|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 5 March 2008
doi: 10.1242/dev.017624
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Developmental Biology, Max-Planck Institute of Immunobiology,
Freiburg, Germany.
2 Department of Genetics and Tumor Cell Biology, St Jude Children's Research
Hospital, Memphis, TN, USA.
3 Faculty of Human and Medical Sciences, Stopford Building, The University of
Manchester, Manchester M13 9PT, UK.
* Author for correspondence (e-mail: Nicoletta.Bobola{at}manchester.ac.uk)
Accepted 25 February 2008
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Six2, Hoxa2, Branchial arch, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Hoxa2 and Hoxb2 exhibit the most-anterior expression domain,
in the cranial neural crest that migrates to the second branchial arch
(Prince and Lumsden, 1994;
Nonchev et al., 1996;
Mallo, 1997). In mice,
Hoxa2 loss-of-function leads to a transformation of second branchial
arch derivatives into the more anterior first branchial arch derivatives
(Gendron-Maguire et al., 1993;
Rijli et al., 1993;
Barrow and Capecchi, 1999). In
addition, ectopic expression of the homeobox gene Six2 was also
observed in these mutant embryos in territories normally controlled by
Hoxa2 (Kutejova et al.,
2005). This result, together with the demonstrated ability of
Hoxa2 to bind to the Six2 promoter in vitro
(Kutejova et al., 2005),
suggest that repression of Six2 by Hoxa2 is a crucial step in the
developmental pathway leading to second branchial arch formation.
Despite extensive genetic analysis, the molecular basis of Hox function is
proving difficult to understand. Together with Six2, other likely Hox
downstream targets have been identified in vertebrates
(Pearson et al., 2005;
Svingen and Tonissen, 2006),
but it remains unclear for most of these genes whether they are regulated
directly or indirectly by Hox proteins. With the exception of Hox genes
themselves, it is also currently unknown how the activities of the few genes
proven to be directly regulated by Hox proteins in vertebrate embryogenesis
contribute to the function of Hox proteins
(Serpente et al., 2005;
Salsi and Zappavigna, 2006;
Shaut et al., 2007). A
conclusive characterization of the nature of direct downstream genes is
essential to explain how Hox gene activities are converted into morphogenetic
processes and to understand the transcriptional properties of Hox proteins as
exerted on their target promoters. In addition, insight into the organization
and the hierarchy of the pathways controlled by Hox proteins in vertebrates
requires the analysis of the functional role of the direct downstream targets
in the Hox pathway.
Here, we conclusively show that Hoxa2 directly controls Six2 transcription in the second branchial arch. Lack of control over Six2 transcription contributes to the generation of the Hoxa2 mutant phenotype, with analysis of Six2; Hoxa2 double mutants indicating that Hoxa2 controls additional downstream targets. We identify components of the IGF molecular pathway as targets of Hoxa2 regulation and correlate the changes in Six2 expression with those in the expression of the gene encoding Igf-binding protein, Igfbp5, suggesting a role of Six2 in mediating Hoxa2 control over the IGF system.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed according to a standard
protocol (Upstate Biotechnology, Lake Placid, NY) with the following
modifications. Branchial arches and frontonasal mass of embryos were dissected
in PBS. After fixing in 1% formaldehyde for 23 minutes at 4°C, embryonic
tissues were desintegrated with a 25-gauge needle. The cross-linked material
was sonicated to 200-1000 bp fragments (Vibracell sonicator; seven times for
10 seconds at 40% output) and the immunoprecipitations were performed starting
with second branchial arches (50 pairs of second branchial arches from E10.5
embryos, or ten pairs of second branchial arches from E11.5 embryos) or five
pairs of first branchial arches together with frontonasal mass and 3 µg of
anti-Hoxa2 antibodies (43 or 44), 1 µg of anti-polymerase II antibody
(Santa Cruz Biotechnology, Santa Cruz, CA), 1 µg of anti-Pbx1 antibody
(Santa Cruz Biotechnology) or 3 µg of normal rabbit IgG. PCR amplifications
were performed using the following primers: forward,
5'-CTCGGGTTACCGGTGACTGACAGCGTCTCC-3' and reverse,
5'-CTCTCCCTCCCGTCTAGCTCGCTTGCAGCT-3' for the Six2
promoter; 5'-GGCTGACTTTGGAGATGACTC-3' and reverse,
5'-GAATGCCTGCTCTAACTGTTCAC-3' for the IP10
(Cxcl10 - Mouse Genome Informatics) promoter.
