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Keywords:

  • autophagy;
  • atg gene;
  • C. elegans;
  • D. melanogaster;
  • embryogenesis;
  • organogenesis;
  • vertebrates

Abstract

  1. Top of page
  2. Abstract
  3. Autophagy in the Development of Invertebrates
  4. Autophagy in the Development of Vertebrates
  5. Conclusions
  6. Acknowledgments
  7. References

Autophagy is a lysosome-mediated degradation pathway used by eukaryotes to recycle cytosolic components in both basal and stress conditions. Several genes have been described as regulators of autophagy, many of them being evolutionarily conserved from yeast to mammals. The study of autophagy-defective model systems has made it possible to highlight the importance of correctly functioning autophagic machinery in the development of invertebrates as, for example, during the complex events of fly and worm metamorphosis. In vertebrates, on the other hand, autophagy defects can be lethal for the animal if the mutated gene is involved in the early stages of development, or can lead to severe phenotypes if the mutation affects later stages. However, in both lower and higher eukaryotes, autophagy seems to be crucial during embryogenesis by acting in tissue remodeling in parallel with apoptosis. An increase of autophagic cells is, in fact, observed in the embryonic stages characterized by massive cell elimination. Moreover, autophagic processes probably protect cells during metabolic stress and nutrient paucity that occur during tissue remodeling. In light of such evidence, it can be concluded that there is a close interplay between autophagy and the processes of cell death, proliferation and differentiation that determine the development of higher eukaryotes.

Autophagy is a lysosome-mediated, bulk degradative process by which eukaryotes recycle organelles and long-lived proteins. Three main forms of autophagy have been described in higher eukaryotes, named macroautophagy, microautophagy and chaperone-mediated autophagy. The major distinction between these forms is the degradative mechanisms involved. Microautophagy and chaperone-mediated autophagy degrade portions of cytosol and proteins by directly delivering them to lysosomes in a chaperone-dependent or independent way. By contrast, macroautophagy (hereafter referred to as autophagy) represents a more complex process involving the formation of intermediate membrane-surrounded structures, so-called autophagosomes, that engulf the substrates and deliver them to lysosomes for degradation by moving along cytoskeletal structures (1,2). Many autophagy regulatory genes (Atg) have been discovered and characterized in yeast (3,4) and some of them are conserved in higher eukaryotes. In these multicellular organisms, specific Atg genes, together with their regulators, control different stages of autophagosome formation and maturation. Atg1, Atg6, Ambra1 and Vps34, among others, are involved in the formation of the early autophagosome membrane (5); Atg5-9 and Atg12-14, in the maturation of the autophagosome membrane (1). The protein kinase target of rapamycin (TOR) is a crucial upstream regulator of autophagy: it acts as a nutrient sensor which activates or represses, by phosphorylation, the activity of some proteins regulating protein translation, e.g. the eukaryotic initiation factor 4E binding protein and the ribosomal protein S6 kinase (6). In nutrient-poor conditions, TOR repression shifts the cellular metabolism toward autophagy, thus allowing cells to recycle cytosolic components and provide energy by a mechanism that has not yet been elucidated. Studies conducted in several model eukaryotic systems delineate a role for autophagy in both physiological and pathological conditions. In particular, autophagy is involved in the maintenance of tissue homeostasis, as demonstrated by the observation that many neurodegenerative diseases are characterized by a failure in the autophagic clearance of protein aggregates or toxic molecules (7).

During embryogenesis, cells undergo various processes, including proliferation, cell death and differentiation, thus allowing the embryo to transform into an adult organism. In particular, cells require the appropriate tools to suddenly modify their protein content and morphology in order to respond to intrinsic and external stimuli. In this context, autophagy has recently been proposed as a crucial mechanism for the cellular remodeling that occurs during the development of multicellular organisms (8). Here we describe some specific examples of autophagy during embryogenesis.

