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Shaping Limbs by Apoptosis
Department of Cell and Molecular Biology, Tulane University, New Orleans,
Louisiana 70118
Molecular and Cell Biology Graduate Program, Tulane University, New
Orleans, Louisiana 70118
Center for Bioenvironmental Research, Tulane University, New Orleans,
Louisiana 70118
Programmed cell death has long been recognized as an important mechanism of
normal embryonic development. In the developing limb, massive programmed cell death apparently plays a critical role in controlling the amount of mesodermal tissues, sculpting overall limb
shape, and defining the digits. Cell death in the limb bud has been shown to be regulated by a
number of factors, including environmental conditions and epithelial–mesenchymal interactions.
Removing the ectoderm overlying the interdigital region results in inhibition of cell death and
ectopic cartilage formation in the subjacent mesenchyme. Recently, several signaling factors and
genes have been implicated in the control of cell death in the limb bud. FGFs may acts as trophic
factors that interfere with cell death. There is evidence that BMPs and Msx genes participate in
regulating cell death. BMPs may in some cases trigger the cell death cascade, while Msx gene
products regulate Bmp expression. A correlation between FGF, BMP and Msx in interdigital cell
death is discussed. J. Exp. Zool. 282:691–702, 1998. © 1998 Wiley-Liss, Inc.
Programmed cell death (PCD) is an active and
genetically well-controlled process to eliminate
unwanted cells during embryogenesis, metamorphosis, and tissue turnover. This process
occurs predictably at a defined time and place
in both invertebrates and vertebrates. It is now
widely accepted that PCD plays a critical role
in controlling precise cell populations and
sculpting the shape of many developing organs.
In addition to its role in normal embryonic development, PCD might be important in a variety of human disorders, including Alzheimer’s
and Huntington’s diseases.
Apoptosis was originally introduced as one form
of PCD (Kerr et al., ’72). Although the original
definitions of the terms programmed cell death
and apoptosis are slightly different (reviewed in
Sanders and Wride, ’95), they seem to have become synonymous. The apoptotic process is detectable at morphological and biochemical levels.
Morphologically, the apoptotic process can be observed and followed by light, transmission, and
scanning electron microscopy (reviewed in Hurle
et al., ’96). In most cases, the nuclear and cytoplasmic alterations of apoptotic cells are accompanied by a characteristic condensation of the
chromatin, cytoplasmic blebbing, and DNA fragmentation through cleavage by endonucleases.
Eventually, apoptotic cells become fragmented
into membrane-bound pieces, and are engulfed
by neighboring phagocytic cells (Steller, ’95). At
biochemical and cytochemical levels, it has been
shown that DNA fragmentation in the affected
cells is a consequence of activation or de novo
synthesis of endonuclease(s) which causes the
internucleosomal cleavage of DNA (Wyllie, ’80;
Compton, ’92; Schwartzman and Cidlowski, ’93).
This internucleosomal cleavage of DNA was believed to be a hallmark of apoptosis, distinguishing it from necrosis (Compton, ’92). However,
apoptosis was recently shown to occur in the absence of endonuclease activity and DNA fragmentation (Cohen et al., ’92; Schultze-Osthoff et al.,
’94). Activation, and increased activity of tissue
transglutaminase was also reported in association
with apoptotic cells, although the function of tissue transglutaminase is uncertain (Fesus et al.,
’91; Piacentini et al., ’91; Jiang and Kochhar, ’92).
Changes in the calcium ion balance and elevation
of cAMP levels have also been implicated in
Grant sponsor: National Science Foundation; Grant number: IBN9796321; Grant sponsor: American Heart Association National Center; Grant number: 9750104N.
*Correspondence to: YiPing Chen, Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118. E-mail:
apoptosis (McConkey et al., ’89, ’90; Nicotera et
al., ’94).
