Seminars in Cell and Developmental Biology xxx (xxxx) xxx
Please cite this article as: Pauliina Paloviita, Sanna Vuoristo, Seminars in Cell and Developmental Biology,
https://doi.org/10.1016/j.semcdb.2022.02.010
1084-9521/© 2022 Published by Elsevier Ltd.
The non-coding genome in early human development –
Recent advancements
Pauliina Paloviita, Sanna Vuoristo *
Department of Obstetrics and Gynaecology, University of Helsinki, 00014 Helsinki, Finland
A R T I C L E I N F O
Keywords:
Human embryo
Pre-implantation development
Non-coding genome
Non-coding RNA
Transposable element
Small RNA
A B S T R A C T
Not that long ago, the human genome was discovered to be mainly non-coding, that is comprised of DNA sequences
that do not code for proteins. The initial paradigm that non-coding is also non-functional was soon
overturned and today the work to uncover the functions of non-coding DNA and RNA in human early
embryogenesis has commenced. Early human development is characterized by large-scale changes in genomic
activity and the transcriptome that are partly driven by the coordinated activation and repression of repetitive
DNA elements scattered across the genome. Here we provide examples of recent novel discoveries of non-coding
DNA and RNA interactions and mechanisms that ensure accurate non-coding activity during human maternal-tozygotic
transition and lineage segregation. These include studies on small and long non-coding RNAs, transposable
element regulation, and RNA tailing in human oocytes and early embryos. High-throughput approaches
to dissect the non-coding regulatory networks governing early human development are a foundation for functional
studies of specific genomic elements and molecules that has only begun and will provide a wider understanding
of early human embryogenesis and causes of infertility.
1. Introduction
Pre-implantation embryo development, including embryonic
genome activation (EGA) and cellular lineage commitment, has been
extensively investigated during the past decades and is reviewed in
[1-5]. Studies in Mus musculus, Drosophila melanogaster, Xaenopus laevis,
and other model organisms have provided fundamental mechanistic
insights into early developmental processes [6-10], that are only
partially recapitulated in humans, thus limiting the potential of using
non-primate organisms to investigate human embryonic development.
Considerable interspecies differences range from bonafide measures,
such as distinct sets of genes, to more complex divergencies, like the
timing of embryonic genome activation [8,11]. Enlightening the
mechanisms of early embryo development in humans is of high scientific
and clinical interest and will provide grounds for better understanding
of reproduction-related failures and congenital defects, thus facilitating
the development of improved diagnostics during infertility treatments.
Despite the potential and importance of human embryo research, a
variety of challenges impede its progression. Human embryo research is
typically performed using either supernumerary embryos or gametes
that have been donated for research after termination of infertility
treatments, which limits the number of available samples. Other challenges
related to human embryo research include for instance differences
in legislation and applicable funding between countries, and the
paradigm of open sharing of genetic data while simultaneously protecting
it according to strict interpretation of the General Data Projection
Regulation. We propose that more uniform policies and legislation
would likely facilitate collaborative approaches, open access publishing,
and data sharing across the globe.
Recent improvements in single-cell technologies that enable studying
the rare and unique embryo samples and retaining information on
cellular heterogeneity have led to major breakthroughs in human embryo
research. These landmarks include for instance the identification of
the embryonic transcription start site regions that are upregulated
during EGA [12], and recognition of the first protein-coding transcripts
that are heterogeneously expressed during lineage commitment [13-17].
Development of the CRISPR-Cas9 genome editing technology has
enabled the generation of knockout alleles at nucleotide-level accuracy
[18], even in human embryos [19,20]. Although genome editing approaches
in human germ cells and embryos are technically very challenging
and their ethical justification in human embryo research must be
meticulously evaluated, these techniques are unique in allowing
* Correspondence to: Functional Embryo Genomics Lab, University of Helsinki, Biomedicum Helsinki 1, Haartmaninkatu 8, A430B, 00014, Finland.
E-mail address: sanna.vuoristo@helsinki.fi (S. Vuoristo).
Contents lists available at ScienceDirect
Seminars in Cell and Developmental Biology
journal homepage: www.elsevier.com/locate/semcdb
https://doi.org/10.1016/j.semcdb.2022.02.010
Received 1 December 2021; Received in revised form 8 February 2022; Accepted 8 February 2022
Seminars in Cell and Developmental Biology xxx (xxxx) xxx
2
scientists to investigate functions of specific genes during human early
embryogenesis [19]. Embryonic transcripts have recently been knocked
down in human zygotes and embryos using RNAi technologies that are
expected to affect target mRNA stability and translation [21-23].
Manipulated embryos have typically been analysed for the presence
(expression levels) of protein-coding marker genes either using immunofluorescence
and microscopy in three dimensions or single-cell
RNA-sequencing (scRNA-seq). Although some of the recent pivotal
studies have shed light upon the non-coding genome [21,22,24,25] and
transcripts [26-30] in human gametes and early embryos, the vast majority
of human embryo research has focused on protein coding genes.
The non-coding genome is widely accessible and transcriptionally
active, producing a multitude of RNA classes reviewed in [31] (Fig. 1),
during early human embryo development. Accessible genomic regions
in early human embryos frequently comprise transposable elements,
reviewed in [32,33]. The functions of the non-coding genome have been
studied in human pluripotent stem cells (hPSCs) [34], however here we
do not summarize this information but only refer to it, to exemplify
mechanisms that may be extrapolatable to the human embryo context.
In this review, we discuss recent advances in elucidating the non-coding
regulatory genome in human embryo development and highlight prospects
of this fundamental and challenging area of research.
