It has a single ortholog of most yeast Atg proteins; however, two nematode homologs exist for Atg4 and Atg8.
Autophagy has copious roles during C. The localized induction of autophagy around the site of the penetrated sperm indicates that a selective degradation is occurring rather than the bulk degradation of the embryonic cytoplasm [ 33 ]. During larval development, autophagy is involved in the specification of multiple cell fates controlled by regulating miRNA-mediated gene silencing through the degradation of one of the components of the miRNA-induced silencing complex, AIN-1 [ 32 ].
Autophagy also has an important role during C. Dauer larvae show some metabolic and morphological changes that allow long-term survival, and their cells use autophagy to generate amino acids for the neosynthesis of proteins indispensable for survival during starvation periods [ 16 ]. Autophagy was also suggested to be involved in longevity [ 35 , 36 ]. Finally, during the development of the hermaphrodite germline, autophagy acts coordinately with the core apoptotic machinery to execute genotoxic stress [ 32 ].
It is worthy to note that, unlike in many other model organisms, programmed cell death is not essential for C. Similarly, inactivation or mutations in atg genes do not affect embryonic development and viability. An elegant work [ 38 ] showed that, strikingly, double mutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. These observations suggested that the apoptotic and autophagic processes are redundantly required and share essential developmental functions for C.
The growing appreciation of the conservation of some apoptotic and autophagic responses in insects and mammals is producing an exchange of ideas that is continuing to invigorate this research field. The development of the D. The identification of many central genes required to execute apoptosis provides the tools for the exploration of the phenotypes during development. Genetic manipulation allowed the study of apoptotic regulators and effectors in the context of the whole animal.
Each of these genes is independently regulated allowing developmental apoptosis to be finely controlled [ 39 ]. The role phagocytosis plays in the final stages of apoptosis and the molecular mechanisms guiding the elimination of apoptotic corpses has been outlined [ 40 ].
The initiator genes reaper, grim and hid in turn activate the core apoptotic machinery, including caspases, a conserved family of cysteine proteases [ 39 ]. While caspases have been characterized from many organisms, little is known about insect caspases. The availability of new insect genome sequences will provide a unique opportunity to examine the caspase family across an evolutionarily diverse phylum and will provide valuable insights into their function and regulation.
This concept is supported by numerous genetic studies in D. Importantly, cell death in some cell types occurs in the absence of Dronc and the primary effector caspase, Drice, suggesting that, similar to mammals, redundancies have been built into the cell death system of insects [ 41 ]. The methods used to study apoptotic cell death in the D. These techniques, including acridine orange staining, fragmented DNA in situ analysis TUNEL , cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization, are suitable for analysis of apoptosis in normal and stress conditions [ 42 ].
In Drosophila , many developing tissues require programmed cell death PCD for proper formation. Two bcl-2 genes are encoded in the Drosophila genome and some studies suggested their requirement during embryonic development. However, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development.
By irradiating fly mutants, it has been demonstrated that developmental PCD regulation does not rely upon the Bcl-2 proteins but that it provides an added layer of protection in the apoptotic response to stress [ 43 ]. Cells damaged by environmental insults have to be repaired or eliminated to ensure tissue homeostasis in metazoans. Recent studies on apoptosis induced by stress during embryogenesis of D. JNK signaling is implicated in many processes of normal development, e.
The repression of Dpp Decapentaplegic signalling causes the activation of the proapoptotic role of JNK and the following activation of proapoptotic gene reaper. The protein Schnurri mediates the repression of gene reaper through Dpp.
This arrangement allows JNK to control migratory behavior without triggering apoptosis. Dpp plays a dual role during dorsal closure: it cooperates with JNK in stimulating cell migration and also prevents JNK from inducing apoptosis [ 44 ]. It has been demonstrated that Drosophila lines overexpressing the gene menin or an RNA interference for this gene normally develops but are impaired in their response to several stresses including heat shock, hypoxia, hyperosmolarity and oxidative stress.