A major difference between apoptosis and necrosis is that apoptosis is an active process, with
the endogenous control of cell death involving
the expression of a specific set of genes; while
necrosis is a passive process, and involves cell
lysis caused by external agents (Kerr et al., ’72;
Gerschenson and Rotello, ’92). There are several genes whose expression has been associated with PCD or apoptosis. The first genes
directly linked to PCD were identified in the
nematode C. elegans (Ellis and Horvitz, ’86) and
comprise an 11-gene pathway (Horvitz, ’94). These
genes fall into three categories: genes involved in
killing cells (ced-3, ced-4 and ced-9), genes controlling the phagocytosis of dying cells by neighboring cells (ced-1, ced-2, ced-5-8 and ced-10) and
a gene functioning to degrade the engulfed cells
(nuc-1). Of these ced-3 and ced-4 are essential for
cell death, while ced-9 protects cells from PCD
(Ellis and Horvitz, ’86; Yuan and Horvitz, ’90;
Hengartner et al., ’92). The products of the ced-3
and ced-4 genes act autonomously to cause death
in the cells expressing them. Mutations that inactivate either gene prevent cell death in cells that
otherwise normally die during development. In
vertebrates, three gene families have been identified as playing a central role in apoptotic pathways, including the interleukin-1β-converting
enzyme (ICE) or caspase family of cysteine proteases (Yuan, ’95), the Bcl-2 (B-cell lymphoma/leukemia-2) proto-oncogene family (Boise et al., ’95)
and the tumor necrosis factor (TNF) family and
their receptors (Nagata and Golstein, ’95). The
mammalian homolog of the ced-3 gene product,
ICE, was shown to be able to induce apoptosis
when transfected into rat fibroblasts (Miura et al.,
’93). In contrast, overexpression of bcl-2, the mammalian homolog of ced-9, inhibits cell death. bcl-2
is able to prevent cell death in both vertebrate
cells and C. elegans (Garcia et al., ’92; Vaux et
al., ’92), indicating that these death gene functions are highly conserved in the control of physiological cell death. The apoptotic cascade can be
triggered by internal genetic programs and a variety of physiological stimuli. The pathway that
activates the apoptotic response is therefore different in different cases. For example, in mammalian cells, the Fas receptor mediates a pathway
that triggers apoptosis. Binding of antibody to Fas
protein on the cell surface activates Fas and induces apoptosis (Eischen et al., ’94). The tumor
suppressor p53 and the product of the proto-
oncogene c-myc seem to induce apoptosis through
distinct intracellular pathways (Evan et al., ’94;
Gottlieb et al., ’94).
In vertebrate embryos, apoptosis occurs in many
developing organs and tissues, including heart,
kidney, lens, neural crest, tail bud, somites, and
branchial arches as well as limbs (reviewed in
Sanders and Wride, ’95). PCD is a prominent aspect of limb morphogenesis in amniote embryos,
and has received considerable attention. This article provides an update on cell death during limb
development in birds and mice, with emphasis on
the recent progress in this area.
Vertebrate limbs develop from paired primordial buds that appear on the embryo’s lateral
surface at appropriate levels along its anterioposterior body axis. At early stages, the buds
exhibit a paddle shape and consist of undifferentiated mesenchymal cells and overlying ectoderm. The mesenchymal cells originate mainly
from two distinct sources, lateral plate mesoderm and lateral somites. At the distal tip of
the bud, the ectoderm forms a specialized thickened epithelial structure, known as the apical
ectodermal ridge (AER). The AER is known to
express a number of growth factors and is responsible for the control of limb-bud outgrowth
(reviewed in Caruccio and Fallon, unpubl). As
the bud continues to grow out, mesenchymal
cells at the proximal part of the bud aggregate
to form cartilage blastemal elements of the future definitive skeleton, while cells in the distal part, directly under the AER (i.e., the
progress zone) remain undifferentiated. This
process results in a sequential differentiation
of limb elements along the proximal-distal axis,
with the stylopod (humerus/femur) forming
first, the zeugopod (radius-ulna/tibia-fibula)
forming second and the autopod (carpals/tarsals
and phalanges) forming last. During this phase
of development, large numbers of mesenchymal
cells undergo prominent PCD in defined regions. In the process, the limb’s characteristic
structure and shape are established.
