Morphology and significance of programmed cell death in the developing limb bud of the vertebrate embryo

MICROSCOPY RESEARCH AND TECHNIQUE 34236-246 (1996)

Morphology and Significance of Programmed Cell Death in the Developing Limb Bud of the Vertebrate Embryo JUAN M. HURLE, MARIA A. ROS, VICENTE CLIMENT, AND VIRGIN10 GARCIA-MARTINEZ Departamento de Anatom ta y Biologia Celular, Universidud de Cantabria, 39011 Santander, Spain (J.M.H., MA.R.1; Departamento de Ciencias Morfolcigicas, Universidad de Extremudura, 06071 Badujoz, Spain (V.C., V.G.-M.)

KEY WORDS Apoptosis, Lysosomes, Endonuclease, Extracellular matrix

ABSTRACT Cell death constitutes a basic mechanism accounting for many morphogenetic and histogenetic events during normal and abnormal development of embryonic organs and tissues. This article focuses on the major areas of mesodermal cell death occurring during vertebrate limb development. In early stages of limb development, cell death appears to reduce the amount of mesodermal tissue destined to form the anlage of the autopodium. In later stages, cell death plays a role sculpturing the shape of the digits. The morphology of the dying cells corresponds with apoptosis, but internucleosomal DNA fragmentation by endonuclease activation does not appear to be a precocious feature. The cell death program can be inhibited in vivo and in vitro by changing the environmental conditions of the prospective dying cells up to 6-10 h before death. In this review, we survey possible factors controlling the establishment of the cell death program. Infor- mation concerning the biochemical basis of cell death in the developing limb is also revised. Finally, the possible role of genes whose pattern of expression is coincident with the dying processes is discussed. o 1995 Wiley-Liss, Inc.

INTRODUCTION It is now widely recognized that cell death, as a con-

trolled behavior of the cells, plays a key role in growth, differentiation, and tissue homeostasis. The recogni- tion of the importance of this process is largely due to the early studies carried out on developing organisms. During embryonic development and larval metamorphosis, the formation of many parts of the body involves the elimination by cell death of large cell popu- lations or even all the cellular components of an organ rudiment (Hurle, 1988; Lockshin, 1981; Saunders, 1966; Snow, 1987). In some cases cell death has a direct effect on morphogenesis by sculpturing the shape of an organ. In other cases, cell death is involved in morphogenesis in a more subtle fashion. For example, the elimination of a small number of cells a t a precise location may have an important morphogenetic effect if these cells are required to maintain proliferation in the main cellular constituents of the developing organ. There are other examples in which cell death facili- tates the migration through the developing organs of cells coming from other locations or the rearrangement of the cellular constituents of an organ to achieve an appropriate cytoarchitectural organization. The development of the limb in the vertebrate embryo provides very illustrative examples of these kinds of participation of cell death in morphogenetic and histogenetic processes. It should be mentioned, however, that the involvement of cell death in vertebrate limb morphogenesis appears to be a characteristic only of the amniotas (Cameron and Fallon, 1977; Fallon and Cam- eron, 19771, and, while a general pattern of cell death in limb development can be described (Hinchliffe, 1982), variations among the different amniota species are abundant. These variations involve the spa-

tial distribution of the areas of cell death and are related in most cases with the characteristic limb morphology and skeletal pattern of each species. This article focuses on cell death during limb development in chick embryos, but significant differences with other avian, reptilian and mammalian species will be mentioned.

DISTRIBUTION AND DEVELOPMENTAL SIGNIFICANCE OF CELL DEATH IN THE

EMBRYONIC LIMB BUD The early limb primordium of the amniota embryos

is a simple structure. Each primordium appears as a bud growing on the lateral surface of the embryonic body and consists of an apparently homogeneous core of mesenchymal cells encompassed by ectoderm. At the distal margin of the bud, the ectoderm exhibits a specialized thickened region termed the apical ectodermal ridge (AER) which accounts for the proximodistal growth of the limb. Experimental amputation of the AER results in the arrest of limb outgrowth and massive degeneration of the subridge mesenchyme (Rowe et al., 19821, but normal development occurs when the amputated AER is replaced by the local administration of fibroblast growth factor in an appropriate carrier (Fallon et al., 1994; Niswander et al., 1993).

