Formation of intestinal atresias in the Fgfr2IIIb-/- mice is not associated with defects in notochord development or alterations in Shh expression.
Formation of intestinal atresias in the Fgfr2IIIb-/- mice is not associated with defects in notochord development or alterations in Shh expression.
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Fgfr2IIIb-/- 小鼠肠闭锁的形成与脊索发育缺陷或 Shh 表达改变无关。
DOI：
10.1016/j.jss.2012.04.024
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发表时间：
2012-09
期刊：
影响因子：
--
通讯作者：
Nichol PF
中科院分区：
文献类型：
--
作者：
Reeder AL;Botham RA;Franco M;Zaremba KM;Nichol PF
肠闭锁的病因仍然难以捉摸，但已被归因于许多可能的事件，包括子宫内血管意外，肠腔再通失败和机械压迫。另一个被认为是导致闭锁形成的原因是脊索发育中断。这一假设来自肠闭锁患者脊索异常的临床观察以及肠闭锁药理学动物模型中观察到的脊索发育异常。该模型中的闭锁由子宫内暴露于阿霉素引起，其中在高达80%的表现为肠闭锁的胚胎中注意到脊索缺陷。脊索异常的胚胎被观察到有Sonic Hedgehog（Shh）的异位表达，这反过来被假定为是闭锁形成的原因。我们感兴趣的是确定脊索发育或Shh表达的中断是否发生在已建立的肠闭锁遗传模型中，并利用成纤维细胞生长因子受体2 IIIb纯合突变体（Fgfr 2 IIIb −/−）小鼠模型。这些胚胎发育为结肠闭锁（100%闭锁率）和十二指肠闭锁（42%闭锁率）。在E10.5、E11.5、E12.5和E13.5收获野生型和Fgfr 2 IIIb −/−小鼠胚胎。对Shh的E10.5胚胎进行整体原位杂交。收获每个时间点的胚胎并切片用于H&E染色。专门拍摄脊索切片，并使用Amira软件进行三维重建。从E10.5的野生型和Fgfr 2 IIIb −/−胚胎中分离结肠，然后在存在或不存在外源SHH蛋白的情况下，在具有FGF 10的基质胶中培养48小时。收获外植体，在福尔马林中固定并拍照。Fgfr 2 IIIb −/−小鼠胚胎在E10.5没有表现出Shh表达的中断，此时已知闭锁形成的第一个事件发生。三维重建未能证明任何解剖中断脊索不连续或过度分支。在Shh存在下培养野生型肠未能诱导十二指肠或结肠闭锁形成。培养的Fgfr 2 IIIb −/−肠在存在或不存在Shh蛋白的情况下发生结肠闭锁。虽然脊索发育的中断可能与肠闭锁的形成有关，但在Fgfr 2 IIIb −/−遗传动物模型中，脊索发育的中断和外源性Shh蛋白的存在都不是这些缺陷形成的原因。
The etiology of intestinal atresia remains elusive but has been ascribed to a number of possible events including in utero vascular accidents, failure of recanalization of the intestinal lumen and mechanical compression. Another such event that has been postulated to be a cause in atresia formation is disruption in notochord development. This hypothesis arose from clinical observations of notochord abnormalities in patients with intestinal atresias as well as abnormal notochord development observed in a pharmacological animal model of intestinal atresia. Atresias in this model result from in utero exposure to Adriamycin, wherein notochord defects were noted in up to 80% of embryos that manifested intestinal atresias. Embryos with notochord abnormalities were observed to have ectopic expression of Sonic Hedgehog (Shh) which in turn was postulated to be causative in atresia formation. We were interested in determining whether disruptions in notochord development or Shh expression occurred in an established genetic model of intestinal atresia and utilized the Fibroblast Growth Factor Receptor 2IIIb homozygous mutant (Fgfr2IIIb−/−) mouse model. These embryos develop colonic atresias (100% penetrance) and duodenal atresias (42% penetrance). Wild-type and Fgfr2IIIb−/− mouse embryos were harvested at E10.5, E11.5, E12.5 and E13.5. Whole mount in situ hybridization was performed on E10.5 embryos for Shh. Embryos at each time point were harvested and sectioned for H&E staining. Sections were photographed specifically for the notochord and resulting images reconstructed in 3-D using Amira software. Colons were isolated from wild-type and Fgfr2IIIb−/− embryos at E10.5, then cultured for 48 hours in matrigel with FGF10 in the presence or absence of exogenous SHH protein. Explants were harvested, fixed in formalin and photographed. Fgfr2IIIb−/− mouse embryos exhibit no disruptions in Shh expression at E10.5, when the first events in atresia formation are known to occur. Three-dimensional reconstructions failed to demonstrate any anatomical disruptions in the notochord by discontinuity or excessive branching. Culture of wild-type intestines in the presence of Shh failed to induce atresia formation in either the duodenum or colon. Cultured Fgfr2IIIb−/− intestines developed atresias of the colon in either the presence, or absence, of Shh protein. Although disruptions in notochord development can be associated with intestinal atresia formation, in the Fgfr2IIIb−/− genetic animal model neither disruptions in notochord development nor the presence of exogenous Shh protein are causative in the formation of these defects.
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