Autophagy in the Development of Invertebrates

  1. Top of page
  2. Abstract
  3. Autophagy in the Development of Invertebrates
  4. Autophagy in the Development of Vertebrates
  5. Conclusions
  6. Acknowledgments
  7. References

Drosophila melanogaster

Because of its short life cycle, the well-characterized genetics of the organism, and the expression of atg genes and their regulators, the D. melanogaster model system has proven very useful in dissecting the physiological roles of autophagy. Analogous to what is observed in yeast, worms and mice, atg inactivation may result in severe phenotypes in fruit flies. D. melanogaster development is characterized by the three distinct stages: larva, pupa and adult, each with a unique body plan. Mutation of atg18 and atg6 is lethal at the larval stage, indicating that these genes are essential for development through this stage (9). Atg1 expression is also crucial for development as atg1 mutants show reduced larval viability, and those that do surive cannot develop beyond pupa (Figure 1A) (9). Atg7 mutant flies, on the other hand, just show an attenuation of autophagy, probably because of compensatory mechanisms; they are fully viable, fertile and do not present major morphological defects during metamorphosis. Emerging atg7 mutant adults, however, are hypersensitive to stress and have a reduced life span, potentially because of accumulation of ubiquitin-positive aggregates in degenerating neurons (Figure 1A) (10).

image

Figure 1. Autophagy involvement in the development of invertebrates. In (A), the developmental stages of D. melanogaster are shown. Embryos develop to adult flies, by progressing through three larval stages and one pupal stage. The pupa-to-adult transition is mediated by a burst in 20-hydroxyecdysone levels, as indicated. The atg proteins involved in the progression of life cycle and the result of their deficiency are indicated, as the exact stage affected. In (B), the development of C. elegans to adult, across several larval stages (L1–L4) is shown. In some stress conditions, such as starvation, high population density or temperature increase, L1 larva develops in Dauer larva, which progress to L4 stage when the stress stimulus stops. The role and the stage of Atg involvement in nematode embryogenesis are indicated. bec-1 is the atg6 fly homolog; lgg-1 is the Atg8 homolog; let-512 is the vps34 homolog; F41E6,13 is the Atg18 homolog; unc-51 is the Atg1 homolog.

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During insect metamorphosis, a constant supply of nutrients is required to provide the energy necessary for growth, metabolism and survival, and autophagy plays an important role in this process. Studies conducted in the D. melanogaster fat body under starvation conditions have provided important insights into the mechanisms that regulate autophagy. The larval fat body is a nutrient storage and mobilization organ, analogous to the vertebrate liver. The fat body contains glycogen and lipids from which nutrients are mobilized to imaginal discs in response to amino acid starvation in a process that is regulated by autophagy (9). This starvation pathway is regulated by Tor signaling; when Tor is mutated or repressed there is inhibition of growth, reduction of cell size and decreased viability. Moreover, when atg1, which is not lethal per se, is mutated in Tor mutant flies, there is an increasing severity of their phenotypes resulting in embryonic lethality. These findings clearly indicate that autophagy is required to maintain cell size, viability and normal metabolism in this context (9,10).

There is considerable evidence that autophagy is induced in several D. melanogaster tissues as a normal physiological response to the rise of hormones that occurs during metamorphosis. The steroid hormone 20-hydroxyecdysone (ecdysone) defines the length of the development period and controls larval moulting and metamorphosis during the fruitfly life cycle (Figure 1A) (11). Specifically larval growth is arrested when normal levels of ecdysone are disrupted (12). At the end of the larval period, the level of ecdysone increases and the autophagic process is thought to be responsible for the cellular reorganization necessary to prepare for the maturation of adult tissues. It has been observed that the mRNA levels of some atg genes (and consequently autophagy) increase following the ecdysone burst in the fat body (13). This occurs in degenerating larval structures, in the intestine and in salivary glands (14). In particular, Rusten et al. clearly demonstrated that ecdysone signaling leads to a reduction of phasphatidylinositol-3,4,5-tris-phosphate (PIP3) levels by downregulating phosphoinositide (PI) 3-kinase (PI3K) activity in the fat body (13). This phenomenon is thought to be the mechanism for induction of autophagy. It has also been demonstrated that autophagy, but not apoptosis, is essential in ecdysone-induced degradation of dying midgut cells; therefore, this degradation does not occur in flies devoid of atg1, atg2 or atg18(15,16). In addition, a previous study indicates that atg7 mutants exhibit a slight delay in midgut histolysis (10). This study provides the first evidence that autophagy is strictly related to cell death during midgut histolysis.