Menin, the product of the multiple endocrine neoplasia type I gene, is implicated in several biological processes including gene expression control and apoptosis, modulation of mitogen-activated protein kinase pathways and DNA damage sensing or repair.
In the Drosophila embryo subjected to heat shock, this impairment was characterized by a high degree of developmental arrest and lethality. The gene menin seems to be implicated in the regulation of stress response and in the preservation of protein structure and function, as suggested by the fact that a deletion of the menin gene causes a strong decrease of HSP70 and HSP23 synthesis and an increase of the sensibility to many kinds of stresses [ 45 ].
Concerning autophagy, the fruit fly provides an excellent model system for in vivo studies in the context of a developing organism. Because of its short life cycle, the well-characterized genetics of the organism and the expression of genes and their regulators, the D.
Atg genes and their regulators are conserved in Drosophila and autophagy can also be induced in response to nutrient starvation and hormones during development. Autophagy is induced in Drosophila in starvation conditions or during metamorphosis in specific tissues. Consequently, the regulation of this process may change in these different contexts and circumstances; thus, this field needs further investigations [ 46 ]. During the embryogenesis of Drosophila , autophagy and apoptosis seem to occur contemporarily.
Autophagy characterizes embryonal districts with massive cell elimination but also exerts a protective role against metabolic stress during tissue remodelling [ 16 ]. Analogous to the observations in yeast, worms and mice, Atg inactivation may result in severe phenotypes in Drosophila. Mutation of atg18 and atg6 is lethal at the larval stage.
Atg1 expression is also crucial for development as atg1 mutants show reduced larval viability and those that survive cannot develop beyond pupa [ 47 ]. In Atg7 gene mutant flies, attenuation of autophagy occurs, but there is no impairment of vitality, fertility or morphology.
Nevertheless, atg7 mutant adults show higher sensibility to stress and decrease of life span, probably for the neuronal accumulation of ubiquitinated proteins [ 48 ]. Induction of autophagy has also been observed during two nutrient status checkpoints of oogenesis in the fruit fly: germarium and mid-oogenesis stages [ 49 ].
Mutagenesis experiments in atg7 genes have confirmed these evidences [ 50 ]. The relationship between autophagic cell death and apoptosis has been examined during Drosophila embryogenesis by studying the elimination of an extra-embryonic tissue known as the amnioserosa AS , demonstrating that both processes are required for the its disruption. Interestingly, autophagy seems to be essential for caspase activation in the AS: a reduction of cell death and permanence of AS are related to the down-regulation of autophagy; a caspase-dependent premature AS dissociation is related to autophagic up-regulation [ 51 ].
In recent years, this organism has become a model for the study of cell death both in physiological and stress conditions.
Physiological apoptosis can be considered an important aspect of sea urchin development, which is regulated and controlled by specific genetic programs and is necessary to construct the animal [ 52 ]. Studies on the activation of physiological apoptosis in the sea urchin embryo were conducted for the first time in using different methodological approaches: DNA electrophoresis analysis, morphological observations and TUNEL assay. Physiological apoptosis at pluteus stage the first larval stage was shown by those investigations, especially in cells from specific districts: oral and aboral arms and intestine.
No apoptotic signals were observed at the blastula or gastrula stage. It has been assumed that some embryonic structures known to at least partially disappear after metamorphosis can be somehow eliminated through this pathway [ 53 ]. Further studies showed the occurrence of apoptosis in P. During the stage of metamorphosis corresponding to competent and juvenile larvae , only a few cells trigger apoptosis. Therefore, it has been hypothesized that the removal of inadequate cells was the result of a programmed cell death required for the development of the adult and for the elimination of unnecessary structures [ 54 ].
Regarding early embryogenesis, the available data suggest that apoptosis is not frequent during developmental cleavage, becoming active from gastrula stages onwards. Only necrotic or pathological cell death has been observed during cleavage stages [ 55 ]. The sea urchin embryo, as well as many other embryos of marine organisms, is highly sensitive to several kinds of stressors and is able to activate different defense strategies such as apoptosis.
