Growth of developing multicellular organisms is a complex process which results from the coordination of different cellular events, including proliferation, death, and differentiation [1,2]. During all these processes individual cells often require to eliminate and/or recycle part of their own components to obtain extra energetic supply or to build new components. Autophagy is a self-degradative process first achieved by unicellular organisms to adapt to fluctuating supply of external nutrients and then also employed by multicellular organisms to account for those degradative processes [3]. In the course of development, autophagy also functions as a self-destruction mechanism responsible for the elimination of cells and/or tissues, which need to be removed. Although it has to be kept in mind that the available molecular data has changed our view of these processes (for example, it has shown ‘apoptosis’ mediated without the accepted biomarkers of apoptosis) and the existence of alternative cell death mechanisms such as entosis have been reported [4,5], historically, three major types of cell death have been described depending on the developmental context: type-I programmed cell death or apoptosis, type-II cell death or autophagy, and type-III cell death or necrosis [6,7] (Figure 1). Autophagy is observed in dying cells, especially during vast elimination of developmental tissues such as during insect metamorphosis [8]. Self-eating of cellular cytosolic constituents, though with a lower intensity, are also crucial for balancing sources of energy in response to different extracellular stimuli and for preventing the accumulation of misfolded proteins or damaged organelles. Autophagy also promotes cell survival by providing the cell with either energy or with the building blocks obtained from aged or defective macromolecules. Therefore, autophagy has been shown to have key roles in development [9,10,11], tumor suppression [12], prevention of neuron degeneration [13], anti-aging [14], and protection against intracellular pathogens and the immune inflammatory response [15,16]. Due to its functions in either eliminating molecules and organelles or providing energy or molecular components, autophagy is an important instrument to enable cellular differentiation. Accordingly, autophagy is involved in invertebrate and vertebrate development. Examples of its functions in development include the sporulation in yeast [17], the metamorphosis in flies [18], the induction of dauer arrest in worms [19], and the process of the mammalian embryo pre-implantation [20] among others [21,22].

Three types of autophagy have been described so far: (i) macroautophagy (herein after referred to as autophagy) that requires the formation of double membrane vacuoles, the autophagosomes, which fuse their content with lysosomes forming the autophagolysosomes; (ii) microautophagy in which the engulfment of cargo occurs by the invagination of the lysosome membrane; and (iii) chaperone-mediated autophagy that has only been reported in mammals, where there is a direct delivery of the substrates to the lysosomes [3,23,24] (Figure 2A).

Although autophagy generally degraded different constituents through an apparently arbitrary mechanism, it can also present cargo specificity and selectively to eliminate specific molecules such as the yeast cytosolic acetaldehyde dehydrogenase Ald6p that has to be removed in a nitrogen-starved medium [25] and damaged organelles such as mitochondria (mitophagy) and peroxisomes (pexophagy) [26,27] (Figure 2B). For instance, defects in the selective clearance of damaged mitochondria are implicated in the pathogenesis of Parkinson’s disease. Mitochondrial kinase PINK1 and cytosolic E3 ubiquitin ligase Parkin act in a common pathway to regulate mitochondrial function, and mutations in these genes are the most common causes of recessive Parkinson’s disease. Moreover, there is recent evidence suggesting that the PINK1/parkin pathway also plays a critical role in mitophagy. Pathogenic Parkin mutations interfere with distinct steps of mitochondrial translocation, ubiquitylation and/or final clearance through mitophagy [28,29]. Briefly, PINK1 induces the translocation of Parkin to depolarized mitochondria, which mediates the formation of two distinct poly-ubiquitin chains. The autophagic adaptor p62/SQSTM1 is then recruited to mitochondrial clusters and is essential for the clearance of mitochondria. VDAC1 (voltage-dependent anion channel 1) is also a target for Parkin-mediated poly-ubiquitylation and mitophagy [28].

The study of the function of these genes by reverse genetics has provided insight into the molecular mechanisms of autophagy and its functions in cell differentiation and organism development, both in vertebrates and invertebrates. We review here the role of autophagy in cell death, survival, proliferation and differentiation during vertebrate development. This review does not intend to be exhaustive but to focus on key examples to illustrate the importance of autophagy during embryonic development. To get further insight into aspects and animal models that are not covered here, we recommend several excellent recent reviews [11,30,31,32,33,34,35].

