Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration

Abstract

BackgroundTissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature “post-mitotic” myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration.ResultsFollowing subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry.ConclusionsReprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4236-y) contains supplementary material, which is available to authorized users.

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

“…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].…”

“…The regenerative blastema can originate from resident, tissue-specific stem cells or extant mature cells that are reprogrammed into a dedifferentiated state [11,12]. Following ablation of muscle, myocytes dedifferentiate and enter the cell cycle to proliferate and regenerate functional tissue [7,13,14,15].…”

“…During development in zebrafish, midkine-a, one of two paralogous midkine genes, is expressed in differentiating somites and the central nervous system [66]. In adults, midkine-a is induced during regeneration of the heart [35], fin [36], skeletal muscle [14] and retina [37,38]. Previously, we generated a Midkine-a-loss of function mutant, mdka mi5001 [39].…”

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

“…Our lab has discovered that in adult zebrafish, extraocular muscles (EOMs)–a subtype of skeletal muscle–can undergo de novo regeneration that is driven by myocyte reprogramming and dedifferentiation [ 6 ]. We have further characterized the early steps of EOM reprogramming, revealing important roles for epigenetic alterations, FGF signaling and autophagy in regulating proliferation by reprogrammed myoblasts [ 7 – 9 ].…”

Identification with SILAC Proteomics of Novel Short Linear Motifs in Demethylase Enzymes Regulated During Myoblast Differentiation

“…Myoblasts are skeletal mononuclear myocytes that are committed to the myogenic lineage but also are capable of proliferation in cell culture. During differentiation, myoblasts elongate and fuse with each other forming multinucleated, terminally differentiated myocytes, capable of contraction [2,3]. Induction of myocyte differentiation is initiated by the successive activation of signaling pathways that cooperate in the inhibition of proliferation and in the induction of differentiation via expression of several muscle differentiation-specific transcription factors, such as the basic-helix-loop muscle regulatory Myf5 protein, which is expressed first in the developing embryo, followed by MyoD, myogenin and MRF5 [4,5].…”