Mutant animals and phenotypic analyses
Hoxa2-null and Six2-null mutant mice have been described
(Gendron-Maguire et al., 1993;
Self et al., 2006).
900Six2-lacZ transgenic mice and the a2-Six2 transgene are
described by Kutejova et al. (Kutejova et
al., 2005). The a2-Six2 transgenic embryos were derived
from a founder with no apparent phenotypic defects, which transmitted the
transgene to the F2, causing perinatal lethality and the skeletal defects
expected by overexpression of Six2
(Kutejova et al., 2005).
Skeletal phenotypes were analyzed by Alcian Blue/Alizarin Red staining as
described (Mallo and Brändlin,
1997). Whole-mount and tissue sections were analyzed by in situ
hybridization as described (Kanzler et
al., 1998), using Igfbp5
(Bobola and Engist, 2008) and
Igf1 (Weger and Schlake,
2005) probes. RT-PCR on second branchial arches of E10.5 embryos
from a2-Six2 transgenics was performed as described
(Kutejova et al., 2005).
Animals experiments were approved by the ethics committee of the
Regierungspräsidium Freiburg.
|
). HEK 293 cells were transfected using the calcium phosphate method, cultured for an additional 36 hours and lysed in buffer comprising 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 1% NP40. Branchial arches and frontonasal mass of embryos were dissected in DMEM (Sigma) and total proteins were extracted using Trizol (Invitrogen) according to manufacturer's instructions. The membranes were probed with anti-Pbx1 antibody (Santa Cruz Biotechnology) diluted 1:100.
Electrophoretic mobility shift assays were performed using T7-coupled TNT
rabbit reticulocytes (Promega). The BstEII/SspI (from -181
to -48) fragment of the Six2 promoter is described by Kutejova et al.
(Kutejova et al., 2005). The
oligonucleotide reproducing the sequence of the Six5 binding site in the
Igfbp5 promoter has been described
(Sato et al., 2002); the
sequence of the mutant oligonucleotide is
5'-TGGGTGTTGGGGAGCGCAAATTGCAGCTA-3'.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Mallo, 1997;
Nonchev et al., 1996), second
branchial arches were isolated from E11.5 wild-type embryos. At this stage,
Hoxa2 function is still required for second arch development
(Santagati et al., 2005) and
Six2 is ectopically expressed in this territory in the absence of
Hoxa2 (Kutejova et al., 2005).
As negative controls, we used two embryonic regions colonized by Hox-negative
cranial neural crest (Le Douarin and
Kalcheim, 1999): first branchial arches and frontonasal mass
(hereafter referred to as first arch, Fig.
1C). Cross-linked, sheared chromatin from second and first arches
was immunoprecipitated using the two Hoxa2 polyclonal antibodies and analyzed
by PCR for the presence of the highly conserved Six2 chromatin region
recognized by Hoxa2 in vitro (Kutejova et
al., 2005) (Fig.
1D). Second branchial arch immunoprecipitated chromatin showed a
substantial enrichment for the most-proximal Six2 promoter region. No
enrichment was detected with chromatin from first arches or from that
immunoprecipitated in the presence of unrelated antibodies. Similar results
were obtained with both of the polyclonal antibodies directed against Hoxa2
(Fig. 1E).
The earliest stage at which we could detect Hoxa2 bound to the
Six2 promoter was E10.5, corresponding to the appearance of ectopic
Six2 expression in the mutant second branchial arch
(Kutejova et al., 2005):
second branchial arch chromatin immunoprecipitated in the presence of Hoxa2
polyclonal antibody showed a significant enrichment for the most-proximal
Six2 promoter region, whereas no enrichment was detected for an
unrelated, control promoter (Fig.
1G). ChIP analysis of E9.5 embryos revealed no enrichment in
Six2 promoter in the presence of the specific antibody (not shown).
