THE JOURNAL OF EXPERIMENTAL ZOOLOGY 282:691–702 (1998) Shaping Limbs by Apoptosis YIPING CHEN1,2,3* AND XIANG ZHAO1 1 Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118 2 Molecular and Cell Biology Graduate Program, Tulane University, New Orleans, Louisiana 70118 3 Center for Bioenvironmental Research, Tulane University, New Orleans, Louisiana 70118 ABSTRACT 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. © 1998 WILEY-LISS, INC. 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: email@example.com 692 Y. CHEN AND X. ZHAO 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. PROGRAMMED CELL DEATH IN DEVELOPING LIMBS 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 CONTROL OF CELL DEATH IN LIMB BUD 693 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. 694 Y. CHEN AND X. ZHAO (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). REGULATION OF APOPTOSIS BY EPITHELIUM 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 CONTROL OF CELL DEATH IN LIMB BUD 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. 695 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. BONE MORPHOGENETIC PROTEINS TRIGGER APOPTOTIC CASCADE 696 Y. CHEN AND X. ZHAO 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). MSX GENES ARE COMPONENTS OF APOPTOTIC PATHWAY 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- CONTROL OF CELL DEATH IN LIMB BUD 697 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 698 Y. CHEN AND X. ZHAO 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). CONCLUSION 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. ACKNOWLEDGMENTS 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. LITERATURE CITED Ahuja, H.S., W. James, and Z. Zakeri (1997) Rescue of the limb deformity in Hammertoe mutant mice by retinoic acidinduced cell death. Dev. Dyn., 208:466–481. Alles, A.J., and K.K. Sulik (1989) Retinoic-acid-induced limbreduction defects: Perturbation of zones of programmed cell death as a pathogenetic mechanism. Teratology, 40:163–171. Barlow, A.J., and P.H. 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