1 Hubrecht Laboratory, The Netherlands Institute for Developmental Biology,
Uppsalalaan 8, 3584CT Utrecht, The Netherlands
2 Departments of Genetics and Pediatrics, Stanford University School of
Medicine, Stanford, CA, USA
Author for correspondence (e-mail:
frits{at}niob.knaw.nl)
Accepted 2 February 2005
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SUMMARY |
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Key words: Aristaless-like genes, Gli3, Mouse mutants, Pax genes, Scapula, Skeletogenesis, Tbx15
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Introduction |
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Relatively few mutants display disturbed pectoral girdle development, and
even less show total absence of one or more elements. Pellegrini et al.
reported a total lack of the scapula blade and ilium in Emx2 mutants,
whereas the remainder of the limb remained mostly intact
(Pellegrini et al., 2001).
Despite this striking observation it was recently concluded from detailed
functional analyses in chick that Emx2 is not sufficient for inducing
shoulder structures (Pröls et al.,
2004
). These authors described its role as providing positional
signals to cells fated to contribute to the skeletal elements of the shoulder.
Loss of function of the forelimb-specific gene Tbx5 can lead to total
absence of the limb, including the scapula
(Ahn et al., 2002
;
Ng et al., 2002
;
Rallis et al., 2003
;
Takeuchi et al., 2003
). As
this phenotype resulted from an experimental design where loss of function was
restricted to limb bud mesoderm, it was suggested that a disturbed recruitment
of cells caused the absence of the scapula
(Rallis et al., 2003
). Both of
these examples underscore the notion that even after the correct
laying down of progenitor structures disruption of positional cues may
suffice to totally ablate skeletal elements of the shoulder.
Genes functionally linked to shoulder formation by less dramatic phenotypes
in loss-of-function mutants include the Paired box gene Pax1, which
is allelic with the classical undulated mutant
(Balling et al., 1988),
Hoxa5 (Aubin et al.,
1998
), the mutant of which has a reduced acromion, Pbx1,
the mutant of which has a reduced scapular blade
(Selleri et al., 2001
), and
the Polycomb homolog M33, the mutant of which has a hole in the scapular blade
(Core et al., 1997
). Such a
scapular foramen may also be present in embryos homozygous for the Extra
toes (Xt) mutation, which is allelic with Gli3
(Hui and Joyner, 1993
;
Johnson, 1967
), and with
near-complete penetrance in de (droopy ear) mutants
(Curry, 1959
). We recently
demonstrated that de is allelic with Tbx15
(Candille et al., 2004
). Very
recently, and after the first submission of the present manuscript, a very
thorough description of the skeletal phenotype of de was published
(Singh et al., 2005
).
Here we show that the aristaless-related genes Alx4 and
Cart1 previously linked to functions in limb and craniofacial
development (Qu et al., 1999;
Beverdam et al., 2001
;
Meijlink et al., 2003
)
are implicated in shoulder girdle development, a role that was previously
concealed by the redundancy of their functions. We investigated in more detail
the phenotype of Tbx15 mutants, exploring its genetic interactions
with Gli3, Alx4 and Cart1. In spite of the similar scapular
phenotypes of Gli3 and Tbx15 mutants, the very strong defect
seen in Tbx15/Gli3 embryos suggests a synergistic functional
relationship between these two genes. By contrast, the phenotype seen in
Tbx15/Alx4/Cart1 triple mutants appears to be a
mere addition of phenotypes seen in the single mutants. This suggests
complementary functions, and a connection to parallel processes of
Gli3 on the one hand, and to Alx4 and Cart1 on the
other. Expression of several genes that represent markers for the different
cell types contributing to the shoulder elements, including the chondrocyte
marker Pax1, were affected in Gli3, Tbx15 and
Alx4/Cart1 mutants. Strikingly, in
Alx4/Cart1 mutants Pax1 expression was ventrally
shifted. In addition, the myogenic marker Pax3, whose expression
domain does not overlap with that of Alx4 and Cart1, was
abnormally expressed in Alx4/Cart1 but not Tbx15 or
Gli3 mutants. These results appear to point to a disruption of local
signals, implying that in these mutants, ectopic positional cues are causative
of the skeletal defects.
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Materials and methods |
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Mice were bred in a mixed genetic background. Most of the phenotypes observed were influenced by this variable background, but this variability was minor when compared with the morphological consequences seen in comparisons between single and double mutants. Moreover, conclusions on genotype-phenotype relationships were always based on, or consistent with, comparisons of embryos within one litter, in which the contribution of alleles of unlinked modifier genes should be essentially identical.