These results demonstrate that at the stages (E10.5-11.5) when Hoxa2 actively
represses Six2 transcription in the second branchial arch
(Kutejova et al., 2005), Hoxa2
is bound to the Six2 regulatory region in vivo.
|
). In
vertebrates, Hox proteins bind in a complex with Pbx1 on a Hox/Pbx bipartite
binding site that is essential for the activity of the Hoxb1 and
Hoxb2 enhancers (Jacobs et al.,
1999; Ferretti et al.,
2000).
The Six2 promoter contains a Pbx/Meis binding site located a few
nucleotides upstream of the binding sites recognized by Hoxa2 in vitro
(Kutejova et al., 2005). ChIP
assay using a Pbx1-specific antibody indicated that Pbx1 is bound to the
Six2 promoter in vivo (Fig.
2A). Moreover, Pbx1 was similarly detected on the Six2
promoter in immunoprecipitated chromatin extracted from embryonic areas where
Hoxa2 is present (second branchial arches) and from areas where Hoxa2, or any
other Hox proteins, are absent (first branchial arches and frontonasal mass)
(Fig. 2A), indicating that the
recruitment of Pbx1 to the Six2 promoter does not depend on Hox
proteins. The presence of the Pbx1 protein isoforms was confirmed in all
embryonic areas examined (Fig.
2B-D).
Does Hoxa2 interfere with a Six2 autoregulatory mechanism?
Transcriptional repression of a target promoter can be achieved by a
variety of mechanisms (Gaston and
Jayaraman, 2003), some of which are difficult to investigate
without identifying the proteins acting as activators of Six2. Six2
is expressed in a large domain in the first branchial arch and in a restricted
one in the second branchial arch. Upon Hoxa2 inactivation, an
identical Six2 expression pattern is observed in first and second
arches (Kutejova et al.,
2005), suggesting that the mechanism of activation is the same in
both domains.
We and others previously showed that 1 kb of Six2 promoter is
sufficient to recapitulate Six2 endogenous expression in various
embryonic sites, including the branchial arches
(Brodbeck et al., 2004;
Kutejova et al., 2005). This
promoter fragment is activated by Six2 and contains conserved Six-binding
sites that are recognized by Six2 in vitro
(Brodbeck et al., 2004) (N.B.
and E.K., unpublished), suggesting that Six2 activity in the
branchial arches might rely on an autoregulatory loop of Six2 protein on its
own promoter. To investigate whether Six2 controls its promoter in vivo, we
introduced the 900Six2-lacZ transgene, containing the first 900 bp of
the Six2 promoter fused to a lacZ reporter gene
(Kutejova et al., 2005), into
the Six2 mutant background (Self
et al., 2006). As shown in Fig.
3, lacZ expression was unchanged in the absence of Six2
in the branchial arches, maxilla and limbs at the stages examined. These
results indicate that Six2 is not necessary to maintain the activity of its
own promoter in these embryonic areas.
The Six proteins share a conserved homeodomain, recognize similar binding
sites and can substitute for each other in vivo and in vitro
(Spitz et al., 1998;
Ando et al., 2005;
Grifone et al., 2005;
Giordani et al., 2007;
Kobayashi et al., 2007). Owing
to the presence of other Six proteins in the branchial arches and their known
capacity to compensate for each other, our experimental system cannot
definitively rule out the possibility that a Six-dependent activation
mechanism is nevertheless in place for the Six2 promoter. Within the
Six2 promoter, two Six2 binding sites overlap with or are in close
proximity to Hoxa2 binding sites (Brodbeck
et al., 2004) (Fig.
3E). In addition, Hoxa2 targets the 1 kb promoter fragment in vivo
(Kutejova et al., 2005).
However, an analysis of whether Hoxa2 might interfere with the binding of Six
proteins to the Six2 promoter revealed no change in the binding of
Six2 to its promoter in the presence of increasing concentrations of Hoxa2
(Fig. 3F), despite the close
arrangement of Six and Hoxa2 binding sites on the promoter.
Ectopic expression of Six2 contributes to the Hoxa2 mutant phenotype
In the absence of Hoxa2, the skeletal derivatives of the second branchial
arch do not form and are replaced by cartilage and bone that resemble, in
shape and position, first arch skeletal derivatives
(Gendron-Maguire et al., 1993;
Rijli et al., 1993;
Barrow and Capecchi, 1999).
