Download Regulation of limb bud initiation and limbtype

DEVELOPMENTAL DYNAMICS 240:1017–1027, 2011
a
SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM
Regulation of Limb Bud Initiation and
Limb-Type Morphology
Developmental Dynamics
Veronique Duboc and Malcolm P. O. Logan*
While the paired forelimb and hindlimb buds of vertebrates are initially morphologically homogeneous,
as the limb progenitors differentiate, each individual tissue element attains a characteristic limb-type
morphology that ultimately defines the constitution of the forelimb or hindlimb. This review focuses on
contemporary understanding of the regulation of limb bud initiation and formation of limb-type specific
morphologies and how these regulatory mechanisms evolved in vertebrates. We also attempt to clarify the
definition of the terms limb-type identity and limb-type morphology that have frequently been used interchangeably. Over the last decade, three genes, Tbx4, Tbx5, and Pitx1, have been extensively studied for
their roles in limb initiation and determining limb-type morphologies. The role of Tbx4 and Tbx5 in limb
initiation is clearly established. However, their putative role in the generation of limb-type morphologies
remains controversial. In contrast, all evidence supports a function for Pitx1 in determination of hindlimb
morphologies. Developmental Dynamics 240:1017–1027, 2011. V 2011 Wiley-Liss, Inc.
C
Key words: limb bud initiation; limb-type morphology; Tbx genes; FGFs
Accepted 20 January 2011
INTRODUCTION
Tetrapods have two sets of paired
appendages that emerge as budding
outgrowths from the lateral plate
mesoderm (LPM) at fixed positions
along the rostro-caudal axis of the
body. Initially, the forelimb and hindlimb buds are morphologically homogeneous bulges of cells. As outgrowth
of the nascent limb continues, the
expanded pools of progenitors differentiate into the inter-connected array
of bones, tendons, muscles, vasculature, and skin. Each individual tissue
element attains a characteristic morphology that ultimately defines the
constitution of a forelimb or hindlimb.
While the bones, tendons, and some of
the limb vasculature are derived from
the LPM, all of the limb muscles are
derived from precursors that migrate
from the epaxial dermomyotomal
compartment of the somites medial to
the limb buds (Pearse et al., 2007).
The emergence of the array of limb
skeletal elements is regulated by signalling molecules expressed in key
signalling centres and their targets
(Butterfield et al., 2010; Duboc and
Logan, 2009; Zeller et al., 2009),
which are thought to be acting equivalently in both forelimb and hindlimb
buds. Therefore, the development of
forelimb and hindlimb-type morphologies is a useful paradigm to study
how diversity of form can be generated through modulation of conserved
gene regulatory networks.
Several classical experiments performed in the chick demonstrate that
the forelimb- or hindlimb-forming
potential is specified prior to limb bud
formation and that the cells of the
limb bud are autonomously deter-
mined. Cells transplanted from a
wing-forming region give rise to a
wing when grafted to an ectopic location and, conversely, comparable leg
grafts develop into a leg (Hamburger;
1938; Stephens et al., 1989; Saito
et al., 2002, 2006). In addition, recombination experiments in the chick
have shown that limb-type morphology is determined by the mesenchyme
and is independent of the overlying
ectoderm. Transplants of wing bud
mesenchyme combined with leg bud
ectoderm transplanted to the flank
will develop into wing structures,
while reciprocal grafts of leg mesenchyme covered with wing ectoderm
produce leg elements (Zwilling, 1959;
Logan, 2003; Saunders and Gasseling, 1968). Both classical embryology
and more recent molecular data indicate that limb muscles are patterned
Division of Developmental Biology, MRC-National Institute for Medical Research, London, United Kingdom
*Correspondence to: Malcolm P. O. Logan, Division of Developmental Biology, MRC-National Institute for Medical
Research, The Ridgeway, London, NW7 1AA, UK. E-mail: [email protected]
DOI 10.1002/dvdy.22582
Published online 28 February 2011 in Wiley Online Library (wileyonlinelibrary.com).
C 2011 Wiley-Liss, Inc.
V
Developmental Dynamics
1018 DUBOC AND LOGAN
Fig. 1. Schematic illustration of the limb initiation process in the mouse. A: From left to right, dorsal view of the right half of a mouse embryo
prior to limb initiation (around [E] 8.75). Axial cues confer limb-forming potential to a region of the LPM. A combinatorial Hox code determines the
levels at which the limbs will form. B: Mouse embryos at the onset of forelimb budding (15–20 somites) (top) and hindlimb budding (30–35
somites) (bottom). Schematic of the presumptive limb-forming areas of the forelimb (top) and hindlimb (bottom) showing the expression domains
of Tbx5 and Pitx1/Tbx4, respectively. C: Initiation of Fgf10 expression in the limb mesenchyme by Tbx factors leads to establishment of the positive feedback-loop between mesenchymal Fgf10 and Fgf8 in the ectoderm/AER. A stable FGF signalling positive feedback-loop marks the end of
the initiation phase and the onset of the outgrowth process. D: Chronologies of the major events starting from a uniform flank to the emergence of
the limb buds.
by extrinsic cues (Hasson et al., 2010;
Christ et al., 1977; Kardon et al.,
2003). Muscle progenitors that
migrate into a forelimb bud develop
into forelimb muscles in response to
environmental signals present in the
forelimb and hindlimb muscle progenitors respond to equivalent cues in the
hindlimb environment.
Two T-box transcription factors,
Tbx5 and Tbx4, and a paired-type
homeodomain transcription factor,
Pitx1, have expression profiles that are
consistent with these genes playing a
role in determining formation of forelimb or hindlimb structures. Tbx5 is
expressed in the forelimb but not the
hindlimb, whereas Pitx1 and Tbx4 are
expressed in the hindlimb buds but not
the forelimb. In addition, each gene is
initially expressed throughout the limb
mesenchyme, but not in the limb ectoderm, and these limb-type restricted
expression patterns are conserved
across vertebrate species (Lamonerie
et al., 1996; Szeto et al., 1996; GibsonBrown et al., 1998; Isaac et al., 1998;
Logan et al., 1998; Ohuchi et al., 1998).
A more historical review of the factors
regulating limb initiation and limbtype morphology has been published
previously (Logan, 2003).
FGFS: A COMMON NODE IN
FORELIMB AND HINDLIMB
FORMATION
One of the most dramatic demonstrations of which factors regulate limb
bud initiation came from experiments
carried out in the chick that showed
that a source of FGF applied to the
interlimb LPM (spanning somites 20 to
26, between stage 13 to 17) of the
embryo is sufficient to induce formation of a complete ectopic limb (Cohn
et al., 1995, 1997). This and subsequent
experiments (Ohuchi et al., 1997) not
only implicate FGF signalling as critical for limb initiation but also demonstrate that FGF ligands, acting during
a relatively brief period, are sufficient
to establish the series of events that
lead to formation of a mature limb.
The identity of the FGF ligand critical for limb initiation was revealed by
analyses of Fgf10 knockout mice (Min
et al., 1998; Xu et al., 1998; Sekine
et al., 1999). Forelimb and hindlimb
buds fail to form in Fgf10 mutants
and all limb skeletal elements are
absent apart from a rudimentary
Developmental Dynamics
REGULATION OF LIMB-TYPE MORPHOLOGY 1019
Fig. 2. Limb-type identity and limb-type morphology. A, a: Representation of the flank of a mouse embryo after limb bud formation. The axial levels at which
the limbs form define their forelimb or hindlimb identity. b, d: 3D renderings of the serially homologous skeletal elements of the forelimb (b) and hindlimb (d),
respectively. Scapula/pelvis (grey), humerus/femur (blue), radius and ulna/tibia and fibula (red), carpals and phalanges/tarsals and phalanges (orange); arrow indicates the patella. Images adapted from the interactive 3D mouse limb anatomy atlas http://www.nimr.mrc.ac.uk/3dlimb (Delaurier et al., 2008). c: Colour coding
used to describe the serially homologous bones of the limb in b and d. e, f: Immunostaining, using an anti-muscle Myosin antibody, illustrating the forelimb (e) and
hindlimb (f) -type muscle patterns in (E) 15.5 Limbs. Fdb, Flexor digitorum brevis; Abq, Abductor quinti; Fds, Flexor digitorum superficialis muscles. g, h: Dermal
pad pattern of a (E) 17.5 forelimb (g) and hindlimb (h). B: Schematic representation the chronology of events regulated by the activities of Tbx5, Tbx4, and Pitx1.
scapula and pelvis (see Fig. 2Ab–d).
