Characterization of migratory primordial germ cells in the aortagonad-mesonephros of a 4.5-week-old human embryo : a toolbox to evaluate in vitro early gametogenesis

STUDY QUESTION: Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? STUDY FINDING: We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). WHAT IS KNOWN ALREADY: The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. STUDY DESIGN, SIZE, DURATION: This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. PARTICIPANTS/ MATERIALS, SETTING, METHODS: We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. MAIN RESULTS AND THE ROLE OF CHANCE: We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. LARGE SCALE DATA: Non-applicable. LIMITATIONS REASONS FOR CAUTION: Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. WIDER IMPLICATIONS OF THE FINDINGS: Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro

Development of the anterior-posterior axis is a self-organising process in the absence of maternal cues in mouse embryo

This work was supported by Wellcome Trust, Grant ID: 098287 (MZG) and EMBO (MB).This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/cr.2015.10

Development of the anterior-posterior axis is a self-organizing process in the absence of maternal cues in the mouse embryo.

This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/cr.2015.104This work was supported by Wellcome Trust, Grant ID: 098287 (MZG) and EMBO (MB)

We Do Not Like It: A Likert-Type Scale Survey on the Attitudes of a Young Population towards the Transhumanistic Theory of Education

Transhumanists assume that future education may be purely based on technological stimulation. The question is: Do potential clients of education “like” such vision? In order to check this, we asked over one thousand two hundred young Poles to evaluate their identification with the transhumanistic theory of education. The results are quite surprising: its show that they disagree with the assumptions of this theory, while they rather agree with the postulates of more traditional (and no technology-based) concepts of education

In vitro culture of mouse blastocysts beyond the implantation stages

The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond

Real time monitoring of BMP Smads transcriptional activity during mouse development

Summary: Bone morphogenetic protein (BMP) signaling is a key pathway in the patterning and development of organisms as diverse as fruit fly and humans. However, the determination of net BMP signaling, paramount to understanding organogenesis, is limited to the analysis of fixed material. We generated a transgenic mouse that reports the transcriptional response of BMP Smad activation by coupling a well established BMP response element (BRE), isolated from the Id1 promoter, to green fluorescent protein (BRE:gfp). We monitored BMP Smad transcriptional activity in fresh and fixed BRE:gfp embryos. GFP expression was observed where expected on the basis of known signaling sites, but also in specific cell populations in which BMP signaling had been implicated but not directly demonstrated. Deletion of Smad5 reduced GFP in vivo as expected. The BRE:gfp transgenic mice are a novel tool, which will facilitate the identification of specific BMP Smad responsive cell types and allow BMP Smad signaling to be monitored in real time, supporting studies in development and disease.status: publishe