Since Hoxa2 in the second arch is found to be associated with the
Six2 promoter and negatively regulates its transcription
(Kutejova et al., 2005), we
asked whether the upregulation of Six2 observed in the absence of
Hoxa2 is responsible for the Hoxa2 mutant phenotype. To test this
possibility, we generated double Hoxa2; Six2-null mice. No obvious
abnormalities are detected in first and second arch skeletal derivatives of
Six2-null pups (Self et al.,
2006) (data not shown). Double Hoxa2; Six2-null mutants
were obtained by crossing compound heterozygotes, as Hoxa2 and
Six2 single mutants die shortly after birth
(Gendron-Maguire et al., 1993;
Rijli et al., 1993;
Barrow and Capecchi, 1999;
Self et al., 2006). Analysis
of middle-ear skeletal preparations from double-null newborns showed that the
removal of Six2 activity partially rescued the Hoxa2
phenotype. As shown in Fig. 4,
the gonial bone, which in the Hoxa2 single mutant abnormally extends
to connect the tympanic ring and its duplication
(Gendron-Maguire et al., 1993;
Rijli et al., 1993;
Barrow and Capecchi, 1999), was
reduced to as much as normal size; the size of the duplicated mallei was also
reduced in some of the double-mutant embryos. We observed incomplete
penetrance in the extent of the rescue of the double-mutant phenotype as well
as variability within the same embryo, ruling out background-dependent effects
(Fig. 4D,
Table 1). Complete rescue of
the ectopic growth of the gonial bone could be observed already upon removal
of a single Six2 allele (Fig.
4E). Other aspects of the Hoxa2 phenotype
(Gendron-Maguire et al., 1993;
Rijli et al., 1993;
Barrow and Capecchi, 1999)
remained unaffected. The partial rescue shows that one of the functional
mechanisms by which Hoxa2 participates in the formation of the second
branchial arch is via its repression of Six2 expression in that
territory.
|
|
|
;
Li et al., 2002;
Lagutin et al., 2003;
Brodbeck et al., 2004;
Ando et al., 2005;
Chai et al., 2006;
Giordani et al., 2007;
Kobayashi et al., 2007),
including Igf2 and Igfbp5, genes encoding components of the
IGF system (Sato et al.,
2002). IGF positively regulates bone growth
(Liu et al., 1993) and removal
of Six2 from the Hoxa2 mutant mainly affects the growth of
the ectopic gonial bone; moreover, IGF is required for the normal development
of intramembranous bones that are part of, or develop in close proximity to,
the middle-ear skeleton (Louvi et al.,
1997). For these reasons, we analyzed the expression of various
IGF components in wild-type and Hoxa2 mutant embryos. Whereas
Igf1 was upregulated in Hoxa2 mutant embryos, in which
ectopic expression was detected below the developing otic vesicle at E11.5
(Fig. 5A,B), expression of
Igfbp5, which appears in the second arch mesenchyme by E11.5, was
strongly reduced in the absence of Hoxa2
(Fig. 5C,D). The other
components of the IGF system examined (Igf2, Igfbp2, Igfbp3, Igfbp4)
showed no change at this stage (data not shown). These results show that Hoxa2
acts upstream of the IGF system in the second branchial arch where it
controls, directly or indirectly, the expression of Igf1 and of the
gene encoding the Igf-binding protein, Igfbp5.
|
), we tested
whether Six2 might act upstream of Igfbp5 and account for the
Igfbp5 downregulation observed in Hoxa2 mutant embryos. For
these experiments, we analyzed Igfbp5 expression in embryos
transgenically expressing Six2 in the second branchial arch, rather
than using the double mutants whose phenotypic variability precludes rigorous
analysis of Six2 molecular function. In the transgenic embryos, Six2
overexpression results in phenotypic characteristics of the Hoxa2
mutant (Kutejova et al.,
2005). Igfbp5 expression was reduced in the second
branchial arch of E11.5 embryos overexpressing Six2 (n=3,
Fig. 5E,F). Igfbp5
expression was also noticeably reduced in the anterior forelimb, an area where
the expression of the transgene generates a strong phenotype (not shown). The
embryos analyzed were derived from the same founder, allowing a direct
correlation between Six2 expression levels
(Fig. 5G), the observed
phenotypic effects (Kutejova et al.,
2005) and the effects on Igfbp5 mRNA levels. These results indicate that Hoxa2 regulates the IGF pathway in the second branchial arch and suggest at least a partial role for Six2 in mediating this Hoxa2 function.
