Modeling X-chromosome inactivation and reactivation during human development

Stem-cell-based embryo models generate much excitement as they offer a window into an early phase of human development that has remained largely inaccessible to scientific investigation. An important epigenetic phenomenon during early embryogenesis is the epigenetic silencing of one of the two X-chromosomes in female embryos, which ensures an equal output of X-linked gene expression between the sexes. X-chromosome inactivation (XCI) is thought to be established within the first three weeks of human development, although the inactive X-chromosome is reactivated in primordial germ cells (PGCs) that migrate to the embryonic gonads. Here, we summarize our current understanding of X-chromosome dynamics during human development and comment on the potential of recently established stem-cell-based models to reveal the underlying mechanisms.


Introduction
X-chromosome inactivation (XCI) is an epigenetic process in female placental mammals that equalizes the expression of X-linked genes between the sexes [1].Achieving dosage compensation during the development of female placental mammals entails dynamic changes between transcriptionally active and inactive X-chromosome states (Xa/Xi).Female murine embryos display imprinted XCI (iXCI) during cleavage stages, whereby the paternal X-chromosome (Xp) is selectively inactivated and remains silenced in extraembryonic lineages [2][3][4].The inactive Xp is reactivated in the pluripotent epiblast (EPI) of the mouse blastocyst [5][6][7].In contrast, human and cynomolgus monkey pre-implantation embryos exhibit two actively transcribed X chromosomes [8][9][10].Upon implantation, either the maternal or paternal X (Xm/Xp) is inactivated through random XCI (rXCI) in humans, mice, and monkeys [8,10,11].A long noncoding RNA called X Inactive Specific Transcript (XIST) is required in cis for XCI to occur, as was first demonstrated using mouse embryonic stem cells (ESCs) [12].
Proper XCI is vital for embryonic development.Studies in mice using a paternally transmitted XIST deletion demonstrated that biallelic expression of X-linked genes causes embryonic lethality due to malfunctioning extraembryonic tissues [13,14].The clinical significance of XCI during early human development is underscored by the unexpectedly high prevalence of male babies born from in vitro fertilization (IVF) [15,16].A similar sex ratio distortion in mouse IVF offspring has been attributed to impaired XCI in female embryos [17].Furthermore, skewed XCI patterns have been linked to infertility, intellectual disability, immune diseases, and cancer [18,19].However, our knowledge of X-chromosome regulation thus far is mainly based on studies in mice, which -based on a limited number of studies performed on human embryos -appear to have substantially different dosage compensation mechanisms [20][21][22].These observations highlight the need for appropriate models of XCI during human development.
The past decade has witnessed remarkable progress in capturing various stages of human embryonic and extraembryonic development using human pluripotent stem cells (hPSCs).Naive hPSCs, which correspond to the preimplantation EPI [23,24], can be reprogrammed into 8-cell embryo-like cells (8CLCs) [25][26][27] or programmed to acquire a formative state of pluripotency akin to the early postimplantation EPI [28].In addition, naive hPSCs have a broad potential for extraembryonic differentiation, contributing to both primitive endoderm (PE) and trophectoderm (TE) derivatives [29][30][31][32][33].In a transformative advance, naive hPSCs have even been shown to self-organize into entire 3D blastocyst-like structures (blastoids) that encompass all three lineages of the preimplantation embryo [34][35][36].The rapidly expanding array of stem-cell-based models of human embryogenesis offers an unprecedented opportunity to investigate the mechanisms of XCI in vitro.

