| 研究実績の概要 |
In the first year of this project (2018), we have completed major work in achieving aim 1 and have started to obtain preliminary results for aims 2-4. More specifically, 1) for aim1, we have obtained quality promoterome data (CAGE-based TSS mapping) on genes expressed in different tissues during CAM fusion. We have also mapped temporal progression of CAM fusion to a 6-12 hour window (E3.75-E4.25 of chick development). We have uncovered involvement in CAM fusion of canonical EMT genes (of the Snail and Zeb families) and of specific signaling pathways (WNT, FGF and CXC chemokine pathways). We have also uncovered involvement of novel genes specific for chorionic ectoderm during this fusion process. 2) for aim2, we have obtained proof-of-principle results that CAM fusion can be studies in voth allantoic explants (in vivo) and allantoic/chorionic explants ex vivo. Fluorescent dyes, electroporation methods and GFP avian strains can be utilized for labeling and imaging studies. 3) for aim3, we have obtained immunofluorescence staining data for canonical epithelial markers, supporting the hypothesis of mesothelial EMT during CAM fusion. For chorionic mesothelial cells, definitive conclusion has not been reached because they are covered by an epithelial layer of ectoderm cells (with their basement membrane facing that of the mesothelium), we have started a electron microscopy-based study of CAM fusion to achieve better resolution. 4) for aim 4, we have evidence that perturbation of the FGF and WNT pathways (using chemical agonists or antagonists) can affect the fusion process.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The project is making progress according to the plan. We have confirmed, with molecular and cellular data, that EMT is indeed involved during CAM fusion. We have obtained a list of enriched genes during CAM fusion and have data to support the involvement of WNT and FGF signaling pathways during CAM fusion. We are currently making progress to narrow down key molecular players that regulate CAM fusion and its EMT. Our aim is to be able to identify a small set of genes and regulators of the involved pathways, and to perturb (induce and inhibit) CAM fusion through targeted modulation of molecular functions of those key players. In the first year of this project, we have also encountered two interesting aspects of CAM fusion. One is that the chorionic ectoderm shows prominent molecular difference in the fused and unfused territories. This indicates that the signals coming from the chorionic ectoderm play a role in CAM fusion. Secondly, the allantoic mesothelium shows molecular asymmetry along the proximal-distal axis of the fusion plane during CAM fusion. This suggests that there is an inherent asymmetry in allantoic mesothelium, either due to original cellular heterogeneity in allantoic mesothelial cell contribution or due to secondary patterning after allantoic expansion and the establishment of allantoic vascular flow. These aspects of CAM fusion will be taken into consideration in the next fiscal year of this project.
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| 今後の研究の推進方策 |
In the second year of this project, we will 1) pinpoint molecular players of the CAM fusion. For chemical perturbation using agonists/antagonists, we aim to distinguish their direct effort on the mesothelial cells and their indirect effect on the endoderm, allantoic vasculature and chorionic ectoderm. This will be achieved by applying agonists/antagonists with several different methods (e.g., inside allantoic cavity, inside extraembryonic coelomic cavity, outside chorion, ex ovo, etc). For molecular candidates, we will construct wild type and mutant versions and test their effect on CAM fusion by ex ovo electroporation. By the end of this fiscal year, we aim to have a clear set of genes that can be used to induce or inhibit CAM-like fusion. 2) establish and optimize imaging of CAM fusion. We have achieved macroscopic level of imaging of CAM fusion. In this fiscal year, we aim to achieve cellular level (either single cell or a group of few cells) in ex ovo CAM culture. This experimental setting will be used to investigate cellular-level control of CAM fusion. 3) complete the characterization of CAM fusion through electron microscopy. We have already prepared EM grade samples for EM study of CAM fusion. The aim this part of the project is to characterize fine cell morphological changes in both chorionic and allantoic mesothelial cells and to associate those with changes in molecular markers. Together, those data will give use an understanding of both cellular-level and molecular-level details of CAM fusion.
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