In the chick embryo, the primordia of the limbs appear by day 2-3 of incubation, and limb morphogenesis

Received June 6, 1994; accepted in revised form September 6, 1994. Address reprint requests to Juan M. Hurle, Departamento de Anatomia y

Biologfa Celular, Facultad de Medicina, Poligono de Cazofia, Santander 39011, Spain.

0 1996 WILEY-LISS, INC.

CELL DEATH IN THE DEVELOPING LIMB 237

Fig. 1. Scanning electron micrographs showing the morphology of the limb in the course of development. A: Initial appearance of the limb bud with its typical paddle shape (chick wing bud at stage 23). x 40. B The limb is elongated and the autopodial, digit-forming seg-

ment of the limb is appreciated in its distal end (chick leg bud at stage 25). X 20. C.D Appearance of the autopodium at the time when the digits become individualized (quail leg buds at stages 31 and 33). C: x 35. D: X 40.

is accomplished by day 8-8.5 of incubation (stage 34- 35) (Murray and Wilson, 1994). At the beginning, the limb bud exhibits a paddle shape (Fig. lA), but it becomes elongated as outgrowth progresses (Fig. 1B). Dif- ferentiation of the skeletal pieces of the limb follows a proximo-distal sequence and involves the condensation of the central mesenchyme in precartilaginous primordia of the definitive skeleton. The proximal part of the limb bud, the stylopodium, forms a single central skeletal piece which corresponds to the femurhumerus (hindlimb/forelimb). The zeugopodium (tibia-fibulaha- dius-ulna) appears next and contains two central condensations separated by undifferentiated mesenchyme. The autopodium is the last segment of the limb to be formed and contains the condensations of the tarsall carpal skeleton and the digital rays separated from each other by undifferentiated interdigital mesenchyme (Fig. lC,D). The muscles and the connective tissue differentiate around the cartilaginous skeleton.

Specification of the pattern of skeletal pieces along the proximo-distal axis (i.e., the sequence stylopodium, zeugopodium, and autopodium) and antero-posterior axis (i.e., the order of location of the five digits and the zeugopodial skeleton along the antero-posterior axis) has been related with the coordinated temporal and spatial expression of homeobox-containing genes of the Howl and H o d cluster respectively (see review by Iz- pisua-Belmonte and Duboule, 1992). However, concomitantly with the condensation of the precartilaginous elements, a large number of mesenchymal cells die in a very patterned fashion. As we will survey here, these dying processes have an important role in the establishment of the form and structure of the limb, but they do not appear to be controlled by the same mechanisms as those proposed for the skeleton (Ros et al., 1994).

In the early chick limb bud, there are three areas of massive mesodermal cell death that have been termed the anterior necrotic zone (ANZ), the posterior necrotic

238 J.M. HURLE ET AL.

zone (PNZ), and the opaque patch (OP). These areas of cell death exhibit a precise pattern of temporal and spatial distribution related with the establishment of the skeletal morphology of the limb (see review by Hinchliffe, 1982).

The ANZ and PNZ are characteristic of the limb buds of avian embryos and extend through the undifferentiated mesenchyme located anteriorly and posteriorly to the central chondrogenic core of the limb. The role of these areas appears to be the reduction of the antero- posterior axis of the limb, thus delimiting the amount of mesenchyme available for digit formation, giving rise to the characteristic three-digit pattern of the wing and four-digit pattern of the avian legs. These areas are not present in the tdpid3 chick mutant, characterized by polydactylous limbs with up to eight digits (Hinchliffe and Ede, 1967). The opposite occurs in the wingless mutant which has a very enlarged ANZ resulting in the loss of the wing (Hinchliffe and Ede, 1973). In the mouse and rat limb buds, which have a typical pentadactyl pattern, as in the tulpid3, there are neither an ANZ nor a PNZ comparable to those of the chick (Milaire and Rooze, 1983).

The OP is located between the two chondrogenic condensations of the skeletal pieces of the zeugopodium and has been observed in the fore- and hind-limb buds of most avian (Dawd and Hinchliffe, 1971) and mammalian (Alles and Sulik, 1989) species. Again this area of cell death is missing in the tulpid chick mutant in which the skeletal pieces of the zeugopodium appear partially fused or forming a single ill-defined chondrogenic mass (Hinchliffe and Thorogood, 1974).