2. Overview of human pre-implantation development
The maturation of oocytes occurs after ovulation, when the prophase
I arrested germinal vesicle (GV) stage oocytes resume meiosis I, undergo
asymmetric division, and exclude the first polar body, which is a haploid
cell with a minimal amount of cytoplasm [35]. The oocytes arrest at
meiosis II and final maturation takes place only at fertilization when the
second polar body is extruded. Mature (MII) oocytes are
transcriptionally quiescent but the composition of their stored RNAs and
proteins changes during the maternal-to-zygotic transition (MZT)
(Fig. 2). At fertilization the maternal and paternal pronuclei are
assembled in a clustered configuration [36,37]. After DNA replication,
the pronuclei membranes break and the first cleavage division can take
place [38]. Maternal RNAs and proteins contribute to the programming
of the totipotent fertilized oocyte, as elaborated by the classical cloning
approach where the maternal cocktail in the oocyte cytoplasm can
reprogram a somatic nucleus sufficiently enough to contribute to the
production of live offspring [39-42]. Moreover, while maternal RNAs
are essential for embryo development before the onset of zygotic RNA
transcription, their clearance is equally important [43].
scRNA-seq of human oocytes and embryos has revealed that the
human EGA essentially occurs by the 8-cell stage [44,45,17]. More
recent findings indicate that the first genes with embryo-specific splicing
patterns are activated soon after fertilisation [46] while other results
reveal that the human EGA commences in a minor and major wave by
the 4-cell, and 8-cell stage, respectively [12]. Some of the well-known
EGA genes, such as LEUTX [47] that is upregulated at the 4-cell stage
[12], were not upregulated in zygotes [46]. Therefore, further studies
are needed to elaborate to what extent the zygotic gene expression[46]
and the minor EGA wave [12] overlap and which mechanisms drive
these early EGA process. Maternal RNAs are degraded along with the
activation of transcription from the embryonic loci.
Acquirement of key morphological features such as blastomere polarization
initiates in parallel with major epigenetic remodelling and
transcriptional activation at the 8-cell stage. In mice, apicobasal polarity
of the cells is driven by intracellular asymmetries in the placement of the
cell adhesion protein E-cadherin and the partitioning defective (Par)
complex, and cytoskeletal components, while the mechanism in humans
remains to be elucidated. Blastomere polarization occurs concomitantly
Fig. 1. Cellular compartmentalization of non-coding
genomic elements and their RNA products.Genomic
transposable elements provide cis-regulatory regions
that can be bound both by non-coding RNAs, such as
micro RNAs (miRNA), and proteins. Non-coding DNA
produces both long and small non-coding transcripts
(lncRNA and small RNA, respectively) that exist in the
nuclear and cytoplasmic compartments. Piwi interacting
RNAs (piRNA) are found both in the cytoplasm and
nucleus where their canonical function is to silence
transposable elements (TE) and transcripts generated
from them. Canonical miRNA function, the repression
of mRNAs, occurs in the cytoplasm but they may also
contribute to gene regulation via binding to promoters
or enhancers in the nucleus.
P. Paloviita and S. Vuoristo
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with compaction, the process of blastomeres forming tight adhesions
with each other, and contributes to cellular lineage segregation [23]. In
the compacted embryo, the cell-cell contact area is maximized and a
compressed morphology is attained. The human embryo reaches the
morula stage typically on the fourth day post fertilization, when it is
composed of approximately 16- to 32 cells. The morula to early blastocyst
transition coincides with the opening of the blastocoelic cavity
and the appearance of the first cellular lineages. Human embryos usually
reach the blastocyst stage around five days post fertilization. At this
stage the blastomeres segregate to form the inner cell mass (ICM) or
trophectoderm lineages. During blastocyst maturation, the blastocyst
expands, the trophectoderm forming trophoblast cells differentiate
further and the ICM is divided into two lineages, the hypoblast (primitive
endoderm in mice) and epiblast. Trophoblast cells form the embryonic
part of the placenta, the pluripotent epiblast cells form the
foetus, and the hypoblast cells form the yolk sac as development proceeds.
The Zona pellucidae, a structure which encloses the blastocyst,
becomes thinner allowing the blastocyst to finally hatch. Implantation in
humans typically takes place around seven days post fertilization. The
human embryo pre- to post-implantation morphology was recently
coupled with embryonic and extra-embryonic cellular lineage segregation
events using an in vitro pre-to post-implantation culture method
[48,49]. These studies revealed that human embryos can self-organize
through the implantation stages in vitro in the absence of maternal tissues
[48,49]. Gastrulation that commences during the third week of
human embryo development, determines the future body axes, reviewed
in [50]. Tyser et al. used single cell RNA-sequencing to characterize cell
populations in a 16–19-day post-fertilization human gastrula [51].
Thorough bioinformatics analyses revealed the specification of the three
germ layers; ectoderm, endoderm, and mesoderm, as well as appearance
of primordial germ cells and red blood cells, among the other further
specified cell types [51]. Human early embryonic development seems to
be far more variable than that of the typical model organisms, like the
mouse, when it comes to for example timing and synchrony of the
cleavage divisions, ratio of aneuploid cells, and cellular lineage
commitment. The interindividual differences between human embryos
call for repetition of central findings by incorporating samples from
different donors and treatment facilities, and in sufficient quantities to
ensure that the results are generalizable.
