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The human natural killer-1 (HNK-1) carbohydrate epitope, composed of a unique sulfated trisaccharide (HSO3–3GlcAβ1–3Galβ1–4GlcNAc-R), is highly expressed during brain development and regulates higher brain function. However, it remains unclear which glycoprotein carries the HNK-1 epitope in the embryonic brain and the functional role it plays. Here, we showed that one of the major HNK-1 carrier proteins in the embryonic brain is tenascin-C (TNC), an extracellular matrix protein that regulates neurite outgrowth by interacting with the GPI-anchored protein contactin-1 (CNTN). Because the alternatively spliced fibronectin type-III (FNIII) repeats in TNC give rise to many isoforms and affect neuronal function, we evaluated neurite outgrowth of primary hippocampal neurons on purified recombinant FNIII repeats with or without the HNK-1 epitope as a substrate. We found that the presence of the HNK-1 epitope on the C domain of TNC promoted neurite outgrowth, and that this signal was mediated by CNTN, which is an HNK-1-expressing neuronal receptor. The neurite-promoting activity of the HNK-1 epitope on TNC required neuronal HNK-1 expression, which was defective in neurons lacking the glucuronyltransferases GlcAT-P and GlcAT-S. These results suggest that the HNK-1 epitope is a key modifier of TNC and CNTN in the regulation of embryonic brain development.

Muscle stem cells, also called muscle satellite cells (MuSCs), are responsible for skeletal muscle regeneration and are sustained in an undifferentiated and quiescent state under steady conditions. The calcitonin receptor (CalcR)-protein kinase A (PKA)-Yes-associated protein 1 (Yap1) axis is one pathway that maintains quiescence in MuSCs. Although CalcR signaling in MuSCs has been identified, the critical CalcR signaling targets are incompletely understood. Here, we show the relevance between the ectopic expression of delta-like non-canonical Notch ligand 1 (Dlk1) and the impaired quiescent state in CalcR-conditional knockout (cKO) MuSCs. Dlk1 expression was rarely detected in both quiescent and proliferating MuSCs in control mice, whereas Dlk1 expression was remarkably increased in CalcR-cKO MuSCs at both the mRNA and protein levels. It is noteworthy that all Ki67+ non-quiescent CalcR-cKO MuSCs express Dlk1, and non-quiescent CalcR-cKO MuSCs are enriched in the Dlk1+ fraction by cell sorting. Using mutant mice, we demonstrated that PKA-activation or Yap1-depletion suppressed Dlk1 expression in CalcR-cKO MuSCs, which suggests that the CalcR-PKA-Yap1 axis inhibits the expression of Dlk1 in quiescent MuSCs. Moreover, the loss of Dlk1 rescued the quiescent state in CalcR-cKO MuSCs, which indicates that the ectopic expression of Dlk1 disturbs quiescence in CalcR-cKO. Collectively, our results suggest that ectopically expressed Dlk1 is responsible for the impaired quiescence in CalcR-cKO MuSCs.

Skeletal muscle has attracted attention as endocrine organ, because exercise-dependent cytokines called myokines/exerkines are released from skeletal muscle and are involved in systemic functions. While, local mechanical loading to skeletal muscle by exercise or resistance training alters myofiber type and size and myonuclear number. Skeletal muscle-resident stem cells, known as muscle satellite cells (MuSCs), are responsible for the increased number of myonuclei. Under steady conditions, MuSCs are maintained in a mitotically quiescent state but exit from that state and start to proliferate in response to high physical activity. Alterations in MuSC behavior occur when myofibers are damaged, but the lethal damage to myofibers does not seem to evoke mechanical loading-dependent MuSC activation and proliferation. Given that MuSCs proliferate without damage, it is unclear how the different behaviors of MuSCs are controlled by different physical activities. Recent studies demonstrated that myonuclear number reflects the size of myofibers; hence, it is crucial to know the properties of MuSCs and the mechanism of myonuclear accretion by MuSCs. In addition, the elucidation of mechanical load-dependent changes in muscle resident cells, including MuSCs, will be necessary for the discovery of new myokines/exerkines and understating skeletal muscle diseases.

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