1 Laboratoire de génétique moléculaire, Institut de
recherches cliniques de Montréal, 110 avenue des Pins Ouest,
Montréal, QC H2W 1R7, Canada
2 Department of Human Genetics, University of Michigan Medical School, Ann
Arbor, MI 48109-0638, USA
* Author for correspondence (e-mail: drouinj{at}ircm.qc.ca)
Accepted 11 October 2002
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SUMMARY |
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Key words: Pitx1, Pitx2, Limb, Patterning, Mouse
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INTRODUCTION |
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En1 and Lmx1b are both transcription factors of the
homeodomain family and they appear to play roles in marking the identity of
ventral or dorsal limb domains, respectively
(Chen and Johnson, 1999).
Indeed, the knockout of these genes lead to the loss of ventral (En1)
(Logan et al., 1997
;
Loomis et al., 1998
) or dorsal
(Lmx1b) structures in mice (Chen
et al., 1998
; Dreyer et al.,
1998
). Similarly, the identity of proximodistal (PD) domains in
the limb appears to be defined very early in the growing limb bud. In
particular, the proximal limb domain where stylopod (femur or humerus) will
form is marked by expression of other homeobox-containing transcription
factors, Meis1 and Meis2, and gain-of-function experiments in chick embryos
have suggested that this restricted expression is required for specification
of both zeugopod and stylopod domains of the limb
(Capdevila et al., 1999
;
Mercader et al., 2000
).
The scheme described above for limb induction, patterning and growth is
thought to be a generic one acting both at forelimbs (FL) and hindlimbs (HL).
However, the appearance of distinct HL during evolution has probably required
a new set of signals and transcription factors to mark the identity of HL by
comparison to FL. The extent to which FL represent a default pathway for limb
formation remains a subject of debate, although FL-specific transcription
factors such Tbx5 have been identified
(Chapman et al., 1996;
Gibson-Brown et al., 1996
;
Gibson-Brown et al., 1998
;
Logan et al., 1998b
) and are
involved in FL formation (Basson et al.,
1997
; Li et al.,
1997
; Rodriguez-Esteban et
al., 1999
; Takeuchi et al.,
1999
). The implication of transcription factors for specification
of HL identity is clearer. Indeed, the homeobox containing transcription
factor Pitx1 has been shown to become specifically restricted to HL mesenchyme
following its early expression throughout posterior lpm
(Lanctôt et al., 1997
).
The role of Pitx1 in HL identity was clearly supported by gene
inactivation experiments in mice that resulted in HLs showing features of FL
in particular at the level of zeugopod and knee joint
(Lanctôt et al., 1999b
;
Szeto et al., 1999
). The
interpretation of these studies were further supported by gain-of-function
experiments using retrovirus-mediated Pitx1 expression in FL buds of
chick embryos: the resulting wings developed with partial features of legs
both at the level of skeleton and muscle
(Logan and Tabin, 1999
).
Another transcription factor, a member of the T-box family Tbx4, was
also implicated in specification of HL identity but its expression appears to
be downstream and, in part, under control of Pitx1
(Lanctôt et al., 1999b
;
Szeto et al., 1999
;
Logan and Tabin, 1999
).
A surprising observation made on Pitx1-/- embryos was a
relatively frequent left-right (LR) asymmetry in the severity of the phenotype
(Lanctôt et al., 1999b).
Indeed, femur length was found to be more often reduced on the right compared
with left HLs. As the Pitx1-related homeobox factor Pitx2
was shown to be an effector for LR asymmetry in the lpm
(Logan et al., 1998a
;
Piedra et al., 1998
;
Ryan et al., 1998
;
Yoshioka et al., 1998
), we
have suggested that redundancy between the Pitx genes may explain the LR
asymmetry in the phenotype of Pitx1-/- embryos. This
redundancy is somewhat counter-intuitive because under normal conditions both
limbs are symmetrical and are not subject to LR patterning. In part to verify
this hypothesis, we generated mice that are double mutants for Pitx1
and Pitx2. The analysis of these mice not only confirmed an apparent
redundancy between the two factors but unexpectedly highlighted a co-operative
role of both Pitx genes in formation of HL buds.
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MATERIALS AND METHODS |
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Skeletal preparation and staining
E17.5 or E16.5 embryos were stained with Alcian Blue and Alizarin Red, and
younger embryos (E13.5) were only stained with Alcian Blue as described
(McLeod, 1980).
