Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth.

Abstract

Morphological differences between forelimbs and hindlimbs are thought to be regulated by Tbx5 expressed in the forelimb and Tbx4 and Pitx1 expressed in the hindlimb. Gene deletion and misexpression experiments have suggested that these factors have two distinct functions during limb development: the initiation and/or maintenance of limb outgrowth and the specification of limb-specific morphologies. Using genetic methods in the mouse, we have investigated the roles of Tbx5, Tbx4, and Pitx1 in both processes. Our results support a role for Tbx5 and Tbx4, but not for Pitx1, in initiation of limb outgrowth. In contrast to conclusions from gene misexpression experiments in the chick, our results demonstrate that Tbx5 and Tbx4 do not determine limb-specific morphologies. However, our results support a role for Pitx1 in the specification of hindlimb-specific morphology. We propose a model in which positional codes, such as Pitx1 and Hox genes in the lateral plate mesoderm, dictate limb-specific morphologies.

Full text

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Developmental Cell, Vol. 8, 75–84, January, 2005, Copyright 2005 by Elsevier Inc.DOI 10.1016/j.devcel.2004.11.013
Tbx5 and Tbx4 Are Not Sufficient to Determine
Limb-Specific Morphologies but Have
Common Roles in Initiating Limb Outgrowth
prospective hindlimb region but not in the developing
forelimb (Lamonerie et al., 1996; Logan et al., 1998;
Shang et al., 1997). The limb-type restricted expression
patternofthesegenesisretainedthroughoutlimbdevel-
opment. Misexpression experiments in the chick have
suggestedthatthesegenesareinvolvedinspecification
of limb-specific morphologies. Ectopic expression of
Tbx5 in the chick leg bud can induce a partial leg-to-
wingtransformation.Conversely,misexpressionofTbx4
in the chick wing bud is able to cause a partial wing-
to-leg transformation (Rodriguez-Esteban et al., 1999;
Takeuchi et al., 1999). Similarly, misexpression of Pitx1
in the wing bud leads to the development of limbs with
leg-like characteristics (Logan and Tabin, 1999; Szeto
et al., 1999; Takeuchi et al., 1999). Accordingly, in Pitx1
mutant mice, hindlimbs show a loss of hindlimb charac-
teristics (Lanctot et al., 1999; Szeto et al., 1999).
Gene deletion and knockdown experiments have
shown that Tbx5, Tbx4, and Pitx1 are required for the
initiation and/or maintenance of limb bud outgrowth.
Functional knockdown of zebrafish tbx5 results in a fail-
ure to initiate pectoral fin bud formation (Ahn et al.,
2002). Similarly, all skeletal elements of the forelimb are
missing in a limb-restricted Tbx5 knockout (Rallis et al.,
2003). One of the earliest molecular read-outs of limb
initiation is the expression of Fgf10 in the prospective
limb fields (Min et al., 1998; Sekine et al., 1999). When
Tbx5 is inactivated, Fgf10 is never expressed in the
prospective forelimb region (Agarwal et al., 2003). Tbx5
is therefore required for the induction of Fgf10 in the
LPM at prelimb bud stages, which leads to forelimb
bud initiation. In Tbx4?/?embryos, induction and initial
patterning of the hindlimb appears normal, but it fails
to develop further, and Fgf10 expression is not main-
tained in the hindlimb bud mesenchyme (Naiche and
Papaioannou,2003). Pitx1mutantmouse hindlimbsalso
display an outgrowth defect, although much less severe
than that observed in either Tbx5 or Tbx4 mutants.
Pitx1?/?hindlimbs are smaller than wild-type, yet the
skeletal elements, with the exception of the ilium, are
present (Lanctot et al., 1999; Szeto et al., 1999).
