Introduction

Autophagy has been the subject of growing interest in the scientific community in recent decades, with the number of publications on the topic increasing steadily. Research that began with the identification of autophagy genes in yeast has allowed the analysis of samples from organisms that lack key autophagy genes or autophagy markers. This has led to the gradual elucidation of signaling pathways that regulate autophagy and autophagic cell death. The progress in this field has not only broadened our understanding of the basic processes that regulate cell survival and death, but has also led to a better understanding of human diseases by elucidating the role of autophagy in pathological conditions in a variety of organisms from plants to humans.

In 1972, Kerr et al. [1] classified cell death into two major types: apoptosis and necrosis. Over the past 30 years, the term “apoptosis” has been used to refer to programmed cell death (PCD), and numerous studies have been conducted on the molecular mechanisms of apoptosis. PCD is an evolutionary phenomenon that occurs in multicellular organisms and plays a critical role in several important functions, including developmental morphogenesis, tissue homeostasis, and defense against pathogens. Because the concepts of apoptosis and necrosis have been so dominant in the understanding of cell death, early references to forms of PCD other than apop-tosis were largely ignored by the scientific community [2]. However, in recent years, there has been increasing interest in forms of PCD that are different from the traditional concepts, and molecular studies of these forms have become possible. One of these is autophagic cell death, and the identification of mammalian orthologues of yeast autophagy genes has recently been a hot topic of research. As a result of several experiments, autophagic cell death is now recognized as a major form of cell death that is distinct from apoptosis or complementary to it.

Autophagy is a physiological and evolutionary phenomenon that is one of the ways to maintain homeostasis through protein degradation and cell organelle turnover. It is particularly common in cells that are exposed to stressful environments, such as nutrient or growth factor deprivation, and a shortage of substrates that can produce energy necessary for cell survival. There is still not much information on the mechanisms of planned cell death, which is the concept of “autophagic cell death” that is independent of caspase production. However, autophagic cell death is becoming a focus of attention as a form of cell death other than apoptosis, and genetic studies of it are also being actively conducted. Now, autophagic cell death is taking its place as a new method of cell death along with apoptosis.

Autophagy has been shown to play a role in cell differentiation and development. When autophagy is activated in response to environmental or hormonal cues, it can lead to rapid cell changes that are necessary for proper differentiation and development. This has been shown in organisms that lack autophagy, such as insects, protozoa, and fungi [3]. In mammals, autophagy plays an important role in the developmental process, survival during starvation, and cell differentiation during the formation of erythrocytes, lymphocytes, and adipocytes. These remodeling functions indicate that autophagy plays a critical role in cell regeneration, and the maintenance of homeostasis is particularly important for the health of differentiated cells, such as neurons.

In this paper, we will examine the morphological features and regulatory pathways of autophagy. Then, we will explore how autophagy interacts with apoptosis. Finally, we will discuss the role of autophagy in development in relation to its role in helping cells to survive.

Autophagy

1. Types of programmed cell death

Building upon the research of Schweichel and Merker [2], Clarke investigated three major forms of PCD, which occur during embryogenesis or after toxic treatments [4]. Among Clarke’s categorized forms of cell death, the first is apoptosis. Apoptosis is characterized by cell contraction, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies fragments through which the cell disintegrates. Caspases are pivotal mediators of apoptosis, initiating the breakdown of crucial target proteins within the cell through the action of cysteine proteases. The second form of cell death classified by Clarke is autophagic cell death. This distinctive form involves the appearance of double or multiple membrane vesicles enclosing portions of the cytoplasm, mitochondria, or other organelles within the cellular cytoplasm. These vesicles, also known as autophagosomes, merge with lysosomes, preparing their contents for degradation by lysosomal enzymes. This process is referred to as autophagy, and it encompasses the previously referred to process of macroautophagy. While observations of dying cells have revealed distinctive features of autophagic phenomena, whether autophagy serves as the cause of cell death remains uncertain. One hypothesis suggests that under specific stimuli, varying forms of cell death can occur depending on the nature of the stimulus, its duration, and the extent of autophagic activity. From this perspective, cells might detach themselves for the reuse of intracellular components, ultimately leading to their demise. Notably, nuclear changes such as chromatin condensation are more prevalent in cells undergoing autophagic cell death than in cells undergoing apoptosis, and in such cases, DNA fragments or apoptotic bodies do not form. Residues of autolyzed cells, resulting from lysosomal digestion, tend to appear later compared to those from apoptotic cells. The third form of cell death classified by Clarke involves the formation of vesicles without lysosomal involvement, with subsequent degradation. Research in this area is still limited. This form is characterized by a simultaneous increase in morphological changes seen in the two aforementioned forms of cell death within specific tissues or under particular circumstances. This suggests the simultaneous activation of multiple cell death mechanisms within a single cell. This mixture of PCD mechanisms hints at the existence of yet another distinct form of cell death.