Six2 interacts directly with the Igfbp5 promoter in vitro
Regulation of Igfbp5 by Six5, a Six2 homolog, is mediated by
direct binding to a short, perfectly conserved sequence in the Igfbp5
proximal promoter (Sato et al.,
2002). To test whether Six2 directly represses Igfbp5 via
binding to the Igfbp5 promoter, we performed an electrophoretic
mobility shift assay (EMSA) using the Six5 binding site identified in the
Igfbp5 promoter as a probe. Whereas no binding was detected when the
probe was incubated with unprogrammed reticulocytes, incubation of the probe
with in vitro translated Six2 resulted in the formation of a retarded complex
(Fig. 6A). This complex
represents the specific interaction of Six2 with the probe because its
formation was competed by an excess of cold wild-type oligonucleotide at
different molar concentrations. The same molar excess of a mutant
oligonucleotide, containing one nucleotide substitution in the Six5 binding
site (Fig. 6C), left the
complex unaffected (Fig. 6A).
Finally, no binding was detected when Six2 was incubated in the presence of
the mutant oligonucleotide used as a probe
(Fig. 6B).
These data show that Six2 recognizes the Six5 binding site in the Igfbp5 promoter and that this interaction is sequence-specific, and suggest that Six2 could directly repress Igfbp5 transcription.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
),
indicate that Pbx1 and Hoxa2 bind independently of each other on the
Six2 promoter, where they recognize two separate sites. Although this
finding is in apparent contrast with previous studies in vertebrates, in which
Pbx and Hox proteins bind in a complex on specific Hox/Pbx bipartite sites to
regulate the Hoxb1 and the Hoxb2 enhancers, it should be
noted that: (1) the number of Hox targets analyzed in vertebrates to date is
too small to extract general rules about the transcriptional properties of Pbx
and Hox proteins on their target promoters; and (2) Hoxb1 and
Hoxb2 are activated by Hox proteins and repression might require a
different binding architecture of Pbx and Hox proteins on the promoter.
Pbx1 mutant mice display defects in the neural crest-derived
skeletal elements of the second branchial arch. One of these defects, the
elongation of the lesser horn of the hyoid bone, is phenocopied by transgenic
embryos overexpressing Six2 in the second arch
(Selleri et al., 2001;
Kutejova et al., 2005). This
is suggestive of a role for Pbx1 in controlling Six2 levels in the
second branchial arch. Insight into the functional relevance of Six2
promoter occupancy by Pbx1 in the absence of Hox proteins, and of the
simultaneous presence on Six2 promoter of Pbx1 and Hoxa2 for
Six2 regulation, awaits analyses in single Pbx1 and combined
Hoxa2/Pbx1 mutants.
The molecular mechanism responsible for Six2 activation in the
first branchial arch (and most likely in the second branchial arch of the
Hoxa2 mutant) is unknown. Although activation of Six2
expression through the cooperation of Eya1, Pax2 and Hoxa11 has recently been
described in kidney development (Gong et
al., 2007), the situation is rather different in the branchial
arches, where Hoxa2 acts as a repressor. In addition, Eya1 expression is
mainly restricted to the epithelia in this embryonic area, whereas
Six2 is expressed in the mesenchyme
(Kutejova et al., 2005) (data
not shown). The capacity of Six2 to activate the promoter fragment that
recapitulates Six2 expression in the branchial arches instead
suggests the involvement of Six proteins in Six2 promoter activation
in vivo (Brodbeck et al., 2004;
Kutejova et al., 2005). We
have ruled out any Six2 requirement for Six2 transcription, but we
cannot exclude the in vivo relevance of Six-mediated activation owing to the
likely compensatory effect between other Six proteins present in the branchial
arches (Grifone et al., 2005).
However, even if such a mechanism does activate the Six2 promoter,
Hoxa2 repression of Six2 transcription does not appear to be the
result of mutually exclusive binding of Hoxa2 and Six proteins to their
closely spaced binding sites on the Six2 promoter. The discovery and
analysis of additional Hoxa2 targets will undoubtedly shed light on the
transcriptional properties of Hoxa2 and the requirements to switch from a
repressor to an activator role.
