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Programmed cell death in the embryonic vertebrate limb
Seminars in Cell & Developmental Biology 16 (2005) 261–269
Review
Programmed cell death in the embryonic vertebrate limb Vanessa Zuzarte-Luis, Juan M. Hurle ∗ Departamento de Anatomia y Biologia Celular, Universidad de Cantabria, C/Cardenal Herrera Oria, s/n, 39011 Santander, Cantabria, Spain Available online 16 January 2005
Abstract The developing limb bud provides one of the best examples in which programmed cell death exerts major morphogenetic functions. In this work, we revise the distribution and the developmental significance of cell death in the embryonic vertebrate limb and its control by the BMP signalling pathway. In addition, paying special attention to the interdigital apoptotic zones, we review current data concerning the intracellular death machinery implicated in mesodermal limb apoptosis. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Caspases; BMPs; Morphogenesis
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell death in the limb bud mesoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ectodermal cell death. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BMPs are the apoptotic triggering signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of BMP signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell death machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
2. Cell death in the limb bud mesoderm
The vertebrate limb has always been an important developmental context for the study of programmed cell death during embryogenesis as in the developing appendages cell death is a constant feature. The correlation between the areas of cell death and limb morphogenesis has been reported in amniote embryos including birds, reptiles and mammals (see Ref. [1]). In contrast, in the anamniote embryos limb development occurs in absence of cell death [2].
The most remarkable apoptotic areas of the limb bud are observed in the undifferentiated mesodermal cells. In the avian embryo, all these areas seem to have the role of regulating the amount of pre-skeletal cells accounting for skeletal morphogenesis. The elimination of interdigital cells (Interdigital Necrotic Zones; INZ) in species with free digits constitutes a paradigmatic example of morphogenetic cell death. In amniota embryos, digits develop as chondrogenic condensations separated by interdigital regions containing undifferentiated mesenchymal cells. The fate of these interdigital cells is dependent on the final morphology of the digits in each species (Fig. 1). In species with free digits such as chicken [3,4], lizard [5], and mouse or human [6,7], apop-
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Corresponding author. Tel.: +34 942 201922; fax: +34 942 201903. E-mail address: [email protected] (J.M. Hurle).
1084-9521/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2004.12.004
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Fig. 1. Pattern of interdigital cell death in different species after neutral red vital staining and ectopic interdigital chondrogenesis resulting from inhibition of interdigital cell death in the chick (lower right panel).
tosis extends through all the interdigital space. In species with webbed digits such as duck [3,8], or tortoise [5] apoptosis is limited to the distal part of the interdigit. In species with autopods of singular morphology, such as the moorhen (Gallinula chloropus) or the coot (Fulika atra) [9], which have digits with lateral membranous lobulations or the split autopod of the chameleons [10], the pattern of interdigital cell death correlates closely with the specific phenotype of each species. Numerous experimental approaches have shown that the interdigital mesoderm contains cells with chondrogenic potential and when cell death is inhibited they are able to form an extra digit (Fig. 1; [11]). In addition to the INZ there are other well characterized areas of apoptosis which are also correlated with the skeletal pattern of the limb. In the early avian limb two areas of cell death, the anterior necrotic zone (ANZ) and the posterior necrotic zone (PNZ) have been related with the reduced number of digits in birds, since they are absent in the poly-
dactylous avian mutants [12]. In accordance with this interpretation in the mouse pentadactylous limb ANZ and PNZ are not present [13]. In contrast with that avian limb, the early limb bud of mouse and rat embryos exhibit a pattern of mesodermal cell death functionally associated with limb outgrowth and with the regression of the AER ([14,15] and see below). A further area of cell death is also present in the central limb mesenchyme of avian embryos. This area which has been termed the opaque patch (OP) accompanies the formation of independent rudiments for the zeugopodial bones (tibia-fibula; ulna-radius). The talpid3 chick mutant lacks this area of cell death and exhibits fusion of the zeugopodial skeletal pieces [16]. Although ANZ, PNZ, INZ and OP are the best characterized areas of cell death that occur during limb development, programmed cell death is observed in other limb domains with distinct and often unclear functions.
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It has been proposed that programmed cell death could play an important role in joint development. During digit development in early stages of joint formation the areas corresponding to presumptive phalangeal joints undergo apoptosis [17]. This cell death starts in the metacarpophlangeal joint and proceeds to more distal joints. The occurrence of cell death in this context suggests that it is important to determine the position of the future joint. It has been also proposed that cell death could play a role in the process of joint cavitation, however recent studies appear to discard this interpretation [18]. A further function proposed for mesodermal cell death is to delineate the axonal pathways. In 1988, Tosney et al. [19] reported a correlation between the spatial distribution of axon pathways and dying cells and phagocytes at the stage when growth cones first enter the avian hind limb, proposing the cell death is essential for growth cone guidance providing directional cues to the different axon populations. Additionally, cell death is detected in tissue sections of limb at late stages of development but their potential functions have been so far neglected.
