Seo Hwa-Seon, Serra Rosa
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Dev Biol. 2009 Oct 15;334(2):481-90. doi: 10.1016/j.ydbio.2009.08.015. Epub 2009 Aug 21.
Transforming growth factor beta (TGFbeta) is known to play important roles in multiple developmental processes. One of the main functions is in skeletal development. Our previous studies demonstrated that loss of Tgfbr2 in Prx1Cre-expressing limb mesenchyme results in defects in the long bones and joints of mice. Here we show that loss of Tgfbr2 also results in defects in the development of the skull vault indicating Tgfbr2 has a critical role in intramembranous bone formation as well as endochondral bone formation. Mutant mice did not survive after birth and demonstrated an open skull. The first signs of skull defects were observed at E14.5 day. Prx1Cre(+)/Tgfbr2(f/f) embryos showed significantly reduced cell proliferation in the developing mesenchyme of the skull by E14.5 day without any detectable alteration in apoptosis suggesting that reduced cell proliferation in Prx1Cre(+)/Tgfbr2(f/f) embryos was at least partially responsible for the defects observed. Immunofluorescent staining showed a significant reduction in the expression of Runx2/Cbfa1 and Osterix/Sp7 in Prx1Cre(+)/Tgfbr2(f/f) embryos suggesting that osteoblast differentiation was also altered in Prx1Cre(+)/Tgfbr2(f/f) embryos. To distinguish between the effects of losing Tgfbr2 on mesenchymal proliferation versus osteoblast differentiation, osteoprogenitor cells from the skulls of Tgfbr2(f/f) embryos were cultured under conditions of high cell density and Tgfbr2 was deleted from the cells using Adeno-Cre virus. RT-PCR analysis showed that the mRNA level of Runx2 and Osterix as well as Dlx5 and Msx2 were down-regulated in Tgfbr2-deleted cultures compared to control cultures indicating that Tgfbr2 regulates osteoblast differentiation independent of regulating proliferation. Together, these results suggest that Tgfbr2 is required for normal development of the skull.
已知转化生长因子β（TGFβ）在多个发育过程中发挥重要作用。其主要功能之一是在骨骼发育方面。我们之前的研究表明，在表达Prx1Cre的肢体间充质中Tgfbr2缺失会导致小鼠长骨和关节出现缺陷。在此我们表明，Tgfbr2缺失还会导致颅顶发育缺陷，这表明Tgfbr2在膜内成骨以及软骨内成骨过程中都起着关键作用。突变小鼠出生后无法存活，并表现出颅骨开放。在胚胎发育第14.5天观察到颅骨缺陷的最初迹象。到胚胎发育第14.5天，Prx1Cre(+)/Tgfbr2(f/f)胚胎在发育中的颅顶间充质中细胞增殖显著减少，且未检测到细胞凋亡有任何改变，这表明Prx1Cre(+)/Tgfbr2(f/f)胚胎中细胞增殖减少至少部分是观察到的缺陷的原因。免疫荧光染色显示，Prx1Cre(+)/Tgfbr2(f/f)胚胎中Runx2/Cbfa1和Osterix/Sp7的表达显著降低，这表明Prx1Cre(+)/Tgfbr2(f/f)胚胎中的成骨细胞分化也发生了改变。为了区分Tgfbr2缺失对间充质增殖与成骨细胞分化的影响，将Tgfbr2(f/f)胚胎颅骨中的骨祖细胞在高细胞密度条件下培养，并使用腺病毒Cre病毒从细胞中删除Tgfbr2。逆转录聚合酶链反应分析表明，与对照培养相比，Tgfbr2缺失的培养物中Runx2、Osterix以及Dlx5和Msx2的mRNA水平下调，这表明Tgfbr2独立于调节增殖来调节成骨细胞分化。总之，这些结果表明Tgfbr2是颅骨正常发育所必需的。