Whole-mount embryo staining
Whole-mount in situ hybridization and immunohistochemistry was done as
described in protocols from Dr Janet Rossant's laboratory. These two protocols
used can be found at
http://www.mshri.on.ca/develop/rossant/protocols.html
Immunohistochemistry
Section immunohistochemistry was performed as described
(Lanctôt et al., 1999a)
using previously characterized Pitx1 and Pitx2 primary antibodies
(Tremblay et al., 1998
;
Hjalt et al., 2000
). MyoD
antibody was purchased from Pharmingen. Biotinylated anti-rabbit (Vector Labs,
1/150), was used as secondary antibody and revealed using streptavidin-HRP
(NEL750, NEN, 1/1000) and DAB. Slides were counter-stained with Methyl
Green.
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RESULTS |
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In order to ascertain the putative redundancy between Pitx1 and
Pitx2 genes, Pitx1+/- mice were crossed with mice
carrying either a hypomorphic (neo) or null allele of the Pitx2 gene
(Fig. 1A) (Gage et al., 1999). To obtain
double mutant mice, we crossed Pitx1+/- mice with
Pitx2+/- mice. Surprisingly, we did not get the expected
Mendelien ratio of 25% double heterozygotes
(Pitx1+/-,Pitx2+/-) but only 2%. We
cannot explain the poor viability of these mice. This was not observed with
the Pitx2neo allele, which gave close to the expected yield (20%)
when crossed with Pitx1+/- mice. Double mutant embryos
(Pitx1-/-,Pitx2+/-) were obtained by
crossing Pitx1+/- mice with
Pitx1+/-,Pitx2+/- mice. We only ever
got one Pitx1-/-,Pitx2-/- embryo by
intercrossing double heterozygotes and a few
Pitx1-/-,Pitx2neo/- embryos were obtained by
crossing double heterozygotes of each Pitx2 allele.
Mutant (Pitx1-/-,Pitx2neo/neo) mice with the most
extreme phenotype showed a much more extensive phenotype than single mutant
mice (Fig. 2). Whereas
Pitx1-/- mice exhibit the patterning defects described
above, Pitx2 mutant embryos do not exhibit any obvious limb defect
(Gage et al., 1999;
Kitamura et al., 1997
;
Lin et al., 1999
;
Lu et al., 1999
). By contrast,
double mutant mice have lost three HL skeletal elements. Indeed, both right
and left femur, tibia and digit one are missing in these embryos
(Fig. 2). The pelvis is not
more severely affected than in Pitx1-/- mice. The
identification of the only remaining zeugopodal element as fibula is based on
the contact between this bone and the calcaneus. Except for the loss of digit
one, it is striking how the autopod is unaffected by the double gene
mutation.
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In agreement with the hypothesis of a gene dose-dependent phenotype, the
loss of HL skeletal elements followed a reproducible pattern in series of
embryos deficient for Pitx1, either carrying the
Pitx2neo/neo alleles (data not shown) or the
Pitx2+/- alleles (Fig.
3). The order of bone loss with progressive penetrance of the
phenotype is as follows. Right digit 1 was the most sensitive to loss of Pitx
function (Fig. 3B), as was
observed in some Pitx1-/- mice
(Fig. 1C). In more affected
embryos, the right tibia partially or completely failed to develop
(Fig. 3C) and then the right
femur was lost (Fig. 3D). On
the left side, dependence on Pitx function followed a similar sequence: digit
1 (Fig. 3D), tibia
(Fig. 3E), followed by
reduction (Fig. 3F) and loss of
left femur (Fig. 2). All
skeletal preparations examined (over 20 embryos) fit within this sequence of
bone losses. The phenotype of these double mutant mice is in part reminiscent
of embryos deficient for limb AER Fgf8 expression. Indeed, these mice
also failed to develop femur and digit 1 and the tibia is hypoplastic
(Lewandoski et al., 2000).
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Analysis of early limb bud development revealed smaller HL buds (in all of
over 100 embryo pairs examined), both in Pitx1-/- and
Pitx1-/-,Pitx2+/- embryos compared
with wild-type littermates (Fig.