Modeling X-chromosome regulation during human preimplantation development
The divergence in dosage compensation mechanisms between mice and humans is best exemplified during preimplantation development.Female mouse embryos display iXCI, in which the Xp is inactivated and marked by upregulation of XIST (Xa XIST-Xi XIST+ ), shortly after zygotic genome activation (ZGA) [6].In contrast, RNA fluorescence in situ hybridization (FISH) and single-cell RNA sequencing (scRNA-seq) indicate that both X chromosomes are actively transcribed in human pre-implantation embryos [8,9,37].Lanner and colleagues reported that X-linked gene expression first increases from embryonic day E3 to E4 and then gradually decreases until E7 in all three lineages of the blastocyst (EPI, PE, and TE) [9] (Figure 1a-b).These observations led to the proposition that dosage compensation in human preimplantation embryos is achieved by lowering gene expression from both X chromosomes, a process named X-chromosome dampening (XCD).Surprisingly, XIST is expressed by both actively transcribed X chromosomes in human preimplantation embryos (Xd XIST+ Xd XIST+ ) [8,9,37].This provides another important distinction with the mouse embryo in which XIST is downregulated in the preimplantation EPI [5][6][7].
A subsequent study reanalyzed XCI dynamics in human preimplantation embryos using a different bioinformatic pipeline and reported a decrease in biallelic and a concomitant increase in monoallelic X-linked gene expression, suggestive of rXCI, rather than dampening, in the human blastocyst [38].However, analyzing an independent scRNA-seq dataset of human embryos, Tang and colleagues concluded that the majority of single cells in E6 blastocysts still express X-linked genes in a balanced manner from both the maternal and paternal alleles [11].Regardless of the exact time when rXCI is completed, it is evident from these studies that human embryos are subject to unique dosage compensation mechanisms that cannot be adequately modeled in mice, such as the lack of iXCI and the uncoupling of XIST expression from XCI.
Conventional or 'primed' hPSCs resemble the post-implantation EPI on the cusp of gastrulation [39,40], a stage of human development when XCI is close to completion [11].Consequently, most female primed hPSCs have already initiated XCI, although a subset of primed hPSC lines harbor an Xi that becomes partially reactivated ('eroded') with prolonged culture (XaXe) [41][42][43] (Figure 2a, right).This eroded XCI status has been linked to poor differentiation behavior and hampers disease modeling for X-linked mutations [44].Recently developed protocols for inducing naive pluripotency have enabled the generation of female hPSCs in which a majority of cells exhibit X-chromosome reactivation (XCR) [45][46][47].Encouragingly, naive hPSCs derived using one of two conditions, t2i/L/Gö [23] or 5i/L/A [24], also express XIST from the actively transcribed X-chromosome [37,45] (Figure 2a, left).However, most naive hPSCs show monoallelic expression of XIST and only a minority of cells (5-30%) fully mirror the Xd XIST+ Xd XIST+ pattern seen in the human blastocyst.This fraction varies depending on the naive culture medium and derivation method (primed-to-naive resetting vs. de novo derivation from the blastocyst).Furthermore, Plath and colleagues reported a downregulation of X-linked genes as cells transition from an XIST-negative XaXa intermediate state to the XIST-positive XaXa naive state [45].These data suggest that naive hPSCs are subject to XCD, as previously reported in human embryos [9], and that this process may be dependent on XIST.Further studies are required to clarify whether XCD affects only one X-chromosome in naive hPSCs with monoallelic XIST and both X chromosomes in cells with biallelic XIST.Thus, while currently available naive hPSCs do not capture all facets of X-chromosome regulation in the early human embryo, they provide a starting point for mechanistic studies into human-specific forms of dosage compensation.
How can naive hPSCs help us unravel the mechanisms of X-chromosome regulation during human preimplantation development?First, the role of XIST in XCD can be further investigated by comparing the levels of X-linked gene expression from the XIST-expressing X-chromosome to that of the XIST-negative X-chromosome at the single-cell level [45].More direct evidence about the role of XIST could be obtained by deleting one or both of its copies in naive hPSCs and measuring the impact on X-linked gene expression.Second, Rougeulle and colleagues reported that the hominid-specific lncRNA X Active Coating Transcript (XACT) co-accumulates, yet barely overlaps, with XIST on actively transcribed X chromosomes in both human pre-implantation embryos and naive hPSCs [37].These observations raised the suspicion that XACT might compete with XIST accumulation in cis.This hypothesis can be explored further by genetically perturbing XACT in naive cells and determining the impact on XIST localization and X-linked gene expression.It is worth pointing out, however, that XACT seems to be dispensable for maintaining Xi erosion, since its deletion does not alter the expression of X-linked gene in female-primed hPSCs [48].Third, it will be of interest to determine whether histone modifications participate in achieving dosage compensation in the human preimplantation embryo.Elsässer and colleagues recently reported that inhibition of the polycomb subunit EZH2 does not cause global upregulation of X-linked gene expression in naive cells [49], excluding an active role for H3K27me3 in XCD.Finally, naive hPSCs provide a unique model system for investigating the impact of 3D conformational changes along the X chromosome.A recent study reported that naive hPSCs derived in 5i/L/A display a shift towards more Xa-like folding conformations, but surprisingly this did not correlate with XIST expression state [80].