3. Ectodermal cell death The most remarkable area of apoptosis observed in the limb ectoderm is located in the apical ectodermal ridge (AER). This area of cell death is particularly important since the AER directs the growth of the limb bud and its degeneration has dramatic consequences to the final morphogenesis of the limb. Different experimental approaches have shown that the AER is the signaling center, which regulates the outgrowth and the distal pattern of the limb. Removal of this structure is followed by the arrest of limb outgrowth while if it is experimentally duplicated two limbs instead of one are formed [20]. Physiologically, numerous dying cells are observed in the apical ectodermal ridge of all the studied species. Furthermore, Todt and Fallon [21] have revealed that the amount of cell death regulates the size of this structure resulting in a finetuning of the AER function throughout the different stages of limb development. An abnormal increase of cell death in the AER results in partial or total truncation of the limb [22] and cell death in the AER explains the absence of limbs in serpentiform reptiles [23]. Also of remarkable interest are the observations in mouse embryos showing that increasing or decreasing cell death in the AER results in changes in the number of digits [24,25]. A further function of AER concerning cell death is its influence on survival of the subjacent mesoderm. The AER is the source of FGFs, which maintain proliferation and survival in the underlying mesoderm (Progress Zone mesoderm, PZ; [26]). When the AER is excised the PZ mesoderm undergoes apoptosis by a mechanism involving the MAPK pathway [27].
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4. BMPs are the apoptotic triggering signals Programmed cell death in the limb bud shares many control mechanisms with proliferation and differentiation. Initial studies in chicken embryos unraveled the molecular mechanism responsible for the induction of apoptosis during limb development. The bone morphogenetic proteins (BMPs), a family of secreted proteins that belong to the transforming growth factor  superfamily, were identified as the triggering apoptotic signals for both the ectoderm of the AER [28,29] and mesodermal cells [30–34]. More recently, transgenic studies and in vitro experimental approaches confirmed these data in the mouse [35,36]. The BMP family members BMP-2, BMP-4, BMP-5 and BMP-7 [15] are expressed in the limb-undifferentiated mesoderm, in interdigital mesoderm and in the AER, in coincidence with the areas of cell death. However, these BMPs are also involved in the control of limb patterning [37] and in the regulation of chondrogenic differentiation [34]. Local treatments with any of the above mentioned BMPs result in intense growth and differentiation of the prechondrogenic mesenchyme as well as massive apoptosis in the undifferentiated mesoderm [34]. BMPs exert their function through serine/threonine receptor kinases composed of type I and type II receptors. The type IA and IB receptor mediate the chondrogenic effect of BMPs [38] while the receptor implicated in the control of apoptosis awaits clarification (but see Ref. [39]). Although overexpression experiments using dominant negative type IB and type IA BMP receptors result in an inhibition of apoptosis [31,32] it is likely that the phenotype is caused by the depletion of BMPs, which are sequestered by the overexpressed receptors. Furthermore, interdigital induction of the type IB BMP receptor gene by application of TGF1 beads is followed by inhibition of apoptosis and formation of an ectopic digit [40], but overexpression of this receptor promotes apoptosis in early limb mesoderm [39]. The BMP intracellular signaling pathway is not completely known. There are at least two pathways by which BMPs exert their apoptotic effect (Fig. 2; [15]). One implies the Smad proteins. Upon binding of the BMP ligand with the receptor Smads 1, 5 and 8 are phosphorylated coassembled with a cofactor, Smad 4, and translocated into the nucleus where they activate gene transcription. The other pathway described to date is the mitogen activated protein kinase (MAPK) pathway. Thus, the p38 kinase is expressed in the interdigital tissue and is activated by BMP during interdigital tissue regression [15]. BMPs are also able to activate other MAPKs in different cell types (see Ref. [41]). For example, TAK1 (TGF activated kinase) can be activated by BMP-2 and BMP-4 and plays a role inducing apoptosis in some model systems [42]. TAK1 activates the Jnk signaling which in turn activates cjun protein that is able to induce apoptosis. It was also shown that Jnk mediates BMP induction of Dickkopf-1 (Dkk), an inhibitor of the Wnt/-catenin signalling, which has been im-
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Fig. 2. Schematic representation of BMP signaling pathway implicated in the control of interdigital apoptosis. Only factors reported to be active in the control of interdigital tissue regression have been represented. Inhibitory molecules are colored in grey. Active signaling molecules are colored in black.
plicated in the control of limb cell death ([43] and references therein). There is evidence that favor an interaction between the canonical pathway that involves Smad proteins phosphorylation and the MAPK pathway [15,42,44]. This interaction would explain the different cellular responses to BMP signaling in different physiological contexts.