2. Definition and classification of autophagy

1) Definition of autophagy

In 1963, the Christian de Duve first defined the process of cellular component degradation through lysosomes as autophagy [5]. Lysosomes can attach to aged or damaged organelles within cells and eliminate them, and through a process known as selective self-digestion, referred to as autophagy (where ‘auto-’ stands for ‘self’ and ‘-phagy’ means ‘eating’), new replacement components are generated from lysosomes [6]. Autophagy, in conjunction with the ubiquitin proteasome system, not only participates in intracellular protein degradation but also plays a crucial role in digesting organelles, macromolecules, and other materials in times of nutrient deficiency or starvation, recycling them into essential cellular substances [7]. Moreover, autophagy is implicated in the progression of diseases and is known to be involved in the crosstalk between reactive oxygen species/reactive nitrogen species during cellular signaling and protein damage processes [5].

2) Classification of autophagy

Autophagy is classified into 3 types based on how substrates reach lysosomes. The first type is macroautophagy, which is commonly referred to as autophagy itself. Macroautophagy involves the formation of autophagosomes. The second form is microautophagy, where lysosomes directly engulf and digest cellular components. The third form is chaperone mediated autophagy. Chaperone bound protein complexes bind to LAMP- 2A receptors, facilitating the translocation of target proteins to lysosomes for degradation [5].

Role of Autophagy in Cell Fate Determination

The question of whether autophagy contributes to the demise of dying cells has sparked significant controversy. This skepticism stems from initial doubts about whether autophagy would perform well established roles at the end of a cell’s life. Even under normal growth conditions, cells utilize autophagy as a major pathway to degrade and recycle long lived proteins, certain ubiquitinated proteins, and cellular organelles like mitochondria [8,9]. When stresses such as nutrient deprivation or growth factor deficiency are imposed on cultured cells or organisms, autophagy becomes more prevalent to sustain survival [10]. The role of autophagy in promoting cell survival has been demonstrated through various experimental examples, including yeast [11] and plant [12-14] experiments conducted under nutrient deficient conditions, autophagy during the dauer stage of Caenorhabditis elegans (C. elegans) [15], autophagy observed in human cancer cells under nutrient deprived conditions [16], autophagy in Bax/Bak knockout hematopoietic cells under growth factor deficiency conditions [17], and autophagy in mice born under conditions of postnatal nutrient deprivation due to lack of placental blood supply and milk intake [9]. Disrupting or knocking down key autophagy genes during nutrient deprivation does not protect cells from cell death but rather tends to enhance cell death, resembling autophagic cell death functions. However, given the multitude of experimental results, one might question whether survival and death occur by the same mechanism of autophagy. Both autophagy associated with survival and autophagic cell death exhibit morphological features and formation of autophagosomes. Yet, they differ in several molecular aspects. For instance, autophagic cell death induced by zVAD in L929 mouse fibroblasts triggers selective autophagic degradation of the antioxidant enzyme catalase, while nutrient deprivation induced autophagy in the same cells does not lead to catalase removal [18]. Additionally, ceramide treatment and nutrient deprivation activate autophagy through sphingolipid signaling, inhibit the pro-survival PKB/ Akt pathway, induce autophagic cell death, and upregulate Beclin- 1 and BNIP3 expression [19]. Ultimately, the relative levels of the two sphingolipids SK1 and S1P might be key factors in determining whether a cell lives or dies. Accumulation of S1P favors cell survival, a concept referred to as the “S1P rheostat” [20]. These experimental findings underscore how autophagy, by selectively degrading proteins through activation/inactivation, altering the levels of critical intracellular regulators, and controlling cell status from protective mechanisms to cell death induction, can drive cells toward survival or demise. Therefore, while the morphological features of autophagy may appear similar in all cases, the molecular mechanisms underlying survival promoting autophagy and autophagic cell death reveal significant differences at the molecular level.