Fgf10 expressed by cells of the limb
mesenchyme signals via receptors in
the apical ectodermal ridge (AER), a
specialised ridge of cells in the overlying ectoderm, to induce and maintain
Fgf8 expression. In turn, the Fgf8
ligand expressed by cells of the AER
signals via receptors in the limb mesenchyme to positively regulate Fgf10
expression in the limb mesenchyme
(Fig. 1C). Thus, a positive feedbackloop of FGF signalling common to
both forelimb and hindlimb is established that drives limb bud outgrowth
(Xu et al., 1998).
A BROAD REGION OF THE
LPM HAS LIMB-FORMING
POTENTIAL
FGF-soaked bead implantation experiments in the chick also demonstrated
that a strip of the LPM, encompassing
the forelimb- and hindlimb-forming
regions as well as the intervening
interlimb LPM, has limb-forming
capacity (Cohn et al., 1997) (Fig. 1A).
This territory is further subdivided
into a rostral, forelimb-forming region
and a caudal, hindlimb-forming region.
A source of FGF placed in the rostral
region will produce an ectopic wing
while a bead placed in the caudal
region produces an ectopic leg. Strikingly, an ectopic limb that forms at a
level that straddles the junction of
these two territories will be comprised
of wing elements in the anterior and
1020 DUBOC AND LOGAN
Developmental Dynamics
hindlimb elements in the posterior
reflecting the origin of the progenitors.
Consistent with these observations,
the pattern of Tbx4/Pitx1 and Tbx5
expression in the ectopic buds correlates with the morphology of the structures that subsequently develop. Ectopic wings induced close to the wing
express Tbx5. Hindlimbs induced near
the level of the hindlimb express
Tbx4/Pitx1. An ectopic hybrid limb
that forms at a level that straddles the
junction of these two territories
expresses Tbx5 in the anterior and
Tbx4/Pitx1 in the posterior (GibsonBrown et al., 1998; Isaac et al., 1998;
Logan et al., 1998; Ohuchi et al., 1998).
Therefore, the expression of Tbx5 and
Tbx4/Pitx1 is correlative to the ultimate limb-type morphology and, thus,
these genes serve as clear markers of
forelimb and hindlimb progenitors.
RETINOIC ACID IS
REQUIRED FOR LIMB BUD
INITIATION
Retinoic acid (RA) appears to have
multiple, temporally distinct roles
during limb development. It is first
required for limb bud initiation and,
subsequently, influences anterior-posterior and proximal-distal patterning,
although these later roles are subject
to controversy (Lewandoski and
Mackem, 2009). Here, we focus solely
on the role of RA during limb
initiation.
The requirement for RA signalling
during forelimb initiation is conserved in vertebrates. In chick as well
as in zebrafish, application of chemical inhibitors of retinaladehyde dehydrogenases, enzymes essential for RA
synthesis, prevents limb bud formation (Stratford et al., 1996; Grandel
et al., 2002; Gibert et al., 2006). Mice
lacking the retinaladehyde dehydrogenase, Raldh2, do not form forelimb
buds and die by embryonic day (E) 10
(Niederreither et al., 1999). Similarly,
the zebrafish raldh2 mutants, no fin
and necklace, fail to form pectoral fins
(the equivalent of the forelimb in fish)
(Grandel et al., 2002; Gibert et al.,
2006). Zebrafish raldh2 mutants die
during early larval stages (Grandel
et al., 2002) prior to the stage of pelvic
fin budding that occurs after three
weeks of development (Grandel and
Schulte-Merker, 1998) and, consequently, the role of RA during pelvic
fin (the equivalent of the hindlimb in
fish) development has not been
addressed. Significantly, transplantation of wild-type cells that contribute
to the somites in raldh2 mutant hosts
is sufficient to rescue pectoral fin formation, suggesting that this axial
tissue is an important source of RA
signal during normal pectoral fin
induction (Gibert et al., 2006). More
recent work using the zebrafish
raldh2 mutant has proposed that RA
is required for fin bud formation at
two time-points. The first phase
occurs much earlier than originally
predicted, during gastrulation, and
specifies a population of precursors to
a fin-forming fate. Subsequently, RA
is required to regulate expansion of
these progenitors during initiation of
the fin bud (Grandel and Brand,
2010).
Classical experiments performed in
chick demonstrate that initiation of
wing formation is dependent upon
signals from adjacent axial structures
(Kieny, 1970; Kieny et al., 1972).
Placement of a foil barrier between
the somites and LPM at forelimb levels blocks wing bud formation
(Murillo-Ferrol, 1965; Sweeney and
Watterson, 1969; Stephens and
McNulty, 1981). Equivalent experiments placing a foil barrier between
LPM and somites at hindlimb levels
has not been reported and, therefore,
whether axial tissues are the source
of an inductive signal required for
hindlimb initiation remains an open
question that should be addressed in
the future.
Together these observations identify RA as a candidate for the axial
influence on limb bud initiation. RA
may act as a competence factor that
confers limb-forming capacity to a
broad region of the LPM encompassing both forelimb- and hindlimb-forming regions as well as the inter-limb
LPM (Fig. 1A).
ROSTRO-CAUDAL HOX
CODE DEFINES THE
LEVELS OF FORMATION OF
APPENDAGES
Genes of the HoxC cluster exhibit restricted expression patterns along the
LPM of the flank and are thought to
specify the levels at which the limb
bud will appear and, thus, their identity (Cohn et al., 1997; Cohn and
Tickle, 1999) (Fig. 1A). For example,
Hoxc4-5 is expressed mostly in the
mesenchyme of the presumptive forelimb area (Savard et al., 1988; Tabin,
1989; Molven et al., 1990; Burke
et al., 1995), Hoxc9, Hoxc10, Hoxc11
are, predominantly, restricted to the
mesenchyme of the hindlimb domain
(Simon and Tabin, 1993; Peterson
et al., 1994), whereas Hoxc6 and
Hoxc8 expression correlates with the
interlimb flank. Hox9 paralogues are
also believed to contribute to the combinatorial code that specifies forelimb
and hindlimb territories (Rancourt
et al., 1995; Cohn et al., 1997). Hox
transcription factors have been suggested to be responsible for the restricted expression patterns of Tbx4
and Tbx5 (Minguillon et al., 2005)
(Fig. 1B). Currently, no functional evidence has been presented demonstrating a direct role of Hox genes in
the regulation of limb initiation. Mice
that lack the entire HoxC cluster do
not exhibit obvious defects in limb
position (Suemori and Noguchi,
2000). This observation does not, however, rule out the possibility that they
participate in this process in collaboration with other functionally redundant Hox genes. Other indirect observations are consistent with such a
model. Alteration of Hox gene expression patterns correlates with, and has
been implicated in, the evolution of
limbless in snakes (Cohn and Tickle,
1999) while a more recent study suggests that different downstream interpretation of the Hox code is important
in the loss of paired appendages in
this lineage (Woltering et al., 2009).
