INDUCTIVE TISSUE INTERACTIONS IN THE BEAK OF A CHICK EMBRYO *

Series of homologous and heterologous recombination experiments were made between the beak and other skin derivatives, by means of a modified chorioallantoic membrane grafting, to investigate inductive tissue interactions involved in the upper beak of a developing embryo and a hatched chick. 6-day beak epidermis, as well as 6-day cephalic skin epidermis, differentiated into typically normal epidermis of the beak, when they were associated with the mesenchyme taken from 6-day upper beak region. These epithelia, when grafted without association with beak mesenchyme, failed to differentiate into the beak epidermis. 6-day beak epidermis differentiated into typical down feathers when combined with 7-day back skin dermis, prospective feathered area. The inductive potency of the beak mesenchyme was not limited in embryonic life, but persisted even after hatching. These findings were discussed in relation to the role of inductive tissue interactions involved in the expression and stabilization of the differentiated characters of the epidermis in both embryos and adults.     

THE METABOLISM OF TISSUE CULTURES : I. PRELIMINARY STUDIES ON CHICK EMBRYO

1. The metabolism of chick embryo tissues has been followed by analysis of the culture media after various periods of incubation in roller bottles. 2. The initial rate of glucose utilization is increased by increasing glucose in the medium from 100 to 500 mg. per cent. Total glucose used can be increased in the same way or by daily addition of small amounts. Glucose is used in greatest amount when the medium containing 100 mg. per cent is replaced daily. 3. Although glucose consumption appears necessary for survival of cultures it may be used at a rate far in excess of that required for life and maximal growth. Complete blocking of mitosis by colchicine does not alter the rate of glucose utilization. 4. Proteolytic activity of the cultures is shown by an increase in the amino nitrogen of the peptone medium after incubation with tissue. 5. Utilization of nitrogen from an amino acid medium is shown by a decrease in the amino nitrogen of this medium. Cells obtaining their nitrogen from amino acids proliferate as rapidly as those grown in a medium identical except for the substitution of peptone, but the cell type is markedly different, in that embryo muscle forms cells resembling regenerating adult muscle. 6. Lactic acid was formed in both the presence and absence of glucose. Its formation increased with increased glucose utilization. There is some evidence that lactate may be utilized, and that it favors growth in the absence of glucose. 7. Added pyruvate was rapidly metabolized by the tissues. It, too, favors growth slightly in the absence of glucose.     

THE EFFECTS OF 5-BROMODEOXYURIDINE ON YOLK SAC ERYTHROPOIESIS IN THE CHICK EMBRYO

The effects of the thymidine analog, 5-bromodeoxyuridine (BUdR), on the formation of red cells in the yolk sac of the chick embryo were examined. The prospective area opaca vasculosa from a definitive primitive streak embryo was excised, disaggregated, and deposited into a cell clump, and the cell clump was placed in organ culture. Hemoglobin synthesis is detectable after about 16 hr in culture. The formation of erythropoietic foci and incorporation of ⁵⁵Fe into heme were used to measure the extent of erythropoiesis. Exposure to 40 µg/ml of BUdR within 6 hr after explantation almost completely eliminated red cell formation; subsequent transfer to thymidine medium showed that the inhibition was reversible, and there was no histological evidence of analog toxicity. Between 6 and 12 hr after initiation of organ culture, the tissue became completely refractory to BUdR. DNA synthesis, as monitored by thymidine-³H and BUdR-³H pulses, was extensive both during and after the period of BUdR sensitivity. Hence, during both BUdR sensitive and insensitive periods the analog was incorporated into DNA of cells which had not yet synthesized hemoglobin. It is proposed that between 6 and 12 hr a crucial regulatory event for terminal differentiation is perturbed by the presence of BUdR in the chromosomes.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FUSION OF CHICK MYOBLASTS IN VITRO

The process of myoblast fusion was observed in embryonic chick skeletal muscle cells grown in monolayer cultures at the fine structural level. At the first step, the sarcolemmas of cells destined to fuse are closely applied to each other. They are linked in some places by fasciae adherentes; in other places, engulfment of small processes of one cell by another is seen. At a somewhat more advanced stage of myogenesis, vesicles and tubules are formed between the adjacent cytoplasms; presumably, the apposed membranes have opened at several points and their edges have fused to each other. Finally, remnants of cell membranes (vesicles and tubules) disappear completely, and the confluent cytoplasm is formed. The cytoplasmic contents of the multinucleated cells are often poorly admixed, giving the cytoplasm a mosaic appearance in which different zones can be designated as arising from separate cells. This observation suggests, however, that there is slow diffusion of myoblast contents (ribosomes and, possibly, other materials) into the myotube. In agreement with the previous works at the light microscopic level, the present study suggests the occurence of fusion between mononucleated cells, between mononucleated cells and multinucleated myotubes, and between nascent multinucleated myotubes.     

