Muscle Stem Cell Markers Proteins Background
Skeletal muscle makes up a large portion of human body mass. For adult males, on average more than 40% of the body mass is skeletal muscle and for adult females the percentage is ~35%. Skeletal muscle, together with neural system, is responsible for body movement. More than 600 muscles compose the skeletal muscle system and usually these muscles work in groups to achieve precise movements. Skeletal muscle cell, also known as single myofiber, is a terminal differentiated cell, which is multinucleated and acts as the smallest complete unit for muscle contractile. In adult skeletal muscles, upon injury or mechanical stress, regeneration is activated and this process is mediated by muscle satellite cells (SCs). Muscle SCs were discovered in 1961. The mononucleated cells which locate closely to myofibers were discovered by electron microscope and named as satellite cells. After the identification of SCs, their functions in skeletal myogenesis have been studied in the past half century.
Muscle SCs, also known as muscle stem cells, reside under the basal lamina of single myofibers and stay in a quiescent state for most of their lifetime. Muscle SCs are the source of postnatal muscle growth and adult muscle regeneration. In early postnatal mouse skeletal muscles, about 30% of the sublaminal nuclei of myofibers are SCs while this percentage decreases to only ~4% in adult mouse muscles. Upon muscle damage, the quiescent satellite cells (QSCs) can be activated and re-enter cell cycle for proliferation. A small portion of the activated satellite cells (ASCs) maintain the self-renewal ability and return to their niche under the basal lamina, for which SCs are considered as bona fide adult stem cells. The majority of ASCs are committed to express myogenic genes and undergo myogenic differentiation, forming new fibers to fix the muscle damages. Muscle SCs express distinctive transcription factors, surface markers and adhesion molecules such as Pax7, Vcam, CXCR4, Integrin β1, m-cadherin etc.
Adult SCs specifically expresses transcription factor Pax7, which is crucial for SCs biogenesis, proliferation, maintenance and the regulation of myogenic differentiation. Myogenic regulatory factors (MRFs), including MyoD, Myf5, Myogenin and Mrf4, are a group of basic helixloop-helix(bHLH) transcription factors which recognize specific DNA binding domain E-box and form heterodimers with E-protein to bind with DNA. MRFs are vital in initiating and regulating transcription of muscle lineage genes and muscle differentiation in committed ASCs. Majority QSCs only have Pax7 expression without MRF expression. Committed ASCs express MyoD and Myf5, followed by activation of Myogenein and Mrf4, which induce expression of myogenic genes and terminal differentiation. During the ASCs differentiation, the level of Pax7 decreases significantly and no Pax7 is expressed in myofibers. This adult muscle regeneration process involves the dynamic activation and repression of a lot of genes related with cell cycle and myogenesis. Gene transcription is tightly orchestrated by the transcriptional and epigenetic regulatory networks involving transcription factors, chromatin modification and remodeling, DNA methylation and non-coding RNAs.
Pax7 in muscle stem cells
Pax7 was identified as the hallmark of adult muscle satellite cells by immunotypic analysis. Pax gene family is a group of conserved transcription factors which play important role in organogenesis and tissue homeostasis. Both Pax3 and Pax7 are closely related with specification and maintenance of skeletal muscle progenitors but their expression are not restricted to skeletal muscle lineage. Pax3 is expressed in myogenic progenitor cells and mainly regulates embryonic skeletal myogenesis during development. The expression level of Pax3 decreases before birth and in adult Pax3 expression is restricted to a subpopulation of SCs in specific muscles groups. In the embryo Pax7 only exists in the central domain of dermomyotome. In adult muscle Pax7 is expressed in QSCs and ASCs and is rapidly down-regulated when cells enter terminal myogenic differentiation. Functionally Pax7 is important for postnatal myogenesis and adult muscle regeneration. Inducible inactivation of Pax7 in SCs at postnatal stage resulted in dramatic impair of muscle regeneration caused by cardiotoxin. Inducible deletion of Pax7 in Pax7 expressing cells of adult mice resulted in significant loss of the number of Pax7+ SCs on myofibers. The number of CD34+ SCs did not change in the short time after induction but it was declined strikingly at 30 and 60 days post induction, suggesting SCs lacking Pax7 could maintain some features of SCs for several weeks but not in the long run. Deletion of Pax7 in SCs impaired adult muscle regeneration, indicating that continuous expression of Pax7 in SCs during regeneration was necessary for complete muscle repair. The inactivation of Pax7 in SCs also inhibited cell proliferation, cell cycle progression and promoted terminal differentiation. Moreover, deletion of Pax7 in adult SCs resulted in increasing number of atypical SCs with reduced heterochromatin condensation, suggesting that Pax7 might affect chromatin structure at the epigenetic level.
By genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) techniques, the target binding sites of Pax7 were identified and several studies provided mechanistic insights into the regulation of Pax7 on muscle regeneration. In myoblast Pax7 can target MyoD promoter and recruit Pol II to form a preinitiation complex to activate the transcription of MyoD. Pax7 can also directly regulate Myf5 transcription by binding at the promoter region of Myf5 and recruiting Wdr5- Ash2L-MLL2 histone methyltransferase complex. This interaction causes H3 lysine 4 methylation and activates the expression of Myf5. Pax7 ChIP-seq revealed several conserved binding sites across the Myf5 regulatory region and the binding locus at the -110kb was essential for Myf5 expression in adult QSCs. A novel protein Pax7- and Pax3-binding protein (Pax3/7BP) was identified in myoblast, which served as an indispensable adaptor bridging Pax7 and Wdr5, helping the recruitment of Wdr5-Ash2L-MLL2 histone methyltransferase complex. Together with Pax7, Pax3/7BP could regulate the transcription of Id3 and Cdc20. Pax7 has also been shown to directly regulate Id2 and Id3, the repressor of bHLH transcription factors, preventing the initiation of myogenesis in QSCs. Pax7 ChIP-seq data in myoblast derived from cultured SCs provides massive information of transcriptional regulation by Pax7 at the genome-wide level. The genes which may be regulated by Pax7 were significantly enriched in GO terms associated with cell proliferation, hepatocyte growth factor receptor signaling, mitosis, regulation of glucagon secretion and apical junction assembly. Microarray data of Pax7 over-expressing myoblast showed that indeed many genes related to cell growth and proliferation, such as Fgfr2, Egfr, BMP4 etc., were upregulated by Pax7 and muscle specific genes were down-regulated such as Myh1, Mef2c, Myogenin etc. Although current studies suggest Pax7 may regulate many genes related with cell proliferation and myogenesis in SCs during adult muscle regeneration, much remains to be understood at the molecular mechanistic level.