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).
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).