At more advanced stages of limb development, an- other set of areas of cell death is observed in the mesoderm located between the digital rays (Fig. 2). These areas of cell death have been termed the interdigital necrotic zones (INZ) and are characteristic of all the amniota species. The temporal and spatial distribution of INZs is highly dependent on the morphogenesis of the digits. In species with free digits such as the chick (Fig. 2A) (Pautou, 1975; Saunders and Fallon, 1967), quail (Climent, 1986; Fallon and Cameron, 1977), lizard (Eurneces fasciatis) (Fallon and Cameron, 1977), mouse (Fig. 2D), or rat (Ballard and Holt, 19681, the INZs extends through almost all the interdigital mesoderm. In species with webbed digits such as the duck (Fig. 2B) (Saunders and Fallon, 1967; Hurle and Colvee, 1982) or the turtle (Chelydru serpentina and Chrysernyspicta) (Fallon and Cameron, 19771, INZs are limited to the distal part of the interdigit. In syndac- tylous mutant species, INZs are inhibited (Hinchliffe and Thorogood, 1974). Syndactyly induced by drug administration also involves inhibition of cell death in the INZs (Fernandez-Teran and Hurle, 1984; Tone et al., 1983). In avian species having free digits but with a membranous lobulation along the digital margins such as the moorhen (Gallinula chloropus) (Fig. 2C) or the coot (Fuliku atra), INZs are restricted to the central part of the interdigital tissue (Hurle and Climent, 1987). Finally, in species with peculiar patterns of digit distribution, INZs exhibit a distribution closely related with the morphological appearance of the digits. This is the case of the split autopodium of the common chame-

leon (Chamaeleo charnaeleo) (Hurle et al., 1987). On the basis of these observations, the formation of the free digits is usually referred to as a typical model of sculpturing morphogenesis carried out by cell death.

In addition to these major areas of mesodermal cell death, dying ectodermal cells are also present in the AER of the avian limb bud, where they appear to play a role in the control of the extension of that important structure (Todt and Fallon, 1986). This role of cell death in controlling the development of the AER seems to be more relevant in mammalian embryos (Milaire and Roze, 1983). In these species, ectodermal cell death forms well-defined foci, and its inhibition or delay in- creases the length of the AER and the amount of subridge mesenchyme, resulting in the induction of polydactyly (Naruse and Kameyama, 1982, 1986; Scott et al., 1977).

Other events of chick limb development involving cell death are the establishment of the axon pathways (Tosney et al., 19881, cavitation of the developing joints (Mitrovic, 19771, and probably the remodelling of the vascular pattern (Feinberg, 1991; Hurle et al., 1985). TISSUE AND CELL INTERACTION IN THE

ESTABLISHMENT OF THE AREAS OF CELL DEATH

The precise temporal and spatial patterns of distribution of cell death is a relevant characteristic of embryonic systems. Numerous studies have been made to clarify the basis of such a precise distribution of cell death. Early studies by Saunders and Fallon (Fallon and Saunders, 1968; Saunders and Fallon, 1967) on the PNZ in the chick limb bud showed that prospective PNZ cells undergo cell death on schedule after being excised and explanted to organ culture up to 30-40 h before death, suggesting the existence of an internal death clock in these cells. However, more recent studies have revealed that cell death in the limb bud is a more plastic process, and the death program can be inhibited by changing the environmental conditions of the prospective dying cells up to 6-10 h before death (Brewton and MacCabe, 1988; Hurle and Gaiian, 1986; Hurle et. al, 1991; MacCabe et. al., 1991).