A ROLE FOR INTEGRIN
SIGNALLING IN LIMB
INITIATION
The frog Xenopus differs from many
other vertebrates in that its limbs develop at the free-swimming tadpole
stage after several weeks of development and that its hindlimbs develop
prior to the forelimbs. An insertional
mutation disrupting nephronectin
(npnt) in Xenopus tropicalis results in
a frog lacking all forelimb elements
Developmental Dynamics
REGULATION OF LIMB-TYPE MORPHOLOGY 1021
while the hindlimb develops normally
(Abu-Daya et al., 2010). This phenotype can be reproduced by knockdown of npnt using morpholino-oligo
nucleotides. Npnt is a small, secreted
integrin ligand and is necessary for
metanephros development in the
mouse (Muller et al., 1997; Linton
et al., 2007). The amphibian kidney
(pronephros) is located adjacent to the
presumptive forelimb-forming region
and although npnt is ubiquitously
expressed during frog development at
the time forelimb outgrowth is initiated, transcripts are enriched in
pronephros. The correlation with pronephros is potentially significant
since extirpation experiments in the
chick have implicated nephrogenic
mesoderm in forelimb initiation
(Geduspan and Solursh, 1992),
although this model has been recently
challenged by studies in chick (Fernandez-Teran et al., 1997) and mouse
(Perantoni et al., 2005). In the npnt
mutant, Tbx5 expression in the presumptive forelimb area is not initiated, suggesting that nephronectinintegrin signalling acts upstream of
the molecular events leading to forelimb formation. This study demonstrates a novel mechanism required
for forelimb initiation in X. tropicalis
and reveals a difference in the regulatory pathways controlling forelimb
and hindlimb initiation in the frog. It
remains to be determined if integrin
signalling is a conserved mechanism
regulating vertebrate limb initiation
or if it has been independently coopted in the amphibian lineage.
TBX GENES ESTABLISH THE
FGF SIGNALLING LOOP IN
THE NASCENT LIMB BUD
Loss-of-function
experiments
in
mouse and zebrafish have demonstrated that Tbx5 and Tbx4 play critical, conserved roles in forelimb and
hindlimb bud initiation, respectively.
In mouse embryos mutant or conditionally deleted for Tbx5, the forelimb
buds fail to form (Agarwal et al.,
2003; Rallis et al., 2003). In the absence of Tbx5, Fgf10 expression is not
initiated and as a result the FGF signalling loop between limb mesenchyme and ectoderm is never established. Fgf10 appears to be a direct
target of Tbx5 (Ng et al., 2002; Agarwal et al., 2003). In contrast to the
Tbx5 conditional mutant, which lack
all forelimb skeletal elements, a rudimentary scapula forms in the Fgf10
mutant, suggesting that Tbx5 makes
an additional contribution to forelimb
development beyond the positive regulation of Fgf10 expression (Rallis
et al., 2003). In both the zebrafish
tbx5 mutant (heartstrings) and following targeted knockdown using morpholino-oligonucleotides, pectoral fin
formation is blocked (Ahn et al., 2002;
Garrity et al., 2002; Ng et al., 2002).
The absence of the pectoral fin is
caused, at least in part, by the failure
of migration of tbx5 -positive limb
precursors.
The genetic hierarchy leading to
bud induction in zebrafish differs
slightly from other vertebrates. tbx5
regulates the expression of fgf10 indirectly through the expression of the
teleost fish-specific gene, fgf24 (Fischer et al., 2003) and does so via a
feed-forward model of transcriptional
regulation (Harvey and Logan, 2006).
tbx5 is required for the expression of
the FGF ligand fgf24. It also regulates
expression of zinc finger transcription
factors, sall1a and sall4, that are
required for expression of the FGF receptor, fgfr2, through which fgf24 signals. tbx5, therefore, has a positive
input into both expression of FGF
ligand and its receptor to establish the
FGF signalling loop that is required
for fin outgrowth. A similar feed-forward regulatory relationship between
Tbx5, Sall genes, FGF ligands and
receptors is thought to occur in
amniotes (Harvey and Logan, 2006;
Koshiba-Takeuchi et al., 2006).
In gene deletion-gene replacement
experiments, Tbx4 can replace the
function of Tbx5 in forelimb initiation
suggesting that Tbx4 has an equivalent role in hindlimb initiation (Minguillon et al., 2005). In contrast to the
absence of forelimb observed in the
Tbx5 knock-out, in Tbx4 null mice a
hindlimb bud does form although it is
drastically reduced in size. The
resulting hindlimbs fail to grow due
to improper initiation and maintenance of Fgf10 expression (Naiche
and Papaioannou, 2003). Therefore,
although Tbx4 is necessary for normal
Fgf10 expression, it is not exclusively
required and other factor(s) have an
input into the establishment of FGF
signalling in the emerging hindlimb
bud (Fig. 1C).
A role for Tbx5 and Tbx4 in the initiation of limb outgrowth is corroborated by the limb defects found in
human syndromes associated with
mutations in these genes. Mutations in
TBX4 and TBX5 are associated with
Small Patella syndrome (SPS; OMIM
no. 147891) and Holt-Oram syndrome
(HOS; OMIM no. 142900), respectively
(Basson et al., 1997; Li et al., 1997;
Bongers et al., 2004). HOS is a dominant disorder affecting the heart and
upper limbs. Haplo-insufficiency of
TBX5 causes a range of limb abnormalities, the mildest phenotype being triphalangeal (three-jointed) thumb but
that most commonly results in the failure of limb elements to form. SPS, similarly, is a dominant disorder that is
characterised by osteodysplasia of the
knee, pelvis, and foot.
TBX4 AND TBX5 HAVE
TEMPORALLY DISTINCT
ROLES DURING LIMB
DEVELOPMENT
Two independent studies have
investigated the temporal requirement of Tbx4 and Tbx5 during
mouse limb bud outgrowth using
conditional deletion strategies. Both
studies reach identical conclusions:
Tbx4 and Tbx5 are required during
the initial phase of limb initiation
but are dispensable for continued
limb outgrowth (Hasson et al., 2007;
Naiche and Papaioannou, 2007)
(Fig. 2B). During the first phase,
Tbx5 and Tbx4 are required to establish Fgf10 expression and the
FGF signalling positive feedbackloop. Once established, the FGF signalling loop is self-maintaining and
the input from Tbx4 or Tbx5 is no
longer required (Fig. 1C). During a
brief second phase, when the limb
bud can develop autonomously, deletion of Tbx5 in forelimbs or Tbx4 in
hindlimbs has no effect on outgrowth of the limb skeleton but
rather specifically affects muscle
and tendon morphogenesis (Hasson
et al., 2010). It is likely that Tbx5
and Tbx4 proteins have distinct targets during these two phases of
activity.
1022 DUBOC AND LOGAN
In summary, limb bud initiation
describes events that lead to establishment of the FGF signalling positive feedback-loop between limb mesenchyme and the AER. In contrast,
limb bud outgrowth encompasses
events during subsequent expansion
of the limb bud after the FGF positive
feedback-loop has been established.
Forelimb initiation is a Tbx5 -dependent process and although a small
hindlimb bud can form in Tbx4
mutants, Tbx4 is required for normal
hindlimb initiation. In contrast, limb
outgrowth can occur independently of
Tbx5 and Tbx4 activities.
Developmental Dynamics
DEFINING LIMB-TYPE
IDENTITY AND LIMB-TYPE
MORPHOLOGY
The terms limb-type identity and
limb-type morphology have frequently
been used interchangeably in studies
describing the establishment of the
specific structures of the forelimb and
hindlimb. Limb-type identity in tetrapods is primarily defined by the position along the rostro-caudal axis at
which the limbs emerge; the pair of
limbs positioned at the anterior of the
embryo are the forelimbs while the
limbs at the posterior are the hindlimbs (Ruvinsky and Gibson-Brown,
2000). This distinction is independent
of the morphology of the limbs. There
is no generic forelimb or hindlimbtype morphology since there can be
tremendous diversity in the equivalent appendages between different
species, for example, the wing of a
bird and forelimb of a mouse. Many of
the characteristic features that define
particular limb-type morphologies
are, therefore, specific to the species
considered.
