RESEARCH ARTICLE 4465
Development 138, 4465-4473 (2011) doi:10.1242/dev.065359
© 2011. Published by The Company of Biologists Ltd
Islet1-mediated activation of the -catenin pathway is
necessary for hindlimb initiation in mice
Yasuhiko Kawakami1,2,3,4,5,*, Merce Marti6, Hiroko Kawakami1,2,3, Junji Itou2,3, Thu Quach2, Austin Johnson2,
Setsuko Sahara7, Dennis D. M. O’Leary7, Yasushi Nakagawa3,4,8, Mark Lewandoski9, Samuel Pfaff1,
Sylvia M. Evans10 and Juan Carlos Izpisua Belmonte1,6,*
SUMMARY
The transcriptional basis of vertebrate limb initiation, which is a well-studied system for the initiation of organogenesis, remains
elusive. Specifically, involvement of the -catenin pathway in limb initiation, as well as its role in hindlimb-specific transcriptional
regulation, are under debate. Here, we show that the -catenin pathway is active in the limb-forming area in mouse embryos.
Furthermore, conditional inactivation of -catenin as well as Islet1, a hindlimb-specific factor, in the lateral plate mesoderm
results in a failure to induce hindlimb outgrowth. We further show that Islet1 is required for the nuclear accumulation of catenin and hence for activation of the -catenin pathway, and that the -catenin pathway maintains Islet1 expression. These two
factors influence each other and function upstream of active proliferation of hindlimb progenitors in the lateral plate mesoderm
and the expression of a common factor, Fgf10. Our data demonstrate that Islet1 and -catenin regulate outgrowth and Fgf10Fgf8 feedback loop formation during vertebrate hindlimb initiation. Our study identifies Islet1 as a hindlimb-specific
transcriptional regulator of initiation, and clarifies the controversy regarding the requirement of -catenin for limb initiation.
INTRODUCTION
During vertebrate development, a variety of progenitor cells are
generated and multiple organs are constructed by coordinated
proliferation and differentiation. A critical step during
organogenesis is the initiation of organ development, where genetic
mechanisms control growth and differentiation in a stereotypical
morphogenetic process that results in a specific function and
morphology for each organ. A thorough understanding of the
processes and mechanisms that regulate organ initiation is,
therefore, crucial for the study of developmental biology. The
vertebrate limb has served as an ideal model system for
understanding the mechanisms of organogenesis, including
initiation, outgrowth, patterning and morphogenesis (Towers and
Tickle, 2009; Zeller et al., 2009). It also provides a unique setting
in which to study the development of the forelimb versus the
hindlimb, which are two morphologically distinct but serially
homologous organs (Duboc and Logan, 2011; Kawakami et al.,
2003b; Logan, 2003).
1
Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 N.
Torrey Pines Road, La Jolla, CA 92037, USA. 2Department of Genetics, Cell Biology
and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St SE
Minneapolis, MN 55455, USA. 3Stem Cell Institute, University of Minnesota, 2001
6th SE, Minneapolis, MN 55455, USA. 4Developmental Biology Center, University of
Minnesota, 321 Church St SE Minneapolis, MN 55455, USA. 5Masonic Cancer
Center, University of Minnesota, Minneapolis, MN 55455, USA. 6Center of
Regenerative Medicine in Barcelona, Doctor Aiguader 88, 08003 Barcelona, Spain.
7
Molecular Neuroscience Laboratory, The Salk Institute for Biological Studies, 10010
N. Torrey Pines Road, La Jolla, CA 92037, USA. 8Department of Neuroscience,
University of Minnesota, 321 Church St SE Minneapolis, MN 55455, USA. 9Cancer
and Developmental Biology Laboratory, National Cancer Institute, NCI-Frederick,
Frederick, MD 21702, USA. 10Skaggs School of Pharmacy, and Department of
Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
*Authors for correspondence ([email protected]; [email protected])
Accepted 5 August 2011
During initiation of the vertebrate limb, cells in the lateral plate
mesoderm (LPM) proliferate to initiate outgrowth, and,
concomitantly, a signal emanating from the LPM acts on the
overlying ectoderm (Capdevila and Izpisua Belmonte, 2001). It has
been revealed that, both in the forelimb and hindlimb fields,
fibroblast growth factor 10 (Fgf10) in the LPM activates the
expression of another Fgf gene, Fgf8, in the surface ectoderm (Min
et al., 1998; Ohuchi et al., 1997; Sekine et al., 1999), which then
leads to the formation of a mesenchymal Fgf10-ectodermal Fgf8
feedback loop to maintain limb outgrowth. Although it is known
that the Fgf10-Fgf8 feedback loop is an evolutionarily conserved
molecular component that is essential for maintaining
mesenchymal cell proliferation and limb outgrowth, two major
questions remain with respect to vertebrate limb initiation. One is
the involvement of the canonical Wnt/-catenin pathway in this
process, and the other is the difference between forelimb and
hindlimb initiation.
Depending on the species, there remains some controversy as to
the involvement of the canonical Wnt/-catenin pathway. Our
previous studies have demonstrated that Wnt/-catenin signaling
in the limb-forming region is an upstream regulator of Fgf10 in
chick and zebrafish embryos (Kawakami et al., 2001; Ng et al.,
2002). Wnt2b is expressed in the forelimb and pectoral fin-forming
area in the chick and zebrafish, respectively, and Wnt8c is
expressed in the hindlimb-forming area in chick embryos. These
Wnts signal through the -catenin-dependent pathway. Consistent
with this, ectopic activation of the -catenin pathway in the chick
flank can induce ectopic Fgf10 expression and the formation of an
extra limb. The reciprocal experiment, in which the Wnt/-catenin
pathway is downregulated, resulted in loss of limb and fin buds in
the chick and zebrafish, respectively. In contrast to these studies,
the expression of a Wnt ligand in the limb-forming area of mouse
embryos has not been reported so far. Moreover, mice mutant in
Lef/Tcf factors, which are transcription factors that mediate Wnt
signaling by forming a complex with -catenin, have not exhibited
DEVELOPMENT
KEY WORDS: Islet1, -catenin, Mouse limb initiation, Fgf10
4466 RESEARCH ARTICLE
MATERIALS AND METHODS
Mouse lines
The Ctnnb1flox/flox line (Ctnnb1tm2Kem) was purchased from the Jackson
Laboratory. Islet1flox/flox (Song et al., 2009; Sun et al., 2008), T-cre
(Perantoni et al., 2005) and Hoxb6Cre (Lowe et al., 2000) lines have been
reported. Fgf10 mutant mice (Min et al., 1998; Sahara and O’Leary,
2009) and the BATgal reporter line (Maretto et al., 2003) have been
described.
