Download Role of chick cux1 and cux2 during limb development

5133
Development 127, 5133-5144 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV2577
Evidence that members of the Cut/Cux/CDP family may be involved in AER
positioning and polarizing activity during chick limb development
Ana Teresa Tavares1,2, Tohru Tsukui1 and Juan Carlos Izpisúa Belmonte1,*
1The
Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, California 92037,
USA
2Instituto Gulbenkian de Ciência, Rua da Quinta Grande, n.6, Apartado 14, 2780-156 Oeiras, Portugal
*Author for correspondence (e-mail: [email protected])
Accepted 14 September; published on WWW 2 November 2000
SUMMARY
In vertebrates, the apical ectodermal ridge (AER) is a
specialized epithelium localized at the dorsoventral
boundary of the limb bud that regulates limb outgrowth.
In Drosophila, the wing margin is also a specialized region
located at the dorsoventral frontier of the wing imaginal
disc. The wingless and Notch pathways have been
implicated in positioning both the wing margin and the
AER. One of the nuclear effectors of the Notch signal in
the wing margin is the transcription factor cut. Here we
report the identification of two chick homologues of the
Cut/Cux/CDP family that are expressed in the developing
limb bud. Chick cux1 is expressed in the ectoderm outside
the AER, as well as around ridge-like structures induced
by β-catenin, a downstream target of the Wnt pathway.
cux1 overexpression in the chick limb results in scalloping
of the AER and limb truncations, suggesting that Cux1
may have a role in limiting the position of the AER
by preventing the ectodermal cells around it from
differentiating into AER cells.
The second molecule of the Cut family identified in this
INTRODUCTION
The apical ectodermal ridge (AER), a specialized epithelial
structure that forms at the tip of the vertebrate limb bud, is
essential for limb outgrowth (Saunders, 1948). Embryological
manipulation experiments have shown that when the AER is
excised, limb bud outgrowth is inhibited (Rowe et al., 1982;
Summerbell, 1974; Todt and Fallon, 1987). In the last decade,
we have begun to understand at the molecular level how the
AER controls limb outgrowth. Some of the pathways
implicated include the Fibroblast Growth Factor (FGF), Notch
and Wnt signaling pathways. Members of the FGF family play
a critical role in AER function. In particular, three Fgfs (Fgf2,
Fgf4, and Fgf8) are expressed in the AER and can fulfill the
outgrowth functions of the AER (Cohn et al., 1995; Fallon
et al., 1994; Laufer et al., 1994; Niswander et al., 1994;
Niswander et al., 1993; Vogel and Tickle, 1993).
An important player in the Notch signaling pathway is
fringe. During chick limb outgrowth, overexpression studies
study, cux2, is expressed in the pre-limb lateral plate
mesoderm, posterior limb bud and flank mesenchyme, a
pattern reminiscent of the distribution of polarizing
activity. The polarizing activity is determined by the ability
of a certain region to induce digit duplications when
grafted into the anterior margin of a host limb bud. Several
manipulations of the chick limb bud show that cux2
expression is regulated by retinoic acid, Sonic hedgehog
and the posterior AER. These results suggest that Cux2
may have a role in generating or mediating polarizing
activity. Taking into account the probable involvement of
Cut/Cux/CDP molecules in cell cycle regulation and
differentiation, our results raise the hypothesis that chick
Cux1 and Cux2 may act by modulating proliferation versus
differentiation in the limb ectoderm and polarizing activity
regions, respectively.
Key words: Apical ectodermal ridge, Polarizing activity, cux, Limb,
Chick
indicate that the radical fringe (R-fng) gene is involved in
directing the initial positioning of the AER (Laufer et al., 1997;
Rodriguez-Esteban et al., 1997). The ridge forms at the
boundary between cells that express R-fng (dorsal ectoderm)
and those that do not (ventral ectoderm). However, loss-offunction of mouse R-fng does not result in any limb defects,
suggesting that other members of the Fringe family may
functionally overlap with R-fng during limb development
(Moran et al., 1999). serrate 2 (ser2) is initially expressed
throughout the limb ectoderm, but becomes restricted to the
AER soon after its formation (Laufer et al., 1994; Shawber et
al., 1996). In addition, both the syndactylism mouse mutant and
the knockout of serrate 2 display abnormal thickening of the
AER (Jiang et al., 1998; Sidow et al., 1997). Notch 1
expression also localizes to the AER (Myat et al., 1996). R-fng
is thought to act by inducing Notch 1 signaling, probably in
response to activation by Serrate 2 (Fleming et al., 1997; Panin
et al., 1997). Based on all of these observations, it has been
suggested that Notch signaling in the limb ectoderm may serve
5134 A. T. Tavares and others
to regulate the number of AER progenitor cells (Tickle
and Altabef, 1999). Therefore, restricting the activity and
expression of notch 1 and serrate 2 to the AER is probably
necessary for maintaining the structure and function of the
ridge throughout limb development.
In addition to FGF and Notch, the Wnt signaling pathway
has also been shown to be implicated in AER formation
(Galceran et al., 1999; Kengaku et al., 1998). Wnt3a is
initially expressed in the presumptive chick AER, and
subsequently in the mature AER. Gain-of-function
experiments indicate that Wnt3a can induce Fgf8 expression
in the chick AER. The induction appears to be mediated by
β-catenin and Lef1 (Kengaku et al., 1998). Although all of
these experiments have revealed how the establishment of the
AER is regulated by several secreted factors, receptors and
cytoplasmic mediators, most of the nuclear targets of these
pathways are still unknown.
One of the most fruitful approaches to studying vertebrate
limb development has been to pursue parallels of the molecular
mechanisms that pattern the Drosophila appendages. In
Drosophila, the gene cut, a member of the Cut/CDP/Cux
family of transcription factors has been shown to have an
important role during the development of the dorsoventral
(DV) boundary in the wing margin. The DV boundary is
established at the junction between dorsal, fringe-expressing
cells and ventral, non-expressing cells (Irvine and Wieschaus,
1994). Fringe protein modulates the interactions between the
Notch receptor and its ligands Serrate and Delta (Fleming et
al., 1997; Panin et al., 1997). In the wing margin, Notch
signaling is activated by Delta and is inhibited by Fringe to
respond to the dorsal Serrate-expressing cells. At later stages,
Notch activity induces the expression of cut in the wing margin
(Micchelli et al., 1997; Neumann and Cohen, 1996). Cut
promotes the expression of wingless (wg) and inhibits the
expression of serrate and delta in the margin (de Celis and
Bray, 1997; Neumann and Cohen, 1996). At this stage, Notch
activation no longer depends on the dorsoventral boundary,
but is regulated by a feedback loop between margin cutexpressing cells and flank delta- and serrate-expressing cells.
