Division of Developmental Biology, National Institute for Medical Research, London, NW7 1AA, UK e-mail: mlogan{at}nimr.mrc.ac.uk
SUMMARY
Despite their obvious similarities, the forelimbs and hindlimbs of tetrapod vertebrates have evolved distinct structural elements to carry out their discrete functions. Many genes required for limb initiation and patterning are involved in regulatory networks common to both limb-types. Other genes are differentially expressed between forelimb and hindlimb, and have been implicated in the initiation of limb bud outgrowth and the specification of limb-type identity. In this review, I will discuss the current understanding of how genes that control limb identity interact with regulatory networks common to both appendages to produce the fingers of the hand and toes of the foot.
Introduction
The mature limb is a complex structure comppsed of many different tissues types. To produce the interconnected array of bones, muscles and tendons that make up the limb, processes such as cell fate specification, proliferation, apoptosis and migration must be tightly controlled. Many of the genes required for initiating limb development, and for subsequent patterning of the limb bud, are now known. In most cases, these genes are expressed in identical patterns in both the developing forelimb and hindlimb, and are believed to play identical roles in generating homologous limb elements; for example, fingers in the hand and toes in the foot. This raises the important and general question in developmental biology of how two different structures are produced by common signalling cascades. Recent work has begun to explain how genes that specify limb-type identity interact with signalling cascades common to both forelimb and hindlimb. Limb-type specification therefore provides an ideal model for studying the way in which the embryo produces serially homologous structures by modulating the outcome of conserved gene regulatory networks.
The mechanisms that control the development of both limb-types have been
reviewed comprehensively elsewhere
(Capdevila and Izpisua Belmonte,
2001; Tickle,
2003
). The aim of this review is to cover recent developments that
have advanced our understanding of how the differences between forelimbs and
hindlimbs are established in vertebrates. I will discuss the roles of genes
that are expressed exclusively in one limb-type and not the other during
embryonic development, and outline the current understanding of how these
genes interact with regulatory networks common to both limb-types to produce
structures unique to the forelimb and hindlimb.
Limb patterning: an overview of key events
The forelimb and hindlimb buds are derived from regions of the lateral
plate mesoderm (LPM). The LPM comprises two strips of tissue that run along
the length of the main body axis, lateral to the somites
(Fig. 1A). The territories of
the LPM that give rise to limbs are located at defined rostrocaudal levels
along the main body axis (Fig.
1A). Limb bud outgrowth is initiated when cells within the
prospective limb-forming regions respond to cues from more medial tissues
(reviewed by Capdevila and Izpisua
Belmonte, 2001). Initially, the limb buds are morphologically
uniform collections of cells, and these subsequently develop into structurally
distinct limb elements. The limb musculature is derived from myotomal cells
that originate in the somites medial to the limb bud and then migrate into the
limbs.
|
Lessons from classical embryological studies
The first morphological sign of limb development is a budding of tissue
from the LPM. The first transplant experiments to explore the determination of
limb-type identity were carried out in the newt. Harrison demonstrated that
cells of the limb-forming region were able to give rise to fully developed
limbs when transplanted to ectopic sites along the flank of the embryo
(Harrison, 1918). Similar
experiments using chick embryos demonstrated that limb-type identity is
determined by the origin of the graft
(Hamburger, 1938
): tissue
taken from the wing-forming region produces an ectopic wing and cells
transplanted from the leg-forming region always produce a new leg. Tissue
grafts from the prospective limb-forming tissue therefore behave as
classically defined developmental fields in that they have the properties of
self-determination and self-organisation
(Jacobson and Sater, 1988
).
Building upon these original observations, more extensive transplantation
experiments in the chick have mapped the temporal and spatial sequence of the
limb-forming capacity of the LPM (Stephens
et al., 1989
). Transplanted LPM tissue taken from prospective
limb-forming regions forms a limb when placed in an ectopic location at stages
just prior to overt limb outgrowth. Similar transplants of LPM tissue from the
neck or inter-limb flank at the same stage will not form a limb. The limb-type
potential of the grafts is fixed by this stage; prospective wing and leg
forming regions will only develop into wing or leg, respectively.
