Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA, 15219-3143, USA.
Present Address: Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
BMC Dev Biol. 2021 Jul 5;21(1):10. doi: 10.1186/s12861-021-00241-9.
Mice with a loss of function mutation in Wdpcp were described previously to display severe birth defects in the developing heart, neural tube, and limb buds. Further characterization of the skeletal phenotype of Wdpcp null mice was limited by perinatal lethality.
We utilized Prx1-Cre mice to generate limb bud mesenchyme specific deletion of Wdpcp. These mice recapitulated the appendicular skeletal phenotype of the Wdpcp null mice including polydactyl and limb bud signaling defects. Examination of late stages of limb development demonstrated decreased size of cartilage anlagen, delayed calcification, and abnormal growth plates. Utilizing in vitro assays, we demonstrated that loss of Wdpcp in skeletal progenitors lead to loss of hedgehog signaling responsiveness and associated proliferative response. In vitro chondrogenesis assays showed this loss of hedgehog and proliferative response was associated with decreased expression of early chondrogenic marker N-Cadherin. E14.5 forelimbs demonstrated delayed ossification and expression of osteoblast markers Runx2 and Sp7. P0 growth plates demonstrated loss of hedgehog signaling markers and expansion of the hypertrophic zones of the growth plate. In vitro osteogenesis assays demonstrated decreased osteogenic differentiation of Wdpcp null mesenchymal progenitors in response to hedgehog stimulation.
These findings demonstrate how Wdpcp and associated regulation of the hedgehog signaling pathway plays an important role at multiple stages of skeletal development. Wdpcp is necessary for positive regulation of hedgehog signaling and associated proliferation is key to the initiation of chondrogenesis. At later stages, Wdpcp facilitates the robust hedgehog response necessary for chondrocyte hypertrophy and osteogenic differentiation.
先前已经描述过，WDPCP 功能丧失突变的小鼠在发育中的心脏、神经管和肢芽中表现出严重的出生缺陷。WDPCP 缺失小鼠骨骼表型的进一步特征受到围产期致死性的限制。
我们利用 Prx1-Cre 小鼠生成肢芽间质特异性的 WDPCP 缺失。这些小鼠重现了 WDPCP 缺失小鼠的附肢骨骼表型，包括多指和肢芽信号缺陷。对肢芽晚期发育的检查表明软骨原基体积减小、钙化延迟和生长板异常。利用体外实验，我们证明了骨骼祖细胞中 WDPCP 的缺失导致 Hedgehog 信号反应性丧失以及相关的增殖反应丧失。体外软骨生成实验表明，这种 Hedgehog 和增殖反应的丧失与早期软骨形成标记物 N-钙粘蛋白表达减少有关。E14.5 前肢表现出骨化延迟和成骨细胞标记物 Runx2 和 Sp7 的表达。P0 生长板表现出 Hedgehog 信号标记物的丧失和生长板肥大区的扩张。体外成骨实验表明，WDPCP 缺失的间充质祖细胞对 Hedgehog 刺激的成骨分化减少。
这些发现表明，WDPCP 及其对 Hedgehog 信号通路的调节如何在骨骼发育的多个阶段发挥重要作用。WDPCP 是 Hedgehog 信号正向调节所必需的，而相关的增殖是起始软骨形成的关键。在后期，WDPCP 促进了肥大软骨细胞和成骨分化所需的强烈 Hedgehog 反应。