Cell death during limb development has been a
focus of research into the mechanisms of morphogenesis for several decades (Saunders and Fallon,
’67). Much of the experimental work has been performed on the embryonic chick limb, due to its
easy access to surgical manipulation in ovo. In
the chick wing buds, massive mesenchymal cell
death appears first in the superficial mesoderm
of the anterior edge of the limb bud and adjacent
body wall at stage 21 (Hamburger and Hamilton,
’51; Saunders et al., ’62). This region has been
termed the anterior necrotic zone (ANZ). Subsequently at stage 24, two other regions start to exhibit massive mesenchymal cell death (Saunders
et al., ’62; Hinchliffe and Ede, ’73). They have been
named the posterior necrotic zone (PNZ) and the
opaque patch (OP). The PNZ is located in the posterior junction of limb bud and the body wall,
while the OP is located between the two chondrogenic condensations and functions to separate the
ulna from radius or fibula from tibia. At later
stage (around chick stage 31), cell death is observed in the mesenchyme between the forming
digits. These areas of cell death are known as
interdigital necrotic zones (INZ) and are responsible for separating the chondrifying digits of the
developing wing or leg autopodia. The INZ, found
in all amniotes, occurs extensively in the interdigital regions in species with free digits, but is
reduced in the interdigital regions of webbed species, such as ducks (Hurle and Colvee, ’82).
In the mouse limb bud, cell death can be detected in the ANZ as early as embryonic day 10.
5 (E10.5) (Zakeri et al., ’94). Cell death in the ANZ
becomes more obvious at E11.5 (Fig. 1A). Interdigital cell death begins in the AER at E12.5 (Lee
et al., ’94; Fig. 1B). Meanwhile, weak cell death
can also be observed in the ANZ and the PNZ.
However, cell death in the mouse ANZ and PNZ
never becomes as intense as that in the chick.
Massive interdigital cell death appears at E13.5
and reaches a peak at 14.5 (Fig. 1C), leading to
the separation of the digits. In both chick and
mouse, cell death in the limb buds is apoptotic,
as evidenced by the occurrence of nuclear condensation and internucleosomal DNA fragmentation
in the dead cells (Garcia-Martinez et al., ’93;
Zakeri et al., ’94; Mori et al., ’95).
Genetic control of cell death in the developing
limb buds has been demonstrated in spontaneous
chick and mouse mutants with limb abnormalities. For instance, in the talpid3 chick mutant,
which is characterized by polydactylous limbs with
up to eight digits, the ANZ and PNZ are absent
(Hinchliffe and Ede, ’67). In contrast, precocious,
and abnormally extensive, cell death occurs in the
ANZ of wingless chick mutant limb buds, leading
to digit loss (Hinchliffe and Ede, ’73). In the mouse
Hammertoe mutant, interdigital apoptosisis suppressed between digit 2 and digit 5, resulting in
soft tissue syndactyly of all four limbs (Zakeri et
al., ’94). Although most of the specific mutations
responsible remain to be determined, it was recently revealed that a mutation in the Gli3 gene,
which encodes a transcription factor, corresponds
to the mouse extra-toes mutation which causes
syndactyly (Hui and Joyner, ’93).
Certain exogenous factors, when administered
to chick or rodent embryos, can induce syndactyly or polydactyly. The effects correlate with the
prevention of cell death in the interdigital regions
and/or in the preaxial mesenchyme, although the
underlying mechanisms are completely unknown.