5. Regulation of BMP signaling The regulation of BMP signaling is highly complex and far from being totally known. There are a number of antagonists that modulate the intensity and/or the spatial distribution of the BMP signal at extracellular level. BAMBI [15] which is a pseudo receptor that lacks the intracellular domain, and the secreted factors Noggin [45–47] Gremlin [48,49], DAN [50], Chordin [51], Follistatin [52], Drm [50], are some of the antagonists that are expressed in the developing limb (see Fig. 2
for details of their distribution in interdigits). Gain of function experiments of these antagonists result in the inhibition of cell death both in mesoderm and ectoderm [29,47,49]. The extracellular matrix (ECM) is another potential modulator of BMP signaling regulating the local delivery or the availability of the BMP proteins [53,54]. BMP signaling may be also positively regulated by other secreted signals. It was reported that crossvein2 (cv2) is expressed in the regressing interdigital tissues and enhances Smad phosphorylation by BMPs [55]. Regulation of BMP signaling may also act at the intracellular level. In this regard zfhx 1a and zfhx 1b are intracellular factors, expressed in the interdigital mesenchyme, which may modulate the transcriptional activity of Smad protein (Fig. 2; [56,57]). Furthermore, the BMP signaling also interacts with other signaling pathways implicated in limb development. The FGF signaling is responsible for limb outgrowth and is also involved in the control of mesenchymal cell death mediated by BMPs. Montero et al. [58], clearly showed that FGFs co-
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operate with BMPs in the control of mesodermal apoptosis although the nature of this cooperation remains unclear. BMPs are not able to trigger apoptosis upon FGF-signaling blockage by specific inhibitors. Furthermore, it was suggested that the reduced pattern of interdigital apoptosis observed in the interdigital webs of the duck is due to a decrease in FGFsignaling rather than caused by absence of BMPs [28,58]. Retinoic acid signaling is another pathway that interacts with BMPs in the control of apoptosis, besides its major role in limb patterning. In mouse, inhibition of interdigital cell death and subsequent syndactyly has been reported in a variety of mutations of retinoic acid receptor genes (see Ref. [59]). Furthermore, the phenotype of the hammertoe mutant caused by defective apoptosis can be partially rescued by administration of retinoic acid to the pregnant females [60]. In the chick, we have observed that retinoic acid acts in concert with BMPs to establish the interdigital regions [61]. The function of RA-signaling consists of promoting the apoptotic effect of BMPs and at the same time inhibiting the chondrogenic effect of these factors. This may be of considerable importance for normal morphogenesis since in the developing autopod BMPs not only induce apoptosis but also promote a dramatic growth of the cartilage.
6. Cell death machinery In all areas of cell death of the developing limb dying cells are TUNEL positive and have morphological features of apoptosis (see review [15]). However, the molecular machinery responsible for cell degradation awaits clarification. It is now well established that, in most cell types, there are at least two distinct mechanisms responsible for apotosis: the extrinsic pathway (“receptor pathway”) which is initiated through specific transmembrane receptors belonging to the superfamily of the tumor necrosis factor receptors (TNF-receptors); and the intrinsic pathway (“mitochondrial pathway”) which is triggered by changes in the permeability of the mitochondrial membrane followed by the release of cytochrome c into the cytosol. Both pathways after several intermediated steps activate the so-called effector caspases (caspase 3, 6, or 7), which are proteases responsible for cell degradation (caspase = cysteinyl aspartate − specific protease). The intermediate steps in both pathways implicate the activation of other caspases by proteolytic cleavage of their pro-domain. These caspases are termed the initiator caspases and exhibit specificity for each pathway. Caspase 8 is an initiator caspase of the extrinsic pathway while caspase 9 is specific for the intrinsic pathway. It is remarkable that in many cell populations, the intrinsic and the extrinsic apoptotic pathways can be interconnected at different levels. Thus, activation of caspase 8 in the extrinsic pathway can result in the permeabilization of the mitochondrial membrane through the cleavage of Bid, a BH3 only member of the Bcl-2 family of apoptotic regulators (Fig. 3).