Imaging analysis
-catenin and laminin immunostaining were performed using anti-catenin (BD Bioscience, #610154) and anti-laminin (Sigma, L9393) in
combination with Alexa 488 anti-mouse IgG and Alexa 568 anti-rabbit
IgG (Invitrogen) with a standard protocol. Islet1 immunostaining was
with rabbit anti-Islet1 antibody (1/1000) (Song et al., 2009) or
monoclonal 39.4D5 (Developmental Studies Hybridoma Bank, final
working concentration 4.5 g/ml). Detection of -galactosidase
immunoreactivity in BATgal embryos was by rabbit anti--galactosidase
antibody (MP Biomedicals, #55976, 1/5000). TUNEL analysis and
phospho-histone H3 (pHis3) analysis were performed using rat antipHis3 (Sigma, H6409) and Cy3-labeled anti-rat IgG (Jackson
ImmunoResearch) in combination with the In Situ Cell Death Detection
Kit (Roche) according to the manufacturer’s instruction. Fluorescent
imaging analysis was performed with Leica TCS SP5 and TCS SPE
microscopes. The graphs were constructed with the Leica SP5 software,
and the quantification of the TUNEL/pHis3 analysis was with
MetaMorph software. Scanning electron microscopy analysis was as
previously described (Kawakami et al., 2006) using a Jeol JSM-6390LV
microscope.
Statistical analysis
For statistical significance, sections from embryos of the same genotype
were stained by immunofluorescence and DAPI and expressed as the
percentage of immunopositive cells among DAPI-positive cells for each
embryo. From each embryo, multiple sections were examined, and the
number of embryos for each analysis was three or more. Statistical
significance was examined by independent t-test.
In situ hybridization
Whole-mount and section in situ hybridization were performed following
standard protocols (Bluske et al., 2009; Kawakami et al., 2009; Wilkinson,
1992).
RESULTS
Activation of the -catenin pathway during limb
initiation in mice
-catenin is a crucial component of canonical Wnt signaling.
Whereas -catenin is abundant in the plasma membrane, its
intracellular level is kept low. Cytosolic -catenin is constitutively
subjected to degradation, leading to low levels in the cytosol and
nuclei. Upon stimulation of cells with a Wnt ligand, the degradation
machinery is inhibited, and cytosolic -catenin accumulates, leading
to its translocation into the nucleus. This results in the formation of
a complex with transcription factors such as Lef1 and Tcf, leading to
the transcriptional activation of downstream genes (Nusse, 2005).
Because nuclear accumulation of -catenin is a hallmark of pathway
activation, in order to clarify whether the -catenin-dependent
pathway is involved in mouse limb initiation, we first investigated
the accumulation of nuclear -catenin by fluorescent immunostaining
and confocal imaging. In the prospective hindlimb field at E9.5, just
prior to hindlimb outgrowth, when Fgf10 expression in the hindlimb
field becomes evident (see Fig. S1 in the supplementary material),
we observed two types of cells in the LPM with respect to nuclear
-catenin levels as revealed by fluorescence intensity. Whereas both
types of cells showed high levels of -catenin in the membrane, one
clearly exhibited lower levels of nuclear -catenin, with an elongated
morphology (Fig. 1A,C,C⬘). By contrast, the other exhibited high
levels of nuclear -catenin and a relatively rounded morphology (Fig.
1A,B,B⬘), and constituted ~24% of the total mesenchymal cells in
the LPM at the hindlimb field at E9.5. These two types of cells are
consistently observed in multiple sections from multiple embryos
DEVELOPMENT
a lack of limb initiation (Galceran et al., 1999). Therefore, it
remains to be clarified whether the -catenin pathway regulates
limb initiation in mouse embryos.
The issue of limb type-specific transcriptional regulation of
initiation is complicated. Although the Fgf10-Fgf8 feedback loop
operates on both the forelimb and hindlimb buds, it has become
evident that transcriptional regulation upstream of active
proliferation of LPM cells and mesenchymal Fgf10 expression is
different in these two types of limbs. Tbx5, which encodes a T-box
transcription factor, is exclusively expressed in the forelimb field,
but not in the hindlimb field (Gibson-Brown et al., 1996). Tbx5–/–
embryos show neither forelimb bud outgrowth nor Fgf10
expression in the forelimb field (Agarwal et al., 2003; Rallis et al.,
2003). These reports have established Tbx5 as a crucial regulator
of forelimb initiation. By contrast, the genetic mechanisms for
hindlimb initiation remain elusive. Previous studies have reported
that two transcription factor genes, Tbx4 and Pitx1, are exclusively
expressed in the hindlimb field, but not in the forelimb field
(Gibson-Brown et al., 1996; Szeto et al., 1999). However, genetargeting experiments have revealed that neither is necessary for
initiation of the hindlimb bud (Lanctot et al., 1999; Naiche and
Papaioannou, 2003; Szeto et al., 1999). Tbx4-null embryos start
both initial outgrowth to form the hindlimb bud and Fgf10
expression in the hindlimb-forming region. However, Fgf10
expression in the hindlimb bud is not maintained in the absence of
Tbx4. These studies established that Tbx4 is not required for
induction but for maintenance of Fgf10 expression (Naiche and
Papaioannou, 2003). Pitx1–/– mice have small, but well-patterned
hindlimbs. These reports highlight our lack of knowledge on the
transcriptional regulation of hindlimb-specific initiation, as
compared with the role of Tbx5 in forelimb initiation (summarized
in Table S1 in the supplementary material). A recent report has
demonstrated that Islet1, a LIM-homeodomain transcription factor
gene, is transiently expressed in the hindlimb field, but not the
forelimb field (Yang et al., 2006). Moreover, lineage analysis has
shown that Islet1-expressing cells contribute to a large part of the
hindlimb mesenchyme. These analyses make Islet1 a strong
candidate to be a hindlimb-specific transcriptional regulator.
However, Islet1-null embryos arrest by E9.5, prior to the initiation
of the hindlimb bud in mice (Pfaff et al., 1996). Thus, the role of
Islet1 in the development of the hindlimb remains elusive.
In order to clarify these two major issues in mouse limb initiation,
we carried out conditional inactivation studies on the Ctnnb1 (which
encodes -catenin) and Islet1 genes in the LPM. Both resulted in loss
of the hindlimb bud, demonstrating that the -catenin pathway and
Islet1 are required for initiation of the hindlimb. We show that both
the -catenin pathway and Islet1 lie upstream of active proliferation
and expression of Fgf10 in the mesenchyme of the hindlimb-forming
region. Furthermore, our analysis demonstrates that Islet1 function
is required for the nuclear accumulation of -catenin and for
activation of the -catenin-dependent pathway. -catenin is not
required for initiation of, but rather only for maintenance of Islet1
expression. Our study has clarified a controversy regarding the
requirement of the -catenin pathway and identified Islet1 as a factor
regulating hindlimb initiation, thus clarifying our understanding of
an important process during vertebrate limb initiation.
Development 138 (20)
Fig. 1. Accumulation of nuclear -catenin in the lateral plate
mesoderm (LPM) of the mouse embryo. (A)Fluorescent imaging
analysis showing two types of cells with different levels of nuclear catenin in the LPM. (B,C)Higher magnification of the cells indicated by
a yellow arrow (B) and white arrowhead (C) in A. GFP and DAPI images
are shown in black and white. Merged images with GFP (green) and
DAPI (blue) are also shown. (B⬘,C⬘) Fluorescence intensity analysis of B
and C, with -catenin signal in green and DAPI signal in blue. Higher
DAPI signal indicates the nucleus, and the difference in the levels of
nuclear -catenin is indicated by orange dashed lines and arrows. The
y-axis represents the fluorescence intensity. c, m and n indicate cytosol,
membrane and nucleus, respectively. (D-F⬘) A typical image of E9.5
hindlimb field with -catenin staining (D). Yellow arrows (E) and white
arrowheads (F) represent cells with high and low nuclear -catenin,
respectively. Large numbers of these cells are detected simultaneously,
and clearly have different levels of nuclear -catenin as illustrated in E⬘
and F⬘. (G-H⬘) Accumulation of nuclear -catenin is a characteristic of
the limb-forming area. Fluorescent images of -catenin in the hindlimbforming area and interlimb-forming area of the E9.5 mouse embryo.
