www.elsevier.com/locate/ydbio
Developmental Biology 272 (2004) 39–52
A new role for BMP5 during limb development acting through the
synergic activation of Smad and MAPK pathways
V. Zuzarte-Luıs,a J.A. Montero,a J. Rodriguez-Leon,b R. Merino,c
J.C. Rodrıguez-Rey,c and J.M. Hurlea,*
aDepartamento de Anatomıa y Biologıa Celular, Facultad de Medicina, Universidad de Cantabria, Santander 39011, Spainb Instituto Gulbenkian de Ciencia, Oeiras, Portugal
cDepartamento de Biologıa Molecular, Facultad de Medicina, Universidad de Cantabria, Santander 39011, Spain
Received for publication 27 February 2004, revised 20 April 2004, accepted 20 April 2004
Available online 28 May 2004
Abstract
In an attempt to identify new genes implicated in the control of programmed cell death during limb development, we have generated a
cDNA library from the regressing interdigital tissue of chicken embryos. We have analyzed 804 sequences from this library and identified 23
genes involved in apoptosis in different models. One of the genes that came up in the screening was the Bone Morphogenetic Protein family
member, Bmp5, that has not been previously involved in the control of apoptosis during limb development. In agreement with a possible role
in the control of cell death, Bmp5 exhibited a regulated pattern of expression in the interdigital tissue. Transcripts of Bmp5 and BMP5 protein
were abundant within the cytoplasm of the fragmenting apoptotic interdigital cells in a way suggesting that delivery of BMPs into the tissue is
potentiated during apoptosis. Gain-of-function experiments demonstrated that BMP5 has the same effect as other interdigital BMPs inducing
apoptosis in the undifferentiated mesoderm and growth in the prechondrogenic mesenchyme. We have characterized both Smad proteins and
MAPK p38 as intracellular effectors for the action of BMPs in the developing limb autopod. Activation of Smad signaling involves the
receptor-regulated genes Smad1 and -8, and the inhibitory Smad6, and results in both the upregulation of gene transcription and protein
phosphorylation with subsequent nuclear translocation. MAPK p38 is also quickly phosphorylated after BMP stimulation in the limb
mesoderm. Treatment with the inhibitor of p38, SB203580, revealed that there are interdigital genes induced by BMPs in a p38-dependent
manner (DKK, Snail and FGFr3), and genes induced in a p38-independent manner (BAMBI, Msx2 and Smads). Together, our results suggest
that Smad and MAPK pathways act synergistically in the BMP pathway controlling limb development.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Programmed cell death; Chondrogenesis; Smad; MAPK; p38; c-jun; BAMBI
Introduction rays in all amniota embryos and have the function of
The developing vertebrate limb provides a paradigm
model for analyzing programmed cell death. Several areas
of cell death, including the anterior (ANZ) the posterior
(PNZ), the opaque patch (OP) and the interdigital areas
(INZs) are present in the developing avian limb (see Chen
and Zhao, 1998; Zuzarte-Luis and Hurle, 2002). INZs are
observed in the mesoderm between the developing digital
0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2004.04.015
* Corresponding author. Departamento de Anatomıa y Biologıa
Celular, Facultad de Medicina, Universidad de Cantabria, C/Cardenal
Herrera Oria s/n, Santander 39011, Spain. Fax: +34-942-201903.
E-mail address: [email protected] (J.M. Hurle).
sculpting the shape of the digits.
A variety of morphological and molecular approaches
have established that cell death in INZs is executed by
apoptosis through the activation of caspases (Garcia-Marti-
nez et al., 1993; Jacobson et al., 1996; Nakanishi et al.,
2001). Several genes of the apoptotic machinery, including
the proapototic factors: Apaf-1 (Cecconi et al., 1998); Bax
(Dupe et al., 1999); Bak (Lindsten et al., 2000); DIO-1
(Death Inducer-Obliterator-1; Garcia-Domingo et al.,
1999); Gas1 and Gas2 (Growth Arrest Specific; Lee et al.,
1999, 2001), have been implicated in this process. In
addition, some antiapoptotic factors, including Bcl-2, Bcl-x
and A1, are expressed in the digital rays but not in the
interdigital spaces of the mouse autopod (Carrio et al., 1996;
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5240
Novack and Korsmeyer, 1994). Moreover, the interdigital
regions before the onset of apoptosis express Bag-1 which
encodes an antiapoptotic protein that binds to Bcl-2 (Crocoll
et al., 2002). Another antiapoptotic factor, Dad-1 (Defender
Against apoptotic cell Death), has been implicated in the
control of limb programmed cell death since heterozygous
mutant mice for this gene display soft-tissue syndactyly
(Nishii et al., 1999).
It has been shown that BMP2, BMP4 and BMP7 are
triggering signals for limb programmed cell death (Ganan et
al., 1996; Guha et al., 2002; Kawakami et al., 1996; Yokouchi
et al., 1996; Zou and Niswander, 1996; Zou et al., 1997).
These secreted factors are expressed in the preapoptotic limb
mesoderm, and mesodermal apoptosis is induced and
inhibited in gain- and loss-of-function experiments, respec-
tively. It is well known that other signaling pathways,
including Wnt (Grotewold and Ruther, 2002), FGF (Montero
et al., 2001) and retinoic acid (Dupe et al., 1999; Lussier et al.,
1993; Rodriguez-Leon et al., 1999), interact with BMP
signaling in the control of apoptosis. However, the mecha-
nism by which BMPs trigger apoptosis and the intracellular
signaling pathway operating between BMPs and the activa-
tion of caspases remains largely unknown. This is particularly
important since it has been shown that caspase-independent
necrosis substitutes apoptosis in Apaf1-deficient embryos
(Chautan et al., 1999), suggesting that BMPs are able to
trigger both apoptosis and necrosis. In addition, it is not clear
whether the cell death in different areas of the limb is
triggered by the same signals. In fact, experimental evidence
suggests that this may be not the case. For example, Bmp2 is
not expressed in ANZ, while TGFh2 appears to cooperate
with BMPs in the control of INZ (Dunker et al., 2002).
Furthermore, SHH, which promotes the expression of Bmp2,
is a proapoptotic factor for PNZ while exerts an antiapoptotic
effect in ANZ (Sanz-Ezquerro and Tickle, 2000).
In an attempt to identify new genes implicated in the
control of INZ apoptosis, we have generated a cDNA
library from the regressing interdigital tissue of chicken
embryos. By exploring this library, we have detected the
expression of genes implicated in apoptotic processes in
other models. These genes included: components of the
proteosome; genes implicated in DNA repair; genes pos-
itively implicated in apoptosis; antiapoptotic genes; and
genes of potential importance in phagocytosis. One of the
genes identified in the library, Bmp5, exhibited a temporal
and spatial pattern of expression closely coincident with
INZ and was selected for functional studies. Our results
provide evidence for a role of this BMP member in the
development of limb autopodium through the activation of
Smad proteins and MAPK p38.
Material and methods
Fertilized chicken embryos (Rhode Island), ranging from
day 3 to day 9 of incubation, were employed in this study.
Interdigital mesoderm cDNA library
Six hundred interdigital spaces were carefully micro-
dissected from chicken leg buds at day 7.5 of incubation
and total RNA was extracted using TRIzol Reagent (Gib-
coBRL). mRNA was next isolated with the Dynabeads
Oligo(dT)25 kit (Dynal) and employed to construct a cDNA
library using the Creator SMART cDNA Library Construc-
tion kit (CLONTECH). The obtained cDNAwas cloned into
the pGEM-T easy vector and transformed into competent
cells. The clones were analyzed by enzymatic digestion and
fragments bigger than 600 bp were selected for sequencing.
Sequences were analyzed and blasted against both NCBI
and a chicken database (www.chickest.udel.edu) in search
for homologies.
Probes and in situ hybridization
Digoxygenin-labeled sense and antisense RNA probes
were generated for all the cloned sequences of potential
interest. Sense RNA probes were employed as controls.
Specific RNA probes for Msx2, Dkk, Snail and Fgfr3 were
also generated in addition to those obtained from the library.
