- Email: [email protected]
Untangling early embryo development using single cell genomics
Journal Pre-proof Untangling early embryo development using single cell genomics
Ramiro Alberio PII:
S0093-691X(20)30075-3
DOI:
https://doi.org/10.1016/j.theriogenology.2020.01.062
Reference:
THE 15359
To appear in:
Theriogenology
Received Date:
19 January 2020
Accepted Date:
28 January 2020
Please cite this article as: Ramiro Alberio, Untangling early embryo development using single cell genomics, Theriogenology (2020), https://doi.org/10.1016/j.theriogenology.2020.01.062
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Untangling early embryo development using single cell genomics Ramiro Alberio
School of Biosciences, University of Nottingham, NG2 5RD, UK
Running title: Lineage segregation in mammalian embryos
Correspondence: Ramiro Alberio: [email protected] OrcidID: 0000-0001-6560-3919
Keywords: Embryo; Single Cell Genomics; Cell Lineages; Pluripotency
1
Journal Pre-proof Abstract
The zygote undergoes five cell divisions prior to the first signs of lineage segregation. Blastocyst formation requires segregation of the trophectoderm, needed for implantation, and the inner cell mass, which differentiate towards the major lineages of the fetus. This process is broadly conserved in mammals, however, in recent years investigations using high throughput single cell transcriptomics have provided new insights on the gene regulatory networks and epigenetic mechanisms controlling these processes in different species, highlighting novel unique evolutionary adaptations. Although analysis of single cell datasets is inherently challenging due to stochastic gene expression in single cells, continuous development of novel computational tools have contributed to improving the quality of these datasets. Single cell -omics provides detailed information on discrete cellular states, and when combined with spatial transcriptomics it can inform on the relationship between cellular compartments and fate determination. This technology has recently been used to shed new light into the progression of lineage segregation, establishment of pluripotency, epigenetic regulation and signalling pathways participating in mammalian pre-gastrulation development. The adoption of these new technologies for generating high-resolution maps of embryogenesis will readily
2
Journal Pre-proof translate into biotechnological applications that will have significant impact in livestock production.
1. Introduction
The fertilized egg undergoes a series of cell divisions that result in the formation of a self-organized blastocyst, the first embryonic structure comprised of distinct cell types. Maternal proteins initially regulate these events and after embryonic-genome activation (EGA) the embryo is capable of developing cell autonomously until the blastocyst stage. Broadly speaking these events are similar in most mammals, yet important differences in timing and cellular behaviour have been described [1, 2]. For instance, there are differences in acquisition of cell polarity and compaction in the pre-morula stage, as well as marked differences in the timing of lineage segregation in the blastocyst. With the recent development of single cell genomic technologies we have entered the era of the molecular investigation of development with unprecedented level of detail and temporal resolution[3]. Similar cellular mapping initiatives to those carried out in humans [4] should be done in other mammalian species, particularly in those with important agricultural value and relevant as biomedical models of human development and disease. The variety of techniques available [5] as well as complete genome sequences for most domestic animal
3
Journal Pre-proof species, offer for the first time the opportunity for in depth investigations of the gene regulatory networks and epigenetic contexts controlling development in domestic animal embryos. In this review, I will present a brief overview of the recent advances using single cell genomics in mammals.
1.1. Transcriptional heterogeneity in early cleaving blastomeres
One of the critical events during preimplantation development is the activation of the embryonic genome. Two waves of transcriptional activation, a minor and a major, define EGA, and temporal differences exist [6]. In pigs and human it starts at the 4cell stage, and in bovine at the 8-stage [7-9]. Early studies used pools of in vivo or in vitro produced bovine embryos to define the progression of EGA [10, 11]. Although the largest number of upregulated genes was detected between the 4-8 and 8-16 cell stage in bovine, a smaller subset of genes was also upregulated at the 2- and 16-cell stages. Importantly, a delay in EGA was detected in in vitro produced embryos[11, 12], emphasizing the importance of analysing in vivo derived embryos for gaining accurate representation of gene expression profiles in different species. Gene ontology analysis indicated that most of the early upregulated genes participate in regulation of RNA transcription and translation, and metabolic processes [11]. Analysis of gene modules upregulated during EGA show higher
4
Journal Pre-proof preservation between human and bovine compared to mouse gene expression, suggesting similar kinetics of development between these species [11]. In the pig, RNAseq of pools of embryos confirmed major EGA at the 4-cell stage, and showed that proteins linked to ATP metabolism were highly upregulated [13]. This study also showed that somatic cell nuclear transfer (SCNT) embryos activate the genome one cell cycle later than IVF embryos, indicating defective early development in these embryos. However, questions remain as to whether gene activation during EGA is synchronous in all cells, and whether there is a predisposition to lineage allocation based on the progress of EGA in individual blastomeres. In human, the first study to perform single cell RNA sequencing (scRNAseq) in early embryos confirmed EGA at the 4-8 cell stage, and showed heterogeneity in gene expression between individual blastomeres of the same embryo [14]. This was further supported by another study showing that transcriptional noise between cells of any given stage increased during pre-implantation development, suggesting heterogeneity of developmental programs between individual cells [15]. Another study showed that during human EGA transcriptional regulation, RNA processing and metabolic genes were among the most upregulated genes, consistent with findings in other species [16]. A study in pig embryos also showed onset of EGA at the 4-8 cell stage and demonstrated transcriptional asynchrony between blastomeres from 8 cells to the blastocyst stage