Molecular function of Six2 in the second branchial arch
Ectopic expression of Six2 in the second branchial arch reproduces
a molecular aspect of the Hoxa2 mutant phenotype, i.e. downregulation
of the gene encoding the Igf-binding protein, Igfbp5. A possible
direct regulation of Igfbp5 by Six2 is consistent with the ability of
Six5 and Six1 to activate the Igfbp5 promoter
(Sato et al., 2002), and is
supported by the ability of Six2 to recognize the Six5 binding site in the
Igfbp5 promoter. The opposite effects of Six5 and Six2 on
Igfbp5 expression are in line with the documented ability of Six
proteins to function both as transcriptional activators and repressors
(Li et al., 2003;
Brugmann et al., 2005).
Together with Igfbp5, Igf1 is also affected in the Hoxa2
mutant. The IGF system positively controls bone development and growth, with
cranial and facial bones displaying the most dramatic defects in the absence
of IGF signaling (Liu et al.,
1993; Louvi et al.,
1997). The activity of the IGF system is regulated by six
insulin-like growth factor-binding proteins able to bind Igf1 and Igf2
directly and to control the pool of free IGF proteins available for
interaction with the cognate receptors to transduce the signal in target
cells. In most cases, this interaction leads to down-modulation of IGF
signaling (Clemmons, 1998;
Collett-Solberg and Cohen,
2000). We indeed found that expression of Igfbp5
negatively affects bone development and growth in the craniofacial area
(Bobola and Engist, 2008),
where it partially reproduces the effects of Hoxa2 overexpression
(Kanzler et al., 1998). In
addition, these effects are IGF-dependent
(Bobola and Engist, 2008).
Hoxa2 control of second arch skeletal development could be exerted, at least
in part, via a decrease in IGF signaling, resulting from downregulation of
Igf1 and upregulation of its potential negative regulator
Igfbp5. An increase in IGF signaling is expected to result in
increased bone formation, which is indeed a phenotypic characteristic of the
Hoxa2 mutant (Kanzler et al.,
1998).
Alternative functional organization downstream of Hoxa2
Removal of ectopic Six2 expression from the Hoxa2 mutant
indicates that Six2 is only one of the genes controlled by Hoxa2 and
that Hoxa2 regulates second arch development by activation and/or repression
of additional targets. In addition, the variability observed in the rescue of
the gonial bone phenotype in Hoxa2; Six2-null mutants indicates a
high degree of redundancy, with other genes able to substitute for
Six2 function.
The double-mutant phenotype can be explained by either of two models. The
simplest interpretation of the rescue observed in the double mutant is that
the ectopic expression of Six2 specifically promotes the growth of
the gonial bone. The rescue of the double mutant is limited because
Six2 function is restricted to the control of a specific process.
Hoxa2 also regulates other as yet unknown genes, the loss- or gain-of-function
of which in the absence of Hoxa2 would promote some of the various specific
phenotypes observed in Hoxa2 mutant mice. However, because the
ectopic gonial bone is also the phenotypic component most sensitive to Hoxa2
dosage (Santagati et al.,
2005), a rescue in this aspect of the phenotype might simply
reflect the immediate readout of the phenotype to any change introduced into
the Hoxa2 mutant (in this specific case, the loss of Six2
ectopic activity). In this alternative model, Six2 has broader
effects on the development of the second branchial arch and the limited rescue
does not rest in the control by Six2 of a restricted aspect of the
phenotype, but rather in Six2 acting redundantly with other genes
that can partially compensate for modifications in its activity. Additional
observations are in support of a broader function for Six2 in the
generation of the Hoxa2 phenotype, beyond that in gonial bone growth.