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In the developing limb, caspase 3 has been identified as the major effector caspase during physiological cell death [62,63] and in apoptosis induced by treatments with 4hydroxycyclophosphamide [63], and retinoic acid administration [64]. It is however remarkable that treatment of the limb bud with specific caspase 3 inhibitor peptides only reduces, but do not block, physiological or induced apoptosis [63]. Furthermore, mice deficient for caspase 3 lack a limb phenotype [65,66]. And, most important, it has been reported that interdigital cell death still occurs by a caspaseindependent pathway when caspases are fully inhibited [67]. Another controversial question in limb apoptosis concerns the pathway employed by the dying cells to activate caspase 3. In favor of a role of the extrinsic pathway it can be mention that a TNF-␣-like protein exhibits a specific expression in the regressing interdigits [68], and FLASH, a protein which interacts with the death effector domain of caspase 8 [69] is expressed in the regressing interdigital tissue of chick embryos [15]. However, on the basis of a reduced apoptotic response to cyclophosphamide of mice embryos lacking TNFalpha (TNF-␣−/− ) it has been suggested that in the developing limbs this factor may act as a protector against cell death rather than an apopototic signal [70]. In addition, Huang and Hales [63] failed to detect the activation of pro-caspase 8 (the specific caspase of the extrinsic apoptotic pathway) during interdigital apoptosis. The involvement of the intrinsic apoptotic pathway in the control of limb cell death has a stronger experimental support but there are still aspects, which remain confuse. In favor of its implication it can be mentioned the specific expression of Bax in the areas of interdigital cell death [59] and the occurrence of soft tissue syndactyly in mice deficient for both Bax and Bak [71]. These factors are apoptotic regulators belonging to the BH3-only homologues of Bcl-2, and promote the release of cytochrome c from the mitochondria [72]. Similarly, mice null for Apaf-1 are characterized by a deficient regression of the interdigital tissue [73]. Apaf-1 together with caspase 9 and the cytochrome c delivered by the mitochondria constitute the apoptosome, a central element of the apoptotic machinery of the intrinsic pathway. In support of the mitochondrial implication in apoptosis it must be also mentioned the interdigital expression of VDAC2 [15] a gene responding to the Bcl-2 family of apoptotic regulators involved in the control of mitochondrial permeability [74] and the presence of reactive oxygen species in the areas of limb programmed cell death [75]. In contrast with those findings, Huang and Hales [63] were unable to detect the activation of the procaspase 9 (the specific caspase of the intrinsic pathway) in normal limb apoptosis or after 4-hydroxycyclophosphamide treatments, and mice null for caspase 9 lack a limb phenotype [76]. However, vimentin fragments specifically cleaved by caspase 9 are present in the regressing interdigits of mouse embryos [77]. Additional evidence in support of the regulation of limb programmed cell death through the mitochondrial pathway is the specific absence during the stages of degeneration of anti-
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Fig. 3. Schematic representation of the extrinsic and the intrinsic death pathways accounting for apoptotic cell death.
apoptotic factors of this pathway, including Bcl-2 [78], Bag-1 [79] and A1 [80]. Another antiapoptotic factor proposed to be involved in the control of limb cell death is Dad-1 (Defender against apoptotic cell death) as mice null for this gene are syndactylous [81]. In addition of the above mentioned apoptotic molecules many other different factors have been implicated in the control of limb cell death or are specifically expressed in the areas of apoptosis, including: DIO-1 (death inducer obliterator-1, [82]); Gas1 and Gas2 (growth arrest specific; [83,84]; Zfp145 [85]; c-Fos [86]; c-jun [87]; Cyclin dependent kinase5 [88]; p53 [89,90,59]; Msx-2 [91]; c-rel [92]; Dickkopf-1 [43]; Snail and FgfR3 [58]; Tissue transglutaminase [93,59]; TRPM-2 [94]; Tissue plasminogen activator [95]; Insulin growth factor [96,97]; Transforming growth factor-2 and -3 [98]; Wnt signalling [99]; Shh [100]; Iroquois [101]; Dynein [102]; Slit ligand and Robo receptors [103] and components of the proteosome [104,15]. These factors may act through different mechanisms, such as regulating cell proliferation, cell adhesion; modulating the expression or the local distribution of BMPs, or acting as intermediate factors between BMPs and the apoptotic machinery. An increased amount of lysosomal components have been also detected in the areas of cell death [105–107]. Although, such increase may reflect the involvement of macrophages in the elimination of dead cells, its implication in the death
machinery can not be fully discarded, as Chautan et al. [67] reported the spontaneous activation of a necrotic cell death pathway when caspases are inhibited.
7. Concluding remarks The developing vertebrate limb provides several examples of morphogenesis mediated by apoptosis. Some of them, such as the formation of the digits, are illustrative for the role of cell death in the massive elimination of embryonic tissue to sculpt the shape of adult structures from initial primordia that contains an excess of cells. Interestingly, it has been well documented that the factors responsible for the growth and differentiation of the skeleton are also the factors, which trigger the apoptotic processes. In contrast, the molecular cascade responsible for the execution of apoptosis remains largely unknown.
Acknowledgements This work was supported by a grant from the Ministerio de Ciencia y Tecnolog´ıa to J.M.H. (BMC2002-02346). V.Z.L. was supported by a grant from the Fundac¸a˜ o para a Ciˆencia e a Tecnologia, Portugal (SFRH/BD/5834/2001).
