice
(Laclef et al., 2003b
).
Indeed, Six1 inactivation provided the first direct evidence
implicating Six genes in the development of the facial skeleton; the strong
craniofacial phenotype of the Eya1 mutant and the reported genetic
interaction of Eya and Six genes give additional, indirect hints
(Xu et al., 1999
).
Here we show that Hoxa2 negatively regulates Six2 expression during second arch development. Ectopic expression of Six2 in the second branchial arch causes distinctive phenotypic features seen in the Hoxa2 mutant. Furthermore, gain-of-function experiments show that Hoxa2 is sufficient to downregulate Six2 expression in the neural-crest-derived mesenchyme. We also show that a 0.9 kb fragment of the Six2 promoter is the target of Hoxa2 regulation and that Hoxa2 physically interacts with the proximal region of this promoter.
Together, our results show that Six2 is genetically downstream of Hoxa2 in the second branchial arch and suggest that regulation of Six2 may be one of the mechanisms utilized by Hoxa2 to pattern the second arch skeleton.
Finally, we propose that the Six2 gene could be a target of Hox proteins in different developmental processes in addition to patterning the second arch.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RT-PCR
Second arches of E10.5 embryos from Hoxa2+/-
intercrosses and a2-Six2 transgenics were dissected, frozen and
pooled according to genotype. RT-PCR was performed using Superscript
Preamplification System (Invitrogen) and Taq polymerase (PeqLab) with the
following primers: Six2F 5'-CAGCCGCCACCATGTCCATGCTG-3'; Six2R
5'-GAACTTGCGCCGCACGCGGTAC-3'; Six1F
5'-AAGAACCGGAG-GCAAAGAGACC-3'; Six1R
5'-CCAATATCTCCCCACTTAGGAA-CC-3'; Six4F
5'-AACCAGTATGGCATTGTCCAGATCC-3'; Six4R
5'-ACTGCAGAACCAAGCGCTGTTCTC-3'; Six5F
5'-GAGTGACT-GCGCTGCAACTTCCCTCG-3'; Six5R
5'-AGGGCTCCTCCACG-GGTACCGAC-3'; GadphF
5'-ACCACAGTCCATGCCATCAC-3'; GadphR
5'-TCCACCACCCTGTTGCTGTA-3'; Hoxa2F
5'-GCCT-GAGTATCCCTGGATG-3'; Hoxa2R
5'-ACCCTTCCCTCTCCAG-AAG-3'. First-strand cDNA was subjected to 24
amplification cycles. The specificity of each PCR product was confirmed by
sequencing.
Mutant and transgenic animals and embryos
Hoxa2 mutant mice were described
(Gendron-Maguire et al.,
1993). Transgenic embryos were generated by pronuclear injection
of the following transgenes: a2-Six2, containing Six2 cDNA,
amplified with primers Six2F 5'-CAGCCGCCACCATGTCCATGCTG-3' Six2R
5'-CTCTAGGAGCCCAGGTCCACAAGG-3' cloned downstream the
Hoxa2 enhancer (Kanzler et al.,
1998
); Msx2-Hoxa2
(Kanzler et al., 1998
);
900Six2-lacZ containing BamHI (-893) - Sph1 (+18)
Six2 promoter fragment obtained by screening the RPCI mouse PAC
library 21 (Osoegawa et al., 2000) (provided by UK HGMP Resource Centre),
cloned into pCMVbeta (Clontech). 900Six2-lacZ was injected to
generate both transgenic embryos and transgenic lines. A line showing high
expression in the first branchial arch was crossed to
Hoxa2+/- mice to obtain 900Six2-lacZ;
Hoxa2+/-, which were mated to Hoxa2+/-
to generate 900Six2-lacZ;Hoxa2-/- embryos.
In vitro transcription/translation and electrophoretic mobility shift assay
Mouse Hoxa2 cDNA, containing a HA tag inserted in frame before the stop
codon, mouse Meis1 cDNA and human Pbx1a cDNA
(Di Rocco et al., 1997) were
cloned in pcDNA3 (Invitrogen) and transcribed/translated using T7-coupled TNT
rabbit reticulocytes (Promega).
BstEII/SspI probe, probe 1 and probe 2 were labeled with
32P-dCTP. The binding reaction was performed as described
(Scheidereit, 1987).