The above studies need not be restricted to naive hPSCs under self-renewing conditions.By reverting naive hPSCs into an 8-cell embryo-like state [25-27], it is now possible to establish an in vitro model system for dosage compensation mechanisms at an even earlier stage of human development when XIST is first induced [38] (Figure 2b).In addition, by applying culture conditions that support the differentiation of naive hPSCs into PE [50] or TE [30,33], we can model human-specific dosage compensation mechanisms in extraembryonic lineages of the preimplantation embryo (Figure 2c).Finally, the generation of blastoids from naive hPSCs [34][35][36] offers an integrated embryo model in which the functional significance of candidate regulators of XCI, such as XIST and XACT, on all three blastocyst lineages can be interrogated simultaneously (Figure 2d).

Modeling random X-chromosome inactivation during human postimplantation development
Early postimplantation development is the executive phase of XCI when a random choice is made to silence either the Xm or Xp, which is then clonally propagated through cell division.Currently, our only direct insights into the kinetics of rXCI during human embryogenesis come from the study by Tang and colleagues, who performed scRNA-seq analysis of human embryos cultured in vitro up to E12 [11].They concluded that allelespecific expression of X-linked genes in female embryos gradually accumulates between E8 and E12 in the EPI, PE, and TE lineages (Figure 1c).In addition, they reported that the expression ratio of X-linked and auto-somal genes (X:A ratio) in male embryos was close to 1.This observation is consistent with Ohno's hypothesis that genes located on the single X-chromosome in male cells are upregulated by nearly twofold to compensate for dosage differences with autosomes, a process named X-chromosome upregulation [51].On the other hand, the X:A ratio in female embryos was above 1, suggesting that rXCI had been initiated but not completed by E12.
Saitou and colleagues recently performed a detailed analysis of the dosage compensation program in cynomolgus monkey embryos [10].They noted the formation of an intermediate XCI state in female embryos in which both X chromosomes are marked by repressive histone modifications and a compacted chromatin structure, but nonetheless remain actively transcribed.This active intermediate XCI state was already present in the TE around implantation, but emerged more slowly in other postimplantation lineages [10], suggesting that XCI may be accelerated in the primate TE.It remains unclear why the XIST-coated Xa in human preimplantation embryos does not accumulate repressive histone modifications in a similar manner, but the authors speculate that this difference may be attributed to hominid-specific expression of XACT [37].
Can we use human stem cells to model the initiation of rXCI?Currently available naive hPSCs initiate de novo XCI upon differentiation, but inactivation is biased toward the X-chromosome that was silenced in the parental primed cells [45,46] (Figure 3a).Wang and colleagues reported that overcoming autocrine FGF signaling enhances the fraction of naive cells with biallelic XIST expression and facilitates rXCI upon differentiation [52] (Figure 3b).However, supplementing the 5i/L/A cocktail with an FGF receptor inhibitor was associated with reduced cell survival and did not support induction of naive pluripotency.We recently showed using a dual-color fluorescent reporter system that naive hPSCs initiate rXCI at the locus of a gene called Mutations in methyl CpG binding protein 2 (MECP2) upon differentiation into human trophoblast stem cells (hTSCs) [53].These experiments were performed using an alternative naive induction cocktail containing PD0325901, XAV939, GDC-0994, Gö 6983, Y-27632, and Activin A (PXGGY/A), which also promotes biallelic MECP2 reporter activity in naive cells [54].Hence, hTSC derivation from naive hPSCs may provide a tractable system for studying the initiation of rXCI during early human development.It will be important to define the extent of rXCI at multiple X-linked genes, the relative kinetics of XCI during embryonic and extraembryonic differentiation, and the influence of specific naive culture conditions on the XCI process.