The larval salivary gland is another tissue that is degraded (within 4 h) during metamorphosis, showing the morphological characteristics of ‘type II cell death’, as autophagy is sometimes referred to (17). Cell death in the salivary gland is induced by an increase in ecdysone levels 10–12 h after puparium formation (apf), and is maintained at that level for up to 16 h apf. The presence of autophagosomes and absence of phagocytosis in the cytosol have been observed in these cells (14). Remarkably, the lack of atg genes, including atg1, atg2, atg3, atg6, atg7, atg8, atg12 and atg18, leads to a failure to degrade the salivary glands. In addition, the overexpression of atg1 is sufficient to induce caspase-independent cell death, indicating that the induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner (17). Recently, it has been observed that the dynein light chain 1 (ddlc1), a component of the dynein motor complex, is required for cell death of salivary glands, because ddlc1 mutants show an impairment of their degradation. In ddlc1 salivary glands, caspases are still active but autophagy is attenuated, thus indicating an involvement of ddlc1 in regulating autophagy but not apoptosis in dying cells (18). The mechanism by which ddlc1 regulates autophagy remains unclear, but overexpression of atg1 is sufficient to rescue salivary gland degradation in mutant flies. Notably, studies conducted in mammalian cells have demonstrated the importance of the dynein motor complex for the transport of autophagosomes along microtubules (2). However, ddlc1 could also regulate autophagy in a motor-independent manner, given that dynein heavy chain mutations do not result in an impairment of autophagy in salivary gland (15).

In light of this evidence, it appears clear that apoptosis and autophagy work in parallel in the disruption of certain tissues during fly development. For example, it has recently been observed that both autophagy and caspases are required for the disruption of the D. melanogaster amnioserosa (AS), an extraembryonic membrane that is eliminated during embryogenesis (19). The AS shows autophagic hallmarks during the final stages of embryogenesis; however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue disassembly and engulfment by phagocytic macrophages.

Induction of autophagy during the D. melanogaster development has also been observed during Drosophila oogenesis. The autophagic process accompanies cell death in the ovary at two stages during oogenesis, the germarium and mid-oogenesis. These stages represent two nutrient status checkpoints. By using a fluorescent autophagy marker, it has been demonstrated that autophagy occurs in degenerating midstage egg chambers and also in germaria of nutrient-deprived D. melanogaster(20). Genetic inactivation of atg7 results in a significant decrease of autophagy in dying midstage egg chambers and in germaria of starved flies, further supporting the induction of autophagy during these stages of oogenesis (21). In some aspects, cell death taking place during D. melanogaster oogenesis is similar to that observed in larval salivary glands. Both nurse and salivary gland cells are large and polyploid, and the entire tissues undergo cell death simultaneously.

In conclusion, during Drosophila development, autophagy is induced in response to different stimuli in different tissues such as fat body, intestine, salivary glands and ovary and co-operates with canonical apoptosis to model developing tissues.

Development and autophagy in Caenorhabditis elegans

The involvement of autophagy in development has also been demonstrated in the nematode C. elegans, in which many of the components of the autophagic machinery are conserved. Similar to other eukaryotic organisms, different autophagy genes play distinct roles during embryonic development in C. elegans and some of these genes show partial redundancy to each other (Figure 1B). For example, worms deficient in the yeast Atg6 homolog bec-1 exhibit a lethal phenotype, dying before or during the first larval stage and exhibiting increased vacuolization and molting defects (22). In addition to the involvement in viability, bec-1 activity also seems to be involved in fertility because mutant worms that reach adulthood are sterile. Furthermore, a lethal phenotype is observed in worms carrying a mutation in the C. elegans ortholog of Class III PI3K Vps34 (let-512), and in atg8 (lgg-1) or atg18 (F41E6,13) knockdown worms (23,24). In contrast to D. melanogaster atg1 mutants, C. elegans unc-51 mutants develop into mature organisms although they exhibit axonal defects that impair motor neuron function (25).

