Migration Of Primordial Germ Cells Research Articles

Examination of the effects of Nanos gene knockdown on fecundity and reproductive modes in the brine shrimp, Artemia parthenogenetica

Artemia is not only a crucial live food source in fish and crustacean larviculture but also an ideal experimental organism for biological studies. Female Artemia subjects release either diapause-encysted embryos (cysts) via oviparous reproduction or nauplii via ovoviviparous reproduction. As a maternal effector gene, nanos is involved in the differentiation and migration of primordial germ cells. In the present study, the open reading frame (ORF) of the nanos gene of Artemia parthenogenetica was screened and analyzed. Nanos cDNA contained a 933-bp ORF, encoding 310 amino acids with a conserved zinc-finger motif. The expression patterns of nanos in various tissues and during embryo development in Artemia were analyzed by qRT-PCR. The results showed that nanos was specifically expressed in the ovary, with the highest expression being observed during early ovary development. Moreover, nanos knockdown induced by dsRNA injection significantly decreased the gene and protein expression levels of nanos. In addition, knockdown of nanos resulted in an obvious shift from ovoviviparity to oviparity and a reduced number of offspring, corresponding to irregular oocyte shape, cell membrane damage, and cytoplasmic vacuolization. Our study suggests that nanos not only regulates the ovary development and reproductive performance of Artemia but also play a critical role in determining its reproductive mode. This study will improve our knowledge of the role of nanos in the reproductive process of Artemia and the molecular mechanisms underlying reproduction in this shrimp.

Observation of the Expression of Dazl and Vasa Suggests that Chinese Soft-shelled Turtle Use the Inductive Mode of Primordial Germ Cell Specification

The origin, migration and differentiation of primordial germ cells (PGCs) have always been an important issue in developmental biology. At present, studies on the specification of PGCs in amniotes mainly focus on birds and mammals. Turtles are reptiles known as "living fossils", and their sex determination is influenced by both genetic and environmental factors, making them an ideal model animal for the study of vertebrate sex differentiation. However, the study of PGCs specification in turtles is very limited. Here we reported using RNA probes for the germ cell-specific markers Dazl and Vasa to examine growing oocytes, early embryos and adult ovaries in the Chinese soft-shelled turtle. The results shows that the expression of Dazl and Vasa could not be detected in the growing oocytes and early embryos, and the specification of germline begins around E7, indicating that the Chinese soft-shelled turtle do not contain germ plasm and belongs to the inductive specification.

Human DND1-RRM2 forms a non-canonical domain-swapped dimer.

RNA Recognition Motifs (RRMs) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Human DND1 (dead end protein homolog1) also known as microRNA-mediated repression inhibitor 1 is an RNA binding protein containing two RRMs in tandem. It is essential for migration and viability of primordial germ cells during development, its complete loss causes lethality, and mutations in the gene are associated with perturbed cellular regulation and eventually cancer. Using x-ray crystallography we have determined 2.3 Å structure of the human DND1 RRM2 domain. The structure revealed an interesting non-canonical RRM fold. This is maintained by the formation of a 3D domain-swapped dimer between β1 and β4 strands across protomers. We have delineated the structural basis of the stable domain-swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our results indicate that monomer to dimer switch is governed by several determinants such as hinge loop residues, its length, hydrophobicity, concentration of protein, disulphide bond, and so forth. Several variations in the canonical βαββαβ topology have been observed so far. However, this is the first report of domain swapping in the RRM domain, which may increase the binding surface area and allow this protein to exhibit multifaceted role during post-transcriptional regulation. Our structural and dynamics studies substantiate major determinants and molecular basis for domain-swapped dimerization observed in the RRM domain that could allow varied roles for its RRMs.

Histogenesis of intracranial germ cell tumors: primordial germ cell vs. embryonic stem cell.