If heterotopic grafts in the chick are performed after a limb bud has
formed, a chimaeric limb develops that contains elements of both limb-types
(Saunders, 1948;
Kieny, 1964
). Furthermore,
when prospective thigh mesoderm is grafted beneath the AER of the developing
chick wing (Saunders et al.,
1957
; Saunders et al.,
1959
) it is re-specified in its proximodistal fate, but its
limb-type identity remains fixed. These grafts form hindlimb toes and not wing
digits, demonstrating that the identity of the developing limb bud is stably
determined by this stage and remains unaltered if grafted to a new position
within the embryo.
Tissue recombination experiments in the chick have also demonstrated that
limb-type identity is determined by the mesodermal, rather than the
ectodermal, component of the limb
(Zwilling, 1956a;
Zwilling, 1956b
). If wing-bud
mesenchyme is combined with leg-bud ectoderm and then transplanted to the
flank, a normally patterned wing is produced. The reciprocal recombination,
leg-bud mesenchyme combined with wing-bud ectoderm, produces a normally
patterned leg. Furthermore, when surface ectoderm from one limb-type is
grafted to a host bud of another, the ectoderm assumes the identity of the
host mesenchyme (Saunders and Gasseling,
1968
). Limb-type identity is therefore a property of the mesoderm,
which provides instructions to the overlying ectoderm.
Classical embryological experiments have therefore established the criteria that candidate specifiers of limb-type identity need to meet. Such candidates are expected to be expressed prior to overt limb-bud formation and to show maintained expression during subsequent limb bud outgrowth. Their expression is also expected to be restricted to the limb mesenchyme.
Candidate specifiers of limb-type identity
Most factors thought to play important roles in vertebrate limb patterning
have common expression patterns in both forelimb and hindlimb, but there are
exceptions to this general rule. The first genes found to be expressed
exclusively in either forelimb or hindlimb were members of the Hox gene family
(Nelson et al., 1996;
Oliver et al., 1988
;
Simon and Tabin, 1993
;
Tabin, 1989
). However, none of
these genes satisfy the necessary criteria to qualify as candidates for
specifying limb-type identity; they are expressed at relatively late stages of
limb development and are not expressed throughout the limb mesenchyme.
Instead, they may be downstream targets, and perhaps downstream effectors, of
factors that specify limb-type identity. Other data indicate that Hox genes
also act upstream of factors that control limb-type identity. Hox9 paralogues
and other genes of the four main Hox clusters are believed, from their nested
expression patterns along the main body axis, to create a combinatorial code
that may specify the molecular identity of the prospective forelimb and
hindlimb territories (Cohn et al.,
1997
; Rancourt et al.,
1995
). However, gene deletion analysis in the mouse has not been
informative regarding the potential roles for these genes in positioning the
limb-forming regions.
Studies on a range of vertebrate species have identified three genes with
temporal and spatial expression patterns consistent with their having roles in
specifying limb-type identity. Two T-box family transcription factors,
Tbx5 and Tbx4, are expressed in the forelimb and hindlimb
buds, respectively (Gibson-Brown et al.,
1996; Ruvinsky et al.,
2000
; Simon et al.,
1997
; Takabatake et al.,
2000
) (Fig. 2A,B).
A third gene, the paired-type homeodomain factor Pitx1, is expressed
in the developing hindlimb but not in the forelimb
(Lamonerie et al., 1996
;
Szeto et al., 1996
)
(Fig. 2C). Each of these three
genes are expressed throughout the limb mesenchyme and not in the limb
ectoderm (Gibson-Brown et al.,
1998
; Isaac et al.,
1998
; Logan et al.,
1998
; Ohuchi et al.,
1998
). Furthermore, these studies show that the limb-type
restricted expression pattern of each gene is retained in grafts of wing
tissue into leg bud, or leg tissue into wing bud in chick embryos.
|
Insights into limb-type specifiers in the chick
Direct evidence that Tbx5, Tbx4 and Pitx1 have roles in
specifying limb-type identity has been obtained from misexpression experiments
in the chick. Ectopic expression of Tbx5 in the developing chick
hindlimb bud can, at least partially, transform the identity of the leg to a
more wing-like morphology
(Rodriguez-Esteban et al.,
1999; Takeuchi et al.,
1999
). In the limb bud, ectopically expressed Tbx5 can
induce the expression of forelimb markers, such as Hoxd9, and repress
the expression of hindlimb markers, for example Hoxc9. If allowed to
develop to later stages, the resulting limbs produce elements with
morphological characteristics of a wing, such as wing digits and feather buds.