3. The interplay of transposable elements, DNA methylation and
oocyte short piRNAs during human MZT
During MZT, the control of embryonic development is transferred
from stored maternal RNAs and proteins to zygotic RNAs transcribed
from the newly organized genome. Maternal protein-coding genes,
including for instance GROWTH AND DIFFERENTIATION FACTOR 9
[52] and the ZONA PELLUCIDA genes [53], are relatively
well-characterized. Although most retrotransposons, reviewed in
[54-56], in the human genome are non-mobile and transcriptionally
silenced, some of these elements are transcribed and seem to have
retained the ability to transpose in human oocytes [57,26,58-60,28].
Human GV oocytes express at least LINE1, HERV-K10, as well as SINE-R,
VNTR and ALU (SVA) elements [58,61]. Long interspersed nuclear elements
(LINE) have been suggested to regulate the expression of proximal
protein coding genes, such as AR and TMEFF2 [61]. Long terminal
repeat (LTR) elements, particularly the endogenous retrovirus (ERV)
ERV1 and the primate-specific LTR12C and LTR7 elements, are
expressed and provide alternative transcription start sites in mouse and
human oocytes [62,26,24,63]. Whether transcription from the retroelement
loci and the generated transcripts are required for MZT and EGA
in human oocytes is yet to be elucidated.
Oocyte maturation and MZT entail genome-wide changes in chromatin
accessibility and epigenetic landscapes [24,64,25]. Although
large-scale epigenetic changes seem to be conserved between mouse and
human MZT, some characteristics differ between these two species,
reviewed in [65]. Human oocyte maturation from the GV to MII stage is
characterized by relatively stable and pre-established CpG methylation
(occurring in CpG dinucleotides) levels, particularly in gene bodies [66].
In addition, increased non-CpG methylation explains the observed
overall accumulation of methylation during oocyte maturation. Interestingly,
most of the increasingly methylated regions [67] and non-CpG
regions [61] in MII oocytes are located at transposable element (TE) loci.
Moreover, increased methylation at several non-CpG distal regions that
overlap with LINEs correlates negatively with the expression of nearby
coding genes [61], while increased non-CpG methylation at gene bodies
correlates positively with the expression of the corresponding genes in
MII oocytes [67]. This suggests that non-CpG methylation levels during
oocyte maturation may play a role in gene expression regulation and
potentially involve TE-mediated mechanisms. After fertilization, the
paternal pronucleus immediately initiates dramatic global demethylation,
followed by that of the maternal pronucleus. Demethylation proceeds
until the 2-cell stage [67]. At the 4-cell to 8-cell stage de novo DNA
methylation commences and is enriched at evolutionary young transposable
elements [68]. In conclusion, the methylation dynamics during
human MZT seem to be extremely complex, involving abundant
methylation level changes in transposable element regions.
Transposition may be deleterious to the genome and therefore
human oocytes and embryos are likely to employ multiple mechanism to
regulate TE silencing. Global re-methylation takes place at the postimplantation
stage [67,68], but for instance LTR5-HS and LTR7B
become accessible at the 8-cell stage and are rapidly downregulated at
the morula stage [24], indicating efficient suppression. Small RNAs
(sRNAs) contribute to the regulation of TEs in oocytes and the earliest
embryonic stages. A novel sRNA class that is specifically expressed in
primate oocytes, oocyte short Piwi-interacting RNAs (os-piRNAs), was
Fig. 2. Human early embryo developmental stages.In humans, the maternal-to-zygotic transition entails oocyte maturation, fertilisation, and embryonic genome
activation at the 4- to 8-cell stages. This is followed by the morula stage during which cells acquire heterogeneity that leads to the establishment of the first embryonic
lineages at the blastocyst stage. The developmental timeline is shown above the illustration.
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found to account for over 70% of the total sRNA-seq reads in human
oocytes and are likely to contribute to the silencing of TEs, especially the
L1 family members [30]. Conventional Piwi-interacting RNAs (piRNAs)
are also found in human oocytes although at lower overall levels [29,30]
and they preferably target ERV and LTR family members suggesting
different regulatory roles for the two piRNA classes [30]. Further differences
between the two piRNA classes are detailed in the glossary. The
piRNA pathway is dispensable to mouse oogenesis and embryo development
[69], where another sRNA class, endogenous short interfering
RNAs (endo-siRNAs), is likely to be responsible for TE silencing.
Endo-siRNAs are abundant in mammalian oocytes and early embryos
[70] but lacking in human counterparts [30] and the contrary is true in
the case of os-piRNAs [29,30]. This limits the potential to study human
sRNA-TE interactions in model organisms. KRAB zinc-finger proteins
have also been associated with TE silencing in the context of early embryonic
genes [71] and future experimental work is needed to better
elucidate how the co-evolved coding and non-coding transcripts and
their products ensure controlled TE activity during MZT.
4. Modifications of maternal coding and non-coding transcripts
in MZT
Transcriptomic heterogeneities can be accomplished in multiple
ways, for instance through invocation of alternative transcription start
sites, RNA-editing, and alternative splicing, resulting in transcript isoforms,
reviewed in [72]. Differences in transcript isoform sequences,
including the three prime untranslated region (3’ UTR) regulatory elements,
modulate transcript stability, degradation, and translation efficacy.
Findings across species have emphasized selective regulation of
maternal mRNAs to occur during MZT, reviewed in [73]. Maternal
mRNAs can be roughly classified based on the timing of their degradation;
RNAs belonging to the ‘maternal RNA decay pathway’ are downregulated
(degraded) between the GV oocyte and zygote stage, while
RNAs belonging to the ‘zygotic decay pathway’ are degraded in a
delayed manner between the GV oocyte or zygote and 8-cell stage [74].
