Programmed cell death in animal development and disease

Programmed cell death (PCD) plays a fundamental role in animal development and tissue homeostasis. Abnormal regulation of this process is associated with a wide variety of human diseases, including immunological and developmental disorders, neurodegeneration, and cancer. Here, we provide a brief historical overview of the field and reflect on the regulation, roles, and modes of PCD during animal development. We also discuss the function and regulation of apoptotic proteins, including caspases, the key executioners of apoptosis, and review the nonlethal functions of these proteins in diverse developmental processes, such as cell differentiation and tissue remodeling. Finally, we explore a growing body of work about the connections between apoptosis, stem cells, and cancer, focusing on how apoptotic cells release a variety of signals to communicate with their cellular environment, including factors that promote cell division, tissue regeneration, and wound healing.     

Apoptosis in cardiac development

Cell degeneration, as a phenomenon accompanying developmental processes, was originally described over a century ago. Apoptosis, a term introduced approximately three decades ago, has occupied investigators particularly with respect to cell and tissue kinetics, emphasizing its role in the disposal of supernumerary, malinstructed or damaged cells. Although apoptosis is mostly related to developmental processes, evidence has been gathered indicating that it may also perform other roles. In this review, which concentrates on cardiac development, we examine focal apoptosis and subsequent signal cascades in combination with timed morphogenetic events. Apoptosis mainly occurs in the non-myocardial compartment of the embryonic heart, a compartment that consists of cells derived from the endocardium, the epicardium and the neural crest. The last-mentioned population invades the outflow tract and the atrioventricular endocardial cushions. The signalling cascade seems to involve the activation of latent transforming growth factor beta, resulting in cardiomyocyte migration and subsequent myocardialization of the endocardial cushions. Aberrant apoptosis accompanies cardiac anomalies. Furthermore, an apoptotic population is found surrounding the developing conduction system. A possible role for differentiation is suggested.     

Apoptosis in the nervous system]

Apoptosis (from Greek apoptosis, i.e., falling of leaves) is the phenomenon of programmed cell death, which plays an important role in the normal embryonic development and maintenance of homeostasis of the differentiated tissues of adult organisms. Completion of the apoptosis process is accompanied by specific morphological and biochemical changes in the involved cells. Various disturbances in the control of apoptosis underlie various neurodegenerative diseases, the formation of malignant tumors, autoimmune disturbances, and developmental abnormalities. A deficit of neurotrophic factors leads to apoptosis of neurons. Survival of specific cell populations of neurons is controlled by neurotrophic factors and their combinations. Oncogene bcl-2, a repressor of cell death, belongs to the better studied factors controlling apoptosis. The terminal stages of cell death, including death of neurons, depend on the activation of caspases, specifically caspase-1 (interleukin-1 beta-converting enzyme). Ca2+ and reactive forms of oxygen play an important role in the initiation of apoptosis by changing mitochondrial permeability. Neuregulin, a factor of neuronal origin, is the main controlling factor in apoptosis of Schwann cells, and this process determines the size of their definitive population. Fibroblast growth factor b diminishes apoptosis of Schwann cells in regenerating nerve fibers.     

Disturbed morphogenesis of cardiac outflow tract and increased rate of aortic arch anomalies in the offspring of diabetic rats

BACKGROUND: Maternal diabetes (MD) is a risk factor for offspring to develop cardiovascular anomalies; this is of growing clinical concern since the number of women in childbearing age with compromised glucose homeostasis is increasing. Hyperglycemia abrogates cardiovascular development in vitro; however, a link to cardiovascular defects in diabetic offspring remains to be investigated. METHODS: We have studied cardiovascular development in offspring of MD rats by examining serial histological sections of GD 12.0-18.0 offspring. Development of pharyngeal arch artery malformations was analyzed and related to intracardiac anomalies. RESULTS: Pharyngeal arch artery and intracardiac defects were present in 27 of 37 MD GD 13.0-18.0 offspring. Early sixth arch arteries showed abrogated arteriogenesis, whereas fourth arch artery defects developed as a result of abnormal remodeling. Morphometrical analysis showed increased apoptosis in regressing artery segments and reduced apoptosis in persisting artery segments. Double outlet right ventricle with infundibular stenosis (tetralogy of Fallot) was predominantly found in combination with sixth artery defects and pulmonary atresia. As confirmed by morphometric analysis and three-dimensional (3D)-reconstructions, outflow tract defects coincided with endocardial cushion hypoplasia. Cases with teratology of Fallot additionally showed a shorter outflow tract. No relation with apoptosis or disturbed neural crest cell migration was found. CONCLUSIONS: Our data uniquely demonstrate mechanistic differences involved in the development of sixth and fourth artery anomalies. Whereas increased apoptosis induces fourth artery anomalies, pulmonary outflow obstruction abrogates sixth artery differentiation independent of apoptosis. The model presented allows analysis of diabetic conditions on cardiovascular development in vivo, essential for elucidating this teratology.     