A question of particular interest is to identify regulators of rXCI during the differentiation of naive hPSCs.The XIST antisense RNA transcript (Tsix) is a well-known repressor of XIST on the Xa in mice [55], but its locus is poorly conserved in humans, and human TSIX appears to colocalize with XIST on the Xi [56].Studies in mouse ESCs have also identified several putative XIST activators on the future Xi, such as Just proximal to XIST (Jpx) and Ring finger protein 12 (Rnf12) [57,58].Rougeulle and colleagues recently reported that inhibition of JPX reduces the expression of XIST in primed hPSCs [59].Therefore, it will be instructive to genetically perturb candidates such as JPX or RNF12 in naive hPSCs and determine whether these factors contribute to the initiation of rXCI upon differentiation.Ongoing efforts to culture human blastoids through implantation stages may soon allow investigation of rXCI in multiple embryonic and extraembryonic lineages simultaneously.

Modeling X-chromosome reactivation in the human germline
Although XCI is completed during early postimplantation development, studies in mice, cynomolgus monkeys, and humans have shown that the Xi becomes reactivated in female primordial germ cells (PGCs), the precursors of the gametes [60,61].During the first 2-3 weeks of development, human primordial germ cells (hPGCs) are specified from the posterior EPI or early amnion [62] (Figure 1d).They migrate to the embryonic gonads during weeks 4-5 where they differentiate into oogonia or gonocytes in the ovaries and testes [63,64].During week 10, oogonia and gonocytes begin their sexual differentiation: in females, oogonia differentiate into oocytes through meiotic prophase, while gonocytes differentiate into fetal spermato-gonia in males [63,64].These stages of hPGC development are accompanied by extensive epigenetic reprogramming, including DNA demethylation, imprint erasure, and XCR in females (Figure 4a-d) [65][66][67].
Payer and colleagues investigated X-chromosome dynamics in detail during mouse germ cell development in vitro and reported that XCI is crucial for proper germ cell differentiation, while XCR is necessary for meiotic progression [68].These findings highlight the importance of timely XCI and XCR for germ cell maturation and suggest a potential link between X-chromosome dosage compensation and developmental progression in germ cells.Several studies concluded that XCR has taken place in human PGCs (hPGCs) by the fourth week of development based on biallelic expression of X-linked genes and an absence of H3K27me3 domains [66,69].However, Chouva de Sousa Lopes and colleagues reported that roughly 30% of female hPGCs still exhibit incomplete or ongoing X reactivation between weeks 4 and 9 of development (Figure 4b) [70].More recently, Clark and colleagues demonstrated using RNA FISH that female hPGCs harbor two actively transcribed X chromosomes by week 7 (Figure 4c) [71].Interestingly, these actively transcribed X chromosomes were also coated by XACT and XIST, mimicking the X-chromosome profile seen in preimplantation EPI [8,9,37].Clark and colleagues also noted a reduced X:A ratio in female hPGCs, indicating that the female germline is subject to XCD (Figure 4b-c).This Xd XIST+ Xd XIST+ state lasts until hPGCs enter into meiosis during week 10, at which point female germ cells silence both XIST and XACT and upregulate X-linked gene expression (Figure 4d).