Recent data provide insights into these processes and show that embryonic
transcription is necessary for the degradation of some maternal
mRNAs in early human embryos, as alpha amanitin treatment causes
accumulation of maternal mRNAs that belong to the ‘zygotic decay
pathway’ [74]. One of the means to regulate maternal RNAs during MZT
is through modifications in poly(A) tail sequence and length at the
mRNA 3’UTR. Poly(A) tails of maternal mRNAs are deadenylated in
mouse oocytes, where mRNA deadenylation coincides with mRNA
storage and translational inactivity instead of leading to immediate
mRNA degradation. [75]. Global poly(A) tail de-adenylation in human
oocytes is suggested to be mediated by BTG4 that interacts with the
CCR4-NOT de-adenylation complex [76-78] also shown to be active in
the mouse [79,75,80].
mRNAs with short poly(A)tails are more likely to be either rapidly
degraded or modified further by terminal nucleotidyltransferases
(TENT), including terminal uridylyltransferases (TUT), that alter RNAs
post-transcriptionally, reviewed in [81]. Recent methodological developments
have allowed detailed investigation of poly(A) tail lengths
and sequences also during human MZT [76-78]. Shortened poly(A) tails
provide substrates for the incorporation of uridine or other nucleotides.
TUT4/7 can add uridines to the 3’ end of the poly(A) tails, suggesting
that uridines located at the 5’ end or internal parts of the poly(A) tail
have been added prior to (re)-polyadenylation [76-78]. Addition of U
residues seems to mark the transcript for degradation [82-84], however
recent findings indicate that 3’ uridylation may be temporally decoupled
from degradation during human MZT and that these transcripts may be
degraded only post EGA [76-78]. Transcripts can be further modified by
TENT4 A and B-mediated addition of nucleotide residues. TENT4 proteins
have somewhat relaxed nucleotide preferences and can incorporate
also non-adenosine residues, especially guanosine, to poly (A) tails
[81]. This incorporation of guanosine and adenosine residues may occur
in a sequential manner [76-78]. Guanosine residues in the internal parts
of the poly(A) sequences are suggested to stabilize re-adenylated mRNAs
in human zygotes [76-78]. In conclusion, recent data indicate that poly
(A) tail length and nucleotide composition are rigorously regulated
during MZT in humans.
Maternal micro RNAs (miRNAs) also undergo post-transcriptional
adenylation, which is conserved with temporal differences from fly to
human early development [70,85]. TENT2 has been suggested to carry
out the majority of miRNA 3’ adenylation in this context [86]. In
humans, miRNA 3’ adenylation peaks at the zygote stage and reduces in
subsequent embryonic stages [29]. sRNA adenylation during early
development seems to be class specific, as in mice endo-siRNAs are
highly adenylated and piRNAs are not [70]. Additionally, 3’ adenylation
levels in humans and mice are highly miRNA species specific and both
oligo- and polyadenylation has been reported [29,70]. Many maternal
miRNAs seem to undergo degradation at the zygote stage in humans [29,
30] and other organisms [87,88,70] indicating that they belong to the
‘maternal decay pathway’. Whether adenylation is linked to this process
remains controversial as it may both prevent and promote miRNA
degradation [89-91,85,70] and the outcome is likely to be context
dependent. We speculate that RNA modifications and particularly adenylation
introduce another significant layer of regulation in human
MZT. Adenylation provides a rapid way to regulate RNA stability and
mRNA translational outputs without requiring changes in chromatin
accessibility or transcriptional activity. Some of the post-transcriptional
modifications in mRNAs and miRNAs are mediated by the same factors,
such as TENT4 A/B, and TUT4/7 [81]. Despite the valuable information
gained from recent findings, further studies are needed to dissect how
the modified target RNA species are selected spatially and temporally in
the context of MZT. It is also interesting to speculate whether some long
non-coding RNAs (lncRNAs), including transcripts originating from
transposable element loci, might be modified by the factors that regulate
mRNAs and miRNAs, and what might be the outcome.
5. Transposable elements may provide functionally important
cis-regulatory regions for EGA
A recent study revealed that human EGA commences at the time of
fertilization as some genes are upregulated although at relatively minor
levels [46]. Comparison of the transcriptomes of human MII oocyte and
cleavage stage embryos has revealed that while the absolute number of
RNA molecules reduces dramatically between the transition from MII
oocytes to 4-cell stage embryos [12,92], some transcripts are significantly
upregulated [12]. Among the transcripts upregulated during this
minor EGA stage are for example ZSCAN4, a well-known protein-coding
EGA gene also expressed in mouse [93] and bovine early embryos [94].
