Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

Comparison of Four Protocols for In Vitro Differentiation of Human Embryonic Stem Cells into Trophoblast Lineages by BMP4 and Dual Inhibition of Activin/Nodal and FGF2 Signaling.

Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments.

A Comparative Study of Endoderm Differentiation Between Activin A and Small Molecules.

Small molecules such as ROCK inhibitors (Fasudil) and inducer of definitive endoderm 1 (IDE1) can promote differentiation of definitive endoderm, but their effects remain controversial. Therefore, we attempted to verify the effect of these small molecules on promoting definitive endoderm differentiation and found that Fasudil or IDE1 alone could not achieve a similar effect as activin A. On the contrary, CHIR99021 could efficiently promote definitive endoderm differentiation. Nearly 43.4% of experimental cells were SRY-box transcription factor 17 (SOX17)-positive under the synergistic effect of IDE1 and CHIR99021, but its ability to differentiate towards definitive endoderm was still insufficient. Transcriptional analysis and comparison of IDE1 and CHIR99021 synergistic groups (IC) and activin A and CHIR99021 synergistic groups (AC) showed significantly down-regulated definitive endoderm markers in the IC group compared with those in the AC group and the differences between the two groups were mainly due to bone morphogenetic proteins (BMP4) and fibroblast growth factor 17 (FGF17). Further single-cell transcriptome analysis revealed lower expression of BMP4 in SOX17-positive populations, while mothers against decapentaplegic homolog (SMAD) protein translation signal and FGF17 in the AC group were higher than that in the IC group. Western blot analysis showed a significant difference in levels of p-SMAD2/3 between AC and IC groups, which suggests that regulating p-SMAD2/3 may provide a reference to improve the differentiation of definitive endoderm.

Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

Transcriptional Profiling of TGF-ß Superfamily Members in Lumbar DRGs of Rats Following Sciatic Nerve Axotomy and Activin C Inhibits Neuropathic Pain.

BACKGROUND: Neuroinflammation and cytokines play critical roles in neuropathic pain and axon degeneration/regeneration. Cytokines of transforming growth factor-ß superfamily have implications in pain and injured nerve repair processing. However, the transcriptional profiles of the transforming growth factor-ß superfamily members in dorsal root ganglia under neuropathic pain and axon degeneration/regeneration conditions remain elusive. OBJECTIVE: We aimed to plot the transcriptional profiles of transforming growth factor-ß superfamily components in lumbar dorsal root ganglia of sciatic nerve-axotomized rats and to further verify the profiles by testing the analgesic effect of activin C, a representative cytokine, on neuropathic pain. METHODS: Adult male rats were axotomized in sciatic nerves, and lumbar dorsal root ganglia were isolated for total RNA extraction or section. A custom microarray was developed and employed to plot the gene expression profiles of transforming growth factor-ß superfamily components. Realtime RT-PCR was used to confirm changes in the expression of activin/inhibin family genes, and then in situ hybridization was performed to determine the cellular locations of inhibin α, activin ßC, BMP-5 and GDF-9 mRNAs. The rat spared nerve injury model was performed, and a pain test was employed to determine the effect of activin C on neuropathic pain. RESULTS: The expression of transforming growth factor-ß superfamily cytokines and their signaling, including some receptors and signaling adaptors, were robustly upregulated. Activin ßC subunit mRNAs were expressed in the small-diameter dorsal root ganglion neurons and upregulated after axotomy. Single intrathecal injection of activin C inhibited neuropathic pain in spared nerve injury model. CONCLUSION: This is the first report to investigate the transcriptional profiles of members of transforming growth factor-ß superfamily in axotomized dorsal root ganglia. The distinct cytokine profiles observed here might provide clues toward further study of the role of transforming growth factor-ß superfamily in the pathogenesis of neuropathic pain and axon degeneration/regeneration after peripheral nerve injury.

Activin A rescues preterm brain injury through a novel Noggin/BMP4/Id2 signaling pathway.

Activin A (Act A) has been reported to promote oligodendrocyte progenitor cell (OPC) differentiation in vitro and improve neurological outcomes in adult mice. However, the roles and mechanisms of action of Act A in preterm brain injury are unknown. In the present study, P5 rats were subjected to hypoxia­ischemia to establish a neonatal white matter injury (WMI) model and Act A was injected via the lateral ventricle. Pathological characteristics, OPC differentiation, myelination, and neurological performance were analyzed. Further, the involvement of the Noggin/BMP4/Id2 signaling pathway in the roles of Act A in WMI was explored. Act A attenuated pathological damage, promoted OPC differentiation, enhanced myelin sheath and myelinated axon formation, and improved neurological performance of WMI rats. Moreover, Act A enhanced noggin expression, which, in turn, inhibited the expression of bone morphogenetic protein 4 (BMP4) and inhibitor of DNA binding 2 (Id2). Furthermore, upregulation of Id2 completely abolished the rescue effects of Act A in WMI rats. In conclusion, the present findings suggested that Act A rescues preterm brain injury via targeting a novel Noggin/BMP4/Id2 signaling pathway.