In the converse experiment, hindlimb-specific genes Tbx4 or
Pitx1 misexpressed in the forelimb can transform the wing to a more
hindlimb character (Logan and Tabin,
1999
; Takeuchi et al.,
1999
). Similarly, Tbx4 can transform the identity of
Fgf-induced ectopic limbs induced in the interlimb region of the LPM
(Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
). Again, genes that are normally restricted to the hindlimb,
such as Hoxc10 and Hoxc11, are ectopically induced in the
targeted forelimb or ectopic limb and, if these limbs are allowed to develop
further, they produce characteristic leg-like elements such as clawed toes and
scales. In each case, the transformed limb contains elements that are derived
from both the mesenchyme, such as muscle and skeletal elements, and the
ectoderm, such as feather buds and scales. This indicates that ectopically
expressed Tbx5 and Tbx4 alter cell fate by acting directly
on the mesoderm, and indirectly on the ectoderm.
Although the limb phenotypes obtained in chicks following the ectopic
expression of Tbx5, Tbx4 or Pitx1 are dramatic, they are not
completely penetrant, and the resulting limbs are made up of constituent
forelimb, hindlimb or intermediate limb-type characteristics. This can be
explained in part by the observation that, following the misexpression of
Tbx4 or Pitx1, the endogenous expression of Tbx5 in
the forelimb is unchanged (Logan and
Tabin, 1999; Rodriguez-Esteban
et al., 1999
; Takeuchi et al.,
1999
). This observation indicates that in these misexpression
experiments, cells in the limb are presented with two competing programmes; in
some cases one programme may dominate, whereas in others the cells may assume
a mixed or intermediate fate.
The function of Tbx5
The requirement for Tbx5 during vertebrate forelimb initiation has
been confirmed by knockdown experiments in the chick and by targeted gene
inactivation in the mouse. Mouse embryos mutant for Tbx5 have no
detectable forelimb bud outgrowth and no morphologically distinct AER
(Agarwal et al., 2003;
Rallis et al., 2003
).
Tbx5 is required for normal heart development and therefore
Tbx5 mutant mice do not survive beyond early limb bud stages
(Agarwal et al., 2003
). To
overcome this mid-gestation lethal phenotype, a conditional approach has been
taken to delete Tbx5 function in the developing limbs while leaving
the gene intact in the heart; this generates embryos that survive until birth
(Rallis et al., 2003
). In
these embryos, all the elements of the limb, including the shoulder girdle,
are absent, demonstrating that Tbx5 is required for the formation of
all skeletal elements of the forelimb.
Misexpression experiments in the chick have indicated that Tbx5
specifies forelimb identity, in part, by repressing expression of the hindlimb
genes Tbx4 and Pitx1
(Rodriguez-Esteban et al.,
1999; Takeuchi et al.,
1999
). However, in mouse embryos mutant for Tbx5, the
hindlimb genes Tbx4 and Pitx1 were not induced in the
presumptive forelimb-forming region. These genetic studies indicate that, in
the mouse, Tbx5 does not normally repress Tbx4 in the
forelimb (Agarwal et al.,
2003
).
Tbx5 and forelimb bud initiation
Targeted inactivation of Tbx5 in the mouse, and knockdown of
Tbx5 in zebrafish and chick, have recently provided new insights into
the function of this gene during limb development. To understand the role of
Tbx5 in early limb formation, it is necessary to review our current
understanding of the molecules involved in limb initiation. A number of
experimental approaches in a range of model organisms indicate that
Tbx5 is essential for forelimb bud formation, and that it interacts
with the Fgf and Wnt signalling pathways to initiate limb
bud outgrowth (Agarwal et al.,
2003; Ahn et al.,
2002
; Garrity et al.,
2002
; Ng et al.,
2002
; Rallis et al.,
2003
; Takeuchi et al.,
2003
). In the following sections, the current understanding of the
interactions between Tbx5 and Fgf and Wnt family proteins, which initiate and
maintain limb bud formation, is outlined.
Members of the Fgf family of secreted proteins are thought to play
essential roles in both forelimb and hindlimb bud initiation. As outlined
earlier, several Fgfs are able to induce the formation of ectopic limbs from
the flank of the embryo (reviewed by
Martin, 1998). In addition,
deletion of Fgf10 in the mouse results in the absence of limbs
(Min et al., 1998
;
Sekine et al., 1999
).