Interestingly, human CDP and mouse cux cDNAs were shown
to rescue Drosophila ‘cut wing’ phenotype and have a similar
effect on embryonic sensory organ development as the fly gene
(Ludlow et al., 1996). Based on the putative role of Cut in
mediating wing margin formation and limb outgrowth in
Drosophila, we were interested in identifying vertebrate Cut
homologues to study their possible involvement in the
positioning and establishment of the vertebrate AER.
We report the identification of a member of the Cut/Cux/CDP
family of transcription factors, cux1, that is specifically expressed
during chick limb outgrowth in both the dorsal and ventral
limb ectodermal cells bordering the AER. Gain-of-function
experiments indicate that cux1 regulates AER formation.
Furthermore, we show that cux1 expression is regulated by AER
signals. Our results indicate that cux1 may have a role in
establishing the AER at the distal tip of the limb ectoderm.
In addition to cux1, we report the identification of another
member of the Cut/Cux/CDP gene family, cux2. The study of
its spatiotemporal pattern of expression indicates that cux2
could be involved in establishing the Zone of Polarizing
Activity (ZPA), a group of cells located at the posterior region
of the developing limb bud that control the posterior
patterning of the vertebrate limb (Saunders and Gasseling,
1968).
A key molecular component of the ZPA is the gene sonic
hedgehog (Shh) (Riddle et al., 1993). Shh mRNA transcripts colocalize with the ZPA (Riddle et al., 1993; Yonei et al., 1995),
and loss- and gain-of-function experiments have shown that Shh
is required for distal outgrowth and anteroposterior patterning of
the vertebrate limb (Chiang et al., 1996; Lopez-Martinez et al.,
1995; Riddle et al., 1993). Two other molecules involved in
the polarizing activity are retinoic acid (RA) and Hoxb8.
Application of a RA bead to the anterior side of a limb
reorganizes its anteroposterior patterning and results in digit
duplications (Tickle et al., 1982). This effect appears to be
mediated by the induction of Shh expression (Riddle et al.,
1993). Retinoic acid is also able to induce the expression of
Hoxb8 (Helms et al., 1996; Lu et al., 1997a; Stratford et al.,
1997), and ectopic expression of Hoxb8 in the anterior region of
the mouse forelimb induces digit duplication (Charité et al.,
1994). Whilst these results implicate Hoxb8 in the establishment
of the ZPA in the forelimb, they do not correlate with the
spatiotemporal pattern of expression of Hoxb8 in the
presumptive hindlimb. Moreover, Hoxb8 knockout mice do not
display any limb defects, suggesting that Hoxb8 is not essential
for the anteroposterior patterning of the forelimb (van den Akker
et al., 1999). In this manuscript we show that cux2 mRNA
co-localizes with regions shown to have polarizing activity
(Hornbruch and Wolpert, 1991), including the ZPA in both the
vertebrate fore and hindlimb. In addition, cux2 can be regulated
by both Shh and RA. These results suggest that cux2 may be a
nuclear target of some of the known pathways implicated in the
anteroposterior patterning of the vertebrate limb.
MATERIALS AND METHODS
cDNA cloning
Chick cux1 clones were identified in the screening of a chick stage
12-16 embryonic λZAPII cDNA library (David Wilkinson) with a
chick cerebellum EST clone as a probe (GenBank accession number
T25685). The full coding sequence of chick cux1 (2673 nucleotides)
was identified. A 3′ fragment of chick cux2 (1218 nucleotides) was
cloned from the screening of a stage 20-22 chick limb bud λZAPII
DNA library using a 490 bp PstI clone of cux1 3′ sequence as a probe.
The positive clones were sequenced with an automated sequencer.
Sequence comparison was performed using the software DNASISMac, version 3.0 (Hitachi Software Engineering Co., Ltd.) The
GenBank accession numbers of the protein sequences used in the
alignment are: P53564 (mouse Cux1), P39880 (human CDP), P10180
(Drosophila Cut), 6681089 (mouse Cux2), and BAA22962 (human
CUX2).
Northern blot analysis
Total RNA was extracted from limb buds and bodies (embryos
without limb buds) of stage 21-23 chick embryos using Trizol (Gibco
BRL). Polyadenylated mRNA was selected using a Nucleotrap Mini
Kit (Clontech) according to the manufacturer’s instructions.
Polyadenylated mRNA (10 µg per lane) was size fractionated on a 1%
agarose/2.2 M formaldehyde gel and transferred onto a Nytran
membrane (Schleicher & Schuell). A 3′ fragment of chick cux1 cDNA
(nucleotides 2521 to +305) was labeled by random priming using [α32P]dCTP. The membrane was hybridized overnight at 55°C in 0.25
M Na2HPO4 (pH 7.2) and 7% SDS, washed at 60°C in 2× SSC and
0.1% SDS, and analyzed both by autoradiography and by a
PhosphorImager (Molecular Dynamics).
Role of chick cux1 and cux2 during limb development 5135
Whole-mount in situ hybridization, histology and cartilage
staining
Chicken embryos were obtained from MacIntyre Poultry (San Diego,
California). Eggs were incubated at 38°C and staged according to
Hamburger and Hamilton (HH; Hamburger and Hamilton, 1951).
Whole-mount in situ hybridization was carried out as described
(Wilkinson, 1993). The riboprobe used for cux1 encompasses
nucleotides 147-646 (HindIII-NcoI subclone). The cux2 probe
includes the whole sequence cloned (1218 nucleotides). The probe for
C-ser2 was a kind gift from C. Tabin (Laufer et al., 1997). The
remaining probes have been described elsewhere: Fgf8 (Vogel et al.,
1996) and Bmp2 (Francis et al., 1994). In some cases, the embryos
were dehydrated in 30% sucrose, embedded in gelatin, frozen and
sectioned in a cryostat.