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Review
Programmed cell death in the embryonic vertebrate limb Vanessa Zuzarte-Luis, Juan M. Hurle ∗ Departamento de Anatomia y Biologia Celular, Universidad de Cantabria, C/Cardenal Herrera Oria, s/n, 39011 Santander, Cantabria, Spain Available online 16 January 2005
Abstract The developing limb bud provides one of the best examples in which programmed cell death exerts major morphogenetic functions. In this work, we revise the distribution and the developmental significance of cell death in the embryonic vertebrate limb and its control by the BMP signalling pathway. In addition, paying special attention to the interdigital apoptotic zones, we review current data concerning the intracellular death machinery implicated in mesodermal limb apoptosis. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Caspases; BMPs; Morphogenesis
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell death in the limb bud mesoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ectodermal cell death. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BMPs are the apoptotic triggering signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of BMP signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell death machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
2. Cell death in the limb bud mesoderm
The vertebrate limb has always been an important developmental context for the study of programmed cell death during embryogenesis as in the developing appendages cell death is a constant feature. The correlation between the areas of cell death and limb morphogenesis has been reported in amniote embryos including birds, reptiles and mammals (see Ref. [1]). In contrast, in the anamniote embryos limb development occurs in absence of cell death [2].
The most remarkable apoptotic areas of the limb bud are observed in the undifferentiated mesodermal cells. In the avian embryo, all these areas seem to have the role of regulating the amount of pre-skeletal cells accounting for skeletal morphogenesis. The elimination of interdigital cells (Interdigital Necrotic Zones; INZ) in species with free digits constitutes a paradigmatic example of morphogenetic cell death. In amniota embryos, digits develop as chondrogenic condensations separated by interdigital regions containing undifferentiated mesenchymal cells. The fate of these interdigital cells is dependent on the final morphology of the digits in each species (Fig. 1). In species with free digits such as chicken [3,4], lizard [5], and mouse or human [6,7], apop-
∗
Corresponding author. Tel.: +34 942 201922; fax: +34 942 201903. E-mail address: [email protected] (J.M. Hurle).
1084-9521/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2004.12.004
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Fig. 1. Pattern of interdigital cell death in different species after neutral red vital staining and ectopic interdigital chondrogenesis resulting from inhibition of interdigital cell death in the chick (lower right panel).
tosis extends through all the interdigital space. In species with webbed digits such as duck [3,8], or tortoise [5] apoptosis is limited to the distal part of the interdigit. In species with autopods of singular morphology, such as the moorhen (Gallinula chloropus) or the coot (Fulika atra) [9], which have digits with lateral membranous lobulations or the split autopod of the chameleons [10], the pattern of interdigital cell death correlates closely with the specific phenotype of each species. Numerous experimental approaches have shown that the interdigital mesoderm contains cells with chondrogenic potential and when cell death is inhibited they are able to form an extra digit (Fig. 1; [11]). In addition to the INZ there are other well characterized areas of apoptosis which are also correlated with the skeletal pattern of the limb. In the early avian limb two areas of cell death, the anterior necrotic zone (ANZ) and the posterior necrotic zone (PNZ) have been related with the reduced number of digits in birds, since they are absent in the poly-
dactylous avian mutants [12]. In accordance with this interpretation in the mouse pentadactylous limb ANZ and PNZ are not present [13]. In contrast with that avian limb, the early limb bud of mouse and rat embryos exhibit a pattern of mesodermal cell death functionally associated with limb outgrowth and with the regression of the AER ([14,15] and see below). A further area of cell death is also present in the central limb mesenchyme of avian embryos. This area which has been termed the opaque patch (OP) accompanies the formation of independent rudiments for the zeugopodial bones (tibia-fibula; ulna-radius). The talpid3 chick mutant lacks this area of cell death and exhibits fusion of the zeugopodial skeletal pieces [16]. Although ANZ, PNZ, INZ and OP are the best characterized areas of cell death that occur during limb development, programmed cell death is observed in other limb domains with distinct and often unclear functions.
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It has been proposed that programmed cell death could play an important role in joint development. During digit development in early stages of joint formation the areas corresponding to presumptive phalangeal joints undergo apoptosis [17]. This cell death starts in the metacarpophlangeal joint and proceeds to more distal joints. The occurrence of cell death in this context suggests that it is important to determine the position of the future joint. It has been also proposed that cell death could play a role in the process of joint cavitation, however recent studies appear to discard this interpretation [18]. A further function proposed for mesodermal cell death is to delineate the axonal pathways. In 1988, Tosney et al. [19] reported a correlation between the spatial distribution of axon pathways and dying cells and phagocytes at the stage when growth cones first enter the avian hind limb, proposing the cell death is essential for growth cone guidance providing directional cues to the different axon populations. Additionally, cell death is detected in tissue sections of limb at late stages of development but their potential functions have been so far neglected.