Autophagy begins with the formation of a phagophore that expands to form a double-membrane autophagosome that engulfs intra-cellular cargo, such as protein aggregates, organelles and ribosomes. The autophagosome fuses then with a lysosome and the acid hydrolases degrade its contents and the resulting macromolecules are recycled by the permeases. Numerous studies have revealed that there are more than 30 different autophagy-related proteins (ATG) conserved from yeast to humans [36,37] that participate in a coordinate fashion at different stages of the process [3] (Figure 2C). The serine/threonine kinase Atg1 was the first ATG identified in yeast. Vertebrate genomes encode five closely related kinases, of which UNC-51-like kinase 1 (ULK1) and ULK2 are both involved in the initiation of autophagy [38]. However the role of ULK1 in the initiation of mammalian autophagy is under discussion, as Ulk1 knockout mice are viable and do not show any apparent autophagy related phenotype. It seems that ULK1 and ULK2 might show redundant functions, which is supported by the observed lethality of the double-knockout mice [38,39]. ULK1 is involved in mitophagy in reticulocytes [40]. ULK1 and ULK2 complexes (that include the ULK1/2, Atg13 and FIP200 proteins) are activated by AMP activated protein kinase (AMPK), which functions as an energy sensor [41]. These complexes are then translocated to the membrane of certain regions of different intracellular organelles that will form an expanding membrane structure: the phagophore. The source of the membrane that generates the phagophore is under intense debate. Potential membrane origins include the endoplasmic reticulum, Golgi complex, mitochondria, endosomes and the plasma membrane [42]. The autophagic stimulus also contributes to the membrane source as has been proposed with mitochondria and its central role in starvation-induced autophagy [43]. In mammalian cells there are two types of phosphatidylinositol 3-kinases (PI3K): Class I and Class III. The Class III PI3K is known to participate in various membrane trafficking events and it forms a complex, the PI3KCIII, in which the core proteins are Beclin-1, Atg14, p150 and Vps34. Amino acid deprivation leads to autophagy activation through mTOR inhibition. In that sense, a major pathway by which amino acids control mTOR is mediated through the Class III PI3K, through the regulating actions of the ULK complexes. On the other hand, Class I PI3K acts through an insulin signaling cascade to activate mTOR and PKB; hence it has an inhibitory effect on autophagy [44]. Autophagy is positively and negatively regulated through Beclin-1 interactions by UVRAG and Rubicon, respectively [45,46,47]. Ambra-1, the product of a gene only found in vertebrates, also positively regulates autophagy by promoting Beclin-1 interaction with Vps34 [48]. Atg9 is the only known transmembrane protein essential in the autophagy pathway. Atg9 is involved in the autophagosome biogenesis, acting as a membrane deliverer that cycles between membrane organelles but does not stably integrate on the autophagosome [49,50]. Autophagosome elongation requires evolutionary conserved ubiquitin-like conjugation systems and is carried out by lipidic modifications by the action of phosphatidylethanolamine (PE) of microtubule-associate protein 1 light chain 3 (MAP1LC3/LC3), the mammalian homolog of Atg8 in yeast [51]. This process is orchestrated, among others, by Atg7 (an E1-like ubiquitin conjugating enzyme), Atg3 (an E2-like ubiquitin conjugatin enzyme [52]), and Atg4C, to which LC3 is bound at first. Atg7 also acts in an ubiquitin-like conjugation system involving the E2-like ubiquitin enzyme, Atg10 and Atg12/Atg5, which, at the end of the process, are transferred to Atg16L. The complex Atg12/Atg5/Atg16L mediates LC3-PE by binding to the autophagosome membranes [53,54] and promotes the elongation and isolation of the autophagosome [3,30] (Figure 2C). Atg4/autophagin cleaves LC3 into its cytosolic version, also known as LC3-I. LC3-I generation is started by Atg7, transferred to Atg3, and finally modified with a lipidic attachment to bind with the autophagosome membrane, constituting the membrane-bound form LC3-II [55,56,57]. This conversion is considered as a hallmark to detect active autophagy (Figure 2C). Both LC3 and Atg4 proteins have been genetically abrogated in mice [58,59], and interestingly, none of them showed developmental abnormalities, which probably implies redundancy in the Atg4 family and the existence of at least two murine forms of LC3 (LC3α and LC3β) [58].