Intracranial germ cell tumor (iGCT) is a rare disorder and often occurs during childhood and adolescence. iGCTs are frequently localized in pineal region and hypothalamic-neurohypophyseal axis (HNA). In spite of well-established clinical and pathological entity, histogenesis of iGCTs remains unsettled. Current theories of histogenesis of iGCTs include germ cell theory (from primordial germ cells (PGCs) of aberrant migration) and stem cell theory (transformed embryonic stem (ES) cells). In order to comprehend the histogenesis, we revisit the origin, migration, and fate of the human PGCs, and their transformation processes to iGCT. In "germ cell theory," transformation of ectopic PGCs to iGCT is complex and involves multiple transcription factors. Germinoma is derived from ectopic PGCs and is considered a prototype of all GCTs. Non-germinomatous germ cell tumors (NGGCTs) develop from more differentiated counterparts of embryonic and extra-embryonic tissues. However, there is a distinct genomic/epigenomic landscape between germinoma and NGGCT. ES cells transformed from ectopic PGCs through molecular dysregulation or de-differentiation may become the source of iGCT. "Stem cell theory" is transformation of endogenous ES cells or primitive neural stem cell to iGCTs. It supports histological diversity of NGGCTs because of ES cell's pluripotency. However, neural stem cells are abundantly present along the subependymal zone; therefore, it does not explain why iGCTs almost exclusively occur in pineal and HNA locations. Also, the vast difference of methylation status between germinoma and NGGCT makes it difficult to theorize all iGCTs derive from the common cellular linage. Transformation of PGCs to ES cells is the most logical mechanism for histogenesis of iGCT. However, its detail remains an enigma and needs further investigations.

Conditions for transplantation of primordial germ cells in the yellowtail tetra, Astyanax altiparanae.

Biotechniques, including surrogate propagation derived from primordial germ cell (PGC) transplantation, are valuable tools for the reconstitution of endangered fish species. Although promising, there are no previous studies reporting such approaches using neotropical fish species. The aim of this study was to establish germline chimeras in neotropical fish by using the yellowtail tetra Astyanax altiparanae as a model species of the order Characiformes. Germline chimeras were obtained after transplantation of PGCs cultivated under different conditions: saline medium and supplemented with DMEM, amino acids, vitamins, glutamine, pyruvate, and fetal bovine serum, and subsequently transplanted into A. altiparanae triploids and triploid hybrids from the cross between A. altiparanae (♀) and A. fasciatus (♂). The results indicate ectopic migration in host embryos after transplantation of PGCs cultivated in saline medium. However, PGCs cultivated in supplemented medium migrated to the region of the gonadal ridge in 4.5% of triploid and 19.3% in triploid hybrid. In addition, the higher expression of dnd1, ddx4 and dazl genes was found in PGCs cultivated in supplemented culture medium. This indicates that the culture medium influences the maintenance and development of the cultivated cells. The expression levels of nanos and cxcr4b (related to the differentiation and migration of PGCs) were decreased in PGCs from the supplemented culture medium, supporting the results of ectopic migration. This is the first study to report the transplantation of PGCs to obtain germline chimera in neotropical species. The establishment of micromanipulation procedures in a model neotropical species will open new insights for the conservation of endangered species.

The anticancer effects of Metformin in the male germ tumor SEM-1 cell line are mediated by HMGA1.

