Zhang Guodong, Boyle Daniel L, Zhang Yuntao, Rogers Austin R, Conrad Gary W
Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA.
Mol Vis. 2012;18:348-61. Epub 2012 Feb 5.
To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS).
The anterior halves of whole eyeballs from chickens on embryonic (E) days E10 to E21 and Japanese quail on embryonic days E8 to E17 were fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence. Propidium iodide (PI) was used to detect the nuclei of osteocytes in scleral ossicles. FESEM and EDS were then used to show areas of mineralization and to identify differences in the elemental composition of different regions of the ossicles.
Using Giemsa as a fluorescence stain, it was possible to observe the detailed morphology and development of both chicken and quail scleral ossicles. In chickens, bone microporosities first became visible at E15. Each microporosity contained a single nucleus, likely that of an osteocyte. The amount of carbon in ossicles steadily decreased during embryogenesis and post-hatching, while the concentration of oxygen showed a distinct increase over this time period. Calcium and phosphate levels in the ossicles increased gradually during embryonic and post-hatching stages.
A novel approach to study the development and mineralization of avian scleral ossicles during embryogenesis is presented. This methodology was validated by studying two different species, both important models for avian developmental research.
结合场发射扫描电子显微镜（FESEM）和能谱仪（EDS），利用荧光显微镜研究鸟类巩膜骨的发育和矿化。
将胚胎期（E）第10至21天的鸡以及胚胎期第8至17天的日本鹌鹑的整个眼球前半部分固定于100%甲醇中1分钟，用吉姆萨溶液染色5分钟，用蒸馏水脱色30分钟，然后通过落射荧光观察。碘化丙啶（PI）用于检测巩膜骨中骨细胞的细胞核。随后使用FESEM和EDS显示矿化区域，并识别巩膜骨不同区域元素组成的差异。
使用吉姆萨作为荧光染料，可以观察到鸡和鹌鹑巩膜骨的详细形态和发育情况。在鸡中，骨微孔在E15时首次可见。每个微孔包含一个单核，可能是骨细胞。在胚胎发育和孵化后，巩膜骨中的碳含量稳步下降，而在此期间氧浓度明显增加。在胚胎期和孵化后阶段，巩膜骨中的钙和磷水平逐渐升高。
提出了一种研究胚胎发育过程中鸟类巩膜骨发育和矿化的新方法。通过研究两个不同物种（均为鸟类发育研究的重要模型）验证了该方法。
