Formation Of Primordial Germ Cells Research Articles

Ubiquitination plays an important role during the formation of chicken primordial germ cells.

As an important post-translational modification, ubiquitination plays an important role in regulating protein homeostasis in eukaryotic cells. In our previous studies, both the transcriptome and proteome suggested that ubiquitination is involved in the formation of chicken primordial germ cells (PGCs). Here, affinity enrichment combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) was used to analyze the ubiquitome during the differentiation from embryonic stem cells (ESCs) to PGCs, and we identify that 724 lysine ubiquitinated sites were up-regulated in 558 proteins and 138 lysine ubiquitinated sites were down-regulated in 109 proteins. Furthermore, GO and KEGG enrichment analysis showed that ubiquitination regulates key proteins to participate in the progression of key events related to PGC formation and the transduction of key signals such as Wnt, MAPK and Insulin signals, followed by the detailed explanation of the specific regulatory mechanism of ubiquitination through the combined proteome and ubiquitome analysis. Moreover, both the activation and inhibition of neddylation were detrimental to the maintenance of the biological characteristics of PGCs, which also verified the importance of ubiquitination. In conclusion, this study provides a global view of the ubiquitome during the formation of PGCs by label-free quantitative ubiquitomics, which lays a theoretical foundation for the formation mechanism and specific application of chicken PGCs.

DDX5 Can Act as a Transcription Factor Participating in the Formation of Chicken PGCs by Targeting BMP4.

As an RNA binding protein (RBP), DDX5 is widely involved in the regulation of various biological activities. While recent studies have confirmed that DDX5 can act as a transcriptional cofactor that is involved in the formation of gametes, few studies have investigated whether DDX5 can be used as a transcription factor to regulate the formation of primordial germ cells (PGCs). In this study, we found that DDX5 was significantly up-regulated during chicken PGC formation. Under different PGC induction models, the overexpression of DDX5 not only up-regulates PGC markers but also significantly improves the formation efficiency of primordial germ cell-like cells (PGCLC). Conversely, the inhibition of DDX5 expression can significantly inhibit both the expression of PGC markers and PGCLC formation efficiency. The effect of DDX5 on PGC formation in vivo was consistent with that seen in vitro. Interestingly, DDX5 not only participates in the formation of PGCs but also positively regulates their migration and proliferation. In the process of studying the mechanism by which DDX5 regulates PGC formation, we found that DDX5 acts as a transcription factor to bind to the promoter region of BMP4-a key gene for PGC formation-and activates the expression of BMP4. In summary, we confirm that DDX5 can act as a positive transcription factor to regulate the formation of PGCs in chickens. The obtained results not only enhance our understanding of the way in which DDX5 regulates the development of germ cells but also provide a new target for systematically optimizing the culture and induction system of PGCs in chickens in vitro.

Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

LncCPSET1 acts as a scaffold for MLL2/COMPASS to regulate Bmp4 and promote the formation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the ancestors of female and male germ cells. Recent studies have shown that long non-coding RNA (lncRNA) and histone methylation are key epigenetic factors affecting PGC formation; however, their joint regulatory mechanisms have rarely been studied. Here, we explored the mechanism by which lncCPSET1 and H3K4me2 synergistically regulate the formation of chicken PGCs for the first time. Combined with chromatin immunoprecipitation (CHIP) sequencing and RNA-seq of PGCs transfected with the lncCPSET1 overexpression vector, GO annotation and KEGG enrichment analysis revealed that Wnt and TGF-β signaling pathways were significantly enriched, and Fzd2, Id1, Id4, and Bmp4 were identified as candidate genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ASH2L, DPY30, WDR5, and RBBP5 overexpression significantly increased the expression of Bmp4, which was up-regulated after lncCPSET1 overexpression as well. It indicated that Bmp4 is a target gene co-regulated by lncCPSET1 and MLL2/COMPASS. Interestingly, co-immunoprecipitation results showed that ASH2L, DPY30 and WDR5 combined and RBBP5 weakly combined with DPY30 and WDR5. lncCPSET1 overexpression significantly increased Dpy30 expression and co-immunoprecipitation showed that interference/overexpression of lncCPSET1 did not affect the binding between the proteins in the complexes, but interference with lncCPSET1 inhibited DPY30 expression, which was confirmed by RNA immunoprecipitation that lncCPSET1 binds to DPY30. Additionally, CHIP-qPCR results showed that DPY30 enriched in the Bmp4 promoter region promoted its transcription, thus promoting the formation of PGCs. This study demonstrated that lncCPSET1 and H3K4me2 synergistically promote PGC formation, providing a reference for the study of the regulatory mechanisms between lncRNA and histone methylation, as well as a molecular basis for elucidating the formation mechanism of PGCs in chickens.