EPIDERMAL METAPLASIA OF THE CORNEAL ANTERIOR EPITHELIUM IN THE CHICK EMBRYO

The corneal anterior epithelium of younger chick embryos can be changed into a keratinized epidermis, when it is cultured in vitro combined with 6 1/2-day dorsal dermis. Even if a Millipore filter is inserted between the corneal anterior epithelium and underlying dorsal dermis, the epithelium undergoes similar metaplastic changes. In older embryos, however, the epithelium gradually loses the competence for the keratinization. Cultivation of cornea (anterior epithelium, stroma and endothelium) of 6 1/2- or 10-day embryos results in maintenance of its original pattern, and the epithelium fails to differentiate into a keratinized epidermis. The dermis isolated from 8 1/2-day dorsal or 12 1/2-day tarsometatarsal skin is not so effective in inducing the epidermal metaplasia. The mesenchyme of 5 1/2-day proventriculus or 5 1/2-day gizzard fails to bring about any endodermal metaplasia of the corneal epithelium. The corneal stroma, on the other hand, has no inhibitory action on the keratinization of the epidermis obtained from 6 1/2-day dorsal skin.     

鸡胚视网膜基膜的免疫电镜的研究

扫描电镜观察到鸡胚视网膜基膜(RBM)上的菊花样结构,经免疫胶体金透射电镜证实为Müller胶质细胞的末端足,层粘连蛋白(laminin)分布在末端足的辐射臂及其周围的基膜上。培养的视网膜细胞,其神经纤维在RBM伸长,收缩了的Müller细胞末端足呈球状颗粒分布在神经纤维及其生长锥的上面和侧面。推测分布在Müller细胞末端足的辐射臂及其周围基膜上的层粘连蛋白在体内可能与神经纤维生长的导向有关。     

CELL SURFACE ALTERATIONS BY TAXOL ASSOCIATED WITH ABNORMAL MORPHOGENESIS IN THE CHICK EMBRYO

The anticancer drug taxol brings about its biological effects by altering the stability of microtubules. We have examined the effects of taxol on early morphogenesis in chick embryos culturedin vitro. Taxol induced various abnormalities in the developing nervous system, heart and somites as well as general retardation of development. SEM studies revealed that taxol treatment leads to dramatic alterations in the embryonic cell surfaces. Time-course experiments demonstrated that the action of taxol is very rapid and becomes evident within a few minutes at the ultrastructural level. Taxol thus throws embryonic cell adhesion and motility out of balance. This appears to be the major cause of abnormal morphogenesis in taxol-treated embryos.     

鸡胚发育中肝脏类脂滴液晶态及体外结晶态的相变特性研究

观察到鸡胚发育中肝脏液晶类脂滴在降低温度时,可向结晶态转变,类脂滴液晶——结晶——各向同性态三相之间可互相转变。肝脏组织中液晶类脂滴在0℃左右转变为结晶;结晶在39—43℃时转变成各向同性类脂滴(正交偏光下无双折射);后者在热的铜样品台上,随样品台缓慢自然降温至35—36℃时又转变成结晶态;各向同性类脂滴在较快速度降温时,则转变成液晶态类脂滴。     

STRUCTURAL VARIATIONS DURING MITOSIS IN THE CHICK EMBRYO

Selected tissues from chick embryos were fixed in 2% glutaraldehyde and 1% OsO₄, both buffered at pH 7.6 with Veronal-acetate, and were embedded in Maraglas or Araldite. Two types of cell division have been noted. Generally, epithelial cells divide predominantly by a shortening of the chromosome-to-pole distance rather than by spindle elongation; mesenchymal cells undergo extensive spindle elongation. The presence of numerous continuous microtubules in cells that undergo extensive spindle elongation functionally implicates these tubules in the elongation process. In most embryonic epithelia, the cleavage furrow converges to a fixed site forming a mid-body near the anchoring desmosomes at the free surface; symmetrical furrow formation is typical of mesenchymal cells which lack desmosomes. The hypothesis of cleavage furrow formation and the fate of the mid-body that is formed during cytokinesis are discussed.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

鲢疯狂病病原体鲢碘泡虫孢子形成各阶段的观察研究

链碘泡虫是鲢疯狂病的病原体。本文报道鲢碘泡虫营养体细胞的核型、各期有丝分裂相、孢子形成过程的早期、即二个生殖细胞的联合以及孢子的产生。     

C-H_3加强神经生长因子对培养的鸡胚背根神经节细胞的作用

在培养的鸡胚背根神经节上观察了 C-H_3(GhineseH_3)、神经生长因子(NGF)以及 C-H_3和 NGF 的混合剂对神经细胞的影响。主要结果如下:1.C-H_3处理的标本和对照组没有差别。培养3d 的节神经细胞数平均约420个;6d 时,部分细胞衰退,降到200个。2.NGF 能促进节神经纤维外长,增加神经母细胞向神经元的转变。培养3d 的节神经细胞数平均约700个;6d 时720个。NGF 维持神经元的存活,延缓神经元的衰退。3.C-H_3和NGF 的混合剂不仅增进节神经纤维的长出,而且还延缓了长出纤维的消退,消失时程比NGF 组延长一倍。培养3d 的节神经细胞数平均约1000个,是2.4倍于对照,1.4倍于 NGF组;培养6d 时1120个,为对照组的5倍,NGF 组的1.5倍。混合剂的效果比 NGF 更好。结果表明,C-H_3增强 NGF 对培养的节神经细胞存活的效应。