Single‐Nucleus RNA Sequencing Identifies CREM as a Brake on Myofiber Hypertrophy in Chickens

ABSTRACT Myofiber hypertrophy is central to economically important agricultural traits and vertebrate development, yet the underlying cellular and molecular programs remain elusive. Chickens have undergone substantial divergence in muscle growth following decades of artificial selection, making them a unique model for dissecting these mechanisms at single‐cell resolution. Here, we performed singlenucleus RNA sequencing on 91 144 nuclei from postnatal chicken pectoralis major. Eight major conserved cell populations were resolved, with temporal dynamics exhibiting divergence between fast‐growing broilers and slow‐growing layers. A novel terminally differentiated myocyte subpopulation ( AK1 + myocytes) was markedly enriched in slow‐growing birds. Analysis revealed that accelerated myoblast expansion and delayed terminal differentiation are primary drivers of myofiber hypertrophy, supported by a pro‐growth niche involving IGF‐signaling FAPs and macrophage‐myogenic crosstalk. Further comprehensive analyses identified CREM as a master repressor of myofiber hypertrophy. Mechanistically, CREM directly binds and transcriptionally activates the JUN promoter, and both factors cooperatively sustain the expression of growth inhibitors MSTN, FBXO32, and TRIM63. In vivo and in vitro functional validation confirmed that CREM deletion promotes myofiber hypertrophy, increasing pectoralis major weight and percent by 29.96% and 20.50%, respectively. This study uncovers CREM as a novel brake on myofiber hypertrophy, offering fundamental insights into vertebrate skeletal muscle development.