For the evaluation of cartilage structures, embryos were collected
7 days after infection, fixed in 5% trichloracetic acid, stained in 0.1%
Alcian Green, dehydrated in ethanol and cleared with methyl
salicylate.
For the histological analysis of the AER, embryos were collected
2 days after virus injection, fixed in Bouin’s, dehydrated in ethanol
and embedded in paraffin wax. Embryos were then serially sectioned
at 7 µm, stained with Haematoxylin/Eosin (H&E), and mounted with
Permount.
Retrovirus and recombinant adenovirus production
A constitutively active mutant form of Xenopus β-catenin containing
the internal Armadillo repeats (Funayama et al., 1995) was cloned into
the shuttle vector pSLAX-12 and subcloned into the retroviral vector
RCAS(BP)A. Retroviruses were produced and harvested as described
by Morgan and Fekete (1996). Briefly, primary chick embryonic
fibroblasts were transfected and the supernatant was collected and
concentrated by ultracentrifugation.
Three recombinant adenoviruses were constructed containing the
full coding sequence of chick cux1 (rAd-Cux1), a histone 2B/GFP
fusion (Kanda et al., 1998); rAd-GFP or chick Shh (Cohn et al.,
1995; rAd-Shh). All genes were driven by a CAG promoter (Niwa
et al., 1991). The recombinant adenoviruses were constructed
according to the COS-TPC method previously described (Miyake et
al., 1996), and were produced by homologous recombination in 293
cells. Briefly, the cells were cultured in 6 cm plates and cotransfected with partial viral genome fragments and the cosmid
DNA using a CellPhect transfection kit (Amersham Pharmacia
Biotech, New Jersey, USA). On the next day, the cells were split
onto collagen-coated 96-well plates (BIOCOAT, Becton Dickinson,
New Jersey, USA). After 10 days, some wells contained dead cells
as a result of viral propagation. Recombinant viruses from each of
these wells were amplified in four 225 cm2 collagen-coated flasks,
and purified by CsCl step-gradient centrifugations (Kanegae et al.,
1994).
Stage 10-13 embryos were microinjected in the fore- or hindlimb
primordia and harvested either after 2-3 days, for the analysis of
changes in gene expression, or after 7 days, for the evaluation of the
limb skeletal structures.
Bead implantation and experimental manipulations of the
limb in ovo
Heparin acrylic beads (Sigma) were soaked in 1 mg/ml of FGF-2
(R&D Systems) for 1 hour, and grafted into the lateral plate
mesenchyme of stage 14-15 chick embryos in ovo.
AG1-X2 ion exchange beads were soaked for 1 hour in either 0.1
mg/ml all-trans retinoic acid (Sigma; diluted in dimethyl sulfoxide,
DMSO) or in 10 µM retinoids receptor antagonist AGN 193109
(Johnson et al., 1995; Alergan Pharmaceuticals, Irvine, California,
USA) under constant agitation. The beads were then briefly rinsed in
phosphate-saline buffer (PBS) containing 10 mg/l Phenol Red. RA
beads were implanted in the lateral plate of stage 13-15 embryos at
somite level 15, or under the anterior side of the AER of stage 20 limb
buds. Antagonist beads were implanted in the lateral mesoderm at
somite level 18 of stage 13-14 embryos.
Surgical removal of the posterior half of the AER in wing buds of
stage 20 embryos was performed using fine tungsten needles.
Additionally, in some of these limb buds, Affi-Gel blue beads
(BioRad) soaked in a 7.5 mg/ml Shh-N protein solution (Ontogeny,
Inc., Massachusetts, USA) were grafted into the posterior
mesenchyme.
After the operations, the embryos were harvested at different time
points and analyzed for changes in gene expression by in situ
hybridization.
RESULTS
Cloning of chick cux1
Chick cux1 coding sequence was cloned by screening a stage
12-16 HH chick embryo cDNA library using a chicken EST
clone similar to mouse cux1 as a probe. The longest clones of
chick cux1 obtained had 2673 nucleotides of coding sequence
and contained two cut repeats and one homeodomain (Fig. 1A).
Northern blot analysis of RNA isolated from stage 20-23 chick
embryo revealed that two cux1 transcripts of about 10 and 13
kb in size are present in both limb buds and embryo bodies
(Fig. 1B). All human, mouse and fly Cut proteins exist as splice
variants with sizes ranging from 2.4 to 13 kb (Blochlinger et
al., 1988; Neufeld et al., 1992; Vanden Heuvel et al., 1996b).
RT-PCR experiments using primers located immediately
downstream of the first exon and upstream of the second exon
did not identify any other sequence than the cDNA cux1
sequence reported here (data not shown). This suggests that the
splice variant containing the additional Cut repeat reported in
other species and indicated in Fig. 1A does not appear to be
present in the chick limb bud. A bacterial fusion protein
containing the third Cut repeat and the homeodomain of CDP
has been shown to bind to native CDP target sites (Aufiero et
al., 1994). Moreover, the second Cut repeat was shown to have
overlapping binding specificities with the third Cut repeat
(Aufiero et al., 1994). These observations suggest that the
protein encoded by the cloned chick cux1 cDNA, which is
lacking the second Cut repeat, might recognize similar DNA
binding sites as the full-length protein.
The percentage of amino acid identity within the cut repeats
and homeodomain of chick cux genes and their homologues is
summarized in Fig. 1C, and the alignment of the chick Cux1
sequence with those of other Cut proteins is shown in Fig. 1D.
The chick cux1 deduced amino acid sequence shows high
homology to the mouse and human counterparts, especially
within the cut repeats, the homeodomain, and the most N- and
C-terminal portions. The conserved N-terminal portion contains
a potential coiled-coil domain thought to mediate dimerization
between Cut proteins (Blochlinger et al., 1988). The C-terminal
region has high homology immediately downstream of the
homeodomain and in the last 70 residues. In spite of the
divergence in between these subdomains, the C terminus is likely
to act as a repression domain (Mailly et al., 1996).