We have used loxP/Cre technology in combination
with transgenic methods in the mouse to disrupt and
replaceTbx5functionintheforelimb.Ourassayinvolves
attempting to rescue the no-forelimb phenotype of the
Tbx5 limb-restricted knockout (Rallis et al., 2003) by
expressing either Tbx4 or Pitx1, or both genes simulta-
neously,intheforelimb-formingregionwhereTbx5func-
tion has been specifically deleted. This genetic assay,
which we refer to as the limb-rescue assay, allows us
to test the properties of these factors in two processes
of limb development: (1) initiation of limb outgrowth and
(2) specification of limb-specific morphologies. We
show that Tbx4 can replace the function of Tbx5 and
rescue limb outgrowth, whereas Pitx1 cannot. In contrast
to previous chick misexpression studies, Tbx4-rescued
limbs have a forelimb-like phenotype, suggesting that
Tbx4 alone is not able to dictate hindlimb-specific mor-
phology and that forelimb characteristics can develop
in the absence of Tbx5. To determine whether Pitx1 can
Carolina Minguillon, Jo Del Buono,
and Malcolm P. Logan*
Division of Developmental Biology
National Institute for Medical Research
Mill Hill
London NW7 1AA
United Kingdom
Summary
Morphological differences between forelimbs and
hindlimbs are thought to be regulated by Tbx5 ex-
pressed in the forelimb and Tbx4 and Pitx1 expressed
in the hindlimb. Gene deletion and misexpression ex-
periments havesuggested thatthese factorshave two
distinct functions during limb development: the initia-
tion and/or maintenance of limb outgrowth and the
specificationoflimb-specific morphologies.Usingge-
netic methods in the mouse, we have investigated the
roles of Tbx5, Tbx4, and Pitx1 in both processes. Our
results support a role for Tbx5 and Tbx4, but not for
Pitx1, in initiation of limb outgrowth. In contrast to
conclusions from gene misexpression experiments in
the chick, our results demonstrate that Tbx5 and Tbx4
do not determine limb-specific morphologies. How-
ever, our results support a role for Pitx1 in the specifi-
cation of hindlimb-specific morphology. We propose
a model in which positional codes, such as Pitx1 and
Hox genes in the lateral plate mesoderm, dictate limb-
specific morphologies.
Introduction
Vertebrate forelimbs and hindlimbs are serially homolo-
gousstructures.Althoughthelimbbudsfromwhichthey
are derived are patterned by common signals during
embryonic development, they ultimately form morpho-
logically distinct structures. A question that arises is
how cells exposed to common signals can respond dif-
ferentially and give rise to distinct morphologies.
Vertebrate forelimbs and hindlimbs arise from regions
of the lateral plate mesoderm (LPM) at defined locations
along the rostral-caudal axis of the embryo. Trans-
plantation experiments in the chick have demonstrated
that limb-type specification, the process by which cells
oftheprospectivelimb-formingterritoriesareinstructed
to form either forelimb or hindlimb, occurs prior to the
initiation limb bud outgrowth (reviewed in Logan, 2003;
Saito et al., 2002; Stephens et al., 1989).
Three genes have been identified that fulfill many of
the criteria to be candidates to specify limb-type iden-
tity. Two T-box transcription factors, Tbx5 and Tbx4,
are expressed in the LPM of either the prospective fore-
limb or hindlimb region, respectively (Chapman et al.,
1996; Gibson-Brown et al., 1996). In addition, a paired-
related homeodomain factor, Pitx1, is expressed in the
*Correspondence: mlogan@nimr.mrc.ac.uk

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Developmental Cell
76
Figure 1. Transgenic Mouse Lines Express-
ing Tbx4 or Pitx1 in the Limbs
(A) Schematic of the Prx1-Tbx4 and Prx1-
Pitx1 transgenic constructs. The ORF frag-
ments (blue box) contain the HA epitope
(black box) as a 3? fusion. The numbers in
brackets denote the size of each fragment
in Kbp.
(B–E) Whole-mount in situ hybridization for
transgene-derived expression in the devel-
oping limbs at E10.5. Expression is also de-
tected in the head and flank in all lines. Tbx4
expression in Prx1-Tbx4(4.1) (B) and Prx1-
Tbx4(4.48) (C). Pitx1 expression in Prx1-
Pitx1(P1.1) (D) and Prx1-Pitx1(P1.2) (E). Lat-
eral views are shown.
(F) Western blot analysis of protein extracts
from transgenic lines. Transgene-derived
protein is detected by virtue of the HA tag.
