Acetylcholinesterase in neural tube defects: A model using chick embryo amniotic fluid

Acetylcholinesterase was measured in amniotic fluid from normal chick embryos and embryos with neural tube defects. Neural tube defects were induced in the chick embryos by three procedures, removal of albumen, mechanical disruption of the closed neural tube or injection of tetanus toxin.The concentration of acetylcholinesterase in amniotic fluid from untreated normal embryos changed throughout the period examined (5–14 days incubation) but was stable at 0.5 Ul^?1 over the time period 6–11 days. Amniotic fluid taken from treated embryos with neural tube defects at 8 days always contained a higher concentration of acetylcholinesterase than fluid from sham operated but otherwise normal embryos, mean 40.9 Ul^?1,S.E.M. = 10.1 Ul^?1,versus 1.0 Ul^?1,S.E.M. = 0.2 Ul^?1. The range of values (6.1–393 Ul^?1) was clearly separated from the normal values, range 0.0–5.5 Ul^?1. In 13 cases with developmental abnormalities other than neural tube defects, the concentration of acetylcholinesterase was elevated in only one. Two different forms of acetylcholinesterase, as shown by gel electrophoresis, were present in fluid form both normal and defective embryos. These forms were also present in blood plasma, cerebrospinal fluid and in the high speed supernatant from brain extracts, the latter tissue contained an additional form of greater electrophoretic mobility.After irreversible inhibition, enzyme activity in amniotic fluid recovered slowly; only half the control value was reached by 140 h compared with complete recovery in the tissues of the embryo within 19 h. Histochemical staining for acetycholinesterase showed that the spinal cord in the region of the lesion contained high concentrations of the enzyme. The possible sources of acetylcholinesterase in amniotic fluid are discussed.This chicken model of neural tube defects provides support for the use of acetylcholinesterase tests in the detection of neural tube defects clinically, and provides a model for experimentation with this system.     

Interstitial bodies in the early chick embryo

Chick embryos of graded ages, ranging from freshly laid eggs to one week incubation, were prepared for electron microscopy. Interstitial bodies are expressions of “ground substance” that resemble structureless masses of cytoplasm without enclosing plasmalemma. They measure from 0.1 to ca. 1 μ in diameter. Toward the end of the first day of incubation they are found in the tissue space near to or in contact with the ectodermal boundary (basement) membrane. They seem to contribute to its increasing amorphous component. Microfibrils first appear close to or in contact with the ectodermal boundary membrane and are similarly related to interstitial bodies. At 44 hours interstitial bodies are especially numerous where the neural tube is separating from the ectoderm. Here boundary membranes have become intermittent and interstitial bodies appear to contribute to their repair. By the fourth day interstitial bodies are less numerous. Many appear to break up. Their edges tend to become dispersed into clouds of finely granular material, especially in areas of the tissue space occupied by wisps of microfibrils. The close association of amorphous ground substance and extracellular fibrils persists indefinitely.     

Natural wound formation: Endodermal responses in experimental primary neural induction in the chick embryo

Natural wound formation in experimental primary neural induction has been studied by SEM and in paraffin wax sections in embryos from 0 minutes to 10 hours of re-incubation. Stage 4 host and graft embryos were removed from hen's eggs and mounted as for New culture. Graft Hensen's nodes were transplanted into “pockets” created in the host area pellucida and re-incubated for up to 10 hours. Initially the cut edges of the graft establish contact with the host ectoderm layer. After 4 hours the cut edges of the graft move from the host ectoderm to the host endoderm layer. Several small openings form in the host endoderm over the graft tissue. By 6 hours, these openings join to form a single natural wound through which the underlying graft is exposed to the external environment. At 8 hours the graft forms a head-fold and neural folds are evident. During 8 to 10 hours of re-incubation the edges of the graft which attach to the edges of the host edoderm meet in the midline and close the opening in the host endoderm; simultaneously, the graft forms a neural tube. The endodermal wounds form by cell re-arrangement and by a minor contribution from cell loss. © 1993 Wiley-Liss, Inc.     

Morphological study of surgically induced open neural tube defects in chick embryos--postoperative 24 hours.