4). In most cases (60% of pair comparisons), limb bud size
reduction was greater for double than single mutant embryos
(Fig. 4A). Greater reduction
was observed on right compared with left side in about 50% of either
Pitx1-/- or double mutant embryos. The reduction in
Pitx1-/- HL bud size is surprising as these embryos show
patterning defects but no loss of skeletal elements, except for reduction in
femur size. This observation could however be consistent with a joint role of
Pitx1 and Pitx2 genes in early expansion of 1pm in the HL
field and of early limb bud mesenchyme. When measured relative to somites
(Fig. 4A), the reduction in HL
bud size observed in mutant embryos is striking because it results from a
narrowing of the HL bud from a length of about 3.5/4 somites (approx. somites
24.5 to 28.5) to a length of 2.5 somites in Pitx1-/-
embryos (approx. somites 25.5 to 28.0) and to a length of about 2 somites in
Pitx1-/-,Pitx2+/- embryos (approx.
somites 26 to 27.5-28.0). In all cases, the limb bud is centered on somite 27.
This narrowing along the AP axis was best revealed in embryos labeled by
whole-mount in situ hybridization with a probe for Tbx4, a
HL-specific marker that has previously been shown to be decreased in
Pitx1-/- embryos
(Lanctôt et al., 1999b
;
Szeto et al., 1999
) and which
is similarly decreased in double mutant embryos
(Fig. 4A). Thus, HL bud size
reduction affects both outgrowth and width of the bud along the AP axis.
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Bud outgrowth is thought to be controlled by growth factors produced by the
AER. In particular, Fgf8 is the earliest growth factor to mark the AER and at
E10.5, this Fgf8 expression appears similar in single and double
mutant embryos compared with wild-type
(Fig. 4B). Fgf10
expressed throughout the mesenchyme of the limb bud is also thought to play a
role in growth control (Ohuchi et al.,
1997). Fgf10 expression did not appear to be affected in
the single or double mutant embryos (Fig.
4C). Although expression of Fgf8 and Fgf10 are
not grossly affected in mutant embryos, the loss of skeletal elements in
double mutant embryos may reveal a failure to specify limb bud segments, for
example, the proximal segment from which the stylopod (femur) develops. As
this proximal segment is marked by expression of Meis genes, we investigated
Meis gene expression in embryos mutant for Pitx1 or for
Pitx1 and Pitx2. In both, Meis2 expression was
similar to that in wild-type embryos (Fig.
4D); similar results were obtained for Meis1 (data not
shown). These data suggest that failure to develop stylopod (femur) in
Pitx1-/-,Pitx2+/- embryos does not
result from a failure to specify the proximal limb domain.
However, limb outgrowth could be curtailed if early expression of Fgf genes
was delayed (Min et al., 1998;
Sekine et al., 1999
;
Lewandoski et al., 2000
;
Moon and Capecchi, 2000
). For
this reason, we investigated early expression of Fgf10, Fgf8 and
other markers. As shown in Fig.
4E, early HL expression (25 somites) of Fgf10 was not
significantly altered in either Pitx1-/- or
Pitx1-/-,Pitx2+/- embryos. Examination
of 5-10 embryos/genotype suggested a slight decrease of Fgf10
expression, but this proved difficult to substantiate objectively. Similarly,
early HL expression of Bmp7 was not different in double compared with
single mutant embryos (Fig. 4F) and AER expression of Msx2 was also unaffected in mutant embryos
(Fig. 4G). AER expression of
Fgf8 in HL starts at stage 27 somites in wild-type embryos. A similar
onset was observed for Pitx1-/- embryos, although
expression could be slightly reduced (Fig.
4H). AER expression of Fgf8 was delayed in
Pitx1-/-,Pitx2+/- embryos with an
onset at stage 30 somites (Fig.
4H). Hence, a delay and/or reduction in AER expression of
Fgf8 may account in part for the phenotype of double mutant embryos,
as proposed to explain the differential effect in FL or HL of conditional
Fgf8 knockout (Lewandoski et al.,
2000
).
The reduction in HL bud size along the AP axis suggests that AP patterning of the limb bud might be altered. In order to assess this within the context of global AP patterning, the expression in HL of posterior Hox genes was ascertained by whole-mount in situ hybridization. At E11.5, the anterior border of Hoxc11 expression was found to be on the rostral side of somite 27, which lies in the middle of the developing HL buds (Fig. 5A). In Pitx1-/- and Pitx1-/-,Pitx2+/- embryos, the anterior border of Hoxc11 expression was the same relative to somite 27 (Fig. 5A) but the narrowing of the HL bud in mutant embryos appeared to result in loss of anterior bud mesenchyme. In agreement with this, the strong band of Hoxc11 expression observed in the posterior third of wild-type HL buds is similarly posterior in mutant limb buds but the band now accounts for about half of the bud mesenchyme, as if anterior bud mesenchyme was missing (Fig. 5A). Expression of Hoxc9 and Hoxc10 was not affected in these mutant embryos (data not shown).