Activin A Modulates Betaglycan Shedding via the ALK4-SMAD3-Dependent Pathway in Endometriotic Cells.

The TGF-ß superfamily members, activins and inhibins, are mainly involved in cell proliferation, cell survival, invasion, immune surveillance, and lesion growth in endometriosis. Herein, we investigated the modulation of the TGF-ß type III receptor (betaglycan or BG) by activin A and inhibin A in endometriosis in vitro. Often, BG undergoes ectodomain shedding releasing soluble BG (sBG) which frequently antagonizes TGF-ß signaling. The effects of activin A on BG shedding and signaling pathways involved were evaluated with the inhibitors LY364947 and SIS3, siRNA knockdown in human endometrial cells (12Z, THESC, Ishikawa, and primary stromal cells) and were quantified with BG ELISAs. The effects of activin A and inhibin A on the secretion of MMP2 and MMP3 were analyzed using ELISAs. The effects of activin A on the BG expression were analyzed using RT-qPCR and western blot. The CCK-8 and BrdU assays were used to evaluate the effects of the recombinant BG on cell viability and proliferation. Activin A stimulation resulted in a significant time- and dose-dependent reduction in BG shedding, which was found to be activin A/ALK-4/SMAD3- but not SMAD2-dependent. Activin A increased the BG mRNA expression but had no effect on the protein expression. Likewise, inhibin A was found to block BG shedding. Activin A, but not inhibin A, significantly enhanced the secretion of MMP2 and MMP3. The recombinant BG had no effect on the viability and proliferation of endometriotic cells. Together, these observations support a novel role for activin A with BG in modulating the TGF-ß superfamily ligands in endometrial cells in vitro.

LncRNA-Smad7 mediates cross-talk between Nodal/TGF-ß and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells.

Transforming growth factor ß (TGF-ß) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-ß and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-ß and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

Somatic cell fate maintenance in mouse fetal testes via autocrine/paracrine action of AMH and activin B.

Fate determination and maintenance of fetal testes in most mammals occur cell autonomously as a result of the action of key transcription factors in Sertoli cells. However, the cases of freemartin, where an XX twin develops testis structures under the influence of an XY twin, imply that hormonal factor(s) from the XY embryo contribute to sex reversal of the XX twin. Here we show that in mouse XY embryos, Sertoli cell-derived anti-Mullerian hormone (AMH) and activin B together maintain Sertoli cell identity. Sertoli cells in the gonadal poles of XY embryos lacking both AMH and activin B transdifferentiate into their female counterpart granulosa cells, leading to ovotestis formation. The ovotestes remain to adulthood and produce both sperm and oocytes, although there are few of the former and the latter fail to mature. Finally, the ability of XY mice to masculinize ovaries is lost in the absence of these two factors. These results provide insight into fate maintenance of fetal testes through the action of putative freemartin factors.

Mechanisms underlying WNT-mediated priming of human embryonic stem cells.

Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context-dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs). Our previous work demonstrated that during early stages of hESC differentiation, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening but, instead, acts by inducing the expression of the SMAD2 co-factor EOMES. Expression of EOMES is sufficient to replace WNT upstream of activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

Activin B-activated Cdc42 signaling plays a key role in regulating adipose-derived mesenchymal stem cells-mediated skin wound healing.

BACKGROUND: In our previous study, activin B in combination with ADSCs enhances skin wound healing. However, the underlying molecular mechanisms are not well studied. Cdc42 is recognized to play a critical role in the regulation of stem cells. METHODS: Pull-down assay was performed to investigate the activity of Cdc42. The dominant-negative mutant of Cdc42 (Cdc42N17) was used to explore the role of Cdc42 in activin B-induced ADSCs migration, proliferation, and secretion in vitro. Cdc42N17-transfected ADSCs were injected into a full-thickness excisional wound model to explore their efficiency in wound healing in vivo. The wound healing efficacy was evaluated by the wound closure rates and histological examination. The neovascularization and wound contraction were detected by immunohistochemistry staining of CD31 and α-SMA. Finally, the underlying mechanisms were explored by RNA sequencing. RESULTS: Cdc42N17 inhibited ADSCs migration, proliferation, and secretion induced by activin B. Furthermore, Cdc42N17-transfected ADSCs inhibited the wound closure rate and suppressed the expression of CD31 and α-SMA induced by activin B in vivo. The RNA sequencing showed that the differentially expressed genes in Cdc42N17-transfected ADSCs versus ADSCs were associated with cell migration, proliferation, and adhesion. Further study revealed that the Cdc42-Erk-Srf pathway was required for activin B-induced proliferation in ADSCs. CONCLUSIONS: Our study indicates that Cdc42 plays a crucial role in ADSCs-mediated skin wound healing induced by activin B.

