Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2.

The ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2-mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1(high)PDGFRα(-). Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2-dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability.

ER71 specifies Flk-1+ hemangiogenic mesoderm by inhibiting cardiac mesoderm and Wnt signaling.

Two distinct types of Flk-1(+) mesoderm, hemangiogenic and cardiogenic, are thought to contribute to blood, vessel, and cardiac cell lineages. However, our understanding of how Flk-1(+) mesoderm is specified is currently limited. In the present study, we investigated whether ER71, an Ets transcription factor essential for hematopoietic and endothelial cell lineage development, could modulate the hemangiogenic or cardiogenic outcome of the Flk-1(+) mesoderm. We show that Flk-1(+) mesoderm can be divided into Flk-1(+)PDGFRα(-) hemangiogenic and Flk-1(+)PDGFRα(+) cardiogenic mesoderm. ER71-deficient embryonic stem cells produced only the Flk-1(+)PDGFRα(+) cardiogenic mesoderm, which generated SMCs and cardiomyocytes. Enforced ER71 expression in the wild-type embryonic stem cells skewed toward the Flk-1(+)PDGFRα(-) mesoderm formation, which generated hematopoietic and endothelial cells. Whereas hematopoietic and endothelial cell genes were positively regulated by ER71, cardiac and Wnt signaling pathway genes were negatively regulated by ER71. We show that ER71 could inhibit Wnt signaling in VE-cadherin-independent as well as VE-cadherin-dependent VE-cadherin/ß-catenin/Flk-1 complex formation. Enforced ß-catenin could rescue cardiogenic mesoderm in the context of ER71 overexpression. In contrast, ER71-deficient Flk-1(+) mesoderm displayed enhanced Wnt signaling, which was reduced by ER71 re-introduction. We provide the molecular basis for the antagonistic relationship between hemangiogenic and cardiogenic mesoderm specification by ER71 and Wnt signaling.

Effect of nicotinamide mononucleotide on osteogenesis in MC3T3-E1 cells against inflammation-induced by lipopolysaccharide.

Objective: Nicotinamide mononucleotide (NMN) is extensively utilized as an anti-aging agent and possesses anti-inflammatory properties. Lipopolysaccharide (LPS) activates Toll-like receptor 4, a process modulated by intracellular signaling pathways such as the Wnt/ß-catenin pathway. This study investigated the impact of NMN on osteogenesis in the presence of LPS. Methods: To elucidate the role of NMN in osteogenesis in the context of Gram-negative bacterial infection after LPS treatment, we cultured a mouse pre-osteoblast cell line (MC3T3-E1) and subsequently incubated it with NMN and/or LPS. We then evaluated osteogenic activity by measuring alkaline phosphatase activity, assessing gene expression and protein levels, and performing Alizarin Red S staining and immunocytochemistry. Results: MC3T3-E1 cells underwent successful differentiation into osteoblasts following treatment with osteogenic induction medium. LPS diminished features related to osteogenic differentiation, which were subsequently partially reversed by treatment with NMN. The restorative effects of NMN on LPS-exposed MC3T3-E1 cells were further substantiated by elucidating the role of Wnt/ß-catenin signaling, as confirmed through immunocytochemistry. Conclusion: This study showed that infection with Gram-negative bacteria disrupted the osteogenic differentiation of MC3T3-E1 cells. This adverse effect was partially reversed by administering a high-dose of NMN. Drawing on these results, we propose that NMN could serve as a viable therapeutic strategy to preserve bone homeostasis in elderly and immunocompromised patients.

Ovastacin: An oolemma protein that cleaves the zona pellucida to prevent polyspermy.

Monospermy occurs in the process of normal fertilization where a single sperm fuses with the egg, resulting in the formation of a diploid zygote. During the process of fertilization, the sperm must penetrate the zona pellucida (ZP), the outer layer of the egg, to reach the egg's plasma membrane. Once a sperm binds to the ZP, it undergoes an acrosomal reaction, which involves the release of enzymes from the sperm's acrosome that help it to penetrate the ZP. Ovastacin is one of the enzymes that is involved in breaking down the ZP. Studies have shown that ovastacin is necessary for the breakdown of the ZP and for successful fertilization to occur. However, the activity of ovastacin is tightly regulated to ensure that only one sperm can fertilize the egg. One way in which ovastacin helps to prevent polyspermy (the fertilization of an egg by more than one sperm) is by rapidly degrading the ZP after a sperm has penetrated it. This makes it difficult for additional sperm to penetrate the ZP and fertilize the egg. Ovastacin is also thought to play a role in the block to polyspermy, a mechanism that prevents additional sperm from fusing with the egg's plasma membrane after fertilization has occurred. In summary, the role of ovastacin in monospermic fertilization is to help ensure that only one sperm can fertilize the egg, while preventing polyspermy and ensuring successful fertilization.

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity.

OBJECTIVE: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. METHODS: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. RESULTS: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. CONCLUSION: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

Oct-4 controls cell-cycle progression of embryonic stem cells.

Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the 'stemness' of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation.

Comparison of the Thermal Degradation of Heavily Nb-Doped and Normal PZT Thin Films.

The degradation of niobium-doped lead zirconate titanate (PZT) and two types of PZT thin films were investigated. Undoped PZT, two-step PZT, and heavily Nb-doped PZT (PNZT) around the morphotropic phase boundary were in situ deposited under optimum condition by RF-magnetron sputtering. All 2- [Formula: see text]-thick films had dense perovskite columnar grain structure and self-polarized (100) dominant orientation. PZT thin films were deposited on Pt/TiOx bottom electrode on Si wafer, and PNZT thin film was on Ir/TiW electrode with the help of orientation control. Sputtered PZT films formed on microelectromechanical system (MEMS) gyroscope and the degradation rates were compared at different temperatures. PNZT showed the best resistance to the thermal degradation, followed by two-step PZT. To clarify the effect of oxygen vacancies on the degradation of the film at high temperature, photoluminescence measurement was conducted, which confirmed that oxygen vacancy rate was the lowest in heavy PNZT. Nb-doping PZT thin films suppressed the oxygen deficit and made high imprint with self-polarization. This defect distribution and high internal field allowed PNZT thin film to make the piezoelectric sensors more stable and reliable at high temperature, such as reflow process of MEMS packaging.

Enhanced hemangioblast generation and improved vascular repair and regeneration from embryonic stem cells by defined transcription factors.

The fetal liver kinase 1 (FLK-1)(+) hemangioblast can generate hematopoietic, endothelial, and smooth muscle cells (SMCs). ER71/ETV2, GATA2, and SCL form a core transcriptional network in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage in mouse embryonic stem cells (ESCs) robustly enhanced hemangioblast generation by activating bone morphogenetic protein (BMP) and FLK-1 signaling while inhibiting phosphatidylinositol 3-kinase, WNT signaling, and cardiac output. Moreover, etsrp, gata2, and scl inhibition converted hematopoietic field of the zebrafish anterior lateral plate mesoderm to cardiac. FLK-1(+) hemangioblasts generated by transient coexpression of the three factors (ER71-GATA2-SCL [EGS]-induced FLK-1(+)) effectively produced hematopoietic, endothelial, and SMCs in culture and in vivo. Importantly, EGS-induced FLK-1(+) hemangioblasts, when codelivered with mesenchymal stem cells as spheroids, were protected from apoptosis and generated functional endothelial cells and SMCs in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage. ESC-derived, EGS-induced FLK-1(+) hemangioblasts could provide an attractive cell source for future hematopoietic and vascular repair and regeneration.