5-bromodeoxyuridine (BrdU), a thymidine analog,
causes syndactyly in chick leg buds by suppressing interdigital apoptosis. Incorporation of BrdU
into the DNA of mesenchymal cells otherwise programmed to die may alter their transcriptional
pattern and thus modify their developmental fates
Fig. 1. Apoptosis in the developing mouse limb buds detected by Nile blue staining. A: An E11.5 mouse limb showing cell death in the ANZ (arrow). B: An E12.5 mouse limb
bud showing cell death in the AER (arrows). C: An E13.5
mouse limb bud showing massive cell death in the interdigital
regions, as indicated by arrows.
(Toné et al., ’83). This repression of interdigital
cell death by exogenous drugs is sometimes accompanied by the presence of ectopic interdigital
cartilages (Fernandez-Teran and Hurle, ’84). On
the other hand, retinoic acid (RA) administration
causes mouse limb defects in part by inducing excessive cell death in developing limb bud (Zakeri
and Ahuja, ’94). Increased cell death caused by
exogenous RA is primarily seen in the AER and
INZ (Sulik and Dehart, ’88; Alles and Sulik, ’89;
Lussier et al., ’93; Lee et al., ’94). Interestingly, it
was reported that RA can partly reverse the suppression of cell death in mouse Hammertoe mutant limbs (Ahuja et al., ’97). RA’s teratogenic
effects on developing limbs may also involve a
more general promotion of precocious phenotypic
expression (Paulsen, ’94), with apoptosis being one
phenotype of particular importance to morphogenesis. RA effects on the cell death appear to be associated with RA receptor-beta (RAR-β), since
RAR-β transcripts are detected in the interdigital
mesenchyme and the OP in mouse limbs just prior
to the onset of, and throughout the whole process
of the apoptosis (Dollé et al., ’89; Mendelsohn et
al., ’92). RAR-β may mediate this RA effect, since
its expression is dramatically enhanced in the limb
bud, including the AER and OP, after RA treatment
(Noji et al., ’91; Harnish et al., ’92; Shen et al., ’92;
Mendelsohn et al., ’92; Kochhar et al., ’93).
A classic experiment carried out by Saunders et
al. (’62) demonstrated that cell death can be suppressed if mesenchyme from the limb bud’s prospective PNZ is transplanted to the dorsal surface of a
host wing bud. These observations indicated that
the cell death program can be repressed by changing the environmental condition of the prospective
dying cells. Indeed, removing a small piece of AER
or ectoderm overlying the interdigital regions inhibits apoptosis in the adjacent interdigital mesenchymal cells, and induces the formation of ectopic
cartilage in the interdigital region (Hurle and
Gañan, ’86, ’87). These observations imply that tissue interactions are crucial for apoptosis in the mesenchyme. Since the distal cells are homogeneously
chondrogenic in standard culture conditions (Cottrill
et al., ’87), it has been proposed that the whole
autopodium has digit-forming potential, and that
the default phenotype of its mesenchymal cells is
chondrogenic. From this perspective, the ectoderm
overlying the interdigital mesenchyme seems to provide signals that divert the mesenchyme’s program
of differentiation from chondrogenesis to cell death
(Hurle et al., ’91; Gañan et al., ’94). Indeed, interdigital mesenchyme prior to occurrence of cell death
are able to form cartilage when cultured in vitro
(Hurle et al., ’91; Lee et al., ’93, ’94). Solursh (’84)
suggested that the antichondrogenic effect of
ectoderm is mediated by a diffusible factor. This ectodermal antichondrogenic effect has been demonstrated in tissue culture (Solursh et al., ’81; Solursh
and Reiter, ’88), and is probably mediated by a peptide produced from ectoderm that behaves biologically like fibronectin (Zanetti et al., ’90). It was
recently found that in addition to the ectoderm, the
developing digits may also exercise an inhibitory
effect on chondrogenesis in the adjacent interdigital
mesenchyme (Lee et al., ’93, ’94; Gañan et al., ’94).