Apoptosis in the Nervous System

Apoptosis (from the Greek apoptosis, i.e., falling of leaves) is the phenomenon of programmed cell death, which plays an important role in the normal embryonic development and maintenance of the homeostasis of the differentiated tissues of adult organisms. Completion of the apoptosis process is accompanied by specific morphological and biochemical changes in the involved cells. Various disturbances in the control of apoptosis underlie various neurodegenerative diseases, the formation of malignant tumors, autoimmune disturbances, and developmental abnormalities. A deficit of neurotrophic factors leads to apoptosis of neurons. The survival of specific cell populations of neurons is controlled by neurotrophic factors and their combinations. Oncogene bcl-2, a repressor of cell death, belongs to the better-studied factors controlling apoptosis. The terminal stages of cell death, including the death of neurons, depend on the activation of caspases, specifically caspase-1 (interleukin-1-converting enzyme). Ca²⁺and reactive forms of oxygen play an important role in the initiation of apoptosis by changing the mitochondrial permeability. Neuregulin, a factor of neuronal origin, is the main controlling factor in apoptosis of Schwann cells, and this process determines the size of their definitive population. Fibroblast growth factor b diminishes the apoptosis of Schwann cells in regenerating nerve fibers.     

The balance of two opposing factors Mad and Myc regulates cell fate during tissue remodeling

Cell proliferation and differentiation are two distinct yet coupled processes in development in diverse organisms. Understanding the molecular mechanisms that regulate this process is a central theme in developmental biology. The intestinal epithelium is a highly complex tissue that relies on the coordination of cell proliferation within the crypts and apoptosis mainly at the tip of the villi, preservation of epithelial function through differentiation, and homeostatic cell migration along the crypt-villus axis. Small populations of adult stem cells are responsible for the self-renewal of the epithelium throughout life. Surprisingly, much less is known about the mechanisms governing the remodeling of the intestine from the embryonic to adult form. Furthermore, it remains unknown how thyroid hormone (T3) affects stem cell development during this postembryonic process, which is around birth in mammals when T3 level increase rapidly in the plasma. Tissue remodeling during amphibian metamorphosis is very similar to the maturation of the mammalian organs around birth in mammals and is regulated by T3. In particular, many unique features of Xenopus intestinal remodeling during metamorphosis has enabled us and others to elucidate how adult stem cells are formed during postembryonic development in vertebrates. In this review, we will focus on recent findings on the role of Mad1/c-Myc in cell death and proliferation during intestinal metamorphosis and discuss how a Mad1–c-Myc balance controls intestinal epithelial cell fate during this T3-dependent process.     

Apoptosis--a death-inducing mechanism tightly linked with morphogenesis in Hydractina echinata (Cnidaria, Hydrozoa).

Programmed cell death is not only known as a mechanism mediating tissue destruction, but also as an organismic tool for body shaping and regulation of morphological events during development. Here we report the tight and vital link of the most prominent form of programmed cell death, apoptosis, to one of the oldest, most basic, and most radical developmental processes, the metamorphosis of the marine hydrozoon Hydractinia echinata. Apoptosis, represented by DNA fragmentation, appears very early during metamorphosis, approximately 20 minutes post induction. It is then executed in a very distinct spatial and temporal pattern, including the removal or phagocytosis of a large number of larval cells prior to the appearance of stolons and tentacles. Our data indicate a developmental program striving to reduce all body parts that are no longer necessary, before reaching a distinct turning point, when the development of adult features is initiated. During these events, morphogenesis of basal and apical structures correlates with recycling of that particular larval region, indicated by the presence of apoptosis. Based on these data, the necessity of apoptosis for normal development of adult patterns is inferred and a fundamental association of apoptosis with developmental processes can be stated.     