5
Journal Pre-proof [17]. In bovine, Lavagi et al., (2018) showed differences in gene expression in individual blastomeres of day 2 (8-cell) and day 3 (16-cells) and identified six unique gene signatures. Furthermore, they also report differences in the timing of EGA between blastomeres of single embryos, pointing to a degree of asynchrony in this major developmental process. It is however not clear whether asynchronous EGA influences the allocation of cell towards specific lineages at the blastocyst stage. In the mouse, early cleavage blastomeres can form an entire blastocyst. Transcriptional asynchrony between 2- and 4-cell stage blastomeres within an embryo was reported after high throughput sequencing, suggesting predisposition for inner cell mass (ICM) or trophectoderm (TE) differentiation [18]. Future studies combining fluorescent gene reporter expression and cell tracking using time-lapse will help elucidate whether asynchronous development impacts cell fate allocation. 1.2. Lineage segregation in the blastocyst The compact morula contains inner and outer cells, whose destiny is partially determined by their polarity. Inner apolar cells have tendency to give rise to ICM, whereas outer cells tend to become TE [19]. The transcriptional network that determines these outcomes is well known for the mouse: Tead4 is highly expressed in the outer cells and promotes activation of the TE markers Gata3 and Cdx2 [20, 21]. At the same time Sox2 is inactivated in outer cells as a result of Tead4 expression [22]. These findings suggest that blastomeres of the morula are subject to binary decisions
6
Journal Pre-proof based on integrating mechano-sensory responses and lineage specific transcriptional activation in advance of two key events in mammalian embryogenesis, implantation and gastrulation. Implantation is mediated by the TE, which establishes of a maternalfetal interphase supporting the transport of nutrients and protects the fetus. The development of the TE and the process of implantation in diverse between mammals [23], suggesting that TE fate may be under different control between species. Indeed, with the use of scRNAseq we are beginning to gain detailed understanding of the unique events controlling TE segregation. For instance, the role of TEAD4 in TE differentiation in other mammals is unresolved. TEAD4 protein translocation has been shown to determine the TE or ICM delineation in the blastocyst of different mammalian embryos [24]. However, bovine blastocysts can develop normally after TEAD4 knock-down [25]. Interestingly, scRNAseq showed that TEAD4 is not differentially expressed in bovine [26], pig [27] and human embryos [28]. Instead, other TEAD proteins (TEAD1 and 3) are differentially expressed in pig and human TE, suggesting that different members of this family may be participating in trophectoderm commitment [27, 28]. Similarly, differences in the regulation of Cdx2 and Oct4 have been observed between mice and other mammals. In mice Cdx2 represses Oct4 in the TE lineage [29, 30], whereas in pig, cattle and human embryos CDX2 and OCT4 are co-expressed in the TE [31, 32]. Zygotic OCT4, in contrast, seems critical for
7
Journal Pre-proof maintaining expression of NANOG in the epiblast of bovine blastocysts, rather than supporting the TE development [33, 34]. Single cell analysis offers the opportunity for tracking the developmental progression of a lineage and identify asynchronic events even within an embryo. The segregation of TE, epiblast (Epi) and hypoblast (Hypo) in mammalian embryos has been a subject of intense debate following reports showing significant differences between mice and humans [35]. The first report using this technology in the mouse validated classic experimental observations showing gradual segregation of TE and ICM, followed by segregation of Epi and Hypo [36]. The study in human embryos carried out shortly after proposed that a tripartite mechanism of lineage segregation initiated after the morula stage in this species. A key observation made in this study was that the specification of these lineages occurred after a period of co-expression of lineage markers and then resulted in the segregation of TE-Epi and Hypo on Day 7 embryos [35]. These findings led to a re-evaluation of the data in this study, which determined that embryo staging differences may have accounted for the differences reported in lineage segregation between human and other species [37]. Furthermore, a recent study used live imaging and single cell transcriptomics to show that human embryos are highly asynchronous [38]. Thus, using day after fertilization as a staging parameter can lead to inaccuracies that do not reflect precise developmental staging, in contrast to mice. Importantly, this study showed that an initial segregation of TE and
8
Journal Pre-proof ICM can be defined by the transcriptional profile, prior to epiblast and hypoblast in the human embryo [38]. These findings were also recently confirmed in the pig embryo using an in vivo developmental time series for the first time. We showed that a significant time window delineates the emergence of the ICM and TE on day 6, followed by the segregation of the hypoblast and formation of the Epi on day 7-8, persisting until day 11 [27]. The evidence from these studies suggests that whilst there are differences in the timing of these events, gradual segregation of TE followed by the epiblast and hypoblast in later stages reflects the paradigm of development of mammals. The diversity in the gene networks controlling these lineages may be highlighting differences in cell fate determination. For example, expression of CDX2 is delayed in human, pig and bovine compared to the mouse TE, suggesting that canalization of cells towards the TE lineage may be subject to tighter control in the mouse that in other species. In fact experimental evidence shows that bovine TE cells are able to differentiate into hypoblast cells in blastocysts [31], suggesting higher developmental plasticity [39].
