Cell Death and Morphogenesis During Early Mouse

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 basement 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 epiblast morphogenesis from pre- to post- implantation stages [2, 3].

Introduction diversity of tissues that undergo morphogenetic 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

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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.

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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. the 2- to 4-cell stage transition was also found to influence Using high-speed time-lapse imaging the patterns of developmental success. The zygote and 2-cell stage cytoplasmic movements have recently been related to the blastomeres can divide either meridionally (M) or equato- developmental potential of the zygote. Frequent speed rially (E). These sequential divisions generate four different peaks, as well as a low mean basal speed of cytoplasmic kinds of embryos: ME, EM, MM or EE (Fig. 1C). In contrast movement, were found to be associated with a low rate of to the other three classes, the development of EE embryos development to the blastocyst stage in vitro and to birth in to term is significantly reduced [64]. To elucidate the vivo (Fig. 1B). Thus, the dynamics of cytoplasmic flows underlying molecular mechanism and potentially identify a triggered by the sperm are predictive of the embryo’s unique EE transcriptional signature, it will be important to potential to develop to term [60]. correlate the transcriptional profile of individual 4-cell stage The regulative development of pre-implantation embry- blastomeres with their division pattern. It also remains to os enables adaptation to reductions in cell number. If one of be determined whether the history of the EE divisions can the 2-cell stage blastomeres is destroyed, an individual be traced back and correlated with the cytoplasmic totipotent blastomere can still complete embryonic devel- movements of zygotes that have predicted reduced opment [61]. Within one round of cell divisions, from the 2- developmental potential.

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translocate to the correct position for Interestingly, inactivation of genes as- ment. Only embryos that successfully their fate during the segregation of the sociated with neoplastic transforma- attach and implant stand a chance of PE and EPI cell populations [22–24] tions in adult tissues, such as Brca1 completing embryogenesis. The process hn again Think (Fig. 1E). However, the mechanism by and 2, lead to growth arrest of the early of implantation also boosts cell prolif- which cells sense their “incorrect” posi- egg cylinders [34–36]. Another example eration and, for the first time, embryo tion remains unknown. A potential is the loss of function of Mdm2 oncogene growth is initiated. As the egg cylinder autocrine loop of Egfr activation by the that results in a complete elimination of emerges the EPI dramatically changes Tgf-a ligand is proposed to modulate the all cells in the embryo shortly after its morphology from a simple ball of levels of PCD in the ICM [25]. This is also implantation. Mdm2 binds directly to unpolarised cells into a cup-shaped indicated by the depletion of Egfr, which p53 and inhibits the expression of p53 pseudostratified epithelium, surround- results in complete ICM degeneration in target genes (Fig. 1G). In the absence of ing the proamniotic cavity. the CF-1 mouse genetic background [26]. Mdm2, p53 activity is not regulated and Cavitation and hollowing are the Maintenance of the PE depends on pro- the intrinsic apoptotic program is trig- two major paths through which a solid survival cues downstream of Pdgfra. gered, killing the embryo by E5.5. The cohort of cells can be transformed into Genetic ablation or pharmacological Mdm2–p53 regulatory circuit can be a tube by the generation of a central inhibition of this receptor result in bypassed by combined deletion of both luminal space. Cavitation is an apopto- increased Caspase-3 activity and deple- genes, rescuing the double knockout sis-dependent mechanism of cell elimi- tion of the PE layer [27]. embryos [37, 38]. Deletion of p53 alone nation in the core of a coherent mass of After the second lineage segregation does not affect embryonic development cells. Hollowing is apoptosis-indepen- is complete, the mature blastocyst in general, although a subset of later dent, but requires separation of apical hatches out of the zona and initiates (E13.5–E16.5) embryos exhibit exence- membranes in a radially polarized implantation. The mural TE mediates phaly (location of the brain outside structure [4, 5]. In vitro, the same type the first interactions of the implanting the cranial cavity) [39]. Thus, hyper- of cells are able to follow either of these blastocyst with the maternal environ- activation of p53 leads to global PCD and alternative paths, depending on cell ment. The direct contact between the peri-implantation lethality, whereas in- density and the efficiency of establish- TE cells and the uterine epithelium activation of p53-dependent apoptosis ing apical-basal polarity. For example, induces apoptosis at the attachment has no effect on early embryogenesis. MDCK cells grown at low density and site, allowing penetration of the embryo receiving strong polarization cues po- into the underlying stroma. This apo- larize rapidly and form a central lumen ptotic process is suggested to be a result via hollowing. However, when MDCK of TNF-receptor I activation that triggers Does peri-implantation cells are grown at high density, in the Caspase-3 mediated local PCD of the morphogenesis depend on absence of strong polarization signals, luminal epithelium [28, 29]. the process of apoptosis the central space is gradually formed by apoptosis [44]. Which mechanism is or is there an alternative utilized by the embryo to establish the mechanism? cup-shaped EPI after implantation? Multiple signaling According to a long-standing simple pathways regulate Following implantation the polar TE and elegant two-step model, the EPI was apoptosis and survival forms the extraembryonic ectoderm thought to be reshaped by an apoptosis during the peri- (ExE) at the proximal region of the driven process of cavitation (Fig. 2A). egg cylinder. The ExE contains the This model proposes that at E5.0 the EPI implantation and early multipotent progenitors of the tropho- of the early egg cylinder is a coherent post-implantation stages blast lineage that form the embryonic mass of pluripotent cells. As the egg portion of the placenta. The PE layer cylinder elongates, the VE provides an As the embryo invades the maternal differentiates into parietal endoderm apoptotic signal to eliminate the cells in environment the surrounding stroma (PE) that migrates over the mural TE the core. The EPI cells in direct contact proliferates and transforms into the surface and visceral endoderm (VE) that with the surrounding basement mem- decidua that supports the growth of engulfs the developing egg cylinder. brane (BM) are rescued and form the developing egg cylinder and ensures Signalling centers of the VE pattern the polarized epithelium at the same time foetomaternal immune tolerance [30]. underlying EPI, breaking its symmetry as the cavity is established [2, 3]. The Following implantation, multiple signal- to establish the anterior – posterior evidence supporting this model comes ing cascades regulate the processes of axis [40–43]. from studies using embryoid bodies cell death, survival and proliferation in The process of EPI re-organization (EBs) composed of embryonic stem the embryo. The Igf pathway promotes during peri-implantation stages has (ES) or embryonic carcinoma (EC) cells. survival and cell proliferation by acti- been a long-standing mystery. Embryos Cavitation in EBs never starts from the vating mTOR downstream of the PI3K/ at those stages are no longer free- center, instead multiple peripheral cav- Akt cascade [31] (Fig. 1F). Genetic floating and as they invade the maternal ities are established that coalesce as the inactivation of class IA or class 3 PI3K tissues they become relatively inacces- core is gradually eliminated by PCD. EBs results in embryonic lethality at the time sible. The time of implantation is one of contain hundreds to thousands of cells or shortly after implantation [32, 33]. the most critical periods of develop- and the processes of cavity formation

