Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emergning as key regulatory proteins with great promise as therapeutic agents for bone disorders. Until recently Sost and its paralog Sostdc1 have been described as growth factors with highly restricted expression in the adult where Sost was assumed 'osteocyte-' and Sostdc1 'kidney-' specific. Here we show that these two proteins emerged throgh ancestral genome duplication and their expression patterns have diverged to span complimentary domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1-/- mice lack any obvious limb and skeletal defects, Sost-/- mice recapitulate the hand defects described for sclerosteosis patients. However, elevated WNT signaling in Sost-/-; Sostdc1-/- mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and ultimately preaxial polydactyly. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost-/- and Sost-/-; Sostdc1-/- mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanisms and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling. Five arrays were analyzed, consisting of two total embryonic fore-limb RNA (experimental) and three total embryonic forelimb RNA (reference) samples at E11.5 DPC mouse (C57Bl6 strain). Embryos were not pooled to generate samples. Each time point has 3 to 5 biological replicates for limb bud samples, duplicates for whole embryos. Comparisons were made between limb bud samples and whole embryo at the same stage, fore-limb samples of different stages, hind-limb samples of different stages, and fore-limb samples compared to hind-limb samples at the same or the next stage.WNT信号通路（WNT signaling）在骨骼发育与稳态的绝大多数环节中均发挥关键作用，而WNT信号通路的拮抗剂正逐渐成为一类关键调控蛋白，在骨骼疾病的治疗领域展现出巨大应用前景。此前学界普遍认为，硬化蛋白（Sost）及其旁系同源蛋白Sostdc1属于生长因子，在成体组织中表达范围高度受限，其中Sost被认为是“骨细胞特异性”蛋白，Sostdc1则为“肾脏特异性”蛋白。本研究证实，这两种蛋白起源于祖先基因组复制事件，且二者的表达模式已发生分化，在肌肉骨骼、心血管、神经、消化、生殖与呼吸等绝大多数器官系统中形成互补的表达域。在发育中的肢体中，Sost与Sostdc1呈现动态的表达模式：Sost仅局限于远端外胚层，而Sostdc1则分布于近端外胚层与间充质中。Sostdc1敲除（Sostdc1-/-）小鼠未表现出明显的肢体与骨骼缺陷，而Sost敲除（Sost-/-）小鼠则重现了硬化性骨病（Sclerosteosis）患者所报道的手部缺陷。然而，Sost-/-; Sostdc1-/-双敲除小鼠中升高的WNT信号通路活性会导致音猬因子（Sonic Hedgehog, SHH）信号通路调控紊乱，在第1指域出现Sox9的异位激活，最终引发轴前多指畸形（preaxial polydactyly）。此外，本研究发现，硬化性骨病（Sclerosteosis）中记录的并指畸形在Sost-/-单敲除小鼠与Sost-/-; Sostdc1-/-双敲除小鼠中均存在，其致病机制源于顶外胚层嵴（apical ectodermal ridge, AER）区域中成纤维细胞生长因子8（Fibroblast Growth Factor 8, Fgf8）的表达调控紊乱——而该区域并不表达Sost与Sostdc1。本研究揭示了WNT信号通路在骨骼生物学与疾病中的复杂性，并强调了冗余调控机制与非细胞自主效应如何协同作用，揭示出由WNT信号通路异常升高所引发的新型复杂表型。本研究共分析了5组芯片微阵列数据，样本取自C57Bl6品系小鼠胚胎发育第11.5天（days post coitum, DPC），其中包含2份胚胎前肢RNA样本（实验组）与3份胚胎前肢RNA样本（对照组）。样本未通过混合胚胎制备得到。同一时间点的肢芽样本设置3~5次生物学重复，全胚胎样本设置2次生物学重复。研究中开展了多组比较：同一发育阶段的肢芽样本与全胚胎样本的比较、不同发育阶段的前肢样本之间的比较、不同发育阶段的后肢样本之间的比较，以及同一发育阶段或下一发育阶段的前肢样本与后肢样本之间的比较。