Basal autophagy is a survival process that contributes to cell homeostasis. Autophagy acts as a fast-response pathway against nutrient deprivation or oxidative stress, favoring cell homeostasis. In addition, autophagy can also constitute a cell death mechanism, namely type-II cell death or autophagic cell death. It is characterized by an increased number of large autophagic vacuoles that digest cytoplasmic material, and also by its independence of phagocytosis [7]. However, the role of autophagy as a cell death mechanism is a controversial issue, specifically in the mammalian system [60]. The presence of autophagosomes in dying cells has been taken as a feature of cell death, but it may represent an epiphenomenon that coexists with cell death [61,62]. Autophagosomes can be an initial attempt of the cells to survive, thus, a damaged cell might trigger autophagy as a protective mechanism, but finally die. Therefore, vacuolated dead cells may die with autophagy, but not by autophagy [60]. However, autophagy might ultimately cause cell death by massive cytosolic self-digestion or by selective elimination of protective proteins such as the catalase [63]. Even considering the presence of autophagosomes in dying cells as a controversial subject, autophagy as a cell death mechanism has been demonstrated in lower eukaryotes, where there are many examples. For example in Dictyostelium discoideum, where the absence of the apoptotic machinery facilitates the understanding of the molecular autophagic pathway [64], and in Drosophila melanogaster. Drosophila larvae experience large cellular remodeling during metamorphosis to achieve tissue maturation: Several structures such as the fat body and the salivary glands have to be degraded to generate the adult organism. Loss of function mutations in Atg genes shows the permanence of the glands for at least 24 h longer [65], which provides evidence for the role of autophagy in developmental cell death. These events are regulated by an increase in the levels of the hormone ecdysone, which triggers the autophagic program in a variety of tissues, which starts with the rise of some ATG mRNA levels [66]. Mutant flies in Atg1, Atg2 or Atg18 show that the elimination of midgut cells is also dependent on autophagy [67]. However, the possibility that autophagy is acting in a non-cell autonomous fashion has to still be excluded, and thus the cells that show autophagy markers are not the cells that will eventually die.

Although autophagic cell death is observed in mammalian cells in culture [68] and accompanies other degradation processes in areas of massive cell death during development [21], cell death by autophagy is infrequent in vertebrates [60]. The lack of cell death defects in distinct autophagy-deficient mutant mice raises the question of whether autophagy per se or only part of the autophagy pathway is involved in type-II cell death during development [59,69,70]. Due to its high adjustability, apoptosis might be the manager of removing individual cells while autophagy might be useful to remove high quantities of tissues as required in larval metamorphosis [5].

Additionally, autophagy and apoptosis may contribute together to cell elimination and indeed they are strictly connected. Thus, a dying cell may show features of both types I and II cell death, as for example, the HIV-infected CD4+ T lymphocytes that undergo apoptotic cell death induced by autophagy [71]. Autophagy can also assume the killer role when apoptosis is unavailable. For example, autophagy mediates cell death in apoptosis-deficient BAX^−/− BAK^–/– cells in response to genotoxic or endoplasmic reticulum stress stimuli [72,73,74]. In some cases, autophagy might ultimately cause cell death by massive cytosolic self-digestion in response to cellular stress during apoptosis-deficient conditions. Related to this, it is interesting to point out that autophagy and apoptosis are strongly connected through the BH3-only proteins, Bcl-2 and Beclin-1. Bcl-2 is an antiapoptotic protein, a key player of the intrinsic apoptotic pathway that regulates mitochondrial permeabilization. Beclin-1 has a BH3 domain [75] that interacts with Bcl-2, and this interaction prevents the initiation of autophagy [72,76], which reflects the convergent regulation of apoptosis and autophagic cell death [77]. Relative amounts of Beclin-1 and Bcl-2 seem to regulate the transition from cell homeostasis to cell death. Accordingly, the absence of autophagy genes increases cell death during nutrient deprivation and other forms of cellular stress [10]. Such studies raise the possibility that autophagic cell death might be induced in a similar manner to that of apoptosis. Remarkably, there is increasingly more data showing the crosstalk of apoptosis and autophagy and the dual-role of their components. Indeed, many core proteins of one of the processes have been reported to regulate the other. For example, caspases inhibit autophagy by the cleavage of Beclin-1 and Vsp 34 [78,79,80]. The recent role of Atg12 in this apoptosis-autophagy crosstalk is also very interesting. Atg12 acts as a positive apoptosis regulator by interacting with members of the Bcl-2 family by a predicted BH3-like motif [81].

