Function of GDFs in Tooth Development

我们利用简并引物从大鼠牙(切牙)牙髓RNA中克隆并鉴定了BMP/TGFb超家族的新成员GDF11。Gdf11编码的成熟羧基末端结构域与小鼠生长分化因子8(Gdf8)有85%的同源性。Northern印迹分析表明，Gdf11在成人牙髓和脑中有表达。切片和整体胚胎的原位杂交表明，GDF11早在交配后8.5天(DPC)就在尾芽中表达。在10.5dpc时，在臂弓、肢芽、尾芽和后背侧神经管有表达。随后，它在终末分化的成牙本质细胞、鼻上皮、视网膜和大脑的特定区域表达。这些研究表明，Gdf11与其他BMP/TGFb超家族成员在多个发育过程中具有协同作用。我们还克隆了锌指转录因子G23…的一个新成员利用简并引物进行RT-PCR，从切牙牙髓中提取更多的RNA。小鼠G23编码的五个锌指结构域与Gli1、Gli2和Gli3的氨基酸序列同源性接近65%。Northern印迹分析表明，G23在牙髓、肾脏和睾丸中均有表达。切片和整体胚胎的原位杂交表明，G23在交配后10.0天(DPC)的前肢和后肢芽中首次弥漫检测到。在10.5dpc时，表达于鳃弓、肢芽、颅面缘和尾部腹侧。后来，它在胡须滤泡、间盘、肾脏和睾丸中表达。G23的表达与Sonic Hedgehog(Shh)信号通路相关基因Shh、PTC、Gui1、Gli2和Gli3在牙齿发育过程中的表达进行了比较。G23在牙乳头中的表达在时间上和空间上与PTC、Gli1、Gli2和Gli3重叠，而Shh不表达。为了探讨G23在Shh信号通路中的可能作用及其与Glis的相互作用，我们检测了G23在牙齿发育和肾脏发育过程中的表达。Gli2-/-和Glil+/-Gli2-/-突变型G23转录本在牙胚中表达上调，提示G23在牙齿发育过程中可能对Shh信号通路具有抑制作用。Gli2-/-和Glil+/-；Gli2-/-突变株肾脏中G23的表达无明显变化。在牙齿和肾脏发育过程中，Shh在细胞增殖和维持中的作用可能存在不同的反馈途径。G23可能具有冗余功能，并表现出与Gli成员相似的信号活动。为了更直接地研究G23在胚胎发育中的作用，特别是牙齿和肾脏的发育，我们在小鼠中进行了G23基因的靶向干扰。较少

We have cloned and characterized a new member of the BMP/TGFb superfamily, Gdf 11, from rat dental(incisor) pulp RNA by RT-PCR using degenerate primers. The mature carboxyl-terminal domain encoded by Gdf11 is 85% identical to mouse Growth/differentiation factor 8 (Gdf8). Northern blot analysis revealed Gdf11 is expressed in adult dental pulp and brain. In situ hybridization of sections and whole mount embryos demonstrated Gdf 11 is expressed as early as 8.5 days post coitus (dpc) in the tail bud. At 10.5 dpc, it is expressed in the branchial arches, limb bud, tail bud and posterior dorsal neural tube. Later, it is expressed in terminaliy differentiated odontoblasts, the nasal epithelium, retina and specific regions of the brain. These studies indicate that Gdf11 could cooperate with other BMP/TGFb superfamily members in multiple developmental processes. The mice lack of Gdf 11, however, did not have phenotype.We have also cloned a new member of the zinc finger transcription factor, G23 … More from incisor pulp RNA by RT-PCR using degenerate primers. The five zinc finger domain encoded by mouse G23 has almost 65 % amino acid sequence homology with Gli1, Gli2 and Gli3. Northern blot analysis revealed G23 is expressed in dental pulp, kidney and testis. In situ hybridization of sections and whole mount embryos demonstrated G23 is first detected diffusely in forelimb and hindlimb bud at 10.0 days post coitus (dpc). At 10.5 dpc, it is expressed in the branchial arches, limb bud, craniofacial border, and ventral part of the tail. Later, it is expressed in whisker follicle, intervertebral disc, kidney, and testis. The expression of G23 was compared with the genes involved in the Sonic Hedgehog (Shh) signaling pathway, Shh, Ptc, Gui1, Gli2, and Gli3 during tooth development. G23 was found to be expressed in dental papillae in temporally and spatially overlapped pattern with Ptc, Glil, Gli2, and Gli3 where Shh are not expressed. In order to assess the possible role of G23 in Shh signaling pathway and its interaction with Glis, the expression of G23 in Glil/Gli2 mutant and Shh mutant was examined during tooth development and kidney development. The G23 transcript was upregulated in tooth germ in Gli2-/- and Glil+/- ; Gli2-/- mutant, suggesting G23 might have a role of suppresser on the Shh signaling pathway during tooth development. The expression of G23 in kidney was not changed in Gli2-/- and Glil+/- ; Gli2-/- mutant. There might be different feedback pathways operating to regulate the action of Shh in the cell proliferation and maintenance between tooth and kidney development. G23 might have redundant function and display similar signaling activity with Gli members. To examine more directly the role of G23 in embryogenesis, special reference to tooth and kidney development, the targeted gene disruption of G23 in the mouse was performed. Less