Germ cell tumors (GCTs) are the most common type of cancer in young men. These tumors usually originate from the testis, but they can occasionally develop from extragonadal sites probably due to primordial germ cells (PGCs) migration errors. Cisplatin-based chemotherapy is usually effective for male GCTs, but the risk of toxicity is high and new therapeutic strategies are needed. Although Metformin (Met) has been widely studied as a potential cancer treatment over the past decades, there is limited evidence to support its use in treating male GCTs. Additionally, the mechanism by which it acts on tumor cells is still not entirely understood. SEM-1 cells, a newly established human cell line of extragonadal origin, were treated with Met. Cell viability was studied by MTT assay, while cell migration and invasion were studied by the wound healing assay and the transwell assay, respectively. The effect of Met on 3D spheroid formation was determined by seeding SEM-1 cells in appropriate cell suspension culture conditions, and cell cycle was characterized by flow cytometry. Factors involved in PGCs migration and GCT invasion, such as IGFBP1, IGF1R, MMP-11 and c-Kit, together with cyclin D1 (a key regulator of cell cycle progression), and the upstream factor, HMGA1, were determined by immunoblots. Treatment of SEM-1 cells with Met resulted in a potent and dose-dependent reduction of cell proliferation, as evidenced by decreased nuclear abundance of cyclin D1 and cell cycle arrest in G1 phase. Also, Met prevented the formation of 3D spheroids, and blocked cell migration and invasion by reducing the expression of IGFBP1, IGF1R and MMP-11. Both, IGFBP1 and MMP-11 are under control of HMGA1, a chromatin-associated protein that is involved in the regulation of important oncogenic, metabolic and embryological processes. Intriguingly, an early reduction in the nuclear abundance of HMGA1 occurred in SEM-1 cells treated with Met. Our results document the antiproliferative and antimigratory effects of Met in SEM-1 cells, providing new insights into the potential treatments for male GCTs. The anticancer properties of Met in SEM-1 cells are likely related to its ability to interfere with HMGA1 and downstream targets, including cyclin D1, the IGFs system, and MMP-11.

Tetraspanin Cd9b plays a role in fertility in zebrafish.

In mice, CD9 expression on the egg is required for efficient sperm-egg fusion and no effects on ovulation or male fertility are observed in CD9 null animals. Here we show that cd9b knockout zebrafish also appear to have fertility defects. In contrast to mice, fewer eggs were laid by cd9b knockout zebrafish pairs and, of the eggs laid, a lower percentage were fertilised. These effects could not be linked to primordial germ cell numbers or migration as these were not altered in the cd9b mutants. The decrease in egg numbers could be rescued by exchanging either cd9b knockout partner, male or female, for a wildtype partner. However, the fertilisation defect was only rescued by crossing a cd9b knockout female with a wildtype male. To exclude effects of mating behaviour we analysed clutch size and fertilisation using in vitro fertilisation techniques. Number of eggs and fertilisation rates were significantly reduced in the cd9b mutants suggesting the fertility defects are not solely due to courtship behaviours. Our results indicate that CD9 plays a more complex role in fish fertility than in mammals, with effects in both males and females.

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’.

S2896 A Rare Case of Necrotic Testicular Seminoma Presenting as a Large Retroperitoneal Mass Diagnosed With EUS and Laparotomy

Introduction: Retroperitoneal seminomas are a rare form of cancer that accounts for approximately 2% of all seminomas. Seminomas commonly present with fever, weight loss, abdominal or back pain, and a painless testicular nodule. The differential diagnosis for a retroperitoneal mass is broad, including abscess, lymphoma, sarcoma, metastatic lymph nodes, and extragonadal tumors. The pathogenesis of germ cell tumors is unclear, but the failure of primordial germ cell migration during embryonal development has been suggested. We report an atypical case of a large necrotic retroperitoneal mass diagnosed as a primary retroperitoneal seminoma after EUS and laparotomy. Case Description/Methods: A 33-year-old male with no medical history presented to the ED for new-onset flank pain and night sweats. On admission, the patient was febrile and tachycardic. The testicular exam and US of the testes were normal. CT abdomen and pelvis revealed a well-circumscribed, round mass posterior to the pancreatic head. EUS with FNA was performed, noting a round heterogenous 7.1cm x 6.2cm mass with well-defined borders and a necrotic center located posterior to the pancreatic head. An intact interface was seen between the mass and the duodenum, IVC, pancreas, and right kidney, suggesting a lack of invasion. The FNA yielded 0.5ml of blood-tinged, thick, and purulent fluid with cytology significant for malignant tumors with extensive necrosis. An exploratory laparotomy with excisional biopsy established the diagnosis of extragonadal germ cell tumor with extensive necrosis, consistent with seminoma. The patient completed four cycles of Etoposide followed by a right-sided orchiectomy for residual lesions found on the right testicle. A post-surgical PET scan did not demonstrate recurrence. Discussion: Primary retroperitoneal seminoma is a rare entity that must be considered when evaluating a retroperitoneal mass. GCTs are the most common neoplasm arising from the testes, with < 2% originating in other locations, termed primary extragonadal GCTs. Studies suggest the remission rate with chemotherapy and resection of residual masses was 92%, and the five-year overall survival rate was 88% for primary retroperitoneal tumors. Early identification of primary retroperitoneal seminomas may expand therapeutic possibilities and help improve outcomes for patients with a favorable prognosis. EUS-FNA was attempted in this case to obtain the diagnosis; however, an excisional biopsy was required to confirm the diagnosis.