Expression pattern of cux1 in the developing chick
embryo
The expression of cux1 during chick development was
analyzed by in situ hybridization using a 500 bp 3′ fragment
as a probe. During the early stages of limb budding, cux1
5136 A. T. Tavares and others
Role of chick cux1 and cux2 during limb development 5137
Fig. 1. (A) Schematic representation of the Cux/CDP genes and the
cloned sequences of the chick cux1 and cux2. (B) Northern blot of
chick cux1 transcripts present in RNA isolated from stage 20-23 limb
buds (lb) and embryos without limb buds (emb). Each lane contains
10 µg of polyadenylated RNA. Two bands corresponding to >10 kb
and >13 kb transcripts are detected in both samples (arrowheads).
(C) Percentage amino acid identity of the cut repeats and
homeodomain of the chick Cux1 and Cux2 in comparison with the
mouse, human and Drosophila homologues. (D) Alignment of the
chick Cux1 deduced amino acid sequence with the mouse, human
and Drosophila homologues. (E) Alignment of the deduced amino
acid sequence of the chick Cux2 protein (C-terminal portion) with
the mouse, human and Drosophila homologues. Lines above the
sequence indicate the cut repeats (purple) and the homeodomain
(green). Suppressed portions of the sequences are indicated by purple
dots.
transcripts are found all over the ectoderm except for the AER
cells (Fig. 2A). As limb development proceeds, the expression
becomes stronger in the ectodermal cells immediately adjacent
to the ridge (Fig. 2B,D). At stages 23-25 cux1 transcripts
become confined to cells bordering the AER in both the dorsal
Fig. 2. Expression patterns of cux1 and cux2
during chick limb development by in situ
hybridization. (A-F) Expression of cux1 in the
limb bud of stage 18-31 chick embryos. (A) At
stage 18, cux1 transcripts are found in the limb
ectoderm with the exception of the AER cells
(arrowhead). (B) At stage 21, cux1 expression
becomes higher in the ectoderm lining the ridge
(arrow). (C) At stage 23, cux1 transcripts are
restricted to the ectoderm bordering the AER
(arrows). (D) Transverse section of a stage 21
limb bud showing the ectodermal expression of
cux1 outside of the ridge. (E) Transverse section
of a stage 23 limb bud showing the expression
of cux1 in two regions immediately adjacent to
the AER (arrows). (F) At stage 31, cux1
transcripts are still seen in the ectoderm
flanking the tip of the limb (arrow).
(G-N) Expression of cux2 in stage 14-27 chick
embryos (arrows). (G) At stage 14, cux2
expression is found in the lateral plate
mesoderm (LPM) adjacent to somites 17-22.
(H) At stage 16, the expression of cux2 in the
LPM has a broader domain spanning the region
between somites 19 and 26. The white
arrowheads in G and H indicate the limits of the
wing field, between somites 16 and 20.
(I) Expression of cux2 in stage 19 embryos is
now found in the flank and in the posterior
mesenchyme of the limb buds. (J,K) At stage 20
(J) and stage 22 (K), cux2 expression remains in
the flank and posterior regions of the limb buds.
By stage 24 (L), the expression of cux2 in the
flank is reduced. (M) At stage 25, cux2
transcripts are no longer found in the flank.
(N) Cux-2 expression remains in the posterior
distal mesenchyme of the limb buds until stage
28. Note the expression of cux2 in the dorsal
neural tube. Embryos in panels J and K have an
open neural tube as a result of processing for in
situ hybridization.
and ventral limb ectoderm (Fig. 2C,E), and can still be detected
in this pattern at stage 31 (Fig. 2F).
Chick cux1 is expressed in the primitive myoblasts of the
myotome (Fig. 3A,B) as well as in the developing limb bud. It
is also expressed in the developing mesonephros (Fig. 3C), in
the distal portions of the developing mouth (Fig. 3D), and in
the feather buds (Fig. 3E). The expression is initially restricted
to the posterior mesenchyme of the buds but later adopts a
striped pattern along the longer feather buds, an expression
pattern very similar to that of C-Delta 1 (Chen et al., 1997).
Upregulation of cux1 expression by β-catenin
During chick limb development, β-catenin, which is expressed
in the AER (Lu et al., 1997b), appears to regulate AER
formation in response to Wnt3a signaling (Kengaku et al.,
1998). The misexpression of an activated form of β-catenin in
chick limb buds was shown to induce the formation of ectopic
ridge-like ectodermal structures that express Fgf8 and other
AER markers (Kengaku et al., 1998). In order to investigate
whether cux1 expression is regulated by AER cells, we
misexpressed this constitutively active mutant form of β-
5138 A. T. Tavares and others
catenin in chick limb buds using a replication competent avian
virus (RCAS) as a vector, and analyzed cux1 expression in
relation to the induced ectopic AERs.
Fig. 4A shows an RCAS-β-catenin injected limb bud
displaying ridge-like ectodermal structures that express Fgf8. In
the infected buds, cux1 expression is upregulated by the ectopic
ridges (Fig. 4B-E). cux1 transcripts appear around the nodules
but not in the AER-like cells (Fig. 4B,C). In tissue sections, we
observe that the expression pattern of cux1 around the ectopic
ridges (Fig. 4D; Fig. 4E, arrow) is identical to the one found
bordering the wild-type ridge (Fig. 4E, arrowhead). These
results suggest that cux1 expression is maintained by factors
released by the AER into the surrounding ectoderm.
Misexpression of cux1 in the developing chick limb
In order to investigate the role of cux-1 in limb development, we
injected a recombinant adenovirus containing the cux1 coding
sequence (rAdCux-1) into the presumptive limb region of stage
10-13 chick embryos. After 48-72 hours the infected limb buds
exhibited truncations and various degrees of AER disruption
(55%, n=360; Fig. 5B,D; arrows). In some of the indented areas
the ridge appeared thinner than normal (Fig. 5F in comparison
to Fig. 5E). In more severe cases, part of the AER was completly
absent (Fig. 5N). By co-injecting rAd-Cux-1 with rAd-GFP, the
affected regions were seen to co-localize in the infected portion
of the limb buds (Fig. 5H,I, and data not shown). Note that
several of the GFP-expressing cells seem to have left the
disrupted region of the AER and are found in both the dorsal
and ventral limb ectoderm (Fig. 5I, arrowheads). Abnormal
expression was observed in limb buds infected in the distal
ectoderm or AER but not in the mesenchyme (data not shown).