Forelimb and hindlimb are serially
homologous structures reflecting their
common evolutionary and developmental histories (Fig. 2A, b–d)
(Ruvinsky and Gibson-Brown, 2000).
Homologous structures of the forelimb and hindlimb, such as humerus/
femur and radius-ulna/tibia-fibula,
therefore, share some anatomical similarities but can be distinguished by
characteristic limb-type morphologies. Forelimb muscle and tendons
also have hindlimb homologous counterparts that differ in size, shape, rel-
ative position in the limb, and origin
and insertion sites on the limb skeleton (Fig. 2A, e,f). Defining precisely
what constitutes an unambiguous
characteristic forelimb and hindlimb
feature is important when interpreting the apparent disruption of limbtype morphologies.
CONFLICTING DATA ON
THE ROLE OF TBX4 AND
TBX5 IN ESTABLISHING
LIMB-TYPE
MORPHOLOGIES.
As discussed previously, two T-box
transcription factors Tbx4 and Tbx5
have expression profiles that are consistent with them playing roles in
determining limb-type morphologies.
Tbx5 is expressed in the forelimb,
whereas Tbx4 is expressed in the
hindlimb buds and both 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). Ectopic gene misexpression experiments in the chick
appeared to confirm a role for Tbx5
and Tbx4 in determining forelimb and
hindlimb morphologies, respectively.
Ectopic expression of Tbx5 in the
developing chick hindlimb bud has
been reported to partially transform
the morphology of the leg to a more
wing-like type and induce the ectopic
expression of forelimb markers
(Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999). Conversely, Tbx4
misexpression in the forelimb can
apparently partially transform the
wing to develop some hindlimb features and the expression of hindlimb
markers (Takeuchi et al., 1999). Following the misexpression of Tbx4, the
endogenous expression of Tbx5 in the
forelimb is maintained arguing that
the morphologically transformed forelimbs have retained their forelimb
molecular identity.
The observations in the chick are in
direct contrast with results of genetic
experiments in the mouse using a
combination of a Tbx5 conditional
mouse and transgenic lines to carry
out gene deletion-gene replacement
experiments. Following conditional
deletion of Tbx5 and simultaneous
replacement with transgenically-sup-
plied Tbx4, forelimb outgrowth and
morphologies are rescued (Minguillon
et al., 2005, 2009). These results demonstrate that Tbx4 is sufficient to compensate for Tbx5 in forelimb initiation
and that, in this context, it is not able
to determine hindlimb morphologies.
They also demonstrate that a morphologically recognizable forelimb can
form in the absence of Tbx5 activity
and indicate that other factors must
determine their formation. Moreover,
the Tbx4-rescued forelimbs retain
expression of forelimb markers Tbx5,
Hoxc4, and Hoxc5, and hindlimb
markers are not ectopically expressed.
The Tbx4-rescued forelimbs have,
therefore, retained their forelimb molecular identity. Taken together, these
results demonstrate that in mouse,
Tbx5 and Tbx4share common roles
during limb initiation but do not
determine limb-type morphologies.
LIMB FORMING CAPACITY
OF TBX4 AND TBX5 IS
EVOLUTIONARILY ANCIENT
Tbx4 and Tbx5 are paralogous genes
that arose by duplication of a single,
ancestral Tbx4/5 gene. The extant
cephalochordate amphioxus possesses
a single Tbx4/5 (amphiTbx4/5) and
lacks paired appendages, whereas all
vertebrates with paired appendages
express Tbx5 in the forelimbs and
Tbx4 in the hindlimbs (Gibson-Brown
et al., 1996). It is potentially significant that the timing of this duplication coincides with the acquisition of
paired appendages in vertebrates. In
the background of the conditional
deletion of Tbx5, an amphiTbx4/5
transgene is able to rescue forelimb
formation. This demonstrates that
the ancestral gene, present in limbless organisms, has the ability to initiate limb formation if expressed in the
appropriate
location
(Minguillon
et al., 2009). In transgenic analysis,
constructs containing fragments of
the mouse Tbx5 and Tbx4 genomic
regions are able to drive gene expression in the forelimb- and hindlimbforming regions of the LPM while, in
contrast, the amphiTbx4/5 genomic
locus appears to lack the regulatory
modules enabling expression in the
LPM. In accordance with broadly
accepted evolutionary models, these
REGULATION OF LIMB-TYPE MORPHOLOGY 1023
results suggest that changes at the
level of the regulation of Tbx5 and
Tbx4expression, rather than the generation of novel protein function, was
necessary for the acquisition of paired
appendages during vertebrate evolution (Horton et al., 2008; Minguillon
et al., 2009).
Developmental Dynamics
PITX1 CONTRIBUTES TO
SPECIFICATION OF
HINDLIMB-TYPE
MORPHOLOGIES
One gene, the paired-type homeodomain transcription factor Pitx1, has
been unambiguously implicated in
limb-type specification. Pitx1 is
expressed in the hindlimb-forming
region and hindlimb bud, but not in
the forelimb region (Lamonerie et al.,
1996). Functional studies performed
in both chick and mouse support a
role for Pitx1 in specification of hindlimb morphology. When misexpressed
in the developing wing of a chick
embryo, the muscles, skeletal elements, and skin of the wing adopt
some characteristic leg features
(Logan and Tabin, 1999; Takeuchi
et al., 1999). Consistent with these
observations, Pitx1 misexpression in
the mouse forelimb results in transformation and translocation of specific
muscles, tendons, and bones to acquire a hindlimb-like morphology
(DeLaurier et al., 2006). Significantly,
equivalent transgenic lines that
express ectopic Tbx4 in the forelimb
or Tbx5 in the hindlimb do not produce any alterations to limb-type morphologies again consistent with neither gene having a role in
determining limb-type morphologies
(Minguillon et al., 2005, 2009). In
Pitx1 null mice, the hindlimb skeleton
loses some of its characteristic features (Lanctot et al., 1999; Szeto
et al., 1999; Marcil et al., 2003). This
is particularly clear in the zeugopodal
bones. Normally, the diameter of the
fibula is around half that of the tibia
while the homologous elements in the
forelimb zeugopod (ulna and radius)
are roughly equivalent in diameter.
In the Pitx1 mutant, the fibula and
tibia have equivalent diameters. The
knee joint also lacks a patella and size
and shape of the calcaneous bone in
the ankle are abnormal. The loss of
these hindlimb characteristics has
been interpreted as indicating that
the Pitx1/ hindlimb has adopted a
more forelimb-like morphology. However, the molecular marker of the
forelimb Tbx5 is not ectopically
expressed and the putatively ‘‘transformed’’ hindlimbs do not develop definitive forelimb morphologies (Marcil
et al., 2003). The Pitx1/ hindlimb
phenotype is thus consistent with a
loss of hindlimb attributes rather
than acquisition of forelimb features
and suggests that forelimb is not the
default morphology.
The Pitx1/ mutant hindlimb is
not devoid of all hindlimb characteristics. Although Pitx1 clearly plays an
important role in determining some
hindlimb morphologies, it is unlikely
to be acting alone but rather is part of
the multiple inputs required for the
complete array of hindlimb morphologies to be produced. Ectopic expression of Pitx1 at stages when the forelimb bud has already formed is
sufficient to partially transform the
morphology of limb structures to
those of the hindlimb. This is consistent with Pitx1 acting to establish
hindlimb morphologies, from the
onset of limb budding until limb progenitors are undergoing terminal differentiation to form distinct tissues
(Fig. 2B). In summary, therefore,
Pitx1 appears to act as a ‘‘modifier’’ to
influence the response of progenitors
to signalling/patterning inputs that
are common between forelimbs and
hindlimbs.