Nomenclature and definitions
For the sake of simplicity, throughout this paper we refer to
deH mutants as `Tbx15 mutants', to
LstJ mutants as `Alx4 mutants', and to
XtJ mutants as `Gli3 mutants'. The terms anterior
and posterior with respect to the scapular blade may seem ambiguous, but refer
in this paper to supraspinatus fossa and infraspinatus
fossa, respectively.
Analysis of embryos and newborn mice
Bone and cartilage staining was done essentially as described
(Beverdam et al., 2001).
Whole-mount in situ hybridization using digoxigenin-labeled RNA probes was
performed as described (Leussink et al.,
1995
; Ten Berge et al.,
1998a
), and in situ hybridization on sections was essentially as
described (Gregorieff et al.,
2004
). Probes used included Alx4, Gli3
(Te Welscher et al., 2002
),
Pax1 (Balling et al.,
1988
), Pax3 (Goulding
et al., 1991
), Emx2
(Simeone et al., 1992
),
Scleraxis (Cserjesi et al.,
1995
) and Tbx15. To prepare a Tbx15 in situ
probe, a 762-bp fragment corresponding to part of the last exon was
PCR-amplified from genomic DNA and subcloned in a pGEM-T vector (Promega,
Madison, WI). Primers used were 5'-CCCTTCAACTAATAATCAGC-3'
(forward) and 5'-GAAGCCAAGTCCAGGTGTAGC-3' (reverse).
Proliferating cells were detected using rabbit anti-phospho-histone H3 (Upstate, Charlottesville, VA, USA), according to manufacturer's protocol. Cells in equal areas of controls and mutants were counted and the average number compared. Apoptotic cells were detected using rabbit anti-cleaved Caspase-3 (Cell Signaling, Beverly, MA, USA), according to manufacturer's protocol. For Nile Blue (Sigma) staining, embryos were dissected and directly stained in 2 ml of DMEM culture medium containing 2 µl of 1.5% Nile Blue at 37°C, then washed with PBS for several hours and photographed.
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Results |
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Skeletal staining of E16.5 fetuses showed a severe reduction of the scapular blade in Gli3//Tbx15/mutants (Fig. 3F) when compared to either single mutant (Fig. 3B,C). Only the most anterior part of the scapular blade (supraspinatus fossa) remained, as well as a small part of the edge of the blade. Furthermore, the acromion was more severely reduced (arrowhead in Fig. 3B,D,F), whereas the glenoid fossa (scapular head) was normal. This aggravated shoulder phenotype is clearly dependent on gene dosage because the scapular foramen of Gli3+//Tbx15/ mutants is enlarged in comparison with Tbx15/ single mutants (compare Fig. 3B and 3D). Although we never observed a scapular foramen in the Gli3 mutants used in our study, the shoulder elements were broader than those in wild types, and contained indentations in the scapular blade (white arrowheads in Fig. 3C). In Tbx15+//Gli3/ mutants these malformations were enhanced, the scapular blade was still broader and a foramen in the scapula was now present (Fig. 3E).
In conclusion, the phenotypes of compound mutants suggest that Tbx15 and Gli3 act synergistically during shoulder girdle formation, representing at some level a degree of functional redundancy.
Functions of Alx3, Alx4 and Cart1 in limb girdle development
Previously, we, and others, have studied the roles of Alx3, Alx4
and Cart1, with an emphasis on limb and craniofacial development.
These three genes have strongly overlapping functions in limb and craniofacial
development (Qu et al., 1999;
Beverdam et al., 2001
) (also
A.B., unpublished). We reported severe truncation of the collar bone
(clavicle) of Alx3/Alx4 mutants, but not of either single
mutant (Beverdam et al., 2001
)
(see Fig. 3T); otherwise these
genes have not been linked to pectoral or pelvic girdle development.
Close inspection of the shoulder elements of Cart1 mutants revealed a slight reduction of the anterior blade in this single mutant, although the acromion was normal (Fig. 3H). In Alx4/Cart1 mutants (Fig. 3I,U), not only was the clavicle truncation much more severe than in Alx3/Alx4 mutants, but also the scapular blade rostral from the spine was virtually absent, and the spine itself and the acromion was shortened (arrowhead in Fig. 3I). Analyses of various compound genotypes showed similarly affected phenotypes in all double homozygous mutants (Fig. 3I,J); however, it was clear that inactivation of Cart1 had the most impact and that the impact of Alx3 mutation was much less. In conclusion, Cart1 has a major function in scapula formation that becomes clearly manifest only in the context of mutation(s) in Alx4, with which it shares an overlapping function.
