Cell death during embryogenesis
Cell death during embryogenesis
Reproduction in multicellular organisms functions to pass the cell's genome on to the next generation. The genotype survives and continues through time as the phenotype of the organism itself dies. The organism can be considered the vehicle that the genome uses to get into the next generation. After the fertilization of two gametes, cell proliferation through mitosis must occur to increase the population of cells in the developing organism. However, cell death is also necessary for embryological development, differentiation and morphogenesis to occur. The health of all animals depends not only upon the production of new cells, but also on the orderly death and removal of superfluous cells when they are no longer necessary for the functioning of the whole organism. There are two different types of death that must be considered here. Normally people think of cell death as a traumatic, injurious, often accidental event - this is necrosis. There is another type of cell death that is essential to the normal growth and development of a multicellular organism. Apoptosis, or programmed cell death, is genetically programmed into cells and is activated only under very special circumstances. It plays a vital role in embryological development. It is a normal and necessary process that starts with the formation of the embryo and continues throughout life of the individual. It helps in the formation of body structures, is involved in forming memory and consciousness of the mind, and enables chemical recognition of pathogens by the immune system. Apoptosis works by activating specific genes to synthesize proteins that breakdown and package cytoplasm and chromatin for easy consumption by neighboring cells. For example, when the hand is developing in a human embryo during the fourth week, it appears as a tiny bump at the location of the future arm. By the end of the sixth week, the hand has traces of future finger bones connected by webs of tissue. Between the forty-sixth and fifty-second days of development, the webbing disappears, leaving separated fingers. The inter-digital webbing cells do not move or simply disappear, they actually die by cellular suicide according to a program set down in the DNA of their genome.
This built-in program for self-destruction is also found in other types of cells in adult tissues. Most animal cells are capable of apoptosis using cell-to-cell signaling to rid unwanted cells from the tissues. Apoptosis has many roles in development of an organism; the ridding of cells no longer functional in development or evolution, the elimination of cells needed by only one sex, the decrease in the number of germ cells able to be passed into the next generation, the elimination of cells that migrated to abnormal locations or lacked proper linkages with extra-cellular substance, and the elimination of cells that were originally produced in excess. How apoptosis works on the cellular level is not well understood, but it is known to take about three hours to complete. The vast majority of the cells are in the Go, nonproliferating phase of the cell cycle. A specific set of transcription factors is activated from the highly conserved Hox gene family in cells undergoing apoptosis. Many molecular messengers, cytokines and growth factors, are released to enable cell-to-cell signaling to activate the process. Apoptosis begins with the shrinking of the nucleus and cytoplasm of the cell. Then the chromatin and cytoplasm are partitioned off into apoptotic cell fragments at the cell surface in a process of exocytosis that resembles boiling. The subcellular fragments are then ingested by neighboring cells or macrophages (www.apopnet.com).
In many instances, cell death is observed in developing tissues but its function is not known, and it is unclear whether apoptosis is necessary for these processes to occur or if it is simply coincident with them. The study of Drosophila melanogaster mutants that completely lack developmental apoptosis has allowed this question to be addressed. These studies indicate that in these organisms' apoptosis is not necessary for some aspects of normal development (White et al., 1994). However it is now clear that the components of the apoptotic machinery are expressed in virtually all nucleated animal cells and that ...
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In many instances, cell death is observed in developing tissues but its function is not known, and it is unclear whether apoptosis is necessary for these processes to occur or if it is simply coincident with them. The study of Drosophila melanogaster mutants that completely lack developmental apoptosis has allowed this question to be addressed. These studies indicate that in these organisms' apoptosis is not necessary for some aspects of normal development (White et al., 1994). However it is now clear that the components of the apoptotic machinery are expressed in virtually all nucleated animal cells and that the activation of this machinery is controlled by a set of intracellular regulatory proteins that transduce signals from both inside and outside the cell. Apoptosis is mediated by the activation of a specific class of proteases known as caspases. These are constitutively present in cells as inactive zymogens with variable length amino-terminal prodomains (Cryns and Yuan, 1998). There are two classes of caspases based on their prodomains and roles in cell death. The initiator caspases, when activated, proteolytically activate the second class of caspases, effector caspases. The resulting cascade ends with the cleavage of many cellular targets including structural proteins and enzymes involved in gene expression, DNA replication and metabolic activities. The dying cell eventually fragments into apoptic bodies, which are subsequently engulfed by neighbouring cells or macrophages.
Drosophila melanogaster has proven to be a convenient organism for the study of apoptosis. The genes required for the induction of apoptosis in Drosophila have been cloned, as have numerous other factors involved in the initiation and execution of the cell death pathway (reviewed by Abrams, 1999). Additionally the development of the fruit fly from oogenesis through embryogenesis and metamorphosis into adulthood has been extremely well characterized and described through decades of work.
Microscope examination of acridine orange (AO) stained embryos shows that cell death is prominent and widespread during embryogenesis and that it occurs in relatively predictable spatial and temporal pattern (Abrams et al, 1993). The first dying cells are detected in the dorsal region of the head approximately 7 hours after egg laying, which corresponds to stage 11. As development proceeds, apoptic cell death becomes more prominent throughout the embryo, and corpses are engulfed by circulating macrophages. Time-lapse photography of these embryos shows that while cell death occurs in a consistent pattern, the precise spatial and temporal aspects of this pattern are somewhat variable, indicating that here is a certain degree of plasticity in embryonic cell death (Abrams et al., 1993). Regulators of cell death were identified as three genes, reaper (rpr), grim, and head involution defective (hid). Their gene products are involved in the initiation of all embryonic cell death in Drosophila (White et al., 1994). Over expression of any of these genes in cultured fruit fly cells results in the rapid transduction of cell death (Chen et al., 1996).