PITX1 REGULATES TBX4
EXPRESSION AND
CONTRIBUTES TO NORMAL
GROWTH OF THE
HINDLIMB
Accurately assigning roles for genes
in determination of limb-type morphology based on the characteristics
of abnormally developed limb elements that form following gene deletion or gene misexpression can be
problematic. A definitive set of limbtype-specific criteria needs to be
established that can unambiguously
categorise structures and distinguish
changes in limb-type morphology
from altered morphology arising
through abnormal development. A
good example of this type of problem
is presented by the phenotype of the
Pitx1 mutant. Pitx1 is required for
normal levels of Tbx4expression in
the hindlimb (Lanctot et al., 1999;
Szeto et al., 1999) and misexpression
of Pitx1 in the forelimb of the chick
or the mouse is sufficient to induce
Tbx4 ectopic expression (Logan and
Tabin, 1999; DeLaurier et al., 2006).
Moreover, Tbx4 expression is reduced
in Pitx1/ mutants (Fig. 1B).
Therefore, at least some aspects of
the Pitx1/ phenotype can be
attributed to hypomorphic levels of
Tbx4 transcripts that will lead to the
abnormal development of limb
structures.
Clarification of the epistatic relationship between Pitx1 and Tbx4 and
the effects of hypomorphic Tbx4
expression on hindlimb formation has
come from an analysis of the regulatory elements of Tbx4 in mouse. By
combining a systematic functional
scanning in transgenic mice bearing
BAC-modified lacZ-reporters and
alignment with the human Tbx4 locus
to find evolutionary conserved elements, Menke et al. (2008) identified
two hindlimb enhancer sequences
that regulate Tbx4 hindlimb expression. A region located upstream of the
Tbx4 coding sequence can drive
strong expression in the proximal portion of the hindlimb, but not the distal
half of the autopod at E12.5. An
element, 30 of the coding sequence,
drives
strong
LacZ
expression
throughout the hindlimb and genital
tubercle. Importantly, both enhancers
are active in the hindlimb-forming
region before the onset of hindlimb
bud outgrowth and thus are likely to
be important during hindlimb bud initiation. Three putative Pitx1-binding
sites are present in the 50 enhancer
and mutation of the most conserved
site leads to reduction in enhancer activity. Homologous recombination to
excise the 50 hindlimb enhancer from
the mouse Tbx4 locus results in lowered Tbx4 hindlimb expression and
animals homozygous for the deletion
have smaller hindlimb bones (pelvis,
femur, tibia, and patella). In the autopod, anterior digits are more affected
than posterior digits and distal elements are more reduced than proximal elements. The reduction in the
size of hindlimb bones following loss
1024 DUBOC AND LOGAN
of Pitx1 input in Tbx4 expression suggests that regulation of Tbx4 by Pitx1
is required for normal initiation of
hindlimb outgrowth.
These results demonstrate that a
hindlimb bud expressing hypomorphic levels of Tbx4 does form structures with hindlimb characteristics,
consistent with the phenotypes
observed in human with Small Patella syndrome (SPS). This underlines
the importance of distinguishing a
disruption in normal growth and formation of structures from a disruption in them attaining their correct
limb-type morphology. This can be difficult since normal growth of a structure will ultimately contribute to its
morphogenesis.
Developmental Dynamics
OLD DOGMA THROWN A
NEW BONE
In recent work, Ouimette et al.
adopted a strategy to compare the
abilities of transgenically provided
Tbx5 or Tbx4 to rescue the Pitx1/
hindlimb defects (Ouimette et al.,
2010). In the Pitx1 mutant, the levels
of Tbx4 transcripts are reduced and
the ilium of the pelvic girdle fails to
form normally contributing to a defective rotation of the hindlimb. Delivery
of transgenic Tbx4 can rescue formation of the ilium and, consequently,
rescues the rotation defect. In the
Pitx1/ knee joint, the fibula makes
contact with the femur rather than
the tibia in a manner that has some
similarities to the articulation of
bones at the elbow of the forelimb and
a more hindlimb-like pattern of bone
contacts are re-established in the
Tbx4-rescued knee. In addition, the
normal angle between the calcaneus
and the footplate is also restored.
Aspects of hindlimb-specific muscle
position are also apparently rescued
by the Tbx4 transgene. In contrast, a
Tbx5 transgenic line was unable to
rescue the formation of these structural elements to the same extent.
These morphological changes in the
apparently ‘‘rescued’’ mutant are
interpreted as a rescue of hindlimb
morphology although they may also
be explained by rescue of initiation of
normal
outgrowth.
Interestingly,
some hindlimb features absent in the
Pitx1 mutant, such as the patella and
the small ratio of fibula/tibia, are not
rescued by transgenic elevation of
Tbx4 levels.
The authors (Ouimette et al.,
2010) suggest that these paralogous
proteins have evolved distinct functions and that changes in the Tbx4
protein function explain how it
determines hindlimb morphologies.
In luciferase assays, as previously
reported (Agarwal et al., 2003; Rallis
et al., 2003), Tbx5 functions as a
transcriptional activator. However,
Tbx4 is identified to have transcriptional repressor properties. Both activator and repressor activities were
mapped to the C-terminal domain.
In this work and other studies (Minguillon et al., 2005, 2009), Tbx4
ectopically expressed in the forelimb
is not capable of transforming structures to a more hindlimb-like morphology. An explanation is offered
that Tbx4 is normally acting in cooperation with a hindlimb-specific transcriptional co-repressor and, therefore, in the forelimb context Tbx4 is
an activator and the Tbx4 repressor
activity, correlated with attainment
of hindlimb morphology, is not
functional.
These results have the surprising
conclusion that following duplication
to generate the Tbx5 and Tbx4
paralogues, the Tbx4 protein evolved
a novel functional domain. It also
remains unclear how Tbx4 is acting
simultaneously in hindlimb progenitors as both transcriptional activator,
in its role in hindlimb initiation, and
as transcriptional repressor, directing
formation of hindlimb morphologies.
MUTATION OF CISREGULATORY MODULES
CAN GENERATE
MORPHOLOGICAL
DIVERSITY
Paired appendages have been repeatedly lost in some fish, amphibian, reptile, and mammalian lineages (Shapiro et al., 2006). In stickleback fish,
loss of the pelvic girdle occurs in several freshwater populations likely as
an adaptive response to predators
and/or water chemistry (Protas and
Tabin, 2004). Previous studies have
mapped stickleback pelvic reduction
locus to the Pitx1 gene through
genetic linkage analysis and have
found that Pitx1 expression is absent
in pelvic reduced species (Shapiro
et al., 2004). A recent study by Chan
et al. (2010) further refines these
observations by precisely identifying
the cis-regulatory modification responsible. This study efficiently illustrates how morphological changes can
arise from alteration in a single regulatory region of a key developmental
control gene rather than by modification of the protein sequence (Chan
et al., 2010). F1 hybrid stickleback
fish were generated by crossing fish
with intact pelvic girdles (FRIL) to
those with a reduced pelvis (PAXB).
The presence of the FRIL pitx1 allele
is able to rescue formation of pelvic
structures in the hybrid fish. Furthermore, the PAXB allele of pitx1 is
expressed at lower levels than the
FRIL allele in a pelvic-restricted manner, suggesting that the failure of pelvic development is a consequence of
cis-regulatory changes. Using association mapping in a population of
sticklebacks in which some individuals had a complete pelvis and others
had a reduced pelvis, Chan et al.
(2010) found that a 23-kb region
upstream of Pitx1 was strongly associated with pelvic reduction. Alignment
of the medaka and zebrafish sequences that span this region enabled the
authors to isolate conserved regions
that could act as potential enhancer
elements. Further deletion analysis,
by cloning different fragments
upstream of a reporter gene, identified a 2.5-kb pelvic-specific enhancer
(2.5-kb Pel). Notably, the Pel region is
absent from the Pitx1 loci of pelvicreduced species. This 2.5-kb Pel
region upstream of a Pitx1 transgene
is able to rescue pelvic girdle development in the pelvic-reduced fishes, confirming that regulatory changes in
pitx1 underlies pelvic reduction in the
stickleback. This study demonstrates
how cis-regulatory changes can be
employed to generate diversity in
hindlimb size.