Determining the potency of primordial germ cells by injection into early mouse embryos

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency invitro by forming embryonic germ (EG) cells and invivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation invivo establishes a latent form of pluripotency that restricts chimera contribution.

The Establishment and Optimization of a Chicken Primordial Germ Cell Induction Model Using Small-Molecule Compounds.

In recent years, inducing pluripotent stem cells to differentiate into functional primordial germ cells (PGCs) in vitro has become an important method of obtaining a large number of PGCs. However, the instability and low induction efficiency of the in vitro PGC induction system restrict the application of PGCs in transgenic animal production, germplasm resource conservation and other fields. In this study, we successfully established a two-step induction model of chicken PGCs in vitro, which significantly improved the formation efficiency of PGC-like cells (PGCLCs). To further improve the PGC formation efficiency in vitro, 5025 differentially expressed genes (DEGs) were obtained between embryonic stem cells (ESCs) and PGCs through RNA-seq. GO and KEGG enrichment analysis revealed that signaling pathways such as BMP4, Wnt and Notch were significantly activated during PGC formation, similar to other species. In addition, we noted that cAMP was activated during PGC formation, while MAPK was suppressed. Based on the results of our analysis, we found that the PGC formation efficiency was significantly improved after activating Wnt and inhibiting MAPK, and was lower than after activating cAMP. To sum up, in this study, we successfully established a two-step induction model of chicken PGCs in vitro with high PGC formation efficiency, which lays a theoretical foundation for further demonstrating the regulatory mechanism of PGCs and realizing their specific applications.

Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors

The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.

The Distribution of the Germ Plasm Gene <i>germes</i> among Anurans

The germes gene is a marker of germ plasm and primordial germ cells (PGC) described in the African clawed frog Xenopus laevis. It is known that overexpression of its mutant form negatively affects the formation and migration of PGC. However, until now it was not known how widely this gene is represented in animals of different phylogenetic groups. In this work, we performed bioinformatic analysis of genomic and transcriptome sequences of animals with germ plasm. It turned out that germes homologs are present only in representatives of the genera Xenopus and Hymenochirus of the family Pipidae (order Anura). The obtained results were confirmed by RT-PCR analysis of the expression of germes orthologs in the ovaries of six representatives of different Anura families. Phylogenetic analysis of cloned sequences of germes homologs suggests the appearance of this gene in the ancestors of Pipidae and its secondary loss in the genus Pseudohymenochirus. It is also identified that the amino acid sequences of the functional domains of the Germes protein are rather conservative.

Nutritional and metabolic control of germ cell fate through O-GlcNAc regulation.

Fate determination of primordial germ cells (PGCs) is regulated in a multi-layered manner, involving signaling pathways, epigenetic mechanisms, and transcriptional control. Chemical modification of macromolecules, including epigenetics, is expected to be closely related with metabolic mechanisms but the detailed molecular machinery linking these two layers remains poorly understood. Here, we show that the hexosamine biosynthetic pathway controls PGC fate determination via O-linked β-N-acetylglucosamine (O-GlcNAc) modification. Consistent with this model, reduction of carbohydrate metabolism via a maternal ketogenic diet that decreases O-GlcNAcylation levels causes repression of PGC formation in vivo. Moreover, maternal ketogenic diet intake until mid-gestation affects the number of ovarian germ cells in newborn pups. Taken together, we show that nutritional and metabolic mechanisms play a previously unappreciated role in PGC fate determination.