Role of Autophagy in Embryonic Development and Differentiation

1. Role of autophagy in mammalian embryogenesis

The importance of autophagy during development has been demonstrated in various lower eukaryotes such as yeast, C. elegans , Drosophila , and Dictyostelium discoideum [21-25]. In these organisms, autophagy is responsive to stress and becomes activated during high stress conditions like yeast sporulation or insect metamorphosis. Through a process of hormesis–a response to environmental and hormonal cues– autophagy induced cellular changes play a vital role in promoting appropriate differentiation and development. Deficiency in autophagy has been shown to result in developmental issues in various organisms. Hence, it is important to explore how autophagy contributes to cell differentiation and development.

1) From oocyte to embryo

In mammalian development, early autophagy occurs in modified oocytes, autophagy related genes (Atg) 5 is essential for early pre-implantation stages. One highly differentiated cell type, the oocyte, undergoes rapid reprogramming from a highly differentiated state after fertilization. This reprogramming occurs both in the nucleus and the cytoplasm. Maternal mRNA and proteins are rapidly degraded after the two-cell stage, and newly synthesized mRNA and proteins expressed from the zygotic genome undergo distinct changes in protein species from the four-cell to eight-cell stage onwards. Additionally, degradation of maternal proteins and RNAs may be critical for the activation of the zygotic genome. Although autophagy only occurs at low levels in unmodified oocytes, it is highly induced within 4 hours after fertilization. Induction of autophagy is entirely dependent on fertilization and not due to egg deficiency, as fertilized oocytes that remain unfertilized do not undergo autophagy. This type of autophagy is triggered by calcium oscillations and is also induced by parthenogenetic activation, which initiates autophagy in unfertilized oocytes. Interestingly, autophagy is temporarily suppressed from the one-cell to midtwo- cell stage and reactivated afterward. A similar inhibition of autophagy during mitosis has been observed in cultured mammalian cells and may involve mechanisms to avoid the degradation of crucial nuclear factors during cell division [3]. In general, regular Atg5^-/- mice can survive during early embryogenesis due to the presence of maternally inherited Atg5 protein in the oocyte. However, the elimination of maternally inherited Atg5 protein in oocytes specific Atg5 deficient mice leads to embryonic death from the four-cell to eight-cell stage. The precise role of autophagy during this process remains unclear. Autophagy at normal levels may be essential for producing sufficient amino acids for protein synthesis, given that protein synthesis rates decrease in autophagy deficient embryos. However, autophagy may also be necessary for removing unnecessary proteins and organelles or for promoting remodeling by maternal suppressors of zygotic gene programs. Considering autophagy as an intracellular recycling system, these possibilities are not mutually exclusive [3].

2) Processes in neonate mice

Widespread autophagy in mice is observed during the initial stages after birth. Autophagy is actively occurring in all tissues except the brain within 1 to 2 days after birth. During mammalian embryogenesis, essential nutrients are supplied through the placenta. After birth, this supply is cut off, and neonatal mice face inevitable severe nutrient deprivation. Therefore, despite appearing almost normal at birth, knockout of Atg3, Atg5, Atg7, Atg9, or Atg16L1 in neonatal mice leads to fatal conditions within the first day (sometimes within hours). Amino acid levels in plasma and tissues are reduced in neonatal mice with these Atg knockouts, implying that autophagy may be essential for maintaining amino acid pools during early neonatal stages. However, the precise utilization of the resulting amino acids during early neonatal stages remains uncertain. They may be used to generate energy through autonomous cellular means to meet high energy demands in specific tissues. For instance, autophagy is highly active in the heart and diaphragm of neonatal mice shortly after birth, and adenosine monophosphate-activated kinase, which acts in low energy conditions, is activated in Atg5 knockout hearts with low energy levels. Yet, since there are additional variables of mouse anomalies, it is uncertain whether the reduction in amino acid levels is the sole cause of early neonatal death in Atg knockout mice. First, although autophagy activity in neuronal cells is not enhanced due to deficiency, the absence of fundamental autophagy in neuronal cells contributes to early neonatal death in Atg knockout mice through impaired breastfeeding, worsening their nutritional condition. However, Atg5^-/- and Atg7^-/- neonatal mice die faster than wild type (WT) neonatal mice, even without breastfeeding, indicating that failed suckling alone cannot explain early death. Second, autophagy is vital not only for protein degradation but also for polymeric degradation. For instance, lysosomal glycogen transport in the liver and muscles triggers glucose and energy generation in neonatal mice. Third, Atg5^-/- neonatal mice almost lack fat tissue due to impaired lipid synthesis, hindering sufficient energy production. Fourth, impaired clearance of apoptotic bodies is observed in Atg5^-/- late stage embryos, which might also contribute to developmental anomalies. Finally, as the environmental conditions change drastically at birth, autophagy mediated degradation also contributes to cellular remodeling by generating new proteins and organelles as part of cellular responses to new conditions [3].

