Amnion formation in the mouse embryo: the single amniochorionic fold model

<p>Abstract</p> <p>Background</p> <p>Despite the detailed knowledge obtained over the last decade on the molecular regulation of gastrulation in amniotes, the process of amnion development has been poorly described and illustrated in mice, and conflicting descriptions exist. Understanding the morphogenesis and development not only of the early mouse embryo, but also of its extraembryonic tissues, is crucial for correctly interpreting fate-mapping data and mouse mutants with gastrulation defects. Moreover, the recent isolation from amnion of cells with stem cell features further argues for a better understanding of the process of amnion formation. Here, we revisit the highly dynamic process of amnion formation in the mouse. Amnion development starts early during gastrulation and is intimately related to the formation of the exocoelom and the expansion of the amniotic fold. The authoritative description involves the fusion of two amniotic folds, a big posterior and a smaller anterior fold. We challenged this 'two amniotic folds' model by performing detailed histomorphological analyses of dissected, staged embryos and 3D reconstructions using historical sections.</p> <p>Results</p> <p>A posterior fold of extraembryonic ectoderm and associated epiblast is formed early during gastrulation by accumulation of extraembryonic mesoderm posterior to the primitive streak. Previously called the "posterior amniotic fold", we rename it the "amniochorionic fold" (ACF) because it forms both amnion and chorion. Exocoelom formation within the ACF seems not to involve apoptosis within the mesoderm. The ACF and exocoelom expand without disrupting the anterior junction of epiblast, extraembryonic ectoderm and visceral endoderm. No separate anterior fold is formed; its absence was confirmed in 3D reconstructions. Amnion and chorion closure is eccentric, close to the anterior margin of the egg cylinder: we name it the "anterior separation point".</p> <p>Conclusions</p> <p>Here, we reconcile previous descriptions of amnion formation and provide new nomenclature, as well as an animation, that clarify and emphasize the arrangement of the tissues that contribute to amnion development and the dynamics of the process. According to our data, the amnion and the chorion are formed by a single amniochorionic fold initiated posteriorly. Finally, we give an overview on mutant mouse models with impaired amnion development.</p

The development of the amnion in mice and other amniotes

The amnion is an extraembryonic tissue that evolutionarily allowed embryos of all amniotes to develop in a transient and local aquatic environment. Despite the importance of this tissue, very little is known about its formation and its molecular characteristics. In this review, we have compared the basic organization of the extraembryonic membranes in amniotes and describe the two types of amniogenesis, folding and cavitation. We then zoom in on the atypical development of the amnion in mice that occurs via the formation of a single posterior amniochorionic fold. Moreover, we consolidate lineage tracing data to better understand the spatial and temporal origin of the progenitors of amniotic ectoderm, and visualize the behaviour of their descendants in the extraembryonic-embryonic junctional region. This analysis provides new insight on amnion development and expansion. Finally, using an online-available dataset of single-cell transcriptomics during the gastrulation period in mice, we provide bioinformatic analysis of the molecular signature of amniotic ectoderm and amniotic mesoderm. The amnion is a tissue with unique biomechanical properties that deserves to be better understood. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'

BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells

Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation

Expression of hypoxia-inducible factor-1α and cell cycle proteins in invasive breast cancer are estrogen receptor related

BACKGROUND: The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. Previous studies showed that concentrations of its subunit HIF-1α, as a surrogate for HIF-1 activity, are increased during breast carcinogenesis and can independently predict prognosis in breast cancer. During carcinogenesis, the cell cycle is progressively deregulated, and proliferation rate is a strong prognostic factor in breast cancer. In this study we undertook a detailed evaluation of the relationships between HIF-1α and cell cycle-associated proteins. METHODS: In a representative estrogen receptor (ER) group of 150 breast cancers, the expression of HIF-1α, vascular endothelial growth factor, the ER, HER-2/neu, Ki-67, cyclin A, cyclin D(1), p21, p53, and Bcl-2 was investigated by immunohistochemistry. RESULTS: High concentrations (5% or more) of HIF-1α were associated with increased proliferation as shown by positive correlations with Ki-67 (P < 0.001) and the late S–G2-phase protein cyclin A (P < 0.001), but not with the G1-phase protein cyclin D(1). High HIF-1α concentrations were also strongly associated with p53 positivity (P < 0.001) and loss of Bcl-2 expression (P = 0.013). No association was found between p21 and HIF-1α (P = 0.105) in the whole group of patients. However, the subgroup of ER-positive cancers was characterized by a strong positive association between HIF-1α and p21 (P = 0.023), and HIF-1α lacked any relation with proliferation. CONCLUSION: HIF-1α overexpression is associated with increased proliferation, which might explain the adverse prognostic impact of increased concentrations of HIF-1α in invasive breast cancer. In ER-positive tumors, HIF-1α is associated with p21 but not against proliferation. This shows the importance of further functional analysis to unravel the role of HIF-1 in late cell cycle progression, and the link between HIF-1, p21, and ER

A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of integrin-α4 expression

UNLABELLED: Although it is well established that BMP4 plays an important role in the development of hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis and how. Here, we describe a novel mechanism by which BMP4 regulates homing of murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment of murine BM derived c-kitLin-Sca-1 (KLS) and CD150CD48-KLS cells for up to 5 days in vitro prevented the culture-induced loss of Integrin-α4 (ITGA4) expression as well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmia-associated transcription factor (MITF), known to induce ITGA4 expression. Elevated ITGA4 expression significantly enhanced HSPC attachment to bone marrow stromal cells, homing and long-term engraftment of the BMP4 treated cells compared with the cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM derived Lin-CD34 cells in culture, which was associated with improved homing potential. Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution. KEY POINTS: Cytokine-induced loss of murine as well as human HSPC homing during ex vivo culture can be prevented by addition of BMP4. In HSPCs, BMP4 directly regulates Integrin-α4 expression through SMAD-independent p38 MAPK-mediated signaling.status: publishe

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.status: publishe

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.status: publishe

Expression patterns of follistatin and two follistatin-related proteins during mouse development

We compared the expression patterns of follistatin and two follistatin-related proteins (FRP and m7365) during early mouse development. m7365 is expressed continuously during preimplantation development, in contrast to FRP and follistatin. At early postimplantation stages, follistatin and 7365 are expressed from E6.0, while FRP is detected from E7.5 onwards. Although there is some overlap between the expression of these genes in the primitive streak and somites, their overall expression patterns are distinct.status: publishe

The development of the amnion in mice and other amniotes

The amnion is an extraembryonic tissue that evolutionarily allowed embryos of all amniotes to develop in a transient and local aquatic environment. Despite the importance of this tissue, very little is known about its formation and its molecular characteristics. In this review, we have compared the basic organization of the extraembryonic membranes in amniotes and describe the two types of amniogenesis, folding and cavitation. We then zoom in on the atypical development of the amnion in mice that occurs via the formation of a single posterior amniochorionic fold. Moreover, we consolidate lineage tracing data to better understand the spatial and temporal origin of the progenitors of amniotic ectoderm, and visualize the behaviour of their descendants in the extraembryonic-embryonic junctional region. This analysis provides new insight on amnion development and expansion. Finally, using an online-available dataset of single-cell transcriptomics during the gastrulation period in mice, we provide bioinformatic analysis of the molecular signature of amniotic ectoderm and amniotic mesoderm. The amnion is a tissue with unique biomechanical properties that deserves to be better understood. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'