Fgf10 signalling in the limb mesenchyme is required to induce and
maintain normal Fgf8 expression in the AER
(Min et al., 1998
;
Ohuchi et al., 1997
;
Sekine et al., 1999
). A
positive-feedback loop is then established in which Fgf8 expression
in the AER maintains expression of Fgf10 in the underlying mesenchyme
(Fig. 3).
|
In the zebrafish, initiation of the pectoral fin bud, which is analogous to
the forelimb of the chick and mouse, is regulated by both tbx5 and
wnt2b. Functional knockdown of tbx5 in the zebrafish using
antisense morpholino oligonucleotides results in a failure to initiate
pectoral fin bud formation, and the absence of fgf10 and
fgf8 expression in the fin bud mesenchyme and AER, respectively
(Ahn et al., 2002;
Garrity et al., 2002
;
Ng et al., 2002
). A mutation
in the zebrafish tbx5 gene heartstrings, induced by the
chemical mutagen ENU, produces an identical phenotype to the tbx5
morpholino-knockdown phenotype (Garrity et
al., 2002
). Morpholino knockdown of wnt2b produces
phenotypes similar to those obtained following knockdown of tbx5, and
tbx5 expression is downregulated in embryos in which wnt2b
activity is disrupted (Ng et al.,
2002
). Furthermore, although tbx5 partially rescues the
phenotype of wnt2b knockdown, injection of wnt2b RNA could
not rescue the phenotype resulting from tbx5 knockdown. Together, the
results suggest that pectoral fin initiation is co-regulated by wnt2b
and tbx5, and that tbx5 lies downstream of wnt2b
(Ng et al., 2002
).
As yet, no Wnt family member has been described in the mouse that is
expressed in a similar manner to that of Wnt2b and Wnt8c in
the chick, or wnt2b in the zebrafish. Furthermore, in mouse embryos
mutant for two nuclear components of Wnt signalling, Lef1 and
Tcf1, limb bud outgrowth is initiated normally
(Galceran et al., 1999). This
suggests that a comparable role for Wnt signalling in limb bud initiation may
not have been conserved in the mouse. Alternatively, other Wnt genes in the
mouse might be expressed in a comparable pattern to Wnt2b and
Wnt8c in the chick, and other Tcf genes may compensate for the loss
of Tcf1 in Lef1-/-/Tcf1-/-
embryos (Ng et al., 2002
).
Further experiments to determine the signalling hierarchy between Tbx5 and Fgf
and Wnt proteins are discussed later in the review.
A genetic hierarchy in limb initiation and outgrowth
To examine the genetic hierarchy of Tbx5 and Fgfs in limb
initiation, the expression patterns of these genes have been analysed in
different mutant mouse backgrounds. In the forelimbs of embryos mutant for
Fgf10, Tbx5 is initially activated but expression is not maintained,
suggesting that Tbx5 initially acts upstream of Fgf10
(Sekine et al., 1999)
(Fig. 3). Consistent with this
model, deletion of Tbx5 in the mouse results in the loss of
Fgf10 expression in the limb bud mesenchyme
(Agarwal et al., 2003
;
Rallis et al., 2003
).
Similarly, following knockdown of tbx5 function in zebrafish
(Ahn et al., 2002
;
Ng et al., 2002
), or
misexpression of dominant-negative forms of Tbx5 in the chick
(Ng et al., 2002
;
Takeuchi et al., 2003
),
Fgf10 expression is either absent or downregulated in the developing
limb. As a consequence of failure of normal Fgf10 signalling in
Tbx5 mutants, Fgf8 expression is also disrupted in the limb
(Agarwal et al., 2003
;
Ng et al., 2002
;
Rallis et al., 2003
;
Takeuchi et al., 2003
).
A number of observations suggest that activation of Fgf10 by
Tbx5 expression is direct, as T-box binding sites have been
identified in the Fgf10 promoter in human, mouse and zebrafish
(Agarwal et al., 2003;
Ng et al., 2002
). The human
and mouse Fgf10 promoter also contains consensus binding sites of the
nuclear mediators of Wnt signalling Lef1 and Tcf1,
suggesting additional regulation by Wnt family members
(Agarwal et al., 2003
;
Ng et al., 2002
). In in vitro
transactivation studies, Tbx5 can activate a Fgf10 promoter
construct. This promoter can also be activated by a constitutively-active form
of ß-catenin, which is an essential intracellular component of
the canonical Wnt signalling pathway. Furthermore, Tbx5 and
ß-catenin, in combination, have an additive effect on activation
of the Fgf10 promoter, indicating that, in mouse, Tbx5 and
Wnt signalling may act together to regulate Fgf10
(Agarwal et al., 2003
). Direct
positive-feedback loops between T-box genes and Fgfs have been established in
other tissues during development. In the chick, a regulatory loop between
Tbx4 and Fgf10 has been implicated in lung formation
(Sakiyama et al., 2003
),
whereas in Xenopus a positive regulatory loop between eFgf
and Brachyury operates during mesoderm formation
(Schulte-Merker and Smith,
1995
).