Autophagy has also been reported to be essential for apoptosis by providing energy for phosphatidylserine (PS) exposure in the outer leaflet of the apoptotic cell plasma membrane. Apoptosis in mammals is initiated upon different upstream signals that lead to the activation of members of the Bcl-2 family that in turn inhibit anti-apoptotic members, such as Bcl-2, BCL-XL and MCL1 by direct interaction in the outer mitochondrial membrane. This causes the release of the BAX and BAK protein inhibition, which in turn leads to mitochondrial damage and cytochrome c release. Cytochrome c promotes the formation of the apoptosome, composed of APAF1 and caspase-9, which cleaves and activates downstream caspases, including caspase-3, caspase-6 and caspase-7 that carry out the execution phase of apoptosis [82]. Apoptotic cells need to be actively eliminated from the surrounding living cells, and it is known that PS exposure on the cell surface is an ‘eat me’ signal, which triggers engulfment by phagocytes that efficiently remove PS expressing cells. Thus, in this context, autophagy facilitates the clearance of the apoptotic bodies [83,84,85]. Thus, the interaction Beclin-1-Bcl-2 represents a molecular switch between apoptosis and autophagy.

In summary, autophagy in vertebrate development, besides being responsible for cell elimination in the few examples already mentioned, above all accompanies programmed cell death. Moreover, autophagy provides energy for the clearance of apoptotic bodies during mouse embryonic cavitation [85], a role also confirmed in cultures of developing avian retina [22]. The interplay between autophagy and apoptosis is an emerging aspect of developmental biology [86], which has a particular relevance for otic neurogenesis during early inner ear development (Aburto, unpublished observation; Figure 3) [87,88].

Numerous observations suggest that autophagy is strongly associated with cell cycle regulation. The deficiency in autophagy genes causes the miss-regulation of cell proliferation, for example, Atg4C [59], Atg5 [89], Bif-1 (Bax-interacting factor 1) a positive inductor of PI3KCIII and of autophagy in mammalian cells) [90] or Beclin-1 [91,92], that triggers mutations and tumors in the mouse. Miss-regulation of cell cycle due to defects in autophagy has also been observed during development in the Ambra1 mutant mice and in early otic neurogenesis through pharmacologic inhibition (Aburto, unpublished observation; Figure 3). The Ambra1 deficient mouse embryo exhibits increased proliferation in the neuroepithelium, excessive apoptosis and defects in neural tube closure [48]. These data indicate that Ambra1 is required in cell cycle regulation during nervous system development. In physiological conditions, the complex Ambra1-Beclin-1 is anchored to the microtubule machinery through dynein, whereas under nutrient deprivation when autophagy is induced, the complex is released [93]. Organotypic cultures of otocysts exposed to chemical inhibitors of autophagy show an increase in the fraction of cells that progress through the G₁/S-phase checkpoint but that are unable to complete cell division (Aburto, unpublished observation). Treated otocysts also showed a remarkable accumulation of apoptotic cells that might be due to a failure in cell cycle regulation during inner ear development. Autophagy has an important role in recycling proteins and, thus, essential negative-cell cycle regulators may not be available under autophagy impairment. Additionally, differentiation processes also require cytosolic rearrangements. Without autophagy, developing progenitors may not progress through the differentiation program and thus they could initiate a new unnecessary proliferation round. Moreover, defects in autophagy could deregulate the dynamics of microtubules, which are essential to complete the cell cycle [10]. Considering the high incidence of tumors in mutants of autophagic genes and the important role of cell proliferation in development, there is an emerging interest in understanding the involvement of autophagy in cell cycle regulation and cancer [94,95].

