Epigenetic programming of chicken germ cells: a comparative review

Abstract

Chicken embryos serve as an important model for investigating germ cells due to their ease of accessibility and manipulation within the egg. Understanding the development of germ cells is particularly crucial, as they are the only cell types capable of transmitting genetic information to the next generation. Therefore, gene expression regulation in germ cells is important for genomic function. Epigenetic programming is a crucial biological process for the regulation of gene expression without altering the genome sequence. Although epigenetic programming is evolutionarily conserved, several differences between chickens and mammals have been revealed. In this review, we compared the epigenetic regulation of germ cells in chickens and mammals (mainly mice as a representative species). In mammals, migrating primordial germ cells (precursors for germ cells [PGCs]) undergo global DNA demethylation and persist until sexual differentiation, while in chickens, DNA is demethylated until reaching the gonad but remethylated when sexually differentiated. Prospermatogonia is methylated at the onset of mitotic arrest in mammals, while DNA is demethylated at mitotic arrest in chickens. Furthermore, genomic imprinting and inactivation of sex chromosomes are differentially regulated through DNA methylation in chickens and mammals. Chickens and mammals exhibit different patterns of histone modifications during germ cell development, and non-coding RNA, which is not involved in PGC differentiation in mice, plays an important role in chicken PGC development. Additionally, several chicken-specific non-coding RNAs have been identified. In conclusion, we summarized current knowledge of epigenetic gene regulation of chicken germ cells, comparing that of mammals, and highlighted notable differences between them.

INTRODUCTION

Germ cells represent a unique cell lineage containing the entire genetic information transferable to the next generation. These cells, such as sperms or oocytes, originate from primordial germ cells (PGCs), which are diploid unipotent stem cells capable of generating germ cells. Once PGCs differentiate into gametes, functional haploid gametes are produced, capable of fertilization to form a nascent zygote. The zygote comprises pluripotent stem cells and has the potential to develop into an entire organism, emphasizing the significance of germ cells and their precursor, PGCs, not only in preserving intact genetic information for future generations but also in exploring developmental processes and conserving genetic resources (Han and Park, 2018).

In chickens, PGCs are formed in the central region of the area pellucida before oviposition. This formation occurs in the presence of germplasms containing chicken vasa homologue (CVH) and Deleted in Azoospermia Like (DAZL) (Lee et al., 2016; Tsunekawa et al., 2000). These cells passively migrate from the core region to the anterior region of zona pellucida and then actively migrate to the germinal crescent at Hamburger-Hamilton (HH) stage 4 (Kang et al., 2015). Subsequently, PGCs enter the arteries between HH stage 9 and 10 and infiltrate the epithelium of the genital ridge at HH stages 15 to 18 (Kim and Han, 2018). This migratory pattern facilitates the isolation of PGCs at various developmental stages from the germinal crescent, blood vessels, and genital ridge (Kim and Han, 2018; Kim et al., 2023). The specific gene expression of chicken PGCs, such as DAZL and CVH, during the formation and migration of PGCs, is crucial for their development. Additionally, deletion of DAZL affects chicken PGC formation (Lee et al., 2016), indicating that the regulation of chicken PGCs-specific gene expression is indispensable for PGC specification.

One of the crucial mechanisms governing gene expression is epigenetic programming, which controls gene expression without altering the DNA sequence (Skinner, 2018). Well-characterized examples of epigenetic processes include DNA methylation, histone modification, and post-transcriptional regulation through non-coding RNA (ncRNA). DNA methylation involves the covalent attachment of a methyl group to DNA sequences, predominantly CpG dinucleotides. This process, mediated by DNA methyltransferases (DNMTs), converts cytosines to 5-methylcytosines (5mC). Typically, DNA methylation upstream of the promoter suppresses gene expression by directly impeding transcription factor binding or recruiting methyl-CpG-binding proteins (Moore et al., 2013). DNA methylation is crucial for maintaining gene imprinting and inactivating one of the two X chromosomes in mammals (SanMiguel and Bartolomei, 2018).