Fragments of chicken Smad1 (744 bp), Smad5 (714
bp), Smad6 (702 bp) and Smad8 (972 bp) genes were
obtained by RT-PCR. First-strand cDNA was synthesized
with a random hexamers and M-MulV reverse transcrip-
tase (Fermentas) and 1 Ag of total RNA from a day-7
autopods. The following primers were used to subsequent
PCR amplification: degenerated primers for cSmad1, 5V-GARGARGARAARTGGGC-3V (sense) and 5V-RCAC-CARTGYTTNGGYTC-3V (antisense); degenerated primers
for cSmad5, 5V-GAYGARGARGARAARTGG-3V (sense)
and 5V-RCACCARTGYTTNGGYTC-3V (antisense);
cSmad6, 5V-AGTACAAGCCACTGGATCTGTCCGA-3V(sense) and 5V-CCAGCCCTTGGCGAAGCTGATGC-3V(antisense); and for cSmad8, 5V-AGGTGCCATGGAA-GAACTGG-3V (sense) and 5V-AGTTACAATTGCGGC-TCTGC-3V (antisense).
The PCR conditions were 94jC, 4 min and then 35 cycles
of 94jC, 20 s; 55jC, 30 s; 72jC, 60 s; and final extension by72jC, 10 min. PCR products were run on a 1% agarose gel,
isolated, and subcloned into pGEMT-easy (Promega). Iso-
lated clones were sequenced using dideoxy sequencing.
Digoxygenin-labeled antisense RNA probes were gener-
ated for in situ hybridization analysis.
In situ hybridization was performed in whole mount
specimens of limb buds. Samples were treated with protein-
ase K (10 Ag/ml) for 25–30min at 20jC. Hybridization of thedigoxygenin-labeled probes was performed at 68jC. Reac-tions were developed with BM purple AP substrate (Roche).
Experimental manipulation of the limb
The function of BMP5 in the control of cell death and in
chondrogenesis was studied by analyzing the effects of local
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–52 41
administration into the limb mesoderm of recombinant
BMP5 (R&D Systems) at concentrations ranging from 10
Ag/ml using as carriers heparin acrylic beads (Sigma). The
potential function of Noggin to block cell death mediated by
BMP5 was explored by the implantation of beads incubated
in BMP5 and beads incubated in Noggin at 1 mg/ml
(generously donated by Regeneron Pharmaceuticals Inc.,
Tarrytown). In both experiments, the treated limbs were next
processed to study changes in the skeleton and in the pattern
of apoptosis. To explore if the MAP kinase p38 was the
cellular effector for BMPs, the BMP beads were co-
implanted with beads (affigel blue, heparin or AG1-X2)
incubated in SB203580 (Calbiochem).
In all the experiments, PBS-soaked beads were also
implanted as a control. These treatments did not induce
changes either in the normal development of the limb or in
the expression of the analyzed genes.
Limb skeletal morphology and pattern of cell death
The morphology of the limbs treated with BMP5 beads
was studied in whole mount specimens after cartilage
staining with Alcian green as described previously (Ganan
et al., 1996). The pattern of cell death was analyzed with
neutral red vital staining (see Macias et al., 1997) and by
TUNEL assay performed in paraffin sections following the
instructions of the manufacturer (Roche). To evaluate the
rate between living and dead cells, TUNEL was combined
with DAPI staining or immunolabeling with an anti-panhi-
stone antibody (see confocal microscopy).
Confocal microscopy
Immunolabeling, in situ hybridization and Tdt-mediated
dUTP nick end labeling (TUNEL) were performed in
dissociated interdigital mesodermal cells and examined with
a BioRad MRC-1024 confocal microscope using argon and
HeNe lasers. For this purpose, small squares of the third
interdigit of limbs (of embryos ranging from 5.5 to 8 days of
incubation) fixed in 4% paraformaldehyde were isolated and
carefully squashed with a coverslide to dissociate the
mesodermal cells. To stick the cells to the glass surface,
the slides were then deep frozen over dry ice. The samples
were then processed for the different methodologies.
For immunolabeling experiments, cells were permeabi-
lized with 0.5% Triton X-100 (10 min) before incubation
with the primary antibody, and after several PBS washes,
the cells were incubated with the secondary antibody.
The following primary antibodies were employed: poly-
clonal antibody against phosphorylated Smad1 (a gift from
Dr. C. Heldin); polyclonal antibodies against Smad 1/5/8,
phosphorylated p38 and BMP5/BMP7 from Santa Cruz
Biotechnology; polyclonal antibody against lamina B
(Drouin et al., 1991); monoclonal antibody against panhi-
stone (Anti-Histon, pan, Boehringer) and a polyclonal
antibody against ubiquitin (Dako, Denmark).
TUNEL assay was employed as a specific marker to
distinguish between living and apoptotic cells. We followed
the protocol for isolated cells recommended by the manu-
facturer (Roche).
For in situ hybridization, cells were permeabilized as for
immunolabeling and incubated with the digoxygenin-la-
beled probe for 3 h at 42jC. Detection was made with
rhodamine-labeled anti-digoxygening antibody (Roche).
For double labeling purposes, we performed first the in
situ hybridization followed by the immunolabeling or the
immunolabeling followed by TUNEL assay.
Results
Identification of genes expressed in the regressing
interdigital tissue
By screening a cDNA library of the third interdigital
space of 7.5-day chicken leg buds we expected to
obtain new genes implicated either in the different
stages of the apoptotic process or in all mechanisms
related with tissue regression, including phagocytosis and
extracellular matrix degradation. Analysis of TUNEL-
and DAPI-stained sections (Figs. 1A and B) showed
that at this stage the interdigital mesoderm was com-
posed of living mesenchymal cells negative for TUNEL
(60%); apoptotic, TUNEL-positive cells (30%) and phag-
ocytes (10%). It must be remarked that living meso-
dermal cells die within the next 24–36 h since by day
9 of incubation the interdigital tissue has been totally
removed. Therefore, genes identified in our library could be
potentially implicated in all mechanisms related with tissue
regression.
Eight hundred four clones containing cDNA fragments
of sizes between 600 and 1000 bp were sequenced. From
all the analyzed sequences, 447 did not show any similar-
ity with published sequences, 117 sequences corresponded
to ribosomal and mitochondrial sequences. The remaining
240 sequences were non-ribosomal cDNAs corresponding
to 131 different known genes (see Table 1). Some of these
genes were factors implicated in the regression of the
interdigits, including Wnt5a (Chimal-Monroy et al.,
2002), Msx2 (Ferrari et al., 1998) Bmp7 (Macias et al.,
1997) and BAMBI (Grotewold et al., 2001). We also
detected factors implicated in apoptotic processes in a
variety of systems including: components of the proteo-
some (polyubiquitin; proteosome 28-kDa subunit homo-
logue; ubiquitin C ubiquitin I; ubiquitin II; ubiquitin
specific proteases 8 and 24); genes implicated in DNA
repair (Prkdc gene for DNA-dependent protein kinase;
Mori et al., 2001); genes positively implicated in apoptosis
(FLASH, a caspase 8 binding protein, Imai et al., 1999;
damage-specific DNA binding protein, Sun et al., 2002;
voltage-dependent anion channel, VDAC2, Sugiyama et
al., 2002; spermidine/spermine N1-acetyltransferase, Ha et
Fig. 1. (A, B) Section of the third interdigital space of the leg bud at 7.5 days of incubation double stained with DAPI (A) and TUNEL (B) to show that the
interdigital mesoderm at this stage contains both living cells negative for TUNEL and apoptotic TUNEL-positive cells.
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5242
al., 1997; cytochrome c oxidase subunits I, II and III,
Chandra et al., 2002; cathepsin-L, Boland and Campbell,
2004; SAP18, Schwerk et al., 2003); antiapoptotic genes
(dynein protein inhibitor of neuronal nitric oxide synthase;
Chang et al., 2000; elongation factor 1-alpha 1, Talapatra
et al., 2002; protein-disulfide isomerase, Ko et al., 2002)
and genes of potential importance in phagocytosis (apoli-
ipoprotein A1; Hamon et al., 2000). In addition, Bmp5, a
member of the bone morphogenetic signaling molecules
and Dek that codes for an oncogenic-like protein, which is
activated by BMP signaling (Hollnagel et al., 1999), were
also isolated from the library. In situ hybridizations using
riboprobes generated for each one of these genes that are
potentially implicated in apoptosis revealed that except for
dek, VDAC2 and bmp5, which had interdigital expression
domains, all the remaining genes exhibited a ubiquitous
pattern of expression. In this work, we chose BMP5 for
additional functional studies.