Under stressful conditions, such as starvation, high population density or increased temperature, C. elegans larvae respond by arresting development at the third larval stage (L3). This is referred to as the dauer diapause (26). Dauer larvae undergo a number of metabolic and morphological changes, distinct from normal L3 larvae that permit long-term survival. These changes include increased intestinal fat storage, pharyngeal, intestinal and hypodermal constriction, total body elongation, and the formation of a thick cuticle that seals them from the environment. If external conditions improve, dauer larvae can resume reproductive development, reach the adult stage, and have a normal lifespan (27). Through autophagy, cells generate a sufficient pool of aminoacids for the neosynthesis of proteins essential for survival when the food supply is limited. Although the regulation of dauer development has been extensively characterized, the cellular pathways involved in dauer morphogenesis are less well understood. In dauer larvae with a daf-2 (the insulin-like receptor) mutation there is an increase of autophagy that is detectable by visualization of the active form of green fluorescent protein (gfp)::LGG-1, the C. elegans Atg8/LC3 homolog, in hypodermal precursor cells (24). The depletion of autophagic activity in daf-2 mutants in which bec-1, unc-51, atg7, atg8 and atg18 are also individually knocked down by RNAi results in a defect in dauer formation, with worms dying within a few days. These studies elegantly demonstrate that autophagy acts downstream of the insulin/IGF (insulin-like growth factor) pathway and is required for normal dauer morphogenesis.

In C. elegans, a role for autophagy in the clustering of neurotransmitter receptors in neuronal development has also been reported, suggesting a novel function for autophagy in the degradation of neuronal cell surface receptors (28). The degradation of GABA receptors in muscle cells that are internalized in the absence of presynaptic inputs seems to be specifically mediated by autophagy. Interestingly, the mammalian GABAA-receptor-associated protein Gabarap is an ortholog of the yeast autophagy protein Atg8. The role of autophagy in this selective process was elegantly verified by checking for autophagosome formation in unc-51 mutants (28). Unc-51 is required for C. elegans’ axonal outgrowth along the antero-posterior axis. An increased number of organelles containing tagged GABA receptors in non-innervated muscle cells was observed. Receptor internalization and degradation is associated with autophagy because receptors colocalized both with LGG-1 and BEC-1 in autophagosomes of non-innervated muscles. Trafficking of receptors to autophagosomes most likely occurrs via the endocytic pathway because blocking endocytosis prevented receptors from aggregating in autophagosomes (29).

Very recently, a novel role has been proposed for autophagy in degrading the P granules in somatic cells during the early stages of embryo segmentation in C. elegans(30,31). P granules are cytosolic aggregates containing both proteins and RNA. Initially expressed in all the cells of the worm blastula, they quickly redistribute exclusively into the germ cell, thus determining its fate as a precursor of the germinal lineage. This specific role for autophagy clearly implies that this process plays a relevant function in orchestrating cell lineage differentiation, as previously postulated in mammalian cells (8).

Autophagy in the Development of Vertebrates

  1. Top of page
  2. Abstract
  3. Autophagy in the Development of Invertebrates
  4. Autophagy in the Development of Vertebrates
  5. Conclusions
  6. Acknowledgments
  7. References

Autophagy in early stages of development

It is known that maternal mRNAs and proteins accumulate in oocytes during oogenesis (32). These RNAs are responsible for the synthesis of proteins that guide oocyte maturation and egg activation after fertilization, thus allowing the transition from egg to zygote. After fertilization, however, maternal proteins are largely degraded and the mRNAs encoded by the zygotic genome start to be translated. Concomitantly, maternal mRNAs are also degraded and the organelles remodeled by processes that have not yet been completely characterized. As autophagy is considered a bulk degradation process, it represents a good candidate for eliminating maternal material. In fact, it has recently been demonstrated in an outstanding study by Tsukamoto et al. that autophagy is up-regulated after egg fertilization in the mouse, and that this process is essential for the preimplantation development (33). In particular, a significant increase of autophagosomes visualized by confocal and electron microscopy can be observed in oocytes within a few hours after fertilization compared to unfertilized oocytes. After fertilization, autophagy appearance is accompanied by the mTOR substrate S6 kinase dephosphorylation (33). Thus, inactivation of mTOR signaling, which could be a consequence of the Ca2+ wave following fertilization, could be responsible for autophagy induction. Interestingly, autophagy is suppressed from the late one-cell to the middle two-cell stage, and is newly activated after the late two-cell stage. To understand whether activation of autophagy is critical for early embryogenesis, some models of autophagy-deficient embryos have also been developed. For example, Atg5−/− mice are known to survive embryogenesis. The mice die soon after birth because of an incapacity to feed and overcome the critical starvation perinatal period (34). However, the small quantity of Atg5 protein provided by maternal Atg5 oocytes can be sufficient to support autophagy induction (35). Tsukamoto et al., in order to overcome this problem, generated oocytes completely deficient in autophagy using the cre-lox transgenic system (30). After fertilization of Atg5-deficient cells with an Atg5 sperm, embryonic development stopped at the eight-cell stage, thus demonstrating that this protein and the autophagy pathway are both essential for very early developmental stages (Figure 2). Based on these important results, it can be concluded that induction of autophagy in the very early stages of development of a mouse embryo could be responsible for both degradation of maternal proteins and macromolecules, and for generation of a pool of free aminoacids available for zygotic protein synthesis.