In contrast to Tbx15 mutants, and also not previously reported,
the pelvic girdle of Alx4/Cart1 double mutants is affected
as well. Although Alx3 and Cart1 single mutants were normal
(not shown), we noticed that Alx4 mutants had a reduced pubic bone
(arrowhead in Fig. 4B).
However, the pubic bone was more severely affected in
Alx3/Alx4 and Alx4/Cart1 compound mutants
(Fig. 4C,D). Note that the
ilium of the mutant was normal, and that the scapula and pubic bone serve
non-analogous functions. Therefore, Alx3, Alx4 and Cart1 have
overlapping functions in the formation of elements of both the pectoral and
pelvic girdle, but different structures are affected. By contrast, loss of
function of, for example, Emx2 and Pbx1 affects the scapula
and ilium in similar ways (Pellegrini et
al., 2001; Selleri et al.,
2001
).
Genetic relationship between Tbx15, Alx4 and Gli3 mutants
We generated Alx4/Cart1/Tbx15 mutant embryos to
investigate possible genetic interactions in the way these genes pattern the
shoulder girdle. Upon skeletal staining of
Alx4/Cart1/Tbx15 triple mutants at E16.5, a severe
reduction of the scapular blade was observed
(Fig. 3N). The only remnants of
the scapula were the glenoid fossa (scapular head), a small fraction of the
acromion (arrowhead in Fig. 3N)
and the posterior part of the blade (the infraspinatus fossa). The clavicle
was completely absent, as is shown in an intact skeleton
(Fig. 3O,P).
The severity of the phenotype depends on gene dosage as shown by intermediate phenotypes in a Tbx15+/ background. For example, in Cart1/Tbx15 compound mutants the hole in the scapula is larger than in Tbx15 homozygotes (Fig. 3L), and in Alx4//Cart1//Tbx15+/ the spine was more reduced, the scapular blade was more severely malformed than in Alx4/Cart1 mutants, and a tiny remnant of the clavicle was present (compare Fig. 3I and 3M, and Fig. 3U and 3V). These results demonstrate that Tbx15 acts cooperatively with Alx4 and Cart1 in the development of the scapula. Comparing phenotypes of single and double mutants does not necessarily imply synergism between aristaless-related and T-box genes, as it appears that the defects are largely additive.
Similarly, we generated Gli3/Tbx15 compound mutants. This not only allows the investigation of genetic interactions between these two genes, but also provides an opportunity to compare the impact on shoulder formation, of the Alx4 and Cart1 genes on the one hand and of the similarly expressed Gli3 gene on the other. Alx4/Cart1/Tbx15 triple mutants and Gli3/Tbx15 compound mutants show strikingly contrasting phenotypes. By and large, whereas Alx4/Cart1/Tbx15 homozygous triple mutants lacked the anterior part of the scapular blade, Gli3/Tbx15 double mutants lacked the posterior part of the blade (compare Fig. 3F and 3N). Therefore, against the background of deficient Tbx15 function, the different roles of Alx4 and Cart1, when compared with Gli3, become more conspicuous.
Gene regulation in shoulder mutants
To begin to clarify the molecular pathways underlying shoulder girdle
development in mammals, we studied the expression of genes that are
functionally linked to this process in Alx4, Cart1, Tbx15, and
Gli3 single and compound mutants. First, we set out to study the
expression patterns of Tbx15, Alx4, Cart1 and Gli3 in the
mutants used in this study. Alx4 has previously been reported to be
downregulated in the limb buds of Gli3 mutants; however, this
concerns only the most distal part of the expression domain, which would
therefore not be expected to be relevant for the phenotype of the scapula
(Te Welscher et al., 2002)
(see Fig. 1D,E). We observed
that expression of Tbx15 in Alx4, Cart1 and Gli3
mutant limb buds at E10.5 and E11.5 was not altered, and neither was
expression of Alx4 and Gli3 in
Tbx15/ mutant limb buds at E10.5 (data not
shown).