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In order to further investigate AP patterning within the buds, we assessed
Shh and Gli3 expression by whole-mount in situ
hybridization. Shh labels the ZPA, which is known to play an
important organizer function to define AP polarity in the limb bud and
Gli3 marks the anterior bud mesenchyme. In both wild-type and
Pitx1-/- embryos, Shh expression was similar at
the posterior margin of the limb bud (Fig.
5B). By contrast, Shh expression extended halfway up the
limb bud in Pitx1-/-,Pitx2+/- embryos
(Fig. 5B). Thus, the ZPA of
double mutant embryos appears to extend further anteriorly compared with
wild-type or Pitx1-/- embryos. By contrast, anterior bud
expression of Gli3 was similar in mutant and wild-type embryos
(Fig. 5C), indicating that
anterior signals are still present in Pitx mutant embryos. AER
expression of Fgf4 was also extended anteriorly in mutant embryos
(Fig. 5D). Given the narrowing
of the limb bud, the apparent extension of the ZPA may be secondary to the
loss of mesenchyme and/or extension of posterior signal. This was further
assessed using another marker of posterior limb mesenchyme, Hand2
(dHand), that has previously been associated with AP patterning
defects at the zeugopod and autopod levels
(Charité et al., 1995;
Fernandez-Teran et al., 2000
).
Indeed, overexpression of Hand2 in the HL has resulted in loss of
tibia, similar to our double mutant mice
(Charité et al., 1995
).
Expression of Hand2 was found to extend more into the anterior half
of the HL bud in mutant embryos. A striking example of this is shown in
Fig. 5E, where Hand2
expression extends the entire width of the right limb bud at zeugopod level
but still only covers the posterior side of the left HL. Thus, the effect of
the loss of Pitx genes, in particular at the zeugopod level, might be in part
ascribed to a more anterior expression of Hand2 within the limb
bud.
Clearly, the role of Pitx genes would be best revealed in double null mutant embryos. We only obtained one such embryo in almost two years of breeding and we got a few Pitx1-/-,Pitx2neo/ embryos, which should express less Pitx2 than null heterozygotes. These latter embryos had more severely affected HL, in particular autopods (Fig. 6A-C). Indeed, both embryos shown in Fig. 6 have three remaining digits on the left side and only two on the right, as revealed either by Alcian Blue staining of cartilage (Fig. 6B) or by in situ hybridization for Sox9, which also marks cartilaginous condensations (Fig. 6C). The further loss of digits as Pitx2 gene dose was decreased is suggestive of a dependence on Pitx genes for expansion of limb bud mesenchyme. This idea is further supported by the single Pitx1-/-,Pitx2-/- embryo that we obtained (Fig. 6D). Indeed, at E12.5, this embryo had severely retarded HL development. Furthermore, the left HL bud exhibited some AER expression of Fgf8 and it was bigger than the right HL bud. This LR asymmetry cannot be attributed to Pitx2 and may suggest involvement of other regulators. It thus appears that induction of AER function was not prevented in absence of both Pitx genes, although growth of HL buds was severely curtailed. Total Pitx gene expression level appears to be the most important parameter for HL bud growth as Pitx1+/-,Pitx2-/- embryos from the same litter (Fig. 6D) had relatively normal HL bud development, in agreement with the idea that Pitx1 has the highest expression level and is the most important for HL bud formation.
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The genetic requirement for both Pitx1 and Pitx2 during
growth and patterning of HL is surprising in view of the previously
characterized expression of these genes. Whereas Pitx1 was known to
be expressed from early-on throughout the HL mesenchyme, Pitx2 is not
known to be expressed in this mesenchyme
(Campione et al., 1999;
Kitamura et al., 1997
;
Logan et al., 1998a
;
Mucchielli et al., 1996
;
Piedra et al., 1998
;
Ryan et al., 1998
;
Semina et al., 1997
;
Yoshioka et al., 1998
). It was
therefore surprising to observe such strong genetic requirement for both
genes, and this led us to reinvestigate in detail the expression of both Pitx
genes from early development throughout limb growth. Both whole-mount and
sectioned embryos were analyzed for mRNA expression using in situ
hybridization and for protein using immunohistochemistry. Whole-mount
histochemical analysis of Pitx1 and Pitx2 in early E8.5-E9.0 embryos revealed
that, in addition to their joint expression in the stomodeum, both factors are
also co-expressed in the tail bud region presumed to become the HL field
(Fig. 7). As previously
reported (Lanctôt et al.,
1997
), Pitx1 expression was restricted to the lpm of the posterior
end of the embryo (Fig. 7A-D).