Several experiments have been carried out to determine the hierachical
relationship between Tbx5 and Wnt and Fgf signalling. Lef1 is a transcription
factor that is part of the transcriptional read-out of Wnt signalling during
limb initiation. In in vitro co-transfection experiments, mutation of a
conserved Lef1 binding site in the 5' promoter of both human and mouse
Tbx5 leads to a decrease in the activation of expression constructs
containing this promoter. This indicates that Wnt signalling may normally
activate the Tbx5 promoter. Consistent with this model, in chick
embryos, misexpression of axin, a negative regulator of the Wnt
pathway, leads to the downregulation of Tbx5
(Ng et al., 2002).
However, other experiments carried out in mouse and chick show that Tbx5
acts upstream of Wnt signalling during limb initiation. The expression of
Tbx5 and Fgf10 was examined in
Lef1-/-/Tcf1-/- mouse embryos
(Galceran et al., 1999). In
these embryos, expression of Tbx5 was unaffected, whereas
Fgf10 expression was detected only at low levels
(Agarwal et al., 2003
). These
results indicate that, in the mouse, Tbx5 acts upstream of Wnt
signalling in the forelimb field, and that Wnt signalling, via
ß-catenin, Lef1 and Tcf1, is required to maintain
normal levels of Fgf10 expression. In support of these conclusions,
in the chick, dominant-negative forms of Tbx5 misexpressed in the
limb led to the absence of Wnt2b and Fgf10 expression in the
prospective wing region (Takeuchi et al.,
2003
). In addition, misexpression of a dominant-negative form of
Lef1 initially had no effect on Tbx5 expression
(Takeuchi et al., 2003
). These
observations suggest that Tbx5 acts upstream of Wnt signalling
(Agarwal et al., 2003
;
Takeuchi et al., 2003
),
although contradictory data in chick (Ng
et al., 2002
) indicates that this may have to be confirmed.
Moreover, the evidence from both mouse and chick contrasts with the results
from zebrafish, described earlier (Ng et
al., 2002
), which indicate that in zebrafish Tbx5 and Wnt2b
co-regulate limb initiation.
To understand the relationship between Tbx5 and Fgf signalling
during limb outgrowth, mouse embryos with deleted Fgfr2 have also
been also examined. Two different isoforms of Fgfr2 exist; these
isoforms act as receptors for Fgf8 (isoform Fgfr2c) or Fgf10
(isoform Fgfr2b), and both isoforms are required for Fgf signalling during
limb bud formation (reviewed by Martin,
1998; Xu et al.,
1998
). In the presumptive forelimb buds of Fgfr2 mutant
mouse embryos, Tbx5 expression was initiated but was not maintained,
indicating that the initial expression of Tbx5 is independent of both
Fgf8 and Fgf10, but might be maintained by Fgf signalling. Furthermore,
application of a potent inhibitor of Fgfr, SU5402, led to the downregulation
of Tbx5 expression in the limb
(Ng et al., 2002
), consistent
with a role of Fgf signalling in the maintenance of Tbx5 expression.
Together, these results indicate that Tbx5 acts directly upstream of
Fgf10 to initiate limb outgrowth. Subsequently, Tbx5 and Wnt
signalling act cooperatively to maintain expression of Fgf10. It
appears that a positive-feedback loop is established in which Fgf10
is required to maintain expression of Tbx5.
Although in the mouse, Wnt genes do not appear to be involved in forelimb
initiation, Wnt signalling is required for proper establishment and
maintenance of the AER. In
Lef1-/-/Tcf1-/- double-mutant embryos,
Tbx5 expression is unaffected and limb bud outgrowth is initiated,
but the AER does not form and outgrowth is not maintained
(Galceran et al., 1999). This
is consistent with the proposed roles of Wnt3 in the mouse
(Barrow et al., 2003
) and
Wnt3a in the chick (Kengaku et
al., 1998
; Kengaku et al.,
1997
) in AER formation.