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Figure 2. Autophagy involvement in the development of mammals. The developmental stages of a mouse are shown. The atg proteins involved in different stages of embryogenesis and the results of their inactivation are indicated and related to the exact stage affected. On the right, examples of autophagy involvement in organogenesis and related genes are shown.

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Another crucial role for autophagy activation in the early stages of embryogenesis has been identified during the cavitation of the early embryo. In this phase of gastrulation, apoptosis and removal of the inner ectodermal cells allow the generation of a proamniotic cavity (36). By using the in vitro model of aggregates of inner-cell-mass-derived embryonic stem cells (embryoid bodies, EB), it has been demonstrated that two pro-autophagy genes, Beclin 1 and Atg5, are required for the clearance of cells that have died during the cavitation process. In previous seminal studies, Yue et al. observed that embryos devoid of Beclin 1 exhibited a developmental delay culminating in a severely and lethal reduced size at E7.5 (37). More recently, it has been demonstrated that EB derived from Atg5- or Beclin 1-defective cells fail to cavitate because of accumulation of dead cells (Figure 2) (38). Such accumulation depends not on an increase in cell death in these autophagy-defective cells, but on a defect in the engulfment of dead corpses by phagocytic cells. The lack of clearance of dead cells was confirmed in the embryonic retina and lungs of Atg5 null mouse (38). Qu et al. demonstrated that autophagy-deficient EBs fail to express the ‘eat-me’ [phosphatidylserine (PS) exposure] and the ‘come-get-me’ (lysophosphatidylcholine release) signals necessary for the phagocytic cells to recognize and engulf apoptotic bodies (38). The engulfment defects observed in these models are associated with low cellular ATP levels, thus suggesting that autophagy-dependent ATP production is necessary for completion of the apoptotic program during development. As PS exposure characterizes both apopotic cells and healthy phagocytes, it is possible that phagocytic functions could also be impaired. A massive accumulation of apoptotic bodies, because of the lack of PS exposure, has also been observed during the early stages of retinal development (embryonic day 5, E5) in an embryonic chick model following pharmacological inhibition of autophagy (39). Intriguingly, autophagy inhibition does not result in failure to expose PS and accumulation of cell corpses in later stages of retinal development (E9), thus demonstrating that autophagy is essential for PS exposure only during morphogenetic cell death. As PS exposure is an evolutionarily conserved mechanism by which phagocytes recognize and engulf apoptotic cells, it could also be the phenomenon responsible for accumulation of embryonic cell corpses observed in bec-1 null C. elegans(22). Therefore, autophagy can be defined as a crucial event in the removal of cell corpses accumulated during morphogenetic apoptosis, which leads to embryo cavitation and neural retina formation.

Autophagy in neurogenesis

A lot of evidence outlines the importance of autophagy in the late stages of embryonic and postnatal development. In particular, correct central nervous system (CNS) development depends on intact autophagic machinery. In addition to the above-mentioned role of autophagy in neuroretina formation, it is known that Atg5 or Atg7 deficiency leads to a defect in suckling capability in mice, suggesting that a defect in the CNS (34) might also be responsible for the perinatal lethality of these animals. By using conditional mouse technology aimed at studying the specific role of these genes in multiple tissues, it has been neatly demonstrated that Atg5 and Atg7 genes are necessary for motor function, and that their deficiency results in a progressive and lethal accumulation of ubiquitinated proteins, starting from E15.5, an early symptom of neurodegeneration (40,41). Moreover, Atg7 inactivation in cerebellar Purkinje cells results in degeneration of axon terminals, similar to what is observed in Ulk1-deficient neural cells. Ulk1, the mammalian ortholog of the yeast Atg1, is a protein kinase involved in the early steps of autophagosome maturation by forming and activating a complex with other Atg proteins (42). Retroviral infection of a dominant negative Ulk1 mutant in immature granule cells, where normally Ulk1 starts to be expressed at E16, inactivates Ulk1 function and results in the impairment of axon outgrowth and of differentiation of neurons (42). Ulk1 activity at the growth cones is thus crucial for the formation of fibers, and allows the progression of cerebellar development. As the mechanisms by which Ulk1 controls the phenomenon are still unknown it remains to be clarified whether the role of Ulk1 in neurodevelopment depends or not on its function in regulating autophagy.