In order to gain insights into the cause of the limb bud
alterations observed after cux1 overexpression, we analyzed a
panel of AER and ectodermal markers. The expression of the
AER genes was seen to be reduced or even absent from the
scalloped regions of the infected limb buds. Fgf8 and Bmp2
expression is reduced in the thinner portions of the ridge (Fig.
5L; arrow) or absent where the AER is missing (Fig. 5J,N;
arrows). Chick serrate 2 expression is also absent from the
abnormal ridge portions (Fig. 5P; arrow). Wnt7a is expressed
in the dorsal ectoderm of chick limb buds (Dealy et al., 1993;
Fig. 5Q). In the infected limbs, Wnt7a expression extends
ventrally in the portions where the ridge is absent (Fig. 5R;
arrow), suggesting that these regions have normal ectodermal
cells instead of ridge cells. All mesenchymal markers tested
(Shh, Fgf10, Twist, Hoxd12 and Hoxd13) showed reduced
expression in correlation with the absence of tissue, caused by
the localized failure of proper AER function (data not shown).
At later stages, this phenotype translated into limb
truncations of various degrees with no AP or DV preference
(65%, n=50; Fig. 5T,U and data not shown). In the normal
chick wing (Fig. 5S) the skeletal elements that can be identified
are the humerus, radius, ulna, and three digits. In the infected
limb shown in Fig. 5T, the ulna is reduced and there is only a
piece of one digit. In Fig. 5U, a more severe phenotype reveals
the absence of all skeletal elements with the exception of a very
reduced humerus.
Cloning and expression pattern of chick cux2 in the
developing embryo
We obtained a clone different from cux1 as a result of screening
a chick limb library at low stringency with a cux1 3′ fragment
as a probe. This cDNA shows high homology to the mouse
cux2 gene, which we shall call chick cux2 from here on. The
sequence is 1218 nucleotides long and contains the third cut
repeat, the homeodomain and the rest of the 3′ coding sequence
(Fig. 1A). Further, it has higher homology within the cut repeat
and the homeobox when compared to the mouse and human
counterparts (Fig. 1C,E).
The expression pattern of cux2 was analyzed in the
developing chick embryo by in situ hybridization using the full
clone as a probe. Chick cux2 expression begins at around stage
13 in the lateral plate mesoderm (Fig. 2G,H, arrows, and data
not shown). At stage 14-16 the expression is higher in the
posterior region of the presumptive wing (white arrowheads in
Fig. 2G-H). It can also be detected at lower levels in the lateral
plate mesoderm of the presumptive hindlimb region. From
stages 17-24, expression is excluded from the anterior regions
of the limb buds and remains in the flank and posterior limb
mesenchyme (Fig. 2I-K). Similarly to Hoxb8 (Stratford et al.,
1997), this expression pattern largely overlaps with the area of
polarizing activity. However, Hoxb8 is downregulated at stage
18 and is not expressed in the leg bud, while cux2 transcripts
can also be detected there. After stage 24, cux2 is no longer
expressed in the flank mesenchyme and becomes restricted to
the posterior region of both the fore- and hindlimb buds (Fig.
2M,N). After stage 28, cux2 expression disappears from the
posterior regions of the limb buds.
Other regions where cux2 transcripts can be found include
the mesonephros (Fig. 3F) and the telencephalon (Fig. 3G).
Beginning at stage 17, cux2 expression is found in the dorsal
region of the neural tube (Figs 2H-M, 3I,J). cux2 also appears
to be expressed by populations of migrating neural crest cells
that are presumably melanocyte precursors (Fig. 3H-J; arrows).
In the neural tube, cux2 transcripts become less abundant at
stage 23 (Fig. 3J) and are undetected by stage 28. cux2 has a
very dynamic expression pattern in the developing feather
buds, similar to the one reported for Lunatic fringe (Noramly
and Morgan, 1998). It is first detected in the ectoderm of the
early buds (day 7.5-8 of embryonic development; rings; Fig.
3K), later becomes restricted to the mesoderm (day 9; Fig. 3L)
and disappears by day 10 (data not shown).
Regulation of cux2 expression during limb
development
The expression pattern of cux2 in the lateral plate mesoderm,
the posterior limb buds and flank co-localizes with regions
shown to have potential polarizing activity (Hornbruch and
Wolpert, 1991). To test whether this expression pattern is
regulated by the same factors that induce or maintain
polarizing activity, we analyzed the regulation of cux2
expression by FGF2 (in ectopic limb buds), retinoic acid, Shh
and the posterior AER.
Regulation of cux2 expression in ectopic limb buds
The application of a bead soaked in FGF proteins in the
presumptive flank of a chick embryo can induce the formation
of ectopic limb buds (Cohn et al., 1995; Ohuchi et al., 1995).
Interestingly, the additional limb buds have reversed AP
polarity. This observation appears to be caused by the
activation of Shh in the anterior flank (Cohn et al., 1995) which
possesses a higher potential polarizing activity (Hornbruch and
Role of chick cux1 and cux2 during limb development 5139
Wolpert, 1991; Yonei et al., 1995). In order to determine if cux2
expression would reflect the reversed polarizing activity
observed in the ectopic limb buds, we implanted FGF2-soaked
beads in the flank of stage 13-15 chick embryos and examined
the cux2 expression pattern after 48 hours. In the additional
limb buds, cux2 expression remained in the anterior region and
was reduced in the posterior (Fig. 6A, arrow; compare with
flank expression in the control side). Indeed, this observation
correlates with the distribution of polarizing activity found in
the FGF-induced limbs.
Regulation of cux2 expression by retinoic acid
It has been shown that retinoid signaling acts early in limb
development and is required for the establishment of the ZPA
(Helms et al., 1996; Lu et al., 1997a). The local application of
all trans-retinoic acid to limb buds rapidly induces Hoxb8, and
later Shh and Bmp2. The application of retinoid receptor
antagonists results in the downregulation of those genes, and
the resulting limbs are truncated (Helms et al., 1996; Lu et al.,
1997a; Stratford et al., 1996; Stratford et al., 1997). The
expression pattern of cux2 in the pre-limb flank suggests that
cux2 may act downstream of RA in the establishment of
polarizing activity. Therefore, we looked at cux2 expression
after the introduction of RA in the anterior presumptive limb
field or in the anterior limb bud.
