Insights & Perspectives Cell death and morphogenesis Think again during early mouse development: Are they interconnected? Ivan Bedzhov1)2) and Magdalena Zernicka-Goetz1)2)* Shortly after implantation the embryonic lineage transforms from a coherent ball of the vertebrate limb [1] and until of cells into polarized cup shaped epithelium. Recently we elucidated a recently was considered responsible previously unknown apoptosis-independent morphogenic event that reorgan- for the establishment of the hollow tube of the egg cylinder [2, 3]. Alternatively, izes the pluripotent lineage. Polarization cues from the surrounding basement central luminal space can be formed in a membrane rearrange the epiblast into a polarized rosette-like structure, where solid cell cluster by apoptosis-indepen- subsequently a central lumen is established. Thus, we provided a new model dent mechanism if strong polarization revising the current concept of apoptosis-dependent epiblast morphogenesis. cues are provided [4, 5]. In early embryos Cell death however has to be tightly regulated during embryogenesis to ensure such signals originate from the base- ment membrane that surrounds the developmental success. Here, we follow the stages of early mouse development epiblast, and they drive polarization and take a glimpse at the critical signaling and morphogenic events that and lumenogenesis in the embryonic determine cells destiny and reshape the embryonic lineage. lineage during the peri-implantation stages [6]. This concept is contrary to Keywords: the apoptosis-dependent mechanism .apoptosis; blastocyst; egg cylinder; epiblast; implantation; morphogenesis of the textbook model describing epi- blast morphogenesis from pre- to post- implantation stages [2, 3]. Introduction diversity of tissues that undergo mor- phogenetic transformations, reshaping Apoptosis and necrosis - Sperm entry triggers the “big bang” that the developing embryo. The first two cell transforms a fertilized egg into a new fate decisions set up the embryonic and major mechanisms of cell organism. Cell fate choices generate a extraembryonic lineages. The embryonic death lineage or the epiblast (EPI) contains pluripotent progenitors that give rise to Multiple cell generations maintain and DOI 10.1002/bies.201400147 all tissues of the foetus. The signals that expand the embryonic and extraembry- organise and support the development onic structures throughout embryogene- 1) Department of Physiology, Development and of the EPI are provided by derivatives of sis. This continuity relies on the tightly Neuroscience, University of Cambridge, Cambridge, UK the two extraembryonic lineages - the balanced processes of cell death and 2) Wellcome Trust/Cancer Research UK Gurdon trophectoderm (TE) and the primitive survival. Cell death can occur following Institute, University of Cambridge, Cambridge, UK endoderm (PE) (Fig. 1A). The fitness of the paths of necrosis or apoptosis. *Corresponding author: these early lineages is essential for Necrosis is not a regulated process and Magdalena Zernicka-Goetz embryo survival and development to is usually a result of physical damage or E-mail: [email protected] term. Cells that fail to segregate into ischemia that compromises cell integrity appropriate positions according to their leading to the release of cell contents into Abbreviations: BM, basement membrane; EB, embryoid body; EC cell fate program, or lack survival the external environment. In contrast, cell, embryonic carcinoma cell; ECM, extracellular signals, have to be eliminated to main- programmed cell death (PCD) or apopto- matrix; EPI, epiblast; ES cell, embryonic stem cell; tain tissue integrity and function. Cell sis is a multilevel, heavily regulated ExE, extraembryonic ectoderm; ICM, inner cell mass; PCD, programmed cell death; PE, primitive endo- death also directs morphogenic process- process, activated by external or cell derm; TE, trophectoderm; VE, visceral endoderm. es such as the sculpting of the digits intrinsic stimuli. The intrinsic apoptotic 372 www.bioessays-journal.com Bioessays 37: 372–378, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. .....Insights & Perspectives I. Bedzhov and M. Zernicka-Goetz Think again Figure 1. Determinants of embryo viability during early mouse development.A:Overview of the pre-, peri- and the early post-implantation stages of mouse embryogenesis. B: Patterns of cytoplasmic movements predict the developmental potential of the zygote. C: Cell division patterns of 2- to 4-cell stage transition in relation to the developmental success to term. D: Paracrine and autocrine pro-survival factors during pre-implantation embryogenesis. E: Cell sorting and elimination of mis-positioned cells by apoptosis during the second cell lineage segregation. F: Activation of the mTOR pathway downstream of the PI3K/Akt signalling cascade. G: Mdm2–p53 regulatory loop regulating embryo survival during the peri-implantation stages of development. Bioessays 37: 372–378, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc. 373 I. Bedzhov and M. Zernicka-Goetz Insights & Perspectives..... program can be triggered as a response signals can be provided via integrin- and downstream signaling components to DNA damage, high calcium and extracellular matrix interactions [10], oftheIgf,Egf,Tgf-b and Pdgf families are oxidant levels or lack of survival signals, cadherin mediated cell-cell contacts [11] expressed throughout pre-implantation whereas the extrinsic apoptotic cascade or by soluble factors. Thus, a crosstalk of development [14] (Fig. 1D). In addition, E- is downstream of ligand activated cell multiple signalling pathways tightly cadherin (E-cad) mediated adhesion, surface receptors. Membrane blebbing, regulates the balance between cell death which is essential for proper blastocyst nuclear condensation and formation of and survival. Embryo viability directly formation, also provides pro-survival apoptotic bodies are the typical morpho- depends on the fitness of the zygote and cues. The E-cad extracellular domain logical characteristic of cells undergoing embryo’s capacity for full development to interacts with Igf1r, mediating efficient apoptosis. Subsequently, the dying cells term can be predicted as early as the first activation of the receptor [15, 16]. In turn, Think again and debris are rapidly cleared from the hours post fertilization (Box 1). Observa- Igf1r triggers anti-apoptotic, metabolic tissue by efferocytosis [7, 8]. tions of embryonic development in vitro and mitogenic responses in developing show that embryos cultured in larger embryo through the PI3K/Akt pathway groups or in smaller volumes of medium [17, 18]. Activated Akt phosphorylates develop to the blastocyst stage with and sequesters pro-apoptotic factors How are the processes of higher rates and contain fewer cells such as BAD, thus keeping the intrinsic cell death and survival undergoing apoptosis in comparison to apoptotic program at bay [19]. balanced during pre- individually cultured embryos [12, 13]. The observed incidence of apoptosis The commonly used media are relatively at the blastocyst stage differs between implantation simple, chemically defined solutions, the TE and the inner cell mass (ICM). development? containing no additional growth factors. PCD in the TE is a relatively rare event, Thus, embryos themselves produce solu- whereastheratesofapoptosisintheICM Apoptosis is suggested as a default cell ble ligands acting in auto- and paracrine are significantly higher [20, 21]. It is destiny that has to be continuously manner, and in larger volumes these proposed that the apoptotic process in suppressed by survival signals [9]. Such factors are diluted out. Ligands, receptors the ICM eliminates cells that failed to Box 1 How can early embryo survival be predicted? PCD is involved in the process of oogenesis, where most of to 4- cell stage, single blastomeres lose capacity for full the oocytes are lost during the first stages of meiotic development to term [62]. How can this be explained? On prophase I [57]. Thus, the females are born with a the one hand the 4-cell stage blastomeres are smaller, since substantially lower number of oocytes in comparison to the cleavage divisions during the pre-implantation period the early stages of oogenesis. The mature oocytes can increase the cell number, but the total cell volume remains activate multiple signaling cascades, driving cell death constant. These individual blastomeres are still able to form through apoptosis, necrosis and autophagy [58]. After small blastocyst-like structures, known as trophoblastic fertilization the first typical features of PCD can be vesicles, but they fail to develop further. This could be observed in the polar bodies [59]. Since embryogenesis because a minimum of four EPI cells needs to be present in directly depends on the fitness of the zygote, can the the blastocyst before implantation for successful establish- potential for full development be predicted at that stage? ment of the embryo proper, and this number is never Fertilization of the egg triggers cytoplasmic Ca2þ oscil- reached in embryos derived from individual 4-cell stage lations that induce contractions of the actomyosin blastomeres [63]. On the other hand, the cleavage pattern of network, manifested as rhythmic cytoplasmic movements.