The sequence of the oligonucleotides used as probes and competitors is shown in Fig. 6. For the supershift experiments, 40 ng of anti-HA antibodies (rat monoclonal 3F10, Roche) were added to the reaction.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hoxa2 is expressed in neural crest cells migrating from rhombomere
4 to populate the second arch, later its main expression domain
(Prince and Lumsden, 1994;
Nonchev et al., 1996
;
Mallo, 1997
). Six2
expression in the branchial area is first detected at E9.5, predominantly in
the first arch (Oliver et al.,
1995
). At this stage, no obvious differences in Six2
expression pattern could be observed between wild-type and Hoxa2
mutant second arches (not shown). At E10.5, Six2 expression was
restricted to the posterior area of the second branchial arch. In addition, a
second, more proximal domain of Six2 expression appeared in the
mutant second arch (Fig. 1A,D,
arrow). The spatial and temporal coordinates of Six2 expression in
the mutant second arch, together with the signal intensity, were equivalent to
those of the Six2 expression domain in the first arch mesenchyme. In
Hoxa2 mutant embryos, the first and second arch
Six2-expressing areas were symmetrical with respect to the first
branchial cleft (Fig.
1A,D).
|
In the absence of Hoxa2, a duplicated incus and malleus form in
the second branchial arch. To see if Six2 upregulation in the second
arch was spatially associated with the ectopic cartilaginous condensations, we
examined mutant and wild-type embryos for the expression of the chondrogenic
marker Sox9 (Ng et al.,
1997; Zhao et al.,
1997
). In E12.5 wild-type embryos, Sox9 mRNA was mainly
expressed in the area derived from the first branchial arch, consistent with
the restricted chondrogenesis that takes place in the second branchial arch
(Fig. 1G). In the
Hoxa2 mutant, an equivalent Sox9 expression in the first and
second arch anticipated the formation of incus and malleus in the first arch
and their duplicated counterparts in the second arch
(Fig. 1H). Six2 signal
could be detected in close proximity to Sox9
(Fig. 1C,D). In a more detailed
inspection of the mutual localization of Six2 and Sox9
mRNAs, performed by in-situ hybridization on adjacent sections of branchial
arches of E11.5 Hoxa2 mutant embryos, a defined mesenchymal area,
including the first and second branchial arch, was positive for both
Six2 and Sox9 (Fig.
1I,L, arrows). We conclude that the upregulation of Six2
takes place in the area fated to give rise to the skeletal duplication in the
Hoxa2 mutant.
In addition to Six2, the genes Six1, Six4 and
Six5 are also expressed in the branchial area
(Oliver et al., 1995;
Klesert et al., 2000
;
Ozaki et al., 2001
). To
distinguish if the upregulation of Six2 observed in the
Hoxa2 mutant second arch is specifically restricted to Six2
or if it is a common feature shared by the other Six genes, we performed
semiquantitative RT-PCR on total RNA extracted from E10.5 Hoxa2
mutant and wild-type arches, using Six1-, Six2-,
Six4- and Six5-specific primers. As expected, we observed a
significant increase in Six2 signal in the mutant versus wild-type
second arch, but Six1, Six4 and Six5 levels (as well as
Gadph control) remained unaffected
(Fig. 2). These data indicate
that Hoxa2 negatively controls Six2 expression in the second
branchial arch specifically and does not affect any of the other Six genes
expressed in this area.
|
|
We then analyzed the effects of Six2 overexpression on the skeletal phenotype of E18.5 a2-Six2 embryos. The second arch skeleton is composed of three cartilages (stapes, styloid process and lesser horn of the hyoid bone), which are transformed into different skeletal elements in the Hoxa2 mutant. All these elements displayed morphological changes in transgenic embryos overexpressing Six2 (Fig. 3E,F,H,I).
The stapes, which is lost in the Hoxa2 mutant, was either reduced or absent in a2-Six2 transgenics (2/6). An ectopic cartilage, fused to the proximal part of the styloid process, extended into the oval window and in front of the incus. This cartilage, which in shape and position resembled a stapedial arch, may be an intermediate between loss of the stapes and formation of a duplicated incus, as observed in the Hoxa2 mutant (4/6) (Fig. 3E,F). The styloid process was thicker (Fig. 3E,F) or abnormally elongated and fused to the lesser horn of the hyoid bone, forming a long Meckel-like cartilage (2/6) (Fig. 3H,I). The lesser horn, absent in the Hoxa2 mutant, was misshapen, enlarged and fused to the greater horn (6/6); in most cases, it appeared as a bifurcation of the greater horn. By contrast to the noticeable effects in cartilages, bone formation was relatively unchanged and in only one case tympanic ring growth was mildly affected (Fig. 3H,I).
Molecular analysis of a2-Six2 transgenic embryos showed no noticeable effect of overexpression of Six2 on the levels of Hoxa2 in the second branchial arch (Fig. 3J), thus ruling out Hoxa2 downregulation as a possible cause of the transgenic phenotype.
Compared with the Hoxa2 phenotype, we observed two main defects:
one was as featured in the Hoxa2 mutant (absent stapes, incus
duplication), while the other consisted of enlargements of the elements
patterned by Hoxa2 (lesser horn, styloid process abnormally
elongated). In all cases, overexpression of Six2 in the second
branchial arch resulted in the formation of ectopic cartilage, one of the
phenotypic characteristics of the Hoxa2 mutant
(Kanzler et al., 1998).