While the abovementioned studies have illuminated our understanding of the Xchromosome status in hPGCs starting around 4 weeks of development, it still remains unclear how XCR is initiated in hPGCs during earlier stages.Some insights can be obtained from studies in closely related mammals.A recent study in pigs provided evidence for XIST repression and extensive XCR in PGCs even before they migrate to the embryonic gonads [72].Similarly, Saitou and colleagues noted an initial wave of XIST repression during PGC specification in female cynomolgus monkey embryos from E13 to E17, which coincided with an increase in biallelic X-linked gene expression [10].This was followed by the disappearance of H3K27me3-enriched domains between E22 and E28 and re-expression of XIST at later stages.Based on these studies, we hypothesize that XCR in nascent hPGCs likely involves an initial wave of XIST repression concomitant with a switch to biallelic X-linked gene expression.
Can we model the initiation of XCR in the human germline in vitro?Several protocols have been developed to model human gametogenesis in vitro (Figure 4e-f).Primed hPSCs can be differentiated into hPGC-like cells (hPGCLCs) that express markers such as SOX17, TFAP2C, and BLIMP1, thus corresponding to weeks 2-4 of an early or premigratory hPGC phenotype (Figure 4e) [73,74].Excitingly, Saitou and colleagues were even able to induce the differentiation of hPGCLCs into early meiotic oogonia-like cells in xenogeneicreconstituted ovaries with mouse embryonic ovarian somatic cells [75,76].These oogonialike cells, but not the parental hPGCLCs, showed partial (~20%) XCR without XIST expression.However, since erosion of the Xi in primed hPSCs is maintained during differentiation [42,71], primed cells do not provide an ideal starting point to dissect XCI and XCR dynamics in the human germline (Figure 4e).A potential solution is to prevent erosion of XCI in primed hPSCs, which has been linked to WNT signaling [77].In fact, Saitou and colleagues recently reported that WNT inhibition prevents erosion of XCI in primed cynomolgus monkey PSCs, providing a better model system of XCI dynamics during germ cell differentiation [78].As an alternative approach, Reik and colleagues have shown that naive hPSCs can acquire germ cell fate in response to similar cues as mouse ESCs (Figure 4f) [79].It will be of interest to determine whether hPGCLC induction from naive hPSCs involves successive waves of XCI and XCR, as expected based on studies in animal models (Figure 4f).The establishment of a cellular model that captures X-chromosome dynamics in the human germline will be of tremendous value given the difficulties in investigating weeks 2-4 of human development.Finally, the extended culture of integrated embryo models, such as blastoids, may offer a complementary model system to elucidate XCI dynamics during germ cell induction, provided that conditions can be established that support their development through gastrulation stages.

Discussion
Our understanding of dosage compensation has come a long way since Mary Lyon first proposed that one of the two X chromosomes in female cells is randomly inactivated to balance the dosage of X-linked gene expression between the sexes [1].However, most studies on X inactivation have been performed in the mouse system, which -as we have learned from the handful of studies in human embryos -only partially recapitulates the human dosage compensation program.Given ethical and practical restrictions on manipulating human embryos, stem-cell-based models of early human development offer an unprecedented opportunity to define human-specific dosage compensation mechanisms.It is important to bear in mind that the X-chromosome status of stem-cell-based embryo models is inextricably linked to that of the stem cells from which they originate.If XCI is already established, as in most conventional (primed) hPSCs, the embryo models will likely maintain XCI, as has been demonstrated during in vitro differentiation [42].In contrast, naive hPSCs display two active X chromosomes and are capable of undergoing de novo XCI upon differentiation [37,45,46].Nevertheless, some important differences with the human embryo remain, such as the lack of biallelic XIST coating in the entire cell population and biased XCI during differentiation, requiring further refinement of naive culture media.Coupled with the rapid pace of stem-cell-based embryo modeling, we anticipate that it will soon be possible to reconstitute the complete dynamics of XCI and XCR from the 