1.3 Nascent pluripotency determined by scRNAseq
Despite the broad similarities in blastocyst formation between mammals, there are significant differences in the molecular identity of the different cellular compartments
9
Journal Pre-proof of the pre-implantation embryo. The emergence of pluripotent cells in the ICM and later in the epiblast is marked by the expression of three pluripotency genes Oct4, Sox2, and Nanog. This triad of transcription factors sits atop of a core pluripotency network, which in concert with other genes, including Klf2, Nr0b1, Grb2 and Esrrb, define early pluripotency in the mouse embryo, also known as naïve pluripotency. Core pluripotency genes are also expressed in human and pig embryos, however there are differences in expression of specific naïve pluripotency markers. KLF2, NR0B1 and GBX2 are not expressed in primates and pig embryos, where instead KLF4, KLF5, KLF17, and TCFP2L1 are expressed [27, 40, 41]. The biological significance of the differences in gene expression between naïve pluripotent cells is still unclear. Naïve pluripotent cells exist for a very transient period of mouse development, and is followed by a transition to primed pluripotent gene expression in E5.5 epiblasts (107). Primed pluripotency is characterized by the activation of other genes including Nodal, Fgf5, Dnmt3b, Otx2, and Lef1, whose functions point to a prelude to gastrulation. Importantly, naïve and primed pluripotent cells can be established in vitro and closely resemble the in vivo counterparts. Identification of equivalent compartments in other species has proven much more elusive, although recent studies have demonstrated that in Cynomolgus monkeys and in pigs a signature of naïve and primed pluripotency can be identified at specific developmental stages. A
10
Journal Pre-proof developmental time-series of Cynomolgus monkey embryos analysed by scRNAseq showed expression naïve markers in early pre-implantation embryos followed by primed pluripotency genes in late pre-implantation and post-implantation epiblast [42]. Likewise, pig embryos show expression of naïve markers at the morula early blastocyst stage, and gradually transition towards a primed state of pluripotency, characterized by the expression of PRDM14, NODAL, DNMT3B [27]. Interestingly, in Cynomolgus monkeys and in pigs this period is estimated to last at least 5-6 days, whereas it only lasts 2 days in mice. An extended period of primed epiblast state could contribute to increase the number of cells in preparation for gastrulation. This would be consistent with estimates indicating that the number of epiblast cells required to initiate mouse gastrulation is ~500-600, whereas for baboons and pigs is ~2500 [43]. The mouse epiblast develops into an egg cylinder as a result of the expansion of the polar trophoblast and initiates gastrulation shortly after implantation. In contrast in large mammals, like primates and ungulates, the epiblast forms an embryonic disc prior to gastrulation. Cell interactions with the extraembryonic lineages (TE and hypoblast) are different between mice and large mammals, suggesting that changes in the gene regulatory network of mouse embryos have enabled adaptation to specific mechanisms of gastrulation that may have diverged from other mammals. Conclusions
11
Journal Pre-proof
The use of single cell genomics to study early mammalian development has provided novel insights to long standing questions in developmental biology, and offers new molecular evidence of the divergent and shared mechanisms between different mammalian species. This new knowledge can be used to better understand selective pressures that resulted in novel reproductive strategies and how genes and their related networks are able to change under selective constraints. Availability of complete genome sequences and scRNAseq enables comparative investigations of preimplantation development leading to critical new understanding of lineage segregation, pluripotency establishment and epigenetic regulation of development.
ACKNOWLEDGEMENTS
Work in the author’s laboratory is funded by the Biotechnology and Biological Sciences Research Council (UK) [grant number BB/S000178/1], The Wellcome Trust, and Horizon2020.
REFERENCES
12
Journal Pre-proof [1] Alberio R. Transcriptional and epigenetic control of cell fate decisions in early embryos. Reprod Fertil Dev. 2017;30:73-84. [2] Rossant J, Tam PP. New Insights into Early Human Development: Lessons for Stem Cell Derivation and Differentiation. Cell Stem Cell. 2017;20:18-28. [3] Kelsey G, Stegle O, Reik W. Single-cell epigenomics: Recording the past and predicting the future. Science. 2017;358:69-75. [4] Regev A, Teichmann SA, Lander ES, Amit I, Benoist C, Birney E, et al. The Human Cell Atlas. Elife. 2017;6. [5] Stark R, Grzelak M, Hadfield J. RNA sequencing: the teenage years. Nat Rev Genet. 2019;20:631-56. [6] Jukam D, Shariati SAM, Skotheim JM. Zygotic Genome Activation in Vertebrates. Dev Cell. 2017;42:316-32. [7] Lavagi I, Krebs S, Simmet K, Beck A, Zakhartchenko V, Wolf E, et al. Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos. Sci Rep. 2018;8:4071. [8] Jarrell VL, Day BN, Prather RS. The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis. Biol Reprod. 1991;44:62-8. [9] Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature. 1988;332:45961. [10] Graf A, Krebs S, Zakhartchenko V, Schwalb B, Blum H, Wolf E. Fine mapping of genome activation in bovine embryos by RNA sequencing. Proc Natl Acad Sci U S A. 2014;111:4139-44.
