Programmed Cell Death in Development
by Dr. Michael Wride and Dr. Leon Browder, Department of Biochemistry and Molecular Biology, University of Calgary
Why must some cells die for the good of the embryo?
Programmed cell death (PCD) is an important mechanism in both development and homeostasis in adult tissues for the removal of either superfluous, infected, transformed or damaged cells by activation of an intrinsic suicide program.
One form of PCD is apoptosis, (an ancient Greek word used to describe the "falling off" of petals from flowers or leaves from trees and was proposed by Kerr, Wyllie and Currie in 1972 to refer to the peculiar morphology of physiologically occurring cell death which plays a complementary but opposite role to mitosis in the regulation of animal cell populations).
Apoptosis is characterized by maintenance of intact cell membranes during the suicide process so as to allow adjacent cells to engulf the dying cell so that it does not release its contents and trigger a local inflammatory reaction. Cells undergoing apoptosis usually exhibit a characteristic morphology, including fragmentation of the cell into membrane-bound apoptotic bodies, nuclear and cytoplasmic condensation and endolytic cleavage of the DNA into small oligonucleosomal fragments (Steller, 1995). The cells or fragments are then phagocytosed by macrophages.
Signals that can trigger apoptosis can include
Extrinsic signals may either suppress or promote apoptosis, and the same signals may promote survival in one cell type and invoke the suicide program in others (Steller, 1995). For example, death receptors that are members of the tumour necrosis factor receptor (TNFR) family sit in cell membranes, but their intracellular domains have direct access to the cell death machinery that lies ready and waiting within the cell (Ashkenazi and Dixit, 1998). When members of the TNF ligand family bind to their receptors some of the pathways activated can include those that bring about cell death. Conversely, there are other factors, such as the neurotrophins, that bind to cell surface receptors and which act to prevent cell death!
Invocation of the suicide program involves the synthesis of specific messenger RNA molecules and their translation. PCD can sometimes be suppressed by inhibiting transcription or translation (Steller, 1995), which provides evidence that cell death is mediated by intrinsic cellular mechanisms.
I'm interested in life! Why do I want to think about death?
Cell death is currently the subject of considerable research activity. This interest stems, in part, from the potential for understanding oncogenesis and the possibility of exploiting the cell death program for therapeutic purposes. For example, inhibition of cell death might contribute to oncogenesis by promoting cell survival instead of death. Likewise, triggering cell death might provide the means for eliminating unwanted cells (e.g., tumor cells).
What on Earth has death got to do with development?
Recognition of PCD as a developmental mechanism dates back well over 100 years (Clark and Clark, 1996; this is an excellent account of old descriptions of "apoptosis" before there was "apoptosis"). Developmental processes that involve PCD include:
Elimination of transitory organs and tissues. Examples include phylogenetic vestiges (pronephros and mesonephros in higher vertebrates), anuran tails and gills and larval organs of holometabolous insects.
Tissue remodeling. Vertebrate limb bud development (Fig. 11.42, Saunders, 1982; Fig. 1, Saunders, 1966) is an example. If PCD fails, in formation of the digits, digits remain joined by soft tissue. Compare, for example, the situation in the chick and duck hind limbs. If chick limb mesoderm is combined with duck ectoderm, PCD fails and the digits remain joined (Saunders, 1966). This observation implicates the ectoderm in providing the signal to trigger PCD. Another example is formation of heart loops during vertebrate development. Depletion of cells in spinal ganglia occurs during development of the chick embryo. As shown in Table 11.1 (Saunders, 1982), there is precise chronological and spatial control over this process. Interestingly, injections of nerve growth factor reduce the frequency of cell death in the spinal ganglia. This observation provides a link between growth control and PCD.
Much of our current knowledge about the molecular genetics of PCD comes from work on Caenorhabditis elegans. The adult hermaphrodite C. elegans forms 1090 somatic cells, of which 131 die by apoptosis. There are four stages in apoptosis in worms, which are equally applicable to the sequence of apoptotic events in vertebrates (Steller, 1995):
A number of genes have been identified that regulate these processes in worms (see Fig. 1, Steller, 1995). Mutations affecting the final three stages affect all somatic cells, whereas genes affecting the death verdict affect very few cells. Execution itself is mediated by ced-3, ced-4 and ced-9 (ced = cell death defective).
Cloning of the apoptotic genes of C. elegans and their characterization have led to considerable understanding of the molecular events of apoptosis and to the identification of mammalian homologues of the apoptotic effectors. For example, ced-9 and ced-3 are homologous to the protooncogene bcl-2 and the cytokine processing enzyme ICE (interleukin-1ß-converting enzyme) respectively, while ced-4 is homologous to the apoptotic protease activating factor1 (Apaf1) gene. A further outline of these factors is presented below.
The Bcl-2 Family, Caspases, and Apaf1: Tools for Suicide.
