小白鼠胚胎肢芽尖端增厚外胚层及其邻近普通外胚层的电子显微镜观察

ELECTRON MICROSCOPIC STUDIES ON APICAL ECTODERMAL THICKENING,NEIGHBORING ECTODERM AND UNDERLYING MESENCHYMAL CELLS OF MOUSE LIMB BUD

用小白鼠胚胎阴栓日龄11—13天的前肢芽和后肢芽,锇酸固定,甲基丙烯酸甲酯及甲基丙烯酸丁酯包埋,在SEMⅢ型电子显微镜观察了肢芽尖端增厚外胚层、邻近普通外胚层及间充质细胞。部分材料用过锰酸钾固定或锇酸固定后氢氧化铅染色。各种细胞相互比较的结果如下:1.两种外胚层的高尔基体极相似,均由小泡及小管组成,无大泡,横切时成为成群的小泡。普通外胚层细胞的高尔基体呈S形,增厚外胚层的马蹄形。2.线粒体在普通外胚层卵圆形或圆形,嵴不整齐。增厚外胚层的线粒体在浅层细胞少,结构与普通外胚层相似,但愈至深层则细长的线粒体愈多。细长线粒体的嵴较密,亦较整齐,与表面垂直,基貭亦较致密。在尖端增厚外层细胞见到由胞质新形成的小泡过渡到小圆的线粒体。3.无论在普通外胚层下或在尖端增厚外胚层下的间充质细胞,其线粒体的形状和结构相同,都是圆或卵圆,嵴少而靠近表面作半月状,内室大,基貭密度低,属比较原始的胚胎型。4.嗜碱质在两种外胚层及间充貭细胞均以弥散的核朊粒为主,由5—10余粒组成小群落。这些群落在尖端增厚外胚层较大、较密,粒亦较多。普通外胚层细胞的有粒内貭网或动貭较间充貭为多,二者均系胚胎型,是分散的小泡或小管。尖端增厚外胚层的动貭有独特形态,即池的一端扩张成大泡,内含网状致密物,显然含有较多的,要不是特异的蛋白貭。这种扩张动貭对于胚胎分化及诱导的关系曾加讨论。5.动质的形成有证据表明是先从核朊粒群落出现膜成为动貭小泡或小管,再并合成较大的小泡和较长的小管。6.增厚外胚层细胞的另一特点是比较普逼的存在着形状、大小及密度不同的致密体。有的完全致密,有的泡状,有的是二者的混合体,有的界限清楚,有的处于分散状态。在形态上类似溶酶体和卵子的多泡体。内含类似核朊粒的致密粒、退化线粒体及动质膜。这些致密体和细胞膜、核膜及高尔基体未显有何关系。基膜及细胞膜完好。未见增厚外胚层细胞有排出、吞食或饮液现象,亦未见致密体排至中胚层,故认为这些致密体起于胞貭。对于分化诱导关系曾提出讨论。7.细胞核及核膜在三种细胞未见有显著不同。核膜有孔,其外膜可与动貭膜相连,核仁小,紧靠核膜,未见有排出现象。氢氧化铅染色的标本有时显示染色貭有微丝,这在胞貭核朊粒群落亦可出现。8.细胞膜完整,比较直,膜的内侧有一层致密物貭,无桥粒。

The cells of the apical ectodermal thickening,neighboring ectoderm and underlyingmesenchyme of the limb buds of mouse embryos,11--13 days vaginal plug age,wereused for this study.Most of the specimens were fixed in 1%osmic acid in veronalacetate buffer,pH 7.6,4℃,1 hour and embedded in methylchrylate.Parts of the speci-mens were either fixed in potassium permagnate or stained in lead hydroxide after osmicacid fixation.Observations and photographs were made under SEMⅢelectron micros-cope.The Golgi apparatus appeared to be horse-shoe shaped in the apical ectodermalthickening and S-shaped in the neighboring ectoderm.It consisted of small vesicles andtubules without any large vesicle.The mitochondria in the neighboring ectoderm were usually round or oval in shape,with fewer and irregular cristae and lighter matrix.The mitochondria of the mesenchy-mal cells were even more embryonic in structure with only 2--3 oblique cristae and alarge inner chamber.The mitochondria in the supperficial layers of the apical ectoder-mal thickening were more or less similar to those in the neighboring ectoderm.Rod-shaped and filamentous mitochondria increased in number in the deeper layers of theapical thickening.Such filamentous mitochondria had vertical cristae,denser matrix andoval and light inner chambers.Transitional stages between the very small vesicles withvague cristae and the large mitochondria with distinct cristae were found in our leadstained preparations.Except in the Golgi region,ribosomes grouped into ring or tubular forms weredistributed all over the cytoplasm in the three kinds of the embryonic cells studied.Transitional stages between such ribosome clusters and the granulated vesicles and tubulescould be clearly demonstrated in the lead stained specimens.In the apical ectodermal thickening,granulated endoplasmic reticulum in the formof scattered small vesicles and tubules seemed to be more abundant than in the neighbor-ing ectoderm and underlying mesenchymal cells.But the most striking difference foundin the apical thickening was the expansion of the granulated endoplasmic reticula intolarge saccules,the cisternae of which containing a grayish dense substance.Another interesting phenomenon found in the apical ectodermal thickening was thepresence of dense bodies in the cytoplasm.They were in various sizes and densitieswith dense granules,masses or cords and various forms of vesicles.They might be round,oval or irregular with a complete,partial or entire absence of limiting membrane.Thosewithout membrane were usually irregular in form and could hardly be demarkated from their surrounding cytoplasm whence assembly of ribosome clusters,mitochondria andendoplasmic reticulum from the surrounding cytoplasm to form the dense bodies could befound.Preliminary histochemical studies found them to be positive in alkaline and acid-phosphatases and RNA staining.They were therefore considered to be lysosomes orcytosomes.The significance of the differences in mitochondrial form and structure,abundantand expanded cisternae of the granulated endoplasmic reticulum and the presence of thedense bodies in the apical ectodermal thickening in relation to embryonic differentiationof the limb was discussed.