In this exposure, protein levels in the Prx1-
Tbx4(4.1) are not detectable. Robust levels of
protein are detected in Prx1-Tbx4(4.48) and
Prx1-Pitx1(P1.1) lines. Lower levels of protein
are detected in the Prx1-Pitx1(P1.2) line.
transform a forelimb to a more hindlimb-like character,
we analyzed the morphology of Pitx1 transgenic fore-
limbs (expressing endogenous Tbx5), and we rescued
the no-forelimb phenotype of the Tbx5 limb-restricted
knockout by supplying both Tbx4 and Pitx1 simultane-
ously. In both cases, we observed a partial forelimb-to-
hindlimbtransformation,suggestingthatPitx1doesplay
aroleinthespecificationofhindlimb-specificmorpholo-
gies but that other factors may also be required.
Tbx4, but Not Pitx1, Can Rescue Limb Outgrowth
in the Absence of Tbx5
Tbx5 is required for initiation and outgrowth of the fore-
limb (Agarwal et al., 2003; Ahn et al., 2002; Rallis et al.,
2003). Using a conditional allele of Tbx5 and a Prx1-Cre
deleter transgenic line, we have previously shown that
in the absence of Tbx5 function, the forelimb fails to
form (Figure 2B; Rallis et al., 2003). To investigate
whether Tbx4 or Pitx1 are capable of replacing the func-
tion of Tbx5 in the forelimb, we crossed the Tbx4 and
Pitx1 transgenic lines into the genetic background of
the conditional deletion of Tbx5 in the limb (Tbx5lox/lox;
Prx1-Cre). Heterozygote Tbx5 mice (Tbx5lox/?;Prx1-Cre),
which form normal forelimbs (Rallis et al., 2003), serve
as controls (Figure 2A). One of the Tbx4 lines (4.48) was
capableofrescuing the
Tbx5lox/lox;Prx1-Cre mice (Figure 2C), and a limb formed
in the forelimb region. This demonstrates that Tbx4 can
replace the function of Tbx5 in limb outgrowth. The
Tbx4(4.1) line, which expresses much lower levels of
Tbx4protein (Figure1F),was notableto rescueforelimb
development (Figure 2D), suggesting that insufficient
amountsofTbx4proteinareproduced.Incontrast,none
of the Pitx1-expressing lines were able to rescue limb
outgrowth in the absence of Tbx5 (Figures 2E and 2F),
althoughatleastonelineexpressesPitx1athigherlevels
than Tbx4 in the Tbx4(4.48) line (Figure 1F). These data
demonstrate that Tbx4, but not Pitx1, can replace the
function of Tbx5 in controlling limb outgrowth.
Results
Generation and Characterization
of Transgenic Lines
Conditional deletion of Tbx5 in the developing limbs
leads to the complete absence of all forelimb elements
(Rallis et al., 2003). We have exploited this genetic back-
ground and developed an assay to test the ability of
Tbx4 and Pitx1 to rescue the forelimb defect that results
from the absence of Tbx5 function.
To distinguish transgene-derived expression of Tbx4
and Pitx1 from endogenous expression, the chick
cDNAs of each gene were placed under the regulation
of the Prx1 regulatory element (Martin and Olson, 2000).
To enable detection of transgene-derived protein, the
cDNAs were tagged with the HA epitope (Figure 1A).
Two independent lines were generated for both Tbx4
and Pitx1 and denoted 4.1 and 4.48 and P1.1 and P1.2,
respectively. In all four lines, the transgene is expressed
in the hindlimb and forelimbs as well as the cranial mes-
enchymeandbodywall(Figures1B–1E),consistentwith
previousobservationsofPrx1-driventransgenes(Logan
et al., 2002). Transgene expression in the limbs corre-
sponds to overexpression of either Tbx4 or Pitx1 in the
hindlimbs and ectopic expression of these genes in the
forelimbs. Western blot analyses and detection with an
anti-HA antibody showed differences in the levels of
protein expression. The Prx1-Tbx4(4.48) and Prx1-
Pitx1(P1.1) lines express higher levels of protein than
thePrx1-Tbx4(4.1)andPrx1-Pitx1(P1.2)lines(Figure1F).
