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Behind the paper: accepting “the creative cloud” as part of the process

In this ‘behind the paper’ post, Özge Özgüç explains how curiosity-led side projects helped her keep going after a series of disappointing results, and eventually shaped her research on the rhythm of early-stage embryo development.   

Our new paper is the result of studies that I completed in the lab of Jean-Léon Maître as part of the international PhD program at the Institut Curie, in Paris, France. In the paper, we focused on cleavage stage embryos to understand how actomyosin contractility prepares the embryo for morphogenesis.

As a molecular developmental biologist by training, I was (and still am) enchanted by the transformation that every multicellular organism goes through at the very beginning of their life. It never fails to amaze me that over the course of hours, days, or months, an organism transforms from a single cell called the zygote into a huge, organized collection of cells, tissues, and organs. My fascination was further fueled when I listened to an eye-opening talk by Jean-Léon about how physical forces shape preimplantation mouse embryos. Fortunately, at the time, he had just started his lab and had an open PhD position to follow up on the phenomenon that he discovered during his postdoc: periodic cortical waves of contractions (PeCoWaCo). I gladly joined his group and started to look into the mysteries of PeCoWaCo.

Over the course of my PhD, the project changed a couple of times and, mentally, it was quite challenging to cope with. After spending half a year optimizing a small interfering RNA screen to characterize PeCoWaCo with no success, my initial enthusiasm was gone. Jean-Léon saw my struggle to keep motivated after all the failures and suggested that I explore other aspects as a distraction, hoping to spark my enthusiasm once again. This was when I realized the importance of having an attentive supervisor!

As a distraction, we decided to play with cell sizes and see what happens to PeCoWaCo. As previous studies had described how cell shape may be an important regulator of contractility, we were expecting to see a scaling between PeCoWaCo and cell size. Thanks to this “distracting” side project, I finally had some interesting results to puzzle over. This distraction became the main focus for me, as we saw how robust the PeCoWaCo was to geometrical changes, with neither its period nor its velocity changing with increasing or decreasing cell sizes. How could that be? Finding this intriguing aspect gave me some new motivation, and I wanted to share it with the scientific community to discuss the possible mechanism behind the phenomenon.

Diagram showing how period and velocity of PeCoWaCo are stable across a broad range of cell sizes
Robustness of PeCoWaCo: Period (T) and velocity (V) of PeCoWaCo are stable across a broad range of cell sizes achieved by fusing or fragmentating 16-cell stage blastomeres.
Image credit

Image by the author of this blog post, Ozge Ozguc

Although this robustness was pointing at an interesting aspect of PeCoWaCo and we were getting into lots of interesting discussions with scientists from different fields, after 2 years of trying to understand what this was, we couldn’t find an answer. I felt lost and it was the lowest point for me throughout my PhD. One day when I was discussing this with Jean-Léon, he mentioned “the cloud”, a story from Uri Alon. In his talk, Uri Alon beautifully described exactly what I was going through. Hearing that this is something that everyone can go through was a relief. However, considering that I had a limited time to complete my PhD, I still felt heavyhearted. A lucky breakthrough came 6 months before handing in my thesis when we thought “it would be a nice introduction if we could show the starting point of these oscillations”, expecting this to begin at the 8-cell stage. However, we saw something that we didn’t expect and that led us to rephrase my question. From that point onwards, the project found an exit from the cloud and results rapidly followed each other.

By looking at the cleavage stages during which cells reduce in size in a stepwise fashion, we observed that actomyosin contractions are already present, manifesting themselves through periodic contractions at the zygote stage. Moreover, being able to look at the actomyosin contractility at these stages showed us that there are some tangible developments, such as increased amplitude from the 2-cell to 4-cell stages and the accelerating rhythm of PeCoWaCo throughout cleavage stages. These two points suggested to us that we can use PeCoWaCo that are manifested on different time scales than the other morphogenetic movements as a tool to study changes in the actomyosin cortex during cleavage stages.

Our previous results concerning cell size manipulations also helped us to understand that the characteristics of PeCoWaCo depend on a given developmental stage and not cell size, which also changes during development. To follow up on the developmental stage dependency of PeCoWaCo, we mapped the surface tension of cells, as actomyosin contractility generates a significant portion of the surface tension of animal cells. This revealed cortical softening throughout cleavage stages. Also, artificially softening the cortex can trigger PeCoWaCo earlier and accelerate the rhythm. Following the timely reviewers’ suggestions and an educated guess, we looked at the protein Fmnl3 as a possible molecular explanation for this change in behavior. We found that Fmnl3 down-regulation during cleavage stages is required for the softening of the cortex, which elicits the appearance of PeCoWaCo. 

Diagram showing how PeCoWaCo changes during embryo development
Maturation of actomyosin cortex and its contractility: Surface tension and PeCoWaCo period reduces in a stepwise manner through cleavage stages. Reducing or increasing surface tension artificially is sufficient to trigger or delay PeCoWaCo initiation and also change the PeCoWaCo period accordingly.
Image credit

Image by the author of this blog post, Ozge Ozguc

After all the problems and the cul-de-sac we had gone through with this project, I am happy to see it in its final form. Looking back, this journey has taught me that being in “the creative cloud” is perfectly normal, and that the easiest way out is to accept it as a part of the research experience and to talk to people to get different points of views. It has also taught me to always keep in mind that if the research project had gone according to plan, then the essence of the work would not have been to uncover the unknown. 

Image credit

Image by the author of this blog post, Ozge Ozguc

About the author

Image of the author

Özge Özgüç completed her PhD at Institut Curie and is now a postdoctoral researcher at IBEC. ORCID:  0000-0002-1545-1715  @oozguc

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