Pitx2 immunoreactivity was observed in left lpm as previously reported
(Logan et al., 1998a
;
Piedra et al., 1998
;
Ryan et al., 1998
;
Yoshioka et al., 1998
).
However, this expression appeared to extend throughout the length of the
embryo down to the tail bud and weak expression was also detected on the right
side of the tail bud (Fig.
7A-D). This expression is much weaker than that of Pitx1. The
unexpected observation of co-expression of Pitx1 and Pitx2 in the tail bud
region destined to become HL may offer the explanation for the genetic
interaction between the two Pitx genes. Later in development, Pitx1 expression
is maintained throughout HL mesenchyme
(Fig. 7E,F), whereas Pitx2 is
not present in HL mesenchyme (Fig.
7E). The only limb bud expression of Pitx2 was observed in
myoblasts (Fig. 7F) as
indicated by the similarity with the pattern of MyoD
(Fig. 7F) and Pax3 (data not
shown) expression. It had previously been shown that Pitx2 is
expressed in chick myotomes and myoblasts
(Logan et al., 1998a
;
Piedra et al., 1998
). It is
very unlikely that Pitx2 expression in muscle cells may be an
important determinant for the growth and patterning defects observed in double
mutant mice as splotch mice, which do not form limb muscle, still form all
skeletal elements (Henderson et al.,
1999
). Thus, co-expression of Pitx1 and Pitx2 is
limited to the mesoderm of the very early HL field and both genes appear
required for early expansion of limb bud mesenchyme.
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DISCUSSION |
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Hindlimb specification and patterning role of Pitx1
Pitx1 was identified as the most upstream gene in a cascade that
also includes Tbx4 for specification of HL identity. This model
derived from knockout of the Pitx1 gene in mice
(Lanctôt et al., 1999b;
Szeto et al., 1999
), and
overexpression of Pitx1 (Logan
and Tabin, 1999
) and of Tbx4
(Takeuchi et al., 1999
) in
chick wing buds. The consequences of these manipulations were mostly observed
at the level of zeugopod and at the boundary between zeugopod and stylopod.
Indeed, the autopod is not drastically affected by Pitx1 inactivation
in mice. Other HL-specific factors include Hoxc10 and Hoxc11
(Nelson et al., 1996
;
Peterson et al., 1994
) and
these were shown to be induced by ectopic expression of Pitx1 in FL
(Logan and Tabin, 1999
),
suggesting that they may be downstream of Pitx1. Our results do not
agree with this interpretation as Hoxc10 and Hoxc11
expression is unaffected in Pitx1-/- or double
mutants.
In view of the effect of Pitx1 deficiency on early HL bud
outgrowth (Figs 4,
5), it is worthwhile
re-visiting the phenotype of Pitx1-/- mice in order to
differentiate, if possible, Pitx1 functions that may be truly involved in
specification as opposed to those that involve dose dependence and redundancy
with Pitx2. Two aspects of the Pitx1 knockout qualitatively affect HL
skeletal structures, producing a resemblance to FL structures. These are the
absence of secondary cartilage development leading to the formation of an
articulation that is more elbow than knee like, and the contact of fibula with
femur instead of tibia much like the contact between equivalent bones in FLs
(Lanctôt et al., 1999b).
These transformations are most likely to reflect a true HL specification role
of Pitx1. By contrast, the reduction in femur length may be associated with
defects in growth regulation rather than specification or patterning.
Pitx gene expression in posterior mesoderm and in HL bud
mesenchyme
The demonstration of strong genetic interaction between the Pitx1
and Pitx2 genes poses the question of where and when might the two
genes be co-expressed or, if not co-expressed, what might be the tissues that
interact to account for the phenotype of the double mutants. The expression of
Pitx1 from very early in posterior lpm and throughout the HL bud mesenchyme
was already well established
(Lanctôt et al., 1997).