For the experimental study of neural tube defect (NTD), a surgical model has advantages over other models in a few aspects. It causes less functional derangement of cells and the NTDs can be made selectively by surgery. The authors planned to use the surgical model for the experimental study of NTD. As the first step for the studies, the chronological changes of morphology during the early postoperative period were investigated using postincubation 3-day chick embryos. The objectives of this study are (1) the morphological evaluation of the surgical model as a method for studies of open NTD, and (2) the observation of morphological changes for the first 24 hours after surgery which include ''overgrowth'' appearance and the continuity between the surface ectoderm and the neuroectoderm. The morphological changes were observed by light microscope and scanning electron microscope. Immediately after surgery, typical open NTDs were observed. Morphologically they were very similar to the appearance of spontaneous (non-surgical) open NTDs. The opened neural tubes were everted progressively and they looked rather flat at 24 hours after surgery. Cellular hyperplasia (''overgrowth'' appearance) was noted within 24 hours after surgery and became more prominent during the 24 hours. There was increasing continuity between the surface ectoderm and the neural tissue until 24 hours after surgery when the continuity looked almost complete. In conclusion, surgically induced NTDs are morphologically very similar to spontaneous NTDs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology of experimental spina bifida in the chick embryo.

Open malformations of the central nervous system may involve the brain or spinal cord, or both. Preliminary experiments in which a window was cut in the shell overlying early chick embryos (with removal of 2 ml of albumen) produced a range of neural and non-neural malformations. Exposure of Stage 5--10 embryos at 26 hours of incubation produced open brain and cord defects. Embryos were recovered at 11--12 days for gross examination. Open cord defects in 12 day experimental embryos could be divided morphologically into 2 types. One group showed an everted symmetrical plaque of neural tissue. In the other group the cord defect was more irregular, partly covered by skin, and often combined with rump and trunk defects. Skeletal staining showed that vertebral lesions increased in severity in a cranio-caudal sequence. Spina bifida occulta was found in the cervical and upper thoracic regions; spina bifida manifesta, associated with open cord defects, occurred from the lower thoracic to the sacral regions; vertebral deletions were almost confined to the caudal region. Spina bifida manifesta at the site of open cord defects also showed 2 distinct patterns. Regular cord defects were associated with regular spinal defects, showing loss of spinous processes, reduction of laminae and eversion of the pedicles. Irregular cord defects were associated with more irregular spinal defects showing vertebral deletions or fusions, rumplessness, and pelvic reduction. Neither group, however, showed local kyphosis or scoliosis. Early neurogenesis in the avian and human embryos is very similar with development of the spinal cord from neural plate and tail bud materials which fuse in an overlap zone. These experimental defects in the chick embryo, separable into regular and irregular types thus provide a useful model for investigation of the embryogenesis of spina bifida.     

Cell death in the ventral region of the neural retina during the early development of the chick embryo eye

The present study deals with morphologic and quantitative changes that take place in the area of cell death in the ventral part of the presumptive retinal wall of the chick embryo. These changes were followed from the optic vesicle stage until the first optic fiber fascicles leave the neural retina. Our results show that both the volume occupied by the area of cell death and the density of its pyknotic fragments undergo considerable variation during the period between Hamburger and Hamilton's (1951) stages 12 to 20. In the optic vesicle stages, cell death in the ventral wall of the vesicle was observed in 50 to 75% of the embryos studied. During stages 14 and 15, this zone was seen in more than 90%. By the time invagination of the optic cup was complete, the ventral retinal zone of cell death had disappeared entirely in a large proportion of embryos; in all others, it shrank significantly both in volume and density of pyknotic fragments. In stage 19, when the first optic fiber fascicles begin to emerge from the retina, a dramatic increase occurs in the number of pyknotic fragments in the posterior pole of the retina. The appearance of dying cells, in a region shortly to be traversed by developing ganglion cell axons, supports the hypothesis that cell death processes are apparently somehow related to the creation of a suitable environment for the emergence of fibers toward the optic stalk. Densities of mitotic and interphasic cells as well as the mitotic index were determined in both the retinal zone of cell death and in areas devoid of dead cells. In all developmental stages analyzed, the mitotic index was notably lower in the former than in non-necrotic zones, suggesting that cell proliferation is partially inhibited in retinal areas of cell death.     

A re-examination of mitotic activity in the early chick embryo

Summary As a result of extensive mitotic index analysis in colchicine-arrested chick embryos during gastrulation, it was ascertained that the primitive streak is a region of elevated mitotic index as compared to the surrounding tissue. Along the cephalo-caudal axis, the embryo displays two large peaks of mitotic index, one at the posterior end of the primitive streak and the other just anterior to Hensen's node. The length of the various phases of the mitotic period was determined in vitro by time-lapse filming, and the colchicine-arrested mitotic indices in vivo and in vitro were determined and compared for various regions. Some observations regarding the orientation of mitotic spindles and abnormal mitosis in vitro are also included, and the relevance of the above observations to early embryonic development is discussed.     