In the last few years, we have analyzed possible mechanisms accounting for the establishment of the INZs in the avian limb bud. Our studies have revealed that the interdigital mesenchyme prior to death is highly chondrogenic when excised and explanted to organ culture conditions (Hurle et al., 1991; Macias et al., 1992). This chondrogenic capacity of the interdigital mesenchyme has also been demonstrated in mammalian embryos (Lee et al., 1993). The chondrogenic potentiality of the interdigital mesenchyme can be elic- ited in vivo by several procedures, such as the removal of a small ectodermal fragment from the interdigital space using microsurgical procedures (Hurle and Ganan, 19861, local trypsin microinjection (Hurle et al., 1991), amputation of one of the digital rays adjacent to the interdigit (Gafian et al., 19941, or removal and re- grafting in the original position of a small wedge of interdigital tissue (Gaiian et al., 1994; Hinchliffe and Horder, 1993). In all these in vivo experiments, not only is the interdigital mesenchyme diverted from the


Fig. 2. (Figure appears in color in the Color Figure Section imme- diately following page 258.) Light micrographs showing the pattern of interdigital cell death in leg buds of different species after vital stain- ing with neutral red. A,B,C: Leg buds of chick, duck, and moorhen embryos, respectively, at the stage of maximum development in the areas of interdigital cell death (Fig. 5A shows the appearance of areas of cell death at this stage in tissue sections). D: Pattern of interdigital

cell death in the mouse embryo. Note the different distribution of cell death in the second and third interdigital spaces of the different avian species. In the chick (A), cell death occupies all the interdigit. In the duck (B), cell death is only present in the distal part of the interdigits. In the moorhen (C), cell death is only present in the central part of the interdigits without affecting the mesenchyma of the margins of the digits. A: x 37. B: x 35. C: x 20. D: x 40.


Fig. 3. Chick leg bud at day 12 of incubation showing an extra digit in the third interdigital space (*), induced by surgical removal of the marginal interdigital ectoderm at day 6 of incubation (36 h before the onset of interdigital cell death). X 25.

death program, but it forms fully differentiated extra digits (Fig. 3) (Ganan et al., 1994; Hurle and Gafian, 1987; Hurle et al., 1989). On the basis of these results, we have proposed that the whole autopodium has digit- forming potentiality and that the establishment of each digit is accompanied by inhibition of the chondrogenic potential in the adjacent interdigital spaces followed by cell death (Ganan et al., 1994). A comparable mechanism involving formation of an excessive number of cells followed by regulation by cell death occurs during the development of the nervous system (see review by Oppenheim, 1991). In newal tissues, cells die when they fail to contact the appropriate target tissues which provide the neurons with the growth factors required for survival. It is not clear whether survival of the limb mesodermal cells also involves the participation of growth factors. MacCabe et al. (1991) have reported that the addition of fibroblast growth factor (FGF) to organ cultures of PNZ and OP rescues the cells from the death program. Local administration of FGF also inhibits cell death of the distal limb mesoderm induced by the amputation of the AER (Fallon et al., 1994). However, we failed to inhibit cell death in the interdigital spaces by local microinjection of several growth factors, including FGF, TGF(31, TGFp2, and EGF (Gafian et al., 1993).

Our observations point to a role of the extracellular

Fig. 4. Confocal micrograph of the chick foot at day 6.5 of incubation after elastin immunolabeling. Note the presence of prominent elastin fibrillar bundles in the interdigital spaces. This feature pre- cedes by 24 h the onset of interdigital cell death. x 40.

matrix in the inhibition of interdigital chondrogenesis and subsequent cell death. The formation of digits is accompanied by the establishment in the autopodium of a complex elastic matrix scaffold with specific features for digital rays and interdigital spaces (Fig. 4) (Hurle et al., 1994). The formation of this scaffold pre- cedes the onset of cell death, and all the procedures described above which inhibit interdigital cell death in vivo cause a precocious disorganization of the extracellular matrix in the interdigit (Hurle and Solursh, in preparation). The possible involvement of the extracellular matrix in the establishment of the INZ is supported by observations in other areas of cell death of the developing limb. The cells of the OP undergo cell death when they are explanted under organ culture conditions but not when they are disaggregated and cultured in monolayer conditions (MacCabe et al., 1991). Furthermore, cell death in the PNZ is inhibited by local ectoderm removal, which has an important effect on the organization of the underlaying extracellular matrix (Brewton and MacCabe, 1988). The involvement of the extracellular matrix in the control of cell survival or death has been well documented in other systems of cell death by apoptosis (Frish and Francis, 1994; Meredith et al., 1993) and is discussed in detail elsewhere in this issue.