It should be noted, however, that although ectoderm
clearly has an antichondrogenic effect on this mesenchyme both in vitro and in vivo, recent studies
using serum-free culture medium have shown that
the default phenotype of these cells is not homogeneously chondrogenic and that a significant proportion is induced to form cartilage in standard cultures
by a serum component (Paulsen et al., ’94). Clearly,
an interplay of intrinsic and extrinsic factors is required to regulate the elegant pattern of differentiation that forms a foot, a wing, or a hand.
Ectodermal regulation of interdigital apoptosis
could involve two control mechanisms: diffusible
factors and extracellular matrix. It has been reported that addition of fibroblast growth factor
(FGF) to organ cultures of avian wing bud PNZ
and OP rescues the cells destined to die (MacCabe
et al., ’91). Local FGF administration not only substitutes for the AER in the induction of limb outgrowth, but also inhibits cell death in the distal
limb mesoderm caused by removing the AER
(Fallon et al., ’94). It was suggested that the
onset of apoptosis in INZ is triggered by the
flattening of the AER which produces FGFs, especially FGF2, FGF4 and FGF8 (Niswander and
Martin, ’92a; Suzuki et al., ’92; Crossley and
Martin, ’95; Savage and Fallon, ’95; Macias et
al., ’96). Interestingly, applying FGF-soaked
beads to the chick interdigital regions inhibits
cell death and induces the formation of webbed
digits (Macias et al., ’96). However, this inhibition of cell death in the INZ is not accompanied by ectopic cartilage formation, suggesting
the existence of separate pathways leading to
these two fates which may be coordinately regulated or uncoupled. The proven ability of FGFs
to promote interdigital cell proliferation (Macias
et al., ’96) supports the view that they may
regulate this important process. The inhibition
of apoptosis by FGFs appears different from
that elicited by the removal of ectoderm. It was
noticed that removing the interdigital ectoderm
eliminates of Bmp4 expression, a factor mediating apoptosis (see below), and extracellular
matrix changes in the subjacent mesenchyme
(Hurle et al., ’96; Yokouchi et al., ’96). It seems
likely that upon removal of the overlying ectoderm, inhibition of apoptosis due to local downregulation of Bmp4 occurs first, and is followed
by ectopic cartilage formation induced by extracellular matrix changes. Changes of extracellular matrix components and organization
are known to accompany inhibition of cell death
in INZ and limb chondrogenesis (Solursh et al.,
’84; Zanetti and Solursh, ’86; Hurle et al., ’96).
Nevertheless, the molecular basis underlying
the complex influence of ectoderm on apoptosis
and chondrogenesis in INZ remains unclear and
warrants further study.
In the last few years, the most exciting progress
in research on apoptosis in the developing limb
has been the identification of Bone Morphogenetic
Proteins (BMPs) as signals triggering apoptotic
pathways. The BMP family consists of many secreted members that are conserved in evolution
and have been implicated in many aspects of vertebrate development (reviewed in Hogan, ’96).
Among them, Bmp2, Bmp4, and Bmp7 exhibit
similar temporal and spatial patterns of expression in the developing vertebrate limbs (Lyons et
al., ’90, ’95; Jones et al., ’91; Francis et al., ’94).
In particular, all three genes are expressed in the
interdigital regions prior to and during the occurrence of apoptosis (Fig. 2), suggesting a role in
the cell death.
The first evidence of a role for BMPs in apoptosis came from the study in which BMP4 was
shown to mediate apoptosis of neural crest cells
from rhombomeres 3 and 5 (Graham et al., ’94).