However, Pitx2 did not appear to be present in limb buds, except in myoblasts,
and, when re-assessed using immunocytochemistry, we confirmed that
Pitx2 is not expressed in HL mesenchyme
(Fig. 7E). However, Pitx1 and
Pitx2 were detected with similar patterns of expression on both sides of the
tail bud at the 7-15 somite stages of development (E8.5-E9.0), with Pitx2
showing LR asymmetry (Fig.
7A-D). Thus, this very early co-expression of Pitx factors
probably accounts for their function in limb bud formation. The higher Pitx1
protein levels (compared with Pitx2) in this area would be consistent with the
absence of marked HL phenotype in Pitx2-/- embryos
(Gage et al., 1999
;
Kitamura et al., 1999
;
Lin et al., 1999
;
Lu et al., 1999
) or in
Pitx+/-,Pitx2-/- embryos
(Fig. 6D). In normal
conditions, the function of Pitx genes in the HL field would thus be
primarily served by Pitx1 and it is only in its absence that the
contribution of Pitx2 to limb bud growth becomes evident. This
interpretation would also be consistent with the fact that asymmetrical
development of HL is only observed in the absence of Pitx1.
Mesoderm outgrowth and limb development
The earliest phenotype observed in Pitx-deficient embryos is the reduction
in HL bud size both along the AP and PD axes
(Fig. 4). The observation that
this phenotype is sometimes asymmetrical is consistent with the partial
penetrance of the Pitx2 alleles in the Pitx1-/-
background (Fig. 3). Hence,
this phenotype is correlated with Pitx gene dose effects observed in the
present study. In Pitx1-/- embryos, the variable reduction
in femur length with its strong bias for the right side correlates well with
the reduction of HL bud size (both right side biases observed in 50-60%
embryos). The impairment of HL bud growth was almost complete in absence of
both Pitx genes (Fig. 6D),
despite relatively conserved AER and bud functions in
Pitx1-/-,Pitx2+/- embryos, as revealed
using markers such as Fgf8, Fgf10, Bmp7, Msx2, Fgf4, Hoxc11, Hand2, Shh,
Gli3 and Meis (Figs
4,5,6).
The similarity of HL phenotypes produced by inactivation of both Pitx genes
(Fig. 6D) or of Fgf10
(Min et al., 1998;
Sekine et al., 1999
) suggests
that they may be mediated through similar mechanisms. Although both mutant
mice initiate bud outgrowth, Fgf10-/- embryos did not
exhibit AER function, whereas Pitx mutant embryos do. As Fgf10
expression was not significantly affected in double mutant embryos
(Fig. 4C,E), it may not be the
production of Fgf10 or of another signal [such as Fgf8,
which was still induced in AER of the
Pitx1-/-,Pitx2-/- embryo
(Fig. 6D)], that is dependent
on Pitx genes. Rather, it may be the ability to respond to signals that is
Pitx dependent. The simplest model for the role of Pitx1 and
Pitx2 genes in HL bud formation may thus be that these genes are
required for appropriate growth response of HL field mesenchyme to growth
factors, such as Fgf10 (Fig.
8A). Alternatively, we cannot exclude the possibility that
Pitx genes are required for Fgf10 expression itself
(Fig. 8B) because we could not
assess its expression in a double null mutant.
How could Pitx genes be essential for formation of proximal (femur) and anterior (tibia and first digit) structures? Given their early co-expression, Pitx genes may be required for patterning the proximoanterior domain of the HL field. The Pitx genes would thus be essential for expression of an anterior-specific factor that remains to be identified. Indeed, a factor with the expected expression or function is not currently known. The Pitx genes themselves do not appear to be the anterior-specific signal, as their expression does not show AP differences at the HL level (Fig. 7), but they may nonetheless serve a permissive function. Alternatively, the progressive loss of anterior and proximal structures first on the right and then on the left side (Fig. 3) would be consistent with an impairment of bud mesenchyme growth dependent on Pitx gene dose.
The loss of anterior HL bud mesenchyme
(Fig. 4A) is associated with
loss of anterior skeletal elements, first digit and tibia (Figs
2,
3). These observations
correlate well with excision experiments performed on chick wing buds in which
removal of the anterior half bud resulted in loss of anterior structures, i.e.
anterior digit and radius (FL equivalent of tibia), together with proximal
part of humerus (Warren, 1934;
Saunders, 1948
). Thus, the
primary defects associated with Pitx gene deficiency is the early loss of bud
mesenchyme, which may result in loss of anterior skeletal elements. Because
most signalling appears to be intact in double Pitx mutant embryos, including
Shh and Gli3, their reduced HL buds may be subjected to
disproportionate posteriorizing activity
(Fig. 5) and this may also
contribute to the loss of anterior skeletal elements.
