Satellite cells do not mediate zebrafish extraocular muscle: when an initial negative result guides you to a more scientifically relevant finding

Degenerative and atrophic muscle diseases such as muscular dystrophies, as well as extensive muscle damage or loss related to trauma, tumor resections and other conditions, represent one of the most important public health concerns. It is estimated that the combined cost of the relatively uncommon disorders amyotrophic lateral sclerosis, Duchenne muscular dystrophy, and myotonic muscular dystrophy is $1.07 to $1.37 billion per year in the USA. Although global estimates are difficult, the total cost of degenerative and traumatic muscle conditions would be substantially higher. Right now the only available treatments for these pathologies are palliative therapies that do not solve the underlying cause of the disease.

In contrast to mammals, salamanders and fish has the capacity of regenerating complex tissues. Thus, zebrafish has become a popular model to study tissue regeneration because are highly regenerative and very amenable to both forward and reverse genetic approaches. To provide foundational transferable knowledge to develop new pharmacological and therapeutic targets, we have described a new model of muscle regeneration using adult zebrafish extraocular muscles. We found that after amputating 50% of the lateral rectus (extraocular muscle chosen as a proof of principle) zebrafish regenerate an anatomically correct and functional muscle in 7 to 10 days (Fig. 1):

Mammalian muscles are considered post-mitotic meaning that they have completely differentiated in mature muscle cells and will never enter the cell cycle again. Therefore, muscle growth and repair are achieved by myogenic progenitor cells termed satellite cells. Therefore, as a first step to understand the mechanisms that control zebrafish muscle regenerative response we looked for the satellite cells, the usual suspects in these matters. We used Pax7, a transcription factor expressed only in muscle satellite cells, to identify the satellite cells in regenerating muscles (Fig. 2). We were able to find Pax7-positive cells (satellite cells) in somatic muscle, both embryonic (Fig. 2A) and adult (Fig. 2D), and embryonic extraocular muscles (Fig. 2B, C). Surprisingly, we could not detect satellite cells in the extraocular muscles of adult zebrafish, either uninjured (Fig. 2E, F) or regenerating after amputation (Fig. 2 G, H):

Because extraocular muscles satellite cells do not do not express Pax7 in some species, we tried to identify these cells using a different method. Classic satellite cells localize between the basal lamina and sarcolemma of muscle fibers. Thus, we used electron microscopy to identify putative satellite cells following this morphologic criterion. Again, classic satellite cells could not be detected in adult zebrafish extraocular muscles but we identified rare cells that might represent a type of ‘‘post satellite cells’’. These cells are muscle progenitor cells fully encapsulated by the basal lamina described in newts.

Combining the fact that we could not detect satellite cells using their most typical marker, that even these so-called ‘‘post’’ satellite cells in newts express Pax7 and that newt limb regeneration also appears to be mostly independent of Pax7-positive cells we concluded that the regeneration of adult extracellular muscles is not mediated by classic satellite cells.

This initial negative result forced us to elaborate a multidisciplinary research approach (including whole-mount fluorescent imaging, cellular tracing, molecular and histological techniques) to decipher how zebrafish extraocular muscles regenerate after injury. We found a mechanism that goes against the accepted dogma in the muscle biology field that postulates that muscle fibers are post-mitotic and therefore will never enter the cell cycle again.

More details about these results and the following investigations in zebrafish extraocular muscle regeneration can be found in Dr. Saera-Vila scientific papers (http://bit.ly/2bDI3me, http://bit.ly/2bu69Cz and http://bit.ly/2baG5dm)  

Written by Dr. Alfonso Saera for The All Results Journals.