Pbx proteins as pioneer factors promoting signal specificity in mesodermal differentiation

Project Information

PIOMES

Grant agreement ID: 660958

DOI

10.3030/660958

Project closed

EC signature date 17 April 2015

Start date 1 May 2015

End date 30 April 2017

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 200 194,80

EU contribution

€ 200 194,80

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - PIOMES (Pbx proteins as pioneer factors promoting signal specificity in mesodermal differentiation)

Reporting period: 2015-05-01 to 2017-04-30

Summary of the context and overall objectives of the project

The mesoderm is one of the three primary germ layers in the early embryo and gives rise to a large variety of tissues, including muscle, bone, blood, cartilage and connective tissues. Understanding how mesodermal tissues develop from progenitor cells is critical to develop efficient protocols applicable to tissue repair and regenerative therapies. In mammals, all mesodermal progenitors (MPs) originate during gastrulation from cells of the epiblast that ingress into a transient structure termed primitive streak (PS). In the PS, the coordinated action of different signalling pathways (Nodal, Wnt, Bmp) and transcription factors (TFs) provides the spatio-temporal information specifying distinct mesodermal cell types.
However, how MPs acquire mesodermal fate is still an open question, due to the complexity of deciphering the transcriptional networks at gastrulation (Figure 1).
In PIOMES we found that genes conferring positional identity, like the Pbx proteins, are important for attributing such spatio-temporal coordinates.
Pbx TFs have widespread roles in the formation of lateral mesoderm-derived structures, such as blood, appendicular skeleton, heart, spleen, and body wall. Pbx mutants die at embryonic day (E) 8.5 displaying dilated heart, defective somite formation, short trunk, turned tail and limbs aplasia.
In PIOMES we found that Pbx1 is initially expressed in epiblast cells preceding gastrulation, and becomes upregulated in the posterior PS and emerging mesodermal cells at E6.5 and E7.5. Consistently, we observed that while the anterior PS and the node of E7.5 Pbx mutant embryos were unaffected, the posterior PS was shorter and abnormally shaped.
All above observations raised the possibility that Pbx proteins guide mesoderm specification of the posterior MPs through specific transcriptional networks.
In PIOMES we employed mouse epiblast stem cells (mEpiSCs), which can be differentiated in vitro into different MPs subtypes and thus are an excellent system for understanding how complex transcriptional regulatory networks control lineage determination. mEpiSCs share a common pluripotent state with the human embryonic stem cells (hESCs), and thus represent the closer model for translating the mouse results into the hESCs system. In PIOMES we had implemented protocol for mesoderm differentiation.
Loss of MPs underlies a spectrum of developmental and degenerative diseases, such as congenital heart defects, muscular dystrophies.
In PIOMES we identified critical molecular mechanisms driving mesoderm specification that will help to conceive new protocols for human stem-cell therapy and open new avenues for regenerative medicine.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work progress and achievements during the period:
The main objective of PIOMES is to dissect the molecular mechanisms by which Pbx protein promote mesoderm specification.

WP1. Dissection of Pbx protein roles in mesodermal progenitors.

WP2. Analysis of mesodermal competence of Pbx1+ cells.

WP3. Identification of Pbx-regulated genes in differentiation of EpiSCs into MPs.

WP4. Characterization of Pbx targets and interactors in mesoderm differentiation.


DISSEMINATION ACTIVITY
In 2015, I gave a talk about the benefits and realist applications of stem cell-based therapeutic methods at “The Stem Cell Day 2015” organized by DanStem. Given the extraordinary success of this first event DanStem organized a second event in 2016 at Norre Gymnasium and the third at the Danish Jewish School in 2017. My laboratory with PIOMES participated to all of these events. All events have been published on DanStem website. http://danstem.ku.dk/news/stem-cell-day-2015/ http://danstem.ku.dk/news/stem-cell-day-may-3-2016/ http://danstem.ku.dk/news/stem-cell-day-2017/).
Since 2015 I am the scientific organizer of the Stem Cell Club seminar series, which have the aim of providing a forum to present and discuss ongoing research projects in the stem cell and developmental biology fields in the Copenhagen area. The final goal of the seminars is to stimulate collaborations by providing a milieu for discussions, http://danstem.ku.dk/communication-and-outreach/the-stem-cell-club/).
In May 2015, I gave talk entitled “Tips for a Successful Marie Curie Individual Fellowship (MSCA) Application” at the University of Copenhagen at workshop about Marie Curie to advice postdoctoral fellows on how to write their MSCA-IF application.
In Maj 2016, DanStem organized the “Stem Cell Niche, Development and Diseases” conference where all the aspects of stem cells were addressed: stem cell niches, pluripotency and expansion, cell fate specification, biomechanics, stem cell modelling, cancer stem cells, http://www.cph-bioscience.com/conferences/stem-cell-niche-development-disease. I was part of the scientific committee and I was actively involved into the choice of the speakers and topics for the event. I have also presented a poster with the progress of the PIOMES project.
In September 2016, in collaboration with the NNF Center cluster DanStem organized the PhD course “Introduction to Molecular Bioscience” where I represented PIOMES and talk about gastrulation and mesoderm differentiation. Title of the talk: “Introduction to gastrulation and mechanisms of mesoderm induction”.
In December 2016, I represented PIOMES at the PhD course “Stem Cell Research and Critical Thinking” to the PhD students at the University of Copenhagen. I gave lecture about Epithelial Stem Cells and relationship with Cancer.
I presented PIOMES at ISSCR (International Society for Stem Cell Research), Stockholm, Sweden in June 2015, at the BSDB - British Society for Developmental Biology in April 2015 and 2017 in Warwick, UK, at the Lund-Copenhagen Stem Cell Symposium in August 2015 and the SWEDBO (Swedish Developmental Biology Organization) in Lund in 2016.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The main focus of PIOMES is to dissect the gene regulatory networks controlling mesoderm specification and early pattering during mammalian development.
Our preliminary data suggest that Pbx proteins could operate as regulators of mesoderm identity. We employed mouse epiblast stem cells (mEpiSCs), which can be differentiated in vitro into different MPs subtypes and thus are an excellent system for understanding how complex transcriptional regulatory networks control lineage determination. mEpiSCs share a common pluripotent state with the human embryonic stem cells (hESCs), and thus represent the closer model for translating the mouse results into the hESCs system. The use of murine cells we will allow us to perform intraspecies experiments to validate the in vitro findings in vivo using both wild type and mutant mouse embryos. Human embryos are inaccessible for genetic studies, in particular at gastrulation stage. However, we plan to test whether the characterized mechanisms are conserved using human ESCs in vitro differentiation systems of hESCs. We believe that the combination of in vitro differentiation systems and genetic studies in mice represents a powerful approach that we will allow us to get a deeper understanding of the molecular mechanisms controlling mesodermal specification and patterning in mammals.
Our long-term goal is to generate robust models for mesoderm differentiation, which will help in conceiving new protocols for hESCs mesodermal differentiation.

Figure 1. Specification of mesodermal progenitors in vivo and in vitro.