Behavioral analysis of opiate-mediated inhibition in the early chick embryo

Morphine (opiate agonist) produced a dose-dependent decrease in the spontaneous motility of 5- and 9-day chick embryos. Naloxone (opiate antagonist) appeared to reverse competitively the inhibition of motility caused by morphine. The effects of morphine on spontaneous motility in 5-day embryos were also reversed stereospecifically by the opiate antagonist pairs WIN 44441-3/WIN 44441-2 and levallorphan/dextrallorphan. Levorphanol (opiate agonist) also produced a dose-dependent decrease in the motility of 5-day embryos while its inactive (+)-isomer, dextrophan, was not effective. Etorphine (opiate agonist) was more than 1000-fold more effective than morphine in inhibiting the motility of 5-day embryos. The effectiveness of several opiate agonists and antagonists on the spontaneous motility of 5-day embryos was similar to their effectiveness in radioligand-binding studies on isolated membrane receptors from either adult mammalian brain or ileum. Levorphanol was more effective than dextrophan and etorphine was substantially more effective than morphine in decreasing the spontaneous motility of 4-day embryos. WIN 44441-3 was more effective than WIN 44441-2 in reversing the inhibition of motility in 4-day embryos caused by morphine. Morphine inhibited spontaneous hind-limb motility in both thoracic spinal and sham-operated 7-day embryos; the inhibition of motility caused by morphine was reversed by WIN 44441-3 in both thoracic spinal and sham-operated 7-day embryos. [Leu5]enkephalin-like immunoreactivity in the lumbar spinal cord was concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of immunoreactive processes occurred in the medial and lateral motor columns where labelled varicosities appeared to contact motoneurons. Opiate receptors appear to be present at least as early as day 5 (and perhaps as early as day 4) in the chick embryo. Opiate receptors appear to be present in the lumbar spinal cord of the chick embryo at least as early as day 7. The structural requirements for ligand binding to opiate receptors in the 5-day chick embryo are similar to the requirements for ligand binding to opiate receptors in the adult.     

Mitotic activity during somite segmentation in the early chick embryo

Summary The mitotic activity of the somites, segmental plate and posterior mesoderm were investigated in colchicine-treated and untreated chick embryos at st. 7-14. The mitotic figures in the somites are restricted to the proximity of the lumen and have their spindles orientated predominantly tangentially to the cavity. In the segmental plate there is no pattern in terms of the position or orientation of the mitotic spindles, but there is a single region, often found close to the cranial end of the segmental plate, with an elevated mitotic index. This may indicate a certain degree of synchrony among groups of segmental plate cells. These results are discussed in relation to the process of somite segmentation.     

Chondroid tissue in the early facial morphogenesis of the chick embryo

The calcified tissues involved in the early morphogenesis of the so-called intramembranous bones of the facial skeleton were studied by microradiographic and histological techniques in 22 chick embryos at the 9th, 12th and 14 th days of incubation. On the 9th day, the bones of the upper face and palatal vault are made up of thin sheets of chondroid tissue, deposited in their respective mesenchymal condensations. Woven and lamellar bone formation subsequently takes place in each of them from the 12th day of incubation, mainly on the external side of their chondroid primordia. The same phenomena occur in the lower facial and mandibular bones. These facts indicate that the primitive facial desmocranium of the chick embryo, which is classically considered to be formed by intramembranous ossification, first consists of chondroid tissue. As in the cranial vault, this tissue thus represents the initial modality of the skeletogenic differentiation within the avian facial mesenchyme.     