Emx2 has an essential role during shoulder development. It is
normally expressed in the anterior-proximal region of the forelimb bud and at
the base of the hindlimb bud. Its ablation leads to loss of both the scapula
and the ilium (Pellegrini et al.,
2001). Expression of Emx2 was unaltered in
Alx4/Cart1 single and double mutants, and in
Tbx15/Gli3 single and double mutants
(Fig. 1F-I), suggesting that
Emx2 acts in a parallel pathway or upstream of Alx4, Cart1,
Gli3 and Tbx15.
Pax1 has a well-documented function in shoulder formation
(Timmons et al., 1994;
Dietrich and Gruss, 1995
). In
addition to being a sclerotome marker, Pax1 is normally expressed in
mesenchyme in the anterior proximal part of the fore- and hindlimb bud
(Timmons et al., 1994
). We
therefore studied Pax1 expression in various mutants with an affected
shoulder phenotype. Expression of Pax1 was not altered in
Alx4 and Cart1 single mutants at E10.5
(Fig. 5F,G). Strikingly, in
Alx4//Cart1/
the expression of Pax1 in the limb bud was strongly reduced at the
site of its normal expression (Fig.
5H) and was upregulated in the ventral body wall adjacent to the
limb bud. Images from a different angle are required to demonstrate this
shift: Fig. 5I-L show ventral
aspects of the forelimb region of a wild type, an
Alx4+//Cart1/
mutant and two
Alx4//Cart1/
mutants at E10.5. This clearly reveals a dose-dependent upregulation of
Pax1 in body wall mesenchyme. In situ hybridization on sections
confirmed the downregulation at E10.5 (Fig.
5Q,R). At E11.5, the Pax1 expression pattern is still clearly
abnormal in mutant limb buds, as shown for both fore- and hind limb (compare
Fig. 5M,O to 5N,P, respectively). In Tbx15/ embryos at E10.5, a
weaker yet still significant downregulation of antero-proximal Pax1
expression was seen (Fig. 5B),
whereas in Gli3/ embryos this effect was
somewhat stronger (compare Fig. 5B and
C). In Gli3/Tbx15 double mutants, Pax1
expression was slightly lower than in Gli3 mutants.
(Fig. 5D). However, a shift
like the one seen in Alx4/Cart1 mutants was, at most, weakly
present in Tbx15 embryos and was absent from Gli3
embryos.
|
Although Pax1 is considered to be a marker of the chondrocyte
lineage, Pax3 is thought to mark, in the context of limb development,
the myogenic lineage. Recently, the syndetome was identified as a fourth
component of the somite, consisting of tendon progenitors that are
distinguished by expression of Scleraxis (Scx) as a marker
(Brent et al., 2003). We
compared the expression of Pax3 and Scx in limb buds of
E10.75 Alx4/Cart1 mutant and wild-type embryos. From the
whole-mount in situ hybridization experiment shown in
Fig. 5V, it can be seen that
Pax3 expression is shifted towards the antero-proximal region of the
limb bud in a region where Pax1 is downregulated. In addition,
hybridization of sections of Alx4/Cart1 mutants with
Pax3 demonstrated that Pax3 expression in the body wall
(marking myotonic progenitors from the dermomyotome and heading for the limb)
is abnormally located in mesenchyme, just adjacent to surface ectoderm
(Fig. 5S,T). Scx
expression was changed to a lesser degree, but appeared to be elevated in the
antero-proximal region of the limb bud in the Alx4/Cart1
mutant (green arrowhead in Fig.
5X). Fig. 5Y
summarizes these results schematically. We conclude that different types of
migrating cells that are bound to contribute to the pectoral girdle are
disorganized in shoulder mutants at an embryonic stage prior to visible
anatomical aberrations. Possibly it is cell migration itself that is affected,
but alternatively a transformation of cells involved may take place. By
contrast, no ectopic expression of Pax3 or Scx was seen in
Tbx15 mutants, or in Tbx15/Gli3 mutants (data not
shown).
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Discussion |
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In this study, we have explored the genetic interactions of a number of genes that are implicated in formation of the pectoral (and to a lesser extent pelvic) girdle skeleton. The study of genotype-phenotype relations in complex compound mutants allows genetic relationships between genes to be determined, and the recognition of important gene functions otherwise masked because of redundancy.