Cell differentiation and functional specification are sequentially acquired during development once the proper number of cells has been generated. Autophagy during this developmental stage may facilitate rapid changes in cytosolic composition, accelerating protein and organelle turnover and the recycling of specific factors exposed receptors and cytoskeletal dynamics necessary to promote the different cell fates. By promoting autophagy, cytosolic composition can be rapidly modulated in the differentiating cells, facilitating alternative pathways for protein and organelle rearrangement. Moreover, it constitutes an extra source of energy supplied by the self-degradation of the cytoplasmic material. Autophagy is crucial for vertebrate development at specific time points of embryogenesis. Immediately after birth, trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. In mice, the level of autophagy remains low during embryogenesis; however, it is immediately up-regulated in various tissues after birth and is maintained at high levels for 3–12 h before returning to basal levels within 1–2 days. Regarding that, mice deficient in Atg5 appear almost normal at birth, but die within one day after delivery. These mice exhibit reduced amino acid concentrations in plasma and tissues and display signs of energy depletion, which highlights the importance of autophagy in the maintenance of energy homeostasis during the neonatal starvation period [96].

There are data supporting that autophagy is required for elimination of paternal mitochondria in Caenorhabditis elegans after oocyte fertilization, where immediately after fertilization, sperm-derived components trigger localized induction of autophagy around sperm mitochondria, which leads to its degradation and consequently to the exclusive maternal inheritance of mitochondrial DNA [97,98].

Autophagy has also been associated with cell differentiation during development of several tissues such as the nervous system, the heart, the hematopoietic system, osseous tissue, and adipose tissue (Figure 4) [11,31].

Basal autophagy has an important role in adult post-mitotic cells as a “quality control mechanism” that protects cell survival by refreshing the cytosolic content. Abnormal protein turnover promotes the generation of protein aggregates that need to be eliminated. Macroautophagy is responsible for removing protein aggregates, while chaperon-mediated autophagy is involved in lysosomal degradation of soluble misfolded or transformed proteins [121,122]. By autophagy, damaged organelles such as mitochondria and peroxisomes, are also removed [123,124]. Thus, autophagy acts as a physiological mechanism against neurodegeneration, cancer and several infections [34,125,126]. Accordingly, loss of Atg5 or Atg7 in neurons and cardiomyocytes shows increased poly-ubiquitinated aggregates [69,110]. Moreover, autophagy is a protective mechanism for the cell against metabolic stress, particularly against hypoxia, nutrient starvation and growth factor reduction. Lysosomal degradation serves as a recycling mechanism that provides the cell with free amino acids or fatty acids that can be used for de novo synthesis or to obtain ATP [127,128].

In summary, autophagy plays an important cytoprotective role in basal conditions by the elimination of aberrant proteins and organelles and in consequence, there is a remarkable connection between autophagy malfunction, ageing and disease [14]. Not surprisingly, mechanisms that promote life-span extension also induce autophagy, including caloric restriction, NAD-dependent deacetylase sirtuin-1 activation, p53 suppression, inhibition of insulin/IGF-1 actions, rapamycin-mediated inhibition of mTOR, and treatment with spermidine or resveratrol [14,129,130]. Therefore, autophagy can be envisaged as a novel anti-aging mechanism, but further work is needed to clarify if it is a cause or a consequence.

Current evidence indicates that autophagy contributes to programmed cell death, proliferation, survival and differentiation to modulate cell number and fate during vertebrate development. Autophagy is fundamental after oocyte fertilization, at early neonatal stages and for the differentiation of a variety of tissues. Autophagy is also essential for tissue homeostasis and its deregulation has been associated with human ageing and diseases. Future work will provide insight into novel functions and mechanisms of action, which would help the design of novel strategies against autophagy-related diseases.