3) Other development associated processes

Although fully autophagy deficient embryos derived from oocyte specific Atg5 deficient mice die before implantation, conventional knockout of ATGs including Atg3, Atg5, Atg7, Atg9, and Atg16L1 results in embryonic survival throughout development, giving rise to offspring according to Mendelian ratios. These data suggest that Atg3, Atg5, Atg7, Atg9, and Atg16L1 are not essential for embryogenesis, except possibly in early stages when maternally inherited proteins allow survival in conventional knockout mice. Nonetheless, the roles of these ATGs in embryogenesis cannot be excluded. Despite reporting high levels of autophagy during later stages of embryogenesis in mice expressing autophagy reporter green fluorescent protein LC3, some phenotypic abnormalities of Atg5^-/- mice suggest that autophagy at WT levels may not be critically important for normal embryonic development, except for some tissues such as the developing thymus. Therefore, a detailed analysis of the role of ATG deficiencies in the aspects of embryonic development is required.

In contrast to embryonic survival observed in conventional Atg3^-/-, Atg5^-/-, Atg7^-/-, Atg9^-/-, and Atg16L1^-/- mice, knockout of other ATGs including Beclin-1, Ambra1, and FIP200 produces somewhat different phenotypes. Beclin-1, which is a component of Class III Phosphatidylinositol 3 Kinase (PI3K) complex and a mammalian homolog of yeast Atg6/Vps30, results in rapid embryonic death in Beclin-1^-/- mice, and abnormally small embryos are detected at embryonic day 7.5 (E7.5). These embryos display extensive cell death and fail to close the proamniotic canal. Beclin-1^-/- embryonic stem cells remain viable, indicating that Beclin-1 is dispensable in vitro but crucial for in vivo development. Ambra1, a Beclin-1 interacting protein that positively regulates autophagy and is strongly expressed in developing neural tissues, is deficient in Ambra1 deficient mice generated through gene trap mutagenesis (Ambra 1^gt/gt). These mice exhibit embryonic lethality between E10 and E14, display defective neural tube formation, and show hyperproliferation of neural tissues. FIP200, an interacting protein of focal adhesion kinase (FAK) and a binding partner of the Mr200K protein RB1CC1, also interacts with ULK1 (homologous to Atg1) and exhibits molecular functions similar to yeast Atg17. However, FIP200 lacks homology with any yeast Atg protein. FIP200^-/- mice also show embryonic lethality between E13.5 and E16.5 due to defects in heart and liver development. The diverse phenotypes observed in different ATG knockout mouse models are not entirely understood. Since Beclin-1 has multiple individual functions, and FIP200 has various interacting partners such as FAK, Pyk2, tuberous sclerosis complex 1, and p53, the phenotypes observed in Beclin-1^-/-, Ambra 1^gt/gt, and FIP 200^-/- mice may involve factors other than autophagy deficiency. Instead, the diverse phenotypes of different ATG knockout mouse models likely depend on the stage of autophagy where each component functions. Beclin-1, which includes PI3K complexes and ULK1-FIP200 complexes, functions early in autophagosome nucleation, while Atg3, Atg5, Atg7, and Atg16L1 function later in autophagosome elongation. Therefore, upstream factors may exhibit more severe phenotypes or, as recently reported, downstream factors may be dispensable for certain types of macroautophagy. However, one exception to this general pattern is ATG9, which, although functioning early (according to yeast hierarchical analyses), leads to less severe phenotypes when deficient. Additionally, there might be varying degrees of functional redundancy among different ATG proteins or varying levels of compensatory mechanisms in the knockout of different ATG proteins. Further studies are needed to determine whether the autophagy pathway itself or the components that transition from oocyte to embryo in a self-autonomous pathway are essential for various stages of embryonic development [3].