Activin A specifically suppresses the expression of annexin A5 mRNA and augments gonadotropin-releasing hormone stimulation of A1 expression in LßT2 gonadotrope cells.

Recently, we reported that gonadotropin-releasing hormone (GnRH) stimulates annexin A1 (Anxa1) and A5 (Anxa5) mRNA expression through the GnRH-receptor-mitogen-activated protein kinase cascade in LßT2 cells. As LßT2 cells respond to activin, we examined the effect of activin A on Anxa1 and a5 expression in LßT2 cells. Activin A (0.4 and 4 ng/mL) treatment decreased Anxa5 mRNA levels in a dose-dependent manner, but did not affect Anxa1 mRNA levels at concentrations up to 40 ng/mL. After activin A treatment (4 ng/mL), Anxa5 mRNA levels significantly decreased at 6 h, gradually declined until 24 h, and remained low until 48 h, whereas Anxa1 mRNA levels did not significantly change following treatment. Pretreatment with activin A for 24 h increased GnRH agonist (GnRHa)-induced Anxa1 increase by approximately 7-fold compared with GnRHa stimulation alone, but Anxa5 was not affected. As previously reported, these activin A treatments increased gonadotropin ß subunit and GnRH receptor mRNA levels and slightly decreased common α-glycoprotein subunit mRNA levels. Furthermore, we examined the effect of activin A on Nr4a3, which is repressed by ANXA5 and which reduces Fshb expression, and found that activin A augmented Nr4a3 expression and slightly decreased the GnRHa-induced increase in Nr4a3. These results suggest that in gonadotrope cells, the mechanism regulating Anxa1 and a5 expression is differentially coupled with activin A signal transduction. Activin A suppresses Anxa5 expression under increased Nr4a3 expression, whereas activin A and GnRH synergistically stimulate Anxa1 expression. These GnRH-inducible annexins may have different specific functions in gonadotropes.

CircNMD3 relieves endothelial cell injury induced by oxidatively modified low-density lipoprotein through regulating miR-498/ BMP and activin membrane-bound inhibitor (BAMBI) axis.

Atherosclerosis (AS) is one of the most common vascular diseases. The endothelial injury theory indicates that atherosclerotic plaque is the result of endothelial cell injury. Recent studies have revealed that circRNAs are abnormally expressed in AS cell models, which are implicated in the regulation of various cell behaviors. In this study, we showed the downregulation of circNMD3 in AS, and studied its role in the model of endothelial cell injury by proliferation and apoptosis assay, caspase 3 activity assay, and ELISA. We also identified and validated its downstream targets by luciferase reporter assay, RNA pull-down experiment, Western blot, and RT-qPCR. CircNMD3 overexpression or miR-498 knockdown could inhibit the ox-LDL (oxidatively modified low-density lipoprotein)-induced injury in HUVEC (human umbilical vein endothelial cells), while the co-transfection of miR-498 mimic or siRNA targeting BAMBI (BMP and activin membrane bound inhibitor) attenuated the protective effect of circNMD3 overexpression. Overall, our data suggest that circNMD3 regulates the miR-498/BAMBI axis in endothelial cells to protect ox-LDL-induced damages. As a molecular sponge of miR-498, circNMD3 regulates the level of miR-498, which in turn modulates BAMBI expression and suppresses ox-LDL-induced injury in HUVECs.

Activin A regulates the excitability of hippocampal mossy cells.

Mossy cells (MCs) in the hilus of the dentate gyrus (DG) receive increasing attention as a major player controlling information processing in the DG network. Furthermore, disturbed MC activity has been implicated in widespread neuropsychiatric disorders such as epilepsy and major depression. Using whole-cell patch-clamp recordings from MCs in acute hippocampal slices from wild type and transgenic mice, we demonstrate that activin, a member of the transforming growth factor-ß (TGF-ß) family, has a strong neuromodulatory effect on MC activity. Disruption of activin receptor signaling reduced MC firing, dampened their excitatory input and augmented their inhibitory input. By contrast, acute application of recombinant activin A strongly increased MC activity and promoted excitatory synaptic drive. Notably, similar changes of MC activity have been observed in a rodent model of depression and after antidepressant drug therapy, respectively. Given that a rise in activin signaling particularly in the DG has been proposed as a mechanism of antidepressant action, our data suggest that the effect of activin on MC excitability might make a considerable contribution in this regard.