MORPHOLOGICAL FEATURES OF CELL DEATH

In most cases, dying cells of the developing limb fol- low very closely the degenerative features described by Kerr and coworkers as characteristic of apoptotic processes (Kerr et al., 1972; Wyllie et al., 1980).

Dying cells exhibit both nuclear and cytoplasmic alterations detectable by light, transmission, and scanning electron microscopy (Figs. 5, 6). Early degenera-


tive changes consist of progressive condensation of the nucleus and cytoplasm. Nuclear condensation (pycno- sis) may take place uniformly or may begin in the marginal zone of the nucleus. Cytoplasmic condensation is accompanied by vacuolation of most of the cytoplasmic organelles and rounding of the cellular contour. In avian and reptilian species, the appearance of ribosome crystals (Fig. 5D) in the cytoplasm of dying cells (Mot- tet and Hammar, 1972) has been described, but the significance of this phenomenon remains obscure (Bar- bieri, 1979). Cell condensation is followed by cytoplasmic and nuclear fragmentation. The fragments resulting from this process appear in tissue sections as darkly stained spherules which usually contain a small nuclear fragment (Fig. 5B). The last stages of cell degeneration (Fig. 5C) involve extensive vacuolation of the cell organelles, rupture of the cytoplasmic membranes, and loss of nuclear basophilia (karyolisis). However, cells with these features are scarce in the areas of cell death of the limb since dying cells are quickly phagocytosed by the neighboring mesenchymal cells (Garcia-Martinez and Climent, 1985). Neverthe- less, it should be mentioned that the morphological features of these isolated dying cells a t advanced stages of degeneration are comparable with those characteristic of cell death by necrosis according to the classification of Kerr et al. (1972). In addition to these alterations, cells with healthy appearance but showing prominent cytoplasmic vacuoles with digested material are also observed (Fig. 5E). The possible significance of these cells will be discussed later.

The use of scanning electron microscopy allows degenerative events to be followed closely in the areas of mesodermal cell death (Hurle and Hinchliffe, 1978). The most characteristic feature of dying cells and their resulting fragments is a rounded profile which con- trasts with the stellate morphology of healthy mesenchymal cells (Fig. 6A). In the course of degeneration, the cell surface becomes pitted with small holes. When focal areas of cell death are first established, isolated dying cells and cell fragments are observed to be phagocytosed (Fig. 6A). As degeneration progresses, the presence of large phagocytes having a rounded contour and ovoid protrusions constitutes an addi- tional and prominent feature of the areas of cell death, when observed under scanning electron microscopy (Fig. 6B).

The origin of the phagocytic cells is a controversial question. Early phagocytes containing only one or two engulfed dead cells are morphologically indistinguish- able from limb mesenchymal cells. This suggests that, as described in other embryonic areas of cell death (Garcia-Porrero et al., 1984; Hurle et al., 19781, healthy limb mesenchymal cells become poised to re- move dying cells by phagocytosis. However, experiments by Cuadros et al. (1992) using chick-quail chimeras suggest that at least part of the phagocytic cells present in the areas of cell death of the developing limb are specialized macrophages of hemopoietic origin. This last possibility is strongly supported by recent studies using specific immunolabeling for macrophages (Hopkinson-Woolley et al., 1994; Rotello, et al., 1994).

BIOCHEMICAL BASIS OF THE CELL DEATH PROCESS

In an attempt to determine the cellular mechanisms accounting for the onset of cell death, a number of studies have focused on cytochemical and biochemical mod- ifications possibly involved in these degenerative events. A coherent picture can not yet be given of the cellular mechanism responsible for the initiation of cell death in the developing limb bud, but a number of relevant findings have been reported. DNA, RNA, and protein synthesis decrease in the prospective dying cells prior to the establishment of cell death in the PNZ and INZ (Pollak and Fallon, 1974, 1976; Tone et al., 1988). It has also been found that 24 h before death, the prospective dying cells of the interdigital spaces enter an S-period which could be critical for the commitment of cells to death (Ton6 et al., 1988).

Changes in the cell coat after lectin and ruthenium red labeling are not detected until the cells show advanced signs of degeneration (Hurle et al., 1981). Pos- sible changes in the aerobic energy metabolism due to a precocious mitochondria1 alteration within prospective dying cells have also been ruled out (Fallon et al., 1974).