Another striking example of the influence of autophagy on CNS development comes from the studies conducted on the autophagy regulator Ambra1. This protein is highly conserved in vertebrates although no orthologs have been identified in lower eukaryotes. Ambra1 forms a multimolecular complex with Beclin1 which regulates the activity of its associated kinase Vps34 and promotes the early steps of autophagosome formation both in basal and induced autophagy (5,43). We have found that, under basal conditions, the Ambra1/Beclin 1 complex is recruited to the cytoskeletal motor protein dynein machinery through direct binding to dynein light chains 1/2. When autophagy is induced by environmental stimuli, such as aminoacid deprivation or rapamycin treatment, Ulk1 kinase promotes the dissociation of Ambra1/Beclin 1 from the dynein complex, thus enabling execution of the autophagic program (44). During embryogenesis, Ambra1 is highly expressed in the CNS, especially in the neural plate, where it plays a crucial role in development. Homozygous mutation of Ambra1 in mouse embryos causes embryonic lethality at stage E16.5 and pronounced defects in the CNS, such as hyperproliferation of the neuroepithelium, midbrain and hindbrain exencephaly and defective closure of the neural tube (Figure 2) (43). The lack of autophagy in Ambra1 mouse mutant neural tissue is also associated with an excess of apoptotic cells, thus demonstrating, also in this case, the existence of a strict relationship between autophagy, apoptosis and cell proliferation (45). In light of such evidence, it can be concluded that Ambra1 is an essential protein for the control of cell proliferation and ensuring cell survival during development of the CNS. Notably, the knockout embryos of Ambra1 and Beclin 1 show different phenotypes even though these proteins are members of the same functional complex. This may be because of the fact that Ambra1, a vertebrate specific gene, is highly expressed only in the nervous system during early embryogenesis, while Beclin 1 is a more widespread factor. Also, Ambra1 targeting was obtained by gene trapping, an approach which may give rise to a hypomorphic allele rather than a null allele. Lastly, besides autophagy, these two factors may play different specific roles in other developmental processes, such as cell proliferation and trafficking.

Autophagy in cardiogenesis

The role of autophagy in cardiogenesis has carefully been investigated by Nakai et al., who inactivated the autophagy gene Atg5 in adult, neonate and embryonic cardiac tissue (42). While autophagy impairment because of Atg5 deficiency resulted in cardiac hypertrophy and dysfunction in adult mice, the inactivation of the gene, starting from E8.0, did not result in a pathological embryonic cardiac phenotype, thus implying the presence of some compensatory mechanisms in embryos. However, the Atg5-deficient mice showed a much more severe response to blood pressure overload relative to control mice, exhibiting ventricular dilation and cell death which proved fatal within one week (46). Reduced autophagy in these Atg5-deficient mice resulted in enhancement of both protein synthesis and degradation via proteasome in the blood-pressure-overloaded hearts. If Atg5 was inactivated in the first days after birth, mice showed a rapid onset of heart failure (Figure 2). These results suggest that constitutive autophagy represents a homeostatic mechanism necessary for cardiomyocyte remodeling during cardiogenesis, maintaining cardiac size and function. They also imply that up-regulation of autophagy plays a protective role for the heart in response to hemodynamic stress, increasing protein turnover and preventing the accumulation of abnormal proteins and organelles.