Contrary to the predominant rescue of intramembranous bone formation in the
double mutant lacking Six2, Six2 ectopic expression in the second
branchial arch (Kutejova et al.,
2005) expands the chondrogenic domains and affects the size and
shape of second arch cartilages. The main effect of Six2 on cartilage
when ectopically expressed can be explained by the transient expression of
Six2 in the second arch of these wild-type embryos (supplied with a
functional Hoxa2 protein), in which expression declines before it affects
later developmental processes, such as intramembranous bone formation. The
analysis of Six2 function in different experimental systems
indicates, therefore, that Six2 can control both chondrogenesis and
intramembranous bone formation in the second arch, i.e. the different
processes that contribute to the Hoxa2 mutant phenotype. Indeed,
removal of Six2 from the Hoxa2 mutant partially rescues the
duplication of the malleus, albeit at a lower frequency than for the rescue in
the gonial bone phenotype. A broader function of Six2 in the
generation of the Hoxa2 phenotype is also supported by Six2
spatiotemporal expression in the mutant second arch
(Kutejova et al., 2005).
The proposed molecular function of Six2, i.e. to control Igfbp5,
is compatible with both models. IGF signaling could have global effects on the
development of the second arch, exerted through a direct effect on bone
formation, or mediated through cell proliferation, with a final impact on both
chondrogenesis and intramembranous bone formation. In that case, any change in
IGF signaling would most likely be perceived first by the aspect of the
phenotype most sensitive to changes, i.e. the ectopic gonial bone. It seems
reasonable to assume that control over a broad mechanism such as IGF signaling
is diverse and likely to involve several genes. Six2 could be one of
those genes, acting to decrease the levels of the Igf-binding protein Igfbp5.
Removal of the ectopic Six2 would only partially affect the state of
IGF signaling, owing to the presence of the additional regulators that
compensate for the loss of Six2, explaining the variability of the phenotype.
Alternatively, Igfbp5 function in the second arch could be restricted
to the growth of the gonial bone by local inhibition of IGF signaling, in
which case repression of Igfbp5 by Six2 would directly lead to
increased growth of the gonial bone. Overall, the wide-ranging effects of
Six2 on second arch skeletogenesis, the broad expression of this gene
in the mutant second arch, and the apparent redundancy of its function favor
the hypothesis that Six2 is part of a network of genes with
overlapping functions exerted downstream of Hoxa2. The conclusive
identification of Six2 as a direct target of Hoxa2, together with the
only partial rescue observed in the double mutant, points to the requirement
for Hoxa2-mediated activation and/or repression of other target genes in
addition to Six2. A redundant organization downstream of Hoxa2 would
preclude the identification of its target genes on the basis of analyses of
their separate functions and would require a more complicated experimental
approach, i.e. the generation of triple and quadruple mutants. In the light of
these perspectives, the lack of rescue of the skeletal phenotype in double
mutants of Hoxa2 and in two additional previously identified targets
(Bobola et al., 2003) (M.
Mallo, personal communication) might warrant a revisit.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ando, Z., Sato, S., Ikeda, K. and Kawakami, K. (2005). Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4. FEBS J. 272,3026 -3041.[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.[Abstract]
Bobola, N. and Engist, B. (2008). IGFBP5 is a potential regulator of craniofacial skeletogenesis. Genesis 46,52 -59.[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.
Brodbeck, S., Besesnbeck, B. and Englert, C. (2004). The transcription factor Six2 activates expression of the Gdnf gene as well as its own promoter. Mech. Dev. 121,1211 -1222.[CrossRef][Medline]
Brugmann, S. A., Pandur, P. D., Kenyon, K., Pignoni, F. and Moody, S. (2005). Six1 promote a placodal fate within the lateral neurogenic ectoderm by functioning both as a transcriptional activator and repressor. Development 131,5871 -5881.[CrossRef]
Chai, L., Yang, J., Di, C., Cui, W., Kawakami, K., Lai, R. and
Ma, Y. (2006). Transcriptional activation of the SALL1 by the
human SIX1 homeodomain during kidney development. J. Biol.
Chem. 281,18918
-18926.
Clemmons, D. (1998). Role of insulin-like growth factor binding proteins in controlling IGF action. Mol. Cell. Endocrinol. 140,19 -24.[CrossRef][Medline]
Collett-Solberg, P. F. and Cohen, P. (2000). Genetics, chemistry and function of the IGF/IGFBP system. Endocrine 12,121 -136.[CrossRef][Medline]
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.[Abstract]
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.[CrossRef][Medline]
Giordani, J., Bajard, L., Demignon, J., Daubas, P., Buckingham,
M. and Maire, P. (2007). Six proteins regulate the activation
of Myf5 expression in embryonic mouse limbs. Proc. Natl. Acad. Sci.