When beads releasing all trans-RA were placed in the
anterior region of the stage 14 wing field or stage 20 wing
buds, induction of cux2 expression was observed all over the
mesenchyme surrounding the bead as soon as 2-4 hours
postoperation (Fig. 6C and data not shown). After 21 hours it
remained only close to the ectoderm (data not shown). The
complementary experiment, blocking RA signaling by using
the highly specific RA receptor antagonist AGN 193109
(Johnson et al., 1995), downregulated cux2 expression in both
the flank and limb bud regions (Fig. 6D and data not shown).
These results suggest that RA directly regulates cux2
expression.
Regulation of cux2 expression by Shh
Shh has been identified as a gene expressed in the ZPA that can
mimic its activity and is responsible for the induction of several
posteriorly localized genes (Riddle et al., 1993). After limb
budding, cux2 is expressed in the posterior mesenchyme inside
and around the ZPA. To determine if cux2 expression may be
regulated by Shh, purified recombinant adenovirus containing
the chick Shh coding sequence was introduced in the
presumptive limb region of stage 16-17 chick embryos. Indeed,
cux2 transcripts were induced ectopically in the anterior
mesenchyme of the infected limb buds. cux2 expression
increased dramatically 24 hours after infection (data not
shown) and was still upregulated 48 hours after infection (Fig.
6F). This observation suggests that Shh may be directly or
indirectly regulating the expression of cux2 in the posterior
mesenchyme of the limb.
Regulation of cux2 expression by the AER
The AER is initially induced by the underlying mesenchyme
of the early limb buds. The subsequent outgrowth and
patterning are dependent on the reciprocal interactions between
the AER and the mesenchyme (Laufer et al., 1994; Maccabe
and Parker, 1979; Niswander et al., 1994; Todt and Fallon,
1987; Vogel and Tickle, 1993). Fgf8 is expressed throughout
the AER, and Fgf4 is expressed posteriorly. FGF8 was shown
to maintain the progress zone cells in a proliferative state
(Vogel et al., 1996), and FGF4 was seen to be responsible for
maintaining Shh expression in the ZPA (Laufer et al., 1994;
Niswander et al., 1994). Removal of the AER leads to truncated
limbs (Saunders, 1948) and cell death in the mesenchyme
(Rowe et al., 1982). Since the expression of posterior genes is
regulated by the AER, we analyzed the effect of posterior AER
removal on cux2 expression. In the operated limb buds, cux2
expression disappeared 4 hours after the operation (Fig. 6G)
and remained undetected 24 hours later (Fig. 6I). This
observation suggests that cux2 expression is maintained by
factors released by the posterior AER, either directly or
indirectly (i.e., by factors from the ZPA regulated by the AER).
Although the secreted protein FGF4 is released specifically by
the posterior AER, the absence of limb defects in Fgf4 mutant
mice (Moon et al., 2000; Sun et al., 2000) argues against it
being a direct regulator of cux2 expression.
As mentioned before, it has been established that the
posterior AER is responsible for maintaining Shh expression
in the ZPA (Laufer et al., 1994; Niswander et al., 1994). To
investigate if cux2 downregulation was due to the absence of
Shh in the operated buds, we placed a bead releasing Shh
protein in the posterior mesenchyme of a bud from which the
posterior AER had been removed. The result was similar and
cux2 expression was not maintained by Shh (Fig. 6J). It is,
therefore, likely that cux2 expression is regulated directly by
the posterior AER.
DISCUSSION
Chick cux1 is involved in limb ectoderm/AER
differentiation
In the developing chick limb, cux1 is expressed throughout the
bud ectoderm at stage 16-17, and becomes excluded from the
AER stripe as soon as it forms (Fig. 2A). At stage 20-22, cux1
expression is stronger in the ectoderm immediately next to the
ridge, and after stage 23 it is restricted to the two stripes
bordering the AER (Fig. 2B-E). This expression pattern
suggests that cux1 may play a role in the limb bud ectoderm
outside the ridge.
When we overexpressed cux1 in the developing chick limb
using a recombinant adenovirus as the vector, the injected buds
were reduced in size and showed notched AERs. At later
stages, the infected buds developed into truncated limbs.
Interestingly, this phenotype resembles the scalloped
Drosophila wings induced by either cut overexpression
(dominant-negative effect) or downregulation (Jack et al.,
1991; Ludlow et al., 1996). The reduced regions of the infected
chick limb buds are associated with scalloped portions of the
ridge. In these portions, the morphology of the AER is different
from normal. It appears thinner and resembles the ectoderm
surrounding it (Fig. 5E,F). In addition, the gaps in the ridge
show reduced or absent expression of AER markers such
as Fgf8, Bmp2 and Ser2, and partially express the dorsal
ectodermal marker Wnt7a (Fig. 5J-L). These results suggest
that chick cux1 may be involved in preventing the limb
ectoderm from assuming the pseudostratified phenotype
characteristic of AER cells.
5140 A. T. Tavares and others
Fig. 3. Expression of cux1 and cux2 in
other regions of developing chick embryos
by in situ hybridization. (A-E) Expression
of chick cux1. (A) cux1 transcripts are
found in the myotome (arrows; stage 23
embryo). (B) Transverse section showing
the expression of cux1 in the myotome
(arrows). (C) cux1 expression is also found
in the developing mesonephros (stage 25
embryo). (D) Expression of cux1 in the
distal portions of the fronto-nasal,
maxillary and mandibular processes of a
stage 28 embryo. (E) In the feather buds of
a 9-day old embryo, cux1 expression is
restricted to the posterior mesenchyme
(arrowhead). (F-L) Expression of chick
cux2. (F) cux2 expression is found in the
tubules of the mesonephros (stage 24
embryo). (G) cux2 is also expressed in the
telencephalon (stage 19 embryo; arrow).