In particular, it was demonstrated that a brief treatment with TPA and heat shock or an exposure to very high Cd concentrations are able to trigger apoptosis in P. Other studies showed that long-lasting exposure to lower Cd concentrations, similar to those found in polluted seawaters, caused severe developmental delays and abnormalities in P.
The authors reported that small Cd concentrations, if accumulated in the cells, induce several cytotoxic effects and abnormalities depending on cell loss caused by apoptosis. Numerous apoptotic cells could interfere with the developmental program causing a misregulation of cellular remodeling, which normally occurs in pre-metamorphic stages of development.
Probably, the correct larval feeding behavior is perturbed by defects in arms and ciliary bands [ 60 ]. Concerning autophagy, the activation of this process in sea urchin embryos was reported for the first time in [ 14 ]. In , the P. Studies on whole embryos make it possible to obtain qualitative and quantitative data for autophagy and also to get information about spatial localization aspects in cells that interact among themselves in their natural environment.
In such a system, it is possible to add many autophagic inductors or inhibitors to the media seawater that will subsequently be directly absorbed through the membrane of embryo cells [ 30 ]. Several experimental approaches have been used to detect physiological autophagy: identification of autophagolysosomes by acidotropic dyes such as neutral red NR and acridine orange AO ; immunodetection of LC3-II an autophagic marker by Western blotting and immunofluorescence.
The results showed that autophagy seems to have a crucial role and it is constantly present, reaching peaks in specific points of embryonic development. This aspect was studied by analyzing the molecular autophagic flux and the dynamics of autophagic organelles autophagosomes and autophagolysosomes.
A major activation of autophagy was detected after 18 h of development, probably because there is a reorganization of the embryo at the gastrula stage as the cells begin to take strategic positions and need to recycle metabolites in order to obtain the energy necessary for the completion of development [ 14 ].
Regarding autophagy induced by stress, it has been found that sea urchin embryos are able to modulate this process as a defense strategy against Cd exposure. Analyzing the autophagosomes by LC3, an increase of the levels of this autophagic marker during development was observed in particular at late gastrula stage [ 14 , 64 ].
Specifically, the experiments revealed a higher level of autophagosomes for embryos treated for 18 h with high Cd concentrations, while embryos show lower levels of autophagosomes after 24 h of treatment, probably because the apoptotic process becomes significant [ 13 , 14 ].
Further studies on the role of autophagy during oogenesis and early stages of development and on the possible interplay between apoptosis and autophagy are in progress in our laboratory.
We proposed three different hypotheses about the homeostatic relationship between survival and death pathways in sea urchin embryos exposed to Cd stress: a hierarchical choice of defense mechanisms, b energetic hypothesis and c clearance of apoptotic bodies.
First hypothesis: the embryo tries to face the stress conditions triggering, initially, a less deleterious defense strategy, i. Placenta ; Narayanan V: Apoptosis in development and disease of the nervous system. Naturally occurring cell death in the developing nervous system. Pediatr Neurol ; Anat Rec ; Mutat Res ; Microsc Res Tech ; Fertil Steril ; Scott WJ: Cell death and reduced proliferative rate.
In Handbook of Teratology, Vol. Int J Dev Biol ; J Reprod Immunol in press. Is it an important cell death modality? Br J Cancer ; MHC involvement in the embryo response to teratogens in mice. Am J Reprod Immunol ; J Nutr ;SS.
Rogers MB: Life-and-death decision influenced by retinoids. Curr Topics Dev Biol ; Diabetes ; Nat Med ; Pharmacol Toxicol ; Wilson JG: Current status of teratology-general principles and mechanisms derived from animal studies. Teratogen Mutagen Carcinogen ; J Exp Med ; Diabetologia ; J Reprod Immunol ; J Immunol ; Download references. You can also search for this author in PubMed Google Scholar. Reprints and Permissions.
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However, much remains to be learned about how the striking cellular reorganization and loss of cells affect the surrounding tissue. Here, we review advances in understanding how apoptosis helps in shaping organs and body structure during animal morphogenesis. Digit individualization is the oldest model of programmed cell death in vertebrates and is considered the classical morphogenetic example for how programmed cell death sculpts an organ recently reviewed in Hernandez-Martinez et al.