Shaping organisms with apoptosis

Programmed cell death is an important process during development that serves to remove superfluous cells and tissues, such as larval organs during metamorphosis, supernumerary cells during nervous system development, muscle patterning and cardiac morphogenesis. Different kinds of cell death have been observed and were originally classified based on distinct morphological features: (1) type I programmed cell death (PCD) or apoptosis is recognized by cell rounding, DNA fragmentation, externalization of phosphatidyl serine, caspase activation and the absence of inflammatory reaction, (2) type II PCD or autophagy is characterized by the presence of large vacuoles and the fact that cells can recover until very late in the process and (3) necrosis is associated with an uncontrolled release of the intracellular content after cell swelling and rupture of the membrane, which commonly induces an inflammatory response. In this review, we will focus exclusively on developmental cell death by apoptosis and its role in tissue remodeling.     

Morphogenesis and dysmorphogenesis of the appendicular skeleton

Cartilage patterning and differentiation are prerequisites for skeletal development through endochondral ossification (EO). Multipotential mesenchymal cells undergo a complex process of cell fate determination to become chondroprogenitors and eventually differentiate into chondrocytes. These developmental processes require the orchestration of cell-cell and cell-matrix interactions. In this review, we present limb bud development as a model for cartilage patterning and differentiation. We summarize the molecular and cellular events and signaling pathways for axis patterning, cell condensation, cell fate determination, digit formation, interdigital apoptosis, EO, and joint formation. The interconnected nature of these pathways underscores the effects of genetic and teratogenic perturbations that result in skeletal birth defects. The topics reviewed also include limb dysmorphogenesis as a result of genetic disorders and environmental factors, including FGFR, GLI3, GDF5/CDMP1, Sox9, and Cbfa1 mutations, as well as thalidomide- and alcohol-induced malformations. Understanding the complex interactions involved in cartilage development and EO provides insight into mechanisms underlying the biology of normal cartilage, congenital disorders, and pathologic adult cartilage.     

Proteins That Fuse and Fragment Mitochondria in Apoptosis: Con-Fissing a Deadly Con-Fusion?

During apoptosis, mitochondria undergo multiple changes that culminate in the release of cytochrome c and other proapoptotic cofactors. Recently, a role for previously overlooked morphological changes, fission of the mitochondrial reticulum and remodeling of mitochondrial cristae, has been suggested in mammalian cells and in developmental apoptosis of C. elegans. Mitochondrial morphology is determined by fusion and fission processes, controlled by a growing set of “mitochondria-shaping” proteins, whose levels and function appear to regulate the mitochondrial pathways of cell death. Expression of pro-fusion proteins, as well as of inhibition of pro-fission molecules reduces apoptosis, suggesting a linear relationship between fragmentation and death. Mechanisms by which mitochondrial fragmentation promotes apoptosis and interactions between fragmentation and remodeling of the inner membrane are largely unclear. A tempting, unifying hypothesis suggests that fission is coupled to cristae remodeling to maximize cytochrome c release.     

Preventing retinal apoptosis — Is there a common therapeutic theme?

There is an urgent need for therapies for retinal diseases; retinitis pigmentosa sufferers have no treatment options available and those targeted at other retinopathies have shown limited effectiveness. The process of programmed cell death or apoptosis although complex, remains a possible target for the treatment of retinal diseases. Having identified apoptosis in the vertebrate retina in populations of immature neurons as an essential part of development it was proposed that re-activation of these developmental cell death pathways might provide insight into the death mechanisms operating in retinal diseases. However, the discovery that numerous factors initiate and mediate the apoptotic cascade in mature photoreceptors has resulted in a relatively untargeted approach to examining and arresting apoptosis in the retina. In the last 5 years, mouse models have been treated with a diverse range of drugs or factors including anti-oxidants, growth factors, steroid hormones, calcium/calpain inhibitors and tetracycline antibiotics. Therefore to draw a unifying theme from these broad research areas is challenging. However, this review focusses on two targets which are currently under investigation, reactive oxygen species and mammalian target of rapamycin, drawing together the common themes of these research areas.     

Early neural cell death: dying to become neurons

The importance of programmed cell death (PCD) during vertebrate development has been well established. During the development of the nervous system in particular, neurotrophic cell death in innervating neurons matches the number of neurons to the size of their target field. However, PCD also occurs during earlier stages of neural development, within populations of proliferating neural precursors and newly postmitotic neuroblasts, all of which are not yet fully differentiated. This review addresses early neural PCD, which is distinct from neurotrophic death in differentiated neurons. Although early neural PCD is observed in a range of organisms, from Caenorhabditis elegans to mouse, the role and the regulation of early neural PCD are not well understood. The regulation of early neural PCD can be inferred from the function of factors such as bone morphogenetic proteins (BMPs), Wnts, fibroblast growth factors (FGFs), and Sonic Hedgehog (Shh), which regulate both early neural development and PCD occurring in other developmental processes. Cell number control, removal of damaged or misspecified cells (spatially or temporally), and selection are the proposed roles early neural PCDs play during neural development. Data from developmental PCD in C. elegans and Drosophila provide insights into the possible signaling pathways integrating PCD with other processes during early neural development and the roles they might play.     