forelimb defect inthe
The Tbx4-Rescued Limb Buds
Are Normally Patterned
During limb development, a positive feedback loop be-
tween Fgf8, expressed in the apical ectodermal ridge
(AER), and Fgf10, expressed in the mesenchyme, is es-
sential for proximodistal outgrowth of the limb bud (re-
viewed in Martin, 1998). Shh expression in cells of the
zone of polarizing activity (ZPA) in the posterior limb
mesenchyme is required for the precise anterior-poste-
rior patterning of the limb (Riddle et al., 1993). Expres-
sion of Fgf8, Shh, and Fgf10 in Prx1-Tbx4(4.48)-rescued

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Outgrowth and Identity of the Vertebrate Limb
77
model using genetic techniques in the mouse, we ana-
lyzed the limb-type identity of the Prx1-Tbx4(4.48)-res-
cued limb buds and limbs. In addition to Tbx5, Tbx4,
and Pitx1, certain Hox genes belonging to the HoxC
cluster have a limb-type restricted expression pattern
and have been implicated in limb-type specification
(Nelson et al., 1996; Peterson et al., 1994). Hoxc4 and
Hoxc5 are expressed in the forelimb, while Hoxc9,
Hoxc10, Hoxc11, Hoxc12, and Hoxc13 are expressed in
the hindlimb. Surprisingly, the Tbx4-rescued limb has
a forelimb-type pattern of gene expression. Following
deletion of Tbx5 and replacement with Tbx4, transcripts
from the endogenous Tbx5 conditional allele that has
been disrupted by Cre recombinase activity are still de-
tected using a probe that recognizes sequences still
presentintherecombined,nonfunctionaltranscript(Fig-
ure 4G). Hence, signals that normally restrict Tbx5 ex-
pression to the forelimb (Figure 4A) are still functioning.
Hoxc4andHoxc5,whicharenormallyexpressedinfore-
limb buds (Figures 4B and 4C), are also expressed in
Prx1-Tbx4(4.48)-rescued limb buds (Figures 4H and 4I).
Conversely, genes normally restricted to the hindlimb
are not ectopically expressed in the Prx1-Tbx4(4.48)-
rescued limbs. Tbx4, Pitx1, and Hoxc10 are expressed
in the hindlimbs but not in the Prx1-Tbx4(4.48)-rescued
limb buds (Figures 4J–4L), as seen in control littermates
(Figures4D–4F).Insummary,wedetectgeneexpression
patterns characteristic of a forelimb in the rescued limb.
To further analyze the identity of the Prx1-Tbx4(4.48)-
rescued limbs, we examined the skeletal morphology in
newborn pups and compared them to forelimbs and
hindlimbs from control littermates. The forelimb-type
character of the rescued limb was evident (compare
Figures4Nto4Mand4O).Threemainlimb-type-defining
features are noticeable: the presence of a scapula, the
relative length of the stylopod and zeugopod bones,
and the joint between stylopod and zeugopodal ele-
ments. The scapula of Tbx4-rescued newborn pups is
indistinguishable from that found in control littermates
(Figure4N,arrow).Thebonesofthestylopodandzeugo-
pod articulate to form an elbow-like joint with the distal
end of the humerus-like bone sitting in an apparent
trochlear notch at the proximal end of the ulna-like bone
(Figure 4N, arrowhead). In addition, the relative size of
the stylopod and zeugopodal bones is similar. This is
most comparable to the arrangement in the forelimb,
where the humerus is of equivalent length to the radius
andulnabones whileinthehindlimbthe femurissmaller
than the tibia. Furthermore, both zeugopodal bones in
the rescued limb are of similar length, resembling the
radius and ulna in forelimbs and not the tibia and fibula
in hindlimbs. Although overall the skeletal morphology
of the Tbx4-rescued limb is remarkably similar to that
of the forelimb, it is not completely identical. The deltoid
tuberosity of the humerus is absent, and the flexure of
the wrist is altered such that the hand extends directly
from the wrist and fails to turn inward. In summary, both
analyses of Tbx4-rescued limbs with limb-type re-
stricted markers at limb bud stages and examination of
the limb skeletal morphology in newborn pups demon-
strate their forelimb-like phenotype, refuting the postu-
lated role for Tbx4 in specifying hindlimb identity.