Hoxa2 is sufficient to downregulate Six2 in the head mesenchyme
Six2 is widely expressed in the head mesenchyme
(Oliver et al., 1995). By
contrast, Hoxa2 expression is mainly confined to the second branchial
arch (Prince and Lumsden,
1994
; Nonchev et al.,
1996
; Mallo,
1997
). To gain insight into how Hoxa2 regulates
Six2, we asked whether Hoxa2 repressor activity is restricted to the
second arch, or, alternatively, if Hoxa2 is sufficient to
downregulate Six2 expression in the craniofacial mesenchyme. For
this, we took a transgenic approach and ectopically expressed Hoxa2
under the Msx2 promoter, able to direct gene expression to the head
mesenchyme (Liu et al., 1994
;
Kanzler et al., 1998
).
To detect expression of Six2 and Hoxa2 in the same
embryo, E10.5 control and Msx2-Hoxa2 transgenic littermate embryo
halves were hybridized with either Six2 or Hoxa2 probe.
Six2 mRNA showed an abundant distribution in the maxillary and nasal
mesenchyme of wild-type E10.5 embryos, areas that are negative for
Hoxa2 expression (Fig.
4A). Transgenic E10.5 littermates (Msx2-Hoxa2; n=4)
displayed a markedly reduced expression of Six2 in the maxillary
mesenchyme and to a lesser extent in the periocular mesenchyme, the areas of
Hoxa2 ectopic expression (Fig.
4B). We conclude that Hoxa2 is sufficient to repress
Six2 in the head mesenchyme in vivo; this observation is particularly
interesting because this very same transgenic expression results in reduction
or absence of the facial skeleton (Kanzler
et al., 1998).
|
As a first step in understanding the role of Hoxa2 in regulating Six2, we examined mouse and human genomic Six2 sequences. The sequence from -900 bp to the putative transcriptional start site (identified as the 5' end of the Six2 first exon, GenBank NM_011380) displayed a high conservation between the two species.
To assay whether this promoter region harbors regulatory elements controlling Six2 transcription, we cloned it upstream of a lacZ reporter gene and injected the resulting construct (900Six2-lacZ) into fertilized mouse oocytes. Transgenic embryos, collected at E11.5, showed a ß-galactosidase (ß-gal) staining consistent with the Six2 expression pattern at various embryo locations, including the branchial area (Fig. 5A) (E.K. and N.B., unpublished). Here, the most proximal 900 bp of the Six2 promoter directed lacZ expression in the proximal part of the first branchial arch (arrowhead) and in mesenchyme proximal to the first branchial arch (arrow), a pattern that faithfully recapitulates endogenous Six2 expression. The activity of the transgene in the second branchial arch appeared stronger compared with the restricted Six2 endogenous expression in this area (Fig. 1B). Insertion of the transgene in multiple copies may have reduced the efficiency of Hoxa2 repressor activity; alternatively, other repressor-responsive elements controlling Six2 transcription independently from Hoxa2 may not be contained in our transgene.
|
These results show that the proximal region of the Six2 promoter is sufficient to direct Six2 expression in the branchial area. More importantly, they demonstrate that Hoxa2 controls the Six2 gene at the transcriptional level and that this control, direct or indirect, is confined to a proximal 900 bp of genomic sequence.
Hoxa2 directly interacts with Six2 promoter sequences
Our data suggest the possibility that Hoxa2 may directly repress
Six2 transcription, interacting with the proximal 900 bp of the
Six2 promoter. As noted above, the first kilobase of Six2
genomic sequences upstream of the transcription start site (TSS; +1) is highly
similar between mouse and human. Sequence conservation is extremely high
between position -249 and -11, reaching 95% similarity between the two
species. To test promoter, we performed an electrophoretic mobility shift
assay (EMSA) using the conserved Six2 region located immediately
upstream of the TATA box (BstEII-SspI fragment;
Fig. 6A) as a probe. Incubation
of the probe with in-vitro translated HA-tagged Hoxa2 resulted in the
formation of two retarded complexes. These bands represent the interaction of
Hoxa2-HA with the probe, as they were supershifted by the addition of the
anti-HA antibody. By contrast, incubation of the probe in the presence of
unprogrammed reticulocytes did not result in any retarded complex, nor did
addition of the antibody have any effect
(Fig. 6B). When the probe was
fragmented and each half-fragment (probe 1 and probe 2) incubated with
Hoxa2-HA, we still observed formation of the characteristic doublet
(Fig. 6C,D). A close inspection
of the BstEII-SspI genomic area revealed the presence of two
conserved 5'-GAATAAT-3' motifs, one in each of the two fragmented
probes. According to in vitro binding experiments, the Hox consensus sequence
contains a TAAT core (Graba et al.,
1997); to test whether Hoxa2 recognizes the GAATAAT sequence, we
performed competition experiments using wild-type and mutant oligonucleotides.
