鸡胚发育期中“细胞从卵黄球形成”问题的研究 Ⅰ.卵黄球在发育期中的形态变化以及血岛的来源及血管血细胞的发生问题

AN INVESTIGATION INTO THE PROBLEM OF“CELL FORMATION FROM YOLK SPHERE”DURING THE DEVELOPMENT OF THE CHICK EMBRYO I.ON THE MORPHOLOGICAL CHANGES OF THE YOLK SPHERES,THE ORIGIN OF THE BLOOD ISLANDS AND THE DEVELOPMENT OF BLOOD VESSELS

一.绪言鸡胚卵黄囊是胚胎发育期中的营养器官,而囊内的卵黄球则是一种营养物质,这是胚胎学界公认的事实。近年来О.Б.勒柏辛期卡娅发表了她的“活质”学说,这个学说的主要论据之一是关于她在鸡胚卵黄球方面的研究工作。

The yolk spheres have long been considered by embryologists to be a source of nutriment for the developing chick embryo.Recently.O.B.Lepeshinskaya,on the basis of her histological observations on early chick embryos and her findings on the cultivation of yolk spheres in vitro,postulated that the yolk spheres which entered the subgerminal cavity could develop into entodermal cells,while those found in the space between ectoderm and entoderm in the germ wall could develop into blood islands.The functional significance of the yolk spheres to the development of the embryo is thus again a problem of controversy in embryology and cytology.Our experiments are a contribution to the study of the morphology,properties and the possible changes of the yolk spheres during embryonic development.We were also concerned with testing whether or not any living element was to be found among them,and how great,if at all,was the possibility of their develop-ing into cells under normal and experimental conditions.In addition we also aimed to study tbe developmental processes of blood islands and blood vessels to discover whether or not the yolk sphere took any part in their formation.The material and methods used in these experiments are a series of histologi-cal sections,whole mounts of chick embryos and yolk sac membranes,the culti-vation of yolk spheres and the entire blastoderm or fragments of it in vitro and in vivo,local cantery of the blastoderm,and the observation on the changes of the yolk sac inclusions at different developmental stages.1)Morphology and properties of the yolk sphere.The yellow and white yolk spheres are distinguishable by their color and ap-pearance after fixation with Bouin’s solution and washing in 70%alcohol.After such treatment the yellow yolk spheres are white and contain homogeneous fine yolk granules,whereas the white yolk spheres are semitransparent and contain coarse and refractile yolk granules(Fig.3,4).Both kinds of yolk sphere are normally spherical in shape but may be altered into oblong or filamentous structures,and sometimes present pseudopodia-like ex-tensions under the influence of changes of the external environment.(Fig.2).This can be easily demonstrated by placing the yolk spheres under artificially ap-plied pressure.Care must be taken not to derive conclusions as to the nature of the normal activity of the yolk spheres from observations in such artificial condi-tions.For,in normal conditions no active movement of any kind was even observed.Although staining reactions of both kinds of yolk sphere with methylene blue,hematoxylin,eosin,SudanⅢ,osmic acid,Wright’s stain,light green and Feulgen’s nuclear reaction are basically the same,however,in our opinion this does not mean that their chemical composition is identical.2)Changes of the yolk sphere during embryonic development.During the course of emhryonic development the whole yolk sac inclusion(yolk materials)iquefies and disintegrates.This phenomenon is closely correlated with increase of liquid within the yolk sac and with the formation of the yolk sac villi.(Fig.1)The yolk spheres which enter the subgerminal cavity and the space between the entoderm and ectoderm in the germ wall region are destined to break down into granules and small droplets.They do not develop into any kind of embryonic cell nor into blood islands.Observations of living material and histological pre-parations made at different stages of development all point to this same conclusion.(Fig.5,6,15,16).Both yellow and white yolk spheres,cultivated in vitro,also show the pheno-mena of disntegration and liquefaction.The following main features are noted during this process:increase of Brownian movement of granules within the sphere;swelling and vacuolization of the granules into small droplets;fusion of the gran-ules or droplets into larger ones;liquefaction of the whole sphere;breakdown of the yolk sphere membrane and dispersion of their inclusions.(Fig.7,8,9,10,11,12,13,14).We could not observe any trace of formation of cells nor of blood islands from the yolk sphere during in vitro cultivation,regardless of whe-ther we used subgerminal cavity fluid or embryonic extract as culture medium.When the yolk spheres were cultivated in vivo(implanted into the brain ve-sicle or amnionic cavity)the results were the same as those obtained in vitro ex-periments.