Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas

Abstract

Background: Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population. Methods: In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers. Results: We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7 −/− teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7−/− teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7−/− ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7−/− teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7−/− teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7−/− teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7−/− teratomas was impaired.

CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation

“…The first striking observation was that even after 3 days in differentiation media, NRMT1 knockout (KO) C2C12 cells lacked expression of the master regulator of postnatal myogenesis, Pax7 (Figure 5(b)). The transcription factor Pax7 and its paralog Pax3 regulate myogenic precursor cell differentiation through their downstream targets, the myogenic regulatory factors, which include myogenic factor 5 (Myf5), myogenic differentiation 1 (MyoD), muscle-specific regulatory factor 4 (Myf4), and myogenin (MyoG) 51 . Accordingly, MyoG expression was also minimally expressed over the differentiation period (Figure 5 progenitor pool that can also give rise to osteoblasts, adipocytes, and chondrocytes 52 .…”

Mislocalisation of Pax7 and Its Interaction with Importin-α1 (KPNA2) in Dystrophin-Deficient Myoblasts

“…It has been reported that Pax7 expression is crucial for the maintenance and self-renewal of myoblasts (19,20). Maintaining Pax7 expression retains the transcriptional regulation of Pax7 for myogenesis and for downregulation at the onset of muscle differentiation (21,22).…”

PAX7 Balances the Cell Cycle Progression via Regulating Expression of Dnmt3b and Apobec2 in Differentiating PSCs

“…Many studies validated this model as a tool to test pluripotency of mouse [ 17 , 18 ] or human cells [ 19 , 20 , 21 ], including first iPSCs derived (e.g., [ 22 , 23 ]). It was also applied to study genetically modified PSCs (e.g., [ 24 , 25 ]).…”

“…In mice, ablation of this factor leads to early postnatal lethality [ 26 ]. Lack of the functional PAX7, however, does not prevent myogenesis but affects its progression ([ 4 , 25 , 27 , 28 ]). Importantly, in Pax7 -null mice the number of muscle-specific stem cells, i.e., satellite cells (SCs), is lower in skeletal muscles [ 29 , 30 ] and tunica muscularis of the esophagus [ 31 ].…”

“…Moreover, PAX7 was shown to inhibit apoptosis [ 28 ] since its absence leads to increased mortality of SCs [ 34 ] as well as rhabdomyosarcoma cells [ 44 ]. Cell cycle phenotype of Pax7 −/− ESCs was also suggested by in vivo analyses of teratomas, e.g., in the absence of functional PAX7 teratoma weight increased [ 25 ]. However, detailed studies on the cell cycle regulation using teratoma in vivo model was not presented, so far.…”