Expression of Bmp5 in the developing limb
Bmp5 exhibited a regulated pattern of expression
throughout the different stages of the developing limb
bud. Transcripts were detected between days 3 and 4.5 of
incubation in the distal mesoderm of the limb bud (Figs. 2A
and B). From day 5, Bmp5 was also expressed in the dorsal
and ventral muscular masses of the developing limb (Figs.
2C and D). By days 5.5 and 6, transcripts were abundant in
the proximal part of the interdigital regions (Fig. 2E). From
day 7, the interdigital domains were displaced to the most
distal part of the interdigits (Figs. 2F and G) reaching the tip
of the digit by day 8.5 of incubation (Figs. 2G and H).
Interestingly, the evolution of the interdigital pattern of
expression resembles the pattern of interdigital cell death
in the chick limb autopod (not shown). An additional
domain of Bmp5 expression was found in the developing
flexor tendons of the autopod from day 6.5 of incubation
(Figs. 2F and G). At later stages of development, transcripts
of Bmp5 were detected in the perichondrium of the pha-
lanxes (2H).
The presence of Bmp5 transcripts and protein in the
regressing interdigital mesenchymal cells was further ana-
lyzed by confocal microscopy. Using this approach, we
found that the interdigital mesoderm expresses Bmp5 tran-
scripts up to the initiation of apoptosis (Fig. 3A). Using an
antibody that cross-reacts with BMP5 and BMP7, we
found that these proteins are abundant in the degenerating
mesenchyme (Fig. 3B). Furthermore, intense labeling was
conserved within the cytoplasm of the dying cells positive
for TUNEL, suggesting that BMP release into the extra-
cellular medium may be enhanced during apoptotic cell
fragmentation (Figs. 3C–E). In an attempt to check
whether BMP5/7 in the apoptotic cells undergoes proteo-
somal degradation, we performed double labeling with
anti-BMP5/7 and anti-ubiquitin antibodies. In agreement
with our initial interpretation, we found that there is no co-
localization between BMP5/7 and ubiquitin both in healthy
(Fig. 3F) and in apoptotic cells (Fig. 3G), suggesting that
BMPs might remain biologically active in degenerating
cells.
BMP5 promotes apoptosis of undifferentiated mesoderm
and cartilage growth
To check the involvement of BMP5 in apoptosis, we
analyzed the effects of local application of BMP5 protein in
the undifferentiated mesenchyme of the developing limb.
For this purpose, heparin beads incubated in human recom-
binant BMP5 protein were implanted at different locations
within the limb bud. In these experiments, an intense area of
apoptosis was induced 10 h after the treatment, when the
beads were implanted in the undifferentiated mesoderm of
the anterior or posterior margin of the limb between days 3.5
and 5 of incubation (Figs. 4A and B), or in the interdigital
Table 1
List of the genes identified in the screening of a cDNA library of the third
interdigital space of 7.5-day chicken leg buds with correspondent
homologous specie
Actin related protein 2/3 complex subunit
3 (ARP2/3)
Homo sapiens
Acyl-CoA dehydrogenase (Long-chain) Sus scrofa
Adhesion protein RA175C Gallus gallus
Alpha-D globin Gallus gallus
Apolipoprotein AI Gallus gallus
ATP synthase, H+ transporting,
mitochondrial F0 complex, subunit c
(subunit 9)
Mus musculus
BAMBI protein Xenopus laevis
Basic cytosolic protein Bos taurus
Beta-actin Anser anser
Bithoraxoid-like protein/HSPC162 protein Homo sapiens
Bone Morphogenetic Protein-5 Gallus gallus
Bone Morphogenetic Protein-7 Gallus gallus
Bromodomain-containing protein BP75 Mus musculus
Cathepsin L Engraulis japonicus
Cct5-prov protein Xenopus laevis
CDC10 cell division cycle 10 homolog Homo sapiens
Cellular nucleic acid binding protein
(CNBP)
Gallus gallus
Centrin (CDC31 homolog) Homo sapiens
CGI-152 protein Homo sapiens
Chaperonin subunit 5 (epsilon) Mus musculus
Chromobox protein (CHCB2) Gallus gallus
Chromosome 13 open reading frame 12
(C13orf12)
Homo sapiens
Clone cerebral inh no syntase Homo sapiens
Clone WAG-69H2 Gallus gallus
Clone XXbac-97F19 Gallus gallus
Cofactor required for Sp1 transcriptional
activation
Gallus gallus
Collagen Type-1 Gallus gallus
CpG island associated sequence Homo sapiens
Cytochrome b Gallus gallus
Cytochrome c oxidase subunit I Gallus gallus
Cytochrome c oxidase subunit II Gallus gallus
Cytochrome c oxidase subunit III Gallus gallus
Damage-specific DNA binding protein 1
(DDB1)
Rattus norvegicus
DC6 protein Rattus norvegicus
DEAD-box RNA helicase Gallus gallus
Degrnerin Caenorhabditis elegans
DEK oncogene Homo sapiens
Diazepam binding protein DBIgene Gallus gallus
Dolichyl-phosphate mannosyltransferase
polypeptide 1 (DPM1)
Homo sapiens
Dynein/protein inhibitor of neuronal
nitric oxide synthase (PIN) gene
Homo sapiens
EDRK-rich factor Homo sapiens
Elongation factor 1-alpha 1 Gallus gallus
Enhancer of rudimentary homologue
(ERH)
Xenopus laevis
Equarin-S Gallus gallus
Erythropoietin 4 Mus musculus
EURL protein homolog (early undifferentiated
retina and lens)
Gallus gallus
FLASH Mus musculus
Glucosamindase Homo sapiens
Golgi complex associated protein 1 Homo sapiens
Growth-related translationally controlled tumor
protein (pCHK23)
Gallus gallus
GTP binding protein (ARL3) Homo sapiens
Heat shock protein 1 (chaperonin) Mus musculus
Heat shock protein 10 Gallus gallus
hematological and neurological expressed
sequence 1
Mus musculus
Hemoglobin alpha-A chain Gallus gallus
Hemoglobin alpha-D chain Gallus gallus
Hemoglobin beta chain Gallus gallus
Heterogeneous nuclear ribonucleoprotein
H3 isoform
Mus musculus
High mobility group 1 protein Gallus gallus
Homeobox protein MSX-2 (CHOX-8) Gallus gallus
HSPC036 protein Homo sapiens
Hypothetical prot 121 Homo sapiens
Hypothetical protein HSPC014 Homo sapiens
Hypothetical protein HSPC148 Homo sapiens
Inosine monophosphate dehydrogenase
1 isoform b
Homo sapiens
Intersectin short isoform Homo sapiens
Lactate dehydrogenase B Gallus gallus
LIM homeobox protein cofactor (CLIM-1) Homo sapiens
Maternal G10 transcript Homo sapiens
Mesoderm development candidate 1 Mus musculus
MHC class II associated invariant chain Gallus gallus
Mitogen-activated protein kinase 6 Homo sapiens
NADH dehydrogenase (ubiquinone) 1 beta
subcomplex, 9,
Homo sapiens
NADH-ubiquinone oxidoreductase 30 KDA Homo sapiens
Non-muscle caldesmon Gallus gallus
Nuclease sensitive element binding protein 1
(Y box binding protein-1)
Gallus gallus
Nucleolar prot #38 Gallus gallus
Okadin; basic helix-loop-helix transcription
factor 6
Mus musculus
Ornithine decarboxylase antizyme inhibitor Homo sapiens
Pericentriolar material-1 (PCM-1 gene) Gallus gallus
Perioedoxin-1 proliferation associated Homo sapiens
Phosphotidylinositol transfer protein, beta Homo sapiens
PNAS-110 Homo sapiens
Poly(A) binding protein, cytoplasmic 1
(PABPC1)
Homo sapiens
Polyglutamine-containing C14ORF4 gene Homo sapiens
Polyubiquitin gene Ub II Gallus gallus
Prematurely terminated mRNA decay
factor-like
Homo sapiens
Prkdc gene for DNA-dependent protein
kinase catalytic Subunit
Gallus gallus
PRO0529 protein Homo sapiens
Pro-alpha-1 (I) collagen Gallus gallus
Probable protein Medicago sativa
Propionyl CoenzymeA carboxylase, beta
polypeptide (Pccb)
Rattus norvegicus
Proteosome 28 kDa subunit homolog Gallus gallus
Protein disulfide isomerase (PDI) Gallus gallus