Differential impact of TGFß/SMAD signaling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells.

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFß pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFß family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFß pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFß/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFß/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

Sequential Treatment with Activin and Hepatocyte Growth Factor Induces FOXM1 to Promote Colorectal Cancer Liver Metastasis.

Background: Cancer stem cells (CSCs) are involved in liver metastasis in colorectal cancer (CRC). Activin and hepatocyte growth factor (HGF) are important regulators of stem cell properties. This study was performed to explore the effect of activin and HGF on CRC invasion and metastasis. The key genes involved in the action of activin and HGF in CRC were identified. Methods: HCT116 CRC cells were sequentially treated with activin and HGF and examined for migration and invasion in vitro and liver metastasis in vivo. RNA sequencing was performed to identify differentially expressed genes in response to activin and HGF. Results: Sequential treatment with activin and HGF-enhanced CRC cell migration, invasion, and metastasis. CXCR4 and AFP expressions were increased by activin and HGF treatment. Knockdown of FOXM1 blocked liver metastasis from HCT116 cells pretreated with activin and HGF and suppressed CXCR4 and AFP expression. Activin alone increased the mRNA and protein expression of FOXM1. In contrast, HGF alone enhanced the phosphorylation of FOXM1, without altering the total protein level of FOXM1. SMAD2 was required for activin-mediated FOXM1 induction. FOXM1 transactivated CXCR4 by directly binding to the promoter of CXCR4. Additionally, CXCR4 regulated AFP expression through the NF-κB pathway. Conclusions: Sequential treatment with activin and HGF accelerates CRC invasion and liver metastasis, which involves the upregulation and activation of FOXM1 and induction of CXCR4 and AFP.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin ß-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The ßA-subunit and activin-A increased, whilst ßB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

Maternal and zygotic activin signaling promotes adequate pattern and differentiation of mesoderm through regulation of pluripotency genes during zebrafish development.

To investigate the role of maternal Activin-like factors in the preservation of stemness and mesendoderm induction, their effects were promoted and inhibited using synthetic human Activin A or SB-505124 treatments, respectively, before the maternal to zygotic transition (MZT). To study the role of zygotic Activin-like factors, SB-505124 treatment was also used after the MZT. Promoting the signaling intensity of maternal Activin-like factors led to premature differentiation, loss of stemness, and no mesendoderm malformation, while its alleviation delayed the differentiation and caused various malformations. Inhibition of the zygotic Activin-like factors was associated with suppressing the ndr1, ndr2, oct4 (pou5f3), mycb and notail transcription as well as differentiation retardation at the oblong stage, and a broad spectrum of anomalies in a dose-dependent manner. Together, promoting the signal intensity of maternal Activin-like factors drove development along with mesendodermal differentiation, while suppression of the maternal or zygotic ones maintained the pluripotent state and delayed differentiation.

Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment.

Activin, a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Elevated retrotransposon activity and genomic instability in primed pluripotent stem cells.

BACKGROUND: Naïve and primed pluripotent stem cells (PSCs) represent two different pluripotent states. Primed PSCs following in vitro culture exhibit lower developmental potency as evidenced by failure in germline chimera assays, unlike mouse naïve PSCs. However, the molecular mechanisms underlying the lower developmental competency of primed PSCs remain elusive. RESULTS: We examine the regulation of telomere maintenance, retrotransposon activity, and genomic stability of primed PSCs and compare them with naïve PSCs. Surprisingly, primed PSCs only minimally maintain telomeres and show fragile telomeres, associated with declined DNA recombination and repair activity, in contrast to naïve PSCs that robustly elongate telomeres. Also, we identify LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz to define primed PSCs, and their transcription is differentially regulated by heterochromatic histones and Dnmt3b. Notably, genomic instability of primed PSCs is increased, in association with aberrant retrotransposon activity. CONCLUSIONS: Our data suggest that fragile telomere, retrotransposon-associated genomic instability, and declined DNA recombination repair, together with reduced function of cell cycle and mitochondria, increased apoptosis, and differentiation properties may link to compromised developmental potency of primed PSCs, noticeably distinguishable from naïve PSCs.