Comparison of 2 ADAMTS metalloproteases in closure of mouse palate - versican proteolysis in regulating palatal mesenchyme proliferation

We identify a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motif), ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days of gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was established in bt/bt:Adamts9+/- mice, which have increased white spotting relative to bt mice, as previously reported, and a fully penetrant cleft palate. Palatal closure was delayed, although eventually completed, in both bt/+;Adamts9+/- and bt/bt mice, demonstrating a cooperative role of these related genes. Adamts9 and Adamts20 are both expressed in palatal mesenchyme, with Adamts9 expressed exclusively in microvascular endothelial cells. Palatal shelves from bt/bt:Adamts9+/- mice fused in culture, suggesting an intact TGFbeta signaling pathway in palatal epithelium, and indicating a temporally specific delay in palatal shelf elevation and growth toward the midline. Palatal shelf mesenchymal cells showed a statistically significant decrease of cell proliferation at E13.5 and E14.5, as well as decreased processing of versican, an ADAMTS substrate, at these stages. Vcan haploinsufficiency led to a greater penetrance of cleft palate in bt mice, and impaired proliferation was also seen in palatal mesenchymal cells of these mice, suggesting a role for ADAMTS-mediated versican proteolysis in palatal closure. In a parallel with recent work identifying a role for a bioactive ADAMTS-generated versican fragment in regulating apoptosis during interdigital web regression, we propose that versican proteolysis may influence palatal mesenchymal cell proliferation. Palatal shelves were dissected from four E13.75 Adamts9+/-:bt/bt embyos (correspond to the 4 samples: Palate_Adamts9+/-:bt/bt_Rep1, Palate_Adamts9+/-:bt/bt_Rep2, Palate_Adamts9+/-:bt/bt_Rep3 and Palate_Adamts9+/-:bt/bt_Rep4) and age-matched 3 wild-type C57Bl/6 embryos (correspond to the 3 samples: Palate_WT_Rep1, Palate_WT_Rep2, and Palate_WT_Rep3) that were used as the controls我们明确了ADAMTS家族（A disintegrin-like and metalloprotease domain with thrombospondin type-1 motif，含血小板反应蛋白1型基序的去整合素样金属蛋白酶结构域）中两种进化相关的分泌型金属蛋白酶——ADAMTS20与ADAMTS9在腭发育中的功能作用。Adamts20突变可诱发小鼠出现白斑突变表型belted（简称bt），而Adamts9则是小鼠妊娠7.5天（E7.5）后存活的必需基因。既往研究证实，Adamts9与Adamts20存在功能冗余：bt/bt:Adamts9+/-小鼠相较于bt小鼠白斑症状更为显著，且表现出完全外显的腭裂表型。bt/+;Adamts9+/-与bt/bt小鼠均出现腭帆闭合延迟，但最终可完成闭合，这证明了这两个同源基因的协同作用。Adamts9与Adamts20均在腭间充质中表达，其中Adamts9仅特异性表达于微血管内皮细胞。bt/bt:Adamts9+/-小鼠的腭突在体外培养中可正常融合，这提示腭上皮的转化生长因子β（transforming growth factor beta, TGFβ）信号通路完整无缺，同时表明腭突向中线抬升与生长存在时间特异性延迟。在E13.5与E14.5阶段，腭突间充质细胞的增殖水平出现具有统计学意义的显著下降，同时ADAMTS底物——Versican（Vcan）的加工过程也受到抑制。Vcan单倍体剂量不足（haploinsufficiency）可使bt小鼠腭裂的外显率进一步升高，且此类小鼠的腭间充质细胞同样出现增殖受损现象，这表明ADAMTS介导的Versican蛋白水解过程在腭突闭合中发挥关键作用。结合近期研究发现ADAMTS生成的活性Versican片段可调控指间蹼消退过程中的细胞凋亡，我们推测Versican的蛋白水解过程可能通过影响腭间充质细胞增殖参与腭发育。我们从4只E13.75的Adamts9+/-:bt/bt胚胎中分离获取腭突，对应4个样本：Palate_Adamts9+/-:bt/bt_Rep1、Palate_Adamts9+/-:bt/bt_Rep2、Palate_Adamts9+/-:bt/bt_Rep3及Palate_Adamts9+/-:bt/bt_Rep4；同时以3只同月龄的野生型C57Bl/6胚胎作为对照，其分离得到的腭突对应3个样本：Palate_WT_Rep1、Palate_WT_Rep2及Palate_WT_Rep3。