Putative transmembrane protein (NMA) Homo sapiens
RAC GTPase-activating protein Homo sapiens
Radixin pseudogene 2 (RDXP2) Homo sapiens
RAN protein Homo sapiens
Reelin Gallus gallus
Restricted expression proliferation
associated protein-100
Gallus gallus
Reticulocalcin Gallus gallus
RNA polymerase II Rattus norvegicus
RUN and FYVE domain-containing 1 Homo sapiens
Table 1 (continued)
(continued on next page)
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–52 43
SAP18, sin3 associated polypeptide p18 Gallus gallus
SET translocation (myeloid leukemia-associated) Homo sapiens
Small EDRK-rich factor 1A
(telomeric)
Homo sapiens
Small nuclear ribonucleoprotein
polypeptide A
Mus musculus
Smooth muscle caldesmon Gallus gallus
Spermidine/spermine N1-acetyltransferase
(SSAT)
Gallus gallus
Stathmin Gallus gallus
Tax responsive element binding protein Homo sapiens
TB2 gene Homo sapiens
T-cell leukemia virus enhancer factor Homo sapiens
THOC4 protein Homo sapiens
TIP120 protein Homo sapiens
Toll-like receptor 9 Felis catus
Translocating chain-associated membrane
protein (TRAM)
Gallus gallus
Tyrosine 3-monooxygenase/tryptophan
5-monooxygenase activation protein
Homo sapiens
Ubiquitin C Rattus norvegicus
Ubiquitin I (UbI) gene Gallus gallus
Ubiquitin specific protease 24 (USP24) Homo sapiens
Ubiquitin specific protease 8 (USP8) Homo sapiens
Ubiquitin-conjugating enzyme E2 variant 2 Homo sapiens
Ubiquitin-fusion protein Gallus gallus
Variant of TSC-22 Gallus gallus
Virus similar to variola C15Land vaccinia F11 Molluscum contagiosum
Voltage-dependent anion channel (VDAC2) Gallus gallus
Voltage-gated K channel beta subunit 4.1 Homo sapiens
Wnt-5a Gallus gallus
WSB-1 form Homo sapiens
X Fragile Chromosome 17/11 Homo sapiens
Table 1 (continued)
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5244
spaces (Figs. 4D and E) before the onset of interdigital cell
death (days 5.5 and 6 of incubation). The intensity of
apoptosis in the early limb after implantation of BMP5
beads was followed by a deficient development of the
skeleton (Fig. 4G).
Local application of the BMP antagonist Noggin into the
interdigital mesoderm has been found to cause an intense
inhibition of INZ leading to severe soft tissue syndactyly
(Merino et al., 1998). To confirm the hypothesis that BMP5
could be physiologically involved in the control of apopto-
sis, we checked the ability of Noggin to block apoptosis
induced by exogenous BMP5. For this purpose, a BMP5
bead and a Noggin bead were implanted into the mesoderm
close to each other. As expected, this double treatment
caused a strong inhibition of the area of apoptosis induced
by the BMP5 bead (Figs. 4C and F).
Previous studies have shown that those BMPs implicated
in the control of limb programmed cell death have an
opposite effect promoting growth and differentiation when
they are applied in the prechondrogenic aggregates. To test
whether this effect was also shared by BMP5, beads bearing
recombinant protein were implanted at the tip of differen-
tiating digits (days 5.5 and 6 of incubation). This treatment
caused a dramatic enlargement of the digital cartilages (Figs.
4H and I).
BMP5 upregulates genes implicated in interdigital
apoptosis
To discard the possibility that apoptosis induced by
BMP5 was mediated through the activation of Bmp2,
Bmp4 or Bmp7, we analyzed whether interdigital implanta-
tion of BMP5 beads regulated the expression of those genes.
None of them were regulated during the first 12 h after the
treatment (not shown). In contrast, the expression of the
pseudoreceptor BAMBI (BMP and activin membrane-bound
inhibitor) that is supposed to constitute a synexpression
gene group with Bmp4 (Grotewold et al., 2001) and nega-
tively modulates BMP signaling (Onichtchouk et al., 1999)
was intensely upregulated by BMP5 beads (Fig. 5A). To
further characterize the function of BMP5 in INZ, we next
explored the effect of BMP5 beads on the expression of
Msx2, Dkk, Snail and Fgfr3 (Montero et al., 2001; Grote-
wold and Ruther, 2002) which were all previously impli-
cated in interdigital tissue regression acting under the
control of BMP signaling. As shown in Figs. 5B–E, all
these genes were intensely upregulated during the first 10
h after interdigital implantation of BMP5 beads.
Interdigital function of BMP5 implicates both Smad and
MAPK signaling
Recently, it has been found that in addition to the well-
characterized Smad signaling pathway, BMPs may also
exert their functions through the activation of the MAPK
pathway (reviewed by Derynck and Zhang, 2003). Here,
we have found evidence for a role of BMP5 activating
both pathways in the interdigital mesoderm (similar experi-
ments were also performed with BMP7 with identical
results).
The cytoplasm of interdigital mesodermal cells was
intensely labeled with a polyclonal antibody against Smad1,
5 and 8 both before and during the apoptotic process (Fig.
6A). In contrast, nuclear labeling for phospho-Smad1 was
almost absent before tissue regression (day 6 of incubation
or earlier; Fig. 6C). However, phospho-Smad1 expression
was significantly increased in the following developmental
stages, concomitantly with the establishment of the apopto-
tic process (Fig. 6B), and upon interdigital application of
BMP5 beads (Fig. 6D). This increase concerned both the
intensity of nuclear labeling in each cell and the number of
labeled cells.
To study the implication of Smads at RNA level, we
cloned the chick BMP-responsive Smad genes (1, 5 and 8)
and the inhibitory Smads. Smad1 was expressed at low
levels in the interdigital mesoderm (Fig. 7A) and this
expression was not affected by BMP treatments. Expression
of Smad5 was undetectable by whole-mount in situ hybrid-
ization both in normal and BMP-treated interdigits (not
shown). In contrast, Smad8 was highly expressed through-
out the whole process of interdigital tissue regression (Fig.
7E). In addition, its expression was intensely downregulated
Fig. 2. (A–H) Expression of Bmp5 in the developing leg bud at days 3.5 (A), 4 (B), 5 (C, dorsal view; D, lateral view), 6 (E), 7.5 (F), 8 (G) and 9 (H). At early
stages, transcripts are observed in the undifferentiated distal mesenchyme (A, B). Next in development in addition of being expressed in the distal mesoderm,
transcripts are also observed in the dorsal and ventral muscle masses (arrows in C and D). As digits become identifiable, well-defined domains are
progressively observed in the interdigital regions (E–G) which become displaced towards the tip of the digits in coincidence with the elimination of the most
distal mesenchyme of the limb (H). Note the presence of additional domains of expression in the developing tendons (F, G) and in the perichondrium of the
differentiating phalanxes (arrows, H).
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–52 45
by Noggin beads (Fig. 7F) and upregulated by application of
BMP beads (Fig. 7G). The upregulation caused by BMP
beads was also observed in early limb buds (not shown)
preceding the induction of mesodermal apoptosis. Finally,
the inhibitor Smad6 was expressed in the interdigital meso-
derm (Fig. 7B), as previously described (Yamada et al.,
1999), and as Smad8, its expression was upregulated by
BMP beads (Fig. 7C) and downregulated by Noggin (not
shown).
