Hu C C, Sakakura Y, Sasano Y, Shum L, Bringas P, Werb Z, Slavkin H C
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles 90033.
Int J Dev Biol. 1992 Dec;36(4):505-16.
The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.
牙器官为短时间发育过程中离散的形态发生模式提供了一个模型。本研究旨在验证以下假说：内源性表皮生长因子（EGF）在小鼠胚胎发育过程中发挥作用，调节多尖磨牙的牙齿形态发生。通过逆转录聚合酶链反应（RT-PCR）分析，测定了成釉器上皮和牙外胚间充质中内源性EGF和EGF受体（EGFR）转录本的相对水平。通过免疫组织化学对EGF和EGFR进行定位；在培养的牙齿外植体中，两种抗原决定簇均在相同的牙源性细胞上得到证实。为了研究EGF介导的信号转导，将帽状期小鼠磨牙器官（E16）在无血清、化学成分明确的培养基中培养，分为以下几组：（i）对照组；（ii）添加曲磷汀（一种EGF受体激酶抑制剂）；（iii）曲磷汀加外源性EGF；（iv）外源性EGF。采用反义寡脱氧核苷酸（ODN）策略，通过以下分组研究内源性EGF的功能：（i）未处理的对照组；（ii）正义ODN对照组；（iii）反义ODN组；（iv）外源性EGF组；（v）正义ODN加外源性EGF组；（vi）反义ODN加外源性EGF组。曲磷汀抑制DNA合成，并使外植体体积显著减小。添加外源性EGF可恢复这些效应。反义ODN抑制导致牙尖形成异常、DNA合成减少、总DNA、RNA和蛋白质含量降低，以及星网状层和牙齿外植体体积减小。牙齿尺寸减小并不均匀，最明显的影响发生在星网状层。当反义ODN处理补充外源性EGF时，未观察到这种变化模式。这些结果表明，在牙发生的帽状期，内源性EGF起到刺激DNA合成的作用，这增加了成釉器上皮内特定表型的细胞数量，从而调节磨牙的形态发生。