The level of -catenin is shown in G⬘ and H⬘ for the cells indicated by
yellow arrows and white arrowheads, respectively. Dashed orange lines
indicate differences in the nuclear -catenin levels between the two
types of cell.
(Fig. 1D-F), indicating that the LPM is a heterogeneous tissue. These
two types of cells are also observed in the forelimb field at E9.0-10.0
(the stage when forelimb buds initiate outgrowth), but are barely
detected after E10.5 (data not shown). The levels of nuclear catenin suggest that stabilization, and hence activation, of the catenin pathway is temporally regulated in the LPM.
RESEARCH ARTICLE 4467
Next, in order to clarify whether the nuclear localization of catenin is associated with limb initiation, we examined the LPM at
the interlimb area of E9.0, E9.5 and E10.0 embryos using the same
approach. In contrast to the LPM at the hindlimb-forming area, we
have not detected any cells with high levels of nuclear -catenin
out of 3581 cells counted in the LPM from 32 sections (compare
Fig. 1G,G⬘,H,H⬘), indicating that nuclear accumulation of -catenin
is spatially linked to limb initiation in the mouse embryo. These
data suggest that the -catenin pathway is active in the limbforming area in the LPM at the time of limb outgrowth initiation.
To confirm the activation of the -catenin pathway in the limb
field in mouse embryos, we utilized the BATgal reporter mouse
line. This line has been used to visualize -catenin-dependent
pathway activity by lacZ staining (Maretto et al., 2003). In addition
to an intense signal in the tail bud, dorsal neural tube and somites,
we also detected patchy signals in the hindlimb-forming area (see
Fig. S2E in the supplementary material). This confirms activation
of the -catenin pathway in the limb-forming area.
Next, we examined the role of mesenchymal -catenin by
conditionally inactivating -catenin, as -catenin-null embryos
exhibit severe gastrulation defects (Haegel et al., 1995).
Inactivation of -catenin as early as E7.5 using the T-cre line
causes severe truncation of the body posterior to the heart level
(Dunty et al., 2008) (see also Fig. S3 in the supplementary
material), precluding it from being used in the analysis of limb
initiation. Instead, we used the Hoxb6Cre line, in which Cre is
activated in the LPM after E8.5 (Lowe et al., 2000). Since Cre
activation takes place completely in the hindlimb-forming field, but
only in the posterior half of the forelimb-forming field, we focused
our analysis on hindlimb development. In order to confirm
elimination of -catenin we utilized both immunofluorescence
confocal imaging and the BATgal reporter line, as they were used
to demonstrate activation of the -catenin pathway in the limbforming area. In both cases, we observed significant reduction of
mesenchymal -catenin activity in the hindlimb field at E9.5, the
stage just prior to hindlimb outgrowth, demonstrating an efficient
inactivation of -catenin in a mesoderm-specific manner (see Fig.
S2A-F in the supplementary material) (hereafter, the Hoxb6Cre;
Ctnnb1flox/flox embryos are referred to as Ctnnb1 cKO). The
outgrowth of the hindlimb bud is evident at E10.0 in control
littermate embryos (Hoxb6Cre; Ctnnb1+/flox), whereas the Ctnnb1
cKO embryos showed no hindlimb outgrowth in seven embryos
examined by scanning electron microscopy between E9.5 and
E10.0 (Fig. 2A,B).
Additionally, no Fgf10 expression in the LPM and no Fgf8
expression in the surface ectoderm were observed in the cKO
embryos (Fig. 2C-F). These results demonstrate that the
mesenchymal -catenin pathway lies upstream of Fgf10 for
hindlimb initiation. Unlike the complete loss of the hindlimb bud,
we observed small forelimb buds in the Ctnnb1 cKO embryos (see
Fig. S2G,H in the supplementary material). This could be because
Hoxb6Cre-mediated inactivation takes place only in the posterior
half of the forelimb region in the LPM, and the cells that escaped
from Ctnnb1 gene inactivation contributed to proliferation and
Fgf10 expression, leading to the formation of small forelimb buds.
A similar phenotype has been observed in Lef1–/–; Tcf1–/– mutant
embryos (Galceran et al., 1999). The Lef/Tcf family of factors form
complexes with -catenin and activate target gene expression. The
smaller limb buds, but not the complete lack of limb buds, in
Lef1–/–; Tcf1–/– embryos might be due to a functional redundancy
with other transcription factors, such a Tcf3 and Tcf4. The Ctnnb1
cKO embryos die by E11.5, consistent with the fact that -catenin
DEVELOPMENT
-catenin and Islet1 in limb initiation
Fig. 2. -catenin is required for limb initiation in the mouse
embryo. (A-H)Scanning electron microscopy analysis (A,B) and wholemount mRNA expression analysis (C-H) of Ctnnb1 cKO embryos
(B,D,F,H) and control littermate embryos (A,C,E,G). Hindlimb buds are
visible in E10.0 control embryos (A, arrows), but are not formed in the
Ctnnb1 cKO embryo (B, arrowhead). Fgf10 expression is detected in
the hindlimb bud in the control embryo at E10.0 (C, arrows), but not in
the Ctnnb1 cKO embryo (D, arrowhead). Fgf8 expression is detected
in the forelimb (blue arrow) and hindlimb (red arrow) buds in the E10.0
control embryo (E). In the Ctnnb1 cKO embryo, Fgf8 expression is not
detected in the hindlimb area (red arrowhead), and weak,
discontinuous Fgf8 expression is detected in the forelimb bud (blue
arrowhead). Pitx1 expression in the hindlimb-forming area is detected
in both control (G, arrow) and Ctnnb1 cKO (H, arrow) embryos at E9.5.
f, forelimb bud; h, hindlimb bud or hindlimb area; t, tail bud.
(I,J)Quantitative analysis of multiple sections shows significant
reduction of proliferation (I) and increased cell death (J) in the Ctnnb1
cKO embryo. Error bars indicate s.d.
activity is essential for the development of many other organs
(Grigoryan et al., 2008). These reports concur with our
observations and support the requirement of -catenin in vertebrate
limb initiation.
Our previous report on the differential expression of Wnt ligand
genes in chick embryos suggested that the Wnt/-catenin pathway
might also have a role in the development of the differences
between these limbs, namely forelimb versus hindlimb identity.
Since Ctnnb1 cKO embryos develop no hindlimb, we examined
Development 138 (20)
this possibility by expression of the hindlimb-specific gene Pitx1,
the expression of which starts prior to hindlimb bud outgrowth.
