Improved culture of non-human primate embryos reveals the establishment of the crucial framework for subsequent development of bodily tissues and the germline in fetuses, bringing us closer to comprehending the elusive development of early human embryos

Neupane, Jitesh

Surani, M Azim

Wong, Frederick CK

English

Apollo (Cambridge)

 1RESEARH HIGHLIGH  1 The unfolding body plan of primate embryos in culture 2 Jitesh Neupane1,2, Frederick C. K. Wong1,2 and M. Azim Surani1,2 3  4 1Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, 5 Cambridge CB2 1QN, United Kingdom; 2Physiology, Development and Neuroscience 6 Department, University of Cambridge, Cambridge, United Kingdom 7  8 Correspondence: M. Azim Surani (a.surani@gurdon.cam.ac.uk) 9  10 Improved culture of non-human primate embryos reveals the establishment of the 11 crucial framework for subsequent development of bodily tissues and the germline in 12 fetuses, bringing us closer to comprehending the elusive development of early human 13 embryos. 14  15 Most of what we know about very early mammalian development after blastocyst implants in 16 the womb comes from mouse studies [1]. Little is known about development in primates, and 17 even less so for human development, because of many challenges and constraints on studies 18 on early human embryos. Accordingly, studies on non-human primate embryos might 19 provide information relevant to human development. Two recent studies now report on the 20 development of cynomolgus monkey embryos for 20 days post fertilization, and their 21 progress through gastrulation in culture [2, 3]. They compare their observations on 22 embryonic development in culture by referring to a previous study on the developing monkey 23 embryos in vivo [4]. 24  25  2Shortly after implantation, the inner cell mass (ICM) of humans and non-human primates 26 develop into epithelial cells, followed by the formation of a lumen, which gives rise to the 27 amniotic cavity. The embryonic tissues themselves develop as a bilaminar disc comprising 28 the epiblast on top of the hypoblast; the latter originates from the primitive endoderm. The 29 epiblast and amnion epithelium encapsulate the amniotic cavity while the hypoblast 30 endoderm and extraembryonic mesoderm encapsulate the primary yolk sac. The formation of 31 a primitive streak along the midline of the epiblast disc marks the onset of gastrulation. 32 Gastrulation is a pivotal event during early development when the three primary germ layers, 33 endoderm, ectoderm, and mesoderm, appear for the first time. Primordial germ cells (PGCs), 34 which eventually develop into sperm or eggs, also emerge around this time, either in the 35 amnion according to some studies, epiblast or in both tissues [5-7]. The anterior and posterior 36 ends of the embryos now become distinguishable before the germ layers undergo further 37 differentiation towards organogenesis, in which the anterior cells,  e.g., develop into neuronal 38 cells. 39  40 In the recent studies on the cynomolgus monkey embryos, Ma et al. [2] and Niu et al. [3] 41 adopted in vitro culture methods previously used to culture mammalian embryos. These 42 studies reveal aspects of non-human primate embryo development after embryo implantation, 43 through gastrulation. These independent studies showed the development of cynomolgus 44 monkey embryos up to 20 days post fertilization (dpf). Niu et al. used the culture system 45 described elsewhere [8]. In contrast, Ma et al. added some novel aspects to their culture 46 conditions containing different serum concentrations, which was first optimized using mouse 47 blastocysts [2]. 48  49  3Broadly speaking, the development of the monkey embryos in both studies recapitulated key 50 developmental events previously described for embryonic development in vivo [4, 6]. They 51 observed the formation of the bilaminar disc structure comprising amniotic and yolk sac 52 cavities and the establishment of an anterior-posterior axis. They also saw the emergence of 53 the primitive streak, and the appearance of the PGC-like cells and of the structures 54 resembling neural plate folding [2]. In the future, it will be essential to conduct a rigorous, 55 detailed analysis of development using approaches such as lineage tracing and live imaging 56 to establish the precise origin and destiny of diverse cell types in the embryo, at a time when 57 the embryo undergoes extensive morphogenetic changes.     58  59 For now, both studies opted to conduct single-cell transcriptome analysis in the hope to 60 characterize the diverse cell types present in the developing embryos.  Ma et al. and Niu et al. 61 compared different stages of embryo development, which they compared with the single-cell 62 transcriptome analysis of cynomolgus monkey embryos in vivo described previously [4]. 63 Although cells of the majority of embryonic and extraembryonic lineages found in vivo were 64 detected in embryos in culture, some disparities in the clustering of cell types were apparently 65 observed. The discrepancies might be due to the appearance of transient and intermediate cell 66 lineages, but the presence of aberrant cells in embryos in culture cannot be excluded. 67 Nonetheless, Ma et al. were able to annotate early (11-14 dpf) and late (16-17 dpf) 68 gastrulating or amniotic cells, where the transcriptional profile of amniotic cells in embryos 69 were not reported before [4]. Niu et al., on the other hand, investigated the chromatin 70 accessibility of the post-implantation embryo at the single-cell level, which, for example, can 71 detect enhancer elements in different cell types during development. Overall, the cell 72 identities from these two studies overlap with those found in vivo, suggesting that embryos in 73 culture broadly follow events observed in vivo. Note that the culture methods used in these 74  4studies can support up to 22% of embryos to the gastrulation stage without feto-maternal 75 interactions. Whether the efficiency of development observed reflects inherent differences in 76 the quality of the embryos or a consequence of culture conditions is unknown. If it is the 77 latter, further optimization of culture conditions, might result in better development at a 78 higher efficiency. 