Histone modifications represent another epigenetic mechanism. Specifically, covalent modifications occur on the tails of H3 and H4 by attaching methyl or acetyl groups. Depending on the location and type of molecule attached, histone modification can serve as a cue for transcriptional activation or repression. For instance, the attachment of a trimethyl group to histone H3 lysine 9 (H3K9me3) serves as a suppressive histone marker (Ninova et al., 2019), while acetylation on the same residue serves as an active histone marker (Sadoul et al., 2008).

Non-coding RNAs, although not translated into proteins, function as post-transcriptional regulators. For example, microRNAs (miRNAs), 18–23 nucleotides in length, complementarily bind to the 3′ untranslated region (UTR) of the target mRNA, thereby repressing transcription by degrading mRNA (O'Brien et al., 2018). Another example is piwi-interacting RNAs (piRNAs), 23 to 30 nucleotides long, transcribed from transposable element (TE) copies or repetitive elements in genomic regions. These piRNAs interact with PIWI proteins, guiding them to cleave transposable element mRNAs (Ozata et al., 2019). Long non-coding RNAs (lncRNAs) are a type of ncRNAs longer than 200 nucleotides, transcribed from sense or antisense transcripts, as well as intergenic regions. They are usually capped at the 5′ ends by 7-methyl guanosine and have a 3′ poly A tail, similar to mRNA (Statello et al., 2021). Through the epigenetic regulation of gene expression, germ cells maintain their properties and integrity, conveying intact genetic information to the next generation.

Although the epigenetic programming of germ cells is highly conserved among species, clear differences exist between avian species and mammals (Skinner, 2018), which necessitates the comparative review of epigenetic regulation of germ cells between birds and mammals. In this review, we compared the common and distinct epigenetic mechanisms for regulating gene expression for proper germ cell development and summarized the current knowledge on the epigenetic programming of chicken germ cells. The major differences in epigenetic regulation between chickens and mammals are shown in Table 1. In the following section, we will compare the epigenetic regulation of germ cells in mammals and chickens in terms of DNA methylation, histone modifications, and ncRNA expression.

Table 1.

Summary of distinct epigenetic regulation of germ cells gene expression between mouse and chicken.

Epigenetic programming	Species	Main research	References
DNA methylation	Mouse	DNA demethylation in migrating PGCs occurs and persists until sexually differentiated	(Guibert et al., 2012) (Kim and Han, 2018) (Rengaraj et al., 2011) (Yu et al., 2019) (Rengaraj et al., 2022b)
	Chicken	DNA demethylation in migrating PGCs occurs until reaching the gonad and re-methylated upon arriving genital ridge
	Mouse	Mitotic arrested spermatogonia undergoes DNA methylation	(Guo et al., 2017) (Choi et al., 2022)
	Chicken	Mitotic arrested spermatogonia is demethylated and kept until 1-wk post-hatch and re-methylated from 2 to 3-wk post-hatch
	Mouse	Sex chromosome of male: XY, that of female: XX X chromosome inactivation of female germ cells	(Ezaz et al., 2006) (Cotton et al., 2014) (Wu and Xu, 2003)
	Chicken	Sex chromosome of male: ZZ, that of female: ZW Z chromosome inactivation of male germ cells is not likely to occur
	Mouse	Igf2r, Igf2, Ascl2, Mash2, Ins2, Dlk1, Ub3ea gene imprinted	(Colosi et al., 2006) (Shin et al., 2010) (Yokomine et al., 2005)
	Chicken	Igf2r, Igf2, Ascl2, Mash2, Ins2, Dlk1, Ub3ea gene not imprinted
Histone modification	Mouse	H3K4me2 is increased during PGC migration	(Zhang et al., 2021b)
	Chicken	H3K4me2 is decreased in PGC compared to blastoderm, but H3K4me2 is concentrated in genes essential for PGC formation
	Mouse	H3K9me3 is increased when embryonic stem cells (ESCs) differentiate into primordial germ cells (PGCs)	(Meshorer et al., 2006) (Luo et al., 2009)
	Chicken	H3K9me3 is already present in chicken embryonic stem cells
	Mouse	H3K27me3 is distributed in mammalian ESCs chromatin
	Chicken	H3K27me3 is concentrated at pericentric heterochromatin in chicken ESCs
	Mouse	Both H3K9 methylation, H3K27 methylation are important for PGCs	(Kress et al., 2016) (Kress et al., 2024)
	Chicken	H3K9 methylation plays more important role in chicken PGCs than H3K27 methylation
Non-coding RNA	Mouse	miR-302 is upregulated at epiblast but almost absent in mouse PGCs on their specification	(Paikari et al., 2017) (Parchem et al., 2014) (Lázár et al., 2021) (Rengaraj et al., 2013)
	Chicken	miR-302 family plays important role in PGCs formation and proliferation
	Mouse	miR-290 degrades maternal mRNA for zygotic genome activation (ZGA)	(Tang et al., 2007) (Hwang et al., 2018)
	Chicken	miR-302 family degrades maternal mRNA for ZGA
	Mouse	-	(Zhang et al., 2021a)
	Chicken	Chicken PGC specific non-coding RNAs were reported

