Proliferation Of Primordial Germ Cells Research Articles

Research progress of nanog gene in fish.

Nanog is a crucial regulatory factor in maintaining the self-renewal and pluripotency of embryonic stem cells. It is involved in various biological processes, such as early embryonic development, cell reprogramming, cell cycle regulation, the proliferation and migration of primordial germ cells. While research on this gene has primarily focused on mammals, there has been a growing interest in studying nanog in fish. However, there is a notable lack of comprehensive reviews regarding this gene in fish, which is essential for guiding future research. This review aims to provide a thorough summary of the gene's structure, expression patterns, functions and regulatory mechanisms in fish. The findings suggest that nanog probably has both conserved and divergent functions in regulating cell pluripotency, early embryonic development, and germ cell development in teleosts compared to other species, including mammals. These insights lay the foundation for future research and applications of the nanog gene, providing a new perspective for understanding the evolution and conserved charactristics of teleost nanog.

Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants.

Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. We previously found that interaction with different somatic niches regulates PGC proliferation along the migration route. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5; these differences include migration-associated actin polymerization machinery and epigenetic reprogramming-associated genes. We furthermore identified changes in signaling with various somatic niches, notably strengthened interactions with hindgut epithelium via non-canonical WNT (ncWNT) in posterior PGCs compared to anterior. Reanalysis of a previously published dataset suggests that ncWNT signaling from the hindgut epithelium to early migratory PGCs is conserved in humans. Trajectory inference methods identified putative differentiation trajectories linking cell states across timepoints and from posterior to anterior in our mouse dataset. At E9.5, we mainly observed differences in cell adhesion and actin cytoskeletal dynamics between E9.5 posterior and anterior migrants. At E10.5, we observed divergent gene expression patterns between putative differentiation trajectories from posterior to anterior including Nodal signaling response genes Lefty1, Lefty2, and Pycr2 and reprogramming factors Dnmt1, Prc1, and Tet1. At E10.5, we experimentally validated anterior migrant-specific Lefty1/2 upregulation via whole-mount immunofluorescence staining for LEFTY1/2 proteins, suggesting that elevated autocrine Nodal signaling accompanies the late stages of PGC migration. Together, this positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency, and epigenetic reprogramming.

Adamts9 is required for the development of primary ovarian follicles and maintenance of female sex in zebrafish.

Previous studies have suggested that adamts9 (a disintegrin and metalloprotease with thrombospondin type-1 motifs, member 9), an extracellular matrix (ECM) metalloprotease, participates in primordial germ cell (PGC) migration and is necessary for female fertility. In this study, we found that adamts9 knockout (KO) led to reduced body size, and female-to-male sex conversion in late juvenile or adult zebrafish; however, primary sex determination was not affected in early juveniles of adamts9 KO. Overfeeding and lowering the rearing density rescued growth defects in female adamts9 KO fish but did not rescue defects in ovarian development in adamts9 KO. Delayed PGC proliferation, significantly reduced number and size of Stage IB follicles (equivalent to primary follicles) in early juveniles of adamts9 KO, and arrested development at Stage IB follicles in mid- or late-juveniles of adamts9 KO are likely causes of female infertility and sex conversion. Via RNAseq, we found significant enrichment of differentially expressed genes involved in ECM organization during sexual maturation in ovaries of wildtype fish; and significant dysregulation of these genes in adamts9 KO ovaries. RNAseq analysis also showed enrichment of inflammatory transcriptomic signatures in adult ovaries of these adamts9 KO. Taken together, our results indicate that adamts9 is critical for development of primary ovarian follicles and maintenance of female sex, and loss of adamts9 leads to defects in ovarian follicle development, female infertility, and sex conversion in late juveniles and mature adults. These results show that the ECM and extracellular metalloproteases play major roles in maintaining ovarian follicle development in zebrafish.

Role of PI3K/AKT signaling pathway involved in self-renewing and maintaining biological properties of chicken primordial germ cells

Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population.

Comparative study of PGCs cultivation systems HiS and FAcs: a transcriptomic and cellular biology perspective