Pitx1 has been demonstrated to be required and sufficient to
determine hindlimb-specific characteristics in mice (DeLaurier et
al., 2006; Lanctot et al., 1999; Szeto et al., 1999). Contrary to
previous predictions, we observed normal expression of Pitx1 in
Ctnnb1 cKO as compared with control embryos (Fig. 2G,H),
suggesting that -catenin activity is unlikely to be required for the
genetic program that drives limb type specificity (Duboc and
Logan, 2011; Kawakami et al., 2003b; Logan, 2003).
In the Ctnnb1 cKO embryos, we found increased cell death and
reduced proliferation of cells in the LPM (Fig. 2I,J). Compared
with littermate control embryos (n4 embryos), both proliferation
and cell death showed significant alterations in the Ctnnb1 cKO
embryos (n4 embryos), suggesting that -catenin serves as a
survival factor as well as a proliferation factor in LPM cells. Given
that we detected normal expression of Pitx1, neither a general
failure to activate genes in the LPM nor a simple loss of cells by
apoptosis is a likely cause of the lack of hindlimb outgrowth.
Rather, these observations suggest that a -catenin-dependent
genetic program is specifically required for hindlimb initiation as
an upstream regulator of Fgf10, independent from serving as a cell
survival factor in the LPM. This is further supported by our
observation that neither cell proliferation nor cell death is changed
in Fgf10–/– embryos (see Fig. S4 in the supplementary material).
This suggests that these alterations in Ctnnb1 cKO embryos are not
due to loss of Fgf10 expression in the LPM and are likely to be
independent of the Ctnnb1-Fgf10 genetic cascade. Although the
Fgf10-Fgf8 feedback loop is known to be required for cell survival
in the developing limb bud (Sun et al., 2002), -catenin-dependent
cell proliferation and cell survival in the LPM seem to be regulated
independently of FGF activity.
Taken together, our results demonstrate that -catenin is required
for the initiation of hindlimb bud outgrowth in mouse embryos,
upstream of the Fgf10-Fgf8 feedback loop.
Islet1 regulates hindlimb-specific limb initiation
The limb initiation process seems to have a strong link to
forelimb and hindlimb specificity. Tbx5 is specifically expressed
in the forelimb field, and Tbx5–/– embryos fail to induce forelimb
bud outgrowth and Fgf10 expression (Agarwal et al., 2003).
Therefore, Tbx5 is required for forelimb initiation; however, the
transcriptional regulation required for hindlimb initiation is still
unclear. Tbx4 and Pitx1, two genes previously identified as
expressed exclusively in the hindlimb field, are not required for
initiation of hindlimb bud outgrowth or initiation of Fgf10
expression in the LPM (Lanctot et al., 1999; Naiche and
Papaioannou, 2003; Szeto et al., 1999). Thus, our understanding
of the transcriptional regulation of limb-type-specific initiation
is limited to Tbx5 in the forelimb, whereas hindlimb-specific
transcriptional regulation remains elusive.
We have previously performed several screenings based on the
expression pattern in chick embryos (Kawakami et al., 2004;
Kawakami et al., 2003a; Trelles et al., 2002), by which we isolated
Islet1 as a gene that is specifically expressed in the hindlimb field
and not in the forelimb field (see Fig. S5A in the supplementary
material). Although this same expression pattern has been reported
in mouse embryos (Yang et al., 2006), its functional significance
during limb development is unclear owing to the lethality of
Islet1–/– embryos by E9.5 (Pfaff et al., 1996). As such, we
inactivated Islet1 using an Islet1 conditional allele (Du et al., 2009;
Song et al., 2009). Since Islet1 expression in the posterior body
DEVELOPMENT
4468 RESEARCH ARTICLE
Fig. 3. Islet1 is required for hindlimb initiation in the mouse
embryo. (A-L)Scanning electron microscopy analysis (A-D) and wholemount mRNA expression analysis (E-L) of Islet1 cKO embryos
(B,D,F,H,J,L) and control littermate embryos (A,C,E,G,I,K). Hindlimb
buds are visible in E10.0 control embryos (A,C, arrows), but are not
formed in the Islet1 cKO embryo (B,D, arrowheads). Asterisks indicate
similarly developed forelimb buds in both embryos. Expression of Fgf10
(E,F) and Fgf8 (G,H) is detected in the control embryo (E,G, arrows), but
not in the Islet1 cKO embryo (F,H, arrowheads). Pitx1 expression in the
hindlimb-forming area (I, arrows) is reduced in the Islet1 cKO embryo (J,
arrowheads). (K)Lateral view of normal Tbx4 expression (arrow) in the
control embryo. (L)Tbx4 expression in the LPM is reduced in the Islet1
cKO embryo (arrowhead). (M-N⬘) In situ hybridization of Tbx4 on crosssections at the hindlimb-forming region at E9.5. M⬘ and N⬘ are
magnifications of the boxed areas in M and N. Tbx4 is strongly
expressed in the LPM (arrow) and the ventral body wall (bracket) in
control embryos (M,M⬘). Tbx4 expression in the LPM is significantly
downregulated (arrowhead), whereas the signal in the ventral body
wall is clearly detectable (bracket) in the Islet1 cKO embryo.
(O,P)Quantitative analysis of multiple sections shows significant
reduction of proliferation (O) but no alteration of cell death (P) in the
Islet1 cKO embryo. Error bars indicate s.d.
starts as early as E8.5 (see Fig. S5A in the supplementary material),
we used the T-cre line, which activates Cre in the broad region of
the mesoendoderm at E7.5 (Dunty et al., 2008; Yang et al., 2006).
This strategy allowed us to successfully delete Islet1 from the LPM
(see Fig. S5B in the supplementary material). T-cre; Islet1–/flox
(hereafter referred to as Islet1 cKO) embryos exhibited no hindlimb
outgrowth at E10.0, whereas forelimbs developed normally (Fig.
3A-D). Although we did not obtain Islet1 cKO embryos beyond
E11.5, this result demonstrates a requirement of Islet1 for initiating
hindlimb outgrowth. The Islet1 cKO embryo showed no Fgf10
expression in the LPM and no Fgf8 expression in the surface
ectoderm, indicating that Islet1 acts upstream of the Fgf10-Fgf8
feedback loop (Fig. 3E-H). Thus, these data demonstrate that Islet1
is specifically required for initiation of the hindlimb, upstream of
the Fgf10-Fgf8 feedback loop.
RESEARCH ARTICLE 4469
Islet1 regulates expression of Tbx4 but not Pitx1
in the LPM
Our findings compare and contrast two different regulators
upstream of limb outgrowth and expression of Fgf10 – Islet1, a
hindlimb-specific transcription factor, and Tbx5, a forelimb-specific
transcription factor (Agarwal et al., 2003) – in the induction of
hindlimb and forelimb bud outgrowth, respectively. Since the entire
hindlimb bud is lost, it is not possible to examine other roles of
Islet1 in hindlimb specification by morphological analyses.
