Objectives Given the role of growth and differentiation factor 5 (GDF5) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how its regulatory regions control knee formation and morphology. Methods The brachypodism (bp) mouse line, which harbours an inactivating mutation in Gdf5, was used to survey how Gdf5 loss of function impacts knee morphology, while two transgenic Gdf5 reporter bacterial artificial chromosome mouse lines were used to assess the spatiotemporal activity and function of Gdf5 regulatory sequences in the context of clinically relevant knee anatomical features. Results Knees from homozygous bp mice (bp/bp) exhibit underdeveloped femoral condyles and tibial plateaus, no cruciate ligaments, and poorly developed menisci. Secondary ossification is also delayed in the distal femur and proximal tibia. bp/bp mice have significantly narrower femoral condyles, femoral notches and tibial plateaus, and curvier medial femoral condyles, shallower trochlea, steeper lateral tibial slopes and smaller tibial spines. Regulatory sequences upstream from Gdf5 were weakly active in the prenatal knee, while downstream regulatory sequences were active throughout life. Importantly, downstream but not upstream Gdf5 regulatory sequences fully restored all the key morphological features disrupted in the bp/bp mice. Conclusions Knee morphology is profoundly affected by Gdf5 absence, and downstream regulatory sequences mediate its effects by controlling Gdf5 expression in knee tissues. This downstream region contains numerous enhancers harbouring human variants that span the osteoarthritis association interval. We posit that subtle alterations to morphology driven by changes in downstream regulatory sequence underlie this locus’ role in osteoarthritis risk.

中文翻译：

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广泛调控区域对膝关节发育和骨关节炎易感性中 Gdf5 表达和功能的影响

目的 鉴于生长和分化因子 5 (GDF5) 在膝关节发育和骨关节炎风险中的作用，我们试图表征 Gdf5 功能丧失导致的膝关节缺陷，以及其调节区域如何控制膝关节形成和形态。方法 使用含有 Gdf5 失活突变的短足 (bp) 小鼠系来研究 Gdf5 功能丧失如何影响膝关节形态，同时使用两个转基因 Gdf5 报告细菌人工染色体小鼠系来评估 Gdf5 的时空活性和功能。临床相关膝关节解剖特征背景下的 Gdf5 调控序列。结果 纯合 bp 小鼠 (bp/bp) 的膝盖表现出股骨髁和胫骨平台发育不全，没有十字韧带，半月板发育不良。股骨远端和胫骨近端的二次骨化也被延迟。bp/bp 小鼠的股骨髁、股骨切迹和胫骨平台明显更窄，股骨内侧髁更弯曲，滑车更浅，胫骨外侧坡度更陡，胫骨棘更小。Gdf5 上游的调控序列在产前膝关节中活性较弱，而下游调控序列在整个生命周期中都处于活跃状态。重要的是，下游但不是上游的 Gdf5 调控序列完全恢复了 bp/bp 小鼠中被破坏的所有关键形态特征。结论 Gdf5 缺失严重影响膝关节形态，下游调控序列通过控制膝关节组织中 Gdf5 的表达来介导其影响。该下游区域包含许多含有跨越骨关节炎相关区间的人类变异的增强子。我们认为，由下游调控序列的变化驱动的形态的微妙改变是该位点在骨关节炎风险中的作用的基础。