Erk, c-jun and p38 are the major components of the
MAPK signaling pathway. The involvement of Erk kinase
in limb development has been previously studied by Kawa-
kami et al. (2003) that showed that it is implicated in
apoptosis and that its effect is dependent on FGF signaling.
Here, we analyzed whether the other MAPK pathways,
namely, c-jun and p38, are involved in the apoptotic BMP
pathway responsible for interdigital tissue regression.
Immunolabeling for phospho-c-jun was positive mainly
within the cytoplasm of the interdigital mesoderm but its
expression did not change either during physiological de-
generation or after BMP treatments (not shown). In contrast,
immunolabeling for the MAPK p38 in interdigital meso-
derm revealed that phophorylated p38 (phospho-p38) was
present within the nucleus of the interdigital mesodermal
cells. Before the onset of apoptosis, nuclear phospho-p38
was not abundant in the interdigital mesodermal cells (Fig.
8A); however, the intensity of the immunolabeling increased
in the subsequent stages, and by day 7.5 of incubation,
immunolabeling was very intense (Fig. 8B). To test whether
this expression was regulated by BMPs, we analyzed
changes in phospho-p38 upon interdigital application of
BMP at day 5.5 of incubation. As shown in Figs. 8C and
D, application of BMP dramatically increases the amount of
nuclear phospho-p38 (after 1 and 6 h, respectively).
The functional implication of p38 in interdigital tissue
regression was analyzed by local treatment with the p38
inhibitor, SB203580. Our results reveal that there are two
categories of genes, p38 dependent and p38 independent,
induced by BMPs in the interdigital mesoderm. Interdigital
BMP-mediated upregulation of Dkk, Snail and Fgfr3 was
severely impaired after the double treatments with a BMP
bead and a bead incubated in SB203580 (Figs. 5H–J), while
the expressions of BAMBI, Msx2, Smad6 and Smad8 were
not affected in these experiments (Figs. 5F, G and 7D, H).
The inhibitory effect of SB203580 beads over gene
expression was only observed during the first 10 h after
the treatment. Neither cell death nor chondrogenesis were
altered in long-term in vivo treatments, suggesting that none
of the beads tested (affigel blue, heparin and AG1-X2) were
efficient retaining and slowly delivering the drug. In accor-
Fig. 3. Confocal images of squashed interdigital mesodermal cells showing the expression of bmp5 transcripts at day 6.5 of incubation (A). (B)
Immunolabeling for BMP5 (red) combined with TUNEL (green) at day 7 of incubation. Note the intense cytoplasmic labeling both in living and in TUNEL-
positive cells. (C–E) Apoptotic mesodermal cell after TUNEL and BMP5 immunolabeling. D and E show independent channels for BMP5 and TUNEL,
respectively. (F, G) Double immunolabeling for ubiquitin (red) and BMP5 (green). Note the lack of co-localization between the two proteins both in the
rounded apoptotic cell (F) and in the healthy stellate cell (G).
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5246
dance with this interpretation, local application of SB203580
beads was unable to disturb digit chondrogenesis but addi-
tion of SB203580 to micromass cultures severely impaired
both spontaneous chondrogenesis and BMP-mediated chon-
drogenesis (not shown). However, other explanations for the
absence of long-term effects of SB203580 beads can be
proposed. Thus, it is possible that not all forms of p38
present in the interdigit were inhibited by SB203580 (Stan-
ton et al., 2003); alternatively, it is also possible that after the
first 10 h of treatment, the SMAD pathway restores normal
BMP function.
Discussion
The third interdigit of the chick leg bud was selected in
this study to obtain a cDNA library due to its potential
advantages to detect genes responsible for apoptosis. First,
the spatial arrangement of the area of interdigital cell death
is very well defined and can be isolated surgically without
risk of including nonregressing tissues. Second, apoptosis
exhibits a constant and relatively short (48 h) temporal
pattern of distribution. And third, cells destined to die
constitute a uniform population of undifferentiated meso-
derm with potential to differentiate into cartilage. At the
stage selected for the generation of the cDNA library, the
interdigital mesoderm contained both preapoptotic cells
committed to die within the next few hours and apoptotic
cells at different stages of degeneration. By exploring this
library, we identified several genes potentially implicated in
the regulation of interdigital cell death. These genes in-
cluded: components of the proteosome which is an struc-
ture responsible for protein degradation implicated in
apoptosis (see Lee and Peter, 2003); genes implicated in
DNA repair (Prkdc gene for DNA-dependent protein ki-
nase; Mori et al., 2001); genes implicated positively in
apoptosis (caspase8 binding protein FLASH, Imai et al.,
1999; damage-specific DNA binding protein, Sun et al.,
2002; voltage-dependent anion channel, VDAC2, Sugiyama
et al., 2002; spermidine/spermine N1-acetyltransferase, Ha
et al., 1997; cytochrome c oxidase subunit I, II and III,
Chandra et al., 2002; cathepsin-L, Boland and Campbell,
2004; SAP18, Schwerk et al., 2003); antiapoptotic genes
(dynein protein inhibitor of neuronal nitric oxide synthase;
Chang et al., 2000; elongation factor 1-alpha 1, Talapatra
et al., 2002) and genes of potential importance in phago-
cytosis (apolipoprotein A1; Hamon et al., 2000). By in situ
hybridization, we noted that except for VDAC2, a gene
Fig. 4. (A– I) Effects of local application of beads bearing BMP5 into the limb bud. (A, D) TUNEL-positive apoptosis induced by BMP5 beads (*) implanted in
the anterior margin mesoderm of the wing bud at day 3.5 of incubation (A) and in the interdigital mesoderm before the onset of physiological apoptosis (D). (B,
C) Experimental limbs vital stained with neutral red to show the area of cell death induced after implanting a BMP5 bead (*; B) and its inhibition by co-
implantation of a Noggin bead (white *; C). E–F show similar results obtained when a BMP5 bead alone (E) or in combination with a Noggin bead (F) is
implanted into the third interdigit. (G) Skeletal pattern of the wing bud lacking the radius (arrow) consequence of the experiment illustrated in B. (H, I) Neutral
red staining of cell death (H) and skeletal staining with Alcian green (I) of limbs after implantation of a BMP5 bead at the tip of digit 3. Note that cell death is
restricted to the most distal mesenchyme of the tip of the digit (H) while the digit cartilage exhibits intense overgrowth (*, I).
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–52 47
responding to the Bcl-2 family of apoptotic regulators
involved in the control of mitochondrial permeability and
Dek, an oncogenic-like gene, which have interdigital ex-
Fig. 5. (A–E) In situ hybridizations for BAMBI (A), Msx2 (B), Dkk (C), Snail (D)
showing upregulation of all the genes. (F–J) Effect of beads soaked in SB203580
BMP-mediated upregulation of Dkk (H), Snail (I) and Fgfr3 (J) is inhibited by the a
(F) and Msx2 (G) remains unchanged.
pression domains, the remaining genes were ubiquitously
expressed in the limb. Therefore, further studies are re-
quired to clarify their function in the apoptotic cascade of
and Fgfr3 (E) 8 h after the application of a BMP5 bead in the third interdigit
co-implanted with BMP5 beads in the expression of these genes. Note that
pplication of the p38 inhibitor SB203580, while the upregulation of BAMBI
Fig. 6. Interdigital mesodermal cells immunolabeled for Smad proteins. (A) Double labeling for non-phosphorylated Smads 1, 5, 8 (red) and panhistone (green)
used as a nuclear marker, of 7.5 days interdigital mesodermal cells. (B) Double labeling for phosphorylated Smad1 (green) and panhistone (red) at day 7.5 of
development showing co-localization of both proteins in several mesodermal cells (yellow nuclei). (C, D) Control (C) and BMP-treated (D) interdigital
mesodermal cells at day 6 of incubation. Note the increase in labeling intensity for phospho-Smad1 and in the number of labeled cells 90 min after BMP treatment.