In chicken, primordial germ cells (PGC) are crucial for the preservation and manipulation of genetic resources in poultry production. The HiS and FAcs culture systems are two important methods for the in vitro cultivation of chicken PGCs. The purpose of this study was to compare and analyze the two cultivation systems for PGCs (His and FAcs culture systems) to assess their efficacy and applicability in supporting PGC growth, maintaining PGC characteristics, and lineage transmission ability. The study found that both HiS and FAcs culture systems could maintain the basic biological characteristics of chicken PGCs, including the simultaneous expression of pluripotency and reproductive marker genes, as well as the presence of abundant glycogen granules. Subsequently, we identified 2,145 differentially expressed genes (DEG) through RNA sequencing. GO and KEGG analysis revealed a large number of DEGs enriched in the cell adhesion and calcium ion binding pathways, and the analysis found that these genes maintained a higher level in HiS-PGCs. Further personalized analysis found that the regulatory genes for maintaining PGC pluripotency were highly expressed in HiS-PGCs, while germ cell-related genes showed similar expression in both systems. Additionally, through RNA sequencing data and cell proliferation ability, it was found that PGCs in the FAcs system had a higher proliferation rate and a faster cell cycle. Finally, it was discovered that the expression of cell migration-related genes was maintained at a higher level in HiS-PGCs, but the migration efficiency of HiS-PGCs did not show a significant difference compared to FAcs-PGCs. These results suggest that both HiS and FAcs culture systems can maintain the proliferation and basic characteristics of chicken PGCs, but differences exist in cell proliferation, pluripotency regulation, and cell adhesion. These findings provide new information for optimizing PGC cultivation systems and are important for the preservation and genetic improvement of chicken PGCs.

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development

BackgroundExploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases.MethodsDiaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development.ResultsBased on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3β-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated.ConclusionsOverall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

Depletion of placental brain-derived neurotrophic factor (BDNF) is attributed to premature ovarian insufficiency (POI) in mice offspring

IntroductionPremature ovarian insufficiency (POI) is one of the causes of female infertility. Unexplained POI is increasingly affecting women in their reproductive years. However, the etiology of POI is diverse and remains elusive. We and others have shown that brain-derived neurotrophic factor (BDNF) plays an important role in adult ovarian function. Here, we report on a novel role of BDNF in the Developmental Origins of POI.MethodsPlacental BDNF knockout mice were created using CRISPR/CAS9. Homozygous knockout (cKO(HO)) mice didn’t survive, while heterozygous knockout (cKO(HE)) mice did. BDNF reduction in cKO(HE) mice was confirmed via immunohistochemistry and Western blots. Ovaries were collected from cKO(HE) mice at various ages, analyzing ovarian metrics, FSH expression, and litter sizes. In one-month-old mice, oocyte numbers were assessed using super-ovulation, and oocyte gene expression was analyzed with smart RNAseq. Ovaries of P7 mice were studied with SEM, and gene expression was confirmed with RT-qPCR. Alkaline phosphatase staining at E11.5 and immunofluorescence for cyclinD1 assessed germ cell number and cell proliferation.ResultscKO(HE) mice had decreased ovarian function and litter size in adulthood. They were insensitive to ovulation induction drugs manifested by lower oocyte release after superovulation in one-month-old cKO(HE) mice. The transcriptome and SEM results indicate that mitochondria-mediated cell death or aging might occur in cKO(HE) ovaries. Decreased placental BDNF led to diminished primordial germ cell proliferation at E11.5 and ovarian reserve which may underlie POI in adulthood.ConclusionThe current results showed decreased placental BDNF diminished primordial germ cell proliferation in female fetuses during pregnancy and POI in adulthood. Our findings can provide insights into understanding the underlying mechanisms of POI.

P38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells.

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Non-invasive dsRNA delivery via feeding for effective gene silencing in teleost fish: A novel approach in the study of gene function analysis

RNA interference (RNAi) is a powerful technique for gene silencing, involving post-transcriptional regulation of gene expression. Successful application of RNAi has been demonstrated in various organisms like nematodes, insects, and oysters by ingesting double-stranded RNA (dsRNA)-expressing bacteria. In this study, we attempted a non-invasive feeding method in the marine economy teleost fish, Sebastes schlegelii. To validate the feasibility of this approach in S. schlegelii, we utilized rotifers, Artemia nauplii, and commercial pellet feed as vectors to deliver Escherichia coli strain HT115 engineered to express dsRNA, targeting dead end (dnd) gene, known to be important for primordial germ cells (PGCs) migration and proliferation in fish. Our experimental results consistently showed that knockdown of the dnd gene effectively inhibited the development of PGCs in S. schlegelii, resulting in gonadal dysplasia and even sex reversal. This study marks a significant advancement in understanding gene function in teleost fish, laying the groundwork for future large-scale research in this field.

Novel compound heterozygous variants in FANCI cause premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common reproductive aging disorder due to a dramatic decline of ovarian function before 40 years of age. Accumulating evidence reveals that genetic defects, particularly those related to DNA damage response, are a crucial contributing factor to POI. We have demonstrated that the functional Fanconi anemia (FA) pathway maintains the rapid proliferation of primordial germ cells to establish a sufficient reproductive reserve by counteracting replication stress, but the clinical implications of this function in human ovarian function remain to be established. Here, we screened the FANCI gene, which encodes a key component for FA pathway activation, in our whole-exome sequencing database of 1030 patients with idiopathic POI, and identified two pairs of novel compound heterozygous variants, c.[97C > T];[1865C > T] and c.[158-2A > G];[c.959A > G], in two POI patients, respectively. The missense variants did not alter FANCI protein expression and nuclear localization, apart from the variant c.158-2A > G causing abnormal splicing and leading to a truncated mutant p.(S54Pfs*5). Furthermore, the four variants all diminished FANCD2 ubiquitination levels and increased DNA damage under replication stress, suggesting that the FANCI variants impaired FA pathway activation and replication stress response. This study first links replication stress response defects with the pathogenesis of human POI, providing a new insight into the essential roles of the FA genes in ovarian function.