The complex formed in the presence of probe 1 (BstEII-SmaI,
Fig. 6A) and Hoxa2-HA was
competed at two different concentrations of a cold oligonucleotide reproducing
the GAATAAT sequence and flanking nucleotides of probe 1. A similar effect was
observed upon adding a molar excess of oligonucleotide wt2, which reproduces
the GAATAAT motif and flanking nucleotides contained in probe 2
(SmaI-SspI, Fig.
6A). By contrast, the addition of the same molar excess of m1 or
m2 oligonucleotides, containing three or four nucleotide substitutions in the
GAATAAT, left the complex unaffected (Fig.
6C). Incubation of Hoxa2-HA in the presence of probe 2 resulted in
the formation of the same retarded complexes, and these were efficiently
competed by wt1 or wt2 oligonucleotides, but not by mutant oligonucleotides
(Fig. 6D). The ability of Hoxa2
to recognize the two sites contained in the Six2 promoter was further
confirmed by using the wild-type and mutant oligonucleotides as probes
(Fig. 6E). Hoxa2-HA formed
slower migrating complexes when incubated with oligonucleotides wt1 and wt2,
while no higher complex formation was observed when the mutant
oligonucleotides were used as probes in the same assay.
These data show that Hoxa2 interacts with the proximal region of the Six2 promoter and that this interaction is sequence-specific. Both the identified sites are bound with similar affinity and contain a GAATAAT sequence. Moreover, disruption of their ATAAT core abolishes Hoxa2 binding.
The interaction of Hox proteins with their target promoters often requires
co-factors (Mann and Affolter,
1998), and we have indeed detected Pbx and Meis
binding to the Six2 promoter in close proximity to the Hoxa2 binding
sites. Pbx1a alone bound the BstEII/SspI fragment with very
low affinity, while no binding was detectable for Meis1 alone with the probe.
When the two proteins were co-translated, an intense retarded band was seen.
In contrast to a previous study of Hoxb1 auto-regulatory element
(Ferretti et al., 2000
), we
could not detect the formation of a slower molecular complex by simultaneous
incubation of the probe with Hoxa2, Pbx1a and Meis1
(Fig. 6F).
Six2 overexpression affects development of the third and more posterior arches
Along with second arch-derived structures, transgenic mice overexpressing
Six2 displayed abnormal growth and morphology of the thyroid and
cricoid cartilages and of the hyoid bone, derived from the third and more
posterior pharyngeal arches.
The hyoid bone was malformed, curved and fused to the thyroid cartilage, bilaterally or unilaterally (Fig. 7B,C, arrow). Fusion of the greater horn to the lesser horn and to the thyroid cartilage in a structure independent of the main body of the hyoid was often observed (Fig. 7B,C, arrowhead). The thyroid and cricoid cartilages were abnormally thickened, and larger areas of fusion were observed (Fig. 7B,C). Occasionally, the rings of the trachea were abnormally fused to each other and disorganized (Fig. 7C).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In patterning the embryo, Hox gene activity has been proposed to be
transduced by a battery of genes, termed the realizator genes, that directly
influence cell processes such as cell adhesion, apoptosis or rate of cell
division (Garcia-Bellido,
1975). Like Hoxa2, Six2 is a transcription factor. The next
crucial question will therefore be to identify the cellular processes
controlled by Six2 in the developing branchial arches.
Hoxa2 and second arch patterning
The Hoxa2 mutation affects skeletal development of the second
branchial arch (Gendron-Maguire et al.,
1993; Rijli et al.,
1993
; Barrow and Capecchi,
1999
). Second arch skeletal elements are lost and replaced by
first arch duplicated elements arranged in a mirror image disposition with
respect to their first arch counterparts. How does Hoxa2 pattern
second arch skeleton? Previous reports have shown that Hoxa2
negatively interferes with the development of the facial skeleton, and in the
second arch Hoxa2 surrounds, but is excluded from, the endochondral
ossification centers required to form the hyoid cartilage
(Kanzler et al., 1998
;
Creuzet et al., 2002
). The
most likely mechanism of action is that Hoxa2 restricts skeletogenesis in the
second arch, preventing the formation of first arch duplications, while, at
the same time, shaping second arch-specific elements. Six2 could be
one of the genes regulated by Hoxa2 to restrict skeletogenesis in the
second arch. In support of this, Six2 overexpression in the second
branchial arch interferes with the normal patterning of the second
arch-derived skeleton; endochondral ossification is increased overall,
producing a skeletal phenotype reminiscent of the Hoxa2 phenotype.
Discrepancies in the shape of the skeletal elements between transgenic and
mutant embryos indicate that other factors, acting in parallel or in concert
with Six2, are required to generate the full Hoxa2 phenotype.