Aristaless-related genes
We show that Cart1 and Alx4 single mutants have slight,
not previously reported, defects in skeletal elements of the pectoral and
pelvic girdle, respectively, that become more severe in double mutants
carrying additional mutations in either one of the three redundant genes
Alx3, Alx4 and Cart1. Whereas Emx2 appears to be
essential for the formation of structures of both girdles (scapula and ilium)
that look superficially similar
(Pellegrini et al., 2001),
Alx4/Cart1 mutants, as well as Tbx15 mutants, have very different
phenotypes in the shoulder and pelvis. There is no sound basis for describing
both girdles in terms of homology; clearly evolution of the girdles has
evolved separately and under different species-specific constraints. During
evolution, the aristaless-related genes may have been recruited for different
functions in development of both types of girdle. Any speculation on such
topics is rather futile in the absence of both mutants from appropriate
different species and expression data from extinct ancestors. The pubic bone
is also affected in mutants for the aristaless-related genes Prx1 and
Prx2 (Ten Berge et al.,
1998b
), which have overlapping but broader expression patterns and
are generally linked to different phenotypes.
The prevailing function of Cart1 that emerges from comparing
compound mutants involving Alx3, Alx4 and Cart1 is also
unexpected because Cart1 is expressed only at low levels in mesoderm
of the limb buds and flank (Zhao et al.,
1994; Beverdam and Meijlink,
2001
). This paradox was previously noted for the polydactyly in
Alx4/Cart1 double mutants
(Qu et al., 1999
).
Distinct functions of Gli3 and Alx4 revealed in Tbx15 double mutants
The striking scapular foramen characteristic of Tbx15 mutants
shows that this gene is essential for appropriate shoulder formation.
Gli3 mutants kept in certain genetic backgrounds have a similar
foramen. The combined deficiency of these two genes, however, resulted in a
unique phenotype in which the anterior part of the scapula remained, leaving a
structure that resembled a typical long bone (e.g.
Fig. 3F). Such an element also
resulted from Alx4/Cart1/Tbx15 triple homozygous
mutants (Fig. 3N), but in this
case through ablation of the anterior part of the scapula. These contrasting
phenotypes reveal complementary functions of Gli3 and
Alx4/Cart1, linking Gli3 to the posterior part of
the scapular blade (infraspinatus fossa), and Alx4 and Cart1
to the anterior part (supraspinatus fossa), as depicted in the schematic
representation in Fig. 3W,X. Interestingly, Alx4 and Gli3 have strongly overlapping
expression patterns in the anterior mesoderm of the limb bud. Both genes are
also examples of genes whose functions are linked to formation of both the
shoulder and distal limb elements. Furthermore, Alx4 expression is
partly dependent on Gli3 function (see
Fig. 1D,E) (see also
Te Welscher et al., 2002), but
the region of Alx4 downregulation in Gli3 mutants is not
likely to be relevant for shoulder formation (e.g.
Vargesson et al., 1997
).
Presumably Alx4-associated phenotypes, including radial/tibial
dysplasia and polydactyly, relate to different aspects of its expression
pattern, and it is possible that the expression that disappears in
Gli3 mutants corresponds to the zeugopodal and autopodal
functions.
Defects of the clavicle
In contrast to the scapula, the clavicle arises through intramembranous
rather than endochondral ossification. It evolved in primitive fish as a
skeletal element at the base of the skull (see
McGonnell et al., 1998), and,
in mammals, is the only membranous bone outside of the skull. Alx4
and related genes have a function in shaping the neural crest-derived
craniofacial skeleton, and we can therefore not exclude that the clavicle
phenotypes we describe should be seen in that context. Nevertheless, a degree
of redundancy can be seen between Tbx15 and
Alx4/Cart1, even leading to complete suppression of the
clavicle in the triple homozygous mutant. We therefore favor the notion that a
similar disorganization locally in the shoulder girdle that also causes the
scapular defect is the basis of the interference with clavicle formation.
Pax1 and Pax3 dysregulation in shoulder mutants
In the differentiating somite, the Pax1 gene is a sclerotomal
marker. In the context of formation of the scapula, however, which is of
dermomyotomal derivation, it is primarily considered to be a chondrocyte
marker. Pax1 is linked to shoulder formation by the phenotype of
undulated and other mutant alleles
(Balling et al., 1988;
Deutsch et al., 1988
).
Pax1 mutants lack part of the spine and the cartilaginous acromion.