To determine whether these contradictory mouse/
chick results were due to differences in the expression
Figure 2. Transgene-Derived Tbx4, but Not Pitx1, Can Rescue the
Limb Defect in the Conditional Deletion of Tbx5
All panels show lateral views of E17.5 mouse embryos with the
exception of (E), which is P0.
(A) No limb defect is observed following deletion of one copy of
Tbx5 in the limbs.
(B) No forelimbs form following conditional deletion of Tbx5 in the
limbs.
(C) In the absence of Tbx5, limb formation is rescued by transgene-
derived Tbx4 using the Prx1-Tbx4(4.48) line (arrow).
(D) Limb formation is not rescued by the Prx1-Tbx4(4.1) trans-
genic line.
(E and F) In the absence of Tbx5, limb formation is not rescued by
Pitx1 from either the Prx1-Pitx1(P1.1) (E) or the Prx1-Pitx1(P1.2)
(F) lines.
limb buds is identical to that in control littermates
(Tbx5lox/?;Prx1-Cre) (compare Figures 3A with 3D for
Fgf8, 3B with 3E for Shh, and 3C with 3F for Fgf10).
This demonstrates that in Prx1-Tbx4(4.48)-rescued limb
buds, key signaling centers essential for normal limb
development are established and appear to function
normally, consistent with our observation that limb out-
growth is rescued at E17.5 (Figure 2C). The failure of
Pitx1 and low doses of Tbx4 protein to sustain limb
development in Tbx5lox/lox;Prx1-Cre mice was confirmed
in E10.5 embryos. Fgf8 expression is absent in the fore-
limb-forming region following attempted rescue with ei-
therthePrx1-Tbx4(4.1),thePrx1-Pitx1(P1.1),orthePrx1-
Pitx1(P1.2) lines (Figures 3G–3I). Similarly, although ex-
pression of Fgf10 can be detected in the hindlimbs in
Tbx5lox/lox;Prx1-Cre;Prx1-Pitx1(P1.1)embryos(Figure3J),
it is absent from the forelimb region at late E9.5 stage.
This demonstrates that Tbx4, but not Pitx1, can initiate
and maintain limb outgrowth in the absence of Tbx5.
Forelimb-like Identity of the Tbx4-Rescued Limbs
Genemisexpressionstudiesinthechickhavepreviously
suggested a role for Tbx5 and Tbx4 in specification of
forelimb and hindlimb identity, respectively (Rodriguez-
Esteban et al., 1999; Takeuchi et al., 1999). To test this

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Developmental Cell
78
Figure 3. Signaling Centers in the Limb Are
Established Normally in Tbx4-Rescued Limb
Buds
(A–F) Tbx5lox/?;Prx1-Cre control littermates
(A–C) and Tbx5lox/lox;Prx1-Cre;Prx1-Tbx4(4.48)
embryos (D–F) at E10.5. All are lateral views,
except (C) and (F), which are dorsal views.
Fgf8 is expressed in the AER (A), Shh in cells
of the ZPA (B), and Fgf10 in the limb mesen-
chyme (C). Fgf8 (D), Shh (E), and Fgf10 (F)
are all expressed normally in Tbx5lox/lox;Prx1-
Cre;Prx1-Tbx4(4.48) embryos.
(G–I) Fgf8 is not expressed in the forelimb-
forming region (indicated with an asterisk) in
embryos in which transgene-derived Tbx4 or
Pitx1 has failed to rescue the limb defect fol-
lowing deletion of Tbx5: (G) Prx1-Tbx4(4.1),
(H) Prx1-Pitx1(P1.1), (I) Prx1-Pitx1(P1.2).
(J) Fgf10 is expressed in the hindlimb bud
but is not expressed in the forelimb-forming
regioninwhichtransgene-derivedPitx1(P1.1)
hasfailed torescuelimb outgrowth(asterisk).