13
Journal Pre-proof [11] Jiang Z, Sun J, Dong H, Luo O, Zheng X, Obergfell C, et al. Transcriptional profiles of bovine in vivo pre-implantation development. BMC Genomics. 2014;15:756. [12] Duan J, Zhu L, Dong H, Zheng X, Jiang Z, Chen J, et al. Analysis of mRNA abundance for histone variants, histone- and DNA-modifiers in bovine in vivo and in vitro oocytes and embryos. Sci Rep. 2019;9:1217. [13] Cao S, Han J, Wu J, Li Q, Liu S, Zhang W, et al. Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing. BMC Genomics. 2014;15:4. [14] Yan L, Yang M, Guo H, Yang L, Wu J, Li R, et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol. 2013;20:1131-9. [15] Piras V, Tomita M, Selvarajoo K. Transcriptome-wide variability in single embryonic development cells. Sci Rep. 2014;4:7137. [16] Xue Z, Huang K, Cai C, Cai L, Jiang CY, Feng Y, et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature. 2013;500:593-7. [17] Wei Q, Li R, Zhong L, Mu H, Zhang S, Yue L, et al. Lineage specification revealed by single-cell gene expression analysis in porcine preimplantation embryos. Biol Reprod. 2018;99:283-92. [18] Biase FH, Cao X, Zhong S. Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res. 2014;24:1787-96. [19] Maitre JL, Turlier H, Illukkumbura R, Eismann B, Niwayama R, Nedelec F, et al. Asymmetric division of contractile domains couples cell positioning and fate specification. Nature. 2016;536:344-8.
14
Journal Pre-proof [20] Ralston A, Cox BJ, Nishioka N, Sasaki H, Chea E, Rugg-Gunn P, et al. Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2. Development.137:395-403. [21] Home P, Ray S, Dutta D, Bronshteyn I, Larson M, Paul S. GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression. J Biol Chem. 2009;284:28729-37. [22] Wicklow E, Blij S, Frum T, Hirate Y, Lang RA, Sasaki H, et al. HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genet. 2014;10:e1004618. [23] Roberts RM, Green JA, Schulz LC. The evolution of the placenta. Reproduction. 2016;152:R179-89. [24] Home P, Saha B, Ray S, Dutta D, Gunewardena S, Yoo B, et al. Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment. Proc Natl Acad Sci U S A. 2012;109:7362-7. [25] Sakurai N, Takahashi K, Emura N, Hashizume T, Sawai K. Effects of downregulating TEAD4 transcripts by RNA interference on early development of bovine embryos. J Reprod Dev. 2017;63:135-42. [26] Negron-Perez VM, Zhang Y, Hansen PJ. Single-cell gene expression of the bovine blastocyst. Reproduction. 2017;154:627-44. [27] Ramos-Ibeas P, Sang F, Zhu Q, Tang WWC, Withey S, Klisch D, et al. Pluripotency and X chromosome dynamics revealed in pig pre-gastrulating embryos by single cell analysis. Nat Commun. 2019;10:500. [28] Xia W, Xu J, Yu G, Yao G, Xu K, Ma X, et al. Resetting histone modifications during human parental-to-zygotic transition. Science. 2019;365:353-60.
15
Journal Pre-proof [29] Niwa H, Toyooka Y, Shimosato D, Strumpf D, Takahashi K, Yagi R, et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell. 2005;123:917-29. [30] Huang D, Guo G, Yuan P, Ralston A, Sun L, Huss M, et al. The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation. Sci Rep. 2017;7:17156. [31] Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M, et al. Trophectoderm lineage determination in cattle. Dev Cell. 2011;20:244-55. [32] Kirchhof N, Carnwath JW, Lemme E, Anastassiadis K, Scholer H, Niemann H. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol Reprod. 2000;63:1698-705. [33] Simmet K, Zakhartchenko V, Philippou-Massier J, Blum H, Klymiuk N, Wolf E. OCT4/POU5F1 is required for NANOG expression in bovine blastocysts. Proc Natl Acad Sci U S A. 2018;115:2770-5. [34] Daigneault BW, Rajput S, Smith GW, Ross PJ. Embryonic POU5F1 is Required for Expanded Bovine Blastocyst Formation. Sci Rep. 2018;8:7753. [35] Petropoulos S, Edsgard D, Reinius B, Deng Q, Panula SP, Codeluppi S, et al. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell. 2016;165:1012-26. [36] Deng Q, Ramskold D, Reinius B, Sandberg R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science. 2014;343:193-6. [37] Stirparo GG, Boroviak T, Guo G, Nichols J, Smith A, Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast. Development. 2018;145:1-18.
16
Journal Pre-proof [38] Meistermann D, Loubersac S, Reignier A, Firmin J, Francois-Campion V, Kilens S, et al. Spatio‐temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm. BioRxiv2019. doi.org/10.1101/604751. [39] Alberio R. Regulation of Cell Fate Decisions in Early Mammalian Embryos. Annu Rev Anim Biosci. 2019. doi: 10.1146/annurev-animal-021419-083841. [40] Blakeley P, Fogarty NM, del Valle I, Wamaitha SE, Hu TX, Elder K, et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development. 2015;142:3151-65. [41] Boroviak T, Loos R, Lombard P, Okahara J, Behr R, Sasaki E, et al. LineageSpecific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis. Dev Cell. 2015;35:366-82. [42] Nakamura T, Okamoto I, Sasaki K, Yabuta Y, Iwatani C, Tsuchiya H, et al. A developmental coordinate of pluripotency among mice, monkeys and humans. Nature. 2016;537:57-62. [43] Snow MH. Growth and its control in early mammalian development. Br Med Bull. 1981;37:221-6.
17
Journal Pre-proof Highlights
Gradual lineage segregation determined by single cell transcriptomics of mammalian embryos.
Distinct cells populations of the early embryo display unique pluripotency features in different mammals.