Internucleosomal DNA fragmentation by endonuclease activation is only detected in INZ concomitantly with the appearance of cells with clear apoptotic features, but nonspecific DNA fragmentation is also present at the same time (Garcia-Martinez et al., 1993; Zakeri et. al., 1993). ,

Jiang and Kochhar (1992) have recently reported an increase in tissue transglutaminase activity in the initial stages of mesodermal cell death of the limb bud induced by retinoic acid administration. Whether this feature depends on the administration of retinoic acid or is a general feature of dying cells of the normal limb bud remains to be clarified. However, it has been reported that activation of tissue transglutaminase is a characteristic and precocious phenomenon in some cases of cell death by apoptosis, which prevents cellular disintegration prior to phagocytosis (Fesus et al., 1987; Piacentini, et al., 1991).

The formation of small phagocytic vacuoles (1-2 pm in diameter) accompanied by increased lysosomal acid phosphatase activity has been reported in mesenchy- ma1 cells prior to the establishment of areas of cell death (Hurle and Hinchliffe, 1978). Cells with these features but otherwise resembling viable cells are also present within the areas of cell death (Fig. 5E). The interpretation of these observations in relation with the death process is difficult. One interpretation may be that the irreversible commitment of cells to death is preceded by an initial autophagic process. However, it is also possible (Ballard and Holt, 1968) that cells containing acid-phosphatase positive vacuoles correspond to the hemopoietic macrophages described by Cuadros et al. (1992).

GENETIC CONTROL OF CELL DEATH It is now generally accepted that programmed cell

death is an active process requiring the expression of specific genes. In the nematode C. elegans, several

Fig. 5. Light (A) and transmission electron micrographs W E ) illustrating the morphological features of the dying cells of the INZ. A Semithin section of the third interdigital space of the chick leg bud at day 8 of incubation stained with toluidine blue. Note the abun- dance of isolated dark dead cells and large phagocytes in the mesodermal core of the interdigit. X 600. B Three dark cell fragments with typical features of apoptotic cells. Several nuclear fragments are identified in one of the apoptotic bodies (N). In the cytoplasm, in addition to the intense condensation, the presence of small vacuolations is also

a remarkable degenerating feature. x 7,000. C Detail of INZ at an advanced stage of degeneration (day 8 of incubation). Note that in addition to apoptotic cells (a), several dying cells (arrows) exhibit morphological features such as rupture of the cell membranes which are characteristic of necrosis. x 6,000. D Detailed view of an apoptotic cell from INZ showing the presence of ribosome crystals in the cytoplasm (arrowheads). x 36,000. E: Dividing mesenchymal cell of the INZ containing a prominent phagosome. This feature is often found in the otherwise healthy cells of the INZ. x 3,000.


Fig. 6. Scanning electron micrographs of the third interdigital space of the chick leg bud at the onset of cell death (A) (day 7 of incubation) and at an advanced stage of degeneration (B) (day 8 of incubation). A An isolated cell fragment (arrow) and a cell fragment being engulfed by a mesenchymal cell (arrowheads). Note the typical

stellate morphology of the still healthy mesenchymal cells contrast- ing with the rounded profile of the degenerating cells in B. B Most cells appear in the course of degeneration. Note also the presence of a large macrophage (F). x 3,000.


Fig. 7. Dark-field views of frontal sections depicting the autopodium of the chick leg bud at days 6 and 6.5 of incubation hybridized with msx-1 (A,C) and msx-2 (B) probes. A ,B Normal limbs showing the precise spatial pattern of expression of these genes in the interdigital spaces at the stages preceding cell death. C: The marginal

genes accounting for different events in programmed cell death have been identified: genes required for the onset of cell death (Yuan and Horvitz, 1992), genes protecting cells from programmed cell death (Hengart- ner et al., 1992), and genes required for the engulfment and degradation of dead cells (Ellis et al., 1991). In vertebrates, genes involved in the activation and inhibition of cell death have also been identified, but no general rule has been discovered which governs their participation in the different processes involved in cell death with morphological characteristics of apoptosis (see review by White, 1993). In the developing limb, several genes have been found to exhibit a temporal and spatial pattern of expression coincident with cell death. However, their role in the control of cell death remains obscure.