3. Ectodermal cell death The most remarkable area of apoptosis observed in the limb ectoderm is located in the apical ectodermal ridge (AER). This area of cell death is particularly important since the AER directs the growth of the limb bud and its degeneration has dramatic consequences to the final morphogenesis of the limb. Different experimental approaches have shown that the AER is the signaling center, which regulates the outgrowth and the distal pattern of the limb. Removal of this structure is followed by the arrest of limb outgrowth while if it is experimentally duplicated two limbs instead of one are formed [20]. Physiologically, numerous dying cells are observed in the apical ectodermal ridge of all the studied species. Furthermore, Todt and Fallon [21] have revealed that the amount of cell death regulates the size of this structure resulting in a finetuning of the AER function throughout the different stages of limb development. An abnormal increase of cell death in the AER results in partial or total truncation of the limb [22] and cell death in the AER explains the absence of limbs in serpentiform reptiles [23]. Also of remarkable interest are the observations in mouse embryos showing that increasing or decreasing cell death in the AER results in changes in the number of digits [24,25]. A further function of AER concerning cell death is its influence on survival of the subjacent mesoderm. The AER is the source of FGFs, which maintain proliferation and survival in the underlying mesoderm (Progress Zone mesoderm, PZ; [26]). When the AER is excised the PZ mesoderm undergoes apoptosis by a mechanism involving the MAPK pathway [27].
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4. BMPs are the apoptotic triggering signals Programmed cell death in the limb bud shares many control mechanisms with proliferation and differentiation. Initial studies in chicken embryos unraveled the molecular mechanism responsible for the induction of apoptosis during limb development. The bone morphogenetic proteins (BMPs), a family of secreted proteins that belong to the transforming growth factor  superfamily, were identified as the triggering apoptotic signals for both the ectoderm of the AER [28,29] and mesodermal cells [30–34]. More recently, transgenic studies and in vitro experimental approaches confirmed these data in the mouse [35,36]. The BMP family members BMP-2, BMP-4, BMP-5 and BMP-7 [15] are expressed in the limb-undifferentiated mesoderm, in interdigital mesoderm and in the AER, in coincidence with the areas of cell death. However, these BMPs are also involved in the control of limb patterning [37] and in the regulation of chondrogenic differentiation [34]. Local treatments with any of the above mentioned BMPs result in intense growth and differentiation of the prechondrogenic mesenchyme as well as massive apoptosis in the undifferentiated mesoderm [34]. BMPs exert their function through serine/threonine receptor kinases composed of type I and type II receptors. The type IA and IB receptor mediate the chondrogenic effect of BMPs [38] while the receptor implicated in the control of apoptosis awaits clarification (but see Ref. [39]). Although overexpression experiments using dominant negative type IB and type IA BMP receptors result in an inhibition of apoptosis [31,32] it is likely that the phenotype is caused by the depletion of BMPs, which are sequestered by the overexpressed receptors. Furthermore, interdigital induction of the type IB BMP receptor gene by application of TGF1 beads is followed by inhibition of apoptosis and formation of an ectopic digit [40], but overexpression of this receptor promotes apoptosis in early limb mesoderm [39]. The BMP intracellular signaling pathway is not completely known. There are at least two pathways by which BMPs exert their apoptotic effect (Fig. 2; [15]). One implies the Smad proteins. Upon binding of the BMP ligand with the receptor Smads 1, 5 and 8 are phosphorylated coassembled with a cofactor, Smad 4, and translocated into the nucleus where they activate gene transcription. The other pathway described to date is the mitogen activated protein kinase (MAPK) pathway. Thus, the p38 kinase is expressed in the interdigital tissue and is activated by BMP during interdigital tissue regression [15]. BMPs are also able to activate other MAPKs in different cell types (see Ref. [41]). For example, TAK1 (TGF activated kinase) can be activated by BMP-2 and BMP-4 and plays a role inducing apoptosis in some model systems [42]. TAK1 activates the Jnk signaling which in turn activates cjun protein that is able to induce apoptosis. It was also shown that Jnk mediates BMP induction of Dickkopf-1 (Dkk), an inhibitor of the Wnt/-catenin signalling, which has been im-
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Fig. 2. Schematic representation of BMP signaling pathway implicated in the control of interdigital apoptosis. Only factors reported to be active in the control of interdigital tissue regression have been represented. Inhibitory molecules are colored in grey. Active signaling molecules are colored in black.
plicated in the control of limb cell death ([43] and references therein). There is evidence that favor an interaction between the canonical pathway that involves Smad proteins phosphorylation and the MAPK pathway [15,42,44]. This interaction would explain the different cellular responses to BMP signaling in different physiological contexts.