High resolution developmental maps can be generated using single cell RNAseq.
Ramiro Alberio PII:
S0093-691X(20)30075-3
DOI:
https://doi.org/10.1016/j.theriogenology.2020.01.062
Reference:
THE 15359
To appear in:
Theriogenology
Received Date:
19 January 2020
Accepted Date:
28 January 2020
Please cite this article as: Ramiro Alberio, Untangling early embryo development using single cell genomics, Theriogenology (2020), https://doi.org/10.1016/j.theriogenology.2020.01.062
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Untangling early embryo development using single cell genomics Ramiro Alberio
School of Biosciences, University of Nottingham, NG2 5RD, UK
Running title: Lineage segregation in mammalian embryos
Correspondence: Ramiro Alberio: [email protected] OrcidID: 0000-0001-6560-3919
Keywords: Embryo; Single Cell Genomics; Cell Lineages; Pluripotency
1
Journal Pre-proof Abstract
The zygote undergoes five cell divisions prior to the first signs of lineage segregation. Blastocyst formation requires segregation of the trophectoderm, needed for implantation, and the inner cell mass, which differentiate towards the major lineages of the fetus. This process is broadly conserved in mammals, however, in recent years investigations using high throughput single cell transcriptomics have provided new insights on the gene regulatory networks and epigenetic mechanisms controlling these processes in different species, highlighting novel unique evolutionary adaptations. Although analysis of single cell datasets is inherently challenging due to stochastic gene expression in single cells, continuous development of novel computational tools have contributed to improving the quality of these datasets. Single cell -omics provides detailed information on discrete cellular states, and when combined with spatial transcriptomics it can inform on the relationship between cellular compartments and fate determination. This technology has recently been used to shed new light into the progression of lineage segregation, establishment of pluripotency, epigenetic regulation and signalling pathways participating in mammalian pre-gastrulation development. The adoption of these new technologies for generating high-resolution maps of embryogenesis will readily
2
Journal Pre-proof translate into biotechnological applications that will have significant impact in livestock production.
1. Introduction
The fertilized egg undergoes a series of cell divisions that result in the formation of a self-organized blastocyst, the first embryonic structure comprised of distinct cell types. Maternal proteins initially regulate these events and after embryonic-genome activation (EGA) the embryo is capable of developing cell autonomously until the blastocyst stage. Broadly speaking these events are similar in most mammals, yet important differences in timing and cellular behaviour have been described [1, 2]. For instance, there are differences in acquisition of cell polarity and compaction in the pre-morula stage, as well as marked differences in the timing of lineage segregation in the blastocyst. With the recent development of single cell genomic technologies we have entered the era of the molecular investigation of development with unprecedented level of detail and temporal resolution[3]. Similar cellular mapping initiatives to those carried out in humans [4] should be done in other mammalian species, particularly in those with important agricultural value and relevant as biomedical models of human development and disease. The variety of techniques available [5] as well as complete genome sequences for most domestic animal
3
Journal Pre-proof species, offer for the first time the opportunity for in depth investigations of the gene regulatory networks and epigenetic contexts controlling development in domestic animal embryos. In this review, I will present a brief overview of the recent advances using single cell genomics in mammals.
1.1. Transcriptional heterogeneity in early cleaving blastomeres
One of the critical events during preimplantation development is the activation of the embryonic genome. Two waves of transcriptional activation, a minor and a major, define EGA, and temporal differences exist [6]. In pigs and human it starts at the 4cell stage, and in bovine at the 8-stage [7-9]. Early studies used pools of in vivo or in vitro produced bovine embryos to define the progression of EGA [10, 11]. Although the largest number of upregulated genes was detected between the 4-8 and 8-16 cell stage in bovine, a smaller subset of genes was also upregulated at the 2- and 16-cell stages. Importantly, a delay in EGA was detected in in vitro produced embryos[11, 12], emphasizing the importance of analysing in vivo derived embryos for gaining accurate representation of gene expression profiles in different species. Gene ontology analysis indicated that most of the early upregulated genes participate in regulation of RNA transcription and translation, and metabolic processes [11]. Analysis of gene modules upregulated during EGA show higher
4
Journal Pre-proof preservation between human and bovine compared to mouse gene expression, suggesting similar kinetics of development between these species [11]. In the pig, RNAseq of pools of embryos confirmed major EGA at the 4-cell stage, and showed that proteins linked to ATP metabolism were highly upregulated [13]. This study also showed that somatic cell nuclear transfer (SCNT) embryos activate the genome one cell cycle later than IVF embryos, indicating defective early development in these embryos. However, questions remain as to whether gene activation during EGA is synchronous in all cells, and whether there is a predisposition to lineage allocation based on the progress of EGA in individual blastomeres. In human, the first study to perform single cell RNA sequencing (scRNAseq) in early embryos confirmed EGA at the 4-8 cell stage, and showed heterogeneity in gene expression between individual blastomeres of the same embryo [14]. This was further supported by another study showing that transcriptional noise between cells of any given stage increased during pre-implantation development, suggesting heterogeneity of developmental programs between individual cells [15]. Another study showed that during human EGA transcriptional regulation, RNA processing and metabolic genes were among the most upregulated genes, consistent with findings in other species [16]. A study in pig embryos also showed onset of EGA at the 4-8 cell stage and demonstrated transcriptional asynchrony between blastomeres from 8 cells to the blastocyst stage