The Bcl-2 family: homologues of ced-9
The bcl-2 gene was initially cloned and characterized as a candidate proto-oncogene involved in the t(14:18) translocation that is characteristic of the human B-cell malignancy follicular lymphoma (Tsujimoto et al. 1984) and is the vertebrate homologue of the C. elegans ced-9 gene. Bcl-2 facilitates survival of cells in which it is expressed and possesses a hydrophobic tail that allows it to associate with various cellular membranes, including those of the mitochondrial outer membrane, the endoplasmic reticulum and the nuclear envelope.
Opposing factors exist in the bcl-2 family
A number of ced-9/bcl-2 family members have been identified. Some of which, like bcl-2, protect cells from apoptosis (these include bcl-xl, bcl-w, and mcl-1) while others actually promote apoptosis (these include bax, bcl-xs, bad, and bak).
The members of the bcl-2 family are characterized by conserved amino acid sequence motifs. These are called bcl-2 homology (BH) domains and there are at least 4 different BH domains (BH 1-4) some of which confer pro-apoptotic activity, while others confer anti-apoptotic activity to the proteins in which they are present.
It is apparent from a number of studies that the various bcl-2 family members are able to dimerize with each other to reinforce or cancel out their cell death or cell survival promoting activities. During development, the expression of these molecules will influence whether cells in particular tissues at certain stages of development will survive or die.
A full discussion of bcl-2 family members can be found in Adams and Cory (1998).
Caspases: homologues of ced-3
Caspases are the vertebrate homologues of the product of the C. elegans ced-3 gene. The vertebrate prototype member of this family is ice (interleukin-1ß-converting enzyme). At least eleven vertebrate ICE-like proteins have been identified in vertebrates and these have subsequently been named caspases to signify the fact that they are proteolytic enzymes that specifically cleave at aspartate residues in target proteins within the cell during apoptosis.
For example, caspases cleave proteins called lamins that are associated with the nuclear matrix and which are essential for the structural support of the nucleus. As soon as the lamins are removed by caspases, the nucleus undergoes the characteristic morphological changes that are associated with apoptosis (described above). Although caspases do not directly degrade DNA (they are proteases), they can degrade a protein called DFF45, which is an inhibitor of the nuclease (the DNA munching molecule) responsible for the DNA fragmentation that generally accompanies apoptosis (Mitamura et al.,1998). The various caspases act in a cascade to activate each other (also by proteolytic cleavage). In fact, the involvement of caspases in apoptosis has been called "death by a thousand cuts" because proteins and DNA are rapidly sliced up into bits after the initial apoptosis-inducing stimulus!
A full discussion of caspases can be found in Thornberry and Lazebnik (1998).
Apafs: homologues of ced-4
For a long time there was no identified vertebrate homologue of the nematode ced-4 gene. However, recently a human homologue has been identified (Zou et al 1997). Various forms of cellular stress that promote apoptosis result in activation of pro-apoptotic bcl-2 family members like bax. Bax moves from the cytoplasm to the mitochondria and punctures holes in the outer mitochondrial membrane. This results in the release of cytochrome c from mitochondria and subsequently the binding of cytochrome c to Apaf1 to create an active complex, which in turn activates caspase-9 and finally caspase-3 (reviewed by Green, 1998).
The mouse Apaf1 gene has recently been identified and transgenic mice have been made that lack Apaf1 (Cecconi et al. 1998; Yoshida et al. 1998). These mice exhibited dramatic developmental defects associated with alterations in apoptosis during development and did not survive past about day 16 of development. The phenotype of these mice included severe craniofacial malformations, brain overgrowth, the persistance of the interdigital webs, and dramatic alterations in the development of the lens and retina! An important gene indeed!
When fibroblasts from these mice lacking Apaf1 were grown in culture, they showed an enhanced potential to survive in the presence of drugs such as staurosporine which would normally cause mouse fibroblasts to die. However, Apaf -/- thymocytes and T lymphocytes could be killed by drugs in culture showing that different systems for apoptosis exist in different tissues.
Finally, another fascinating and important finding from these studies is that it was shown that the human Apaf1 gene maps to a region of human chromosome 12 that has been implicated in a human genetic syndrome called Noonan syndrome. Individuals that possess this syndrome have many of the characteristic defects exhibited by the Apaf1 -/- mice, including craniofacial, limb, and retinal abnormalities.
How do Cells in Flies Die?
Recently, Drosophila has entered the apoptosis field (McCall and Steller, 1997) and it is certain that the combined use of the powerful genetic, molecular, biochemical, and cell biological techniques available in Drosophila will provide important insights into the genes involved in and the molecular mechanisms of apoptosis.
Cell death is important during embryonic development and metamorphosis in Drosophila. During embryonic development, apoptosis occurs in the head, epidermis and central nervous system. Further molecular characterization of mutants with defects in cell death has led to the identification of a region of the Drosophila genome (cytological region 75C) which contains a number of genes involved in cell death: reaper, head involution defective (hid), and grim.
This gene is specifically expressed in cells destined to die and its pattern of expression anticipates the pattern of cell death in the Drosophila embryo. Furthermore, the reaper gene can be induced by X-ray irradiation in cells of the Drosophila embryo that do not normally undergo cell death. The reaper gene encodes a protein that contains a putative sequence that has been called the death domain and this appears to be essential for conferring the cell death-promoting activity of the reaper protein.