DNA METHYLATION

Regulation of DNA Methylation During Germ Cell Development

DNA methylation decreases across almost the entire genome during the migration of mouse PGCs to the genital ridge (Guibert et al., 2012). Unlike mammals, avian PGCs circulate through the blood vessels to reach the genital ridge (Kim and Han, 2018). During this period, chicken PGCs also undergo global DNA demethylation until they reach the gonads. Studies have indicated that circulating PGCs in the blood display lower DNA methylation compared to that in gonadal PGCs, and chicken PGCs are globally DNA demethylated via ten-eleven translocation 1 during HH stage 21 to 28, suggesting that DNA demethylation occurs during the migration of PGCs (Rengaraj et al., 2011; Yu et al., 2019). More recently, a germ cell tracing chicken model (DAZL-GFP chicken model) through precise tagging of green fluorescent protein (GFP) downstream of the DAZL gene via CRISPR/Cas9 was created to isolate GFP-positive germ cells (Rengaraj et al., 2022a) with the expression of DAZL, serving as germplasm components and phenotypic markers for chicken germ cells (Lee et al., 2016). Using this model, gene expression profiles related to DNA methylation were tracked by monitoring GFP⁺ germ cells during embryonic development. Although several genes showed differential expression between male and female PGCs, genes involved in de novo DNA methylation and maintenance of methylation, such as MAEL, BM1, HELLS, DNMT3A, DNMT3B, and DNMT1, were enriched in both male and female PGCs from E2.5 to E8, indicating that expression of DNA methylation-related genes was increased to induce DNA re-methylation after reaching the genital ridge (Rengaraj et al., 2022b).

Another study revealed the unique epigenetic programming of mitosis-arrested male germ cells using the DAZL-GFP chicken model (Choi et al., 2022). In mammals, mitotic arrest occurs in prospermatogonia for the proper differentiation of male germ cells (Du et al., 2021). Although the timing of mitotic arrest differs depending on mammalian species, the DNA methylation level is lowest at the onset of mitotic arrest along with global demethylation of PGCs and increases during the mitotic arrest (Guo et al., 2017). In contrast, chicken germ cells are re-methylated after reaching the gonad and the DNA of prospermatogonia becomes demethylated when entering mitotic arrest. Unlike mammals, its demethylation continues during the mitotic arrest, and DNA becomes re-methylated until 3 wk post-hatch (Choi et al., 2022).

Monoallelic Gene Expression Mediated by DNA Methylation

The sex chromosomes of most mammals include X and Y, where males harbor XY while females harbor XX. In chickens, sex chromosomes include Z and W where males possess ZZ while females possess ZW (Ezaz et al., 2006). In females, X chromosome inactivation prevents biallelic expression of genes on X chromosomes during germ cell development, and DNA methylation plays a major role in inactivating the X chromosome (Cotton et al., 2014). However, in male chickens, Z chromosome inactivation does not occur (Wu and Xu, 2003).