Metzincin metalloproteases in PGC migration and gonadal sex conversion

Development of a functional gonad includes migration of primordial germ cells (PGCs), differentiations of somatic and germ cells, formation of primary follicles or spermatogenic cysts with somatic gonadal cells, development and maturation of gametes, and subsequent releasing of mature germ cells. These processes require extensive cellular and tissue remodeling, as well as broad alterations of the surrounding extracellular matrix (ECM). Metalloproteases, including MMPs (matrix metalloproteases), ADAMs (a disintegrin and metalloproteinases), and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), are suggested to have critical roles in the remodeling of the ECM during gonad development. However, few research articles and reviews are available on the functions and mechanisms of metalloproteases in remodeling gonadal ECM, gonadal development, or gonadal differentiation. Moreover, most studies focused on the roles of transcription and growth factors in early gonad development and primary sex determination, leaving a significant knowledge gap on how differentially expressed metalloproteases exert effects on the ECM, cell migration, development, and survival of germ cells during the development and differentiation of ovaries or testes. We will review gonad development with focus on the evidence of metalloprotease involvements, and with an emphasis on zebrafish as a model for studying gonadal sex differentiation and metalloprotease functions.

Potassium perchlorate effects on primordial germ cells of developing medaka larvae

Perchlorate is a chemical compound commonly used in military artillery and equipment. It has been detected in drinking water, air, soil, and breast milk. Exposure of humans to perchlorate can occur in the theater of war and areas adjacent to military training grounds. A high concentration of perchlorate has been found to affect reproduction in vertebrates, including fish. However, whether environmental concentrations of perchlorate can affect primordial germ cells (PGCs), the founders of sperm and eggs, is not clearly understood. In the present study, we examined the effects of 0, 10, 100, and 1000 μg/L potassium perchlorate exposure on the embryonic development of medaka and their PGCs. Perchlorate exposure delayed hatching time, reduced heartbeat, inhibited migration of PGCs, and increased developmental deformities in the larvae. The 10 and 20 mg/L concentrations of perchlorate were lethal to embryos, whereas vitamin C co-treatment (1 mg/L) completely blocked perchlorate-induced mortality. RNA-seq analysis of isolated PGCs showed a non-linear pattern in expression profiles of differentially altered genes. Significantly upregulated genes were found in PGCs from the 10 and 1000 μg/L groups, whereas the 100 μg/L groups showed the highest number of significantly downregulated genes. Gene ontology analysis predicted differentially expressed genes to be involved in proteolysis, metabolic processes, peptides activity, hydrolase activity, and hormone activity. Among the cellular components, extracellular, intracellular, sarcoplasmic, and 6-phosphofructokinase and membrane-bounded processes were affected. Ingenuity Pathway Analysis of PGC transcriptomes revealed thyroid hormone signaling to be affected by all concentrations of perchlorate. The present results suggested that perchlorate affected the development of medaka larvae and vitamin C was able to ameliorate perchlorate-induced embryo mortality. Additionally, perchlorate altered the global transcriptional network in PGCs in a non-linear fashion suggesting its potential effects on developing germ cells and fertility.

Primordial germ cell identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus.

Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.

Impact of germ cell ablation on the activation of the brain-pituitary-gonadal axis in precocious Atlantic salmon (Salmosalar L.) males.

The germ cells are essential for sexual reproduction by giving rise to the gametes, but the importance of germ cells for gonadal somatic functions varies among vertebrates. The RNA-binding dead end (Dnd) protein is necessary for the specification and migration of primordial germ cells to the future reproductive organs. Here, we ablated the gametes in Atlantic salmon males and females by microinjecting dnd antisense gapmer oligonucleotides at the zygotic stage. Precocious maturation was induced in above 50% of both germ cell-depleted and intact fertile males, but not in females, by exposure to an off-season photoperiod regime. Sterile and fertile males showed similar body growth, but maturing fish tended to be heavier than their immature counterparts. Pituitary fshβ messenger RNA levels strongly increased in maturing sterile and fertile males concomitant with the upregulated expression of Sertoli and Leydig cell markers. Plasma concentrations of 11-ketotestosterone and testosterone in maturing sterile males were significantly higher than the basal levels in immature fish, but lower than those in maturing fertile males. The study demonstrates that germ cells are not a prerequisite for the activation of the brain-pituitary-gonad axis and sex steroidogenesis in Atlantic salmon males, but may be important for the maintenance of gonadal somatic functions.

Migration of Primordial Germ Cells Is Regulated by miR-430 During Embryonic Development of Japanese Flounder

The origin and migration of primordial germ cells (PGCs) were regulated by miRNA during embryogenesis, but the underlying mechanism in marine teleost species was still unclear. Here, we studied the function of miR-430 in the development and migration of PGCs in Japanese flounder. By co-injecting miR-430 with eGFP-vasa 3’UTR mRNA, we found that miR-430 could cause the mis-localization of PGCs in flounder. Besides, the overexpression of miR-430 also inhibited the expression of three PGC-related genes (nanos3, piwil2 and tdrd7a), both target gene prediction and dual-luciferase reporter assay showed that miR-430 were directly binding on the canonical target site of their 3’UTRs. Significantly, the miR-430 target site of piwil2 3’UTR was necessary for PGC specific expression, where the binding seeds “GCACTTT” sequence mutation lead the distribution of eGFP-piwil2 mRNA turned from PGC-specific (wild-type) to ubiquitous (mutant). These findings will provide further evidence on the role of miR-430 in the regulation of the development and migration of PGCs.

Oct4 dependent chromatin activation is required for chicken primordial germ cell migration.

Primordial germ cells (PGCs) are the undifferentiated progenitors of the gametes. Unlike the poor maintenance of cultured mammalian PGCs, the avian PGCs can be expanded in vitro indefinitely while preserving pluripotency and germline competence. In mammals, the Oct4 is the master transcription factor that ensures the stemness of pluripotent cells such as PGCs, but the specific function of Oct4 in chicken PGCs remains unclear. As expected, the loss of Oct4 in chicken PGCs reduced the expression of key pluripotency factors and promoted the genes involved in endoderm and ectoderm differentiation. Furthermore, the global active chromatin was reduced as shown by the depletion of the H3K27ac upon Oct4 suppression. Interestingly, the de-activated chromatin caused the down-regulation of adjacent genes which are mostly known regulators of cell junction, chemotaxis and cell migration. Consequently, the Oct4-deficient PGCs show impaired cell migration and could not colonize the gonads when re-introduced into the bloodstream of the embryo. We propose that, in addition to maintaining pluripotency, the Oct4 mediated chromatin activation is dictating chicken PGC migration.

Primordial Germ Cell Development in the Poeciliid, Gambusia holbrooki, Reveals Shared Features Between Lecithotrophs and Matrotrophs.