Studies carried out on both chick and mammalian embryos revealed that several genes have a characteristic pattern of expression in the interdigital mesenchyme during the period of establishment of the INZ. These genes are TRPM-2 (testosterone-repressed pros- tate message-2) (Buttyan et al., 19891, RAR-p gene (retinoic acid receptor p), CRBP-I gene (cellular retinol binding protein), and CRABP I1 gene (cellular retinoic acid binding protein) (Ruberte et al., 1992), BMP-2A gene (bone morphogenetic protein 2A) (Lyons et al., 19901, c-re1 (Abbadie et al., 19931, and the homeobox containing genes msx-1 and msx-2 (formerly Hox-7 and Hoz-8) (Coelho et al., 1991, 1992; Robert et al., 1989; Suzuki et al., 1991). Among all these genes, the TRPM-2 and c-re1 have also been reported in other biological models of cell death. TRPM-2 encodes a protein (SGP-2) secreted by the testicular Sertoli cells, and its possible role in INZ can be deduced by its expression in several tissues undergoing apoptotic cell death (But- tyan et al., 1989). The possible significance of TRPM-2 expression in the death process remains obscure (Ro- senberg et al., 1993). The lack of involvement of this gene in some cases of developmentally regulated cell death (Garden et al., 1991) indicates that its expression is not a general feature of all embryonic cell death processes.

ectoderm and the underlying mesenchyme of the interdigit have been removed from the region delimited by the arrows and sacrificed 10 h later. This procedure changes the fate of the interdigital mesoderm from death to chondrogenesis, but the expression pattern of msx genes is not modified. x 35.

High levels of c-re1 expression are characteristic of many areas of cell death in the developing chick embryo, including ANZ, PNZ, OP, and INZ. Furthermore, induced overexpression of this gene in bone marrow cells leads to cell death (Abbadie et al., 1993). But again, the possible significance of c-re1 in the dying process has not been clarified.

The relation between the expression of msx genes and cell death in the limb bud has often been proposed on the basis only of their concurrent pattern of distribution (Coelho et al., 1991, 1992; Robert et al., 1989; Suzuki et al., 1991). However, we have recently observed (Ros et al., 1994) that expression of these genes is not modified when cell death is inhibited in the interdigital spaces (Fig. 7).

CONCLUSIONS AND FUTURE PROSPECTS In amniotas, the development of form and structure

of the growing limb bud is accompanied by precise patterns of massive mesodermal cell death. Most evidence suggests that the massive areas of mesodermal cell death eliminate undifferentiated cells which are required only for a limited period of limb development. The relation of these areas of cell death with limb morphogenesis is established by the occurrence of predict- able skeletal anomalies of the limb when the pattern of cell death is modified in mutant species or under experimental conditions. Increased cell death results in the deletion or rudimentation of the limb skeleton, while inhibition of cell death causes increased chondrogenesis eventually leading to the formation of extra skeletal pieces.

Limb mesodermal cells appear to die due to the absence of survival factors rather than by the triggering of a precocious death clock as previous thought. The characteristics of the extracellular matrix and local concentrations of growth factors are envisaged as the most important survival factors for limb mesodermal cells. The precise characterization of these survival factors and their mode of action at the cellular and molecular levels constitute a major task in future studies of limb mesodermal cell death. Most recent studies

CELL DEATH IN TH

point to FGF-2 and FGF-4 as key survival factors for the developing limb mesoderm.

As in other programmed cell death processes, in absence of the survival factors, the prospective limb dying cells appear to activate a well-defined molecular path- way of cell death involving the expression of specific genes. However, in spite of the accessibility of the areas of cell death of the limb bud, the molecular biology of this topic is only at a preliminary stage of study. Ques- tions about the possible involvement of genes such as bcl-2 or p-53, which have been well characterized in the control of cell survival/death in several vertebrate tissues, are still awaiting an answer. Obviously, the characterization of the dying molecular machinery constitutes a further major task in future studies of cell death in the developing limb bud.

ACKNOWLEDGMENTS This work has been supported by a grant from the

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Morphology and significance of programmed cell death in the developing limb bud of the vertebrate embryo