Transcriptome-based analysis of key signaling pathways affecting the formation of primordial germ cell in chickens

Bone morphogenetic protein 4 (BMP4) can induce the formation of chicken primordial germ cells (PGCs) in vitro; however, its regulatory mechanism in poultry remains unknown. This study aimed to use RNA-seq to analyze PGCs in chicken embryos and iPGCs induced by BMP4 in vitro, clarify the internal regulatory factors of PGCs, analyze the mechanism of the formation of PGCs, and lay a theoretical foundation for the further optimization of PGCs induction systems. Embryonic stem cells (ESCs), PGCs and iPGCs induced by BMP4 in vitro were collected. The transcriptional maps of the three cell types were studied using RNA-seq. The results showed 6,142 genes differentially expressed between PGCs and iPGCs, of which 2,728 were upregulated in iPGCs and 3,414 were downregulated in iPGCs. Compared to that in ESCs, BMP4 was significantly upregulated in PGCs and iPGCs. KEGG results showed that both the TGF-β and Wnt signaling pathways were activated during the formation of PGCs in vitro and in vivo, and the activation was more significant during iPGCs induced by BMP4. The expression of Nodal, an inhibitory factor of TGF-β signaling, was significantly decreased in PGCs and iPGCs, but was not expressed in iPGCs, which further supports our conclusion. Additionally, the Lysosome and PI3K-AKT signaling pathways were significantly enriched in PGCs and iPGCs, respectively. Further, transmission electron microscopy (TEM) results showed that the number of autolysosomes was significantly higher after the addition of BMP4, which is consistent with the KEGG results. Furthermore, the number of PGCs was significantly reduced after ATG14 was interfered in vivo and in vitro. In conclusion, this study screened out the key signaling pathways during the formation of PGCs, aiming to provide help for enriching the mechanism network regulating PGCs formation in chicken and laying a theoretical foundation for further improving the efficiency of inducing PGCs in vitro.

Gastruloid-derived primordial germ cell-like cells develop dynamically within integrated tissues.

Primordial germ cells (PGCs) are the early embryonic precursors of gametes - sperm and egg cells. PGC-like cells (PGCLCs) can currently be derived in vitro from pluripotent cells exposed to signalling cocktails and aggregated into large embryonic bodies, but these do not recapitulate the native embryonic environment during PGC formation. Here, we show that mouse gastruloids, a three-dimensional in vitro model of gastrulation, contain a population of gastruloid-derived PGCLCs (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of specific exogenous PGC-specific signalling or extra-embryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, owing to their inherent co-development, serve as a novel model to study the dynamics of PGC development within integrated tissue environments.

NRF1 promotes primordial germ cell development, proliferation and survival.

Primordial germ cells (PGCs) are the germline precursors that give rise to oocytes and sperm, ensuring the continuation of life. While the PGC specification is extensively studied, it remains elusive how the PGC population is sustained and expanded after they migrate to embryonic gonads before birth. This study demonstrates that NRF1, a known regulator for mitochondrial metabolism, plays critical roles in post-migrating PGC development. We show that NRF1 protein level gradually increases in post-migrating PGCs during embryonic development. Conditional Nrf1 knockout from embryonic germ cells leads to impaired PGC proliferation and survival. In addition, NRF1 may also actively drive PGC derivation from pluripotent stem cells. Using whole genome transcriptome profiling and ChIP-seq analyses, we further reveal that NRF1 directly regulates key signalling molecules in PGC formation, transcription factors in proliferation and cell cycle and enzymes in mitochondrial metabolism. Overall, our findings highlight an essential requirement of NRF1 in regulating a broad transcriptional network to support post-migrating PGC development both in vitro and in vivo.

Identification and Expressional Analysis of Putative PRDI-BF1 and RIZ Homology Domain-Containing Transcription Factors in Mulinia lateralis.