Spatial and temporal expression of perlecan in the early chick embryo

Perlecan is a major heparan sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules. The expression and spatiotemporal distribution of perlecan was studied by RT-PCR, immunoprecipitation and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Combined RT-PCR and immunohistochemistry demonstrated the expression of perlecan as early as stage X and its presence may be fundamental to the first basement membrane assembly on the epiblast ventral surface at stage XIII (blastula). Perlecan fluorescence was intense in the cells ingressing through the primitive streak and was strong lining the epiblast ventral surface lateral to the streak at stage HH3-4 (gastrula). At stage HH5-6 (neurula), perlecan fluorescence was low in the neuroepithelium and stronger in the apical surface of the neural plate. At stage HH10-11 (12 somites), perlecan fluorescence was intense in the neuroepithelium and was then essentially nondetectable in the neuroepithelium, and the intensity had shifted to the basement membranes of encephalic vesicles by stage HH17. Perlecan immunofluorescence was intense in neural crest cells, strong in pharyngeal arches, intense in thymus and lung rudiments, intense in aortic arches and in dorsal aorta, strong in lens and retina and intense in intraretinal space and in optic stalk, strong in the dorsal mesocardium, myocardium and endocardium, strong in dermomyotome, low in sclerotome in somites, intense in mesonephric duct and tubule rudiments, intense in the lining of the gut luminal surface. Inhibition of the function of perlecan by blocking antibodies showed that perlecan is crucial for maintaining basement membrane integrity which mediates the epithelialization, adhesive separation and maintenance of neuroepithelium in brain, somite epithelialization, and tissue architecture during morphogenesis of the heart tube, dorsal aorta and gut. An intriguing possibility is that perlecan, as a signaling molecule that modulates the activity of growth factors and cytokines, participates in the signaling pathways that guide gastrulation movements and neural crest cell migration, proliferation and survival, cardiac cell proliferation and paraxial mesoderm (somitic) cell proliferation and segmentation.     

Extragonadal distribution of primordial germ cells in the early chick embryo

Chick primordial germ cells (PGCs), after separation from the endoderm in early embryonic development, temporarily circulate via the blood vascular system and finally migrate into the gonadal anlagen. It has been noted by some authors that some PGCs are present in extragonadal sites in some vertebrates. In the present study, we examined the distribution and localization of PGCs in extragonadal sites in the chick embryo. PGCs were identified by periodic acid-Schiff staining with light microscopy. In embryos at stages 20-24 (PGCs are in the settlement stage in the gonadal primordium), approximately 20% of the total number of PGCs were observed in extragonadal regions. Approximately 90% of these ectopic PGCs were found in the head, mainly in the mesenchyme surrounding the neural tube. Even at stage 14 when PGCs were usually circulating in the blood vessels, some of the PGCs had emerged from the blood vessels and were detected in the extragonadal site. This pattern of distribution of ectopic PGCs in the head area is probably attributed to the earlier, dominant development of the capillary network, and to the sluggish capillary blood flow in that region, which allows intravascular PGCs to escape into the tissue.     

Cellular mechanisms of neural fold formation and morphogenesis in the chick embryo

The mechanisms underlying neural fold formation and morphogenesis are complex, and how these processes occur is not well understood. Although both intrinsic forces (i.e., generated by the neuroepithelium) and extrinsic forces (i.e., generated by non-neuroepithelial tissues) are known to be important in these processes, the series of events that occur at the neural ectoderm-epidermal ectoderm (NE-EE) transition zone, resulting in the formation of two epithelial layers from one, have not been fully elucidated. Moreover, the region-specific differences that exist in neural fold formation and morphogenesis along the rostrocaudal extent of the neuraxis have not been systematically characterized. In this study, we map the rostrocaudal movements of cells that contribute to the neural folds at three distinct brain and spinal cord levels by following groups of dye-labeled cells over time. In addition, we examine the morphology of the neural folds at the NE-EE transition zone at closely-spaced temporal intervals for comparable populations of neural-fold cells at each of the three levels. Finally, we track the lateral-to-medial displacements that occur in the epidermal ectoderm during neural groove closure. The results demonstrate that neural fold formation and morphogenesis consist of a series of processes comprising convergent-extension movements, as well as epithelial ridging, kinking, delamination, and apposition at the NE-EE transition zone. Regional differences along the length of the neuraxis in the respective roles of these processes are described.     

胚胎时期小鼠小肠的组织发生

目的　观察小鼠胚胎各个时期小肠组织的形态结构及杯状细胞在小肠内的分布规律,为小鼠小肠的组织发生提供形态学依据。 方法　采用HE染色和PAS染色,对小鼠胚胎第13.5天（E13.5d）至出生后第1天（P1d）胚胎的石蜡切片染色并行光学显微镜观察。 结果　(1)小鼠肠壁于E13.5 d已分化出现黏膜层、黏膜下层、肌层及浆膜。(2)肠绒毛于E15.5 d分化形成,杯状细胞于E16.5 d逐渐发育分化出现,肠腺于E18.5~P1 d发育分化形成,此时小肠基本结构形成。(3)杯状细胞主要分布于小肠绒毛上皮,其中以回肠末端最多,回肠、空肠、十二指肠顺次递减（P<0.05）。杯状细胞数量随胎龄逐渐增加而逐渐增多,于P1 d最多（P<0.05）。 结论　小肠上皮分化于E15.5 d至E17.5 d最为迅速,胚胎时期小肠基本结构形成,其吸收消化功能基本建立完成。