Community Research and Development Information Service - CORDIS

FP7

QUANT CELL STATE Report Summary

Project reference: 330880
Funded under: FP7-PEOPLE

Final Report Summary - QUANT CELL STATE (A quantitative analysis of single cell variation in transcription during the stabilisation of neural mesodermal cell states in vivo and in vitro.)

In order to investigate the hypothesis that gene expression heterogeneity acts to maintain neuromesodermal progeniors in a bipotential state (i.e. able to generate both neuroectodermal and mesodermal derivatives), this project consists of three interrelated objectives:
1) To establish imaging and image analysis techniques to quantify neural and mesodermal markers within fixed zebrafish embryos at successive stages of posterior body elongation.
2) To generate live reporter lines to follow transcriptional dynamics in live zebrafish embryos.
3) To apply the above techniques generated in zebrafish embryos to monitor cell fate decisions within mouse embryonic stem cells.
These objectives have been met or altered as described below:
Establishment of imaging and image analysis techniques to quantify neural and mesodermal markers within fixed zebrafish embryos at successive stages of posterior body elongation.
We have successfully implemented a multiplexed in situ hybridization technique to quantify mean expression levels of several markers of tailbud stem cells, including sox2, brachyury, snail2, tbx16, nkx1.2 and oct4. In addition, we have monitored the downstream early paraxial mesodermal gene mesogenin. This technique utilized Hybridisation Chain Reaction (HCR), developed in collaboration with the host lab of the outgoing phase (Choi et al., 2010; Choi et al., 2014). HCR makes use of DNA labelled hairpins that, in the presence of probe binding, form chains of approx. 200 hairpins in length that provide a significant amplification of signal. Data from the lab has revealed a linear amplification of signal that allows for a precise quantification of gene expression levels (Trivedi, Trinh, Pierce and Fraser, in prep).
An Image analysis workflow has been set-up with which to calculate local regions of interest approximating individual cells within 3D confocal stacks of whole mount zebrafish embryos. This allows for the automated quantification of mean transcript levels and the local coefficient of variation for each cell. Furthermore, we have development an analysis workflow with which to compare cytoplasmic vs. nuclear localized transcript abundance.
Generation of live reporter lines to follow transcriptional dynamics in live zebrafish embryos.
We were unsuccessful in generating live reporter lines to monitor transcriptional dynamics. However, in order to understand how the gene expression changes that were observed in objective one are mapped to cellular movements and divisions, we generated a series of live imaging datasets in which individual cell behaviours can be quantified. Furthermore, in collaboration with the Cambridge Advanced Imaging Centre, we have developed a tool that automatically adjusts the position of the x, y, z stage in order to keep the progenitor region within the centre of the field of view during long-term light-sheet imaging. These results reveal that zebrafish tailbud progenitors are a closed system with no cells being added and no cells leaving up until late stages of somitogenesis.
Together with our single-cell gene expression analyses, this suggests that cells gradually resolve into distinct neural and mesodermal cell states prior to exiting the progenitor domain. This is distinct from amniotes such as the mouse, where cells undergo continuous divisions and exit the progenitor domain to provide a continuous fuel of neural and mesodermal progenitors to generate posterior growth.
Application of single-cell quantitation techniques to monitor cell fate decisions within mouse embryonic stem cells.
The incoming host laboratory has been successful in generating mouse ES cell aggregates that undergo symmetry breaking and begin to elongate, recapitulating the early events of the developing mouse embryo. In bringing my expertise generated during the outgoing phase of this fellowship, we have been successful in addressing two key questions that relate to the overall objectives of this proposal:
1) Do the ES cell aggregates elongate their body axis in a manner similar to that of the normal embryo? In this regard it was important to ascertain whether they possess cells akin to the self-renewing, bipotent neuromesodermal progenitors as seen in vivo. To address this, I have applied the specific expertise derived from the outgoing phase of this fellowship (i.e. quantitative gene expression analyses in situ and the required imaging/analysis techniques) in order to determine whether they express a combination of sox2 and brachyury. This is now enabling us to address how this heterogeneous cell state resolves over time in line with the primary objectives of this proposal.
2) Are the neuromesodermal cells produced in these cellular aggregates able to self-renew and generate large clones of neural and mesodermal derivatives, as seen for the endogenous cell population? This question is of particular importance, as it was not clear whether similar cells produced by adherent ES cell culture techniques could propagate themselves before undergoing overt differentiation. In order to assess this, I have established a mouse-chick chimera assay within the host laboratory in order to assess the developmental potential of transplanted cells by live imaging. This study has revealed important findings relating to differences between neuromesodermal progenitors either generated by adherent of aggregate cell culture that we intend to publish in the coming year.
In conclusion, this project has enabled me to generate important results relating to the gene expression and cellular dynamics that generate neural and mesodermal tissue during axis elongation n both zebrafish embryos and mouse ES cell aggregates. Importantly, this has also provided a series of bespoke imaging and analysis techniques that allow me to probe these events at an unparalleled level of detail in a quantitative manner. In addition to generated important results and conclusion that are currently in preparation for publication, it also provided the required preliminary data for obtaining my first large independent funding stream, in the form of a Sir Henry Dale career development fellowship based within the incoming host organization. This will enable me to build upon the results obtained from this post-doctoral fellowship both as an independent researcher and through further collaboration with the both incoming and outgoing hosts.

Contact

Schaeffer, Renata (European Policy Manager)
Tel.: +441223 333543
Fax: +441223 332988
E-mail

Subjects

Life Sciences
Record Number: 189271 / Last updated on: 2016-09-14