Keratocyte-mediated establishment of lamellar structure in corneal development
Keratocyte-mediated establishment of lamellar structure in corneal development
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角膜发育中角膜细胞介导的层状结构的建立
DOI：
10.1111/j.1755-3768.2010.203.x
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发表时间：
2010
影响因子：
3.4
通讯作者：
YOUNG R
中科院分区：
文献类型：
--
作者：
YOUNG R
目的：目前尚不清楚角膜发育中的角化细胞如何影响透明基质的三维板层基质。为了更好地了解角膜基质的生物合成、透明和修复过程，我们采用体积扫描电镜观察了禽类胚胎角膜板层的形成过程。方法从E10和E14岁的鸡胚中获得胶原纤维束，当疏松结构的胶原纤维束凝结成成熟基质的板层组织。醛固定角膜在铁氰化锇和单宁酸中增强，并包埋在杜库潘树脂中。甲苯胺蓝染色切片用于定位合适的位点，随后在Jeol 1010透射电子显微镜下在未染色的超薄切片中进行检查。然后使用量子3D FEG FIB/SEM中的BSE探测器通过交替切片和视图协议对树脂块的抛光面进行成像。用聚焦镓离子束进行50nm切片，并在18h内收集图像序列。使用ImageJ软件对图像栈进行处理。结果透射电镜和体积扫描电镜显示发育中的雏鸡角膜基质存在复杂的区隔。角化细胞丝状足从细胞延伸到细胞外空间许多微米。这些胶原原纤维具有不同的方向，并随着基质凝聚而持续存在，似乎协调了组成片层的空间和方向组织。结论体积扫描电镜（SEM）三维显示了具有广泛丝状足的雏鸡角膜发育过程中潜在角质细胞的复杂细胞形态，这些细胞可能引导和引导胶原原纤维束形成新兴的板层结构。
PurposeMechanisms by which presumptive keratocytes in the developing cornea engineer the 3‐dimensional lamellar matrix of the transparent stroma remain unknown. We used Volume Scanning Electron Microscopy to observe lamella formation in embryonic avian cornea in order to understand better corneal stromal biosynthesis, transparency and repair.MethodsCorneas were obtained from chick embryos at E10 and E14, when a loose structure of collagen fibril bundles condenses into the lamellar organisation of the mature stroma. Aldehyde‐fixed corneas were contrast‐enhanced in osmium ferricyanide and tannic acid and embedded in Durcupan resin. Toluidine blue‐stained sections were used to locate suitable sites, subsequently examined in unstained ultrathin sections in a Jeol 1010 transmission electron microscope. The polished faces of the resin blocks were then imaged using a BSE detector in a Quanta 3D FEG FIB/SEM via an alternate slice‐and‐view protocol. 50nm slicing was performed with a focused gallium ion beam and image series were collected over 18h. Image stacks were processed using ImageJ software.ResultsTransmission and volume scanning electron microscopy reveal complex compartmentalisation of developing chick corneal stroma. Keratocyte filopodia extend many microns from cells into the extracellular space. These invest collagen fibrils, with varying orientations, and persist as the stroma condenses, appearing to orchestrate the spatial and directional organisation of the component lamellae.ConclusionVolume SEM reveals in 3D the complex cellular morphology of prospective keratocytes in developing chick cornea with extensive filopodia, which presumably channel and direct collagen fibril bundles into an emergent lamellar architecture.