2. The role of autophagy in differentiation processes of various cells

1) Erythrocyte differentiation

An enduring question in developmental biology has been how erythroblasts shed their organelles and the extent to which the autophagy pathway is involved in this process. Erythroblasts contain nuclei and intracellular organelles, which are absent in mature red blood cells, where they are replaced by hemoglobin molecules. During erythroid differentiation, the nucleus is expelled from the cell, but the mechanism by which other organelles are removed is not well understood. The 15-lipoxygenase enzyme, highly expressed during reticulocyte maturation, has been implicated in this degradation process. Electron microscopy studies also suggest a potential role of autophagy in organelle degradation in erythroids. However, erythroid cells from neonatal Atg5 deficient mice appear normal. Thus, the role of autophagy in this process remains uncertain.

Recent studies suggest that mitochondrial clearance in reticulocytes depends on autophagy. This concept first emerged in studies of cells lacking the protein Nix (or BNIP3L), the sole protein present in the outer mitochondrial membrane of cells with impaired mitochondria. Nix is required for selective removal of mitochondria, not ribosomes or nuclei. Nix deficient reticulocytes exhibit autophagosome formation, but lack loss of mitochondrial membrane potential and the subsequent engulfment of mitochondria by autophagosomes. Nix deficient mice have reduced mature red blood cell counts and compensatory increases in erythroid precursor cells (reticulocytosis).

Direct requirements of ATG in mitochondrial removal during erythroid cell maturation have also been demonstrated. Five Atg1 related proteins exist in mammals: ULK1, ULK2, ULK3, ULK4, and STK36. ULK1 and ULK2, but not ULK3, ULK4, or STK36, are considered candidates for the first mammalian Atg1 orthologs due to their interaction with Atg12 and FIP200. ULK1 expression is induced during erythroid differentiation, indicating its significant role in this process. Indeed, ULK1 deficient mice show increased reticulocyte numbers. Interestingly, besides impaired mitochondrial clearance, ULK1 deficient erythroid cells also display defective clearance of RNA bound ribosomes. Similarly, fatal irradiated WT recipients of Atg7^-/- fetal liver transplants exhibit reduced red blood cell counts and delayed mitochondrial clearance in erythroid cells. Moreover, Atg7 deficient hematopoietic progenitor cells (Atg7^flox/flox;Vav-Cre mice) suffer severe anemia and die between 8 and 14 weeks of age. Atg7^-/- erythrocytes accumulate selectively damaged mitochondria, associated with premature cell death. In contrast to ULK1^-/- mice, Atg7^-/- erythroid cells seem to accumulate no other organelles besides mitochondria.

Thus, the autophagy pathway appears to play a role in selective mitochondrial clearance during erythroid differentiation. One proposed mechanism for selective mitochondrial degradation, called mitophagy, involves Nix mediated mitochondrial recognition. Nix carries a typical LC3 interacting WXXI like motif at its amino terminus, allowing it to interact with LC family proteins such as g-aminobutyric acid receptor associated protein and LC3A on autophagosomal membranes. Nix acts as a cargo receptor in mitophagy. However, Nix likely has additional autophagy independent roles in mitochondrial clearance. Treating Nix deficient reticulocytes with an uncoupling reagent restores mitochondrial engulfment by autophagosomes, indicating that Nix mediated effects on mitochondrial membrane potential, rather than cargo receptor function, underlie Nix mediated effects on erythroid maturation.

Detailed characterization of the molecular mechanisms underlying mitophagy and mitochondrial clearance during erythroid differentiation is needed. Understanding how other organelles such as ribosomes and ER are cleared during erythroid degradation is also an interesting question. The normal clearance of ribosomes in Nix^-/- cells and of ribosomes and endoplasmic reticulum (ER) in Atg7^-/- cells suggests the existence of an unknown degradation system, possibly a recently described Atg5/Atg7 independent type of macroautophagy. Alternative forms of mitophagy and macroautophagy may also have broader roles in differentiation of other cell lineages.

