Title: Decoding Smad5-mediated BMP functions in early amnion development by mRNA-Seq
Other Titles: Het decoderen van Smad5-gemedieerde BMP-functies in de vroege ontwikkeling van het amnionvlies door mRNA-Seq
Authors: Dobreva, Mariya; S0186010
Issue Date: 29-Jun-2012
Abstract: Recently, stem cell features were attributed to cells isolated from amnion. The amnion is an avascular extraembryonic membrane that surrounds the fetus of amniotes and contains the amniotic fluid; conferring protection and shock resistance to the conceptus. The so-called amniotic stem cells are immunotolerant and are able to differentiate into all three germ layers in vitro, thus having a great potential in regenerative medicine, especially since amnion is a waste tissue at parturition. However, the precise developmental origin of amnion-derived stem cells is unknown, in part because of the poorly described process of amnion development. A better understanding of (early) amnion development, amnion plasticity and on the eventual origin of so-called amniotic stem cells can be achieved by using model organisms like the mouse (Mus musculus). Furthermore, specific genetic strains lacking certain developmental regulator molecules can help decoding the mechanisms underlying amnion plasticity. Results obtained with mouse models need however to be extrapolated to human and vice versa with care because fundamental differences in the development and positioning of the extraembryonic tissues between human and rodent amnion may lead to misinterpretations when rodent amniotic stem cells are isolated based on procedures established for human tissues and vice versa. The availability of molecular markers that are specifically expressed in amnion and not in the other extraembryonic membranes would be very helpful in the comparative analysis of stem cell populations isolated from different extra-embryonic tissues by different protocols from different species. Genes specifically expressed in amnion had not been documented so far. Therefore, one of the objectives of this PhD project was to identify amnion markers in mouse and human. We propose Periostin and AP-2, two target genes of bone morphogenetic proteins (BMPs), as novel amnion-enriched marker genes.BMP signaling appears pivotal for amnion formation. Mice lacking Bmp2 or Smad5, a mediator of BMP signaling, die around embryonic day (E)9.5 due to multiple developmental defects, including an amnion defect. Several defects of the Smad5 knock-out (KO) embryos have been characterized previously by the laboratory. One peculiar defect, the development of a tumor-like structure in the amnion of Smad5 mutants, has not been associated yet with other loss-of-function models of BMP signaling components. This structure becomes ectopically vascularized and contains red blood cells and cells with stem cell features. Recently, we showed that the reprogramming in the SMAD5-deficient amnion is associated with ectopic NODAL and WNT signaling, cascades that trigger ectopic primitive streak formation/gastrulation, a process of epithelial-to-mesenchymal transition (EMT) in the early embryo. The amniotic structure in SMAD5 mutants shows features of an ectopic primitive streak. Furthermore, in vitro studies support the hypothesis that SMAD5-mediated signaling antagonizes NODAL signaling.To further document the normal amnion signature, but also to identify (novel) target genes of BMP-SMAD5, and to decode the molecular networks that regulate amnion reprogramming/plasticity (including the induction of stem cells) and ectopic EMT in Smad5 mutants, we pursued a more holistic gene expression approach as a major part of this PhD project. Comparative whole transcriptome sequencing (mRNA-Seq) between mutant and wild type amnion appeared very valuable to investigate (impaired) early amnion development. Importantly, amnion samples were collected just before the appearance of the first obvious morphological differences, i.e. soon after amnion formation (E7.5). Given that the amniotic membrane is at this stage a tissue with very few (~200) cells, a microdissection procedure had to be established first, followed by adaptation of a single-cell whole transcriptome cDNA preparation/amplification protocol for single minute amnion samples. Subsequently, proof of concept was achieved by: 1) a pilot mRNA-Seq comparative study on control and Smad5 KO amnions at E9.0, when known features of the KO amnion could be validated, and further validation of the mRNA-Seq results; and 2) a pilot mRNA-Seq and qPCR validation of the ‘single amnion amplification protocolÂ’.Ultimately, six KO and six control littermate amnion samples were isolated from E7.5-E8.0 embryos and were analysed individually by mRNA-Seq. Clustering analysis of the separate transcriptomes resulted in identification of two separate groups of KO amnion samples which showed very dissimilar expression patterns. Differential expression clustering of both KO groups compared to the control group revealed that known target genes of BMP-SMAD signaling were found differentially expressed in one KO amnion sample set, while primitive streak, EMT and stem cell related genes were upregulated in the other set of KO amnion samples. Further validation by qPCR supported that the narrow time window between respectively the first molecular and the first morphological changes in the mutant amnion was indeed captured and is represented by these two separate groups of KO amnion samples.Major results are: · Periostin and AP-2, the two BMP regulated genes that we identified independently in mouse (and human) extra-embryonic tissues as highly enriched in amnion, were found to be downregulated by mRNA-Seq in the Smad5 KO amnions, which may reflect the loss of amnion identity, and/or the direct result of SMAD5 deficiency.· A remarkable result was that Eomes, a crucial gene for EMT and primitive streak formation was highly overexpressed in all KO samples and stood out as a potential novel SMAD5 target gene. Furthermore, the mRNA-Seq results suggested that in the absence of SMAD5, SOX2 and OCT4 may act co-operatively to activate an OCT4-dependent pluripotency program in the mutant amnion. These results opened new hypotheses on the mechanisms of induction of an ectopic primitive streak and stem cell-like cells, alternative to, but not excluding the current hypothesis that ectopic primitive streak is induced by ectopic NODAL signaling in the mutant amnion. · Importantly, the mRNA-Seq data challenged the current model on the origin of the tumor-like structure within the amniotic membrane per se. Indeed, the transient but very robust expression of a whole set of extraembryonic ectoderm specific transcripts in the cluster of KO amnion samples with differential expression of target genes of BMP suggest a putative contribution of the extraembryonic ectoderm to the anterior part of the mutant amnion. This raises new questions on the developmental potential of extraembryonic ectoderm. This tissue normally would have become specified into chorionic ectoderm, but its ectopic location in the amnion resulted potentially in transdifferentiation into more plastic epiblast-like ectoderm cells that can undergo EMT. It is unlikely that this new hypothesis would have been anticipated without the holistic comparative gene expression analysis and the collection of individual amnions around the stage of the development of the first amnion defects. Indeed, the whole transcriptomes sequencing of the 12 individual samples and the subsequent unbiased computational clustering analysis of the transcriptomes of the individual amnion samples gave enormous strength and robustness to the whole analysis and appeared crucial for distilling several new hypotheses. These hypotheses are currently being validated further, and can form the basis of new projects in the lab.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Developmental Signaling
Department of Human Genetics - miscellaneous
Molecular Biology (Celgen) (-)
Embryo and Stemcells (-)

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