Tbx5 and cell migration in zebrafish
Following morpholino knockdown of tbx5 in the zebrafish,
tbx5-expressing cells in the dorsoanterior LPM fail to move into the
pectoral fin-bud producing region, which is situated in a more ventroposterior
position. This indicates that tbx5 is required for the proper
migration of cells that will form the pectoral fin
(Ahn et al., 2002). It is
unclear whether this observation has any significance in higher vertebrates
because, in chick and mouse, Tbx5 is first detected in the region of
the LPM that gives rise to the forelimb buds
(Gibson-Brown et al., 1996
;
Gibson-Brown et al., 1998
),
and these cells do not undergo a similar migration. Zebrafish ikarus
(ika) mutants have mutations in fgf24, which functions in
the genetic cascade responsible for fin initiation and is implicated in the
migration of tbx5-expressing mesenchymal cells into the fin bud
(Fischer et al., 2003
).
ika mutants survive to adulthood but lack pectoral fins, although
they have normal pelvic fins and no other obvious defects.
tbx5-expressing cells fail to migrate normally in ika
mutants, indicating that fgf24 is required for their correct
migration to the pectoral fin primordium. As the fgf24 mutation only
affects the pectoral fin, other molecules must operate during pelvic fin
initiation.
Tbx5 and human disease
Experiments in which Tbx5 function has been disrupted are of
clinical significance because TBX5 mutations in humans are associated
with Holt-Oram Syndrome [HOS; Online Mendelian Inheritance in Man (OMIM)
database number 142900
http://www.ncbi.nlm.nih.gov/omim/],
a dominant disorder that is characterised by upper(fore)limb abnormalities and
heart defects (Basson et al.,
1997; Li et al.,
1997
). Haploinsufficiency of TBX5 causes a range of limb
abnormalities, the mildest phenotype being triphalangeal (three-jointed) thumb
but which most commonly results in the failure of limb elements to form. A
consistent feature of HOS is that abnormalities affect the anterior structures
of the limb, such as the thumb or radius. Consistent with observations in
humans, the heterozygous Tbx5 knockout mouse has defects in digit I
(Bruneau et al., 2001
). The
knockdown of Tbx5 function in zebrafish and chick also produces
phenotypes that are strikingly similar to those observed in HOS patients.
Morpholino knockdown of tbx5 in the zebrafish produces a range of
phenotypes. The most severe cases are identical to those observed in mouse
null mutants: complete absence of the entire pectoral fin. However, by
decreasing the amount of morpholino injected, which is expected to decrease
the effective knockdown of the gene product, progressively milder phenotypes
are produced, such as shortened pectoral fins and a reduced number of rays
(Ng et al., 2002
). In the
chick, a consistent feature of misexpression of dominant-negative forms of
Tbx5 is the disruption of the anterior limb mesenchyme and, as a
consequence, the structures that are ultimately derived from this region of
the limb bud (Ng et al., 2002
;
Rallis et al., 2003
;
Takeuchi et al., 2003
). The
most common phenotype is the absence of the radius and anterior digits, and
this is associated with an observed downregulation of Fgf10 and
Fgf8 in the anterior of the limb, as well as of molecular markers of
the anterior mesenchyme. The analysis of Tbx5 mutants in a range of
vertebrate model organisms from fish to mouse will provide molecular insights
into how the defects observed in HOS arise.
Tbx4 and hindlimb development
Gene deletion, knockdown and misexpression experiments have demonstrated
that Tbx5 is the primary and direct initiator of forelimb bud
outgrowth. In a search for candidate genes with similar roles in the hindlimb,
the simplest model proposes that the closely-related gene Tbx4 plays
an analogous role in the hindlimb. Experiments in the chick have demonstrated
that this might be the case; misexpression of Tbx4 in the flank was
sufficient to induce the formation of an ectopic limb
(Ng et al., 2002;
Takeuchi et al., 2003
).
However, deletion of Tbx4 in the mouse indicates that the action of
Tbx4 in the hindlimb may not simply parallel that of Tbx5 in
the forelimb. In mouse embryos mutant for Tbx4, hindlimb bud
induction and initial patterning occurs normally. However, at later stages of
development Fgf10 expression is not maintained in the mesenchyme and
mutant limbs fail to develop beyond early limb bud stages
(Naiche and Papaioannou,
2003
). In addition to displaying hindlimb defects, Tbx4
mutant embryos fail to undergo chorioallantoic fusion. The failure of this
fusion is thought to cause embryonic lethality by embryonic day (E) 10.5.