Subsequently, implantation of BMP4-soaked beads
in the interdigital regions was shown to accelerate interdigital cell death. Ectopic cell death was
observed when a BMP4 bead was implanted at
the tip of the developing digit pad (Gañan et al.,
’96). The death-inducing effect of BMP4 could be
antagonized by FGF2. A similar antagonism between FGFs and BMPs has been documented in
Fig. 2. Expression of Bmp2, Bmp4 and Bmp7 in E12.5
and E13.5 mouse limb buds. The expression of Bmps was
detected by whole mount in situ hybridization. In all panels,
dorsal view is shown and the anterior is to the top.
the AER’s induction of limb outgrowth (Niswander
and Martin, ’92b). Similarly, BMP2 and BMP7
were also shown to be potent apoptotic signals for
the undifferentiated limb mesenchyme, but not for
the ectoderm or the differentiating chondrogenic
cells (Macias et al., ’97). BMP signaling is mediated by Type I and Type II BMP receptors, which
are transmembrane serine/threonine kinases (reviewed in Massagué, ’96). Once BMP ligands bind
to both receptors, the Type II receptor phosphorylates the Type I receptor which then transduces
the signal into the cell by phosphorylating intracellular targets. Zou and Niswander (’96) and
Yokouchi et al. (’96) have overexpressed dominant
negative BMP receptors in chick leg buds via replication-competent retroviruses to block endogenous BMP signaling pathway. This results in
inhibition of apoptosis in the interdigital mesenchyme, which leads to formation of webbed
chicken feet. These results indicate BMP signaling is necessary for, and may be the actual mediator triggering, the apoptotic cascade in the
interdigital mesenchyme. Unfortunately, Bmp2
and Bmp4 knockout mice die too early to provide
further evidence for the function of these signals
in limb-bud cell death (Winnier et al., ’95; Zhang
and Bradley, ’96). While hindlimb polydactyly involving the presence of a single extra preaxial
digit is indeed observed in Bmp7-null mutant
mice, interdigital cell death occurs normally in
the mutant (Dudley et al., ’95; Luo et al., ’95).
The formation of extra preaxial digit in the Bmp7
mutants correlates with an altered Hoxd-13 expression. It is very likely that the loss of Bmp7
function in the interdigital regions is compensated
by Bmp2 and Bmp4.
Since Bmps are expressed in the interdigital
mesenchyme where apoptosis takes place, inhibition of cell death by blocking the BMP signaling
pathway suggests that BMPs function in an
autocrine manner to induce apoptosis. The correlation of the onset of apoptosis in the INZ with
the cessation of AER function suggests that BMP
signaling for apoptosis is overcome by a signal
from the AER. Once that influence is removed as
the AER flattens, the unopposed BMP signaling
activates the apoptotic cascade. This AER influence is quite possibly mediated by FGFs, as evidenced by the fact that a functional AER expresses
Fgfs and FGF antagonizes BMP’s ability to induce apoptosis (Gañan et al., ’96; Macias et al.,
’97). FGFs may inhibit cell death by activating
the tyrosine kinase activity of its receptor which
plays a critical role in regulating cell survival. It
has been shown that overexpression of a transmembrane tyrosine phosphatase LAR in mammalian cells in culture induces cell death (Weng et
al., ’98).
Induction of apoptosis by BMPs also has been
demonstrated in several other developing organs,
including facial primordia, lung and teeth (Bellusci et al., ’96; Barlow and Francis-West, ’97;
Jervall et al., ’98). However, it should be noted
that not all cells appear to respond to BMP signaling in the same way. This is exemplified by
the fact that BMP2 induces both cell proliferation and cell death in the chick developing facial
primordia (Barlow and Francis-West, ’97). In addition, overexpression/misexpression of Bmp4 also
induces an increase in cell proliferation and in
cell death (Bellusci et al., ’96). These observations
may be explained by the expression of different
type of BMP receptor. It was recently shown that
different Type I BMP receptor isoforms possess
distinct roles in organogenesis, with BMPR-1A
regulating chondrogenesis while BMPR-IB is involved in apoptosis (Zou et al., ’97).