5
Journal Pre-proof [17]. In bovine, Lavagi et al., (2018) showed differences in gene expression in individual blastomeres of day 2 (8-cell) and day 3 (16-cells) and identified six unique gene signatures. Furthermore, they also report differences in the timing of EGA between blastomeres of single embryos, pointing to a degree of asynchrony in this major developmental process. It is however not clear whether asynchronous EGA influences the allocation of cell towards specific lineages at the blastocyst stage. In the mouse, early cleavage blastomeres can form an entire blastocyst. Transcriptional asynchrony between 2- and 4-cell stage blastomeres within an embryo was reported after high throughput sequencing, suggesting predisposition for inner cell mass (ICM) or trophectoderm (TE) differentiation [18]. Future studies combining fluorescent gene reporter expression and cell tracking using time-lapse will help elucidate whether asynchronous development impacts cell fate allocation. 1.2. Lineage segregation in the blastocyst The compact morula contains inner and outer cells, whose destiny is partially determined by their polarity. Inner apolar cells have tendency to give rise to ICM, whereas outer cells tend to become TE [19]. The transcriptional network that determines these outcomes is well known for the mouse: Tead4 is highly expressed in the outer cells and promotes activation of the TE markers Gata3 and Cdx2 [20, 21]. At the same time Sox2 is inactivated in outer cells as a result of Tead4 expression [22]. These findings suggest that blastomeres of the morula are subject to binary decisions
6
Journal Pre-proof based on integrating mechano-sensory responses and lineage specific transcriptional activation in advance of two key events in mammalian embryogenesis, implantation and gastrulation. Implantation is mediated by the TE, which establishes of a maternalfetal interphase supporting the transport of nutrients and protects the fetus. The development of the TE and the process of implantation in diverse between mammals [23], suggesting that TE fate may be under different control between species. Indeed, with the use of scRNAseq we are beginning to gain detailed understanding of the unique events controlling TE segregation. For instance, the role of TEAD4 in TE differentiation in other mammals is unresolved. TEAD4 protein translocation has been shown to determine the TE or ICM delineation in the blastocyst of different mammalian embryos [24]. However, bovine blastocysts can develop normally after TEAD4 knock-down [25]. Interestingly, scRNAseq showed that TEAD4 is not differentially expressed in bovine [26], pig [27] and human embryos [28]. Instead, other TEAD proteins (TEAD1 and 3) are differentially expressed in pig and human TE, suggesting that different members of this family may be participating in trophectoderm commitment [27, 28]. Similarly, differences in the regulation of Cdx2 and Oct4 have been observed between mice and other mammals. In mice Cdx2 represses Oct4 in the TE lineage [29, 30], whereas in pig, cattle and human embryos CDX2 and OCT4 are co-expressed in the TE [31, 32]. Zygotic OCT4, in contrast, seems critical for
7
Journal Pre-proof maintaining expression of NANOG in the epiblast of bovine blastocysts, rather than supporting the TE development [33, 34]. Single cell analysis offers the opportunity for tracking the developmental progression of a lineage and identify asynchronic events even within an embryo. The segregation of TE, epiblast (Epi) and hypoblast (Hypo) in mammalian embryos has been a subject of intense debate following reports showing significant differences between mice and humans [35]. The first report using this technology in the mouse validated classic experimental observations showing gradual segregation of TE and ICM, followed by segregation of Epi and Hypo [36]. The study in human embryos carried out shortly after proposed that a tripartite mechanism of lineage segregation initiated after the morula stage in this species. A key observation made in this study was that the specification of these lineages occurred after a period of co-expression of lineage markers and then resulted in the segregation of TE-Epi and Hypo on Day 7 embryos [35]. These findings led to a re-evaluation of the data in this study, which determined that embryo staging differences may have accounted for the differences reported in lineage segregation between human and other species [37]. Furthermore, a recent study used live imaging and single cell transcriptomics to show that human embryos are highly asynchronous [38]. Thus, using day after fertilization as a staging parameter can lead to inaccuracies that do not reflect precise developmental staging, in contrast to mice. Importantly, this study showed that an initial segregation of TE and
8
Journal Pre-proof ICM can be defined by the transcriptional profile, prior to epiblast and hypoblast in the human embryo [38]. These findings were also recently confirmed in the pig embryo using an in vivo developmental time series for the first time. We showed that a significant time window delineates the emergence of the ICM and TE on day 6, followed by the segregation of the hypoblast and formation of the Epi on day 7-8, persisting until day 11 [27]. The evidence from these studies suggests that whilst there are differences in the timing of these events, gradual segregation of TE followed by the epiblast and hypoblast in later stages reflects the paradigm of development of mammals. The diversity in the gene networks controlling these lineages may be highlighting differences in cell fate determination. For example, expression of CDX2 is delayed in human, pig and bovine compared to the mouse TE, suggesting that canalization of cells towards the TE lineage may be subject to tighter control in the mouse that in other species. In fact experimental evidence shows that bovine TE cells are able to differentiate into hypoblast cells in blastocysts [31], suggesting higher developmental plasticity [39].
