Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors

Abstract

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

Midkine-a functions as a universal regulator of proliferation during epimorphic regeneration in adult zebrafish

“…proliferation of blastema cells. In contrast, during the regeneration of muscle Igf is involved late, during the differentiation of newly generated myoblasts [49,61]. Our results demonstrate that the cytokine growth factor, Midkine-a, is also involved in tissue regeneration, possibly as a generic wound signal to regulate initial burst of proliferation during the regeneration of both fin and muscle ( Fig 7A and 7B and [62]).…”

“…In zebrafish, regeneration of extraocular muscles utilizes the dedifferentiation and proliferation of extant myocytes, which then differentiate into muscle cells [7,13,14,[48][49][50]. An RNAseq screen previously showed the upregulation of midkine-a during regeneration of extraocular muscle [14].…”

Autophagy Activation in Zebrafish Heart Regeneration

“…1). And, as in the tail fin 26 , skeletal muscle [27][28][29] , and infarcted murine heart 34 , increased autophagosome formation is observed under electron microscopy in the cardiac ventricle tissue near the injury margin, and is gradually downregulated to basal levels as regeneration progresses. Moreover, making use of the generated transgenic line we were capable to observe a significant accumulation of the autophagic vesicles within the regenerating tissue.…”

Twist3 is required for dedifferentiation during extraocular muscle regeneration in adult zebrafish

“…First, we determined the timing of twist3 gene expression level by Western blot and found a 1.5-fold induction of twist3 as early as 3 hpi (Fig 3I and 3J). Next, we utilized an EdU incorporation assay to test the number of proliferating myoblasts post-injury, since proliferation of dedifferentiated myoblasts represents the final step of the reprogramming process [25,37]. We found that the percentage of proliferating myoblasts (EdU-positive nuclei) in twist3 MOinjected fish was significantly reduced at both 24 and 48 hpi compared to control MO-injected fish (Fig 3B, 3D, 3F and 3H).…”