Drosophila development begins in the fertilized egg with a series of 13 syncytial divisions, followed by three rounds of cellular divisions for most cells. Cells are allocated and positioned along dorsal-ventral and anterior-posterior axes according to maternally established morphogen gradients. The fate of these cells depends on their positions relative to the axes of the egg. This results in the compartmentalization of the developing embryo into specific domains of cells that will further develop into precise regions of the larva and adult (reviewed in Lawrence and Struhl, 1996). Although cell death does not normally occur during these early stages of development, the stage is set at this point for apoptosis to play a vital role later (Abrams et al., 1993). A general model for animal development includes the accumulation of excess cells required for the formation of a viable embryo. Extraneous cells are then removed at later points by programmed cells death (reviewed in Jacobson et al., 1997). Development in Drosophila is consistent with this model, since early divisions produce more cells than are needed for the subsequent development of specific structures. These extras cells are removed apoptically at precise times along the developmental pathway. For instance, when cell death is blocked in the absence of rpr, grim, and hid, the embryonic nervous system contains a large excess of cells at the end of embryogenesis (White et al., 1994). Although AO staining shows that cell death occurs in a consistent pattern, the precise locations and numbers of dying cells vary, indicating that the number of cells being eliminated depends on the developmental circumstances for a given embryo (Abrams et al., 1993).
The elimination of excess cells appears to be in part related to intercellular signaling via the segment polarity genes. For instance, cell death observed in the epidermal segments at stage 12-14 occurs among cells expressing the segment polarity gene engrailed (Pazdera et al., 1998). When cell signaling is disrupted by mutations in the wingless signaling pathway, cell death among the ENGRAILED expressing cells increases approximately 5 times. This increases death is seen in cells located approximately six rows away from WINGLESS secreting cells but does not occur in the cells that secret WINGLESS or those adjacent to them. A similar situation seems to exist in the embryonic brain. When the wingless gene is deleted in null mutants, the protocerebrum develops initially but is then largely deleted by programmed cell death in later embryonic stages (Richter et al., 1998).
Successful development requires the tight regulation of cell number to assure correct patterning and cell signaling. When the normal regulation of cell number is disrupted, cell death increases to compensate for the extra cells. This has been examined by using ectopic expression of CYCLIN E to extend the normal cycles of cell division by one extra round (Li et al., 1999). When this is done, the cell density in the embryo is nearly doubled within 1 hour following the expression of CYCLIN E. Examination of cell death in these embryos shows that hyperplasia induced by CYLCIN E expression is largely compensated for by increased apoptosis in the first 4 hours following the induction of CYCLIN E. The excess cells are not removed randomly but in a specific pattern, and the dying cells express rpr before they die.
The plasticity of embryogenesis and the role of apoptosis in the plasticity are apparent when examining pattern repair in the Drosophila embryo. It has been shown that the misexpression of the anterior morphogen bicoid results in the mispatterning of a number of morphological markers. Eggs containing inappropriate doses of bicoid have a number of early developmental abnormalities. These include shifting of the cephalic furrow (Driever and Nusslein-Volhard, 1988). This shift results in the enlargement of the head structures in the early embryo. These embryos still frequently develop into healthy adults, showing the plasticity and capacity for repair. Analysis of cell death patterns with inappropriate dosages of bicoid shows that excess cells generated by an expansion of the head domain are deleted by apoptosis (Namba et al., 1997). These extra cell deaths occur on the same schedule as seen for wild type embryos and appear to be by the same mechanism, since increased rpr expression is also observed in this domain. Also, the compressed posterior domain is repaired by a down regulation of cell death. This use of apoptosis regulation to repair both the expansion and compression of developmental domains reflects the efficiency of the system.
However, there are limits to the extent of expansion and compression that the embryo can tolerate. Tumerous structures and breached in the epithelium are observed in domains that have expanded to the point where cell death can no longer completely eliminate the excess. Conversely, if the compression of a developmental domain drops the cell number below a certain threshold, structural problems occur or particular organs fail to develop (Namba et al., 1997).
Drosophila provides a powerful model system to investigate both the mechanisms and roles of apoptosis during animal development. Genetic manipulation allows the study of apoptotic regulators and effectors in the context of the whole animal. This provides the opportunity to understand how different cellular processes are coordinately regulated to form the developed organism. Apoptosis is necessary to establish and maintain the correct cell densities required for efficient and precise patterning mechanisms. Coupled with cell proliferation, cell death ensures a plasticity in developmental processes that allows an embryo to develop dynamically according to circumstances. The investigation of how cell division and apoptosis are coupled is likely to reveal fundamental mechanisms of organogenesis.
Our understanding of apoptosis during animal development is still in its infancy. It is apparent that cell death occurs through development and rudimentary maps detailing the spatial and temporal occurrence of cell death allow us to examine its consequences. The identification of many of the central genes required for apoptosis provide tools for the exploration of how this process is integrate with signaling pathways that specify proliferation or differentiation during development.
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