It is interesting to compare Pitx1 and Pitx2 deficiency
with conditional inactivation of AER Fgf8. In one study, HL knockout
of Fgf8 resulted in loss of femur and first digit, but not tibia
(Lewandoski et al., 2000). In
another study in which Fgf8 knockout was targeted to FL, radius and
first digit were lost in 100% of embryos and the humerus lost in 70% of
embryos (Moon and Capecchi,
2000
). It was proposed that rescue of the zeugopod might be
ascribed to AER expression of Fgf4, which is expressed later and more
posteriorly than Fgf8 (Lewandoski
et al., 2000
; Tickle and
Munsterberg, 2001
). This is consistent with the double knockout of
limb Fgf8 and Fgf4, which abrogated limb bud development
(Sun et al., 2002
); this
latter work also supported a model of sequential growth of bud mesenchyme
pre-specified for PD structures. In Pitx double mutant embryos, the delay in
AER expression of Fgf8 (Fig.
4H) may thus contribute to the reduced size of proximal
structures. However, although AER expression of Fgf8 was delayed from
the 27- to 30-somite stage (Fig.
4H), it is noteworthy that other AER or bud markers are not
significantly affected in mutant embryos. These include Fgf10
(Fig. 4E), which is essential
for Fgf8 expression (Ohuchi et
al., 1997
), Bmp7 (Fig.
4F), Msx2 (Fig.
4G), Gli3 (Fig.
5C) and Fgf4 (Fig.
5D). In addition, the presence of AER Fgf8 in HL bud of
the Pitx1-/-,Pitx2-/- embryo
(Fig. 6D) argues against a
primary role of Pitx genes in establishment of AER function. Taken together
with intact Fgf10 expression in
Pitx1-/-,Pitx2+/- embryos and with the
restricted co-expression of Pitx genes in early limb field lpm, these data are
consistent with a role of Pitx genes in determining the growth capacity of
limb bud mesenchyme (Fig.
8A).
Limb malformations resulting from thalidomide exposure may resemble to some
extent the loss of HL skeletal elements in Pitx-deficient mice. In children
with thalidomide defects, upper limbs are affected more frequently than lower
limbs, but the sequence of limb loss with severity is usually thumb (first
digit), radius, humerus and ulna
(Smithells and Newman, 1992).
In legs, tibia and femur are most often affected. These deficiencies are
similar to those observed for HL in Pitx mutant embryos, suggesting a possible
relationship in mechanism.
What about forelimbs?
The present study suggests an important function for Pitx gene dose in the
growth and patterning of HLs. However, none of the mutant embryos described in
the present work has any phenotype in FL. We must therefore conclude that Pitx
genes do not play any role in FL development and this is consistent with the
absence of Pitx1 or Pitx2 expression in FL buds, except in
myoblasts. This is a somewhat surprising conclusion but the later appearance
of HL during evolution would not be incompatible with the recruitment of Pitx
genes for growth and patterning of HLs, independently of mechanisms acting at
FLs. It is unlikely that another Pitx gene may fulfill a similar function in
FLs as the only other Pitx gene known, Pitx3, is not expressed in
early FL buds (A. M. and J. D., unpublished observations). In this context,
the control of HL bud growth by Pitx genes may be viewed as a recent
function.
Recent work suggests that the Tbx5 gene plays an essential role
for outgrowth of forelimb buds that resembles that of Pitx genes in HL.
Indeed, FL buds do not develop in Tbx5-/- mouse embryos
(Agarwal et al., 2003) (M.
Logan, personal communication). By contrast, the HL-specific Tbx4
gene does not appear to play a similar limiting role for HL bud outgrowth
because Tbx4-/- embryos develop HL buds (V. Papaioannou,
personal communication). The role of Tbx4 thus appears to be
primarily in specification of HL identify. Taken together, these studies
suggest different mechanisms for outgrowth and specification in HL and FL
(Fig. 8C). In FL, the primary
gene controlling both outgrowth and specification appears to be Tbx5,
whereas in HL, these roles are taken by Pitx1, with the downstream
Tbx4 contributing together with Pitx1 only for specification
of HL identity.
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ACKNOWLEDGMENTS |
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