In addition to protein-coding genes, several transcripts upregulated in
zygotes [46] and 4-cell stage embryos [12] were annotated to the
non-coding genome, especially at pseudogene regions and human ERV
elements [46]. Some of the 4-cell stage non-coding transcripts are likely
to be lncRNAs, often enriched at transposable element regions [12]. In
addition to maternally expressed ERVs, expression of the ERV2
(LTR5HS, HERV-K) and ERV3 (LTR7Y) elements [28], which also
harbour DNAse I hypersensitivity sites (DHS) indicating accessible
chromatin loci [22], is obvious at the time of major EGA. LTR elements
provide binding sites for transcription factors [95,96] and participate in
the regulation of their corresponding ERVs. Human HERV-Ks are regulated
by LTR5HSs, which bear binding sites for POU5F1/OCT4, a transcription
factor that is expressed [28] and activates major EGA
transcripts in human EGA stage embryos [22]. OCT4 has been suggested
to bind at LTR5HS loci and recruit P300, which likely leads to the
observed acquisition of H3K27 acetylation [97], H3K4 trimethylation
and HERV-K expression [28]. Another ERV class, HERV-H, that is typically
connected to the maintenance of pluripotency [98], is also
expressed in 8-cell stage human embryos and beyond [22]. HERV-H
elements are flanked by LTR7 elements that are accessible in early
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human embryos [22]. In addition, SVA (SVA-A), L1 (L1 and L1-HS), and
SINE (Alu) elements are expressed [12,28] and located in open chromatin
[22] in human preimplantation embryos. Alu elements are the
most abundant repeat class identified in the human genome [99]. Out of
all Alu elements, particularly AluY and AluS are enriched at the transcription
start sites of many human EGA genes [12], suggesting that they
participate in the regulation of human EGA [12,100]. Alu elements
frequently overlap with a 36-basepair DNA-binding motif, which resembles
a known consensus binding sequences of PRD-like homeobox
genes, T-box and bZIP [12]. Candidate binding factors of this Alu
element overlapping de novo motif include protein products of both
embryonically (e.g. DUXA and LEUTX) and maternally (e.g. OTX2,
PITX2, and GSC) expressed genes [12]. Out of the maternal factors at
least GSC and OTX2 are highly enriched in human embryos at the 2-cell
stage [25], indicating that their binding to Alu elements during the first
days of development is possible.
Transient chromatin opening takes place in human zygotes, prior to
the progressive increase in chromatin accessibility from the 2-cell stage
onwards [24]. Recent studies have indicated that open chromatin does
not automatically denote active transcription in human early embryos
[25]. Alu loci seem to be inaccessible at the 2-cell stage but become
accessible by the 8-cell stage when major EGA takes place in human
embryos [25]. It appears possible that prior to EGA Alu loci might be
accessible, even if not transcribed, and could provide cis regulatory elements
with transcription factor binding sites for the EGA gene activators
as suggested in human 4-cell and 8-cell stage embryos [12]. The
reorganization of three-dimensional (3D) chromatin coincides with EGA
across species, reviewed in [101]. The formation of topologically associated
domains (TAD) seems to depend on polymerase II-mediated
transcription in human embryos, given that the TADs appear at the
8-cell stage and beyond, and that alpha-amanitin treatment of human
zygotes largely abolishes TAD formation at the 8-cell stage [21]. Interestingly,
AluS elements were enriched around some of the very weak
TAD boundaries already at the 2-cell stage and transcribed in later
cleavage stage embryos [21]. Recent data from mouse embryos and
2-cell-embryo-like cells indicate that murine ERVL (MERVL) elements
promote formation of insulating domains [102]. Therefore, it appears
that TEs have a role in the establishment of TAD boundaries in early
cleavage stage embryos.
6. Non-coding RNAs may contribute to cellular heterogeny
preceding the establishment of the first embryonic lineages
The establishment of the first embryonic lineages requires the blastomeres
to retain their capability to proliferate and simultaneously
produce intercellular differences in their molecular composition, that is
heterogeny. Classic protein markers of embryonic lineage segregation
have been studied in humans both to assess the timing of the segregation
events and to identify critical factors in this process. In human embryos,
the depletion of OCT4 [19] and Phospholipase C (PLC) -Protein kinase C
(PKC) [23,103] signalling pathway components has indicated that these
factors are needed for the determination of the ICM and trophectoderm,
respectively. Similarly restricted expression of either NANOG or GATA6
in the ICM cells demarcates the establishment of the human epiblast and
hypoblast [104]. While some mechanism driving lineage determination
are conserved between species, others have diverged. CDX2 drives the
specification and maturation of the trophectoderm and ICM [105] in the
mouse while it may not be necessary for this segregation event in human
embryos [2]. Some of the observed interspecies differences in the
regulation of lineage segregation have been explained by low conservation
of the studied genes, highlighting again the limitations in the use
of model organisms to study human embryogenesis, especially in the
context of the non-coding genome and transcriptome that have greatly
diverged between species.
In humans, cellular heterogeny between blastomeres is observed
initially around the 8-celll to morula stage [13,103] in the compacting
embryo. At that time blastomeres acquire apico-basal cell polarity that
was recently reported to initiate the segregation of the trophectoderm
and ICM via PLC – PKC activity, leading to restricted expression of
trophectoderm-associated factors, such as GATA3, in the outer cells [23,
103]. Morula cells have been described as heterogenous especially in
terms of cell polarity determining proteins, yet it seems that also
non-coding RNAs contribute to the establishment of cellular heterogeny.
lncRNAs, especially those derived from human ERVS, HERV-H and
HERV-K [27,28], are transcribed from the 8-cell stage onwards and
could contribute to the priming of lineage segregation as has been
proposed for the Heterogeneously expressed from the Intronic Plus
Strand of the TFAP2A-locus RNA (HIPSTR) that is unevenly expressed
between blastomeres of human 8-cell stage embryos [106].
In humans [13,15] and other mammals [107–109] the ICM and
trophectoderm segregate prior to the formation of the epi- and hypoblast.