However, even in the scenario with Six2 as the only factor responsible for the Hoxa2 phenotype, the few intrinsic differences that characterize the mutant and transgenic second arches could alone account for discrepancies in the final shape of the skeletal elements. First, being driven by a heterologous promoter, the expression pattern of Six2 in the second arch of transgenic embryos is different from Six2 endogenous expression in the second arch of the mutant. Second, in contrast to the mutant, a functional Hoxa2 protein is present in the second arch of transgenic embryos.
Final evidence that repression of Six2 is one of the mechanisms
employed by Hoxa2 in second arch patterning will require analysis of
a Hoxa2; Six2 double mutant. Our prediction is that
Six2 inactivation should, at least partially, rescue the
Hoxa2 phenotype. The lack of a Six2 mutant hampers the
accomplishment of this experiment, but the inactivation of Six1, the
closest homolog to Six2, supports our prediction. Six1
mutant mice display an evident craniofacial phenotype, thereby identifying Six
genes as important regulators of neural-crest-derived craniofacial skeleton
(Laclef et al., 2003b;
Ozaki et al., 2004
). We found,
both by RT-PCR (see Fig. 2) and
by in-situ hybridization (B.E. and N.B., unpublished), that Six1 is
not regulated by Hoxa2. However, Six1 is required for
development of part of the skeleton that is affected by the Hoxa2
mutation (Laclef et al.,
2003b
; Ozaki et al.,
2004
), suggesting, together with the high similarity in the
encoded proteins and the expression pattern of the Six1 and
Six2 genes (Oliver et al.,
1995
), a functional contribution of Six2 to the
Hoxa2 phenotype.
In the Six1 mutant, second-arch-derived cartilages fail to form
(Laclef et al., 2003b;
Ozaki et al., 2004
). If
Six2 is also required for second arch skeletal growth, a likely
scenario for second arch skeletal patterning is that Hoxa2 `tunes'
the size of the skeletal elements to be produced in the second arch by
regulating the domain of Six2 expression in this area.
Hoxa2 and the formation of the facial skeleton
Evidence accumulated in recent years indicates that Hox genes inhibit
development of the facial skeleton in the areas where they are expressed. Gain
of function of Hoxa2, both in pre-migratory neural crest (Couly,
2002) and in the facial mesenchyme (Kanzler, 1998), prevents the formation of
the facial skeleton. In the second branchial arch, its normal domain of
expression, Hoxa2 negatively regulates skeletal development
(Kanzler et al., 1998). If the
broad effects of Hoxa2 on skeletal development are attributable to a
general mechanism, we should expect the same molecular mediators acting
downstream of Hoxa2 in the facial mesenchyme and in the second
branchial arch. An important requisite for such mediators would be a broad
expression in the area fated to form head cartilages and bones. We would also
expect that gain of function of Hoxa2 would affect the spatial distribution of
such a mediator. Indeed, Six2 displays a widespread expression in the
craniofacial mesenchyme (Oliver et al.,
1995
) and its expression is downregulated following ectopic
expression of Hoxa2. However, while Hoxa2 indistinctly
inhibits bone and cartilage formation, overexpression of Six2 in the
second branchial arch produces ectopic cartilages but does not affect
intramembranous bone growth (the tympanic ring is fairly normal). Does
Six2 specifically promote cartilage formation? The craniofacial
defects of the Six1 mutant are not restricted to cartilage, but
affect bones as well (Laclef et al.,
2003b
; Ozaki et al.,
2004
). In addition, the domain of Six2 upregulation
around the otic vesicle is spatially associated with the squamous bone
duplication observed in the Hoxa2 mutant. Finally, the analysis of
Six mutants indicate that Six genes positively regulate cell proliferation,
which explains how these genes control processes as diverse as muscle
formation, retina development and skeletal development. On this basis, a
likely prediction would be that Six2 promotes both cartilage and bone
development. The lack of effect on bone development in our gain-of-function
experiment could be explained by the absence, in the wild-type second arch, of
a factor acting in concert with Six2. Alternatively, as the process of
intramembranous ossification begins later than chondrogenesis, Six2
overexpression might occur too early to affect bone formation. We favor the
second hypothesis, because a2-Six2 transgenics show a high level of
Six2 mRNA at E10.5; 1 day later, there are barely detectable
differences in Six2 mRNA levels between transgenics and wild-type
embryos (data not shown).
Repression of Six2 by Hoxa2
Despite the vast literature on Hox genes, the nature of the genes regulated
by Hox proteins in vertebrates is still largely unknown. Solving this riddle
is fundamental, if we want to explain how Hox genes control development in the
vertebrate embryo.
We have shown that Hoxa2 controls the Six2 gene at the transcriptional level, as indicated by the identification of a Six2 promoter fragment regulated by Hoxa2. As a transcription factor, Hoxa2 could directly regulate the Six2 promoter, and the observed binding of Hoxa2 to this promoter fragment in vitro strongly suggests that Six2 is a direct target of Hoxa2.