Additionally, the regulation of DNA methylation is required for establishing or erasing genomic imprinting (Hajkova et al., 2002; Seisenberger et al., 2012), which prevents biallelic expression of parentally inherited alleles. In mammals, genomic imprinting is erased during the global DNA demethylation of migrating PGCs. After that, genomic imprinting is re-established through DNA re-methylation in spermatogonia, while a low level of DNA methylation is maintained in oogonia until oocyte growth after birth (Lees-Murdock and Walsh, 2008; Smallwood et al., 2011). In a study of DNA methylation patterns of genes homologous to imprinting genes in chickens, differentially methylated regions (DMRs) of imprinted genes underwent demethylation between E11.5 and E12.5 but were re-methylated in male germ cells. Conversely, female germ cells gradually lost methylation during differentiation (Jang et al., 2013). Moreover, X-linked homologous genes in chickens displayed differential methylation in PGCs compared with chicken embryonic fibroblasts (CEFs), which implies evolutionarily conserved features of DNA methylation in both mammals and chickens (Deakin et al., 2008). However, in chickens, several imprinted genes in mammals, such as Igf2r, Igf2, Ascl2, Mash2, Ins2, Dlk1, and Ub3ea, are expressed in both alleles, implying a differential imprinting mechanism exists in chickens when compared with mammals (Yokomine et al., 2005; Colosi et al., 2006; Shin et al., 2010).

Regulation of Germ Cell-Related Gene Expression Through DNA Methylation

DNA methylation also plays a pivotal role in regulating germ cell-related gene expression. One study explored the role of DNA methylation in regulating gene expression related to pluripotency during embryonic development and highlighted the differential expression of factors crucial for differentiation among embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) in the chicken (He et al., 2018). For instance, the methylation of BCL2 and CSF3R decreased during PGC specification and continued to decrease until SSC development. Conversely, IGF2R showed increased DNA methylation in PGCs, silencing its expression, and was demethylated upon differentiation into SSCs. (He et al., 2018). Additionally, the downregulation of PouV (an avian homolog of Oct4) in embryonic germ cells was correlated with increased DNA methylation when differentiated into embryonic bodies in vitro. Simultaneously, H3 acetylation was reduced, indicating that PouV is epigenetically regulated by DNA methylation and histone modification in chicken germ cells (Jiao et al., 2013).

HISTONE MODIFICATION

Global Histone Modification in Germ Cells

In early mouse PGCs, histone modification occurs in 2 phases: the pre-gonadal and gonadal phases. Before reaching the gonads, repressive and active histone markers synergically act to repress somatic lineage genes while inducing germ cell lineage genes during the migration of PGCs. The level of repressive H3K27me3 increases to suppress somatic cell-lineage gene expression, while active H3K4me2, and H3K9ac increase to activate germ cell-lineage gene expression. In contrast, repressive H3K9me2 levels decrease to release the repressive effect on germ cell-related gene expression, and H3K9me3 levels remain consistent during this period (Seki et al., 2005; Seki et al., 2007; Hajkova et al., 2008). At E11.5, repressive histone marks (H3K9me3 and H2A/H4R3me2) and activating histone marks (H3K9ac) decrease, while H3K27me3 is maintained or lost during this period (Hajkova et al., 2008; Mansour et al., 2012; Kagiwada et al., 2013). After reaching the gonads (E12.5), chromatin recompacts, and repressive H3K9me3 and H3K27me3 increase, while H3K9ac and H2A/H4R3me2 are not re-established after programming (Hajkova et al., 2008; Kagiwada et al., 2013). In chickens, histone H3-R2/H4-R3/H3-K36 demethylation genes, such as JMJD6 and KDM8, are enriched in migrating PGCs but decrease over time, while histone H3-K9/H3-K4/H3-K37/H4-K20/H3-R17 methylation genes, such as SETDB1, KMT2C, ARID4B, and SUV39H2, are enriched over time (Rengaraj et al., 2022b), suggesting differential histone modification profiles of chicken migrating PGCs compared to mammalian PGCs for their proper development.