Mollusca represents one of the ancient bilaterian groups with high morphological diversity, while the formation mechanisms of the precursors of all germ cells, primordial germ cells (PGCs), have not yet been clarified in mollusks. PRDI-BF1 and RIZ homology domain-containing proteins (PRDMs) are a group of transcriptional repressors, and PRDM1 (also known as BLIMP1) and PRDM14 have been reported to be essential for the formation of PGCs. In the present study, we performed a genome-wide retrieval in Mulinia lateralis and identified 11 putative PRDMs, all of which possessed an N-terminal PR domain. Expressional profiles revealed that all these prdm genes showed specifically high expression levels in the given stages, implying that all PRDMs played important roles during early development stages. Specifically, Ml-prdm1 was highly expressed at the gastrula stage, the key period when PGCs arise, and was specifically localized in the cytoplasm of two or three cells of blastula, gastrula, or trochophore larvae, matching the typical characteristics of PGCs. These results suggested that Ml-prdm1-positive cells may be PGCs and that Ml-prdm1 could be a candidate marker for tracing the formation of PGCs in M. lateralis. In addition, the expression profiles of Ml-prdm14 hinted that it may not be associated with PGCs of M. lateralis. The present study provides insights into the evolution of the PRDM family in mollusks and offers a better understanding of the formation of PGCs in mollusks.

Inhibition of Autophagy Maintains ESC Pluripotency and Inhibits Primordial Germ Cell Formation in Chickens.

Autophagy plays an important role in the pluripotency and differentiation of stem cells. Transcriptome data showed that the autophagy genes MAP1LC3A and MAP1LC3B were significantly upregulated in primordial germ cells (PGCs). The Kyoto Encyclopedia of Genes and Genome (KEGG) results showed that the lysosome signaling pathway, which is related to autophagy, was significantly enriched in PGCs. Quantitative RT-PCR, western blotting, and transmission electron microscopy (TEM) results showed that autophagy was expressed in both embryonic stem cells (ESCs) and PGCs but was significantly activated in PGCs. To explore the role of autophagy in the differentiation of chicken ESCs into PGCs, autophagy was activated and inhibited using rapamycin and bafilomycin A1, respectively. Results of qRT-PCR, flow cytometry, and indirect immunofluorescence showed that the efficiency of PGC formation significantly decreased after autophagy inhibition. Our results showed, for the first time, that autophagy plays an indispensable role in the formation of chicken PGCs, which lays the foundation for studying the mechanism of autophagy in chicken PGCs and in bird gene editing and the rescue of endangered birds.

Identification of key events and regulatory networks in the formation process of primordial germ cell based on proteomics.

Currently, studies have analyzed the formation mechanism of primordial germ cell (PGC) at the transcriptional level, but few at the protein level, which made the mechanism study of PGC formation not systematic. Here, we screened differential expression proteins (DEPs) regulated PGC formation by label-free proteomics with a novel sampling strategy of embryonic stem cellsand PGC. Analysis of DEPs showed that multiple key events were involved, such as the transition from glycolysis to oxidative phosphorylation, activation of autophagy, low DNA methylation ensured the normal formation of PGC, beyond that, protein ubiquitination also played an important role in PGC formation. Importantly, the progression of such events was attributed to the inconsistency between transcription and translation. Interestingly, MAPK, PPAR, Wnt, and JAK signaling pathways not only interact with each other but also interact with different events to participate in the formation of PGC, which formed the PGC regulatory network. According to the regulatory network, the efficiency of PGC formation in induction system can be significantly improved. In conclusion, our results indicate that chicken PGC formation is a complex process involving multiple events and signals, which provide technical support for the specific application in PGC research.

Stay on the road: from germ cell specification to gonadal colonization in mammals.

The founder cells of the gametes are primordial germ cells (PGCs). In mammals, PGCs are specified early during embryonic development, at the boundary between embryonic and extraembryonic tissue, long before their later residences, the gonads, have developed. Despite the differences in form and behaviour when differentiated into oocytes or sperm cells, in the period between specification and gonadal colonization, male and female PGCs are morphologically indistinct and largely regulated by similar mechanisms. Here, we compare different modes and mechanisms that lead to the formation of PGCs, putting in context protocols that are in place to differentiate both human and mouse pluripotent stem cells into PGC-like cells. In addition, we review important aspects of the migration of PGCs to the gonadal ridges, where they undergo further sex-specific differentiation. Defects in migration need to be effectively corrected, as misplaced PGCs can become tumorigenic. Concluding, a combination of in vivo studies and the development of adequate innovative in vitro models, ensuring both robustness and standardization, are providing us with the tools for a greater understanding of the first steps of gametogenesis and to develop disease models to study the origin of germ cell tumours.This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