The Msx homeobox containing genes were originally identified on the basis of homeobox sequence
homology to the Drosophila msh gene (Hill et al.,
’89; Robert et al., ’89). In vertebrate developing
embryos, Msx1 and Msx2 are widely expressed in
many tissues and organs, including neural crest,
heart, teeth, hair and limb bud. More specifically,
these two genes are expressed at the sites of epithelial-mesenchymal interactions, suggesting their
role in organ formation by regulating this fundamental process (reviewed by Davidson, ’95). The
Msx genes’ participation in apoptosis in developing limb was first indicated by their expression
pattern (Coelho et al., ’91, ’92a; Suzuki et al., ’91).
Prior to digit ray formation, both Msx1 and Msx2
are expressed in the developing limb’s AER,
progress zone, and in the anterior and posterior
domains where the ANZ and the PNZ reside (Fig.
3A, D). Subsequently, both genes exhibit coincident expression in the ANZ, PNZ and INZ during
and after digits formation (Fig. 3B, C, E, F).
Growth factors such as BMPs and FGFs are capable of inducing and maintaining Msx1 and Msx2
expression in several developing organs, including tooth, face, and limb buds (Vainio et al., ’93;
Vogel et al., ’95; Wang and Sassoon, ’95; Chen et
al., ’96; Barlow and Francis-West, ’97). As discussed above, BMPs are known to induce apop-
Fig. 3. Expression of Msx1 and Msx2 in the developing
mouse limb buds. Whole mount in situ hybridization was used
to show the transcripts of both genes in E11.5, E12.5 and
E13.5 mouse limb buds. Dorsal view of limb buds is shown in
all panels. The anterior is to the top.
tosis in several developing organs. The direct association of Msx genes with BMPs in apoptosis
has been demonstrated in several experimental
systems. For example, BMP4 induces apoptosis
in the neural crest cells of odd-numbered rhombomeres, in the enamel knot of the tooth, and in
cultured P19 cells (Graham et al., ’94; Marazzi et
al., ’97; Jervall et al., ’98). In all these cases the
induction of apoptosis by BMP4 is accompanied
by up-regulation of Msx2 expression. Moreover,
constitutive ectopic Msx2 expression in P19 cells
markedly increases the apoptosis accompanying
aggregation (Marazzi et al., ’97). These results indicate that Msx2 mediates the apoptotic pathway
triggered by BMPs. The strikingly similar expression patterns of Msx genes and Bmp in the limb
bud strongly suggest that Msx genes may play
similar roles in limb apoptosis. Evidence supporting a correlation between Msx genes and cell death
in the developing limb bud came from the observations that expression of both Msx1 and Msx2
are suppressed in the anterior and posterior edges
of the wing buds in two polydactylous mutant
chick embryos, talpid2 and diplopodia-5 (Coelho
et al., ’92b, ’93; Krabbenhoft and Fallon, ’92). In
these mutants, cell death does not occur in the anterior and posterior edges of limb buds, with supernumerary digits developing from the anterior limb
mesoderm (Dvorak and Fallon, ’91). Reduced Msx1
and Msx2 expression was also observed in the duck
autopod (Gañan et al., ’98), where the reduced cell
death leads to webbing.