2) Lymphocyte differentiation

Deletion of lineage specific ATG, including myeloid specific and B cell specific genes, in lymphocyte differentiation reveals specific roles for autophagy. Apart from severe anemia, hematopoietic specific Atg7 knockout mice (Atg7^flox/flox;Vav- Cre mice) also display significant reductions in T cell and B cell numbers (with no change in myeloid lineage cells). Both CD4 and CD8 T cells from these mice possess abundant mitochondria, which correlates with increased mitochondrial mass, elevated reactive oxygen species levels, and heightened apoptosis sensitivity after in vitro culture. Atg7^-/- mice transplanted with fetal liver cells also exhibit lymphocyte reduction. Similarly, T cell specific Atg5 and Atg7 knockout mice (Atg5^flox/flox;Lck-Cre and Atg7^flox/flox;Lck-Cre) show decreased peripheral T cell numbers, accumulation of mitochondria in T cells, and increased apoptosis in mature T cells. Atg5^-/- chimeric mice also exhibit reduced T and B cell counts and impaired proliferative responses to stimuli. During normal thymocyte maturation into circulating mature T cells, mitochondrial content decreases, a change suppressed in autophagy deficient T cells. Thus, mitochondrial clearance emerges as a crucial process for the survival of mature T cells during differentiation. Why erythrocyte and lymphocyte survival, excluding myeloid lineage cells, partially relies on mitophagy remains incompletely understood.

Another recently identified role of autophagy in T cells involves the removal of autoreactive T cells from the thymus. High levels of autophagy were first observed in thymic epithelial cells, the only non-hematopoietic cell type expressing major histocompatibility complex class II (MHC II) molecules, which are crucial for antigen presentation. It was later established that autophagy is essential for transporting endogenously synthesized specific antigens to MHC II loading compartments. Genetic disruption of Atg5 in thymic epithelial cells results in altered selection of specificities in MHC II restricted T cells and the emergence of autoreactivity. Thus, this autophagy dependent antigen presentation pathway is implicated in central tolerance to autoimmunity.

Genetic deletion of ATGs in mice also reduces B cell numbers. B cell specific Atg5 knockout mice (Atg5^flox/flox; CD19- Cre), like Atg7^flox/flox; Vav-Cre and Atg5^-/- chimeric mice, exhibit decreased B cell counts. Beyond its role in B cell development in the bone marrow, Atg5 is also required for survival of certain B cell types in the periphery. The underlying mechanisms of B cell defects remain unclear. Unlike T cells, mitochondrial content does not seem to regulate B cell development.

In summary, ATG in mice play roles in various differentiation processes, including erythrocyte and lymphocyte differentiation. Autophagy is crucial for selective mitochondrial clearance during erythrocyte maturation and survival of mature T cells. The mechanisms and broader implications of these findings warrant further investigation.

3) Adipocyte differentiation

Similar to erythrocyte maturation, adipogenesis also involves significant intracellular remodeling. Adipocytes differentiate from preadipocytes, which arise from multipotent mesenchymal precursors. During adipogenesis, fibroblast like cells differentiate into round cells containing a single large lipid droplet. Autophagy is actively induced during this differentiation process in vitro, and both in vitro and in vivo studies confirm its contribution to cellular remodeling during adipogenesis.

Primary mouse embryonic fibroblasts (MEFs) can differentiate into adipocytes upon the appearance of adipogenic factors. However, this process is significantly delayed or suppressed in primary Atg5^-/- MEFs. Knockdown of Atg7 or Atg5 in 3T3-L1 preadipocytes also suppresses triglyceride accumulation and reduces protein levels of mediators and markers of adipocyte differentiation. Additionally, treating various mammalian cell lines like HeLa, PC12, and HepG2 with short interfering RNA targeting LC3 impairs lipid droplet formation.

Newborn Atg5^-/- mice have less subcutaneous fat than WT neonates, highlighting the importance of autophagy in adipogenesis. The role of autophagy in adipogenesis is further confirmed in adipocyte specific Atg7 knockout mice (Atg7^flox/flox; Ap2-Cre mouse). These mutant mice exhibit reduced white adipose tissue mass, contain small adipocytes with multilocular lipid droplets, and show increased mitochondrial content and cytoplasmic volume. These characteristics resemble those of brown adipose tissue, and the expression of certain brown fat related enzymes like UCP-1 and PGC-1α is observed in the white adipose tissue of these mutant mice. Notably, even in the absence of differences in food intake, adipocyte specific Atg7 knockout mice have less fat than WT mice. Furthermore, they exhibit resistance to diet induced obesity and increased insulin sensitivity, likely due to elevated β-oxidation of fatty acids and lower plasma free fatty acid levels. These mice also show higher basal physical activity. Therefore, autophagy not only plays a role in adipocyte differentiation but also has implications for local and systemic adipose metabolism.