Within the Six2 promoter, Hoxa2 recognizes two GAATAAT motifs near
the transcription start site. The consensus Hoxa2 binding motif has not been
previously described, but the sequence recognized by Hoxa2 on the
Six2 promoter meets the requirements for Hox proteins binding to DNA
(Graba et al., 1997). The
interaction of Hox proteins with their target promoters often requires
co-factors such as Pbx (Mann and Affolter,
1998
), and we have indeed detected Pbx and Meis binding to the
Six2 promoter, in close proximity to the Hoxa2 binding sites. Another hint,
albeit indirect, that Six2 might be a direct target of Hoxa2 is that
the absence of Hoxa2 in the second arch specifically affects Six2
expression, leaving the levels of Six1, Six4 and Six5
unaffected.
Additional analyses will be required to definitely prove that Six2
is a direct target of Hoxa2. However, the experimental analysis of
Six2 regulation by Hoxa2 is complicated by the fact that Hoxa2
behaves as a repressor. Acting as a repressor, Hoxa2 might inhibit the basal
transcription machinery, counteract the activity of a positively acting
transcription factor or, alternatively, interact with proteins that remodel
chromatin (Gaston and Jayaraman,
2003). The elucidation of most of these mechanisms will be greatly
facilitated by the identification of the proteins acting as activators of
Six2. Currently, efforts in this direction are proceeding in our
laboratory.
Six2: a common molecular target of Hox genes?
Regulation of Six2 by Hox genes was described in the Hoxa11;
Hoxc11; Hoxd11 mutant, characterized by loss of metanephric kidney
induction (Wellik et al.,
2002). As in the Eya1 mutant
(Xu et al., 1999
), which has a
remarkably similar phenotype, in the Hox11 triple mutants
Six2 expression disappears
(Wellik et al., 2002
). This
effect is opposite to the one observed in the second arch, where
Hoxa2 represses Six2 expression.
Overexpression of Six2 under the Hoxa2 enhancer resulted in profound effects on the development of the skeleton patterned by Hox genes of paralogous group 3; some of the defects observed in Hox paralogous 3 single and compound mutants were mimicked by Six2 gain of function (consistent with ectopic expression of the transgene in the branchial arches and somitic mesoderm).
All together, these observations raise the possibility that Six2
expression might be under the control of other Hox genes in the development of
the vertebrate embryo. In Drosophila, common targets for different
Hox proteins have been described, as well as the ability of Hox proteins to
behave both as activators and repressors on their target genes (reviewed in
Graba et al., 1997). To learn
whether this holds true in vertebrates and if Six2 is a common target
of different Hox proteins will require a profile of Six2 expression patterns
in different Hox mutants, the entire spectrum of which is currently
available.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alonso, C. R. (2002). Hox proteins: sculpting body parts by activating localized cell death. Curr. Biol. 12,R776 -R778.[CrossRef][Medline]
Barrow, J. R. and Capecchi, M. R. (1999).
Compensatory defects associated with mutations in Hoxa1 restore
normal palatogenesis to Hoxa2 mutants.
Development 126,5011
-5026.
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.
Bonini, N. M., Bui, Q. T., Gray-Board, G. L. and Warrick, J.
M. (1997). The Drosophila eyes absent gene directs ectopic
eye formation in a pathway conserved between flies and vertebrates.
Development 124,4819
-4826.
Burke, A. C. (2000). Hox genes and the global patterning of the somitic mesoderm. Curr. Top. Dev. Biol. 47,155 -181.[Medline]
Carl, M., Loosli, F. and Wittbrodt, J. (2002).
Six3 inactivation reveals its essential role for the formation and patterning
of the vertebrate eye. Development
129,4057
-4063.
Carroll, S. B. (1995). Homeotic genes and the evolution of arthropods and chordates. Nature 376,479 -485.[CrossRef][Medline]
Chambers, D. and McGonnell, I. M. (2002). Neural crest: facing the facts of head development. Trends Genet. 18,381 -384.[CrossRef][Medline]
Chisaka, O. and Capecchi, M. R. (1991). Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5. Nature 350,473 -479.[CrossRef][Medline]
Condie, B. G. and Capecchi, M. R. (1993). Mice
homozygous for a targeted disruption of Hoxd-3 (Hox-4.1) exhibit anterior
transformations of the first and second cervical vertebrae, the atlas and the
axis. Development 119,579
-595.
Condie, B. G. and Capecchi, M. R. (1994).Mice with targeted disruptions in the paralogous genes hoxa-3 and hoxd-3 reveal synergistic interactions. Nature 370,304 -307.[CrossRef][Medline]
Couly, G., Grapin-Botton, A., Coltey, P. and le Douarin, N.
