ASB5 is a specific marker for muscle satellite cells but dispensable for skeletal muscle development and regeneration

Skeletal muscle plays a crucial role in human life, contributing to posture, movement, nutrient storage, and body temperature regulation. Development and regeneration of skeletal muscles rely on embryonic myogenic progenitors and postnatal satellite cells (MuSCs), respectively. Identification of new molecular markers and elucidating their functions in MuSCs will provide better understanding of muscle development and regeneration.

We surveyed single cell RNA-seq (scRNA-seq) data (Tabula Muris and GSE150366) to identify ASB5 (Ankyrin repeat and Suppressor of cytokine signaling Box containing 5) as a marker of MuSCs. We also used CRISPR-CAS9 genome editing and oviduct electroporation to generate a germline knockout (KO) mouse line of Asb5. We then analyzed the muscle growth and regeneration of the KO mice. We further analyzed proliferation and differentiation of MuSCs attached on myofibers. We finally performed Realtime PCR (qPCR) to examine how Asb5 KO affects gene expression in the skeletal muscle.

Analysis of data publicly available at Tabula Muris identified Asb5 as a specific marker of MuSCs. Further analysis of scRNA-seq data on FACS-purified MuSCs at various regeneration time points revealed that Asb5 is highly expressed in MuSCs and their progenies across various stages of muscle regeneration. We then generated a novel Asb5 KO mouse line through CRISPR-Cas9 deletion of Exon 4. The Asb5-KO mice were born normally and exhibited normal postnatal growth. In addition, Asb5-KO MuSCs proliferated, differentiated and self-renewed normally on myofiber explants. Furthermore, the skeletal muscles of Asb5-KO mice regenerated normally after acute injury. qPCR analysis showed that Asb5 KO reduces the expression levels of Tnfa (Tumor Necrosis Factor Alpha) in the skeletal muscles.

These data together identify ASB5 as an abundantly expressed and specific marker of MuSCs and myogenic progenitors. However, Asb5 loss-of-function has no effects on embryonic development and postnatal growth of skeletal muscles, or behavior and regenerative functions of MuSCs under normal physiological conditions.