1.3 Nascent pluripotency determined by scRNAseq
Despite the broad similarities in blastocyst formation between mammals, there are significant differences in the molecular identity of the different cellular compartments
9
Journal Pre-proof of the pre-implantation embryo. The emergence of pluripotent cells in the ICM and later in the epiblast is marked by the expression of three pluripotency genes Oct4, Sox2, and Nanog. This triad of transcription factors sits atop of a core pluripotency network, which in concert with other genes, including Klf2, Nr0b1, Grb2 and Esrrb, define early pluripotency in the mouse embryo, also known as naïve pluripotency. Core pluripotency genes are also expressed in human and pig embryos, however there are differences in expression of specific naïve pluripotency markers. KLF2, NR0B1 and GBX2 are not expressed in primates and pig embryos, where instead KLF4, KLF5, KLF17, and TCFP2L1 are expressed [27, 40, 41]. The biological significance of the differences in gene expression between naïve pluripotent cells is still unclear. Naïve pluripotent cells exist for a very transient period of mouse development, and is followed by a transition to primed pluripotent gene expression in E5.5 epiblasts (107). Primed pluripotency is characterized by the activation of other genes including Nodal, Fgf5, Dnmt3b, Otx2, and Lef1, whose functions point to a prelude to gastrulation. Importantly, naïve and primed pluripotent cells can be established in vitro and closely resemble the in vivo counterparts. Identification of equivalent compartments in other species has proven much more elusive, although recent studies have demonstrated that in Cynomolgus monkeys and in pigs a signature of naïve and primed pluripotency can be identified at specific developmental stages. A
10
Journal Pre-proof developmental time-series of Cynomolgus monkey embryos analysed by scRNAseq showed expression naïve markers in early pre-implantation embryos followed by primed pluripotency genes in late pre-implantation and post-implantation epiblast [42]. Likewise, pig embryos show expression of naïve markers at the morula early blastocyst stage, and gradually transition towards a primed state of pluripotency, characterized by the expression of PRDM14, NODAL, DNMT3B [27]. Interestingly, in Cynomolgus monkeys and in pigs this period is estimated to last at least 5-6 days, whereas it only lasts 2 days in mice. An extended period of primed epiblast state could contribute to increase the number of cells in preparation for gastrulation. This would be consistent with estimates indicating that the number of epiblast cells required to initiate mouse gastrulation is ~500-600, whereas for baboons and pigs is ~2500 [43]. The mouse epiblast develops into an egg cylinder as a result of the expansion of the polar trophoblast and initiates gastrulation shortly after implantation. In contrast in large mammals, like primates and ungulates, the epiblast forms an embryonic disc prior to gastrulation. Cell interactions with the extraembryonic lineages (TE and hypoblast) are different between mice and large mammals, suggesting that changes in the gene regulatory network of mouse embryos have enabled adaptation to specific mechanisms of gastrulation that may have diverged from other mammals. Conclusions
11
Journal Pre-proof
The use of single cell genomics to study early mammalian development has provided novel insights to long standing questions in developmental biology, and offers new molecular evidence of the divergent and shared mechanisms between different mammalian species. This new knowledge can be used to better understand selective pressures that resulted in novel reproductive strategies and how genes and their related networks are able to change under selective constraints. Availability of complete genome sequences and scRNAseq enables comparative investigations of preimplantation development leading to critical new understanding of lineage segregation, pluripotency establishment and epigenetic regulation of development.
ACKNOWLEDGEMENTS
Work in the author’s laboratory is funded by the Biotechnology and Biological Sciences Research Council (UK) [grant number BB/S000178/1], The Wellcome Trust, and Horizon2020.
REFERENCES
12
Journal Pre-proof [1] Alberio R. Transcriptional and epigenetic control of cell fate decisions in early embryos. Reprod Fertil Dev. 2017;30:73-84. [2] Rossant J, Tam PP. New Insights into Early Human Development: Lessons for Stem Cell Derivation and Differentiation. Cell Stem Cell. 2017;20:18-28. [3] Kelsey G, Stegle O, Reik W. Single-cell epigenomics: Recording the past and predicting the future. Science. 2017;358:69-75. [4] Regev A, Teichmann SA, Lander ES, Amit I, Benoist C, Birney E, et al. The Human Cell Atlas. Elife. 2017;6. [5] Stark R, Grzelak M, Hadfield J. RNA sequencing: the teenage years. Nat Rev Genet. 2019;20:631-56. [6] Jukam D, Shariati SAM, Skotheim JM. Zygotic Genome Activation in Vertebrates. Dev Cell. 2017;42:316-32. [7] Lavagi I, Krebs S, Simmet K, Beck A, Zakhartchenko V, Wolf E, et al. Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos. Sci Rep. 2018;8:4071. [8] Jarrell VL, Day BN, Prather RS. The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis. Biol Reprod. 1991;44:62-8. [9] Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature. 1988;332:45961. [10] Graf A, Krebs S, Zakhartchenko V, Schwalb B, Blum H, Wolf E. Fine mapping of genome activation in bovine embryos by RNA sequencing. Proc Natl Acad Sci U S A. 2014;111:4139-44.