DIFFERENCES IN TIMING
OF FORELIMB AND
HINDLIMB DEVELOPMENT
Apart from the differences in morphology, limb-type specific differences
can also be reflected in the timing of
Developmental Dynamics
REGULATION OF LIMB-TYPE MORPHOLOGY 1025
the induction of forelimb and hindlimb budding and their subsequent
development, a phenomenon known
as heterochrony, and this varies in
different species (Richardson et al.,
2009). For example, in mouse and
chick the forelimbs emerge just before
the hindlimbs, while in zebrafish the
relative gap in time between formation of the pectoral and pelvic fins is
much longer. As mentioned earlier, in
the frog Xenopus formation of the hindlimbs occurs before the forelimb.
A dramatic example of heterochronic limb formation is found in
Marsupials. In contrast with placental mammals, marsupials are born
with forelimbs that are developmentally advanced relative to their hindlimbs. Marsupials give birth after a
relatively short period of gestation to
immature neonates that must crawl
to the teat where they attach and continue their development. They are
able to do so due to the presence of
functional forelimbs. The forelimb
bud is first apparent at an earlier
stage compared to the formation of
other embryonic structures in marsupials (Sears, 2009). Perhaps surprisingly, hindlimb formation also occurs
earlier although its subsequent outgrowth is delayed (Sears, 2009).
Consistent with these observations,
expression of markers of forelimb and
hindlimb initiation, Tbx5/Tbx4 and
their targets Fgf10 and Fgf8, are
detected at earlier developmental
stages in the marsupial, the Opossum, Monodelphis domestica when
compared to the mouse (Keyte and
Smith, 2010). It remains unclear
whether the subsequent difference in
timing of forelimb and hindlimb
development arises through acceleration of the forelimb developmental
programme or delay of the hindlimb
programme or though a combination
of these two processes. Further
study will be required to provide a
complete mechanistic explanation for
heterochrony of limb formation in
marsupials.
PERSPECTIVES
The establishment of forelimb and
hindlimb morphologies is likely to be
determined by multiple factors and at
least partially though a modifier mechanism in which the presence (and
therefore also by default, absence) of a
gene alters the response of limb progenitors to patterning signals that are
equivalently expressed in both forelimb
and hindlimbs. Divergence of regulatory networks in forelimbs and hindlimbs also likely contributes to the
diversification of limb-type specific
morphologies. So far the clearest candidate ‘‘modifier’’ contributing to limbtype morphologies is Pitx1 as it is the
only gene unambiguously demonstrated to be part of the limb-type
determination process being both
required for hindlimb morphologies to
develop and also able to transform
limb-type morphologies following ectopic expression in the forelimb. The
Pitx1/ hindlimb loses aspects of
hindlimb morphology but does not acquire forelimb characteristics. Neither
the forelimb nor the hindlimb represent a default limb-type morphology
since forelimb and hindlimb morphologies arise, in part, as a result of the distinct developmental history of the different regions of the LPM from which
the forelimb and hindlimb arise.
The acquisition of forelimb and
hindlimb morphologies is, therefore, a
multigenic process and likely a culmination of the gene expression profiles
in progenitors both at pre-limb
bud stages and limb-bud stages. Teasing apart all the contributing factors
that determine limb-type morphologies is an enormous task but
should provide significant insight into
how an embryo generates morphological diversity with a limited genetic
tool kit.
REFERENCES
Abu-Daya A, Nishimoto S, Fairclough L,
Mohun TJ, Logan MP, Zimmerman LB.
2010. The secreted integrin ligand
nephronectin is necessary for forelimb
formation in Xenopus tropicalis. Dev
Biol 189:246–255.
Agarwal P, Wylie JN, Galceran J,
Arkhitko O, Li C, Deng C, Grosschedl
R, Bruneau BG. 2003. Tbx5 is essential
for forelimb bud initiation following
patterning of the limb field in the
mouse embryo. Development 130:
623–633.
Ahn DG, Kourakis MJ, Rohde LA, Silver
LM, Ho RK. 2002. T-box gene tbx5 is
essential for formation of the pectoral
limb bud. Nature 417:754–758.
Basson CT, Bachinsky DR, Lin RC, Levi
T, Elkins JA, Soults J, Grayzel D,
Kroumpouzou E, Traill TA, Leblanc-
Straceski J, Renault B, Kucherlapati R,
Seidman JG, Seidman CE. 1997. Mutations in human TBX5 [corrected] cause
limb and cardiac malformation in HoltOram syndrome. Nat Genet 15:30–35.
Bongers EM, Duijf PH, van Beersum SE,
Schoots J, Van Kampen A, Burckhardt
A, Hamel BC, Losan F, Hoefsloot LH,
Yntema HG, Knoers NV, van Bokhoven
H. 2004. Mutations in the human TBX4
gene cause small patella syndrome. Am
J Hum Genet 74:1239–1248.
Burke AC, Nelson CE, Morgan BA, Tabin
C. 1995. Hox genes and the evolution of
vertebrate axial morphology. Development 121:333–346.
Butterfield NC, McGlinn E, Wicking C.
2010. The molecular regulation of vertebrate limb patterning. Curr Top Dev
Biol 90:319–341.
Chan YF, Marks ME, Jones FC, Villarreal
G, Jr., Shapiro MD, Brady SD, Southwick AM, Absher DM, Grimwood J,
Schmutz J, Myers RM, Petrov D, Jonsson B, Schluter D, Bell MA, Kingsley
DM. Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science 327:
302–305.
Christ B, Jacob HJ, Jacob M. 1977. Experimental findings on muscle development in the limbs of the chick embryo.
Verh Anat Ges 1231–1237.
Cohn MJ, Tickle C. 1999. Developmental
basis of limblessness and axial patterning in snakes. Nature 399:474–479.
Cohn MJ, Izpisua-Belmonte JC, Abud H,
Heath JK, Tickle C. 1995. Fibroblast
growth factors induce additional limb
development from the flank of chick
embryos. Cell 80:739–746.
Cohn MJ, Patel K, Krumlauf R, Wilkinson DG, Clarke JD, Tickle C. 1997.
Hox9 genes and vertebrate limb specification. Nature 387:97–101.
DeLaurier A, Schweitzer R, Logan M.
2006. Pitx1 determines the morphology
of muscle, tendon, and bones of the
hindlimb. Dev Biol 299:22–34.
Delaurier A, Burton N, Bennett M, Baldock R, Davidson D, Mohun TJ, Logan
MP. 2008. The mouse limb anatomy
atlas: an interactive 3D tool for studying embryonic limb patterning. BMC
Dev Biol 8:83.
Duboc V, Logan MP. 2009. Building limb
morphology through integration of signalling modules. Curr Opin Genet Dev
19:497–503.
Fernandez-Teran M, Piedra ME, Simandl
BK, Fallon JF, Ros MA. 1997. Limb initiation and development is normal in
the absence of the mesonephros. Dev
Biol 189:246–255.
Fischer S, Draper BW, Neumann CJ.
2003. The zebrafish fgf24 mutant identifies an additional level of Fgf signaling involved in vertebrate forelimb
initiation. Development 130:3515–3524.
Garrity DM, Childs S, Fishman MC.
2002. The heartstrings mutation in
zebrafish causes heart/fin Tbx5 deficiency syndrome. Development 129:
4635–4645.
Developmental Dynamics
1026 DUBOC AND LOGAN
Geduspan JS, Solursh M. 1992. A growthpromoting influence from the mesonephros during limb outgrowth. Dev
Biol 151:242–250.
Gibert Y, Gajewski A, Meyer A, Begemann G. 2006. Induction and prepatterning of the zebrafish pectoral fin bud
requires axial retinoic acid signaling.