In agreement with this model, species with webbed limbs, such as the duck 14 and the bat, 15 show scarce cell death in the interdigit region.
Initial data came from the observation of digit malformations linked to an aberrant cell death pattern in the interdigital webs. In these mutants, the lack of cell death in specific web regions leads to the fusion of the corresponding digits or syndactyly.
Interestingly, the defect in cell death pattern has no impact on skeleton formation and can be rescued by retinoic acid-induced cell death. In this process, the role of apoptosis can be compared with the work of a stone sculptor who shapes stone by progressively chipping off small fragments of material from a crude block, eventually creating a form Figure 1.
Here, the major purpose of apoptosis is to eliminate excessive cells, to reveal a new shape in the tissue. It seems that during digits individualization, apoptosis has little effect on the surrounding tissue. However, the expression of matrix metalloproteinases MMP11 and stromelysin , enzymes involved in extracellular matrix degradation, coincides with the appearance of apoptotic cells in interdigital regions, and could be involved in the final remodeling of the digits. For example, it is becoming increasingly clear that the elimination of excess and abnormal neuronal connections during vertebrate nervous system development has direct implications on the organization and maintenance of optimal brain function.
More direct evidence for the influence of apoptotic cells on their microenvironment has come from Cuervo and Covarrubias.
Interestingly, extracellular matrix remodeling also coincides with abundant cell death in other developmental models like amphibian intestine remodeling or digits individualization, suggesting that extracellular matrix redistribution through apoptosis could be a general mechanism involved in morphogenesis.
In both cases, in the absence of apoptosis, fold formation is impaired. In digit formation, the elimination of cells from the whole interdigital zone is sufficient to explain digit sculpting: part of the tissue is destroyed without affecting the living cells forming the digits, thus revealing a new shape. However, in the case of embryo segmentation or leg joint formation, the cellular mechanism must be different.
Here, a few cells are dying in a monolayer epithelium and the epithelial barrier must be maintained while the dying cells are eliminated. Epithelial integrity is maintained by the reorganization of the neighboring cells, filling the gap that would be created by the removal of dead cells and restoring a flat epithelium.
It may rely on cell shape remodeling of the non-dying cells that will create the fold in response to a signal coming from the dying cells. Separation of the digits: an example of morphogenetic apoptosis acting as a stone sculptor. A schematic representation of a developing limb is shown with apoptosis indicated in red up. When cells from the interdigital zones are removed, a new shape is revealed down. Collectively, these studies have revealed that apoptotic cells profoundly influence their environment to promote morphogenetic changes.
However, the precise nature of the underlying signals and their mechanisms remain to be discovered. A number of studies have focused on the communication between dying cells and their neighbors. The fact that phagocytes are often at a certain distance from the place where cells are dying indicates that dying cells are able to send a long range signal to their surroundings.
Another interesting set of data focused on the direct interaction of dying cells and their neighbors. Initially, Rosenblatt et al. Cell extrusion relies on the formation of an actomyosin ring both in the apoptotic cell and in its direct neighbors.
The contraction of the ring squeezes the dying cell out of the epithelium, either apically or basally, depending on the localization of the actomyosin ring. Further insights into the ability of dying cells to communicate came from studies of stress-induced apoptosis in Drosophila. These studies revealed that cells stimulated to undergo apoptosis in response to stress or injury can produce mitogenic signals to promote the proliferation of surrounding living cells.
The ability of apoptotic cells to secrete mitogenic factors has many interesting potential implications for wound healing, tissue regeneration and tumor development. Direct support for the idea that apoptotic cells release mitogenic signals to stimulate tissue regeneration has come from diverse models, including hydra, xenopus, planaria, newts and mice reviewed in. Altogether, these examples illustrate the existence of extensive communication networks between apoptotic cells and their surviving neighbors.