Another different feature of histone modification between chicken and mouse is decreased H3K4me2 in chicken PGCs, while H3K4me2 is increased during mouse PGC migration. However, the levels of H3K4me2 are specifically enriched in chicken PGC lineage-related genes, such as Blimp1. This indicates that a narrow H3K4me2 distribution is essential for chicken PGC formation (Zhang et al., 2021b). In mouse ESCs, differentiation into PGCs involves DNA methylation and repressive histone modifications, such as H3K9me3, in pericentric heterochromatin (PCH) (Meshorer et al., 2006). However, chicken ESCs already possess the H3K9me3 domain, and this modification remains unchanged upon differentiation. Another repressive histone modification, H3K27me3, is broadly distributed in mammalian ESCs (Luo et al., 2009) but is concentrated at PCH in chicken ESCs. When comparing the pattern of H3K9me3 and H3K27me3, global H3K9me3 levels are higher in chicken PGCs than in chicken ESCs, while H3K27me3 levels are lower in PGCs than in chicken ESCs, suggesting that the repressive status of PGC chromatin is primarily mediated by H3K9 methylation rather than H3K27 methylation (Kress et al., 2016). A recent study revealed that H3K9me3 progressively accumulates in migrating chicken PGCs. Unlike in mammals, H3K9me2 is retained in chicken PGCs, highlighting the significance of H3K9 methylation in regulating gene expression in chicken germ cells. Despite these differences, chicken PGCs exhibit several conserved features with mammals, such as the loss of 5-hydroxymethylcytosine, the redistribution of H2A, and an open chromatin structure. These findings suggest that chromatin-mediated regulation of germ cell gene expression is conserved, with distinct histone modification profiles observed in chickens regulating germ cell gene expression (Kress, et al., 2024).

Regulation of Germ Cell-related Genes Through Histone Modification

Several independent studies have explored the epigenetic regulation of genes essential for germ cell formation. In chickens, the CDX2 gene does not undergo DNA methylation during development. Instead, various transcription factors such as STAT1 and TFAP2c are recruited via long intergenic non-coding RNA (lincRNA) to form a complex with RNA polymerase II, regulating CDX2 expression. The recruited TFAP2c induces active H3K4me3 markers, thereby upregulating CDX2 expression. Notably, chicken PGCs and SSCs do not harbor TFAP2c and STAT1, contributing to the repression of CDX2 expression (He et al., 2018). Additionally, BMP4, a key factor in PGC formation, influences the PGC specification by regulating Prdm14 expression, which in turn affects DAZL expression through histone acetylation (Zuo et al., 2019). The expression of another critical regulator of germ cells, NANOG, is controlled by the repression of H3K9ac levels through histone deacetylase (HDAC) complex members, including RE1-silencing transcription factor (REST) and REST corepressor 3 (RCOR3) (Jung et al., 2018), suggesting that the germ cell-related genes are regulated through histone modification.

NON-CODING RNA

MiRNA-Mediated Gene Regulation in Germ Cells

MiRNAs are ncRNAs in the germline with approximately 22 nucleotides. miRNAs play a significant role in mouse PGC specification. Various types of miRNAs, including let-7 miRNAs, the miR-17/92 family, the miR-181 family, and the miR-290 cluster, are involved in PGC specification (Bhin et al., 2015; Fernández-Pérez et al., 2018; Hayashi et al., 2008; West et al., 2009). The let-7 miRNAs target Blimp1 for degradation, which is essential for PGC specification. The degradation of Blimp1 via let-7 is inhibited by Lin28a. Therefore, Lin28a is essential for proper mouse PGC specification (West et al., 2009). miR-290 is highly expressed in mouse PGCs, also observed in the inner cell mass of mouse blastocysts and naïve mouse ESCs (Parchem et al., 2014). Its expression gradually decreases upon specification of the epiblast from the blastocyst; however, it reappears exclusively in germline lineage cells, with continuous expression in the trophoblast. This miRNA expression pattern differs from those of other miRNAs; for instance, miR-302 is upregulated in E5.5–6.5 epiblasts but is almost absent in mouse PGCs upon their specification (Parchem et al., 2014; Paikari et al., 2017). This is in contrast with the miR-302 family in chicken, involved in regulating PGC proliferation (Rengaraj et al., 2013; Lázár et al., 2021). gga-miR-17-302b and miR-17-5p affect PGC proliferation by regulating glucose phosphate isomerase (GPI) expression (Rengaraj et al., 2013). Another study showed that the inhibition of gga-miR-302b-5p (5p) and/or gga-miR-302b-3p (3p) differentially influences PGCs proliferation and the rate of apoptosis, implying the complex influence of the miR-302b family on PGCs proliferation (Lázár et al., 2021).