5. Regulation of BMP signaling The regulation of BMP signaling is highly complex and far from being totally known. There are a number of antagonists that modulate the intensity and/or the spatial distribution of the BMP signal at extracellular level. BAMBI [15] which is a pseudo receptor that lacks the intracellular domain, and the secreted factors Noggin [45–47] Gremlin [48,49], DAN [50], Chordin [51], Follistatin [52], Drm [50], are some of the antagonists that are expressed in the developing limb (see Fig. 2
for details of their distribution in interdigits). Gain of function experiments of these antagonists result in the inhibition of cell death both in mesoderm and ectoderm [29,47,49]. The extracellular matrix (ECM) is another potential modulator of BMP signaling regulating the local delivery or the availability of the BMP proteins [53,54]. BMP signaling may be also positively regulated by other secreted signals. It was reported that crossvein2 (cv2) is expressed in the regressing interdigital tissues and enhances Smad phosphorylation by BMPs [55]. Regulation of BMP signaling may also act at the intracellular level. In this regard zfhx 1a and zfhx 1b are intracellular factors, expressed in the interdigital mesenchyme, which may modulate the transcriptional activity of Smad protein (Fig. 2; [56,57]). Furthermore, the BMP signaling also interacts with other signaling pathways implicated in limb development. The FGF signaling is responsible for limb outgrowth and is also involved in the control of mesenchymal cell death mediated by BMPs. Montero et al. [58], clearly showed that FGFs co-
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operate with BMPs in the control of mesodermal apoptosis although the nature of this cooperation remains unclear. BMPs are not able to trigger apoptosis upon FGF-signaling blockage by specific inhibitors. Furthermore, it was suggested that the reduced pattern of interdigital apoptosis observed in the interdigital webs of the duck is due to a decrease in FGFsignaling rather than caused by absence of BMPs [28,58]. Retinoic acid signaling is another pathway that interacts with BMPs in the control of apoptosis, besides its major role in limb patterning. In mouse, inhibition of interdigital cell death and subsequent syndactyly has been reported in a variety of mutations of retinoic acid receptor genes (see Ref. [59]). Furthermore, the phenotype of the hammertoe mutant caused by defective apoptosis can be partially rescued by administration of retinoic acid to the pregnant females [60]. In the chick, we have observed that retinoic acid acts in concert with BMPs to establish the interdigital regions [61]. The function of RA-signaling consists of promoting the apoptotic effect of BMPs and at the same time inhibiting the chondrogenic effect of these factors. This may be of considerable importance for normal morphogenesis since in the developing autopod BMPs not only induce apoptosis but also promote a dramatic growth of the cartilage.
6. Cell death machinery In all areas of cell death of the developing limb dying cells are TUNEL positive and have morphological features of apoptosis (see review [15]). However, the molecular machinery responsible for cell degradation awaits clarification. It is now well established that, in most cell types, there are at least two distinct mechanisms responsible for apotosis: the extrinsic pathway (“receptor pathway”) which is initiated through specific transmembrane receptors belonging to the superfamily of the tumor necrosis factor receptors (TNF-receptors); and the intrinsic pathway (“mitochondrial pathway”) which is triggered by changes in the permeability of the mitochondrial membrane followed by the release of cytochrome c into the cytosol. Both pathways after several intermediated steps activate the so-called effector caspases (caspase 3, 6, or 7), which are proteases responsible for cell degradation (caspase = cysteinyl aspartate − specific protease). The intermediate steps in both pathways implicate the activation of other caspases by proteolytic cleavage of their pro-domain. These caspases are termed the initiator caspases and exhibit specificity for each pathway. Caspase 8 is an initiator caspase of the extrinsic pathway while caspase 9 is specific for the intrinsic pathway. It is remarkable that in many cell populations, the intrinsic and the extrinsic apoptotic pathways can be interconnected at different levels. Thus, activation of caspase 8 in the extrinsic pathway can result in the permeabilization of the mitochondrial membrane through the cleavage of Bid, a BH3 only member of the Bcl-2 family of apoptotic regulators (Fig. 3).