Meistermann et al. [13] combined live imaging, which is optimal
for human embryo staging [110], to single-cell transcriptomics and reported
that the mRNA profiles of human trophectoderm and ICM
diverge just before blastocyst expansion and prior to epiblast and hypoblast
establishment in the expanded blastocyst. Previous studies of
lineage segregation in humans rely heavily on marker mRNAs/proteins
to distinguish the established lineages [92,111,2,14,104], while it is
known that lncRNAs are more tissue specific and show greater heterogeny
between blastomeres [112,17,106]. ERV-derived RNAs, especially
HERV-H and HERV-K, have been associated with specific lineages
of the human blastocyst [113,92,114,115,28]. Reijo Pera and colleagues
[113] identified a set of long intergenic noncoding RNA (lincRNA) isoforms
that typically incorporate ERVs, especially HERV-H. These Human
pluripotency-associated transcripts (HPATs) are abundant in human
pluripotent cells [113] and almost completely absent in differentiated
adult tissues. In human blastocysts, HPAT-2, − 3 and − 5 expression was
specific to the ICM/epiblast cells and depletion of these HPATs in single
blastomeres excluded the cells from contributing to the ICM [92,114].
HPAT expression specific to also the trophectoderm and hypoblast lineages
has been reported [114,115], altogether indicating that lncRNAs
may be involved in lineage specification and could also be used for
annotating the cell types in human embryos.
Factors associated with the human ICM resembling naïve hPSC state
[34,116], OCT4, NANOG, KLF4, and LBP9, mediate transcriptional
activation of HPATs and other HERV-H containing RNAs via binding to
LTR7 elements in hPSCs, and this axis could function also in human
embryos [92,117]. Conversely, in vitro knockdown of specific ERVs via
RNA interference leads to a reduction in pluripotency markers [59,115].
Furthermore, HPAT5 was suggested to post-transcriptionally inhibit the
let-7 miRNA and this controls the balance between pluripotency and
differentiation in hPSCs [92]. While many discoveries on the function of
non-coding RNAs in balancing pluripotency and differentiation have
been made in hPSCs, it is likely that similar axes govern the segregation
of the different lineages also in human blastocysts, where non-coding
and coding RNAs and their products form elaborate circuitries that
may provide developmental robustness to the early embryo.
The contribution of sRNAs to driving cellular heterogeny that precedes
lineage segregation has not been studied in humans. Current
knowledge on sRNA expression in human cleavage and morula stage
embryos is at the single embryo level [29,30] and blastocysts have been
profiled only for known miRNAs [118]. Differential miRNA expression
has been reported in bovine [119,120] and mouse [87,121] embryos
between the ICM and trophectoderm, and in mouse stem cell models of
embryonic and extra-embryonic tissues [122], suggesting that miRNAs
contribute to the formation of the first differentiated lineages in
mammalian embryos. In humans and mice, miRNA levels increase as
development proceeds [87,29,70]. Additionally, Dgcr8-lacking mouse
oocytes and zygotes arrest at the blastocyst stage or around the time of
gastrulation, respectively, indicating the requirement for an active
miRNA biogenesis pathway in cellular lineage commitment [123-125].
In human oocytes, cleavage stage embryos, and blastocysts,
P. Paloviita and S. Vuoristo
Seminars in Cell and Developmental Biology xxx (xxxx) xxx
6
hsa-miR-372 and its miRNA cluster family members are detected in
abundant levels [29,118]. Hsa-miR-372–3p promotes somatic cell
reprogramming to pluripotency [126] and is found at higher levels in
naive than primed hPSCs [127,128]. The homologous miR-290–295
cluster is an essential regulator of mouse embryonic stem cell pluripotency
[129] and causes partial lethality and infertility upon depletion
[130], altogether suggesting that this conserved miRNA family may
participate in the regulation of mammalian embryogenesis. In addition
to miRNAs and piRNAs, both ribosomal RNA (rRNA)- and transfer RNA
(tRNA)-derived small RNAs are present in human sperm, oocytes, and
early-stage embryos in biologically relevant abundancies [29,30].
Currently, the contribution of miRNAs and other sRNAs to cellular
heterogeny of human blastomeres remains to be elucidated. However, it
is likely that sRNAs may contribute to lineage segregation via the
regulation of the pluripotency/differentiation axis, as has been
described for miRNAs and rRNA- and tRNA-derived small RNAs in PSCs
[131-133].
7. Conclusions
The functions of the non-coding genome, both at the DNA and RNA
level, are essential to early human development and so far, the number
of studies in this context remains limited. The non-coding genome and
transcriptome provide an additional layer of cellular regulation of which
human embryo studies have focused on dissecting the chromatin
conformation and epigenetic landscape at non-coding loci as well as
stage-wise characterisation of the non-coding transcripts and their
modifications. Other aspects such as the coding genome, cell organelles,
mechanical ques [134,135], cellular polarization, and the 3D organization
of the genome (reviewed in [101]), are also crucial to early
(human) development and are likely to be connected to the non-coding
genome with already some examples in literature. For instance, rRNA
biogenesis and nucleolar maturation were recently found to contribute
to the exit from the mouse 2-cell-embryo-like cell state [136,137], via
alterations in the 3D organization of perinucleolar heterochromatin
[137]. Cellular processes essential to early development and cell specification
in general are regulated by extensive crosstalk and competition
between different coding and non-coding molecules, with the highly
tissue specific lncRNAs likely playing especially central roles [138]. This
molecular dialogue is not limited to RNA-RNA/protein interactions but
also includes DNA that is regulated by proteins, lncRNAs and sRNAs. The
best known lncRNA-DNA interaction in the human developmental
context is perhaps X chromosome (in)activation by XIST and XACT
[139-142]. The hybridization of sRNAs to the genome has also been
reported in this context for instance in the case of piRNA mediated
epigenetic TE silencing [143]. Interestingly, TEs as well as other
non-coding DNA sequences located at promoters and enhancers may
also contribute to sRNA-mediated transcriptional activation. The
contribution of sRNAs in regulating transcription of EGA genes by direct
binding to the genome has only recently been discovered in mice [144]
and so far, reports on sRNA-directed regulation of transcription in
humans is limited to miRNAs in somatic tissues [145,146]. The functional
annotation of non-coding DNA and RNA is only at its beginning,
especially in human gametes and embryos, and future discoveries
extending the canonical functions of different RNA classes or genomic
elements is expected.