Therefore, we examined the expression of the hindlimb fieldspecific genes Pitx1 and Tbx4 in order to assess a possible role of
Islet1 in the specification of limb identity. Genetic analysis has
suggested that Tbx4 expression is driven by the hindlimb-specific
program, although its role in hindlimb specificity remains
controversial (Minguillon et al., 2005; Naiche and Papaioannou,
2007; Ouimette et al., 2010). We observed a slightly weaker signal
for Pitx1 expression and significant downregulation of Tbx4 in the
LPM (Fig. 3I-N⬘). Since the slight downregulation of Pitx1 could
be due to a loss of cells in the hindlimb field, we examined
proliferation and cell death. We found that Islet1 cKO embryos
exhibit a significant reduction in the proliferation of cells in the
LPM (n5 embryos, Fig. 3O), as compared with control embryos
(n4 embryos), which could, at least in part, contribute to the
absence of a hindlimb. However, unlike Ctnnb1 cKO embryos, we
did not observe alterations in cell death (Fig. 3P). Such reduced
proliferation might also contribute to the slightly weaker Pitx1
expression. By contrast, Tbx4 expression in the LPM is severely
downregulated, although it is not completely abolished, as
compared with persisting expression in the ventral body wall (Fig.
3M-N⬘). These data suggest that Islet1 is one of factors regulating
Tbx4 expression in the hindlimb field, and other factors might
participate in the regulation of Tbx4 expression. Although
examination of hindlimb-specific morphology in the Islet1 cKO
was not possible, these results indicate that Islet1 function is
unlikely to be linked to determining hindlimb-specific
characteristics in hindlimb progenitors before the formation of the
hindlimb bud. Taken together, our analysis has identified Islet1 as
a missing piece in the transcriptional regulation of hindlimbspecific initiation in the vertebrate embryo.
Relationship between the -catenin pathway and
Islet1
The fact that the losses of -catenin and Islet1 led to the loss of
hindlimb initiation raised the possibility that the -catenin pathway
and Islet1 might interact functionally to control a common process
for limb initiation. In order to address this, we first examined
whether nuclear -catenin and Islet1 colocalize by performing a
double immunostaining for -catenin and Islet1 in sections
prepared from the hindlimb-forming area of wild-type mouse
embryos at E9.5. Among 16 sections from five embryos (total 2371
cells), we observed that, except for just two cells, nuclear -catenin
and Islet1 do not colocalize (Fig. 4A-C⬘). This suggests that the catenin pathway and Islet1 act on different populations in the
hindlimb-forming area.
Because Fgf10 expression is completely lost in both Ctnnb1
cKO and Islet1 cKO hindlimb fields (Fig. 2C,D, Fig. 3E,F), we
next examined which of the Islet1-positive cells or which cells with
active -catenin (BATgal-positive cells) overlap with Fgf10
expression. Fgf10 transcripts were detected in the broad region of
the LPM, with stronger expression on the dorsal side (Fig. 4D). We
observed that BATgal and Fgf10 signals overlapped on the dorsal
side (Fig. 4E), whereas Islet1 immunoreactivity overlapped with
DEVELOPMENT
-catenin and Islet1 in limb initiation
4470 RESEARCH ARTICLE
Development 138 (20)
downregulated in Islet1 cKO embryos as compared with control
littermates at E9.5 (Fig. 5B,C). Examining six sections from two
control embryos showed that 22.0% of cells in the LPM were
BATgal positive, whereas 8.9% of cells in the LPM in six sections
from two Islet1 cKO embryos were BATgal positive (Fig. 5D).
These results suggest that Islet1 functions in the nuclear
accumulation of -catenin and activation of the -catenin pathway.
In the reciprocal experimental setting, we observed Islet1
expression in Ctnnb1 cKO embryos at E9.5, suggesting that the catenin function is not required for Islet1 expression (Fig. 5E,F),
but rather that -catenin seems to be required for the maintenance
of Islet1 expression or for the survival of Islet1-expressing cells.
This is because of the downregulation of Islet1 in the ventralposterior region of the E10.5 embryo, as compared with control
littermates (Fig. 5G,H).
Our analyses have shown that two genetic systems, involving
Islet1 and -catenin, regulate hindlimb initiation in the mouse
embryo as upstream regulators of LPM cell proliferation and Fgf10
expression. Moreover, these two players functionally interact –
Islet1 in the nuclear accumulation of -catenin, and the -catenindependent pathway in maintaining Islet1 expression (Fig. 5I).
Fgf10 signal on the ventral side (Fig. 4F). These results
demonstrate that cells with active -catenin and cells with Islet1,
which constitute two spatially exclusive populations, overlap with
Fgf10 expression.
The mutually exclusive localization of Islet1 and nuclear -catenin
does not rule out the possibility that the -catenin pathway and Islet1
affect each other functionally. To clarify this, we examined whether
the loss of Islet1 affects -catenin pathway activity, and vice versa.
In the hindlimb field of Islet1 cKO embryos, only 1.8% of cells were
nuclear -catenin positive, whereas 24.3% of cells were nuclear catenin positive in control embryos (Fig. 5A). The hindlimb area of
the Islet1 cKO embryos is almost indistinguishable from the
interlimb area, where no nuclear -catenin was detected (Fig. 1H).
We further examined this using the BATgal reporter mice. The
BATgal reporter signal in the hindlimb field is significantly
-catenin is required for hindlimb initiation
It has been controversial whether the -catenin-dependent pathway
is required for mouse limb initiation. Our previous studies have
revealed that the -catenin pathway regulates Tbx5 in the forelimb
field, which in turn upregulates Fgf10 expression, during forelimb
bud initiation in chick and zebrafish embryos (Kawakami et al.,
2001; Ng et al., 2002). Our studies have also demonstrated that the
-catenin pathway regulates hindlimb initiation in the chick
(Kawakami et al., 2001). This scenario has been uncertain in
mammals because, thus far, no Wnt ligand knockout mice have
shown defects in limb initiation (Grigoryan et al., 2008). Similarly,
no Wnt ligand gene expression has been reported in the limbforming area in mouse embryos. Several possibilities might
account for these discrepancies, including the difficulty in detecting
specific Wnt ligands at the low levels at which they are expressed,
the limited sensitivity of our detection system, the short time
window of expression, and rescue by functional redundancy of
multiple ligands. Nonetheless, the possible involvement of the
Wnt/-catenin pathway in limb initiation has remained an open
question. We have focused our study on -catenin, given that its
nuclear accumulation is the hallmark of activation of the signaling
pathway, using confocal imaging analysis to directly assess the
level of nuclear -catenin and examine the activation status of the
pathway. We also utilized a BATgal reporter line, which expresses
lacZ under the control of a multimerized Lef/Tcf element, the
activation of which depends on -catenin (Maretto et al., 2003).
Such a reporter line has proven useful for visualizing specific
signaling activity in a variety of studies, and the confocal imaging
and genetic analyses support each other in demonstrating that the
-catenin pathway is active in the limb-forming area.
In this study, we have revealed that -catenin is required for
mouse hindlimb initiation, upstream of active proliferation and
expression of Fgf10 in the LPM, consistent with our previous
DEVELOPMENT
Fig. 4. Spatial relationship between Islet1-expressing cells and
nuclear -catenin-positive cells during hindlimb initiation.
