The dual roles of geminin during trophoblast proliferation and differentiation

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.     

Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation

Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.     

Aberrant imprinting in mouse trophoblast stem cells established from somatic cell nuclear transfer-derived embryos

Although phenotypic abnormalities frequently appear in the placenta following somatic cell nuclear transfer (SCNT), mouse trophoblast stem cells (TSCs) established from SCNT embryos reportedly show no distinct abnormalities compared with those derived from normal fertilization. In this study, we reexamined SCNT–TSCs to identify their imprinting statuses. Placenta-specific maternally imprinted genes (Gab1, Slc38a4, and Sfmbt2) consistently showed biallelic expression in SCNT–TSCs, suggesting their loss of imprinting (LOI). The LOI of Gab1 was associated with decreased DNA methylation, and that of Sfmbt2 was associated with decreased DNA methylation and histone H3K27 trimethylation. The maternal allele of the intergenic differentially methylated region (IG–DMR) was aberrantly hypermethylated following SCNT, even though this region was prone to demethylation in TSCs when established in a serum-free chemically defined medium. These findings indicate that the development of cloned embryos is associated with imprinting abnormalities specifically in the trophoblast lineage from its initial stage, which may affect subsequent placental development.     

Interdependent fibroblast growth factor and activin A signaling promotes the expression of endodermal genes in differentiating mouse embryonic stem cells expressing Src Homology 2-domain inactive Shb

Abstract The mechanisms controlling endodermal development during stem cell differentiation have been only partly elucidated, although previous studies have suggested the participation of fibroblast growth factor (FGF) and activin A in these processes. Shb is a Src homology 2 (SH2) domain-containing adapter protein that has been implicated in FGF receptor 1 (FGFR1) signaling. To study the putative crosstalk between activin A and Shb-dependent FGF signaling in the differentiation of endoderm from embryonic stem (ES) cells, embryoid bodies (EBs) derived from mouse ES cells overexpressing wild-type Shb or Shb with a mutated SH2 domain (R522K-Shb) were cultured in the presence of activin A. We show that expression of R522K-Shb results in up-regulation of FGFR1 and FGF2 in EBs. Addition of activin A to the cultures enhances the expression of endodermal genes primarily in EBs expressing mutant Shb. Inhibition of FGF signaling by the addition of the FGFR1 inhibitor SU5402 completely counteracts the synergistic effects of R522K-Shb and activin A. In conclusion, the present results suggest that expression of R522K-Shb enhances certain signaling pathways downstream of FGF and that an interplay between FGF and activin A participates in ES cell differentiation to endoderm.     

Neuronal and mesodermal differentiation of P19 embryonal carcinoma cells is characterized by expression of specific marker genes and modulated by activin and fibroblast growth factors

We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx-1 , goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose-dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA-responsive genes, including the three RA receptor genes (RARα, RARβ and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10⁻⁹mol/L RA or a factor produced by visceral endoderm-like cells (END-2 factor). The FGF effect is dose-independent. These results suggest an important function for RA and the END-2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development.     

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.     

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17⁺ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17⁺ cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP⁺ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17⁺ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.     

miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b

Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP-4-induced osteoblastic differentiation of bone marrow-derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR-210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR-210. Furthermore, inhibition of transforming growth factor-β (TGF-β)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR-210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF-β/activin signaling pathway through inhibition of AcvR1b.     

Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals

Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/β-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration.     

Activins and inhibins: Novel regulators of thymocyte development

Activins and inhibins are members of the transforming growth factor-β superfamily that act on different cell types and regulate a broad range of cellular processes including proliferation, differentiation, and apoptosis. Here, we provide the first evidence that activins and inhibins regulate specific checkpoints during thymocyte development. We demonstrate that both activin A and inhibin A promote the DN3-DN4 transition in vitro, although they differentially control the transition to the DP stage. Whereas activin A induces the accumulation of a CD8⁺CD24^hiTCRβ^lo intermediate subpopulation, inhibin A promotes the differentiation of DN4 to DP. In addition, both activin A and inhibin A appear to promote CD8⁺SP differentiation. Moreover, inhibin α null mice have delayed in vitro T cell development, showing both a decrease in the DN-DP transition and reduced thymocyte numbers, further supporting a role for inhibins in the control of developmental signals taking place during T cell differentiation in vivo.