13
Journal Pre-proof [11] Jiang Z, Sun J, Dong H, Luo O, Zheng X, Obergfell C, et al. Transcriptional profiles of bovine in vivo pre-implantation development. BMC Genomics. 2014;15:756. [12] Duan J, Zhu L, Dong H, Zheng X, Jiang Z, Chen J, et al. Analysis of mRNA abundance for histone variants, histone- and DNA-modifiers in bovine in vivo and in vitro oocytes and embryos. Sci Rep. 2019;9:1217. [13] Cao S, Han J, Wu J, Li Q, Liu S, Zhang W, et al. Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing. BMC Genomics. 2014;15:4. [14] Yan L, Yang M, Guo H, Yang L, Wu J, Li R, et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol. 2013;20:1131-9. [15] Piras V, Tomita M, Selvarajoo K. Transcriptome-wide variability in single embryonic development cells. Sci Rep. 2014;4:7137. [16] Xue Z, Huang K, Cai C, Cai L, Jiang CY, Feng Y, et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature. 2013;500:593-7. [17] Wei Q, Li R, Zhong L, Mu H, Zhang S, Yue L, et al. Lineage specification revealed by single-cell gene expression analysis in porcine preimplantation embryos. Biol Reprod. 2018;99:283-92. [18] Biase FH, Cao X, Zhong S. Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res. 2014;24:1787-96. [19] Maitre JL, Turlier H, Illukkumbura R, Eismann B, Niwayama R, Nedelec F, et al. Asymmetric division of contractile domains couples cell positioning and fate specification. Nature. 2016;536:344-8.
14
Journal Pre-proof [20] Ralston A, Cox BJ, Nishioka N, Sasaki H, Chea E, Rugg-Gunn P, et al. Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2. Development.137:395-403. [21] Home P, Ray S, Dutta D, Bronshteyn I, Larson M, Paul S. GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression. J Biol Chem. 2009;284:28729-37. [22] Wicklow E, Blij S, Frum T, Hirate Y, Lang RA, Sasaki H, et al. HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genet. 2014;10:e1004618. [23] Roberts RM, Green JA, Schulz LC. The evolution of the placenta. Reproduction. 2016;152:R179-89. [24] Home P, Saha B, Ray S, Dutta D, Gunewardena S, Yoo B, et al. Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment. Proc Natl Acad Sci U S A. 2012;109:7362-7. [25] Sakurai N, Takahashi K, Emura N, Hashizume T, Sawai K. Effects of downregulating TEAD4 transcripts by RNA interference on early development of bovine embryos. J Reprod Dev. 2017;63:135-42. [26] Negron-Perez VM, Zhang Y, Hansen PJ. Single-cell gene expression of the bovine blastocyst. Reproduction. 2017;154:627-44. [27] Ramos-Ibeas P, Sang F, Zhu Q, Tang WWC, Withey S, Klisch D, et al. Pluripotency and X chromosome dynamics revealed in pig pre-gastrulating embryos by single cell analysis. Nat Commun. 2019;10:500. [28] Xia W, Xu J, Yu G, Yao G, Xu K, Ma X, et al. Resetting histone modifications during human parental-to-zygotic transition. Science. 2019;365:353-60.
15
Journal Pre-proof [29] Niwa H, Toyooka Y, Shimosato D, Strumpf D, Takahashi K, Yagi R, et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell. 2005;123:917-29. [30] Huang D, Guo G, Yuan P, Ralston A, Sun L, Huss M, et al. The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation. Sci Rep. 2017;7:17156. [31] Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M, et al. Trophectoderm lineage determination in cattle. Dev Cell. 2011;20:244-55. [32] Kirchhof N, Carnwath JW, Lemme E, Anastassiadis K, Scholer H, Niemann H. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol Reprod. 2000;63:1698-705. [33] Simmet K, Zakhartchenko V, Philippou-Massier J, Blum H, Klymiuk N, Wolf E. OCT4/POU5F1 is required for NANOG expression in bovine blastocysts. Proc Natl Acad Sci U S A. 2018;115:2770-5. [34] Daigneault BW, Rajput S, Smith GW, Ross PJ. Embryonic POU5F1 is Required for Expanded Bovine Blastocyst Formation. Sci Rep. 2018;8:7753. [35] Petropoulos S, Edsgard D, Reinius B, Deng Q, Panula SP, Codeluppi S, et al. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell. 2016;165:1012-26. [36] Deng Q, Ramskold D, Reinius B, Sandberg R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science. 2014;343:193-6. [37] Stirparo GG, Boroviak T, Guo G, Nichols J, Smith A, Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast. Development. 2018;145:1-18.
16
Journal Pre-proof [38] Meistermann D, Loubersac S, Reignier A, Firmin J, Francois-Campion V, Kilens S, et al. Spatio‐temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm. BioRxiv2019. doi.org/10.1101/604751. [39] Alberio R. Regulation of Cell Fate Decisions in Early Mammalian Embryos. Annu Rev Anim Biosci. 2019. doi: 10.1146/annurev-animal-021419-083841. [40] Blakeley P, Fogarty NM, del Valle I, Wamaitha SE, Hu TX, Elder K, et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development. 2015;142:3151-65. [41] Boroviak T, Loos R, Lombard P, Okahara J, Behr R, Sasaki E, et al. LineageSpecific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis. Dev Cell. 2015;35:366-82. [42] Nakamura T, Okamoto I, Sasaki K, Yabuta Y, Iwatani C, Tsuchiya H, et al. A developmental coordinate of pluripotency among mice, monkeys and humans. Nature. 2016;537:57-62. [43] Snow MH. Growth and its control in early mammalian development. Br Med Bull. 1981;37:221-6.
17
Journal Pre-proof Highlights
Gradual lineage segregation determined by single cell transcriptomics of mammalian embryos.
Distinct cells populations of the early embryo display unique pluripotency features in different mammals.
High resolution developmental maps can be generated using single cell RNAseq.