Ten-eleven translocation 1 mediating DNA demethylation regulates the proliferation of chicken primordial germ cells through the activation of Wnt4/β-catenin signaling pathway.

The objective of this study was to investigate the regulation relationship of Teneleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/β-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/β-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxymethylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/β-catenin signaling pathway. These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/β-catenin signaling pathway.

Effects of Insulin on Proliferation, Apoptosis, and Ferroptosis in Primordial Germ Cells via PI3K-AKT-mTOR Signaling Pathway.

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 μg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 μg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 μg/mL and 100 μg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 μg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures.

First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica)

Japanese quail (JQ, Coturnix japonica) is a farmed animal with a high economic value and has been used extensively as an avian model for research. Germline chimera production based on cryopreserved primordial germ cells (PGCs) is possible for conservation management of quail breeds as successful isolation has been reported of PGCs from their blood and gonads. However, the repeatable cultivation protocol has not been elucidated yet, which has hindered technological development. The current study characterized cultivation of pregonadal PGCs isolated from embryonic parts; embryonic blood (cPGCs), whole embryonic tissues (tPGCs), parts of tail buds (tbPGCs), and a mixture of blood and tail bud tissues (ctbPGCs). The results showed that the cultivation system required the presence of specific embryonic cells to act as a feeder for JQ-PGCs and that such a system facilitated more successful cultivation, as shown by the percentages of isolation and cultivation in tbPGCs (100%, 100%, respectively), tPGCs (60%, 55%, respectively), and ctbPGCs (60%, 30%, respectively), but not in cPGCs (0%) cultured on a mitomycin-treated JQ feeder cell-line. Once the co-culture system had been established, the PGCs could be propagated for at least 5 months. These PGCs expressed germ cell-specific markers (DAZL and CVH) and could colonize embryonic gonads. Conclusively, the isolation of pregonadal PGCs and their long-term cultivation in vitro requires a unique embryonic cell, giant cell feeder, that is indispensable for the proliferation of PGCs. Characterization of cell signaling sustaining a mutual interaction between the PGCs and the specific feeder cells will elucidate a superior environment for in vitro cultivation, as well as support the minimal transfer of used xenobiotics in chimera production.

FAAP100 is required for the resolution of transcription-replication conflicts in primordial germ cells

BackgroundThe maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored.ResultsWe find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice.ConclusionsOur findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability.

Niche cells regulate primordial germ cell quiescence in response to basement membrane signaling.

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates Caenorhabditis elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating that laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germline growth, suggesting that BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors.

Fance deficiency inhibits primordial germ cell proliferation associated with transcription–replication conflicts accumulate and DNA repair defects

Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance−/− mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription–replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance−/− PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance−/− PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance−/− PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance−/− PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.

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.

Coordination of canonical and noncanonical Hedgehog signalling pathways mediated by WDR11 during primordial germ cell development

WDR11, a gene associated with Kallmann syndrome, is important in reproductive system development but molecular understanding of its action remains incomplete. We previously reported that Wdr11-deficient embryos exhibit defective ciliogenesis and developmental defects associated with Hedgehog (HH) signalling. Here we demonstrate that WDR11 is required for primordial germ cell (PGC) development, regulating canonical and noncanonical HH signalling in parallel. Loss of WDR11 disrupts PGC motility and proliferation driven by the cilia-independent, PTCH2/GAS1-dependent noncanonical HH pathway. WDR11 modulates the growth of somatic cells surrounding PGCs by regulating the cilia-dependent, PTCH1/BOC-dependent canonical HH pathway. We reveal that PTCH1/BOC or PTCH2/GAS1 receptor context dictates SMO localisation inside or outside of cilia, respectively, and loss of WDR11 affects the signalling responses of SMO in both situations. We show that GAS1 is induced by PTCH2-specific HH signalling, which is lost in the absence of WDR11. We also provide evidence supporting a role for WDR11 in ciliogenesis through regulation of anterograde intraflagellar transport potentially via its interaction with IFT20. Since WDR11 is a target of noncanonical SMO signalling, WDR11 represents a novel mechanism by which noncanonical and canonical HH signals communicate and cooperate.

LIN28 is essential for the maintenance of chicken primordial germ cells

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

AbstractIn many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF‐β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder‐free substrates. The results showed that TGF‐β signaling agonists (IDE1 and Activin‐A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF‐β, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency‐associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.