FL, forelimb; HL, hindlimb; RL, rescued limb.
levels of Tbx4, we doubled the dose of the Tbx4 trans-
gene by crossing the line to homozygosity in the
Tbx5lox/lox;Prx1-Crebackground.Inthesecases,although
normal limb morphology is severely affected, forelimb-
like characteristics can be still detected. A trochlear
notch and an olecranon process in the ulna-like bone
are clearly identifiable (Figures 4P and 4Q).
AnalysesofTbx5lox/lox;?-actin-Cre;Prx1-Tbx4(4.48)em-
bryos show that Tbx4 is still able to rescue limb out-
growth even when the cells in the prospective forelimb
field have never expressed endogenous Tbx5 (Figure
5A). Consistent with our results using the Prx1-Cre de-
leterline,theseTbx4-rescuedlimbbudshaveaforelimb-
like gene expression pattern. The normally forelimb-
restricted genes Tbx5, Hoxc4, and Hoxc5 are expressed
in the Tbx4-rescued limb in a pattern indistinguishable
from wild-type (Figures 5B–5D), while the normally hind-
limb-restricted markers Tbx4, Pitx1, and Hoxc10 (Fig-
ures5E–5G)arenotectopicallyexpressed.Theseresults
demonstrate that Tbx4 is able to rescue limb outgrowth
in cells in the forelimb-forming region that have never
been exposed to Tbx5 activity and that the resultant
limb expresses genes normally restricted to the fore-
limb. Furthermore, genes normally restricted to the
hindlimbarenotectopicallyinduced.Thisdemonstrates
that an initial pulse of Tbx5 expression is not able to
determine forelimb-specific morphologies.
Forelimb-like Identity of Tbx4-Rescued Limbs
after Ubiquitous Deletion of Tbx5
In the Prx1-Cre deleter line, Cre activity is first detected
at E9.0–E9.5 (Logan et al., 2002). However, Tbx5 tran-
scripts are first detected at E8.5 (Agarwal et al., 2003).
From our previous work (Rallis et al., 2003) and the
results presented here for the 4.1, P1.1, and P1.2 lines
that were unable to rescue limb outgrowth (Figure 2),
we have demonstrated that this transient expression of
Tbx5 is not sufficient to initiate forelimb development.
However, this observation raises the interesting possi-
bility that a short pulse of endogenous Tbx5 transcript
is sufficient to determine forelimb-specific morphology
such that, following deletion of Tbx5 and replacement
with Tbx4, a forelimb develops. To address this issue,
we used the ?-actin-Cre transgenic line (Lewandoski
and Martin, 1997) to disrupt Tbx5 gene function ubiqui-
tously in the early embryo and tested the ability of the
Prx1-Tbx4(4.48) line to rescue limb outgrowth. Although
these embryos die around E10.0 due to heart defects
(Bruneau et al., 2001), they survive long enough to de-
termine whether the appropriate set of normally limb-
type restricted genes are expressed in these Prx1-
Tbx4(4.48)-rescued limbs.
Pitx1 Can Partially Transform Forelimb
to Hindlimb-like Morphologies
Pitx1 is also expressed in a hindlimb-restricted manner
and has been implicated in specifying hindlimb-specific
morphologies (Lanctot et al., 1999; Logan and Tabin,
1999; Szeto et al., 1999; Takeuchi et al., 1999). Pitx1-
expressing transgeniclines were notable torescue limb
outgrowth followingdeletion ofTbx5. Wewere therefore
unable to address the ability of this gene to influence
limb-type identity in the absence of Tbx5. Instead, we
have used our transgenic reagents to test the ability of
Pitx1 to transform the forelimb-like morphology of the

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Outgrowth and Identity of the Vertebrate Limb
79
Figure 4. Tbx4-Rescued Limbs Have Forelimb Characteristics
Tbx5, Hoxc4, and Hoxc5 are normally expressed in the forelimb but not the hindlimb of control littermates (A–C). They are also expressed in
Tbx4-rescued limbs (G–I). Tbx4, Pitx1, and Hoxc10 are normally expressed in the hindlimb but not the forelimb of E10.5 control embryos (D–F).