Unfortunately, this lethality prevents a more detailed analysis of the
hindlimb defects of Tbx4 null mice at later stages of development.
Nevertheless, two aspects of the Tbx4 mouse mutant phenotype raise
particularly intriguing questions. First, what are the mechanisms that allow
hindlimb bud initiation to proceed normally in the absence of Tbx4
when forelimb bud initiation does not occur in Tbx5 mutants? Second,
why do Tbx4 mutant limbs fail to develop beyond early limb bud
stages?
To understand the events surrounding hindlimb bud initiation, markers of
the AER and limb mesenchyme have been analysed in Tbx4 null-mutant
embryos. In these embryos, at early stages of hindlimb initiation,
Fgf8 is expressed normally in the AER. Msx1, a gene normally
expressed in the underlying mesenchyme in response to Fgf signalling from the
AER, is also unaffected (Naiche and
Papaioannou, 2003). The establishment of normal Fgf signalling in
mutant limb mesenchyme was confirmed by the distribution of doubly
phophorylated Erk protein (dpErk), which detects activation of the Map kinase
cascade by the Fgf receptor and serves as a read-out of Fgf signalling. Normal
Fgf signalling in mutant limb mesenchyme may explain why limb bud initiation
proceeds normally in Tbx4 null embryos. Pitx1 expression is
also maintained in mutant embryos, indicating that its expression is not
dependent on Tbx4 (Naiche and
Papaioannou, 2003
). This may also explain how normal hindlimb
initiation occurs in the absence of Tbx4; the loss of Tbx4
in the hindlimbs may be compensated for by a Pitx gene. Compensation for the
loss of Tbx4 by Tbx5 can be ruled out because Tbx5
expression remains restricted to the forelimb, and is not ectopically induced
in the mutant hindlimb. Nevertheless, despite the apparently normal induction
of the hindlimb, and the establishment of the Fgf-feedback loop between the
mesenchyme and ectoderm, Fgf10 expression is not maintained in the
hindlimb of Tbx4 null-mutant embryos. The mechanism of this failure
is not understood but it may explain why Tbx4 mutant limbs fail to
develop beyond early limb bud stages.
Pitx1 and Pitx2 cooperate in hindlimb development
A role for Pitx1 in the specification of hindlimb identity has
been supported by both gene-misexpression and gene-inactivation experiments.
Misexpression of Pitx1 in the developing chick wing can, at least
partially, transform both muscle and skeletal elements of the forelimb and
give them a more hindlimb-like character
(Logan and Tabin, 1999;
Takeuchi et al., 1999
). In
Pitx1 null mice, the hindlimb does form, but features of hindlimb
identity are lost and the limb has a more forelimb-like character
(Lanctot et al., 1999
;
Szeto et al., 1999
). For
example, the diameters of the tibia and the fibula in the Pitx1
mutant limb are similar to those of the homologous elements in the forelimb,
the radius and the ulna, respectively. In addition, the secondary cartilage of
the knee joint, comprising the patella and fabella, fails to form, resulting
in an articulation that is more reminiscent of the elbow joint. Levels of
Tbx4 transcripts are lower in Pitx1-/- hindlimbs
than in wild type, but are nevertheless detectable
(Lanctot et al., 1999
). This
is consistent with the notion that Pitx1 contributes to Tbx4
expression in the hindlimb, but demonstrates that Pitx1 is not absolutely
required for the induction of Tbx4.
A surprising observation to emerge from of the analysis of the
Pitx1-/- null mouse embryos was an occasional left-right
(L-R) asymmetry in the severity of the limb phenotype
(Lanctot et al., 1999). The
right hindlimb was often more severely affected than the left hindlimb,
possibly due to a degree of redundancy between Pitx1 and the
Pitx1-related homeobox factor Pitx2. During embryogeneis,
the Pitx2 gene is asymmetrically expressed at many sites on the left
side of the embryo and is an effector of L-R asymmetry (reviewed by
Capdevila et al., 2000
).
Remarkably, analyses of the
Pitx1-/-;Pitx2-/- double knockout and
Pitx1-/-;Pitx2-/+ mouse
embryos have confirmed this, and have established that Pitx1 and
Pitx2 can co-operate during hindlimb formation
(Marcil et al., 2003
).
