Germline development in amniotes : a paradigm shift in primordial germ cell specification

In the field of germline development in amniote vertebrates, primordial germ cell (PGC) specification in birds and reptiles remains controversial. Avians are believed to adopt a predetermination or maternal specification mode of PGC formation, contrary to an inductive mode employed by mammals and, supposedly, reptiles. Here, we revisit and review some key aspects of PGC development that channelled the current subdivision, and challenge the position of birds and reptiles as well as the binary' evolutionary model of PGC development in vertebrates. We propose an alternative view on PGC specification where germ plasm plays a role in laying the foundation for the formation of PGC precursors (pPGC), but not necessarily of PGCs. Moreover, inductive mechanisms may be necessary for the transition from pPGCs to PGCs. Within this framework, the implementation of data from birds and reptiles could provide new insights on the evolution of PGC specification in amniotes

Current status of in vitro differentiation of stem cells into gametes

From an evolutionary point of view, the gametes are the cells in the body that matter the most as they are the ones who transmit their genes to the next generation ensuring continuation of the species. Being able to generate mature oocytes in vitro is of great interest. Oocytes are the key to totipotency and are able to reprogram somatic cells in approximately one day. In addition, in contrast to a clump of pluripotent stem cells, once the developmental program has started, fertilized oocytes develop into a clump of cells with positional information and the possibility to differentiate into both the embryonic and the extraembryonic lineages that form a complete developing and viable organism. How to instruct pluripotent stem cells to become oocytes in vitro is still unclear and even though the first steps to obtain mouse oocytes have recently been successfully demonstrated, inducing meiosis progression and folliculogenesis in vitro are still far from being understood and have not yet been accomplished. In humans, the specific molecular niche that leads to correct oogenesis is less understood. Here, we discuss the current status of in vitro differentiation of human pluripotent stem cells into gametes, in particular to oocytes

BMPs, TGFbetas and integrins in muscle and germ cell development in mice

We characterized the expression of PSmad1/5/8 (BMP R-Smads) and PSmad2 (TGFbeta R-Smad) during peri-implantation and gastrulation in the mouse, and further characterized the expression of PSmad2 until E12.5, showing were the BMP and TGFbeta Smad-dependent signalling pathways are active (Chapter 2, 3 and 4). The role of ALK2 (BMP signalling) and ALK5 (TGFbeta signalling) in the development of PGCs in the mouse was studied in detail. ALK2 is needed for the formation of PGCs before gastrulation occurs and it signals in the visceral endoderm, a tissue that was previously not associated with primordial germ cell formation (Chapter 3). In contrast to suggestions derived from studies in vitro, ALK5 is probably not involved in PGC migration through the hindgut and to the gonadal ridges and TGF 1 is unlikely to be a chemoattractant for PGCs as proposed (Chapter 4). However, there may be a role for TGF signalling in PGC development in controlling mitotic arrest of PGCs in the gonadal ridges. Beta1 integrin was also implicated in PGC migration to the genital ridges. Beta1 integrin has two isoforms in mice: a more common one beta1A and a muscle specific beta1D that is upregulated late during development. We studied whether the replacement of beta1A by beta1D influenced the development of PGCs, but this occurred normally and we found no evidence of an effect on migration on the genetic background used. Furthermore, beta1 integrin has been implicated in heart development. We showed that the replacement of beta1A by beta1D did not cause heart abnormalities during development. However, we were able to show a clear role for beta1A during myogenesis. The differentiation of primary myoblasts was abnormal when only beta1D was expressed, while secondary myoblasts were formed normally. Furthermore, we showed a novel role of beta1 integrin during placentation, in particular the development of the labyrinthine layer (Chapter 5). CTGF is known to crosstalk with both TGFbeta and BMP in vitro. We studied possible interactions between them, focusing on heart development, where BMP and TGFbeta are known to play prominent roles. However, we were not able to detect direct crosstalk. Both CTGF and TGFbeta are associated with fibrosis. We investigated whether CTGF and TGFbeta were expressed following experimental induction of myocardial infarction in the mouse and showed that both genes are present in particular in the scar tissue and that TGFbeta Smad-dependent signalling is transient 1 week post-infarction and largely restricted to cardiac fibroblasts (Chapter 6)

Proximal visceral endoderm and extraembryonic ectoderm regulate the formation of primordial germ cell precursors.

BACKGROUND: The extraembryonic tissues, visceral endoderm (VE) and extraembryonic ectoderm (ExE) are known to be important for the induction of primordial germ cells (PGCs) in mice via activation of the bone morphogenetic protein (BMP) signalling pathway. We investigated whether the VE and ExE have a direct role in the specification of PGCs, or in an earlier event, namely the induction of the PGC precursors in the proximal posterior epiblast cells. RESULTS: We cultured embryonic day (E) 5.75 to E7.0 mouse embryos in an explant-assay with or without extraembryonic tissues. The reconstituted pieces of embryonic and extraembryonic tissues were assessed for the formation of both PGC precursors and specified PGCs. For this, Blimp1:gfp and Stella:gfp transgenic mouse lines were used to distinguish between PGC precursors and specified PGC, respectively. We observed that the VE regulates formation of an appropriate number of PGC precursors between E6.25-E7.25, but it is not essential for the subsequent specification of PGCs from the precursor cells. Furthermore, we show that the ExE has a different role from that of the VE, which is to restrict localization of PGC precursors to the posterior part of the embryo. CONCLUSION: We show that the VE and ExE have distinct roles in the induction of PGC precursors, namely the formation of a normal number of PGC precursors, and their appropriate localization during early development. However, these tissues do not have a direct role during the final stages of specification of the founder population of PGCs.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Stay on the road: from germ cell specification to gonadal colonization in mammals

The founder cells of the gametes are primordial germ cells (PGCs). In mammals, PGCs are specified early during embryonic development, at the boundary between embryonic and extraembryonic tissue, long before their later residences, the gonads, have developed. Despite the differences in form and behaviour when differentiated into oocytes or sperm cells, in the period between specification and gonadal colonization, male and female PGCs are morphologically indistinct and largely regulated by similar mechanisms. Here, we compare different modes and mechanisms that lead to the formation of PGCs, putting in context protocols that are in place to differentiate both human and mouse pluripotent stem cells into PGC-like cells. In addition, we review important aspects of the migration of PGCs to the gonadal ridges, where they undergo further sex-specific differentiation. Defects in migration need to be effectively corrected, as misplaced PGCs can become tumorigenic. Concluding, a combination of in vivo studies and the development of adequate innovative in vitro models, ensuring both robustness and standardization, are providing us with the tools for a greater understanding of the first steps of gametogenesis and to develop disease models to study the origin of germ cell tumours. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'

Treatment of human embryos with the TGFβ inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation

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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 stria vascularis and potassium regulation in the human fetal cochlea : insights into hereditary sensorineural hearing loss

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9–W18) and show the cochlear expression dynamics of key potassium‐regulating proteins. At W12, MITF+/SOX10+/KIT+ neural‐crest‐derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na(+)/K(+)‐ATPase‐positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1219–1240, 201

The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

Publisher's version (útgefin grein)Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.M.S.A. is supported by the “Göngum saman” cancer fund and the Helga Jonsdottir and Sigurlidi Kristjansson memorial fund; G.V. is supported by the University of Iceland research fund, the Icelandic cancer association, and the Watanabe trust fund at the University of Iceland.Peer Reviewe