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5248
the interdigit. However, it is remarkable the presence in our
library of lysosomal genes, such as cathepsin-L, which
might provide an alternative death pathway to caspases
Fig. 7. Expression and regulation of Smads in the interdigital mesoderm. (A) Ex
Smad6 at day 7 of incubation. The developing joints show also a tenuous expres
3 h after the treatment with a BMP bead that is not modified by co-implantation
of incubation. Note that in addition to the intense interdigital domains, a more r
expression is inhibited by Noggin beads (F) and upregulated by BMP beads (G). T
induced upregulation (H). Arrows indicate SB203580 bead.
(Boland and Campbell, 2004; Dietrich et al., 2003) explain-
ing the maintenance of interdigital degeneration in mice
deficient for Apaf1 (Chautan et al., 1999).
pression of Smad1 at 5.5 days of incubation. (B) Interdigital expression of
sion nonappreciable in the picture. (C) Upregulation of Smad6 expression
with a bead incubated in SB203580 (D). (E) Expression of Smad8 at day 6
educed expression is also observed at the tip of the digits. The interdigital
he double treatment with BMP and SB203580 bead does not inhibit BMP-
Fig. 8. Immunolabeling with phospho-p38 (green) combined with lamina B (red) that stains the nuclear envelope in control and BMP-treated interdigital
mesodermal cells. A and B are control mesodermal cells at days 5.5 and 7.5 of incubation, respectively, showing the increase of the nuclear expression domain
during interdigital tissue regression. (C, D) Interdigital mesoderm at day 5.5 of incubation, 1 h (C) and 6 h (D) after BMP treatment. Note the intense labeling in
C and D in comparison with the nontreated cells at the same stage (A).
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–52 49
In contrast with the predominant ubiquitous expression
of the genes mentioned above, Bmp5 exhibited a pattern of
expression closely coincident with interdigital apoptosis.
Previous studies revealed that BMP2, BMP4 and BMP7
are triggering signals for physiological cell death in the
developing limb (Ganan et al., 1996; Guha et al., 2002;
Kawakami et al., 1996; Macias et al., 1997; Yokouchi et al.,
1996; Zou and Niswander, 1996; Zou et al., 1997). This
conclusion was deduced from their pattern of expression
and by gain- and loss-of-function experiments (Guha et al.,
2002; Merino et al., 1998; Yokouchi et al., 1996; Zou and
Niswander, 1996). By using the same criteria, we found that
BMP5 constitutes a further member of the BMP family
implicated in the control of limb programmed cell death.
Previously, the implication of BMP5 in embryonic cell
death has been reported only in the developing nervous
system (Golden et al., 1999).
Taking into account that at least four different members
of the BMP family (BMP2, BMP4, BMP5 and BMP7) are
involved in the control of limb programmed cell death, it is
not surprising the absence of an interdigital phenotype in
mice deficient for Bmp5 (King et al., 1994; Solloway and
Robertson, 1999). It must be remarked that all these BMPs
share a high identity. BMP2 and BMP4 share 92% identity,
while BMP5 and BMP7 share 87% identity. This high
redundancy in BMPs in the regressing interdigital tissue
suggests that BMPs exert their function by forming hetero-
dymeric complexes as has been found in other biological
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5250
processes (Butler and Dodd, 2003; Katagiri et al., 1998).
Furthermore, on the basis of the phenotypes of single and
double KO mice for Bmp7, Bmp5 and taking into account
their distinct, but rather overlapping, pattern of expression in
the early embryo, it has been proposed that BMP5 might
modulate the effect of BMP7, creating zones of increased
signaling within the Bmp7 expression domain (Solloway
and Robertson, 1999). An interesting observation in our
study was the identification of intense immunolabeling for
BMP5/7 proteins within the cytoplasm of TUNEL-positive
apoptotic cells. This finding suggests that delivery of BMPs
might be potentiated during cell fragmentation associated
with apoptosis explaining why interdigital apoptosis pro-
gresses at advanced stages of degeneration when the amount
of healthy cells, able to secrete BMPs, are dramatically
reduced. A comparable release of growth factors associated
with mechanical disruption of plasma membrane has been
observed in endothelial cell cultures (McNeil et al., 1989).
An additional task of this study was to explore the
intracellular downstream effectors of BMPs in the regress-
ing interdigit. There is now increasing evidence showing
that in addition to the well-characterized Smad signaling
pathway, BMPs, and other members of the TGFh super-
family, may also activate MAP kinases (Derynck and
Zhang, 2003). Furthermore, it has been suggested that in
some cases, the cellular response induced by BMPs may be
the result of the balance between a counteracting function
between Smad and MAPK pathways (see, Massague, 2003).
However, both pathways may act in a cooperative fashion.
Thus, in mouse hybridoma MH60 cells, BMPs induce
apoptosis through p38 kinase and this cell death is inhibited
when smad6 is overexpressed (Kimura et al., 2000).
The presence of Smad1/5/8 proteins in the interdigital
mesoderm along with the progressive abundance of nuclear
phosphorylated Smad1 during regression, and its potentia-
tion by application of BMP beads, indicate that Smad
proteins exert a central role in the control of interdigital
tissue regression by BMPs. In support to such an implica-
tion of Smad signaling in interdigital tissue regression, we
have also found that Smad6 and Smad8 exhibit interdigital
expression domains regulated by BMPs.
Our findings also provide evidence for the activation of
MAPK p38 during interdigital tissue regression in response
to BMPs. Phosphorylated p38 is distributed throughout the
nucleoplasm of the interdigital mesoderm. Furthermore, as
observed for phospho-Smad1, nuclear labeling for phospho-
p38 increases during tissue regression and upon application
of BMP beads. Double treatments with BMPs and the
inhibitor of p38 indicate that this kinase mediates the
upregulation of some of the genes activated by BMPs in
the interdigit, including Dkk, Snail and Fgfr3, but not
BAMBI, Msx2, Smad6 and Smad8.
Although we cannot prove that all forms of p38 were
inhibited by SB203580 in our experiments (see review by
Stanton et al., 2003), the phosphorylation of Smad1 in the
interdigital mesenchyme upon application of BMPs, along
with the occurrence of specific p38-independent interdigital
domains of expression of Smad8 and Smad6, are in favor of
a synergic/cooperative rather then antagonistic function of
both cellular pathways in the regulation of the cellular
response to BMPs in the developing limb.
Although Bmp5 KO mice lack a limb phenotype, our
study is indicative of a function of BMP5 in the differen-
tiation of the limb skeleton. We have found that Bmp5 is
expressed in the perichondrium of the developing phalanxes
and at the tip of the digits, and application of BMP5 in the
differentiating cartilages promotes a dramatic growth. The
intense inhibition of the chondrogenic effect of BMP5 (and
of BMP7) by SB203580 in limb mesoderm micromass
cultures indicates that p38 is an important effector for the
chondrogenic effect of BMPs. This finding is in agreement
with previous studies showing the importance of p38 in
chondrogenesis (Stanton et al., 2003). Furthermore, this
observation argues against a counteracting function between
Smads and p38 regulating the different cellular responses
mediated by BMPs in the limb mesoderm.
Finally, we also show in this study that Bmp5 is expressed
in precise domains in the developing muscle masses and in
the autopodial tendons. Again in these regions, and consis-
tent with the absence of phenotypes in mice deficient for
Bmp5 (King et al., 1994), other BMPs are present controlling
tissue differentiation (Amthor et al., 2002).
Acknowledgments
Thanks are due to A. Economides from Regeneron Phar-
maceuticals for human recombinant Noggin; to C. Heldin
for the antibody against phospho-Smad1; to S. Perez-
Mantecon, M. Fernandez and A. Ibaseta for technical
assistance; and to Hannu Mansukoski for reading this
manuscript. This work was supported by a grant from the
Ministerio de Ciencia y Tecnologıa to J.M.H. (BMC2002-
02346). V.Z.L. and J.R.L. are supported by grants from the
Fundac�ao para a Ciencia e a Tecnologia, Portugal.
References
Amthor, H., Christ, B., Rashid-Doubell, F., Kemp, C.F., Lang, E., Patel, K.,
2002. Follistatin regulates bone morphogenetic protein-7 (BMP-7) ac-
tivity to stimulate embryonic muscle growth. Dev. Biol. 243, 115–127.