(H) Expression of cux2 in the dorsal side
of the embryo, showing stripes that appear
to correspond to migrating neural crest
cells (arrows). (I) Transverse section of a
stage 20 chick embryo showing the
expression of cux2 in the dorsal neural
tube and in populations of migrating neural crest cells (arrows). (J) Transverse section of a stage 23 chick embryo showing the expression of
cux2 in the dorsal neural tube and, presumably, in melanoblasts (arrow). (K) Expression of cux2 in the feather buds of an 8-day old embryo. At
this stage, the expression appears to be restricted to the ectoderm of the buds (rings). (L) At day 9 of development, cux2 expression in the older
feather buds (center of the embryo’s back) has changed to the mesenchyme.
Fig. 4. Regulation of cux1 expression by β-catenin
induced ridges. (A) Fgf8 expression in an RCASβ-catenin infected limb bud. (B-E) Expression of
cux1 in infected limb buds. Both the frontal view
(B) and side view (C) of the ridge-like spikes
show that cux1 expression is concentrated around
them and is excluded from the ridge cells.
(D) Detail of cux1 expression in a cross section of
an ectopic ridge. (E) Section showing cux1
transcripts detected in the main AER (arrowhead)
and in an ectopic ridge (arrow).
It is thought that R-Fng promotes the activation of Notch
signaling at the dorsoventral boundary, probably in response to
Serrate-2 (Laufer et al., 1997; Rodriguez-Esteban et al., 1997).
Notch activation results in a fate change of the ectodermal cells
into AER (Rodriguez-Esteban, personal communication). The
ridge cells begin to express Fgf8 and differentiate into a
pseudostratified epithelium. Wnt3a, a secreted factor from the
wingless family, has also been shown to have a role in the
formation of the AER. Wnt3a is expressed very early by the
cells at the D/V boundary, and overexpression of Wnt3a or its
downstream targets such as β-catenin or Lef1 leads to the
induction of additional ridge-like structures in the limb bud
ectoderm (Kengaku et al., 1998). We have shown that cux1
transcripts are maintained or upregulated in the ectodermal
cells that surround the β-catenin induced ridges. Taken
together, these results suggest that cux1 expression is regulated
by signals emanating from the ridge. Subsequently, Cux1 may
function in the ectodermal cells flanking the AER to prevent
them from responding to differentiating cues, thus restricting
Notch and Wnt3a signaling to the cells in the ridge.
Chick cux2 expression maps to regions of the flank
and limb bud with potential polarizing activity and is
regulated by RA and Shh
cux2 is expressed in the pre-limb lateral plate mesoderm in a
pattern that coincides with the distribution of potential
polarizing activity in the developing chick embryo (Hornbruch
and Wolpert, 1991). cux2 transcripts are also found at later
stages in the posterior mesenchyme of the limb buds and flank,
which are also regions with polarizing activity (Saunders,
1977; Yonei et al., 1995). Unlike Hoxb8 (Lu et al., 1997a;
Stratford et al., 1997), cux2 is expressed in the presumptive
regions of both the fore- and hindlimb and persists during limb
budding stages. Additionally, in FGF2-induced ectopic limbs,
which exhibit inverted AP polarity, cux2 expression follows the
reversed distribution of polarizing activity. These observations
suggest that cux2 may have a role in generating or mediating
polarizing activity.
It has been suggested that retinoid signaling is responsible
for the polarizing potential found in the pre-limb flank (Helms
et al., 1996) and is required for the establishment of the ZPA
Role of chick cux1 and cux2 during limb development 5141
Fig. 5. Effect of cux1 overexpression
on chick limb development. Stage 1114 chick embryos were injected with
recombinant adenovirus carrying the
cux1 coding sequence (rAd-Cux1).
(A,C,E,G,K,M,O,Q,S) Control limbs.
(B,D,F,H,I,J,L,N,P,R,T,U) Limbs
infected with rAd-Cux1. Embryos
were collected at stage 21-23 (A-R)
or at day 10 of development (S-U).
(B,D) rAd-Cux1-infected wing buds
showing distal truncations and
scalloping of the AER (arrows).
(E,F) 7 µm transverse sections of
limb buds stained with H&E. (F) In
the rAd-Cux1 infected regions of the
limb bud, the AER appears thinner
than the wild-type (E). (G-I) Limb
buds infected with rAd-GFP
observed under a flourescence
microscope. (G) Wild-type limb bud
showing the presence of GFP in most
of the mesoderm and ectoderm of the
limb bud. (H) Limb bud infected with
both rAd-Cux1 and rAd-GFP
showing co-localization of GFP with
the scalloped region of the AER
(arrow). (I) Detail of the distal region
of H showing the disrupted region of
the AER and ectodermal cells
expressing GFP (arrowheads) (J-L) In
situ hybridization of the AER marker
Fgf8. (J) Fgf8 expression is severely
reduced in portions of the injected
limb (arrows). (L) In this infected
limb, the chipped region of the ridge
shows a reduction in Fgf8 expression (arrow). (M,N) In situ hybridization of Bmp2. (N) Bmp2 expression is absent in the posterior region where the
ridge is missing and the limb bud is truncated (arrow). (O,P) C-Ser2 expression. (P) C-Ser2 expression is absent from a portion of the distal AER
(arrow). (Q,R) In situ hybridization of Wnt7a. (R) The expression normally restricted to the dorsal ectoderm (Q) is expanded ventrally into the region
where the ridge is missing (arrow). (S-U) Embryos stained for cartilage with Alcian Green. (S) Wild-type wing showing the three digits, radius, ulna
and humerus. (T,U) Infected limb showing truncations (arrows) of the distal skeletal elements (digits and ulna; T), and the severe phenotype of a rAdCux1 infected limb (U) displaying only a portion of the humerus.
Fig. 6. Whole-mount in situ
hybridization of chick embryos
revealing the regulation of cux2
expression by FGF2 (A), RA
(C,D), Shh (F) and the AER
(G,I,J). (B,E,H) Control limb
buds with wild-type expression of
cux2. (A) In the extra limb
induced by FGF2 in the right side
flank, cux2 expression is reduced
in the posterior region (arrow),
which coincides with the inverted
pattern of polarizing activity
present in additional limb buds.