De Novo Nonsense Mutations in KAT6A,a Lysine Acetyl-Transferase Gene,Cause a Syndrome Including Microcephaly and Global Developmental Delay

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129^∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024^∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.     

Congenital malformations among newborns and developmental abnormalities among human embryos in Belarus after Chernobyl accident.

Evaluation of the effects of radioactive contamination on human populations is important for an understanding of the present and future risk for human health, including the genetic risk. This review centers on the results of population monitoring of developmental anomalies among human embryos and congenital malformations among newborn in the Republic of Belarus before and after Chemobyl accident. The data revealed that the incidences of developmental anomalies and congenital malformation from the mostly radionuclide-contaminated rural regions of Belarus reliably exceed the indices in control areas.     

Proteomics investigation of protein expression changes in ouabain induced apoptosis in human umbilical vein endothelial cells

Ouabain is Na(+)/K(+)-ATPase inhibitor and an endogenous regulator of blood pressure, it has dual effect on vascular endothelial cells(VEC) cell growth and VEC apoptosis is contributed to vascular dysfunction involved in vascular remolding. However, the precise mechanisms of apoptosis induced by ouabain remained unclear. The objective of this study was to identify the differently expressed proteins involved in VEC apoptosis induced by ouabain in order to explore cellular and subcellular mechanisms related to ouabain actions. Human umbilical vein endothelial cells (HUVEC) were exposed to increasing concentrations (0.1 nM to 10 microM) of ouabain at 12-48 h intervals. Cell viability tests revealed that high concentrations of ouabain inhibited cell growth. Flow cytometry and caspase-3 activity analysis confirmed that apoptosis was primarily responsible for ouabain induced cell death. Two-dimensional electrophoresis in conjunction with mass spectrometry revealed that the ouabain-induced apoptosis was accompanied by regulated expression of programmed cell death protein 6, cytochrome C1, endothelin converting enzyme, claudin-1, reticulon-4, galectin-1, ras-related protein rab-11B, calnexin, profilin-1 and heat shock protein 60 (HSP60). Further study on cytochrome c and HSP60 demonstrated that levels of mitochondria and cytosol cytochrome c and HSP60 changed in response to ouabain treatment. Data showed that mitochondria proteins such as HSP60 interferes with HSP60-Bax interactions played an important role in ouabain induced apoptosis. These data bring new sights into physiological role for ouabain in VEC apoptosis and vascular remodeling, thus provide new strategies for new anti-cardiovascular disease drug development or the identification of biomarkers for vascular dysfunction in ouabain related hypertension.     

Collagen gel overlay induces apoptosis of polarized cells in cultures: disoriented cell death

In this study, we attempted to investigate the response ofpolarized cells to inappropriate interaction with the extracellular matrix. Cell lines of epithelial [Madin-Darby canine kidney(MDCK) and LLC-PK₁],endothelial [bovine aortic endothelial cells (BAEC)], andmesenchymal (ESK-4 and NIH/3T3) origins were employed. With collagengel overlay, MDCK cells underwent membrane remodeling and graduallydeveloped lumen formation within 24 h. Apoptosis could also be observedfollowing cell remodeling. The ratio of apoptosis was enhanced from12.1 ± 2.4% within 24 h to 58.4 ± 9.8% atday 3, and finally the monolayer wasdisintegrated. Collagen gel overlay-induced apoptosis was not a resultof physical stress, since agarose gel overlay did not induce anymorphological alterations. All epithelial and endothelial cellsexamined developed apoptosis in response to collagen overlay. Incontrast, collagen overlay did not affect growth of fibroblasts at all,although their growth under agarose gel was slightly hindered due tophysical stress. Collagen overlay-induced apoptosis seems to be aunique phenomenon for polarized cells and thus is defined as"disoriented cell death." Furthermore,anti-₂-integrin antibody couldabolish collagen overlay-induced morphological changes and apoptosis inMDCK cells, indicating that signals through₂-integrin on the apicalmembrane are required for disoriented cell death. Finally, Bcl-2overexpression prolonged survival of MDCK cells in response to collagenoverlay, but these cells eventually developed apoptosis due todownregulation of Bcl-2 protein. These findings indicate thatinappropriate cell-matrix interaction results in apoptosis, which mayaccount for cell death mechanisms during developmental processes orunder pathological conditions.