These genes are not ectopically expressed in the rescued limb at E10.5 (J–L). All the embryos are E10.5 and shown in lateral views except
(D), (F), (J), and (L), which are ventral views, and (C) and (I), which are dorsal views. Alcian blue/Alizarin red staining of the control forelimb
(M), Tbx4-rescued limb (N), and control hindlimb (O) skeletal elements at P0. Alcian blue/Alizarin red staining of a rescued limb (E16.5) in
which the Tbx4 transgene is present to homozygosity (P). An outline of the skeletal elements of the limb shown in (P) illustrates that although
abnormal in shape, the articulation is elbow-like (Q). au, autopod; Dt, deltoid tuberosity; F, femur; Fi, fibula; FL, forelimb; H, humerus; HL,
hindlimb; op, olecranon process; R, radius; RL, rescued limb; Sc, scapula; st, stylopod; T, tibia; Tn, trochlear notch; U, ulna; zg, zeugopod.
Tbx4-rescued limb and the wild-type forelimb express-
ing endogenous Tbx5.
We generated double-rescued embryos (Tbx5lox/lox;
Prx1-Cre;Prx1-Tbx4(4.48);Prx1-Pitx1(P1.1)) and Prx1-
Pitx1(P1.1)transgenicsandcomparedtheirlimbskeletal
elements to forelimbs and hindlimbs of control lit-
termates (Figure6) and tothe Tbx4-rescuedlimb (Figure
4N). Following deletion of Tbx5 in cells of the forelimb-
forming region and replacement with Tbx4 and Pitx1,
the limb element that forms shares many morphological
characteristics with a normal hindlimb. The articulation
between the stylopod and zeugopod skeletal elements
is strikingly knee-like (Figure 6B, arrowhead), while be-
tween the zeugopod and autopod it is ankle-like (Figure
6B,double arrowhead)comparedtothe normalforelimb
(Figure 6A). The heads of the stylopod and zeugopod
bones in the double rescue limb have a head-to-head
apposition and the heads of each bone are larger and
broadened, as is found in the knee (Figure 6F, dashed
line). Moreover, the double-rescued limb zeugopodal
elementhasaprotrusionortuberosity(Figure6F,arrow),
similar to that observed in the tibia (Figure 6H, arrow)
thatisnotpresentintheradius(Figure6E).Similarly,this
head-to-head appositionand an extendedtuberosity on
the zeugopodal bone is present in the Prx1-Pitx1(P1.1)
transgenic forelimb (Figure 6G, dashed line and arrow,
respectively). In addition, at this articulation in the Prx1-
Pitx1(P1.1) transgenic forelimb, the olecranon process
and trochlear notch normally present in the ulna (Figure
6E,arrowhead)areabsent(Figure6G,arrowhead).Digits
in the hindlimb are longer than digits in the forelimbs. In
the double-rescued and the Prx1-Pitx1(P1.1) transgenic
limbs, digit lengths are increased and therefore more
similar to those in the hindlimb than the forelimb. To
quantify these differences, we compared the ratio of the
lengths of the second digit metacarpal/metatarsal and
first phalange (yellow continuous versus dashed line in
Figures 6I–6L). The ratio of metacarpal:phalange length
is lower than 2 in the forelimb (Figure 6I, ratio 1.6), while
in the hindlimb the second metatarsal is longer than
twice the length of the first phalange (Figure 6L, ratio
2.5). In the double-rescued limb, this ratio is also above
2 (Figure 6J, ratio 2.6). Similarly, in the Prx1-Pitx1(P1.1)
transgenic autopod, the ratio is also above 2 (Figure 6K,
ratio 2.25). The length of the anterior zeugopodal bone
in the double-rescued and Prx1-Pitx1(P1.1) transgenic
limbs is longer than the stylopodal bone, resembling the
difference in femur/tibia length in the hindlimb rather
than the similar length of the humerus/radius of the
forelimb.
There are also differences between the Tbx4/Pitx1-
rescued limb and the control hindlimb as well as the
Prx1-Pitx1(P1.1) transgenic limb. The most obvious is
the lack of a posterior zeugopodal bone, with the con-
comitant loss of the two posterior-most digits. This
could be the result of gene dosage effects and may