Autophagy in osteogenesis

Much of the vertebrate skeleton develops through the activities of chondrocytes embedded in a cartilagineous structure in which they proliferate and differentiate. As the cartilaginous tissue is devoid of blood vessels, cells proliferate and survive in hypoxic conditions that favor their terminal differentiation and synthesis of calcified extracellular matrix. The extracellular matrix secreted by hypertrophic chondrocytes allows vascular invasion of the tissue, degradation of the calcified matrix and initiation of osteogenesis, when the differentiated chondrocytes are eliminated from the tissue by apoptosis (47). It is noteworthy that terminal chondrocytes exhibit autophagic characteristics, perhaps because of the particular local micro-environment of cartilage (48). The lack of oxygen, nutrients and growth factors in the cartilaginous growth plate is probably responsible for the suppression of mTOR activity and autophagy induction observed in differentiated chondrocytes of E16.5 mice (48). Autophagy observed in these cells seems to be promoted by the activity of an enzyme that desulfates proteoglycans of the extracelllar matrix and that also favors extracellular matrix production, cell proliferation and differentiation (49). Thus, during skeletogenesis, autophagy is initially up-regulated to enable the chondrocytes to survive environmental stress, but can culminate in a severe cannibalistic response that allows the removal of cells from the tissue during osteogenesis. However, if the autophagic chondrocytes encounter a newly vascularized nutrient-rich environment, they could stop autodigestion and survive, thus participating in osteogenesis (50). Very little information is present in the literature about the final fate of the differentiated chondrocytes, and the complexity of the junction between cartilage and bone does not favor any easy conclusions in this regard. The morphological appearance of these cells is cause of the debate, and they are described as cells living in a ‘limbo’ condition, unable to live or to die (47).

Autophagy in hematopoiesis

The selective inactivation of the Atg5 gene in lymphoid precursor cells has been analyzed in chimeric mice by reconstitution of fetal liver after irradiation (51). In this elegant experimental system, autophagy impairment resulted in reduction of both thymocytes and B lymphocytes, and this was initially attributed to a defect in the homeostatic proliferation or activity of lymphoid precursors. By inactivating the Atg5 gene selectively in lymphocytes of the B-cell lineage, it was observed that the pro-B to pre-B transition was impaired (52). Regarding T lymphocytes, a reduction of the number of CD8+ T-cells by apoptosis was found in Atg5-deficient fetal livers (51). As in the case of Atg5-deficient immune cells, lack of Atg7 also results in a serious reduction of T lymphocytes (53). In addition, CD4+ and CD8+ T-cells devoid of Atg7 are more prone to undergo apoptosis and accumulate mitochondria and reactive oxygen species. If removal of mitochondria is not required for lymphocyte development, stringent quality control of the organelles is certainly necessary for long-lived cells, such as lymphocytes.

Lymphocytes are not the only cells that suffer from inactivation of Atg7 in the hematopoietic system. A very interesting observation about the role of autophagy in cell remodeling during erythroid maturation comes from a mouse model system devoid of Atg7 in the entire hematopoietic system. The resulting mice develop severe anemia and die in 12 weeks because of the fact that red blood cell development is prematurely arrested (53). During erythropoiesis, erythroblasts from bone marrow differentiate into anucleated cells, named reticulocytes. These further mature into cells devoid of all organelles, the erythrocytes, whose small size allows them to reach all the blood vessels. The arrest of maturation of Atg7-deficient erythroblasts is accompanied by typical hallmarks of apoptosis and by an accumulation of mitochondria, thus suggesting a defect in the process of their removal, known as mitophagy (53). Autophagy impairment in Atg7-deficient erythroblasts leads to an increase of precursor cells which accumulate damaged mitochondria, reactive oxygen species and signals for apoptosis by exposing PS on their surface.

Based on these results, it can be concluded that autophagy plays an important role for the clearance of mitochondria during development of the erythroid lineage.

Autophagy in folliculogenesis

Another example of the role played by autophagy during organogenesis recently became evident from a study on rat follicular development. In the mammalian ovary, a fraction of follicles mature and are ovulated, whereas most others undergo atresia and death by a mechanism not yet fully understood. Follicular atresia during folliculogenesis has been associated with massive apoptosis, especially in the granulosa cells, where induction of autophagy has also been reported (54). Whereas no LC3 expression is detectable in ovarian oocytes, a strong LC3 immunoreactivity is present in granulosa cells at all stages of development (55). By using rat ovaries primed with pregnant mare serum gonadotropin (PMSG) as a model of follicular development, Choi et al. observed that autophagy induction in granulosa cells is closely related to apoptosis onset (51). In primordial, primary and pre-antral follicles, colocalization of active caspase 3 and LC3 was not detected, as caspase 3 is not active at these stages. In contrast, in antral follicles, cleaved caspase 3 signals were detected in cells showing strong LC3II immunoreactivity, but not in cells expressing an inactive LC3. Moreover, autophagy observed in granulosa cells seems to be dependent on gonadotropin level, because autophagosome formation was suppressed by follicle-stimulating hormone (FSH) treatment (55). Although rather preliminary, these results suggest that autophagy could play a role in shifting the follicular fate from atresia to ovulation during follicular development in the mammalian ovary.

