Abstract
The development of complex musculoskeletal system requires essential interaction between muscles, bone, cartilage, soft connective tissue which includes tendons and ligaments and innervation of muscle by motor neurons. Tendon is a fibrous connective tissue that connects bone to muscle and bone to bone. Axial tendon progenitors arise from the syndetome in somites while limb tendons arise from lateral plate mesoderm. Scleraxis, a bHLH transcription factor is expressed in tendon cells.Meox2 has been shown to express in the limb mesoderm. In this study, we found that Meox2 is also necessary for the normal development of tendons and soft connective tissue. Meox2-/- neonatal mice had brittle, pale and thin tendons. Histological analysis showed mispatterned tendon tissue and reduced tendon mass. Using the Scleraxis-GFP reporter transgene, we found decreased expression of GFP in Meox2-/- in postnatal limb and tail tendons. In situ analysis of Scx RNA expression in Meox2-/- embryos confirmed the earliest tendon defects occurred at E12.5, preceding the defects observed in the Scx-/- mice.
To elucidate the mechanism, Meox2+ cells were analysed by antibody staining and compared with ScxGFP+ cells from E12.5-14.5. At E12.5, when tendon cells organise between prospective muscle and bone in the developing limb and trunk, we found Meox2 expression adjacent to (limbs) or overlapping (somites) but not co-expressed with Scx-GFP. During tendon cell condensation and differentiation at E13.5, Meox2+ cells were found in the connective tissue of the autopod and in non-overlapping expression with Scx-GFP+ cells in the zeugopod. However, at E14.5, the stage when the initial formation of mature tendons is complete, Meox2+ cells were found in Scx-GFP+ autopod tendons, whereas in the zeugopod Meox2+ cells remained non-overlapping with Scx- GFP+ cells revealing tendon diversity in the limb autopod and zeugopod. Analysis of a Meox2-nLacZ knock-in allele showed LacZ+ cells in tendons and connective tissue at E13.5, indicating that although these cells do not express endogenous Meox2 at that stage they are derived from Meox2-expressing progenitors that have contributed to the tendon lineage. We also found reduction of LacZ+ cells in the limb soft connective tissue and tendons at E15.5.
TGFβ signalling plays an essential role in the formation of muscle connective tissues and a severe tendon phenotype in TGFβ2/3 knockouts first manifest at E12.5 which coincides with the earliest tendon defect in Meox2 mutants. Dynamic expression of TGFβ2 in the majority of musculoskeletal tissues and, its involvement in the recruitment of tendon cells led us to hypothesise that the defect in Meox2 mutants may be due to the down regulation of TGFβ2 signalling. Surprisingly, TGFβ2 RNA expression in Meox2-/- at E12.5 showed increased levels in the inter-limb somites and the expression domains were markedly expanded in zeugopod. Additionally, the overexpression of TGFβ2 in vitro showed differential expression of Meox2.
Collectively, these data provide a model on the biphasic necessity of Meox2 wherein it acts extrinsically during tendon induction and organisation while intrinsically during tendon maturation.
| Date of Award | 2016 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Baljinder Mankoo (Supervisor) & Yin-biao Sun (Supervisor) |