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In the developing limb, caspase 3 has been identified as the major effector caspase during physiological cell death [62,63] and in apoptosis induced by treatments with 4hydroxycyclophosphamide [63], and retinoic acid administration [64]. It is however remarkable that treatment of the limb bud with specific caspase 3 inhibitor peptides only reduces, but do not block, physiological or induced apoptosis [63]. Furthermore, mice deficient for caspase 3 lack a limb phenotype [65,66]. And, most important, it has been reported that interdigital cell death still occurs by a caspaseindependent pathway when caspases are fully inhibited [67]. Another controversial question in limb apoptosis concerns the pathway employed by the dying cells to activate caspase 3. In favor of a role of the extrinsic pathway it can be mention that a TNF-␣-like protein exhibits a specific expression in the regressing interdigits [68], and FLASH, a protein which interacts with the death effector domain of caspase 8 [69] is expressed in the regressing interdigital tissue of chick embryos [15]. However, on the basis of a reduced apoptotic response to cyclophosphamide of mice embryos lacking TNFalpha (TNF-␣−/− ) it has been suggested that in the developing limbs this factor may act as a protector against cell death rather than an apopototic signal [70]. In addition, Huang and Hales [63] failed to detect the activation of pro-caspase 8 (the specific caspase of the extrinsic apoptotic pathway) during interdigital apoptosis. The involvement of the intrinsic apoptotic pathway in the control of limb cell death has a stronger experimental support but there are still aspects, which remain confuse. In favor of its implication it can be mentioned the specific expression of Bax in the areas of interdigital cell death [59] and the occurrence of soft tissue syndactyly in mice deficient for both Bax and Bak [71]. These factors are apoptotic regulators belonging to the BH3-only homologues of Bcl-2, and promote the release of cytochrome c from the mitochondria [72]. Similarly, mice null for Apaf-1 are characterized by a deficient regression of the interdigital tissue [73]. Apaf-1 together with caspase 9 and the cytochrome c delivered by the mitochondria constitute the apoptosome, a central element of the apoptotic machinery of the intrinsic pathway. In support of the mitochondrial implication in apoptosis it must be also mentioned the interdigital expression of VDAC2 [15] a gene responding to the Bcl-2 family of apoptotic regulators involved in the control of mitochondrial permeability [74] and the presence of reactive oxygen species in the areas of limb programmed cell death [75]. In contrast with those findings, Huang and Hales [63] were unable to detect the activation of the procaspase 9 (the specific caspase of the intrinsic pathway) in normal limb apoptosis or after 4-hydroxycyclophosphamide treatments, and mice null for caspase 9 lack a limb phenotype [76]. However, vimentin fragments specifically cleaved by caspase 9 are present in the regressing interdigits of mouse embryos [77]. Additional evidence in support of the regulation of limb programmed cell death through the mitochondrial pathway is the specific absence during the stages of degeneration of anti-
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Fig. 3. Schematic representation of the extrinsic and the intrinsic death pathways accounting for apoptotic cell death.
apoptotic factors of this pathway, including Bcl-2 [78], Bag-1 [79] and A1 [80]. Another antiapoptotic factor proposed to be involved in the control of limb cell death is Dad-1 (Defender against apoptotic cell death) as mice null for this gene are syndactylous [81]. In addition of the above mentioned apoptotic molecules many other different factors have been implicated in the control of limb cell death or are specifically expressed in the areas of apoptosis, including: DIO-1 (death inducer obliterator-1, [82]); Gas1 and Gas2 (growth arrest specific; [83,84]; Zfp145 [85]; c-Fos [86]; c-jun [87]; Cyclin dependent kinase5 [88]; p53 [89,90,59]; Msx-2 [91]; c-rel [92]; Dickkopf-1 [43]; Snail and FgfR3 [58]; Tissue transglutaminase [93,59]; TRPM-2 [94]; Tissue plasminogen activator [95]; Insulin growth factor [96,97]; Transforming growth factor-2 and -3 [98]; Wnt signalling [99]; Shh [100]; Iroquois [101]; Dynein [102]; Slit ligand and Robo receptors [103] and components of the proteosome [104,15]. These factors may act through different mechanisms, such as regulating cell proliferation, cell adhesion; modulating the expression or the local distribution of BMPs, or acting as intermediate factors between BMPs and the apoptotic machinery. An increased amount of lysosomal components have been also detected in the areas of cell death [105–107]. Although, such increase may reflect the involvement of macrophages in the elimination of dead cells, its implication in the death
machinery can not be fully discarded, as Chautan et al. [67] reported the spontaneous activation of a necrotic cell death pathway when caspases are inhibited.
7. Concluding remarks The developing vertebrate limb provides several examples of morphogenesis mediated by apoptosis. Some of them, such as the formation of the digits, are illustrative for the role of cell death in the massive elimination of embryonic tissue to sculpt the shape of adult structures from initial primordia that contains an excess of cells. Interestingly, it has been well documented that the factors responsible for the growth and differentiation of the skeleton are also the factors, which trigger the apoptotic processes. In contrast, the molecular cascade responsible for the execution of apoptosis remains largely unknown.
Acknowledgements This work was supported by a grant from the Ministerio de Ciencia y Tecnolog´ıa to J.M.H. (BMC2002-02346). V.Z.L. was supported by a grant from the Fundac¸a˜ o para a Ciˆencia e a Tecnologia, Portugal (SFRH/BD/5834/2001).
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