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Does Wnt promote the switch from cardiac to skeletal muscle programme? – Summer Placement

Posted by , on 13 August 2022

Posting my lab report from last summer again since I can’t log into my last account. Update: I’ll be starting an Mres looking at the mutual suppression of the cardiac versus skeletal muscle programme!

Biomedical research tries to understand among other things, how during development,
gene expression determines cell fates. One of the aspects that we look at is how cells
are recruited to the heart and how to apply this knowledge to cardiac therapy. However,
cell fate decisions in the head mesoderm, the tissue responsible for delivering the heart,
craniofacial muscle, parts of the skull and vasculature, are poorly understood.
Last summer, I had the opportunity to work with Dr. Susanne Dietrich, who studies the
formation of muscles in early development, including, but not only, genes responsible for
committing cells into a mesodermal fate. I was part of ongoing research addressing this
question, at her lab in Portsmouth.
The Dietrich lab has shown that initially, the entire head mesoderm has cardiac
competence. However, at early neurula stages of development, the cardiac inducer
Bmp2 fails to induce the cardiac programme and instead, it induces Msc, a craniofacial
precursor marker. It is not yet clear how this switch in developmental competence is
achieved.
We hypothesize that Wnt may be responsible for the switch, inducing an early expression
of craniofacial precursor markers and downregulating cardiac markers.
To test this idea, I grafted heparin-coated acrylic beads soaked in recombinant Wnt3a,
the Wnt inhibitor Sfrp2 and Bmp2 or bovine serum albumin as control, into HH7/8
embryos. Embryos were cultured for 6 hours, the time sufficient for Bmp to induce Msc.
I then used In situ hybridization to analyse the expression of Msc, the cardiac marker
Nkx2.5 and the Wnt responsive genes Pax3 and Axin2.
We found that Wnt did not upregulate and Sfrp2 did not downregulate Pax3 and Axin2
(data not shown), probably because it takes more than 6 hours to change the expression
of these genes. However, Wnt3a did downregulate Nkx2.5 as expected (Fig.1).
Nonetheless, Wnt did not upregulate Msc (Fig.1), suggesting that the concentration used
and the 6-hour time period might not have been enough for Wnt to participate in the
activation of Msc. This was against our hypothesis, and we wondered why that might be.
So, we decided to test the effect of Wnt on the paraxial head mesoderm marker
Cyp26C1, an inhibitor of retinoic acid signalling. We found that Cyp26C1 was
suppressed (Fig.1). Thus, Wnt may in fact suppress paraxial head mesoderm features.

I am now faced with new questions: What really is the role of Wnt? Does it suppress
heart and paraxial mesodermal features because it posteriorizes the tissue? To answer
this question, I will have to analyse if Wnt causes an ectopic expression of posterior
information markers (e.g: Raldh2 and Hoxb1). And our original question is not answered:
what facilitated the switch from cardiac to skeletal muscle competence? I am intrigued
by these results, and I do want to find out the right mechanism that causes this switch. I
will be working on this project throughout the next academic year, looking at different
embryonic stages, different concentrations of Wnt, different Wnt inhibitors (e.g.: Dkk) and
possibly, longer culture periods. I am hoping to find results that tell us if Wnt signalling is
or not responsible.
If Wnt is not involved, what else could it be? Many other signalling cascades converge
on the head mesoderm, and they could be tested using similar approaches. Alternatively,
I could use small molecule inhibitors of signalling cascades on embryos cultured as
Cornish pasties. We also have to consider that the epigenetic landscape might change
over time, and cardiac genes might be put out of use. This would require a different
approach, chromatin immunoprecipitation. I would love to learn about chromatin
immunoprecipitation and work on this approach during my master’s or PhD.
I want to continue working on these questions with Dr. Susanne throughout the next
years of my academic life. I hope that with my 3rd year module “genes and development”
I will gain more insight into new experimental methods used in developmental biology
and maybe use them as an approach in my project. I would love to work with different
model organisms and upgrade my knowledge with new techniques that may facilitate the
research.
Working with Dr. Susanne and her team made me grow as a scientist. I remember the
week before starting on my project I was so nervous I even had nightmares about it. But
the people in the lab were very kind and helpful and they made me feel at home. During
the summer, I was faced with some of the ups and downs of science. In situs that did not
work, embryos that were accidentally lost, beads not sticking, and all of that (Particularly
the last one), allowed me to develop my problem-solving skills and patience (especially
while grafting beads). Being part of a research group made me realize that I do not see
myself doing anything else. I love planning my experiments and I love the practical part.
I am also very interested in presenting and explaining my results to other people.
I look forward to continue working on developmental biology throughout my studies. I
plan on continue my education with Dr. Susanne, working on finding the mechanism
behind this cardiac to skeletal muscle switch, and other projects.

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