8-cell embryo-like stage to hPGCLC specification in vitro.These approaches will allow investigation of several important questions that have remained elusive thus far, such as the role of the lncRNAs XACT and XIST in XCD, the regulation or rXCI during differentiation, and the events leading to XCR in the human germline.Using chromatin tracing, the authors analysed Xa and Xi-associated 3D chromosome structures in somatic, primed, and 5i/L/A-derived naive hPSCs.They report a shift towards more Xaassociated 3D structures in naive compared to primed hPSCs.The PE and TE contribute to extraembryonic tissues, while EPI contributes to the embryo proper.At this stage, both X chromosomes are in a dampened state where Xlinked gene expression decreases, while XIST is expressed biallelically.However, the extent of XCD in the early human embryo remains the subject of debate.(c) From the implantation of the human embryo at E7 until E14, one of the two X chromosomes is gradually inactivated in all lineages with monoallelic XIST expression from the Xi.Inactivation of one of the X chromosomes takes place independently in each cell where either the maternal or paternal X is randomly inactivated (rXCI), which is clonally maintained thereafter.(d) By the end of week 3, the human embryo forms three germ layers (endoderm, mesoderm, and ectoderm).During this time, PGCs, the precursors of the gametes, develop from the posterior EPI or early amnion and migrate to the developing gonads via the hindgut.At this stage, the XCI pattern established in the early postimplantation EPI is maintained in all three germ layers: monoallelic XIST expression with one Xi.However, the Xi is reactivated in PGCs through the process of XCR.This is thought to entail a gradual process that starts with the specification of PGCs, continues while the PGCs migrate through the hindgut, and is completed after the PGCs complete their migration to the gonads.In the end, XCR in PGCs results in an X-chromosome state very similar to that of preimplantation EPI cells: biallelic XIST expression with dampened X-linked gene expression (XdXd).Stem-cell models of X-chromosome regulation during human preimplantation development.Schematic representation of embryonic and extraembryonic cell identities during human preimplantation development that can be accessed from naive hPSCs to model Xchromosome regulation.(a) hPSCs representing discrete pluripotent states can be isolated from human blastocysts under specific culture conditions: 5i/L/A and t2i/L/Gö capture naive hPSCs that correspond to preimplantation EPI, while basic FGF (bFGF) and activin capture primed hPSCs that correspond to postimplantation EPI.Female-primed hPSCs exhibit an X-chromosome state similar to postimplantation EPI cells: one Xi with monoallelic XIST expression.However, upon prolonged culture, primed hPSCs show X-chromosome erosion, which is characterized by loss of XIST expression and partial reactivation of the Xi.In contrast, female naive hPSCs exhibit two actively transcribed X chromosomes and XCD (XdXd), similar to preimplantation EPI cells.However, the extent of mono-or biallelic XIST expression in naive hPSCs varies between culture conditions and derivation methods (primed-to-naive reversion vs. de novo derivation from human blastocysts).(b) Methods to generate 8CLCs from naive hPSCs as a basis for modeling X-chromosome dynamics at the very beginning of human development.(c) By week 9, hPGCs reach the gonads and, in females, start differentiating into oogonia.Gonadal hPGCs show a homogeneous XCD state with biallelic expression of XIST, XACT, and other X-linked genes [63,64,71].(d) At week 10 of development, female hPGCs enter into meiosis.Before entering into meiosis, hPGCs silence both XIST and XACT and upregulate X-linked gene expression, thereby changing X-chromosome state from XCD to XCR [63,64,71].(e) The most-established model of hPGCLC induction starts from primed hPSCs and passes through an intermediate cell state called incipient mesoderm-like cells (iMeLCs).However, the eroded state of the X-chromosome is known to be maintained during differentiation and may not support XCR during germ cell induction [73].(f) Naive hPSCs have been used to generate hPGCLCs through an intermediate state called epiblastlike cells (EpiLCs).This in vitro method offers an excellent opportunity to study sequential cycles of rXCI (preimplantation to postimplantation stage) and XCR (postimplantation to germ cell development stage) observed during early development of female embryos [79].

Figure 1 .
