Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth.
Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth.
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牙髓细胞中的TGFBR2指导牙齿发育中的神经突生长。
DOI：
10.3389/fcell.2022.834815
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发表时间：
2022
影响因子：
5.5
通讯作者：
Peters SB
中科院分区：
文献类型：
--
作者：
Stanwick M;Barkley C;Serra R;Kruggel A;Webb A;Zhao Y;Pietrzak M;Ashman C;Staats A;Shahid S;Peters SB
转化生长因子β（Transforming growth factor β，TGFβ）在牙齿形态发生和矿化过程中起重要作用。在出生后的发育过程中，牙髓（DP）间充质分泌神经营养因子，引导三叉神经纤维进入并贯穿DP。这一过程与牙本质的形成和矿化密切相关。我们的实验室建立了一个小鼠模型，其中使用Osterix启动子驱动的Cre重组酶（Tgfbr 2 cko）在DP间充质中有条件地缺失Tgfbr 2。这些小鼠出生后存活，骨骼和牙齿有明显缺陷，包括矿化减少和短根。苏木精和伊红染色显示，减少轴突样结构的突变小鼠。报告者成像表明，牙齿内的Osterix-Cre活性在DP和衍生物中是活跃的，但在神经元传入中不是。在出生后第7天和第24天（P7，P24），对对照和突变磨牙的连续冷冻切片进行β3微管蛋白（神经元标记物）的免疫荧光染色。共聚焦成像和像素定量显示，与对照组相比，Tgfbr 2 cko第一磨牙在两个阶段的神经支配减少，表明促进神经突生长所需的信号被Tgfbr 2缺失破坏。我们使用来自P7对照和突变小鼠DP的RNA进行mRNA序列（RNA-Seq）和基因个体学分析，以研究Tgfbr 2介导的牙齿发育中涉及的途径。这些分析确定了与对照组相比，Tgfbr 2 cko DP中几种矿化相关和神经元基因的下调。选择的基因表达模式，证实了定量实时PCR和免疫荧光成像。最后，将三叉神经元在覆盖原代Tgfbr 2 f/f DP细胞的Transwell滤器上共培养。通过腺病毒表达的Cre重组酶删除DP中的Tgfbr 2。通过过滤孔的轴突共聚焦成像显示，与单独培养的神经元相比，与Tgfbr 2阳性DP细胞一起培养的神经元的轴突出芽增加。当Tgfbr 2在DP细胞中被敲低时，轴突发芽减少。牙本质涎磷蛋白在共培养的DP细胞的免疫荧光证实降低矿化的潜力与Tgfbr 2缺失的细胞。我们的蛋白质组学和RNA-Seq分析都表明，轴突引导线索，特别是信号蛋白信号传导，被Tgfbr 2缺失破坏。因此，DP间充质中的Tgfbr 2似乎调节分化和细胞在牙齿矿化和神经支配期间引导神经突生长的能力。
Transforming growth factor β (TGFβ) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2 cko ). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neuronal afferents. Immunofluorescence staining for β3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2 cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2 cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2 f/f DP cells. Tgfbr2 in the DP was deleted via Adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells’ ability to guide neurite outgrowth during tooth mineralization and innervation.
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影响因子：
3.7
作者：
Andersson K;Dahllöf G;Lindahl K;Kindmark A;Grigelioniene G;Åström E;Malmgren B
通讯作者：
Malmgren B
影响因子：
4.6
作者：
Choi H;Ahn YH;Kim TH;Bae CH;Lee JC;You HK;Cho ES
通讯作者：
Cho ES
影响因子：
2.5
作者：
BYERS, MR
通讯作者：
BYERS, MR
影响因子：
2.5
作者：
Alic, Ivan;Kosi, Nina;Mitrecic, Dinko
通讯作者：
Mitrecic, Dinko
影响因子：
4.1
作者：
de Almeida, Jose Flavio A.;Chen, Paul;Diogenes, Anibal
通讯作者：
Diogenes, Anibal