Recent and future novel discoveries on the contribution of the noncoding
genome and transcriptome to human development rely heavily
on sequencing-based methods appropriate for single-cell/embryo
quantities such as PAIS-seq, Cas-seq and other sRNA-seq methods, HiC,
and CUT&Tag [127,147,148,76,77,78,29,30]. While many
sequencing-based methods requiring the homogenization of several
gametes or embryos as starting material are feasible to study early
development, the conclusions are limited as bulk analysis masks biologically
relevant heterogeny between single blastomeres and interindividual
differences between pooled human embryos may cause further
noise in the data. Additionally, both current bulk and single cell methods
to prepare sequencing libraries of non-coding RNAs are like to favour
some RNA classes over others due to the used RNA capture method or
the possible 3D structure and cellular compartmentalization of the
RNAs. Methods allowing for more detailed investigation of the nuclear
and cytoplasmic compartments, such as NET-CAGE [149] and
RD-SPRITE [150] would provide new information on the diverse functions
of nascent and nuclear non-coding RNAs, however, scaling these
methods to the single cell/embryo level remains a challenge.
Challenges involved in bioinformatic analysis the non-coding
genome and transcriptome are not limited to the context of embryology.
Quantification of non-coding RNAs may be obscured by frequent
multimapping of reads, especially short reads from sRNA-seq. Moreover,
classification of non-coding RNAs is challenging as they have diverse
characteristic features, are often lowly expressed and poorly conserved
between species, and may resemble protein-coding RNAs [151]. Interestingly,
translation has been observed beyond protein-coding transcripts
and for instance translated lncRNAs are a potential source of
peptides, some of which may have biological functions [152]. Furthermore,
several mRNAs function on the RNA level in a manner resembling
non-coding transcripts perturbing the division between coding and
non-coding transcripts [152,153]. Consequently, non-coding RNA databases
may include inadequate annotations and the recent discovery of
os-piRNAs in human and primate oocytes exemplifies that the catalogue
of the human developmental transcriptome as well as the genomic
regulatome is far from complete.
Ex vivo human embryo research is also limited to methods that
require a small number of cells. Thus, methods to locate specific molecules
(antibodies for proteins and fluorescence in situ hybridization for
RNA) or perturb a single genomic locus using CRISPR-Cas9 or transcript
using small interfering RNAs (siRNAs) have been employed for functional
studies. In comparison to the coding genome, the functions of the
non-coding genome and transcriptome have been less studied not only in
the field on human development but on a global scale. Recently, the
FANTOM6 project piloted a high-throughput endeavour to functionally
annotate lncRNAs using a cell culture platform coupled with antisense
oligonucleotide (ASO)- and siRNA-mediated knockdown of lncRNAs
[154]. The knockdown method was found in some cases to affect the
outcome of the experiment, likely due to ASOs functioning primarily in
the nuclear and siRNAs in the cytoplasmic compartment. Genetically
engineered animals have also been crucial in functional dissection of
lncRNAs [155] as well as sRNAs [156] and TEs [157], yet the number of
these studies remains very small in comparison to mRNAs. Recent work
characterized a conserved Cdk2ap1 protein isoform that originates from
species-specific TEs and in the mouse has a developmentally reciprocal
expression pattern and opposite biological function with the canonical
Cdk2ap1 [158]. Expression of a human Cdk2ap1 splice isoform originating
from a human-specific TE promoter was sufficient to restore the
development of knockout mouse embryos that lacked the endogenous
TE driving the expression of this isoform in mice [158]. Model organisms
have also been indispensable for discoveries on the contribution of
several non-coding RNA classes found in mammalian gametes, including
miRNAs [159-161], piRNAs [162,163], tRNA-derived small RNAs
[164-167], and lncRNAs [168], to intergenerational inheritance. While
model organisms provide an important in vivo tool to understand the
roles of the non-coding genome in human embryonic development their
use is limited to studying the pathways and molecules that are conserved
between species. Recently established cell models of human embryonic
development, including naïve stem cells [116], blastoids/gastruloids
[76-78,169,170-173], and 8-cell like cells [174,175], are crucial tools
for mechanistic understanding of non-coding elements. However,
meticulousness in the study designs employing these models is imperative
to ensure that the non-coding RNA is studied under the prerequisites
set by observations made in human oocytes and embryos
regarding cellular compartmentalization and dominant transcript isoform
(Fig. 1). As human gametes and embryos are known to employ
P. Paloviita and S. Vuoristo
Seminars in Cell and Developmental Biology xxx (xxxx) xxx
7
regulatory mechanisms elicited by the non-coding genome that are absent
in somatic cell types and may differ in model organism, characterization
of the human gamete and embryo transcriptome and genomic
regulatome at the sequence and on the functional level is likely to be
only at its beginning.
Acknowledgements
We acknowledge Dr Timo Tuuri for critical reading of the manuscript.
This work was supported by the University of Helsinki Doctoral
Programme in Biomedicine (P.P.), the Orion Research Foundation sr (P.
P), the University of Helsinki Three-year grant (S.V.), the Sigrid Jus´elius
Young Investigator Grant (S.V.), and the Jane and Aatos Erkko Foundation
(S.V.).