Figure 1.XCI and XCR dynamics during early human development.Cartoon describing the status of XIST expression (mono-or biallelic), X-chromosome state (Xa, Xi, Xd: dampened), and X-linked gene expression in major cell lineages observed during early development of female human embryos.(a) Shortly after ZGA at E3, both X chromosomes become active with biallelic XIST expression.X-linked gene expression increases until E4 and then starts decreasing until it stabilizes during early postimplantation development.(b)The totipotent cells of the cleavage embryo give rise to the blastocyst, which contains three cell types: EPI, PE, and TE.The PE and TE contribute to extraembryonic tissues, while EPI contributes to the embryo proper.At this stage, both X chromosomes are in a dampened state where Xlinked gene expression decreases, while XIST is expressed biallelically.However, the extent

Figure 3 .
Figure 3. Stem-cell models of XCI during differentiation of naive hPSCs.Schematic representation of two reports describing XCI dynamics during differentiation of naive hPSCs.Naive hPSCs are not directly responsive to embryonic lineage-inductive cues, but can complete germ layer differentiation after transition into a postimplantation EPI identity by repriming or capacitation.(a) Sahakyan et al. reported that repriming of naive hPSCs derived in 5i/L/A or t2i/L/Gö generated a cell state with two active X chromosomes (XaXa) without XIST expression, while further differentiation into fibroblasts resulted in nonrandom XCI (non-rXCI) and monoallelic XIST expression [45].(b) An et al. reported that the additionof an FGF receptor inhibitor to 5i/L/A naive hPSCs enhances the fraction of naive cells with biallelic XIST expression and enables rXCI upon repriming and subsequent fibroblast differentiation[52].

Figure 4 .
Figure 4. Stem-cell models of XCR in the human germline.Schematic representation of XCR in vivo (a-d) and available model systems (e-f) to interrogate the human germline in vitro by generating hPGCLCs from various hPSC states.(a) During weeks 2-3 of development, cells from the posterior EPI or early amnion get specified as hPGC precursor cells, and in females, they show the same XCI status as postimplantation EPI[62].(b) During weeks 4-6, hPGCs begin their migration from the EPI to the gonads through the hindgut and continue to develop.At this stage, female hPGCs show a heterogeneous X-chromosome state: the majority of hPGCs show complete XCR, while about 30% hPGCs remain in full or partial XCI state.The XCR state of hPGCs is marked by XCD together with biallelic expression of XIST, XACT, and other X-linked genes, similar to preimplantation EPI[63,64,66,69,70].
6. Mak W, et al. : Reactivation of the paternal X chromosome in early mouse embryos.Science 2004, 303:666-669.[PubMed: 14752160] 7. Okamoto I, et al. : Epigenetic dynamics of imprinted X inactivation during early mouse development.Science 2004, 303:644-649.[PubMed: 14671313] 8••.Okamoto I, et al. : Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development.Nature 2011, 472:370-374.[PubMed: 21471966] This study was the first to report that human female pre-implantation embryos do not undergo iXCI, but show biallelic expression of X-linked genes and the lncRNA XIST.9••.Petropoulos S, et al. : Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos.Cell 2016, 165:1012-1026.[PubMed: 27062923] By performing scRNA-seq and RNA-FISH analyses, this study confirmed biallelic expression of X-linked genes and XIST in human female pre-implantation embryos.This study also proposed the concept of XCD, whereby gene expression from both X chromosomes first increases from E3 to E4 and then gradually decreases until E7.10••.Okamoto I, et al. : The X chromosome dosage compensation program during the development of cynomolgus monkeys.Science 2021, 374:eabd8887.[PubMed: 34793202] This study analyzed the dosage compensation program in cynomolgus monkey embryos from E7 to E48.Similar to humans, biallelic expression of X-linked genes and XIST is seen in all pre-implantation lineages.Upon implantation, XCI first occurs in the TE.This work also described an intermediate state in which both active X chromosomes accumulate H3K27me3, which is gradually lost from the future active X chromosome.In the germline, the Xi first loses XIST, then the H3K27me3 mark, and finally full XCR takes place coupled with biallelic XIST expression.80•.Patterson B, et al. : Female naïve human pluripotent stem cells carry X chromosomes with Xa-like and Xi-like folding conformations.Sci Adv 2023, 9:eadf2245.[PubMed: 37540754]