Development 133:2649–2659.
Gibson-Brown JJ, Agulnik SI, Chapman
DL, Alexiou M, Garvey N, Silver LM,
Papaioannou VE. 1996. Evidence of a
role for T-box genes in the evolution of
limb morphogenesis and the specification of forelimb/hindlimb identity. Mech
Dev 56:93–101.
Gibson-Brown JJ, Agulnik SI, Silver LM,
Papaioannou VE. 1998. Expression of Tbox genes Tbx2-Tbx5 during chick organogenesis. Mech Dev 74:165–169.
Grandel H, Brand M. 2010. Zebrafish
limb development is triggered by a retinoic acid signal during gastrulation.
Dev Dyn [DOI: 10.1002/dvdy.22461].
Grandel H, Schulte-Merker S. 1998. The
development of the paired fins in the
zebrafish (Danio rerio). Mech Dev 79:
99–120.
Grandel H, Lun K, Rauch GJ, Rhinn M,
Piotrowski T, Houart C, Sordino P,
Kuchler AM, Schulte-Merker S, Geisler
R, Holder N, Wilson SW, Brand M.
2002. Retinoic acid signalling in the
zebrafish embryo is necessary during
pre-segmentation stages to pattern the
anterior-posterior axis of the CNS and
to induce a pectoral fin bud. Development 129:2851–2865.
Hamburger V. 1938. Morphogenetic and
axial self-determination of transplanted
limb primordia of 2-day chick embryos.
J Exp Zool 77:379–399.
Harvey SA, Logan MP. 2006. sall4 acts
downstream of tbx5 and is required for
pectoral fin outgrowth. Development
133:1165–1173.
Hasson P, Del Buono J, Logan MP. 2007.
Tbx5 is dispensable for forelimb outgrowth. Development 134:85–92.
Hasson P, DeLaurier A, Bennett M, Grigorieva E, Naiche LA, Papaioannou VE,
Mohun TJ, Logan MP. 2010. Tbx4 and
tbx5 acting in connective tissue are
required for limb muscle and tendon
patterning. Dev Cell 18:148–156.
Horton AC, Mahadevan NR, Minguillon
C, Osoegawa K, Rokhsar DS, Ruvinsky
I, de Jong PJ, Logan MP, Gibson-Brown
JJ. 2008. Conservation of linkage and
evolution of developmental function
within the Tbx2/3/4/5 subfamily of Tbox genes: implications for the origin of
vertebrate limbs. Dev Genes Evol 218:
613–628.
Isaac A, Rodriguez-Esteban C, Ryan A, Altabef M, Tsukui T, Patel K, Tickle C, IzpisuaBelmonte JC. 1998. Tbx genes and limb
identity in chick embryo development. Development 125:1867–1875.
Kardon G, Harfe BD, Tabin CJ. 2003. A
Tcf4-positive mesodermal population
provides a prepattern for vertebrate
limb muscle patterning. Dev Cell 5:
937–944.
Keyte AL, Smith KK. 2010. Developmental origins of precocial forelimbs in marsupial neonates. Development 137:
4283–4294.
Kieny M. 1970. Part of somitic mesoderm
in the differentiation of chick embryo
limb. C R Acad Sci Hebd Seances Acad
Sci D 270:2009–2011.
Kieny M, Mauger A, Sengel P. 1972.
Early regionalization of somitic mesoderm as studied by the development of
axial skeleton of the chick embryo. Dev
Biol 28:142–161.
Koshiba-Takeuchi K, Takeuchi JK,
Arruda EP, Kathiriya IS, Mo R, Hui
CC, Srivastava D, Bruneau BG. 2006.
Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern
the mouse limb and heart. Nat Genet
38:175–183.
Lamonerie T, Tremblay JJ, Lanctot C,
Therrien M, Gauthier Y, Drouin J.
1996. Ptx1, a bicoid-related homeo box
transcription factor involved in transcription of the pro-opiomelanocortin
gene. Genes Dev 10:1284–1295.
Lanctot C, Moreau A, Chamberland M,
Tremblay ML, Drouin J. 1999. Hindlimb patterning and mandible development
require
the
Ptx1
gene.
Development 126:1805–1810.
Lewandoski M, Mackem S. 2009. Limb
development: the rise and fall of retinoic acid. Curr Biol 19:R558–561.
Li QY, Newbury-Ecob RA, Terrett JA,
Wilson DI, Curtis AR, Yi CH, Gebuhr T,
Bullen PJ, Robson SC, Strachan T, Bonnet D, Lyonnet S, Young ID, Raeburn
JA, Buckler AJ, Law DJ, Brook JD.
1997. Holt-Oram syndrome is caused by
mutations in TBX5, a member of the
Brachyury (T) gene family. Nat Genet
15:21–29.
Linton JM, Martin GR, Reichardt LF.
2007. The ECM protein nephronectin
promotes kidney development via integrin alpha8beta1-mediated stimulation
of Gdnf expression. Development 134:
2501–2509.
Logan M. 2003. Finger or toe: the molecular basis of limb identity. Development
130:6401–6410.
Logan M, Tabin CJ. 1999. Role of Pitx1
upstream of Tbx4 in specification of
hindlimb
identity.
Science
283:
1736–1739.
Logan M, Simon HG, Tabin C. 1998. Differential regulation of T-box and homeobox transcription factors suggests roles
in controlling chick limb-type identity.
Development 125:2825–2835.
Marcil A, Dumontier E, Chamberland M,
Camper SA, Drouin J. 2003. Pitx1 and
Pitx2 are required for development of
hindlimb buds. Development 130:45–55.
Menke DB, Guenther C, Kingsley DM.
2008. Dual hindlimb control elements
in the Tbx4 gene and region-specific
control of bone size in vertebrate limbs.
Development 135:2543–2553.
Min H, Danilenko DM, Scully SA, Bolon
B, Ring BD, Tarpley JE, DeRose M,
Simonet WS. 1998. Fgf-10 is required
for both limb and lung development
and exhibits striking functional similarity to Drosophila branchless. Genes Dev
12:3156–3161.
Minguillon C, Del Buono J, Logan MP.
2005. Tbx5 and Tbx4 are not sufficient
to determine limb-specific morphologies
but have common roles in initiating
limb outgrowth. Dev Cell 8:75–84.
Minguillon C, Gibson-Brown JJ, Logan
MP. 2009. Tbx4/5 gene duplication and
the origin of vertebrate paired appendages. Proc Natl Acad Sci USA 106:
21726–21730.
Molven A, Wright CV, Bremiller R, De
Robertis EM, Kimmel CB. 1990.
Expression of a homeobox gene product
in normal and mutant zebrafish
embryos: evolution of the tetrapod body
plan. Development 109:279–288.
Muller U, Wang D, Denda S, Meneses JJ,
Pedersen RA, Reichardt LF. 1997.
Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis.
Cell 88:603–613.
Murillo-Ferrol NL. 1965. Causal study of
the earliest differentiation of the morphological rudiments of the extremities.
Experimental analysis on bird embryos.
Acta Anat (Basel) 62:80–103.
Naiche LA, Papaioannou VE. 2003. Loss
of Tbx4 blocks hindlimb development
and affects vascularization and fusion
of the allantois. Development 130:
2681–2693.
Naiche LA, Papaioannou VE. 2007. Tbx4
is not required for hindlimb identity or
post-bud hindlimb outgrowth. Development 134:93–103.
Ng JK, Kawakami Y, Buscher D, Raya A,
Itoh T, Koth CM, Rodriguez Esteban C,
Rodriguez-Leon J, Garrity DM, Fishman MC, Izpisua Belmonte JC. 2002.
The limb identity gene Tbx5 promotes
limb initiation by interacting with
Wnt2b and Fgf10. Development 129:
5161–5170.
Niederreither K, Subbarayan V, Dolle P,
Chambon P. 1999. Embryonic retinoic
acid synthesis is essential for early
mouse post-implantation development.