USA 104,11310
-11315.
Gong, K.-Q., Yallowitz, A. R., Sun, H., Dressler, G. R. and
Wellik, D. M. (2007). A Hox-Eya-Pax complex regulates early
kidney developmental gene expression. Mol. Cell. Biol.
27,7661
-7668.
Grifone, R., Demignon, J., Houbron, C., Souil, E., Niro, C.,
Seller, M. J., Hamard, G. and Maire, P. (2005). Six1 and Six4
homeoproteins are required for Pax3 and Mrf expression during myogenesis in
the mouse embryo. Development
132,2235
-2249.
Jacobs, Y., Schnabel, C. A. and Cleary, M.
(1999). Trimeric association of Hox and TALE homeodomain proteins
mediates Hoxb2 hindbrain enhancer activity. Mol. Cell.
Biol. 19,5134
-5142.
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.[Abstract]
Kobayashi, H., Kawakami, K., Asashima, M. and Nishinakamura, R. (2007). Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects. Mech. Dev. 124,290 -303.[CrossRef][Medline]
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.
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, J.-P., Baker, J., Perkins, A. S., Robertson, E. J. and Efstratiadis, A. (1993). Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75,59 -72.[Medline]
Louvi, A., Accili, D. and Efstratiadis, A. (1997). Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev. Biol. 189,33 -48.[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]
Moens, C. and Selleri, L. (2006). Hox cofactors in vertebrate development. Dev. Biol. 291,193 -206.[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.[Abstract]
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.[Abstract]
Pearson, J. C., Lemons, D. and McGinnis, W. (2005). Modulating Hox gene functions during animal body patterning. Nat. Rev. Genet. 6, 893-904.[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.[Abstract]
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]
Salsi, V. and Zappavigna, V. (2006). Hoxd13 and
Hoxa13 directly control the expression of the EphA7 Ephrin tyrosine kinase
receptor in developing limbs. J. Biol. Chem.
281,1992
-1999.
Santagati, F., Minoux, M., Ren, S. Y. and Rijli, F. M.
(2005). Temporal requirement of Hoxa2 in cranial neural crest
skeletal morphogenesis. Development
132,4927
-4936.
Sato, S., Nakamura, M., Cho, D. H., Tapscott, S. J., Ozaki, H.
and Kawakami, K. (2002). Identification of transcriptional
targets for Six5: implication for the pathogenesis of myotonic dystrophy type
1. Hum. Mol. Gen. 11,1045
-1058.
Self, M., Lagutin, O. V., Bowling, B., Hendrix, J., Cai, Y., Dressler, G. R. and Oliver, G. (2006). Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. EMBO J. 25,5214 -5228.[CrossRef][Medline]
Selleri, L., Depew, M. J., Jacobs, Y., Chanda, S. K., Tsang, K. Y., Cheah, K. S., Rubenstein, J. L., O'Gorman, S. and Cleary, M. L. (2001). Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation. Development 128,3543 -3557.[Medline]
Serpente, P., Tumpel, S., Ghyselinck, N. B., Niederreither, K.,
Wiedemann, L. M., Dolle, P., Chambon, P., Krumlauf, R. and Gould, A. P.
(2005). Direct crossregulation between retinoic acid receptor
β and Hox genes during hindbrain segmentation.
Development 132,503
-513.
Shaut, C. A., Saneyoshi, C., Morgan, E. A., Knosp, W. M., Sexton, D. R. and Stadler, H. S. (2007). HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endothelia. Dev. Dyn. 236,951 -960.[CrossRef][Medline]
Spitz, F., Demignon, J., Porteu, A., Kahn, A., Concordet, J. P.,
Daegelen, D. and Maire, P. (1998). Expression of myogenin
during embryogenesis is controlled by Six/sine oculis homeoproteins through a
conserved MEF3 binding site. Proc. Natl. Acad. Sci.
USA 95,14220
-14225.
Svingen, T. and Tonissen, K. F. (2006). Hox transcription factors and their elusive mammalian gene targets. Heredity 97,88 -96.[CrossRef][Medline]
Weger, N. and Schlake, T. (2005). Igf-I signalling controls the hair growth cycle and the differentiation of hair shafts. J. Invest. Dermatol. 125,873 -882.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