79  80 These studies have strengthened our current understanding of the early post-implantation 81 development in primates. They will enhance the technological and conceptual advancement 82 for translating the knowledge to stem cell research and developmental biology. Reliable and 83 efficient culture models could be used to investigate the morphological, molecular, and 84 physiological properties of primate embryos, which could, in principle, be used to study early 85 human peri- to post-implantation development in vitro. There are species-specific differences 86 between early human and non-human primate development, indicating caution when 87 considering extrapolation to human development. The differences include the timing of 88 implantation and amniogenesis between cynomolgous monkey and humans [9], which 89 reflects possible variations due to species evolution [10]. The schedules of gastrulation and 90 establishment of PGCs might also differ, although the regulatory network for PGC 91 specification is apparently broadly conserved in mammals with bilaminar disc embryos [7, 92 10].  Some studies, for example, suggest that PGC-like cells in monkeys can be found in the 93 nascent amnion during the mid-second week (~11 dpf) of development [6]; further work is 94 needed to determine whether amnion and/or epiblast could be the site for the origin of PGCs. 95 Note that PGC specification in the porcine bilaminar disc embryos occurs in the epiblast 96 where the gene regulatory network is more like that in human and not mouse embryos [5]. 97 While the developmental events might be broadly similar amongst mammals, there are more 98 significant differences in the development of the extraembryonic tissues. Combined with 99  5possible timing differences amongst mammalian species and their extraembryonic tissues, 100 differences in the molecular mechanism of embryonic development amongst primates cannot 101 be excluded.   102  103 The recent studies on gastrulating non-human primate embryos in culture represent a 104 significant advance; much will, however, depend on whether the culture models can be used 105 for more in-depth mechanistic studies. While extrapolation from these studies to early human 106 development is possible, crucial species differences amongst primates cannot be entirely 107 excluded. With legal restrictions on similar studies on human embryos, alternative 108 approaches such as establishing in vitro models for early human development with 109 embryonic stem cells, and generating embryoid-like structures is an option [11]. Increasing 110 improvements and sophistication of the in vitro models might for now address some key 111 questions but cannot substitute entirely for direct studies on very early human development. 112 Such studies, if possible, are of potential value for advances in regenerative medicine and 113 treatment of human diseases.   114  115   116  6References 117 1. ROSSANT, J. AND P.P.L. TAM. Science 360, 1075 (2018). 118 2. MA, H. et al. Science 366, eaax7890 (2019). 119 3. NIU, Y. et al. Science 366, eaaw5754 (2019). 120 4. NAKAMURA, T. et al. Nature 537, 57-62 (2016). 121 5. KOBAYASHI, T. et al. Nature 546, 416-420 (2017). 122 6. SASAKI, K. et al. Dev Cell 39, 169-185 (2016). 123 7. IRIE, N. et al. Cell 160, 253-268 (2015). 124 8. DEGLINCERTI, A. et al. Nature 533, 251-254 (2016). 125 9. LUCKETT, P. Am J Anat. 144, 149-167 (1975). 126 10. SYBIRNA, A., F.C.K. WONG, AND M.A. SURANI. In R. LEHMANN. Curr Top Dev Biol, 127 Academic Press. pp35-89 (2019).  128 11. ZHENG, Y. et al. Nature 573, 421-425 (2019). 129 12. HEUSER, C.H. AND STREETER, G.L. CARNEGIE INST. WASH. PUBL. 525 (29): 15-55 130 (1941). 131 13. BOROVIAK, T. AND NICHOLS, J. DEVELOPMENT. 144: 175-186 (2017). 132  133  134   135  7Fig. 1 Schematic illustration showing that post-implantation embryo development of 136 primates might be relevant to humans. a Implantation begins with the attachment of 137 blastocyst to the maternal uterine endometrium, when trophoblast cells proliferate into 138 cytotrophoblast and syncytiotrophoblast, which eventually expand and surround the embryo. 139 b The amniotic cells develop from epiblast cells and form an amniotic cavity, whereas cells 140 from hypoblast form yolk sac endometrium, resulting in a yolk sac cavity. The embryo 141 develops from bilaminar disc epiblast on top of hypoblast cells. Primitive streak appears at 142 the posterior end of embryonic disc, when epiblast starts invading towards hypoblast, thus 143 marking the initiation of gastrulation. Proliferating epiblast cells along the streak migrate to 144 the space between epiblast and hypoblast giving rise to mesoderm cells, thereby converting 145 bilaminar embryonic disc into a trilaminar disc. Primordial germ cells, the precursors of male 146 and female gametes, might originate from the amnion, the epiblast or both tissues during the 147 pre-gastrulation period. They migrate on the wall of the yolk sac close to the allantois, and 148 subsequently along the hindgut before and after colonizing the gonadal ridge. (Figure adapted 149 from Heuser et al., (1941) [12] and Boroviak and Nichols (2017) [13])  150 

The unfolding body plan of primate embryos in culture.

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The unfolding body plan of primate embryos in culture.

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