Boland, B., Campbell, V., 2004. Abeta-mediated activation of the apoptotic
cascade in cultured cortical neurones: a role for cathepsin-L. Neurobiol.
Aging 25, 83–91.
Butler, S.J., Dodd, J., 2003. A role for BMP heterodimers in roof plate-
mediated repulsion of commissural axons. Neuron 38, 389–401.
Carrio, R., Lopez-Hoyos, M., Jimeno, J., Benedict, M.A., Merino, R.,
Benito, A., Fernandez-Luna, J.L., Nunez, G., Garcia-Porrero, J.A., Me-
rino, J., 1996. A1 demonstrates restricted tissue distribution during
embryonic development and functions to protect against cell death.
Am. J. Pathol. 149, 2133–2142.
Cecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A., Gruss, P., 1998.
Apaf1 (CED-4 homolog) regulates programmed cell death in mamma-
lian development. Cell 94, 727–737.
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–52 51
Chandra, D., Liu, J.W., Tang, D.G., 2002. Early mitochondrial activation
and cytochrome c up-regulation during apoptosis. J. Biol. Chem. 277,
50842–50854.
Chang, Y.W., Jakobi, R., McGinty, A., Foschi, M., Dunn, M.J., Sorokin,
A., 2000. Cyclooxygenase 2 promotes cell survival by stimulation of
dynein light chain expression and inhibition of neuronal nitric oxide
synthase activity. Mol. Cell. Biol. 20, 8571–8579.
Chautan, M., Chazal, G., Cecconi, F., Gruss, P., Golstein, P., 1999. Inter-
digital cell death can occur through a necrotic and caspase-independent
pathway. Curr. Biol. 9, 967–970.
Chen, Y., Zhao, X., 1998. Shaping limbs by apoptosis. J. Exp. Zool. 282,
691–702.
Chimal-Monroy, J., Montero, J.A., Ganan, Y., Macias, D., Garcia-Porrero,
J.A., Hurle, J.M., 2002. Comparative analysis of the expression and
regulation of Wnt5a, Fz4, and Frzb1 during digit formation and in
micromass cultures. Dev. Dyn. 224, 314–320.
Crocoll, A., Herzer, U., Ghyselinck, N.B., Chambon, P., Cato, A.C.B.,
2002. Interdigital apoptosis and downregulation of BAG-1 expression
in mouse autopods. Mech. Dev. 111, 149–152.
Derynck, R., Zhang, Y.E., 2003. Smad-dependent and Smad-independent
pathways in TGF-h family signalling. Nature 425, 577–584.
Dietrich, N., Thastrup, J., Holmberg, C., Gyrd-Hansen, M., Fehrenbacher,
N., Lademann, U., Lerdrup, M., Herdegen, T., Jaattela, M., Kallunki, T.,
2003. JNK2 mediates TNF-induced cell death in mouse embryonic
fibroblasts via regulation of both caspase and cathepsin protease path-
ways. Cell Death Differ. 11, 301–313.
Drouin, R., Lemieux, N., Richer, C.L., 1991. Chromosome condensation
from prophase to late metaphase: relationship to chromosome bands and
their replication time. Cytogenet. Cell Genet. 57, 91–99.
Dunker, N., Schmitt, K., Krieglstein, K., 2002. TGF-beta is required for
programmed cell death in interdigital webs of the developing mouse
limb. Mech. Dev. 113, 111–120.
Dupe, V., Ghyselinck, N.B., Thomazy, V., Nagy, L., Davies, P.J., Chambon,
P., Mark, M., 1999. Essential roles of retinoic acid signalling in inter-
digital apoptosis and control of BMP-7 expression in mouse autopods.
Dev. Biol. 208, 30–43.
Ferrari, D., Lichtler, A.C., Pan, Z., Dealy, C.N., Upholt, W.B., Kosher,
R.A., 1998. Ectopic expression of Msx2 in posterior limb bud meso-
derm impairs limb morphogenesis while inducing Bmp4 expression,
inhibiting cell proliferation, and promoting apoptosis. Dev. Biol. 197,
12–24.
Ganan, Y., Macias, D., Duterque-Coquillaud, M., Ros, M.A., Hurle, J.M.,
1996. Role of TGFhs and BMPs as signals controlling the position of
the digits and the areas of interdigital cell death in the developing chick
limb autopod. Development 122, 2349–2357.
Garcia-Domingo, D., Leonardo, E., Grandien, A., Martinez, P., Albar, J.P.,
Izpisua-Belmonte, J.C., Martinez-A, C., 1999. DIO-1 is a gene involved
in onset of apoptosis in vitro, whose misexpression disrupts limb de-
velopment. Proc. Natl. Acad. Sci. U. S. A. 96, 7992–7997.
Garcia-Martinez, V., Macias, D., Ganan, Y., Garcia-Lobo, J.M., Francia,
M.V., Fernandez-Teran, M.A., Hurle, J.M., 1993. Internucleosomal
DNA fragmentation and programmed cell death (apoptosis) in the inter-
digital tissue of the embryonic chick leg bud. J. Cell Sci. 106, 201–208.
Golden, J.A., Bracilovic, A., McFadden, K.A., Beesley, J.S., Rubenstein,
J.L., Grinspan, J.B., 1999. Ectopic bone morphogenetic proteins 5 and 4
in the chicken forebrain lead to cyclopia and holoprosencephaly. Proc.
Natl. Acad. Sci. U. S. A. 96, 2439–2444.
Grotewold, L., Plum, M., Dildrop, R., Peters, T., Ruther, U., 2001. Bambi
is coexpressed with Bmp-4 during mouse embryogenesis. Mech. Dev.
100, 327–330.
Grotewold, L., Ruther, U., 2002. The Wnt antagonist Dickkopf-1 is regu-
lated by Bmp signalling and c-Jun and modulates programmed cell
death. EMBO J. 21, 966–975.
Guha, U., Gomes, W.A., Kobayashi, T., Pestell, R.G., Kessler, J.A., 2002.
In vivo evidence that BMP signalling is necessary for apoptosis in the
mouse limb. Dev. Biol. 249, 108–120.
Ha, H.C., Woster, P.M., Yager, J.D., Casero Jr., R.A., 1997. The role of
polyamine catabolism in polyamine analogue-induced programmed cell
death. Proc. Natl. Acad. Sci. U. S. A. 94, 11557–11562.
Hamon, Y., Broccardo, C., Chambenoit, O., Luciani, M.F., Toti, F., Chas-
lin, S., Freyssinet, J.M., Devaux, P.F., McNeish, J., Marguet, D.,
Chimini, G., 2000. ABC1 promotes engulfment of apoptotic cells
and transbilayer redistribution of phosphatidylserine. Nat. Cell Biol.
2, 399–406.
Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U., Nordheim, A., 1999.
Id genes are direct targets of bone morphogenetic protein induction in
embryonic stem cells. J. Biol. Chem. 274, 19838–19845.
Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K., Yone-
hara, S., 1999. The CED-4-homologous protein FLASH is involved
in Fas-mediated activation of caspase-8 during apoptosis. Nature 398,
777–785.
Jacobson, M.D., Weil, M., Raff, M.C., 1996. Role of Ced-3/ICE-family
proteases in staurosporine-induced programmed cell death. J. Cell Biol.
133, 1041–1051.
Katagiri, T., Boorla, S., Frendo, J.L., Hogan, B.L., Karsenty, G., 1998.
Skeletal abnormalities in doubly heterozygous Bmp4 and Bmp7 mice.
Dev. Genet. 22, 340–348.
Kawakami, Y., Ishikawa, T., Shimabara, M., Tanda, N., Enomoto-Iwamoto,
M., Iwamoto, M., Kuwana, T., Ueki, A., Noji, S., Nohno, T., 1996.
BMP signalling during bone pattern determination in the developing
limb. Development 122, 3557–3566.
