Periodic Reporting for period 1 - SyncyNucDiff (The biology of syncytial cells: Dissecting the mechanisms and functions of nuclear differentiation inside skeletal muscle syncytium)
Periodo di rendicontazione: 2022-12-01 al 2025-05-31
Numerous genetic disorders cause degeneration or severe weakning of our skeletal muscle. Moreover, the function of muscle can be impacted in various physiological situations such as exercise, ageing, diabetes and so on. However, understanding how muscle cells function and fail has been difficult, because these cells are unlike most others in the body: they are giant cells with hundreds of nuclei. In earlier research, we discovered that some of these nuclei take on specialized roles, performing distinct tasks within the same cell. We also found that new types of nuclei emerge in diseased muscles, such as in Duchenne muscular dystrophy, the most common muscle-wasting disease. These findings change how we think about muscle biology and open up new paths for study. Our EU-funded project, SyncyNucDiff, aims to understand how these different nuclear subtypes form and what they do—especially the ones linked to disease. We are also developing new genetic tools that allow us to precisely target genes in specific types of nuclei, which is not possible with conventional approaches. This work will help reshape how we study and eventually treat muscle disorders.
his project aims to uncover how different types of nuclei within a single muscle cell acquire their specialized roles and contribute to muscle health or disease. We focus especially on nuclei found at key regions of the muscle: the neuromuscular junction (NMJ), where nerves connect to muscle, and the myotendinous junction (MTJ), where muscle attaches to tendon. These regions are critical for muscle function and are known to respond to stress, aging, and injury.
To study these nuclei, we first attempted a high-resolution technique called Probe-Seq but found it inefficient and costly. As an alternative, we developed viral tools that fluorescently label NMJ and MTJ nuclei, and engineered new mouse models that enable precise genetic manipulation in these regions. With these tools in hand, we have begun sequencing the RNA of isolated NMJ and MTJ nuclei to identify which genes are active in each subtype. These datasets will guide us in understanding how these specialized identities are established and maintained.
We also explored how the unique features of these nuclei are regulated by external signals. In cultured muscle fibers, we observed that NMJ-specific genes fade quickly, while MTJ markers remain stable. Surprisingly, even well-known NMJ-stabilizing molecules like Agrin could not restore NMJ identity in vitro, suggesting that other, yet-unidentified cues are involved. We are actively investigating whether mechanical forces or specific molecules in the culture environment might play a role.
In parallel, we created new mouse lines that allow us to manipulate gene expression specifically in NMJ or MTJ nuclei. These models are compatible with genome-editing systems like CRISPR-Cas9 and allow us to test the role of key signaling pathways. While we initially focused on TGF-beta and BMP pathways, we have expanded our investigation to include others, such as Notch and Hippo, which are also important for tissue development and repair.
Finally, our work has identified a new type of nucleus that appears in muscles affected by dystrophy. We believe this nuclear subtype helps repair damaged muscle structures. To study it, we established a lab model that mimics muscle damage and repair. This model has helped us identify promising candidate genes, which are now being tested in mice to understand their role. We also attempted to block the activity of this nuclear subtype by targeting a key gene, Polr2a, but encountered technical challenges in developing the necessary mouse model. We are now pursuing an alternative strategy using updated genetic tools to overcome this issue.
Altogether, our project is making significant strides in understanding how the many nuclei inside muscle cells coordinate their roles in both health and disease. These insights—and the new tools we are building—have the potential to transform how muscle disorders are studied and eventually treated.
To study these nuclei, we first attempted a high-resolution technique called Probe-Seq but found it inefficient and costly. As an alternative, we developed viral tools that fluorescently label NMJ and MTJ nuclei, and engineered new mouse models that enable precise genetic manipulation in these regions. With these tools in hand, we have begun sequencing the RNA of isolated NMJ and MTJ nuclei to identify which genes are active in each subtype. These datasets will guide us in understanding how these specialized identities are established and maintained.
We also explored how the unique features of these nuclei are regulated by external signals. In cultured muscle fibers, we observed that NMJ-specific genes fade quickly, while MTJ markers remain stable. Surprisingly, even well-known NMJ-stabilizing molecules like Agrin could not restore NMJ identity in vitro, suggesting that other, yet-unidentified cues are involved. We are actively investigating whether mechanical forces or specific molecules in the culture environment might play a role.
In parallel, we created new mouse lines that allow us to manipulate gene expression specifically in NMJ or MTJ nuclei. These models are compatible with genome-editing systems like CRISPR-Cas9 and allow us to test the role of key signaling pathways. While we initially focused on TGF-beta and BMP pathways, we have expanded our investigation to include others, such as Notch and Hippo, which are also important for tissue development and repair.
Finally, our work has identified a new type of nucleus that appears in muscles affected by dystrophy. We believe this nuclear subtype helps repair damaged muscle structures. To study it, we established a lab model that mimics muscle damage and repair. This model has helped us identify promising candidate genes, which are now being tested in mice to understand their role. We also attempted to block the activity of this nuclear subtype by targeting a key gene, Polr2a, but encountered technical challenges in developing the necessary mouse model. We are now pursuing an alternative strategy using updated genetic tools to overcome this issue.
Altogether, our project is making significant strides in understanding how the many nuclei inside muscle cells coordinate their roles in both health and disease. These insights—and the new tools we are building—have the potential to transform how muscle disorders are studied and eventually treated.
Although these results are not yet published, we believe they mark a major step forward in the field. One of our most important accomplishments is the creation of new genetic tools—specifically, mouse lines that allow us to control genes in very specific types of nuclei within muscle cells. These so-called “Cre driver” lines target nuclei at the neuromuscular and myotendinous junctions, and they fill a long-standing gap in muscle research by enabling precise manipulation within a single muscle cell. Developing these tools exactly as we proposed shows both the feasibility and the potential impact of our approach.
Building on this success, we are now creating even more advanced tools. For example, we’re developing versions of these genetic switches that can be turned on at specific times using a drug called tamoxifen. We’re also working on an improved strategy to study a key gene, Polr2a, by engineering a backup mouse model using artificial genetic elements. These continued innovations not only expand the usefulness of our toolkit but also help maintain the project’s leading role in studying how different nuclei work together inside muscle cells. We believe these tools will benefit not just our own research, but many others working to understand muscle health and disease.
Building on this success, we are now creating even more advanced tools. For example, we’re developing versions of these genetic switches that can be turned on at specific times using a drug called tamoxifen. We’re also working on an improved strategy to study a key gene, Polr2a, by engineering a backup mouse model using artificial genetic elements. These continued innovations not only expand the usefulness of our toolkit but also help maintain the project’s leading role in studying how different nuclei work together inside muscle cells. We believe these tools will benefit not just our own research, but many others working to understand muscle health and disease.