It remains to be determined whether the anti-obesity and insulin sensitizing effects of Atg7 are primarily due to changes in the ratio of brown adipose like tissue to white adipose tissue. While the amount of brown adipose tissue decreases after birth, recent evidence suggests its presence in significant quantities in adult humans. As opinions on the impact of the white to brown adipose tissue ratio on the development of obesity grow, largely driven by increased mass of white adipose tissue, predicting how autophagy disruption affects body weight and insulin sensitivity in fully grown animals is challenging. Therefore, further research using inducible adipocyte specific ATG knockout mice is needed to address this issue.

Involvement of autophagy in hepatocyte lipid droplet accumulation appears to be more complex than its role in adipocyte lipid metabolism. During deficiency, free fatty acids are transported from adipose tissue to the liver, leading to lipid droplet accumulation. Studies suggest that the formation of lipid droplets is diminished in hepatocyte specific Atg7 knockout mice (Atg7^flox/flox;Alb-Cre mice). However, inhibition of autophagy in hepatocytes is known to result in increased hepatic triglyceride storage in Atg7^flox/flox;Mx1-Cre and Atg7^flox/flox;Alb-Cre mouse lines. This may be due to impaired autophagic degradation of damaged lipid droplets in hepatocytes, as small lipid droplets can be selectively sequestered by autophagosomes in cultured hepatocytes. The differences in phenotypes observed in liver specific Atg7 knockout mice in various studies are not entirely clear. It might be that Atg7 is required for rapid lipid droplet formation during acute responses in the liver, while it would generally inhibit chronic lipid accumulation in lipid droplets during feeding states. Furthermore, the mechanistic basis for the differential effects of autophagy on lipid metabolism in the liver and adipose tissue needs to be elucidated.

Role of Autophagy in Tooth Development Process

1. Tooth development process

The process of tooth development is regulated by sequential interactions between the oral epithelium and neural crest derived ectomesenchyme [26-28]. The epithelial mesenchymal interaction governs the development of all epithelial organs, including teeth, hair follicles, and glands. Experiments focusing on tooth development in mice provide valuable samples to study genetic and developmental interactions. These experiments shed light on cellular interactions, differentiation, migration, and apoptosis, among other processes. Over 200 genes related to tooth development, including growth factors, transcription factors, and extracellular matrix components, have been identified through studies in mice [29].

The initial signals observed during early tooth development can be found around E11.5 in mice, where localized thickening of the dental epithelium occurs to form the dental placode. By E12.5, there is an ingrowth of the dental epithelium toward the mesenchyme to form the tooth bud. The transition from bud to cap stage is crucial in tooth formation, as this is when the crown of the tooth is shaped. Cells at the tip of the molar bud cease proliferation and assume a unique morphology, forming the primary enamel knot (PEK), a central signaling center controlling tooth shape. Continued growth and folding of the epithelium determine the shape of the tooth crown during the early bell stage (E16.5). In the late bell stage, the dental papilla gives rise to odontoblasts, which produce dentin, while the inner cells of the dental epithelium differentiate into ameloblasts, which secrete enamel [26,27]. Finally, the tooth matures into an organ with distinct shape, size, and function [28].

Throughout mammalian tooth development, a continuous and extensive morphological transformation occurs, requiring proper cellular proliferation, differentiation, apoptosis, and autophagy [26,27,30]. Maintaining tissue homeostasis is critical, and the precise balance of these processes is necessary for successful tooth development. Thus far, research has focused on understanding cellular proliferation, differentiation, apoptosis, and autophagy that occur during tooth development [30- 32].

2. Autophagy during tooth development process

Autophagy has been observed in various physiological conditions such as nutrient deficiency, hypoxia, differentiation, and development [33-36]. Particularly, based on the fact that autophagy is active during embryogenesis, it has been reported that autophagy also contributes to the process of tooth development.

1) Role of autophagy in early tooth development

Recent studies have reported the presence of autophagy in odontoblast aging and dental pulp pathology [36]. As aging progresses, an increase in autophagic vesicles, serving as a survival mechanism for odontoblasts, is observed, indicating the activation of autophagy [33,34]. However, the role of autophagy in the process of tooth development is still under investigation. ATG such as Atg5, Atg7, and Atg12 have been found in developing tooth buds, and these autophagy markers are clearly present throughout various stages of tooth development [32]. In the early stages of embryogenesis, LC3 is observed in tooth epithelium and dental placodes. Beclin-1 is predominantly expressed in the tooth epithelium of developing tooth buds (Fig. 1) [32,37,38].

