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

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.