     

Gene Regulation by Thyroid Hormone During Amphibian Metamorphosis: Implications on the Role of Cell-Cell and Cell-Extracellular Matrix Interactions

SYNOPSIS. Amphibian metamorphosis is the developmental processinitiated by thyroid hormone which transforms a tadpole intoa frog. This transformation requires extensive remodeling ofalmost every tissue in the animal. One of the more well-studiedtadpole tissues that undergoes remodeling is the small intestine.This tissue requires a shortening in length as well as internalanatomical restructuring to function in the adult frog. Briefly,the tadpole epithelial cells undergo programmed cell death (orapoptosis) and are replaced by a layer of newly formed adultepithelium. About 20 thyroid hormone-regulated genes participatingin this intestinal remodeling have been identified. These genescan be divided into several groups based on the proposed functionsof their products. One of these groups contains several secretedand/or signaling molecules. Most prominent among these are theXenopus homologs of the hedgehog and stromelysin-3 genes. Basedon the expression profiles and cellular localization, hedgehogappears to be involved in adult epithelial morphogenesis. Stromelysin-3may participate in basal lamina modification which is potentiallyinvolved in the apoptosis of the larval epithelium and developmentof the adult epithelium. Here we will review in detail the potentialroles for these secreted factors as well as the proposed molecularmechanisms responsible for their physiological functions. Furthermore,we will examine the effect of these proteins on the extracellularenvironment and how this impacts upon cellular processes involvedin intestinal remodeling.     

The role of autophagy in human endometrium

Autophagy appears to play an important role in the normal development and maintenance of homeostasis in a variety of tissues, including the female reproductive tract. However, the role of autophagy and the association between autophagy and apoptosis in cyclic remodeling of the human endometrium have not been described. Therefore, we investigated the involvement of autophagy during the human endometrial cycle and its association with apoptosis. Endometrial samples were obtained from 15 premenopausal, nonpregnant women who underwent hysterectomies for benign gynecological reasons. The autophagy-associated protein, microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), was immunolocalized, and its expression level was measured by Western blot analysis. Apoptosis was evaluated by measuring the expression level of cleaved caspase 3 protein. MAP1LC3A protein was primarily expressed within the endometrial glandular cells and increased during the secretory phase. The expression level of the membrane-bound form of MAP1LC3A (MAP1LC3A-II) also increased as the menstrual cycle progressed, reaching a maximum level during the late secretory phase. This pattern coincided with the expression of cleaved caspase 3. Furthermore, expression of MAP1LC3A-II and cleaved caspase 3 increased in the in vitro-cultured endometrial cancer cells when estrogen and/or progesterone were withdrawn from the culture media to mimic physiological hormonal changes. These findings suggest that endometrial cell autophagy is directly involved in the cyclic remodeling of the human endometrium and is correlated with apoptosis. In addition, we inhibited autophagic processes using 3-methyladenine (3-MA) or bafilomycin A1 (Baf A1) to evaluate the role of autophagy in apoptosis induction in endometrial cancer cells. While the inhibition of autophagosome formation using 3-MA did not decrease apoptosis or cell death, the inhibition of autophagosome degradation by fusion with lysosomes using Baf A1 increased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces apoptosis. Furthermore, Baf A1-induced apoptotic cell death was decreased by the apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK). In conclusion, these results indicate that autophagy is involved in the endometrial cell cycle affecting apoptosis and is most prominent during the late secretory phase.     

Correcting developmental errors by apoptosis: lessons from Drosophila JNK signaling

Spatio-temporal regulation of the cell death machinery is essential for normal development and homeostasis of multicellular organisms. While the molecular basis for the central cell death machinery driven by caspases is now well documented, its regulatory mechanisms, especially in the context of living animals, remain to be clarified. The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved kinase cascade that regulates the apoptotic machinery. In mammals, JNK signaling has been implicated in stress-induced apoptosis. Drosophila genetics has now provided evidence of a novel role for JNK-mediated cell death signaling in eliminating developmentally aberrant cells from a tissue. The JNK-dependent cell-elimination system is orchestrated by cell-cell communication between normal and aberrant cells and is essential for ensuring developmental robustness, as well as for protecting organisms against fatal abnormalities such as neoplastic development. These processes are mediated by cell competition, morphogenetic apoptosis, and intrinsic tumor suppression. A combinatorial approach using both genetic and live-imaging systems in Drosophila will be extremely powerful to decipher how JNK-mediated apoptosis works within multicellular communities.