Additionally, in chickens, several miRNAs that regulate gene expression in chicken germ cells have been identified. For instance, gga-miR-15c, gga-miR-29b, gga-miR-383, and gga-miR-222 are involved in regulating DNA methylation-related genes, such as DNMT3A and DNMT3B (Rengaraj et al., 2011). Additionally, miR-181a* binds to both homeobox A1 (HOAX1) to repress somatic gene differentiation and nuclear receptor subfamily 6, group A, member 1 (NR6A1) to prevent meiosis initiation in PGCs (Lee et al., 2011).

MiRNA-Mediated Gene Expression During ZGA

After chicken gametes are fertilized, the zygote undergoes transcriptional inactivation until zygotic genome activation (ZGA) (Tadros and Lipshitz, 2009). ZGA is induced by maternal transcription factors, such as Nanog, Pou5f1, and the SoxB1 family, in concert with the action of miRNAs that degrade maternal mRNAs (Tang et al., 2007; Lee et al., 2013; Leichsenring et al., 2013; Yartseva and Giraldez, 2015). In chickens, ZGA occurs in 2 waves: one soon after fertilization and the other at EGK. V–VIII (Rengaraj et al., 2020). During ZGA, the expression of genes related to H3 acetylation increases, while the expression levels of DNMTs, HDACs, methyl binding domains (MBDs), and TETs decrease from the zygote stage to EGK. VIII, suggesting that chromatin structures become open during the first and second waves of ZGA. Additionally, the miR-302 family is highly expressed during EGK.VIII to degrade maternal mRNAs, while the miR-290 family is expressed in mice for ZGA (Tang et al., 2007). These results indicate that ZGA is regulated by the dynamics of epigenetic programming and that miR-302 plays an important role in degrading maternal mRNA in chickens (Hwang et al., 2018).

PiRNAs

PiRNAs, characterized by 2′-O-methyl-modified 3′-ends and a 5′-U bias, interact with PIWI proteins (Weick and Miska, 2014; Czech et al., 2018; Özata et al., 2020). Importantly, their expression is confined to the germline, and their function varies based on their length and binding partners. Unlike miRNAs, piRNAs do not participate in PGC specification but play a role during late sex-specific differentiation. In mice, 3 PIWI proteins (Piwil1, Piwil2, and Piwil4) are specifically expressed in males. The knockout of these proteins leads to defects in spermatogenesis and male infertility (Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004; Carmell et al., 2007). Piwil2 expression begins at E12.5, followed by Piwil4 from E13.5 to postnatal days 3, and Piwil1 at postnatal days 4, when the pachytene of meiosis prophase I begins (Aravin et al., 2008). piRNAs also play a crucial role in repressing TE expression. The knockout of Piwil2 and Piwil4 results in the upregulation of TEs due to the lack of DNA methylation in TEs (Carmell et al., 2007; Aravin et al., 2009). Similarly in chickens, knockdown of testis- and ovary-specific piRNA pathway genes such as PIWI-like protein 1 (CIWI1) and 2 (CILI), increased the expression of piRNA-linked genes, including CR1 and RAP2B, a member of the RAS oncogene family, leading to DNA double-stranded breakage. Hence, piRNA-linked genes play a crucial role in safeguarding germ cells by regulating transposon-mediated DNA double-stranded breakage in chickens (Rengaraj et al., 2014).

LncRNAs

LncRNAs play several important biological roles in regulating genes in mouse germ cells. For example, X-inactive specific transcript (Xist), transcribed from the X chromosome, is necessary for X-inactivation in mouse oocytes. During the migration of female mouse PGCs, DNA demethylation occurs along with a reduction in Xist clouds. This reduction leads to excessive windows of X chromosome dosage until E14.5, ensuring that oocytes possess active X chromosomes post-fertilization (Mak et al., 2004; Sangrithi et al., 2017). The X chromosome: Autosome (X: A) ratio reaches 1 at E15.5 (Li et al., 2017; Sangrithi et al., 2017). Another study examined the differential increase of lncRNAs in mouse PGCs between E12.5 and E15.5 (Bao et al., 2013). However, investigations of the function of these lncRNAs in mouse PGCs are still lacking (Ramakrishna et al., 2021).