More direct evidence for the involvement of Msx
genes in limb apoptosis has been provided by the
knockout studies. Neither Msx1 (Satokata and
Maas, ’94) nor Msx2 knockout mice (Satokata et
al., unpublished data) exhibit limb defects. Since
Msx1 and Msx2 are expressed in overlapping domains in the limb bud, and because their protein
sequence differs by only two amino acids within
the DNA binding homeodomain, functional redundancy may exist during embryogenesis. Indeed,
mice carrying homozygous null mutations for both
Msx1 and Msx2 do exhibit a severely defective
limb phenotype, including webbed digits resulting from inhibition of interdigital apoptosis (Chen
et al., unpublished data). These results support a
role for Msx genes in the apoptotic pathway in
the developing limb. It should be noted that Msx
gene products expressed in the interdigital mesenchyme seem to be necessary but insufficient to
directly trigger apoptotic cascade there. This idea
is supported by the fact that Msx gene expression
in the interdigital mesenchyme is not modified by
removing the overlying ectoderm, an operation
that inhibits apoptosis and induces ectopic cartilage formation (Ros et al., ’94). In addition, inhibition of interdigital cell death by FGF treatment
is also not accompanied by modifications in Msx1
and Msx2 expression (Macias et al., ’96). Additional insight into the precise role of these genes
in the apoptotic cascade may be obtained by comparing signaling pathways in these older developing limbs where the AER is flattening and
interdigital apoptosis occurring, with those in earlier limb buds where removal of the posterior AER
dramatically reduces Msx2 expression in the underlying mesenchyme (Ros et al., ’92) and treatment
with FGF restores the expression (Fallon et al., ’94).
Although the general downstream targets of
Msx genes are unknown, Msx1 has been shown
to regulate Bmp4 expression in the developing
tooth germ (Chen et al., ’96). Similar Msx gene
function is also observed in the interdigital regions
of Msx1 and Msx2 double mutant mice where
Bmp4 is down regulated whereas Bmp2 and Bmp7
are not (Chen et al., unpublished data). Msx genes
thus participate in interdigital cell death by regulating Bmp4 expression which may directly trigger apoptosis. This idea is supported by a recent
finding that ectopic expression of Msx2 in the
chick developing limbs induces Bmp4 expression
and promotes apoptosis (Ferrari et al., ’98). However, it is still unclear how Msx gene products
function at the molecular level. In vitro studies
have indicated that both Msx1 and Msx2 proteins
function equally as general transcriptional repressors (Catron et al., ’96). Msx genes have recently
been proposed to play a critical role in vertebrate
organogenesis by controlling the expression of inductive factors, including BMPs (Chen et al., ’96;
Chen and Maas, ’98).
The developing limb is a classic model where
programmed cell death has been acknowledged for
decades as a fundamental morphogenetic mechanism. It is quite clear that cell death in the limb,
as in other developing organs and tissues, is a
manifestation of apoptosis, a precisely controlled
process involving the participation of many genes.
The rapid accumulation of new insight and tech-
niques has led to substantial progress in the cloning and identification of genes that are involved
in vertebrate apoptotic pathways. It is surprising,
therefore that so little is known about the role of
such “death” genes in the apoptosis occurring in
developing limbs. The assumption that these
genes operate in limb development exactly as they
do in other systems is, at a minimum, premature.
For example, neither Bcl-2 nor ICE knockout mice
exhibit limb defects (Veis et al., ’93; Li et al., ’95).
FGFs have been implicated as trophic factors that
regulate cell death in the limb bud by maintaining cell proliferation. Recent studies indicate that
BMPs may directly trigger apoptotic pathways,
while Msx gene products participate in the apoptotic process by regulating Bmp expression. However, very little is known about the downstream
targets of the apoptotic pathway initiated by BMP
signaling. We also need more information regarding whether Msx genes directly control Bmp expression and what mechanisms are involved at
the molecular level. Obviously, the molecular
study of cell death in the developing limbs is just
beginning. Major breakthroughs are expected in
the next few years from studies in this area. It is
likely that such studies will improve our understanding of the role of apoptosis not only in shaping the limbs, but also our understanding of the
role of this biological phenomenon in a variety of
developmental and disease processes.
Y.P.C. would like to dedicate this paper to the
memory of Michael Solursh, an excellent mentor
and friend, for his enhancement of our understanding of limb development. The authors are
grateful to Dr. Douglas Paulsen of Morehouse
Medical School for his critical reading of the
manuscript. During the preparation of this manuscript, we were supported by grants from the National Science Foundation, the American Heart
Association National Center, and a start-up fund
from Tulane University to Y.P.C.
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