Interestingly, a number of vertebrate proteins containing death domains have been identified, including the cell death promoting members of the tumour necrosis factor-receptor (TNFR) family and proteins that are essential for cell death signaling that associate with TNFRs, including the Fas Associated Death Domain (FADD) and the TNFR Associated Death Domain (TRADD; reviewed by Ashkenazi and Dixit, 1998).
head involution defective (hid)
hid mutants have low levels of cell death, particularly in the head region. hid is predicted to encode a novel protein with limited homology to the putative amino acid sequence of the reaper protein. hid is also expressed in cells that live as well as dying cells, but co-expression of hid and reaper in the midline glia of the Drosophila nervous system results in a dramatic increase in cell death when compared to that seen due to reaper alone. Thus, it appears that, despite the fact that the putative hid protein does not contain a death domain, hid acts in synergy with reaper to regulate Drosophila cell death.
grim appears to have a similar role to hid in modulating reaper activity, since it too does not contain a death domain.
Insights into the effects of the reaper, hid, and grim genes in Drosophila have been provided in studies in which these genes have been ectopically expressed in the Drosophila eye under the control of the eye-specific pGMR promoter. The effect of expression of these genes is complete eye ablation (removal) at high doses, while at lower doses of gene expression various intermediate eye phenotypes are observed.
The phenotype of the eyes is very sensitive to genes acting downstream of reaper, hid, and grim, so this has allowed for screening for mutations that act as genetic modifiers of reaper, hid, and grim activity; i.e. mutations that promote apoptosis are enhancers of eye defects, while mutations that inhibit cell death suppress these phenotypes.
One Drosophila gene identified in this way is the thread gene, which encodes a Drosophila homologue of the baculovirus Inhibitor of Apoptosis (IAP) protein (viral infected cells will try to kill themselves to prevent viral amplification and spread, but through the course of evolution viruses have developed the ability to produce proteins like IAP that inhibit apoptosis in cells that they infect).
Human homologues of IAP have been identified and these include the Neuronal Apoptosis Inhibitory Protein (NAIP), which has been shown to be involved in the muscle wasting disease Spinal Muscular Atrophy (SMA; Roy et al. 1995). Roy et al. showed that in individuals with SMA, mutations in the NAIP locus lead to a failure of normally occurring inhibition of motor neuron apoptosis and this contributes towards the death of motor neurons and brings about the SMA phenotype.
Drosophila homologues of caspases have been identified using degenerate PCR amplification from embryonic cDNA libraries. These Drosophila caspases have been termed DCP-1 and DrICE and they induce apoptosis in cultured cells to which they have been introduced. Mutations to these genes result in defects in the development of the trachea and a complete lack of imaginal discs. So far, no Drosophila homologues of bcl-2 gene family members have been identified, although it seems highly likely that they exist.
Why do some cells die and yet others survive?
There is evidence from work on higher organisms that extrinsic signals may protect cells from apoptosis by suppressing the suicide program (Raff, 1992). For example, the survival of developing neurons may depend upon neurotrophic factors secreted by their targets; a failure to receive sufficient stimulation results in death.
What is the advantage to the organism of using extrinsic signaling to sustain cell survival?
One possibility is that it could provide a simple system to eliminate cells that end up in the wrong place; without a signal to sustain them, rogue cells would be eliminated (Raff, 1992). Consider primordial germ cells, for example. In mammals, they originate in the hindgut and must migrate to the genital ridges, where they form the gametes. Those that fail to reach the genital ridges are eliminated, presumably because they are deprived of the signal (Steel factor) that is required for their survival in the genital ridges (De Felici and Pesce, 1994).
A disadvantage to the organism of the mechanism that necessitates signaling to prevent apoptosis is that its failure by mutation can lead to the survival of unwanted cells, which - paradoxically - can lead to death of the organism itself. On the other hand, an opportunity is presented by such a mechanism to allow investigators to devise means for targeting unwanted cells for destruction. This might be accomplished by harnessing tumor necrosis factor (TNF), which triggers apoptosis in some target cells. Prostate cancer is an example. The survival of prostate cells is dependent upon androgens; androgen depletion leads to a reduction in cell number by apoptosis. Recently, the dependence of prostate cells on androgens to avoid cell death has been exploited therapeutically by the use of androgen ablation to invoke apoptosis in prostate cancer cells and prolong survival in men with prostate cancer.
Significantly, resistance to androgen depletion correlates with overexpression of bcl-2, the human ced-9 homologue that acts as a brake on prostate cancer cell apoptosis (Raffo et al., 1995). Thus, escape from androgen sensitivity by overexpression of bcl-2 in a subset of prostate cancer cells leads to proliferation of these cells and, ultimately, death of the patient. If bcl-2 could be down-regulated, apoptosis of these cells could be invoked and the cancer controlled.
Ironically, there may be an inverse correlation between cell death and survival of the organism. Exploitation of this relationship holds much promise for therapeutic control of diseases such as cancer.
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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Friday, December 4, 1998