Nat Genet 21:444–448.
Ohuchi H, Shibusawa M, Nakagawa T,
Ohata T, Yoshioka H, Hirai Y, Nohno T,
Noji S, Kondo N. 1997. A chick wingless
mutation causes abnormality in maintenance of Fgf8 expression in the wing
apical ridge, resulting in loss of the dorsoventral boundary. Mech Dev 62:3–13.
Ohuchi H, Takeuchi J, Yoshioka H, Ishimaru Y, Ogura K, Takahashi N, Ogura
T, Noji S. 1998. Correlation of wing-leg
identity in ectopic FGF-induced chimeric limbs with the differential
expression of chick Tbx5 and Tbx4. Development 125:51–60.
Ouimette JF, Jolin ML, L’Honore A,
Gifuni A, Drouin J. 2010. Divergent
transcriptional activities determine
limb identity. Nat Commun 1:1–9.
Pearse RV, 2nd, Scherz PJ, Campbell JK,
Tabin CJ. 2007. A cellular lineage analysis of the chick limb bud. Dev Biol
310:388–400.
Developmental Dynamics
REGULATION OF LIMB-TYPE MORPHOLOGY 1027
Perantoni AO, Timofeeva O, Naillat F, Richman C, Pajni-Underwood S, Wilson C,
Vainio S, Dove LF, Lewandoski M. 2005.
Inactivation of FGF8 in early mesoderm
reveals an essential role in kidney development. Development 132:3859–3871.
Peterson RL, Papenbrock T, Davda MM,
Awgulewitsch A. 1994. The murine
Hoxc cluster contains five neighboring
AbdB-related Hox genes that show
unique spatially coordinated expression
in posterior embryonic subregions.
Mech Dev 47:253–260.
Protas ME, Tabin CJ. 2004. Reduce your
pelvis in 10,000 years or less. Dev Cell
6:613–614.
Rallis C, Bruneau BG, Del Buono J, Seidman CE, Seidman JG, Nissim S, Tabin
CJ, Logan MP. 2003. Tbx5 is required for
forelimb bud formation and continued outgrowth. Development 130:2741–2751.
Rancourt DE, Tsuzuki T, Capecchi MR.
1995. Genetic interaction between
hoxb-5 and hoxb-6 is revealed by nonallelic noncomplementation. Genes Dev 9:
108–122.
Richardson MK, Gobes SM, van Leeuwen
AC, Polman JA, Pieau C, Sanchez-Villagra MR. 2009. Heterochrony in limb
evolution: developmental mechanisms
and natural selection. J Exp Zool B Mol
Dev Evol 312:639–664.
Rodriguez-Esteban C, Tsukui T, Yonei S,
Magallon J, Tamura K, Izpisua Belmonte JC. 1999. The T-box genes Tbx4
and Tbx5 regulate limb outgrowth and
identity. Nature 398:814–818.
Ruvinsky I, Gibson-Brown JJ. 2000.
Genetic and developmental bases of serial homology in vertebrate limb evolution. Development 127:5233–5244.
Saito D, Yonei-Tamura S, Kano K, Ide H,
Tamura K. 2002. Specification and
determination of limb identity: evidence
for inhibitory regulation of Tbx gene
expression. Development 129:211–220.
Saito D, Yonei-Tamura S, Takahashi Y,
Tamura K. 2006. Level-specific role of
paraxial mesoderm in regulation of
Tbx5/Tbx4 expression and limb initiation. Dev Biol 292:79–89.
Saunders JW, Gasseling MT. 1968. Ectodermal-mesenchymal interactions in
the origin of limb symmetry. In:
Fleischmajer R, Billingham RE, editors.
Epithetial-mesenchymal
interactions.
Baltimore: Wilfiams and Wilkins. p 78–
97.
Savard P, Gates PB, Brockes JP. 1988.
Position dependent expression of a
homeobox gene transcript in relation to
amphibian limb regeneration. EMBO J
7:4275–4282.
Sears KE. 2009. Differences in the timing
of prechondrogenic limb development in
mammals: the marsupial-placental dichotomy
resolved.
Evolution
63:
2193–2200.
Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, Yagishita
N, Matsui D, Koga Y, Itoh N, Kato S.
1999. Fgf10 is essential for limb and
lung formation. Nat Genet 21:138–
141.
Shapiro MD, Marks ME, Peichel CL,
Blackman BK, Nereng KS, Jonsson B,
Schluter D, Kingsley DM. 2004. Genetic
and developmental basis of evolutionary
pelvic reduction in threespine sticklebacks. Nature 428:717–723.
Shapiro MD, Bell MA, Kingsley DM.
2006. Parallel genetic origins of pelvic
reduction in vertebrates. Proc Natl
Acad Sci USA 103:13753–13758.
Simon HG, Tabin CJ. 1993. Analysis of
Hox-4.5 and Hox-3.6 expression during
newt limb regeneration: differential
regulation of paralogous Hox genes suggest different roles for members of different Hox clusters. Development 117:
1397–1407.
Stephens TD, McNulty TR. 1981. Evidence for a metameric pattern in the
development of the chick humerus.
J Embryol Exp Morphol 61:191–205.
Stephens TD, Beier RL, Bringhurst DC,
Hiatt SR, Prestridge M, Pugmire DE,
Willis HJ. 1989. Limbness in the early
chick embryo lateral plate. Dev Biol
133:1–7.
Stratford T, Horton C, Maden M. 1996.
Retinoic acid is required for the initiation of outgrowth in the chick limb bud.
Curr Biol 6:1124–1133.
Suemori H, Noguchi S. 2000. Hox C cluster genes are dispensable for overall
body plan of mouse embryonic development. Dev Biol 220:333–342.
Sweeney RM, Watterson RL. 1969. Rib
development in chick embryos analyzed
by means of tantalum foil blocks. Am J
Anat 126:127–149.
Szeto DP, Ryan AK, O’Connell SM, Rosenfeld MG. 1996. P-OTX: a PIT-1-interacting homeodomain factor expressed
during anterior pituitary gland development. Proc Natl Acad Sci USA 93:
7706–7710.
Szeto DP, Rodriguez-Esteban C, Ryan AK,
O’Connell SM, Liu F, Kioussi C, Gleiberman AS, Izpisua-Belmonte JC, Rosenfeld MG. 1999. Role of the Bicoidrelated homeodomain factor Pitx1 in
specifying hindlimb morphogenesis and
pituitary development. Genes Dev 13:
484–494.
Tabin CJ. 1989. Isolation of potential vertebrate limb-identity genes. Development 105:813–820.
Takeuchi JK, Koshiba-Takeuchi K, Matsumoto K, Vogel-Hopker A, NaitohMatsuo M, Ogura K, Takahashi N,
Yasuda K, Ogura T. 1999. Tbx5 and
Tbx4 genes determine the wing/leg
identity of limb buds. Nature 398:
810–814.
Woltering JM, Vonk FJ, Muller H,
Bardine N, Tuduce IL, de Bakker MA,
Knochel W, Sirbu IO, Durston AJ,
Richardson MK. 2009. Axial patterning
in snakes and caecilians: evidence for
an alternative interpretation of the Hox
code. Dev Biol 332:82–89.
Xu X, Weinstein M, Li C, Naski M, Cohen
RI, Ornitz DM, Leder P, Deng C. 1998.
Fibroblast growth factor receptor 2
(FGFR2)-mediated reciprocal regulation
loop between FGF8 and FGF10 is
essential for limb induction. Development 125:753–765.
Zeller R, Lopez-Rios J, Zuniga A. 2009.
Vertebrate limb bud development: moving towards integrative analysis of
organogenesis. Nat Rev Genet 10:
845–858.
Zwilling E. 1959. Interaction between
ectoderm and mesoderm in duckchicken limb bud chimaeras. J Exp Zool
142:521–532.