Cell Death during Developmental Processes

Abstract

Embryonic development and differentiation to adult form depends on orchestration of cell division and death. In embryos, programmed death sculpts form, opens lumens, separates or splits tissue layers, allows tissue layers to fuse and removes vestigial organs. Both the central nervous and immune systems overproduce cells and destroy those that do not form successful synapses or produce unusable antibodies. Cell death is first seen in mammalian embryos when the blastocyst expands, but elsewhere, the first deaths are not seen before the maternal–zygotic transition. Abnormal timing, amount or localisation of cell death leads to abnormalities or death of embryos.

Several signalling pathways trigger cell death. Usually, the signals activate caspases (first discovered in embryonic cell death in nematodes) and lead to apoptosis, although apoptosis is not the only form of cell death. The signalling mechanisms that control cell death in embryos are not well understood, but should be if we hope to understand normal and teratological development.

Key Concepts

  • Cell death can be seen in both embryonic development and normal growth of adult tissue.
  • The embryonic cell deaths are highly programmed in that they are predictable in location, time and amount. In the simplest instances, such as in nematodes, control of cell death is under direct control of a small number of genes.
  • Most but not all of the embryonic deaths are apoptotic.
  • Embryonic cell to sculpt the embryo and define the boundaries of tissues and organs. In the central nervous system and the immune system, overgrowth (production of excessive cells) and subsequent pruning by cell death generate the high specificity that characterises these systems.
  • Deregulation of apoptosis can produce many embryonic abnormalities and teratologies and, later in life, produces cancers, autoimmune disease or neurodegenerative disease.
  • There are many means to study cell death, but only a few are directly applicable to the study of cell death in embryos. Nevertheless, further study is needed to understand the signalling mechanisms that decide the death of cells in specific locations and times.
  • Autophagy is increasingly recognised as a factor influencing the likelihood of onset of apoptosis and cell death.
  • New ultra‐resolution fluorescence microscopic techniques, capable of simultaneously analysing energy flux, autophagy and apoptosis, may lead to new insights into these questions.
  • Learning more about cell death in embryos will help us understand how it is controlled in adults.

Keywords: apoptosis; autophagy; caspase; cell death receptor; embryo; embryonic development; gene expression; programmed cell death; techniques

Figure 1. Schematic overview of the major cell death (apoptotic) pathways. Two major pathways lead to apoptosis: external (type I or ligand‐mediated) and internal (type II or metabolically mediated). When apoptosis is initiated through the external pathway, signalling occurs through the interaction between extracellular death ligands such as FasL or tumour necrosis factor α (TNFα) and death receptors such as type I receptor for tumour necrosis factor (TNFR1) and TNF‐related apoptosis‐inducing ligand (TRAIL) receptors followed by activation of the death domain of intracellular part of receptor. Then these components form the death‐inducing signalling complex (DISC), which recruits and activates procaspase‐8. Activated caspase‐8 is an initiator caspase that subsequently cleaves and activates downstream effector caspases such as caspase‐3 and caspase‐7 and finally results in apoptotic cell death. The intrinsic cell death pathway is triggered by a metabolic problem, the nature of which is unknown for embryonic as opposed to experimental models. The signal initiates from within the cell and is mediated by mitochondria, which releases proapoptotic factors from the mitochondria. This release is tightly controlled by the proteins Bak and Bax and BH3‐only protein. The released factors, including cytochrome c and smac/Diablo, lead to the formation of apoptosome in association with Apaf‐1 and procaspase‐9. Activation of caspase‐9 leads to activation of caspase‐3 and apoptotic cell death. The apoptosis‐inducing factor (AIF), released by mitochondria, is capable of inducing apoptosis independent of caspases (Candé et al., ). These steps can be inhibited by the antiapoptotic members of the Bcl‐2 family of apoptosis regulators. Finally, spontaneously for massive and perhaps some other cells, and otherwise if for any reason apoptosis is not evoked, cells destroy the bulk of their contents through autophagy. This may lead to the death of the cell with or without any sign of apoptosis.
Figure 2. Cell death during the morphogenesis of the mouse limb. (a) Embryonic day 13.5 mouse forelimb stained with Nile blue sulphate. Regions of cell death, indicated by the dye picked up by phagocytes, are obvious in the interdigital regions, along the apical ectodermal ridge, and along the anterior (inner) side of the limb. (b) Micrograph of the interdigital region of a similar limb, showing apoptotic cells identified by a positive TUNEL reaction (magenta stain). (c) A semi‐thin section of a similar section, in which apoptotic cells, many within phagocytes, are identified by the condensation of chromatin that gives nuclear material a very dense appearance when stained, as here, by toluidine blue or any other acidophilic dye.
Figure 3. Localised cell deaths during morphogenesis in zebrafish. (a) Dying cells revealed by acridine orange staining in the lens of a 25‐h zebrafish embryo. Focus is at the interface between developing lens and lens epithelium. (b) Lens stalk retained between lens and epithelium if apoptosis is blocked by inhibitor of caspase 3. (c) Caspase‐3 activity, revealed by presence of fluorescent inhibitor zVAD‐FMK (carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone or Carbobenzoxy‐Val‐Ala‐Asp‐Fluoromethylketone), in what will become the vent (anus) of a 48‐h zebrafish embryo. (d) Phase‐contrast image of the same embryo, illustrating the developing vent. If caspase activity is inhibited, the vent will not open. Notes: L, lens and R, retina. Figure courtesy of Nathaniel Abraham.
Figure 4. Cell death along the anterior–posterior axis in the zebrafish larva. (a) Dying cells scattered across an approximately 20‐h zebrafish embryo, revealed by staining with acridine orange. (b) Death of Mauthner cells (large cells regularly spaced along the spine) and a few cells in the tail of an approximately 96‐h zebrafish larva. Photographed by Richard A. Lockshin © R. Lockshin 2010.
Figure 5. What makes a cell die: Not all deaths are apoptosis. Often a massive accumulation of autophagic vacuoles (here observed as green GFP‐LC3 positive structures), which can be seen in close spatiotemporal relationship to the mitochondria (red) precedes the onset of cell death. Scale bar: 20 µm.
Figure 6. A point of no return (PONR) for apoptosis has been previously described in context of mitochondrial depolarisation and is often preceded by changes in the mitochondrial network. These changes can now be morphometrically assessed in great detail, revealing mitochondrial loop formations or shifts in the fission and fusion equilibrium. Mouse hypothalamic GT1‐7 neuronal stained with JC‐1. This ratiometric fluorescent dye indicates polarised (red) and depolarised (green) regions of the mitochondria. SR‐SIM acquisition and three‐dimensional rendering reveals a distinct mitochondrial network that may dynamically shift between a fission (a) or fusion state (b). Depth colour coding (c) reveals the spatial organisation in z, with orange indicating the most superficial mitochondria. Often also circular mitochondria are observed, that can, due to super‐resolution, be quantitatively assessed (d).
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Zakeri, Zahra, Loos, Ben, and Lockshin, Richard A(May 2015) Cell Death during Developmental Processes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022094.pub2]