(A-C⬘) A typical fluorescent image of -catenin (green) and Islet1 (red)
immunoreactivity on a section prepared from the hindlimb-forming
area of an E9.5 wild-type mouse embryo. Insets B and C in A are
shown in B,B⬘ and C,C⬘ at higher magnification. Nuclear -catenin is
predominantly detected in the lateral area (B,B⬘), whereas Islet1 is
detected in the medial-ventral area (C,C⬘). (D-F)Spatial relationship
between Fgf10 transcripts, -catenin activity (BATgal) and Islet1
immunoreactivity. (D)Fgf10 mRNA distribution in a cross-section
prepared from the middle region of the hindlimb-forming area at E9.5.
(E)Spatial relationship between Fgf10 and -catenin activity in a crosssection prepared from the middle region of hindlimb-forming area at
E9.5. Fgf10 mRNA, purple; patchy BATgal signal, orange. The blue and
orange bars represent the Fgf10 and BATgal expression domains,
respectively, along the dorsal-ventral axis. (F)Spatial relationship
between Fgf10 and Islet1 in a cross-section prepared from the middle
region of hindlimb-forming area at E9.5. Fgf10 mRNA, purple; Islet1
immunoreactivity, orange. Blue and orange bars represent the
expression domains of Fgf10 and Islet1, respectively, along the dorsalventral axis.
DISCUSSION
In this study, we have shown that -catenin and Islet1 regulate
hindlimb initiation. Both genes are required for initiating outgrowth
as well as for inducing Fgf10 expression, which provides the signal
from the LPM to activate Fgf8 in the ectoderm, initiating the
Fgf10-Fgf8 feedback loop for maintaining limb bud outgrowth.
-catenin and Islet1 in limb initiation
RESEARCH ARTICLE 4471
Fig. 5. Functional interaction between Islet1 and -catenin during hindlimb initiation. (A)The percentage of cells with nuclear -catenin is
reduced in the LPM of Islet1 cKO mouse embryos at E9.5. (B,C)BATgal reporter activity in the hindlimb-forming area is reduced in the Islet1 cKO
embryo (C, red arrowheads), compared with a littermate control embryo (B, black arrows) at E9.5. (D)Quantitative analysis of BATgal-positive cells
in the LPM. The percentage of cells with BATgal signal is reduced in the LPM of Islet1 cKO embryos at E9.5. Error bars indicate s.d. (E-H) -catenin
activity is not required for expression of Islet1, but rather is involved in the maintenance of Islet1 expression. Islet1 is expressed similarly in control (E)
and Ctnnb1 cKO (F) embryos at E9.5 (arrow indicates expression). Islet1 expression in the control embryo is detected in the very posterior proximal
region of the hindlimb bud (G, arrow), but is not detected in the Ctnnb1 cKO embryo (H, red arrowhead) at E10.5. fl, forelimb bud. (I)Summary of
the analysis. Islet1 functions in the nuclear accumulation of -catenin, which in turn acts to maintain Islet1 expression. Both Islet1 and the catenin-dependent pathway lie upstream of hindlimb outgrowth and Fgf10 expression in the LPM for hindlimb initiation in vertebrates. For more
details, see Discussion.
Fig. S3 in the supplementary material). Interestingly, Hoxb6Cre can
recombine in cells in the posterior half of the forelimb field (Lowe
et al., 2000), and Hoxb6Cre-mediated inactivation of Ctnnb1
resulted in a small forelimb bud compared with control littermates
(see Fig. S2G,H in the supplementary material). This is likely to be
due to limited inactivation of -catenin only in the posterior region
of the forelimb field, and cells that escaped inactivation would
survive and proliferate and turn on Fgf10, leading to the formation
of a small forelimb bud. Although we do not exclude the possibility
of the involvement of an unidentified factor in the forelimb
initiation process, our analysis has revealed that the -catenindependent pathway is active in the limb-forming area, and that it
functions upstream of cell proliferation and Fgf10 expression in the
LPM during hindlimb initiation in the mouse.
Islet1, a hindlimb field-specific factor, is required
for hindlimb initiation upstream of -catenin
activation
Although induction of Fgf10 is required for establishing the Fgf10Fgf8 feedback loop to maintain mesenchymal proliferation for both
forelimb and hindlimb development, the transcriptional programs
that regulate Fgf10 expression are different in these two limbs.
Tbx5 has been shown to be the transcription factor specifically
regulating forelimb initiation in mice (Agarwal et al., 2003);
however, hindlimb-specific transcriptional regulation has been
elusive. Our analysis has revealed that Islet1, a new player in this
field, specifically regulates initiation of the hindlimb, and has
clarified the difference between the two limb-type-specific
transcriptional regulation programs. Based on expression in the
LPM before hindlimb outgrowth, as well as on an immediate
decline in its expression after hindlimb bud outgrowth (see Fig.
DEVELOPMENT
studies in chick and zebrafish. In the absence of -catenin, we
observed increased cell death and reduced proliferation in the LPM.
Since -catenin is a multi-functional factor, it might interact with
many factors to regulate cell survival and proliferation (Min et al.,
1998; Sekine et al., 1999). The increased cell death and reduced
proliferation are likely to be independent from the function of
Fgf10 (Min et al., 1998; Sekine et al., 1999) because Fgf10–/–
embryos exhibit neither increased cell death nor reduced
proliferation (see Fig. S4 in the supplementary material). Although
nuclear -catenin-positive cells constitute ~24% of LPM, the
complete loss of Fgf10 expression and the lack of hindlimb bud
outgrowth strongly suggest that the -catenin pathway regulates
Fgf10 as a genetically upstream factor for limb initiation, and, in
addition, that it controls cell proliferation and survival. Besides the
role of -catenin-dependent signaling discussed here, we should
also consider another possible scenario: a role of -catenin in cell
adhesion, given that only ~24% of LPM cells are nuclear catenin-positive with a salt-and-pepper pattern. In support of this
possibility, a previous study in chick embryos has shown that cellcell adhesion is increased during limb bud formation (Heintzelman
et al., 1978). Although the role of cell-cell adhesion in limb
initiation in the mouse embryo is still to be investigated (Wada,
2011), both functions – in cell-cell adhesion and in the genetic
pathway – might contribute during the -catenin-dependent limb
initiation process.
We speculate that -catenin might also be required in the
forelimb field. Our current genetic tool limits our experiments to
the hindlimb field; very early inactivation of -catenin mediated by
T-cre causes a lack of mesoderm generation (Dunty et al., 2008),
leading to severe truncation of the posterior body structure, which
precludes analysis of -catenin loss during limb development (see
S5A in the supplementary material), Islet1 is likely to be acting
only in the early stage of hindlimb development, similar to how
Tbx5 is required only in the early stage of forelimb development
(Hasson et al., 2007).
Interestingly, these two factors belong to different gene families,
namely the T-box transcription factors (Tbx5) and LIMhomeodomain factors (Islet1). This leads to two possible scenarios.
One is that Fgf10 contains multiple regulatory elements for
expression in the limb-forming region. In this scenario, Tbx5 and
Islet1 regulate different DNA sequences, yet they both genetically lie
upstream of Fgf10 during the limb initiation process. Our
observation that Fgf10 expression overlaps with both Islet1-positive
cells and nuclear -catenin-positive cells, which are located in a
mutually exclusive manner in the hindlimb-forming region, also
supports this scenario. Even in the hindlimb-forming region, Islet1
and -catenin might regulate Fgf10 in different cell populations.