Several lncRNAs contribute to the regulation of chicken PGC formation. For example, the lncRNA PGC regulator (LncPGCR), TCONS_00948124, is significantly associated with germ cell differentiation and is predicted to interact with several miRNAs involved in PGC formation. LncPGCR increases the expression of PGC marker genes (CVH and C-kit) while downregulating the pluripotency-related gene NANOG. The activity of LncPGCR is regulated by histone acetylation and the transcription factor TCF7L2. Additionally, LncPGCR promotes PGC development by upregulating Btrc expression. It competitively binds to Btrc to upregulate its expression by releasing the inhibitory effect of Btrc targeting gga-miR-6577-5p (Jiang et al., 2021). Another lncRNA, termed lncRNA PGC transcript-1 (LncPGCAT-1), also induces the expression of PGC-related genes, such as CVH and C-kit, while inhibiting pluripotency-related genes such as NANOG. This effect is achieved by releasing the gga-miR-1591-mediated inhibition of the MAPK1/ERK signaling cascade and interacting with interleukin enhanced binding factor 3 (ILF3) to activate the JNK signaling pathway, collectively stimulating PGC formation (Zuo et al., 2020). Additionally, H3K4me2 and the transcription factor Jun upregulate another lncRNA, chicken-PGC-specifically-expressed transcript 1 (lncCPSET1), to promote chicken PGC formation (Zhang et al., 2021a). During the differentiation of chicken PGCs into SSCs, the expression of the MAPKAPK5 gene was reduced by removing the binding sites of a lincRNA rather than by DNA methylation (He et al., 2018). These findings identified several lncRNAs specifically expressed in chicken PGCs for regulating their development. Further studies analyzing orthologues of these lncRNAs with those of mammals will unveil evolutionarily conserved or divergent lncRNA-mediated regulation of germ cell development.

Future Perspective of Studying Epigenetics of Chicken Germ Cells

Early chicken embryos have been considered suitable model for studying germ cell epigenetics when compared to mammals (Bednarczyk et al., 2021). When investigating mammalian germ cells, one of the most important issues that should be addressed is germline competence of germ cells when they are cultured in vitro. In studying mechanism of mammalian germ cell differentiation, comparison of epigenetic status of cultured stem cell (or PGCs) and differentiated germ cell is commonly used. However, fundamental question lies that whether cultured germ cells are same as those in vivo (Tang et al., 2016; Bednarczyk et al., 2021). In chickens, however, PGCs are easily isolated from the blood and the gonads, and markers for isolating PGCs are well established (Kim et al., 2023). In this regard, study of chicken PGCs in vivo using early embryo has advantage that intricate epigenetic nature of chicken germ cells can be analyzed without loss of germline competence (Bednarczyk et al., 2021). Additionally, genome edited chicken model allows for efficient isolation of germ cells, facilitating the investigation of epigenetic reprogramming of germ cells during embryo development (Rengaraj et al., 2022a). Thus, chicken embryos are suitable model for researching germ cell epigenetics compared to mammalians in that in vivo germ cells can be easily obtained.

Despite its advantage, there are several remaining challenges to be overcome. One of them is cooperative effects of epigenetic regulation in germ cells. It is noteworthy that regulation of chicken germ cells is not regulated by single epigenetic mechanism, but rather regulated by synergic effects of them. For instance, the expression of CDX2 is regulated by lincRNA, recruiting transcription factors such as STAT1 and TFAP2c. These transcription factors subsequently activate H3K4me3 markers to upregulate CDX2 expression in the early phase of chicken germ cells (He et al., 2018). Additionally, ncRNA such as gga-miR-15c, gga-miR-29b, gga-miR-383, and gga-miR-222 induce expression of DNA methylase genes for DNA methylation (Rengaraj et al., 2011). Furthermore, H3K4me2 and transcription factor Jun cooperate to upregulate ncRNA (lncCPSET1) for chicken PGC development (Zhang et al., 2021a). These findings highlight the cooperative mechanism to regulate gene expression of chicken germ cells for their proper devleopment. Given the synergic effect of each epigenetic mechanism, it is necessary not only to elucidate how each component contributes to germ cell formation and but also how they influence each other to properly accomplish germ cell development.