Kawakami, Y., Rodriguez-Leon, J., Koth, C.M., Buscher, D., Itoh, T.,
Raya, A., Ng, J.K., Esteban, C.R., Takahashi, S., Henrique, D.,
Schwarz, M.F., Asahara, H., Izpisua Belmonte, J.C., 2003. MKP3
mediates the cellular response to FGF8 signalling in the vertebrate
limb. Nat. Cell Biol. 5, 513–519.
Kimura, N., Matsuo, R., Shibuya, H., Nakashima, K., Taga, T., 2000.
BMP2-induced apoptosis is mediated by activation of the TAK1-p38
kinase pathway that is negatively regulated by Smad6. J. Biol. Chem.
275, 17647–17652.
King, J.A., Marker, P.C., Seung, K.J., Kingsley, D.M., 1994. BMP5 and the
molecular, skeletal, and soft-tissue alterations in short ear mice. Dev.
Biol. 166, 112–122.
Ko, H.S., Uehara, T., Nomura, Y., 2002. Role of ubiquilin associated with
protein-disulfide isomerase in the endoplasmic reticulum in stress-in-
duced apoptotic cell death. J. Biol. Chem. 277, 35386–35392.
Lee, J.C., Peter, M.E., 2003. Regulation of apoptosis by ubiquitination.
Immunol. Rev. 193, 39–47.
Lee, K.K., Tang, M.K., Yew, D.T., Chow, P.H., Yee, S.P., Schneider, C.,
Brancolini, C., 1999. Gas2 is a multifunctional gene involved in the
regulation of apoptosis and chondrogenesis in the developing mouse
limb. Dev. Biol. 207, 14–25.
Lee, K.K., Leung, A.K., Tang, M.K., Cai, D.Q., Schneider, C., Brancolini,
C., Chow, P.H., 2001. Functions of the growth arrest specific 1 gene in
the development of the mouse embryo. Dev. Biol. 234, 188–203.
Lindsten, T., Ross, A.J., King, A., Zong, W.X., Rathmell, J.C., Shiels,
H.A., Ulrich, E., Waymire, K.G., Mahar, P., Frauwirth, K., Chen, Y.,
Wei, M., Eng, V.M., Adelman, D.M., Simon, M.C., Ma, A., Golden,
J.A., Evan, G., Korsmeyer, S.J., MacGregor, G.R., Thompson, C.B.,
2000. The combined functions of proapoptotic Bcl-2 family members
bak and bax are essential for normal development of multiple tissues.
Mol. Cell 6, 1389–1399.
Lussier, M., Canoun, C., Ma, C., Sank, A., Shuler, C., 1993. Interdigital
soft tissue separation induced by retinoic acid in mouse limbs cultured
in vitro. Int. J. Dev. Biol. 37, 555–564.
Macias, D., Ganan, Y., Sampath, T.K., Piedra, M.E., Ros, M.A., Hurle,
J.M., 1997. Role of BMP-2 and OP-1 (BMP-7) in programmed cell
death and skeletogenesis during chick limb development. Development
124, 1109–1117.
Massague, J., 2003. Integration of Smad and MAPK pathways: a link and a
linker revisited. Genes Dev. 17, 2993–2997.
McNeil, P.L., Muthukrishnan, L., Warder, E., D’Amore, P.A., 1989.
Growth factors are released by mechanically wounded endothelial cells.
J. Cell Biol. 109, 811–822.
V. Zuzarte-Luıs et al. / Developmental Biology 272 (2004) 39–5252
Merino, R., Ganan, Y., Macias, D., Economides, A.N., Sampath, K.T.,
Hurle, J.M., 1998. Morphogenesis of digits in the avian limb is con-
trolled by FGFs, TGFbetas, and noggin through BMP signalling. Dev.
Biol. 200, 35–45.
Montero, J.A., Ganan, Y., Macias, D., Rodriguez-Leon, J., Sanz-Ezquerro,
J.J., Merino, R., Chimal-Monroy, J., Nieto, M.A., Hurle, J.M., 2001.
Role of FGFs in the control of programmed cell death during limb
development. Development 128, 2075–2084.
Mori, N., Matsumoto, Y., Okumoto, M., Suzuki, N., Yamate, J., 2001.
Variations in Prkdc encoding the catalytic subunit of DNA-dependent
protein kinase (DNA-PKcs) and susceptibility to radiation-induced
apoptosis and lymphomagenesis. Oncogene 20, 3609–3619.
Nakanishi, K., Maruyama, M., Shibata, T., Morishima, N., 2001. Identifi-
cation of a caspase-9 substrate and detection of its cleavage in
programmed cell death during mouse development. J. Biol. Chem.
276, 41237–41244.
Nishii, K., Tsuzuki, T., Kumai, M., Takeda, N., Koga, H., Aizawa, S.,
Nishimoto, T., Shibata, Y., 1999. Abnormalities of developmental cell
death in Dad1-deficient mice. Genes Cells 4, 243–252.
Novack, D.V., Korsmeyer, S.J., 1994. Bcl-2 protein expression during
murine development. Am. J. Pathol. 145, 61–73.
Onichtchouk, D., Chen, G., Dosh, R., Gawantka, V., Delius, H., Massague,
J., Niehrs, J.M., 1999. Silencing of TGF-h signalling by the pseudor-
eceptor BAMBI. Nature 401, 480–485.
Rodriguez-Leon, J., Merino, R., Macias, D., Ganan, Y., Santesteban, E.,
Hurle, J.M., 1999. Retinoic acid regulates programmed cell death
through BMP signalling. Nat. Cell Biol. 1, 125–126.
Sanz-Ezquerro, J., Tickle, C., 2000. Autoregulation of Shh expression and
Shh induction of cell death suggest a mechanism for modulating
polarizing activity during chick limb development. Development
127, 4811–4823.
Schwerk, C., Prasadm, J., Degenhardtm, K., Erdjument-Bromage, H.,
White, E., Tempst, P., Kidd, V.J., Manley, J.L., Lahti, J.M., Reinberg,
D., 2003. ASAP, a novel protein complex involved in RNA processing
and apoptosis. Mol. Cell. Biol. 23, 2981–2990.
Solloway, M.J., Robertson, E.J., 1999. Early embryonic lethality in
Bmp5;Bmp7 double mutant mice suggests functional redundancy with-
in the 60A subgroup. Development 126, 1753–1768.
Stanton, L.A., Underhilll, T.M., Beier, F., 2003. MAP kinases in chondro-
cyte differentiation. Dev. Biol. 263, 165–175.
Sugiyama, T., Shimizu, S., Matsuoka, Y., Yoneda, Y., Tsujimoto, Y., 2002.
Activation of mitochondrial voltage-dependent anion channel by apro-
apoptotic BH3-only protein Bim. Oncogene 21, 4944–4956.
Sun, N.K., Lu, H.P., Chao, C.C., 2002. Identification of rat DDB1, a
putative DNA repair protein, and functional correlation with its dam-
aged-D recognition activity. J. Biomed. Sci. 9, 371–380.
Talapatra, S., Wagner, J.D., Thompson, C.B., 2002. Elongation factor-1
alpha is a selective regulator of growth factor withdrawal and ER
stress-induced apoptosis. Cell Death Differ. 9, 856–861.
Yamada, M., Szendro, P.I., Prokscha, A., Schwartz, R.J., Eichele, G.,
1999. Evidence for a role of Smad6 in chick cardiac development.
Dev. Biol. 215, 48–61.
Yokouchi, Y., Sakiyama, J., Kameda, T., Iba, H., Suzuki, A., Ueno, N.,
Kuroiwa, A., 1996. BMP-2/-4 mediate programmed cell death in chick-
en limb buds. Development 122, 3725–3734.
Zou, H., Niswander, L., 1996. Requirement for BMP signalling in inter-
digital apoptosis and scale formation. Science 272, 738–741.
Zou, H., Wieser, R., Massague, J., Niswander, L., 1997. Distinct roles of
type I bone morphogenetic protein receptors in the formation and dif-
ferentiation of cartilage. Genes Dev. 11, 2191–2203.
Zuzarte-Luis, V., Hurle, J.M., 2002. Programmed cell death in the deve-
loping limb. Int. J. Dev. Biol. 46, 871–876.
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