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Glossary
Maternal-to-zygotic transition: The maternal-to-zygotic transition entails the switch in
control from the maternal to the zygotic genome during embryonic development. This
requires both the transcriptional activation of the embryonic genome that has
assembled from the maternally and paternally inherited haploid genomes and the
degradation of maternal products stored in the oocyte.
Embryonic genome activation: The onset of transcription from the embryo genome, is
called embryonic genome activation. Transcription is initiated in a stepwise manner
with temporal differences between species. In human embryos, the first phase of
transcription, a “minor” wave occurs by the 4-cell stage. The “major” wave occurs at
the 8-cell stage and transcriptional activity is increased.
Non-coding genome: The non-coding genome refers to DNA sequences that do not encode
for proteins. The non-coding genome functions in transcriptional regulation, chromatin
organization, DNA replication, maintenance of chromosomal stability and
segregation, and is also transcribed producing functional non-coding RNA molecules.
Of the human genome 98–99% is considered non-coding and the ratio of coding to
non-coding DNA varies greatly among species.
Transposable element: Transposable elements are mobile DNA sequence that account for
approximately 50% of human DNA. Transposable elements are divided into Class I and
II, that is retrotransposon and DNA transposons, respectively. The former move via
reverse transcription while the latter encode the protein transposase enabling their
independent transposition. Transposition often results in the duplication of the original
moving sequence. Transposable elements provide regulatory sequences and an
efficient means of genomic regulation as they exist in multiple copies across the
genome.
Retrotransposon: Retrotransposons are divided into two groups, distinguished by the
presence or absence of long terminal repeats. The vast majority of human transposable
elements are non-long terminal repeat retrotransposons, mainly long interspersed
nuclear element 1, Alu, and SINE-R, VNTR and ALU (SVA) elements that together
make up approximately one third of the human genome. Human long terminal repeat
elements are endogenous retroviruses and they account for approximately 8% of the
genome.
HERV: Human endogenous retroviruses are DNA sequences of retroviral origin that have
been acquired over the last 100 million years as the result of multiple integrations by
ancient viruses. Most Human endogenous retroviruses have lost their coding potential
due to mutations and produce mainly non-coding RNAs, however some have retained
residual protein coding capacity. Human endogenous retroviruses are typically
composed of two flanking long terminal repeats and an internal portion with viral
genes gag, pro-pol and env.
Non-coding RNA: Non-coding RNAs are molecules that are not transcribed into proteins
but can be functional. Non-coding RNA comprises multiple subclasses with the main
division done according to sequence length into long and small non-coding RNAs.
Non-coding RNAs create networks that include also coding transcripts and proteins to
regulate chromatin conformation, epigenetic state, transcription, translation and RNA
modification and turnover.
Long non-coding RNA: Long non-coding RNAs are over 200 nucleotides long and possess
very little protein-coding potential. The FANTOM5 project reported 27,919 long noncoding
RNAs in various human samples. Most long non-coding RNAs are transcribed
as networks of protein-coding gene overlapping sense and antisense transcripts or are
intergenic.
Small non-coding RNA: Small non-coding RNAs are a class of approximately 30 nucleotides
long molecules with diverse regulatory functions during germ cell and early
embryonic development as well as in somatic tissues. Mammalian oocytes and embryos
primarily express Piwi-interacting RNAs, endogenous small interfering RNAs,
micro RNAs, and ribosomal RNA- and transfer RNA-derived small RNAs.
Micro RNA: Micro RNAs are ~22 nucleotide long RNAs that mediate gene silencing via
binding to partially complementary elements in the 3′
untranslated region of target
mRNAs, resulting in mRNA deadenylation and/or inhibition of translation. Micro
RNAs are generated by sequential DROSHA/DGCR8 and DICER1 cleavage of hairpin
structures embedded in long transcripts and associate with Argonaute proteins to form
the RNA-induced silencing complex.
PIWI-interacting RNA: Piwi-interacting RNAs are generated from primary transcripts that
are cleaved into intermediates by the nuclease Zucchini, subsequently bound by PIWI
proteins, further trimmed to lengths of ~26–30 nt and 2′
-O-methylated at their 3′
termini by Hua enhancer 1. Most mammalian genomes encode four PIWI proteins
(PIWIL1–4). The PIWI proteins form a complex with the primary piwi-interacting
RNAs and cleave target RNAs between positions 10 and 11 of the complementary
sequence. Piwi-interacting RNAs are also generated via a secondary mechanism upon
binding to their target RNAs and this is called the “ping-pong” model for biogenesis.
Primary piwi-interacting RNAs often have an uracil at the 5′
end however, secondary
Piwi-interacting RNAs usually also have an A residue at position 10 and a 10 nucleotide
5′
- end overlap with the primary Piwi-interacting RNAs. Piwi-interacting RNAs
contribute to the silencing of transposable elements primarily in germ line cells.
Oocyte short piRNAs: Oocyte short piwi-interacting RNAs are ~20 nucleotides long,
which is shorter than the piwi-interacting RNAs found in the testes and ovaries of
other species. Oocyte short piwi-interacting RNAs specifically associate with PIWIL3
and lack 2’-O-methylation at the 3’ end. They also have the 1 U/10 A sequence feature
like the long piwi-interacting RNAs in human oocytes however, their overall sequence
characteristics show closer resemblance to the mouse endogenous short interfering
RNAs. Oocyte short piwi-interacting RNAs are primarily expressed in primate oocytes,
lacking in mouse oocytes, and are likely to mediate the silencing of recently evolved
transposable elements.
P. Paloviita and S. Vuoristo