During the bud stage, LC3 is observed around the condensed region where dental epithelium and ectomesenchymal cells are concentrated. Beclin-1 is predominantly distributed towards the oral side of the dental epithelial bud and extends towards the tip of the bud. In the cap stage, the expression pattern of Beclin-1 is similar to that of LC3. LC3 is present both inside and outside the enamel organ, with pronounced expression towards the ectomesenchymal cells, especially evident in structures like the cervical loop and the PEK. In the early bell stage, LC3 is observed in the inner, outer, and middle layers of the dental epithelium, as well as in the vicinity of the dental papilla. Cells within the secondary enamel knots exhibit strong staining for LC3 but weak staining for Beclin-1. Notably, both LC3 and Beclin-1 are strongly immunostained in the cervical loop at this stage. The cervical loop demonstrates high proliferative activity [39]. These findings suggest that autophagy may impact tooth crown formation by promoting the survival of dental epithelium and ectomesenchymal cells during the initial stages of tooth development.

Reused from the article of Yang et al. (J Mol Histol 2013;44:619-27) [38] with original copyright holder’s permission.

2) Role of autophagy in late stage of tooth development

During the late bell stage and postnatal periods, LC3 and Beclin-1 become predominantly distributed in differentiating cytoplasm of cells. These cells undergo differentiation into ameloblasts and odontoblasts (Fig. 2) [38].

Wang et al. revealed through previous research that when the expression of Beclin-1 is limited to a minimal level, cell differentiation is suppressed, and further reduction of Beclin-1 expression beyond this minimal level leads to loss of differentiation function [40]. Autophagy is known as a cellular remodeling mechanism observed during the differentiation of various cells. In differentiating cells, autophagy pathways can generate necessary amino acids and also degrade proteins that might impair the differentiation process [41]. Therefore, autophagy can be considered to play a role in the differentiation of ameloblasts and odontoblasts, while also maintaining protein and organelle regulation, preventing cellular degeneration during embryonic development or postnatally [3]. Given that autophagy is present in differentiated odontoblasts and ameloblasts, it might be involved in maintaining the homeostasis of differentiated cells.

During root formation, the Hertwig’s epithelial root sheath (HERS) contributes to the formation of the dental root by elongating from the vicinity of the dental crown. At this stage, LC3 is abundantly present in the stellate reticulum, stellate intermedium, HERS, dental follicle, ameloblasts, and odontoblasts. Even when the mandibular first molar has partially erupted, LC3 continues to be expressed in ameloblasts and HERS cells. Additionally, cells in the periodontal ligament also exhibit a distinct pattern of immunostaining for LC3 when examined [37].

Conclusions

Autophagy is a crucial mechanism regulating homeostasis in organisms, with various genes and proteins implicated in its regulation. This paper aimed to explore the definition and classification of autophagy, its mechanisms, and regulators. Additionally, it sought to investigate the roles of autophagy in embryonic development, cell differentiation, and specifically in tooth development.

Autophagy emerged as essential during 2 distinct phases of early development: from oocyte maturation until implantation and immediately after birth when nutrient supply from the placenta ceases. The fact that manipulating the expression of certain ATGs led to increased mortality suggested a role for autophagy even during embryogenesis. Moreover, autophagy pathways were observed to participate in the differentiation of cell types such as red blood cells, lymphocytes, and adipocytes. Finally, through the maintenance of homeostasis, autophagy regulates protein and organelle quality by preventing the degradation of post-mitotic cells after embryonic development or postnatally.

In the context of tooth development, autophagy serves various roles: firstly, it impacts the survival of epithelial and ectomesenchymal cells; secondly, it influences the mechanism shaping the final morphology of the tooth crown by regulating the function of enamel knots; thirdly, it contributes to cell differentiation and the maintenance of homeostasis in differentiated ameloblasts and odontoblasts; fourthly, it plays a role in root formation and the regulation of periodontal ligament homeostasis.

Research on autophagy, from cell survival to death, encompasses a wide spectrum of cellular changes. Based on the regulators and mechanisms unveiled in the past decades, further extensive research on autophagy is crucial to advance its applications, potentially ranging from treating human diseases to drug development in the future.