M. (1996). The regeneration of the cephalic neural crest, a
problem revisited: the regenerating cells originate from the contralateral or
from the anterior and posterior neural fold.
Development 122,3393
-3407.
Couly, G., Creuzet, S., Bennaceur, S., Vincent, C. and le Douarin, N. M. (2002). Interactions between Hox-negative cephalic neural crest 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]
Di Rocco, G., Mavilio, F. and Zappavigna, V.
(1997). Functional dissection of a transcriptionally active,
target-specific Hox-Pbx complex. EMBO J.
16,3644
-3654.
Ferretti, E., Marshall, H., Popperl, H., Machonochie, W.,
Krumlauf, R. and Blasi, F. (2000). Segmental expression of
Hoxb2 in r4 requires two separate sites that integrate cooperative
interactions between Prep1, Pbx and Hox proteins.
Development 127,155
-166.
Gammill, L. S. and Bronner-Fraser, M. (2003). Neural crest specification: migrating into genomics. Nat. Rev. Neurosci. 10,795 -805.[CrossRef]
Garcia-Bellido, A. (1975). Genetic control of wing disc development in Drosophila. Ciba Found. Symp. 29,161 -182.[Medline]
Gaston, K. and Jayaraman, P. S. (2003). Transcriptional repression in eukaryotes: repressor and repression mechanism. Cell. Mol. Life Sci. 60,721 -741.[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.[Medline]
Graba, Y., Aragnol, D. and Pradel, J. (1997). Drosophila Hox complex and the function of homeotic genes. Bioessays 19,379 -388.[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.
Grapin-Botton, A., Bonnin, M. A., McNaughton, L. A., Krumlauf,
R. and le Douarin, N. M. (1995). Plasticity of transposed
rhombomeres: Hox gene induction is correlated with phenotypic modifications.
Development 121,2707
-2721.
Heanue, T. A., Reshef, R., Davis, R. J., Mardon, G., Oliver, G.,
Tomarev, S., Lassar, A. B. and Tabin, C. J. (1999).
Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and
Six1, homologs of genes required for Drosophila eye formation.
Genes Dev. 13,3231
-3243.
Helms, J. A. and Schneider, R. A. (2003). Cranial skeletal biology. Nature 423,326 -331.[CrossRef][Medline]
Ikeda, K., Watanabe, Y., Ohto, H. and Kawakami, K.
(2002). Molecular interaction and synergistic activation of a
promoter by Six, Eya, and Dach proteins mediated through CREB binding protein.
Mol. Cell. Biol. 22,6759
-6766.
Kanzler, B., Kuschert, S. J., Liu, Y.-H. and Mallo, M.
(1998). Hoxa2 restricts the chondrogenic domain and
inhibits bone formation during development of the branchial area.
Development 125,2587
-2597.
Kawakami, K., Sato, S., Ozaki, H. and Ikeda, K. (2000). Six family genes-structure and function as transcription factors and their roles in development. Bioessays 22,616 -626.[CrossRef][Medline]
Klesert, T. R., Cho, D. H., Clark, J. I., Maylie, J., Adelman, J., Snider, L., Yuen, E. C., Soriano, P. and Tapscott, S. J. (2000). Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat. Genet. 25,105 -109.[CrossRef][Medline]
Köntges, G. and Lumsden, A. (1996).
Rhomboencephalic neural crest segmentation is preserved throughout
craniofacial ontogeny. Development
122,3229
-3242.
Krumlauf, R. (1994). Hox genes in vertebrate development. Cell 78,191 -201.[Medline]
Laclef, C., Hamard, G., Demignon, J., Souil, E., Houbron, C. and
Maire, P. (2003a). Altered myogenesis in Six1-deficient mice.
Development 130,2239
-2252.
Laclef, C., Souil, E., Demignon, J. and Maire, P. (2003b). Thymus, kidney and craniofacial abnormalities in Six 1 deficient mice. Mech. Dev. 120,669 -679.[CrossRef][Medline]
Lagutin, O. V., Zhu, C. C., Kobayashi, D., Topczewski, J.,
Shimamura, K., Puelles, L., Russell, H. R., McKinnon, P. J., Solnica-Krezel,
L. and Oliver, G. (2003). Six3 repression of Wnt signaling in
the anterior neuroectoderm is essential for vertebrate forebrain development.
Genes Dev. 17,368
-379.
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest. Cambridge, UK: Cambridge University Press.
Li, X., Perissi, V., Liu, F., Rose, D. W. and Rosenfeld, M.
G. (2002). Tissue-specific regulation of retinal and
pituitary precursor cell proliferation. Science
297,1180
-1183.
Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W. and Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426,247 -254.[CrossRef][Medline]
Liu, Y.-H., Ma, L., Wu, L.-Y., Luo, W., Kundu, R., Sangiorgi, F., Snead, M. and Maxson, R. (1994). Regulation of the Msx2 homeobox gene during mouse embryogenesis: a transgene with 439 bp of 5' flanking sequence is expressed exclusively in the apical ectodermal ridge of the developing limb. Mech. Dev. 48,187 -197.[CrossRef][Medline]
Mallo, M. (1997). Retinoic acid disturbs mouse middle ear development in a stage-specific fashion. Dev. Biol. 184,175 -186.[CrossRef][Medline]
Mallo, M. and Brändlin, I. (1997). Segmental identity can change independently in the hindbrain and rhombencephalic neural crest. Dev. Dyn. 210,146 -156.[CrossRef][Medline]
Mann, R. S. and Affolter, M. (1998). Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423-429.[CrossRef][Medline]
Manzanares, M. and Nieto, M. A. (2003). A celebration of the new head and an evaluation of the new mouth. Neuron 27,895 -898.[CrossRef]
Ng, L. J., Wheatley, S., Muscat, G. E., Conway-Campbell, J., Bowles, J., Wright, E., Bell, D. M., Tam, P. P., Cheah, K. and Koopman. P. (1997). SOX9 binds DNA, activates transcription and coexpresses with type II collagen during chondrogenesis in the mouse. Dev. Biol. 183,108 -121.[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.
Ohto, H., Kamada, S., Tago, K., Tominaga, S. I., Ozaki, H.,
Sato, S. and Kawakami, K. (1999). Cooperation of six and eya
in activation of their target genes through nuclear translocation of Eya.
Mol. Cell. Biol. 19,6815
-6824.
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.
Osoegawa, K., Mammoser, A. G., Wu, C., Frengen, E., Zeng, C.,
Catanese, J. J. and de Jong, P. J. (2001). A bacterial
artificial chromosome library for sequencing the complete human genome.
Genome Res. 11,483
-496.
Ozaki, H., Watanabe, Y., Takahashi, K., Kitamura, K., Tanaka,
A., Urase, K., Momoi, T., Sudo, K., Sakagami, J., Asano, M. et al.
(2001). Six4, a putative myogenin gene regulator, is not
essential for mouse embryonal development. Mol. Cell.
Biol. 21,3343
-3350.
Ozaki, H., Watanabe, Y., Ikeda, K. and Kawakami, K. (2002). Impaired interactions between mouse Eya1 harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins. J. Hum. Genet. 47,107 -116.[CrossRef][Medline]
Ozaki, H., Nakamura, K., Funahashi, J., Ikeda, K., Yamada, G.,
Tokano, H., Okamura, H. O., Kitamura, K., Muto, S., Kotaki, H. et al.
(2004). Six1 controls patterning of the mouse otic vesicle.
Development 131,551
-562.
Pasqualetti, M., Ori, M., Nardi, I. and Rijli, F.
(2000). Ectopic Hoxa2 induction after neural crest
migration results in homeosis of jaw elements in Xenopus.
Development 127,5367
-5378.
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. (1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91,881 -891.[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.
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.[Medline]
Ruf, R. G., Xu, P. X., Silvius, D., Otto, E. A., Beekmann, F.,
Muerb, U. T., Kumar, S., Neuhaus, T. J., Kemper, M. J., Raymond, R. M. et
al. (2004). SIX1 mutations cause branchio-oto-renal syndrome
by disruption of EYA1-SIX1-DNA complexes. Proc. Natl. Acad. Sci.
USA 101,8090
-8095.
Santagati, F. and Rijli, F. M. (2003). Cranial neural crest and the building of the vertebrate head. Nat. Rev. Neurosci. 10,806 -818.[CrossRef]
Scheidereit, C., Heguy, A. and Roeder, R. (1987). Identification and purification of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunoglobulin promoter in vitro. Cell 51,783 -793.[CrossRef][Medline]
Trainor, P. A. and Krumlauf, R. (2001). Hox genes, neural crest cells and branchial arch patterning. Curr. Opin. Cell. Biol. 13,698 -705.[CrossRef][Medline]
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.
Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S. and Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23,113 -117.[CrossRef][Medline]
Xu, P. X., Zheng, W., Huang, L., Maire, P., Laclef, C. and
Silvius, D. (2003). Six1 is required for the early
organogenesis of mammalian kidney. Development
130,3085
-3094.
Zakany, J. and Duboule, D. (1999). Hox genes in digit development and evolution. Cell Tissue Res. 296, 19-25.[CrossRef][Medline]
Zhao, Q., Eberspaecher, H., Lefebvre, V. and de Crombrugghe, B. (1997). Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev. Dyn. 209,377 -386.[CrossRef][Medline]
Zheng, W., Huang, L., Wei, Z. B., Silvius, D., Tang, B. and Xu,
P. X. (2003). The role of Six1 in mammalian auditory system
development. Development
130,3989
-4000.