However, much remains to be learned about how apoptosis acts during normal development to actively induce tissue modification, and to what extent signals involved in injury and stress paradigms also contribute to normal development and morphogenesis. A few years ago, an active mechanical function of apoptosis was revealed during Drosophila dorsal closure, a powerful model system to study wound healing in vivo.
Before dorsal closure, only the ventral and lateral surfaces of the embryo are covered by epidermal cells, whereas the dorsal side is covered by the amnioserosa, a transient tissue that is eventually eliminated. During dorsal closure, the lateral epidermis moves dorsalward to cover the amnioserosa. This morphogenetic process is accompanied by cellular elongation and formation of an acting cable in the leading edge, the most dorsal row of lateral epidermis.
Furthermore, apoptosis has an active role in regulating the speed of dorsal closure; if apoptosis is inhibited in the amniosera, closure is delayed and the force produced by the amnioserosa on the dorsal epithelium is reduced. In contrast, ectopic induction of apoptosis can accelerate the movement.
Although the precise underlying molecular mechanisms remain to be elucidated, one can speculate that the modification of the actomyosin network in the neighboring cells through, for instance, the formation of actomyosin extrusion rings, creates a pulling force that is propagated throughout the amnioserosa to the epidermis, thus enhancing the movement Figure 2.
Apoptosis acting as a pulling force. Apoptotic cells are indicated in red in the amnioserosa , the force generated by apoptosis in the lateral epidermis is represented by the green arrows. Successive stages of dorsal closure are represented. The progressive stretching of the neighboring cells is proposed to be the origin of the pulling force generated by apoptosis on the surrounding tissue.
Dorsal closure can be either accelerated when apoptosis is promoted left , or slowed down when apoptosis is inhibited right. Interestingly, a recent study suggests that apoptosis may have a similar function during neural tube closure in mouse.
Large amounts of apoptotic cells were originally observed during neural tube closure. Similar to what is observed during dorsal closure in Drosophila , apoptosis does not seem to be essential for neural tube closure, but is required for accelerating the process and ensure zipping. In the first study, Suzanne et al.
This conclusion was based on the observation that inactivation of apoptosis in either one or the other rotating domain led to similar defects with a genital plate half-rotated. Live imaging of the rotation process using specific markers of each ring domain confirmed that each ring movement depends on the presence of apoptosis at its boundary, suggesting that each ring requires apoptosis to dissociate from the neighboring tissue.
An alternative possibility is that apoptosis may provide a force to actively promote rotation. Although it is difficult to imagine that apoptosis of a relatively small number of cells is sufficient to generate the driving force for the extenisve movement of two large domains, it may contribute force to the movement. Such a role would be consistent with the previous work on dorsal closure.
In support of this idea, inhibition of apoptosis specifically in the inner ring slowed down rotation, but did not completely block movement. Conversely, increasing apoptosis by expression of the pro-apoptotic Reaper protein at the boundary between the two moving rings accelerated genital rotation.
These data suggest that apoptosis may not only separate the rotating rings, but also contribute to the force that drives the movement. Two different explanations could be proposed for the deceleration observed when apoptosis is inhibited in only one rotating domain: either the acceleration of the rotation is affected or the release of the motile domain is compromised.
Scientists estimate that seven to eight million oocytes are formed in the fetus , which are reduced to about , oocytes at birth, and then only a few hundred at the onset of menopause.
Apoptosis occurs not only during embryonic development, but also after birth. In humans for example, brain cells undergo apoptosis prior to and following birth to eliminate excess brain cells and streamline nerve impulses. Apoptosis also occurs in some cells that the body identifies as cancerous to prevent the spread of the cancer and kill the cancerous cells. However, unregulated apoptosis can cause disorders, such as Alzheimer's disease and amyotrophic lateral sclerosis, which is a motor neuron disease.
Keywords: Apoptosis. Apoptosis in Embryonic Development Apoptosis, or programmed cell death, is a mechanism in embryonic development that occurs naturally in organisms. Sources Barres, Ben A. Brenner, Sydney. Clarke, Peter G. Flemming, Walther. Kiel: Honig, Lawrence S. Kerr, John F. Jacobson, Michael D. Lockshin, Richard A.
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