Pitx1 and Pitx2 are co-expressed in the tailbud region,
which includes a region that is destined to become hindlimb, although this
co-expression is not maintained at later stages of development. Hindlimb buds
in Pitx1-/- embryos are smaller than normal limb buds. The
smaller limb size relative to normal hindlimbs is more pronounced in
Pitx1-/-;Pitx2-/+ mouse embryos than
in Pitx1-/-;Pitx2-/- embryos
(Marcil et al., 2003
). The
reduction in limb size is more pronounced in the anterior than the posterior
of the limb bud, and the skeletal defects that ultimately arise are consistent
with the reduction in anterior mesenchyme observed at earlier stages. Although
no dramatic reduction in Fgf10 or Fgf8 expression is
observed in mutant limbs, their onset of expression is delayed and their
expression levels are slightly reduced, which may account, in part, for the
phenotype of
Pitx1-/-;Pitx2-/+ double
mutant embryos. Accordingly, the phenotype of
Pitx1-/-;Pitx2-/- mice is similar to
that observed following conditional deletion of Fgf8 in the AER
(Sun et al., 2002
), and the
phenotypes that result from disruption of Tbx5 and Tbx4 in
the forelimb and hindlimb, respectively. However, the higher levels of
Pitx1 protein compared with Pitx2, and the absence of any
obvious limb phenotype in Pitx2-/- embryos, is consistent
with Pitx1 being the primary determinant of hindlimb identity and its
subsequent development. Only in the absence of Pitx1 does the
contribution of Pitx2 become evident.
The observation that the hindlimbs of Pitx1 null mice possess
forelimb-type characteristics raises some interesting points of
interpretation: does the phenotype correspond to a loss of hindlimb character
and the `acquisition' of forelimb identity, or does it represent a loss of
hindlimb character that results in the reversion to an ancestral limb `ground
state'? Tbx5 expression remains forelimb-specific in
Pitx1-/- embryos, and is not ectopically induced in the
hindlimb. By this criteria at least, the mutant hindlimb has not `acquired'
forelimb identity as such, but rather has lost hindlimb identity. By
extension, forelimb morphology may be most similar to the ancestral ground
state and hindlimb identity the more derived character, perhaps due to the
acquisition of Pitx1 expression in the hindlimb-forming region
(Marcil et al., 2003). Indeed,
Pitx1 may play a pivotal role in defining the difference between
forelimb and hindlimb identity, the two distinct limb-forming territories
being distinguished by whether they express Pitx1 or not. To date no
forelimb specific equivalent to Pitx1 has been identified. A third
member of the Pitx subfamily, Pitx3
(Semina et al., 1997
), is not
expressed in developing limbs (Marcil et
al., 2003
). Further experiments will be required to clarify these
issues.
Summary and future directions
Gene deletion and misexpression studies have demonstrated that Tbx5, Tbx4 and Pitx1 have fundamental roles in limb-type specification (Table 1). Furthermore, gene deletion experiments have shown that all three genes are required for normal limb development and that in their absence limb characteristics are lost. Tbx5 is required for initiation of the forelimb, and interacts with the Fgf and Wnt signalling pathways. Tbx4 is required for hindlimb development, but hindlimb bud formation is initiated in the absence of Tbx4. In Pitx1 mutant embryos, the defects in hindlimb development are more subtle than those in Tbx4 mutant embryos. The limbs are smaller overall and hindlimb characteristics are lost. Furthermore, analysis of Pitx1-/-;Pitx2-/- and Pitx1-/-;Pitx2-/+ double-mutant mice indicates that Pitx genes are required for normal limb outgrowth.
|
Identifing the targets of Tbx5, Tbx4 and Pitx1 will help
elucidate the mechanisms by which common regulatory pathways are modulated to
produce distinct structures in the forelimbs and hindlimbs. Towards this goal,
serial analysis of gene expression (SAGE) of forelimb and hindlimb transcripts
has identified candidate target genes
(Margulies et al., 2001). For
example, Hox genes were identified in the SAGE screen, and may be targets of
these limb identity determinants that act later during the morphogenesis of
forelimb and hindlimb elements. Further work to understand the targets of
Tbx5, Tbx4 and Pitx1, and the molecular basis by which
limb-type morphologies are produced, is certain to enhance our understanding
of the broader issue of how organisms have reused regulatory signalling
cascades in diverse developmental contexts to produce novel structures.
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