The Function And Mechanism Of Dll1 In The Development And Regeneration Of Mouse Skeletal Muscle

Notch signal pathway is one of the major regulatory pathways that regulate multicellular development,mainly by dictating the fate of one cell in relation to that of its neighboring cell.The Notch signaling is an evolutionarily conserved pathway that is of great importance for cell-cell communication and cell-fate determination during development,and is required for adult tissue homeostasis.Canonical Notch signaling is initiated by Notch ligands（Delta1,3,4and Jagged1,2）binding to neighbor cell Notch receptors（Notch1,2,3,4）to release Notch intercellular domain and translocated to nuclear with binding Rbpj and then activate the transcription of target gene.Various studies have demonstrated Notch is crucial for embryonic muscle development including somite.During the fetal development,Notch signaling can regulate the localization of muscle stem cell into basal lamina.After postnatal,Notch signaling maintain the SCs quiescence,SCs self-renewal and inhibit SCs differentiation.As we know,there have 5 ligands of Notch signaling.However,which ligand is the most crucial during muscle development and regeneration is still unknown.Therefore,this study was conducted to investigate which ligand has the most important function in muscle and how it works.First,we analyzed the 5 ligands expression level and pattern in muscle through online database and q RT-PCR.Next,we applied cre-loxp system to knockout Dll1 in mouse muscle tissue and analyze their phenotype.After that we investigate how the ligand affects the muscle stem cell.This study provides us a clear mechanism of Notch pathway in muscle.The results are as follows:1.Through analyzing muscle database,we found Dll1 and Jag1 are highly expressed in muscle tissue and muscle stem cells.During differentiation,Dll1 and Dll4 were upregulated dramatically;Jag1 expression were also increased but exhibited no significant alternation.2.Through“cre-loxp”system,we constructed the MLC^Cre-Dll1 KO(Dll1^MLCKO)mice.The Dll1^MLCKOmuscle develops normally during embryonic and postnatal stage.CTX injury experiment also showed KO mice have the similar regeneration ability and SCs number with WT mice.Treadmill test result suggested that deletion of Dll1 in fiber didn’t influence muscle exercise performance.3.The Myo D^Cre-Dll1(Dll1^{Myo DKO})embryos were dead at～15d.The muscle HE and immunofluorescent staining showed Dll1^{Myo DKO}mice have serve muscle hypotrophy and muscle stem cell exhaustion.Muscle stem cell gradually decreased during later stage somite development and limb-bud development which accompanied with Myo G positive cell proportion transient upregulation.4.Meanwhile,deletion of Dll1 in muscle stem cell after postnatal decreased the muscle weight,muscle fiber size and SCs numbers.CTX induce the juvenile mice TA muscle injury for 5day and 21days showed deletion of Dll1 impaired regeneration capacity at 5 days after injury.Whereas the 21dpi injured muscle in Dll1^Pax7KOmice were regenerated completely and similar with WT mice,but the SCs number significantly decreased in KO mice.SCs specific deletion of Dll1 disrupted the adult mice SCs homeostasis.After CTX injection for 8 and28days,the injured TA muscle in Dll1^Pax7KOmice exhibited the similar regeneration ability with WT mice,but SCs number decreased significantly.With three independent CTX injection trials were repeated in Dll1^Pax7KOmice,we found the muscle regeneration has severe defects accompanied with exhaustion of SCs pool.5.In vivo and vitro experiments suggested that deletion of Dll1 decreased the SCs self-renewal,have no significant influence on myoblast proliferation but increased the SCs differentiation.6.Coculture experiments suggested Dll1 KO phenotype was not rescued when cocultured with WT myoblast.In summary,Dll1 in sarcolemma is dispensable for muscle development and muscle stem cell function.Whereas,the muscle stem cell derived Dll1 is indispensable for the muscle stem cell and its function.Mechanically,Dll1 regulates muscle stem cells by inhibiting their premature differentiation and promoting their self-renewal.Meanwhile,muscle stem cell derived Dll1 does not rely on"cross-talk"with macrophages and endothelial cells feedback muscle stem cell,but depend on a cell autonomous cis-activation manner to active Notch signaling to regulate muscle stem cell.