Examination of the role of the forkhead box L2 (Foxl2) in gonadal and embryonic development in the boring giant clam Tridacna crocea

Tridacna crocea, the smallest and earliest sexually maturing giant clam species, harbours important ecological value in maintaining coral reefs and holds potential for development as an ornamental species. At present, the molecular mechanism of gonadal development of this species remains largely unclear, and this hinders its artificial breeding and subsequent resource proliferation. The transcription factor forkhead box L subfamily member 2 (FoxL2) is one of the most conserved genes involved in the process of ovarian differentiation and development in many vertebrates and invertebrates. In this study, the Foxl2 gene (Tc-Foxl2) was cloned and characterized from T. crocea for the first time. The ORF of Tc-Foxl2 cDNA encoded a putative protein of 384 amino acid residues with a deduced molecular mass of 43.39 kDa and a theoretical isoelectric point (pI) of 8.92. Phylogenetic analysis indicated that the amino acid sequence of Tc-Foxl2 contained a typical FH domain and clustered with molluscan Foxl2 homologues. Tc-Foxl2 was mainly expressed in the gonads of females and hermaphroditic individuals but was fairly low in the gonads of males and resting individuals. Quantitative RT–PCR revealed that the expression of Tc-Foxl2 mRNA was significantly upregulated during vitellogenesis, while in situ hybridization indicated that Tc-Foxl2 mRNA was mainly detectable in follicle cells in the ovary. Furthermore, RNAi-mediated Tc-Foxl2 knockdown impeded the further growth of oocytes on the 14th day postinjection in the process of early vitellogenesis. Whole-mount in situ hybridization and qRT–PCR analyses demonstrated that during embryonic development, Tc-Foxl2 mRNA had a conserved expression profile, which may be involved in the formation of primordial germ cells. These results collectively suggest that Tc-Foxl2 is a female-biased gene that is involved in the vitellogenesis and embryonic development in Tridacna crocea.

Ultrastructure characteristics of primordial germ cells in stage X of pheasant (Phasianus colchicus) embryo.

Stage X is one of the formation stages in birds at which the blastoderm area is distinguished by two areas of area pellucida being responsible for formation of embryonic tissues and primordial germ cells, and area opaca forming the extra-embryonic tissues. Primordial germ cells are multi-potent stem cells giving rise to spermatogonia or oogonia. The present study was carried out to describe the characteristics of primordial germ cells in stage X of pheasants' embryo using a transmission electron microscope. The blastoderm was dissected out from embryos which were already incubated for 12 hr. Toluidine blue was used for staining semi-thin sections; lead citrate and uranyl acetate were also used to stain ultra-thin sections. Images of primordial germ cells elucidated that the nucleus was situated eccentrically and had a compact spherical structure. Moreover, the nucleolus appeared elongated and was located eccentrically. The cytoplasm was composed of yolk granules and glycogen particles. Mitochondria were observed as round structures in the cytoplasm. The most important finding was that the primordial germ cells contained yolk granules, mitochondria and small amount of glycogen at this stage.

A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification

Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.

Dual regulatory actions of LncBMP4 on BMP4 promote chicken primordial germ cell formation.

The unique characteristics of chicken primordial germ cells (PGCs) provide potential strategies for transgenic animal generation; however, insufficient PGC availability has limited their application. Regulation of bone morphogenic protein 4 (BMP4), a crucial factor for PGCs formation, may provide new strategies for PGC generation. We here identify a long noncoding RNA (lncRNA) that targets BMP4 (LncBMP4). LncBMP4 has similar functions as BMP4, in that it facilitates the formation and migration of PGCs. LncBMP4 promotes BMP4 expression by adsorbing the miRNA gga-mir-12211, thus reducing its inhibitory effect on BMP4 expression. In addition, the small peptide EPC5 encoded by LncBMP4 promotes the transcription of BMP4. The competing endogenous RNA (ceRNA) effect of LncBMP4 requires N6-methyladenosine (m6A) modification, in a dose-dependent manner, and high levels of m6A modification hinder EPC5 translation. Understanding the molecular mechanisms through which LncBMP4 promotes BMP4 expression during PGC formation may provide new avenues for efficient PGC generation.