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Figure 2. Models of peri-implantation morphogenesis. A: Apoptosis-dependent two-step signalling mechanism of cavitation in the early embryo. B: Self-organizing mechanism of EPI re-organization into a rosette-like structure, orchestrated by BM-provided polarization cues.

and establishment of epithelial polarity cluster in the center. Although apoptotic the basket-shaped BM can act as a mold, are slow, over the course of several cells and cell debris can be found in transforming the symmetric rosette into days [2, 45]. Does the embryo follow the some embryos, a single central lumen a cup [6, 48]. same path of EPI re-organization? emerges independently of PCD through The BM function in the establish- Using embryos directly isolated from hollowing, most likely via charge repul- ment of apical basal polarity can be the uterus or blastocysts cultured in sion of apical membranes. Fluid ﬁlling substituted in vitro by culturing isolated vitro throughout the corresponding peri- mechanisms such as exocytosis and ICMs in 3D ECM. The EPI cells polarize implantation stages we revealed a pumping are likely to contribute to and initiate lumenogenesis in the ab- strikingly different sequence of events further enlarging the lumen. Similar sence of a surrounding VE layer, (Fig. 2B). The polar TE and the PE secrete morphogenic changes occur in the indicating that a death signal from the ECM proteins that establish a basement ExE, where the processes of polarization VE is not required for this process. membrane (BM) that wraps around the and lumenogenesis generate small inter- Similar morphogenetic changes can be EPI of the late E4.5 blastocyst [46, 47]. membranous spaces that later contrib- induced in ES cells when they are Within 24 hours, polarization cues pro- ute to the mature proamniotic cavity. embedded into 3D ECM at low density. vided by the BM establish apical basal During the differentiation of the polar The ES cell spheres formed in these polarity in the EPI cells, through integrin TE into ExE, the BM that separates conditions efﬁciently polarize and es- mediated signalling. As a result, at this lineage from the EPI is no longer tablish central lumen, mimicking the the time of implantation the EPI globally maintained (Fig. 1A). Thus, at the early pre- to post-implantation transition re-organizes into a radially polarized egg cylinder stage a common BM of the EPI lineage [6]. Together these rosette-like structure. Constriction of the provided by the VE surrounds both the observations lead to a new model actomyosin network reshapes the ini- EPI and the ExE. Since the EPI cells of peri-implantation morphogenesis, tially round cells, as the apical domains are anchored to the ECM by integrins, where the BM established by the

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