In addition, Pax1 is abnormally expressed in a number of shoulder
mutants. Pellegrini showed that Pax1 was upregulated and shifted
dorsally in Emx2 mutants that have totally lost the scapula
(Pellegrini et al., 2001
). By
contrast, we demonstrate a downregulation and ventral shift of Pax1
expression associated with the scapula truncation in
Alx4/Cart1 mutants, whereas loss of Gli3 or
Tbx15 function leads to a similar downregulation, but without the
ventral upregulation. Pax1 is inhibited by the application of beads
containing BMP4 in chick limb buds, which is accompanied by scapula defects
(Hofmann et al., 1998
);
however, it is induced by Sonic hedgehog (SHH) application
(Laufer et al., 1994
). At
later stages, Shh is even upregulated in the anterior limb bud of
Alx4 mutants (Chan et al.,
1995
). At E10.5, both Bmp4 and Shh were normally
expressed in the limb regions of Alx4/Cart1 mutant embryos
(data not shown). This suggests that these signaling pathways are not
downstream of Alx4 and Cart1 in the mechanism leading to
Pax1 deregulation in mutants. Intriguingly, Tbx15 and
Pax1 have complementary rather than overlapping expression patterns
(Fig. 1A,C), basically ruling
out direct interaction. It should be noted that Tbx15, Alx4 (S.K.,
unpublished) and Cart1 (Ten Berge
et al., 1998a
) are not expressed in the dermomytome, which is in
contrast with the high dermomyotomal expression of Gli3 reported by
McDermott and colleagues (McDermott et
al., 2005
). It therefore remains possible that Pax1
downregulation in Gli3 mutants is a direct consequence of interaction
between these genes, but the effect on Pax1 expression seen in
Alx4/Cart1 mutants suggests a model in which Pax1
deregulation reflects the disturbance of positional cues that guide cells
destined to contribute to the shoulder. This would modify the view of
Pax1 as a mere marker of cartilage/bone precursors, although the
affected morphology of the acromion seen in most of the mutants studied here
may be directly related to the loss of Pax1 in the acromioclavicular
region where it is normally expressed. However, ectopic expression of
Pax1 does not lead to ectopic cartilage elements, possibly due to a
lack of permissiveness in its abnormal area of expression. Emx2 marks
the prospective scapular blade region
(Pellegrini et al., 2001
;
Pröls et al., 2004
). The
apparently normal Emx2 expression in all mutants studied shows that
the altered Pax1 expression does not merely reflect the
downregulation of markers for progenitors of structures that are affected.
Only recently has it fully emerged how genes specifically expressed in
different somite compartments mark cells that migrate to the limb to form
corresponding tissues. This process must depend on positional cues and its
disruption is expected to be reflected in the expression of these markers. We
therefore consider the shift in expression of Pax3, and to a lesser
extent Scx, to support a model involving the disruption of positional
signals, as suggested above. It should be noticed that the changes in
expression of Pax1, Pax3 and Scx are seen in regions of
embryos at a stage when anatomical or histological aberrations are not yet
detectable, strongly suggesting that this dysregulation is a part of the
mechanism that leads to the eventual skeletal defects. It remains to be seen
whether it is the process of migration that is disturbed or whether some type
of transformation or abnormal differentiation results in the shoulder
malformations. The latter possibility is not supported by analyses of muscle
tissue in Alx4/Cart1 mutants, as we noted only loss of
muscle tissue, in particular affecting the supraspinatus muscle (not shown).
de mutant embryos also have reduced muscle tissue
(Curry, 1959;
Singh et al., 2005
). Huang et
al. have suggested an essential causal link between the downregulation of
Pax3 in a subset of hypaxial dermomyotomes and the potential of this
tissue to differentiate into cartilage (and to contribute to scapula or ilium)
(Huang et al., 2000
). In this
respect, it is interesting that overexpression of the modulator of
Wnt-signaling Carboxypeptidase Z in the dermomytome of chick embryos leads to
scapular defects, as well as ectopic Pax3 expression
(Moeller et al., 2003
).
Results from grafting studies in chick embryos point to the importance of
signals from the ectoderm to the underlying mesoderm, and the possible
implication of FGF signaling (Ehehalt et
al., 2004; Pröls et al.,
2004
). In Alx4 mutants at E10.5, FGF4 is ectopically
expressed in anterior limb bud ectoderm
(Chan et al., 1995
).
Identifying the exact nature of the signaling cascade underlying scapula
formation is a major challenge and may require combining genetics with the
manipulation of embryo explants.
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ACKNOWLEDGMENTS |
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Footnotes |
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