Such a scenario adds another layer of complexity to the regulation
of Fgf10 expression. Currently, it is not clear whether this is the case,
as enhancer element(s) for Fgf10 expression in the forelimb and
hindlimb fields are unknown.
Our data also provide a second scenario. During hindlimb
initiation, Islet1 function is required for nuclear localization of catenin, and Ctnnb1 cKO showed no hindlimb outgrowth,
suggesting that -catenin mediates Islet1 function in limb initiation.
Then, the -catenin-dependent pathway regulates Fgf10 expression.
In this second scenario, Tbx5 might have a similar role to Islet1 in
the nuclear accumulation of -catenin. Such a model would explain
the requirement for limb-type-specific transcription factors, such
as Islet1 and Tbx5, as well as the -catenin-dependent pathway for
limb initiation. These models need to be studied in the future.
Relationship between Islet1 and -catenin
Our data demonstrate that both Islet1 and -catenin are required for
hindlimb initiation; however, it is still to be elucidated how Islet1
and -catenin interact to initiate the hindlimb bud, given that they
exhibit largely exclusive distributions in the hindlimb field. Both
factors act in nuclei, Islet1 as a DNA-binding transcription factor
and -catenin as a transcriptional co-activator, and thus it is likely
that their interaction is non-cell-autonomous. The finding that Islet1
is required for nuclear -catenin accumulation and -catenin
activity (visualized by BATgal reporter, Fig. 5) suggests that Islet1
might stimulate production of an unknown secreted factor(s),
which triggers activation of the -catenin pathway. If Wntdependent mechanisms are involved, Islet1 may be regulating
multiple Wnt ligands in the posterior hindlimb field to affect the catenin pathway, although specific Wnt ligands involved in this
process are still unknown. Alternatively, Islet1-dependent factor(s)
might be necessary to set up responsiveness to Wnt or other factors
to trigger -catenin activation. Similarly, -catenin-dependent
production of an unknown factor(s) might act on the maintenance
of Islet1 expression. Investigation into these possibilities will be an
important step towards a more detailed understanding of the
mechanisms of limb initiation.
In conclusion, our data demonstrate that Islet1-dependent
nuclear accumulation of -catenin lies upstream of the initiation of
hindlimb outgrowth and Fgf10 expression in the LPM of the
hindlimb field in mouse embryos. Our analyses have clarified a
hitherto controversial role for -catenin in the vertebrate limb
initiation process. Furthermore, we have uncovered transcriptional
regulation of hindlimb initiation by Islet1, which contrasts with that
of the forelimb by Tbx5, and unveiled distinct transcriptional
control in different limb types in vertebrates.
Development 138 (20)
Acknowledgements
We thank Dr Michael Kuehn for the Hoxb6Cre line; Dr Rolf Kemler for making
the Ctnnb1flox/flox line available to the research community; Dr Thomas Neufeld
for access to his microscope in the Developmental Biology Center at the
University of Minnesota; and the B&H platform of the CMRB for their excellent
technical help. The 39.4D5 antibody was obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the NICHD and
maintained by The University of Iowa, Department of Biology, Iowa City,
IA 52242, USA.
Funding
This work was supported by the Minnesota Medical Foundation (3962-921109) and American Cancer Society Institutional Research Grant (IRG-58-001-52IRG04) to Y.K., NINDS (5R37NS037116) and HHMI to S.P., the National
Institutes of Health (R01 NS049357) to Y.N., and CIBER, MICINN, Fundacion
Cellex, the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona
M. and Harry B. Helmsley Charitable Trust and Sanofi-Aventis to J.C.I.B.
Deposited in PMC for release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.065359/-/DC1
References
Agarwal, P., Wylie, J. N., Galceran, J., Arkhitko, O., Li, C., Deng, C.,
Grosschedl, R. and Bruneau, B. G. (2003). Tbx5 is essential for forelimb bud
initiation following patterning of the limb field in the mouse embryo.
Development 130, 623-633.
Bluske, K. K., Kawakami, Y., Koyano-Nakagawa, N. and Nakagawa, Y.
(2009). Differential activity of Wnt/beta-catenin signaling in the embryonic
mouse thalamus. Dev. Dyn. 238, 3297-3309.
Capdevila, J. and Izpisua Belmonte, J. C. (2001). Patterning mechanisms
controlling vertebrate limb development. Annu. Rev. Cell Dev. Biol. 17, 87-132.
DeLaurier, A., Schweitzer, R. and Logan, M. (2006). Pitx1 determines the
morphology of muscle, tendon, and bones of the hindlimb. Dev. Biol. 299, 2234.
Du, A., Hunter, C. S., Murray, J., Noble, D., Cai, C. L., Evans, S. M., Stein, R.
and May, C. L. (2009). Islet-1 is required for the maturation, proliferation, and
survival of the endocrine pancreas. Diabetes 58, 2059-2069.
Duboc, V. and Logan, M. P. (2011). Regulation of limb bud initiation and limbtype morphology. Dev. Dyn. 240, 1017-1027.
Dunty, W. C., Jr, Biris, K. K., Chalamalasetty, R. B., Taketo, M. M.,
Lewandoski, M. and Yamaguchi, T. P. (2008). Wnt3a/beta-catenin signaling
controls posterior body development by coordinating mesoderm formation and
segmentation. Development 135, 85-94.
Galceran, J., Farinas, I., Depew, M. J., Clevers, H. and Grosschedl, R. (1999).
Wnt3a–/– like phenotype and limb deficiency in Lef1(–/–)Tcf1(–/–) mice. Genes
Dev. 13, 709-717.
Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N.,
Silver, L. M. and Papaioannou, V. E. (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.
Grigoryan, T., Wend, P., Klaus, A. and Birchmeier, W. (2008). Deciphering the
function of canonical Wnt signals in development and disease: conditional lossand gain-of-function mutations of beta-catenin in mice. Genes Dev. 22, 23082341.
Haegel, H., Larue, L., Ohsugi, M., Fedorov, L., Herrenknecht, K. and Kemler,
R. (1995). Lack of beta-catenin affects mouse development at gastrulation.
Development 121, 3529-3537.
Hasson, P., Del Buono, J. and Logan, M. P. (2007). Tbx5 is dispensable for
forelimb outgrowth. Development 134, 85-92.
Heintzelman, K. F., Phillips, H. M. and Davis, G. S. (1978). Liquid-tissue
behavior and differential cohesiveness during chick limb budding. J. Embryol.
Exp. Morphol. 47, 1-15.
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Rodriguez Esteban, C. and
Izpisua Belmonte, J. C. (2001). WNT signals control FGF-dependent limb
initiation and AER induction in the chick embryo. Cell 104, 891-900.
Kawakami, Y., Rodriguez-Leon, J., Koth, C. M., Buscher, D., Itoh, T., Raya, A.,
Ng, J. K., Esteban, C. R., Takahashi, S., Henrique, D. et al. (2003a). MKP3
mediates the cellular response to FGF8 signalling in the vertebrate limb. Nat. Cell
Biol. 5, 513-519.