Conclusions

  1. Top of page
  2. Abstract
  3. Autophagy in the Development of Invertebrates
  4. Autophagy in the Development of Vertebrates
  5. Conclusions
  6. Acknowledgments
  7. References

A large body of evidence has emerged to support the important role played by autophagy during the development of eukaryotes. In a plethora of organisms, including Arabidopsis thaliana, D. discoideum, D. melanogaster, C. elegans up to mammals, mutants defective for autophagy show alterations in development that can be lethal (56,57). Owing to the importance of autophagy in the turnover of cellular components, including proteins and organelles, this process is expected to have a crucial role in the events occurring during embryogenesis. Developing cells, in fact, have to adapt constantly and quickly to both intrinsic and environmental changes in order to survive and to differentiate. During this process, the damaged and unnecessary organelles need to be promptly cleared and the cell shape needs to be modified to adapt to new functions. The exact mechanisms by which autophagy contributes to embryonic phenotypes in multicellular eukaryotes are not clear. However, both in invertebrate and vertebrate organisms, it is generally thought that autophagy plays an essential dual role both in the adaptation to stress and starvation phenomena occurring during morphogenesis, and in cell elimination, in concert with the apoptotic machinery. Consequently, in line with the function involved in aging, infection and neurodegeneration, autophagy seems to play a protective role during metabolic stress, such as that occurring in the storage tissues during fly metamorphosis or in the mature chondrocytes, which attempt to survive hypoxic conditions during mammalian osteogenesis. In addition, autophagy is most pronounced in the embryonic stages characterized by massive cell elimination as, for example, insect metamorphosis, glandular and fat body degeneration and lumen formation. In this context, it has been clearly demonstrated that autophagy controls the ATP-dependent expression of the cellular signals necessary for removing the apoptotic corpses during embryo cavitation, and in the first stages of neurogenesis (38,58). Whatever the case, it is now generally accepted that autophagy is not simply an alternative way of programmed death, but acts upstream or in parallel to the apoptotic machinery.

The varying phenotypes of mice harboring mutations in the different atg genes, discussed above, may reflect intrinsic differences in the developmental effects of genes that function at different stages of autophagy. On the other hand, autophagosome maturation is regulated by families of factors and different conjugation systems which are redundant for a long period of embryonic life, but which play a crucial role throughout later stages of development and immediately after birth (Figure 2). However, the possibility that autophagy genes might play autophagy-independent roles must be considered in order to explain the evident variability of phenotypes obtained by the inactivation of several factors.

Research has to yet elucidate the interplay between autophagy and cell proliferation, this also being a crucial process for the formation of a new organism starting from a fertilized egg. Ambra1-deficient mice are, to date, the only known example of an alteration of cell cycle upon autophagy impairment. At embryonic stage E7.0, when Ambra1 is mostly expressed in the neural plate, an increase of mitotic figures can be observed and, at E16.0, the dying mutant shows an excess of proliferation in the neuroepithelium (43). Some other examples of alteration of cell proliferation come from adult models with partial or total impairment of autophagy. The consequence of such alteration is a high incidence of tumors in several organs and an alteration of organ size (59). Given the relevance of the tumor suppressor role for autophagy, an understanding of the mechanisms regulating the crosstalk with cell proliferation and differentiation in both embryogenesis and adulthood is an essential goal for researchers.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Autophagy in the Development of Invertebrates
  4. Autophagy in the Development of Vertebrates
  5. Conclusions
  6. Acknowledgments
  7. References

We thank M. Acuña Villa and M.W. Bennett for editorial and secretarial work. We are indebted with B. Levine and A. Diehl for the expert scientific illustration in Figure 2 and with V. Cianfanelli and C. Fuoco for discussion. The work in our lab is currently supported in part by grants from the Telethon Foundation, AIRC, the Italian Ministry of University and Research, Ricerca Corrente and Ricerca Finalizzata from the Italian Ministry of Health.

References

  1. Top of page
  2. Abstract
  3. Autophagy in the Development of Invertebrates
  4. Autophagy in the Development of Vertebrates
  5. Conclusions
  6. Acknowledgments
  7. References