Metazoans exhibit two modes of primordial germ cell (PGC) specification that are interspersed across taxa. However, the evolutionary link between the two modes and the reproductive strategies of lecithotrophy and matrotrophy is poorly understood. As a first step to understand this, the spatio-temporal expression of teleostean germ plasm markers was investigated in Gambusia holbrooki, a poecilid with shared lecitho- and matrotrophy. A group of germ plasm components was detected in the ovum suggesting maternal inheritance mode of PGC specification. However, the strictly zygotic activation of dnd-β and nanos1 occurred relatively early, reminiscent of models with induction mode (e.g., mice). The PGC clustering, migration and colonisation patterns of G. holbrooki resembled those of zebrafish, medaka and mice at blastula, gastrula and somitogenesis, respectively—recapitulating features of advancing evolutionary nodes with progressive developmental stages. Moreover, the expression domains of PGC markers in G. holbrooki were either specific to teleost (vasa expression in developing PGCs), murine models (dnd spliced variants) or shared between the two taxa (germline and somatic expression of piwi and nanos1). Collectively, the results suggest that the reproductive developmental adaptations may reflect a transition from lecithotrophy to matrotrophy.

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Transcriptional dynamics of the circulating chicken primordial germ cells revealing key genes in cell adhesion and proliferation prior to gonad colonization.

Primordial germ cells (PGCs), precursors to sperms and oocytes, are responsible for the transfer of genetic information to the next generation. The PGCs arise far away from the developing gonad and thus have to migrate across the embryo to reach their site of function. The migration of PGCs from extraembryonic regions to the genital ridges is accomplished through distinct routes among different species. In particular, the birds PGCs utilized the developing circulation system to travel long distance before settling within the gonad. This study screened the transcriptome profile of chicken PGCs isolated from the bloodstream and the genital ridges to identify the cell intrinsic signals that could guide the unique migration path through circulation. We found cell adhesion and extracellular matrix (ECM) associated pathways were highly enriched in the PGCs from blood but not gonads. The platelet-derived growth factor receptors (PDGFRA and PDGFRB) were downregulated during gonad colonization and knockdown of either PDGFRA or PDGFRB inhibit the proliferation of blood PGCs. Furthermore, the migration of blood PGCs was impaired by the suppression of PDGFRA but not PDGFRB. Hence, the chicken PGCs show dynamic transcriptional remodeling during the blood-to-gonad migration and colonization. The free-floating PGCs in the circulation already express genes associated with cell-cell and cell-ECM interactions and therefore prepare for gonadal colonization.

Finer resolution analysis of transcriptional programming during the active migration of chicken primordial germ cells

Primordial germ cells (PGCs) in chickens polarize and move passively toward the anterior region by the morphogenetic movement of the embryo. Further migration of PGCs towards the genital ridge via the germinal crescent region and blood vessels occurs actively through the chemoattractive signals. The mechanisms of initiation of PGCs migration, lodging the PGCs in the vascular system, and colonization of PGCs in the gonads are well-studied. However, transcriptome sequencing-based cues directing the migration of the PGCs towards gonads, some of the relevant molecules, biological processes, and transcription factors (TFs) are less studied in chickens. The current study comprehensively interprets the transcriptional programming of PGCs during their active migration (E2.5 to E8). Current results revealed several vital understandings, including a set of genes that upregulated male-specifically (XPA, GNG10, RPL17, RPS23, and NDUFS4) or female-specifically (HINTW, NIPBL, TERAL2, ATP5F1AW, and SMAD2W) in migrating PGCs, and transcriptionally distinct PGCs, particularly in the gonadal environment. We identified DNA methylation and histone modification-associated genes that are novel in chicken PGCs and show a time-dependent enrichment in migrating PGCs. We further identified a large number of differentially expressed genes (DEGs, including TFs) in blood PGCs (at E2.5) compared to gonadal PGCs (at E8) in both sexes; however, this difference was greater in males. We also revealed the enriched biological processes and signaling pathways of significant DEGs identified commonly, male-specifically, or female-specifically between the PGCs isolated at E2.5, E6, and E8. Collectively, these analyses provide molecular insights into chicken PGCs during their active migration phase.

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.