Kawakami, Y., Tsukui, T., Ng, J. K. and Izpisua Belmonte, J. C. (2003b).
Insights into the molecular basis of vertebrate forelimb and hindlimb identity. In
Patterning in Vertebrate Development (ed. C. Tickle), pp. 198-213. Oxford:
Oxford University Press.
DEVELOPMENT
4472 RESEARCH ARTICLE
Kawakami, Y., Esteban, C. R., Matsui, T., Rodriguez-Leon, J., Kato, S. and
Belmonte, J. C. (2004). Sp8 and Sp9, two closely related buttonhead-like
transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate
embryos. Development 131, 4763-4774.
Kawakami, Y., Rodriguez Esteban, C., Raya, M., Kawakami, H., Marti, M.,
Dubova, I. and Izpisua Belmonte, J. C. (2006). Wnt/beta-catenin signaling
regulates vertebrate limb regeneration. Genes Dev. 20, 3232-3237.
Kawakami, Y., Uchiyama, Y., Rodriguez Esteban, C., Inenaga, T., KoyanoNakagawa, N., Kawakami, H., Marti, M., Kmita, M., Monaghan-Nichols,
P., Nishinakamura, R. et al. (2009). Sall genes regulate region-specific
morphogenesis in the mouse limb by modulating Hox activities. Development
136, 585-594.
Lanctot, C., Moreau, A., Chamberland, M., Tremblay, M. L. and Drouin, J.
(1999). Hindlimb patterning and mandible development require the Ptx1 gene.
Development 126, 1805-1810.
Logan, M. (2003). Finger or toe: the molecular basis of limb identity. Development
130, 6401-6410.
Lowe, L. A., Yamada, S. and Kuehn, M. R. (2000). HoxB6-Cre transgenic mice
express Cre recombinase in extra-embryonic mesoderm, in lateral plate and limb
mesoderm and at the midbrain/hindbrain junction. Genesis 26, 118-120.
Maretto, S., Cordenonsi, M., Dupont, S., Braghetta, P., Broccoli, V., Hassan,
A. B., Volpin, D., Bressan, G. M. and Piccolo, S. (2003). Mapping Wnt/betacatenin signaling during mouse development and in colorectal tumors. Proc.
Natl. Acad. Sci. USA 100, 3299-3304.
Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B. D., Tarpley, J. E.,
DeRose, M. and Simonet, W. S. (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. and Logan, M. P. (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.
Naiche, L. A. and Papaioannou, V. E. (2003). Loss of Tbx4 blocks hindlimb
development and affects vascularization and fusion of the allantois.
Development 130, 2681-2693.
Naiche, L. A. and Papaioannou, V. E. (2007). Tbx4 is not required for hindlimb
identity or post-bud hindlimb outgrowth. Development 134, 93-103.
Ng, J. K., Kawakami, Y., Buscher, D., Raya, A., Itoh, T., Koth, C. M.,
Rodriguez Esteban, C., Rodriguez-Leon, J., Garrity, D. M., Fishman, M. C.
et al. (2002). The limb identity gene Tbx5 promotes limb initiation by interacting
with Wnt2b and Fgf10. Development 129, 5161-5170.
Nusse, R. (2005). Wnt signaling in disease and in development. Cell Res. 15, 2832.
Ohuchi, H., Nakagawa, T., Yamamoto, A., Araga, A., Ohata, T., Ishimaru, Y.,
Yoshioka, H., Kuwana, T., Nohno, T., Yamasaki, M. et al. (1997). The
mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick
limb bud through interaction with FGF8, an apical ectodermal factor.
Development 124, 2235-2244.
Ouimette, J.-F., Jolin, M. L., L’honore, A., Gifuni, A. and Drouin, J. (2010).
Divergent transcriptional activities determine limb identity. Nat. Commun. 1, 1-9.
RESEARCH ARTICLE 4473
Perantoni, A. O., Timofeeva, O., Naillat, F., Richman, C., Pajni-Underwood,
S., Wilson, C., Vainio, S., Dove, L. F. and Lewandoski, M. (2005).
Inactivation of FGF8 in early mesoderm reveals an essential role in kidney
development. Development 132, 3859-3871.
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and Jessell, T. M.
(1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation
reveals a motor neuron-dependent step in interneuron differentiation. Cell 84,
309-320.
Rallis, C., Bruneau, B. G., Del Buono, J., Seidman, C. E., Seidman, J. G.,
Nissim, S., Tabin, C. J. and Logan, M. P. (2003). Tbx5 is required for forelimb
bud formation and continued outgrowth. Development 130, 2741-2751.
Sahara, S. and O’Leary, D. D. (2009). Fgf10 regulates transition period of cortical
stem cell differentiation to radial glia controlling generation of neurons and
basal progenitors. Neuron 63, 48-62.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T.,
Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential
for limb and lung formation. Nat. Genet. 21, 138-141.
Song, M. R., Sun, Y., Bryson, A., Gill, G. N., Evans, S. M. and Pfaff, S. L.
(2009). Islet-to-LMO stoichiometries control the function of transcription
complexes that specify motor neuron and V2a interneuron identity.
Development 136, 2923-2932.
Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF signalling from
the apical ectodermal ridge in limb development. Nature 418, 501-508.
Sun, Y., Dykes, I. M., Liang, X., Eng, S. R., Evans, S. M. and Turner, E. E.
(2008). A central role for Islet1 in sensory neuron development linking sensory
and spinal gene regulatory programs. Nat. Neurosci. 11, 1283-1293.
Szeto, D. P., Rodriguez-Esteban, C., Ryan, A. K., O’Connell, S. M., Liu, F.,
Kioussi, C., Gleiberman, A. S., Izpisua-Belmonte, J. C. and Rosenfeld, M.
G. (1999). Role of the Bicoid-related homeodomain factor Pitx1 in specifying
hindlimb morphogenesis and pituitary development. Genes Dev. 13, 484-494.
Towers, M. and Tickle, C. (2009). Growing models of vertebrate limb
development. Development 136, 179-190.
Trelles, R. D., Leon, J. R., Kawakami, Y., Simoes, S. and Belmonte, J. C.
(2002). Expression of the chick vascular endothelial growth factor D gene during
limb development. Mech. Dev. 116, 239-242.
Wada, N. (2011). Spatiotemporal changes in cell adhesiveness during vertebrate
limb morphogenesis. Dev. Dyn. 240, 969-978.
Wilkinson, D. G. (1992). Whole mount in situ hybridization of vertebrate
embryos. In In Situ Hybridization: a Practical Approach (ed. D. G. Wilkinson), pp.
75-83. Oxford, UK: IRL Press.
Yang, L., Cai, C. L., Lin, L., Qyang, Y., Chung, C., Monteiro, R. M., Mummery,
C. L., Fishman, G. I., Cogen, A. and Evans, S. (2006). Isl1Cre reveals a
common Bmp pathway in heart and limb development. Development 133,
1575-1585.
Zeller, R., Lopez-Rios, J. and Zuniga, A. (2009). Vertebrate limb bud
development: moving towards integrative analysis of organogenesis. Nat. Rev.
Genet. 10, 845-858.
DEVELOPMENT
-catenin and Islet1 in limb initiation