(Fig.17,18).3)Origin and normal development of the blood islands and blood vessels.The blood islands first make their appearance in the germ wall region after 17-18 hours of incubation.The time and place of their appearance are entirely coincident with the arrival of the primary mesoblasts at the germ wall.The pri-mary mesoblasts undergo differentiation into coelomic mesoderm and angioblasts immediately after they arrive at the germ wall region.It seems to us that the differentiation of angioblasts is frow those cells that have“burrowed”into that region.It may be assumed that the cells which were destined to become angio-blasts implanted themselves into the protoplasm of the germ wall,proceeded to proliferate and finally formed a syncytial blood island.Those cells which continued their migration toward the periphery became differentiated into coelomic mesoderm.(Fig.21-24).In the germ wall region,the blood islands were the first to appear and from them the blood vessels were differentiated.This process may be described as fol-lows:Within the blood island a number of vacuoles occur as a result of a process of vacuolization and liquefaction(actually cytolysis).This causes the peripheral cells to separate from the main mass of the island.The former are then flattened and become endothelial cells,while the central mass of syncytial nuclei and their surrounding protoplasm form a spherical erythroblast.(Fig.25-27).On the contrary,in the area pellucida the blood vessels are formed in advance of the blood islands,and the latter are directly differentiated into erythroblasts within the blood vessel.(Fig.36).There is no direct correlation between the yolk sphere and blood island forma-tion nor in the differentiation of blood vessels in either of the processes we have just mentioned.Nor is there blood cell formation from the so-called“living sub-stance”within the blood vessel or blood stream.The mode of proliferation of blood cells is chiefly by mitotic division of the erythroblasts.In addition,endo-thelial cells which undergo mitotic division occasionally discharge cells into the blood stream and become blood cells.The earliest blood vessels are differentiated from the angioblasts,but as soon as the embryonic circulation is established,their mode of development is mainly by the formation of vascular sprouts and branches from previously formed vessels.Contrary to Lepeshinskaya,we failed to observe in the 8-day chick embryo the formation of blood vessels from the yolk spheres and their subsequent communica-tion with the already formed vessels.Beginning from the 5(1/2)th day of incubation,we observed a new type of blood island in the yolk sac membrane,outside the wall of the blood vessels of the villi.(Fig.37,38).They were clusters of cells which were characterized by their nature as centers of hematopoiesis in the yolk sac.At later stages they become connect-ed with each other and finally form a plexus of blood vessels.At the present time we still do not know the origin of this new type of blood island.4)Development of blood islands and blood vessels in experimental conditions.a.Local cauterization.The blastoderm was burned locally with a red-hot metal needle along the border of the area pellucida,inside or outside of the border.This operation was performed on one side of the blastoderm,or surrounding the embryonic axis with a“U”shaped or“O”shaped burn.We found that if the operation was made prior to the appearance of the blood islands in the area outside the burned region in which the mesoderm was absent,blood islands failed to develop subsequently,regardless of the presence of yolk spheres and entoderm.However,inside the burned area an excessive amount of vascular tissues was observed.This was a consequence of the fact that the mesoblasts were prevented from further migration by the cauterized tissue barrier.(Fig.28,29,30,31).On the other hand,if the operation were performed after the blood islands had made their appearance,and the burning took place within the region of the blood islands,the operation mere-ly interrupts the connection of the blood islands with the embryonic axis.The blood islands still develop to some extent,though at a rather slower rate than on the contralateral control side.(Fig.32).These results not only demonstrated that the blood islands originated from mesodern,but also furnished further evid-ence to disprove the contention that the yolk sphere of chick embryo could develop into blood islands.b.In vitro cultivation of the entire posterior blastoderm or fragments of it.The culture of either the entire blastoderm or fragments of posterior or lateral parts of the blastoderm(1-2mm.in size),for 12—14 hours of incubation,produced blood islands.(Fig.34,35).Yolk spheres adherent to the cultivated fragments undergo similar changes as when they are cultivated separately;that is,they dis-integrate and liquefy.These results also show that there is no correlation Between blood island formation and the yolk sphere.From the foregoing results,it is justifiable for us to conclude that the yolk spheres are not a living substance but a kind of nutritious food substance needed for the development of the chick embryo,the blood islands and their derivatives all originate from mesoderm,their development is entirely independent of the yolk spheres.