(C) When a bead releasing RA is
placed in the anterior
mesenchyme of a stage 20 limb bud, cux2 expression is upregulated all over the limb bud mesenchyme (arrowhead) four hours after the
operation. (D) The expression of cux2 is downregulated in the LPM by retinoid receptor antagonist (arrowhead). Cux-2 expression in the
contralateral side is not affected by the treatment. (F) Overexpression of Shh using a recombinant adenovirus (rAd-Shh) induces an
upregulation of cux2 all over the limb bud mesenchyme (arrowhead). (G,I) The surgical removal of the posterior portion of the AER at stage 20
leads to a downregulation of cux2 expression (arrows). The downregulation is observed as early as 4 hours after the operation (G) and is not
restored after 24 hours upon removal of the ridge (I). (J) A bead releasing SHH protein introduced in a limb bud from which the posterior AER
has been removed is unable to maintain cux2 expression 24 hours after the operation (arrow).
5142 A. T. Tavares and others
(Lu et al., 1997a; Stratford et al., 1997). Here we show that
cux2 expression is also rapidly induced by RA and is
downregulated upon blocking RA signaling. These results
suggest that cux2 may be a mediator of RA activity in both the
forelimb and hindlimb. In accordance with experiments in flies
(Johnston et al., 1998), Cux2 may act by regulating the
function or expression of Hoxb8 and other Hox genes. Our
observations suggest that, at later stages, cux2 expression may
be maintained by Shh in the ZPA, and by the factors released
by the posterior AER. Therefore, it is possible that Cux2 may
also be involved in mediating the polarizing activity of the limb
bud, where it acts downstream of ZPA and AER signals.
Chick Cux proteins may have a role in differentiation
during embryonic development
In addition to the limb and flank, cux1 and cux2 transcripts are
also found in a variety of different tissues in the developing
chick embryos, such as the myotome, the mesonephros and the
feather buds. In these tissues, cux transcripts colocalize with
one or more molecules involved in the Notch signaling
pathway and/or in the Wnt pathway. Myoblasts have been
shown to express ser2, L-fng, wnt11 and notch 1 (Cohen et al.,
1997; Hayashi et al., 1996; Marcelle et al., 1997; Nofziger et
al., 1999), the mesonephric tubules express Notch 1, Serrate 1,
Delta 1 and Wnts (Myat et al., 1996; reviewed in Vainio et al.,
1999), and the feather buds express notch 1 and notch 2, serrate
1 and serrate 2, delta 1, L-fng, wnts and β-catenin (Chen et al.,
1997; Noramly et al., 1999; Noramly and Morgan, 1998).
Moreover, Cux1 mouse mutants that have a deletion of the first
cut repeat have been shown to display hair defects (Tufarelli et
al., 1998), and both the Notch and Wnt pathways have also
been implicated in hair formation (Gat et al., 1998; Powell et
al., 1998). These correlations suggest that vertebrate Cux
molecules, like those in Drosophila, may act downstream of
Notch and/or Wnt signaling.
The expression of cux1 in the myotome (Fig. 3A,B), cux2
in neural crest cells (Fig. 3H-J), and of both genes in the
mesonephros (Fig. 3C,F) suggest that Cux molecules may have
a role in committed cell types before terminal differentiation.
This can be seen in other species also as the canine homologue
Clox is expressed by committed chondrocytes and myoblasts,
and a mouse cux1 isoform is found in the developing
mesonephros, and both genes are downregulated in the
terminally differentiated cell types (Andrés et al., 1992;
Vanden Heuvel et al., 1996a).
The morphogenetic processes that take place during
embryonic development are often regulated by proliferation
patterns. The understanding of these patterns requires the
study of genes that control cell cycle in the different
developing organs. Both the Notch and the Wnt signaling
pathways have been shown to regulate cell proliferation
(reviewed by Artavanis-Tsakonas et al., 1999 and Miller et
al., 1999). In the developing limb bud, the cells that will form
the AER differentiate into a specialized epithelium during
ridge induction. Cut proteins can function as cell-cycledependent factors that promote histone H4 expression in
response to certain growth signals (Aziz et al., 1998) and
repress p21 transcription (Coqueret et al., 1998) during early
S phase, and downregulate bone tissue-specific genes in
conjunction with cell-cycle proteins p107 and cyclin A (van
Gurp et al., 1999). These results suggest that Cut proteins
may regulate tissue-specific gene expression in concert with
cell cycle-regulatory signals. It is tempting to speculate that
Cux1 is downregulated in the AER to permit cell
differentiation, and persists in the ectodermal cells outside of
the ridge to promote cell cycle.
The suggested role of cux2 in polarizing activity may be
mediated by its ability to regulate cell cycle. It has been shown
that when the cell cycle length in the anterior limb bud cells
is artificially increased, posterior genes are induced and digit
duplications are observed (Ohsugi et al., 1997). These results
suggest that cell-cycle length regulates pattern formation in
the developing limb buds. Cux2 may be involved in
regulating the adequate cell-cycle length of the posterior limb
mesenchyme, thus allowing for the expression of AP
patterning genes.
In conclusion, our results, together with evidence of the
participation of Cux/CDP proteins in cell cycle regulation
(Aziz et al., 1998; Coqueret et al., 1998; van Gurp et al., 1999),
suggest a role for Cux proteins as translators of developmental
cues into cell proliferation and inhibition of differentiation
responses during limb development. The corroboration of this
hypothesis awaits further investigation using cell cycle markers
and blockers at limb outgrowth.
We are most grateful to T. Kanda and G. M. Wahl for the histone
2B/GFP-fusion construct, and C. Tabin for the C-Ser2 probe. We also
thank D. Büscher for preparing the RCAS-—catenin virus, and J. A.
Belo, S. Campino, S. Marques, J. K. Ng, C. Rodriguez Esteban, K.
Sharma, and K. Tamura for comments, technical support and reagents.
A. T. T. was supported by fellowships from the Programa Gulbenkian
de Doutoramento em Biologia e Medicina (PGDBM), Program
PRAXIS XXI, Fundação Luso-Americana para o Desenvolvimento
(FLAD) and Fundação Calouste Gulbenkian and by the National
Science Foundation. This work was supported by grants from the G.
Harold and Leila Y. Mathers Foundation and the National Institutes
of Health to J. C. I. B.
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