Gene dosage-dependent roles of Mkx in postnatal tendon development and maintenance revealed by conditional deletion

Lin Liu; Tomoki Chiba; Takahide Matsushima; Maiko Inotsume; Tomomi Kato; Yuichi Hiraoka; Hiroshi Asahara

doi:10.1016/j.ydbio.2025.06.022

Gene dosage-dependent roles of Mkx in postnatal tendon development and maintenance revealed by conditional deletion

Dev Biol. 2025 Jun 21:526:15-25. doi: 10.1016/j.ydbio.2025.06.022. Online ahead of print.

Authors

Lin Liu¹, Tomoki Chiba¹, Takahide Matsushima¹, Maiko Inotsume¹, Tomomi Kato¹, Yuichi Hiraoka², Hiroshi Asahara³

Affiliations

¹ Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Institute of Science Tokyo, Tokyo, Japan.
² Department of Molecular Neuroscience, Medical Research Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan; Laboratory of Genome Editing for Biomedical Research, Medical Research Institute, Institute of Science Tokyo, Tokyo, Japan.
³ Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Institute of Science Tokyo, Tokyo, Japan; Department of Molecular and Cellular Biology, The Scripps Research Institute, La Jolla, USA. Electronic address: asahara.syst@tmd.ac.jp.

PMID: 40550378
DOI: 10.1016/j.ydbio.2025.06.022

Abstract

The transcription factor Mohawk (Mkx) contributes to tendon development and differentiation, as demonstrated by conventional knockout studies. However, the temporal requirements and gene dosage effects of Mkx in postnatal tendon maturation and maintenance remain unclear. To address these questions, we generated a novel conditional knockout mouse model harboring a loxP-flanked allele and a Venus-CreERT2 knock-in allele at the Mkx locus by crossing Mkx^{Venus-CreERT2/+} with Mkx^flox/+ lines. Tamoxifen was administered at two distinct stages: early postnatal (P3) and adult (6-week-old). Mkx^{Venus-CreERT2/+} mice exhibited mild reductions in tendon thickness and alterations in collagen fibril organization, while conditional deletion of Mkx (Mkx^{Venus-CreERT2/flox} with tamoxifen induction) resulted in more pronounced defects. Time-course analysis revealed that both early postnatal and adult Mkx deletion led to progressive changes in tendon morphology, with TEM analysis showing a tendency toward reduced collagen fibril diameters. RNA-seq revealed distinct transcriptional changes associated with ECM organization and tendon homeostasis in Mkx-deficient tendons. These findings reveal previously unrecognized gene dosage effects of Mkx and suggest that maintaining appropriate levels of Mkx may be critical for tendon homeostasis, providing new insights into tendon biology and potential therapeutic strategies for tendon-related pathologies.

Keywords: Conditional knockout mouse; Gene dosage effects; Haploinsufficiency; Mohawk (mkx); Tendon development; Tendon homeostasis.