Additionally, environment and genetic background can significantly influence on epigenetic regulation of chicken germ cells. Indeed, there are numerous reports about external factors such as temperature, parental feeding that can affect chicken embryo development. These studies mainly focused on relationship between epigenetic regulation of economic trait, such as muscle content, immunity (David et al., 2019; Willems et al., 2016), and further studies focusing on the effect of environment to the germ cell development remains to be explored. Dunislawska et al. compared epigenetic regulation of PGCs between Green-legged Patridgelike fowl (GP) and White Leghorn and found faster development of GP embryo compared to that of White Leghorn and suggested that epigenetic mechanism of chicken embryo development depends on genetic background, environment, and gender (Dunislawska et al., 2021). Therefore, when investigating epigenetic mechanism of chicken germ cells, external factors, including breeds, environmental factors, and genders, need to be considered.

CONCLUSIONS

The epigenetic programming of chicken germ cells is summarized in Figure 1. While many epigenetic programs share common features with mammals, numerous differences exist between avian and mammalian species. In mammals, DNA demethylation continues after PGCs reach the gonad, while DNA methylation is re-established in chicken and demethylated when the mitotic arrest is initiated in prespermatogonia. Histone markers cooperatively regulate gene expression essential for proper germ cell development, but histone modification profiles between mammals and chickens differ such that chicken germ cells show H3K9me3 rather than H3K27me3 to repress somatic gene expression. Furthermore, several chicken PGC-specific non-coding RNAs regulating germ cell gene expression were identified, and miRNAs that are not involved in the development of PGCs in mammals play a pivotal role in chicken PGC development. Advancements in novel technologies like high-throughput sequencing and the development of genome-edited chicken models substantially contributed to this field. In particular, genome editing has enabled the tracing of chicken PGCs with visible markers, providing extensive insights into epigenetic programming. In conclusion, our understanding of epigenetic programming in chicken germ cells has been clearer due to novel technology and proper genome-edited models, revealing distinct DNA methylation patterns, histone modification profiles, and ncRNA compared to mammals. It is expected to reach a finer resolution with the advancement of sequencing technology, the development of additional appropriate chicken models, and extensive comparative studies with mammals in the future.

Figure 1.

Schematic summary on epigenetic programming in chicken germ cells. The chicken zygote is formed through sperm fertilization with the oocyte in the hen's oviduct at Eyal-Giladi and Kochav (EGK). IX. Primordial germ cells (PGCs) are formed in the presence of germplasms containing germ cell-specific genes, such as CVH and DAZL, regulated by dynamic epigenetic changes. During PGC formation (EGK. IX-EGK. X), there is an increase in DNA methylation. H3K9me3 is increased to suppress somatic cell-related genes rather than H3K27me3. The histone acetylation of the pluripotency-related gene NANOG is downregulated by the REST/CoREST/HDAC complex, while histone acetylation of PGC-specific genes, such as DAZL, is upregulated by BMP4. Additionally, H3K4me2 is specifically upregulated in Blimp1 required for PGC formation. Somatic gene expression is downregulated by miRNAs, while piRNAs maintain the integrity of PGCs. Several long non-coding RNAs are induced to activate genes related to PGC formation, such as CVH and C-kit, and repress pluripotency-related genes such as NANOG. PGCs migrate to the germinal crescent and then enter blood vessels to localize at the germinal ridge. During migration (EGK.X-HH. 15-18), PGCs undergo global DNA demethylation to facilitate PGC-specific gene expression and subsequently undergo re-methylation upon arrival at the genital ridge. H3R2, H4R3, and H3K36 demethylation are downregulated, while H3K9, H3K4, H3K39, H4K20, and H3R17 methylation are upregulated. miR-456, and miR-181a* suppress somatic gene expression and gga-miR-302b and miR-17-5p regulate PGC proliferation. Gonadal PGCs initiate differentiation into sex-specific germ cells. Male germ cells undergo mitotic arrest from E14 to post-hatch while maintaining DNA demethylation, different from mammalian spermatogonia, which exhibits DNA methylation at the mitotic arrest stage. Female germ cells undergo DNA demethylation to initiate meiosis but experience DNA re-methylation at the meiotic arrest phase. During the epigenetic programming of DNA methylation, sex-specific miRNAs differentially downregulate DNA methyltransferase expression. EGK=Eyal-Giladi and Kochav, HH=Hamburger Hamilton.

DISCLOSURES

The authors declare no conflicts of interest.