鸡胚发育中神经钙黏蛋白对头部神经嵴细胞分层的作用

目的:阐明神经钙黏蛋白(N-cadherin)在神经嵴细胞分层中的作用。方法: 利用原位杂交鉴定N-cadherin在神经管上的表达。转染过表达N-cadherin的野生型N-cadherin(wild-type N-cadherin, wt-N-cad)或阻断N-cadherin表达的结构域缺失型N-cadherin(dominant-negative N-cadherin, dn-N-cad),利用免疫组化方法检测头部神经嵴细胞的迁移变化。结果: 无论是过表达N-cadherin还是下调N-cadherin表达,头部神经嵴细胞迁移都受到了影响。结论: 头部神经嵴细胞的分层和迁移依赖于神经管上N-cadherin的表达。     

Association of the cardiac neural crest with development of the coronary arteries in the chick embryo

Background: Chick coronary arteries orginate as penetrating channels from a subepithelial peritruncal ring into the wall of all three aortic coronary sinuses. Two of these capillaries develop a muscular wall and become the definitive coronary arteries. Since cardiac neural crest (CNC) contributes ectomesenchyme to the tunica media (TM) of the aortic arch vessels, we wished to learn if the CNC also contributes to the media of the coronary arteries and if CNC plays an inductive role in determining the site of aortic penetrations and influences which channels persist to hatching. Methods: Quail-to-chick chimeras were made by bilaterally removing the chick CNC and replacing it with quail CNC. The chimeras and unoperated controls were collected on embryonic days (ED) 7–18, fixed in Carnoy's fixative, serially sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. Several ED 18 controls and chimeras were also stained with Gomori's trichrome stain, or labeled with antineurofilament or antivascular smooth muscle alpha actin. Results: The TM of the coronary arteries and the aortic coronary sinuses did not consist of CNC cells. The media of the surviving coronary arteries was disrupted by clusters of CNC cells scattered in the wall of the base of the coronary artery on ED 14 and 18. Persisting coronary arteries were always associated with large neural crest-derived parasympathetic ganglia near their origin. Branches from parasympathetic nerves entered the base of the coronary arteries where the clusters of neural crest cells were located. Quail cells were also associated with tiny vessels exiting the ostia of the coronary arteries and traveling in the outer aortic wall. Labeling with antibodies confirmed a disruption of the TM at the base of the coronary arteries, and showed innervated clusters of quail cells in the disrupted part of the TM. Conclusion: Although the TM of the coronary arteries and the aortic coronary sinuses contained no CNC cells, clusters of innervated quail cells disrupted the TM at the base of the coronary arteries. CNC does not appear to induce capillary penetration direclty; however, the exclusive association of CNC-derived parasympathetic ganglia and nerves with persisting coronary arteries suggests that the presence of parasympathetic ganglia is essential to the survival of the definitive coronary arteries. CNC cells may also be associated with the development of the aortic vasa vasorum. © 1994 Wiley-Liss, Inc.     

不同发育时期鸡胚脊髓内背侧抑制性轴突导向蛋白的表达和神经嵴细胞迁移之间的相关性

目的 探讨不同发育时期的鸡胚脊髓内背侧抑制性轴突导向蛋白（draxin）表达与神经嵴迁移之间在空间分布上的相关性。方法 选
取HH 15-16、HH19-20阶段鸡胚各10只,应用原位杂交、免疫组织化学等方法,观察各阶段鸡胚脊髓内draxin的表达部位及神经嵴细胞迁移路
径,同时比较两者在空间分布上的相关性;应用活组织切片与碱性磷酸酶标记的draxin融合蛋白（draxin-AP）体外孵育的方法,检测迁移路
径内的神经嵴细胞是否具有与draxin蛋白直接结合的能力。结果 随着发育时间的推移,鸡胚脊髓内draxin的表达和神经嵴迁移都具有明显的
由颅侧向尾侧渐进性分布的特性,且draxin的表达部位位于神经管背侧、顶板和背侧生皮肌节之间,这些部位恰均位于神经嵴迁移路径的周围;
同时迁移路径内的神经嵴细胞具有与draxin蛋白体外直接结合的能力。结论 draxin表达在神经嵴迁移路径周围且部分神经嵴具有与draxin蛋白
体外直接结合的能力,推测draxin可能在鸡胚脊髓神经嵴迁移过程中发挥重要的调节作用。     

A segmented pattern of cell death during development of the chick embryo

Summary During the early development of the chick embryo, specific groups of cells die in characteristic patterns. In this study, Nile Blue sulphate staining was used to reveal a novel pattern of segmentally repeated cell death in the paraxial mesoderm of the chick prior to stage 23. This pattern varies according to the developmental stage of the embryo and shifts rostrocaudally, corresponding to progressing somite differentiation. Initially, during early somite differentiation, cell death is restricted to the rostral half of the somite (the rostral pattern of cell death). After the somite has differentiated into dermomyotome and sclerotome, dead cells appear in superficial tissues in a pyramidal pattern which lies in register (rostrocaudally) with the central part of the sclerotome. Finally, small bands of dying cells are seen between the neural tube and the expanding sclerotome. This third pattern (the ventral path) lies in register with the rostral part of the caudal half of the sclerotome. We show by fluorescent labelling of the migrating neural crest that these patterns of cell death correspond to the routes of neural crest migration. In addition, serial sectioning of stage 23 chick embryos confirms that the position of dying cells correlates with the known routes of neural crest migration and with the sites of development of certain neural crest-derived tissues.     

成纤维细胞生长因子信号调控早期鸡胚胎神经嵴细胞的迁移

目的 利用Sprouty2基因阻断成纤维细胞生长因子（FGF）信号,探讨FGF在早期鸡胚胎发育过程中对神经嵴细胞迁移的影响及其机制。方法 通过体内培养的方法孵育鸡胚至HH9期,通过显微注射的方法将Sprouty2-绿色荧光蛋白（GFP）质粒注射入神经管腔内。实验侧使用电穿孔转染的方法转染胚胎半侧神经管,另一侧正常神经管设为对照侧。采用神经嵴细胞特异标记物HNK1免疫荧光的方法检测Sprouty2基因阻断FGF信号后是否影响胚胎头部和躯干部神经嵴细胞的迁移过程。随后,进一步通过检测神经细胞钙黏分子N-Cadherin的表达来观察细胞之间黏附作用的改变。结果 HNK1免疫荧光检测结果显示,Sprouty2转染侧即阻断FGF信号通路后,HNK1在早期鸡胚胎的头部和躯干部的表达量均比对照侧的表达量增多;而神经细胞钙黏分子N-Cadherin检测结果表明,Sprouty2转染侧和正常对照侧N Cadherin在头部和躯干部神经管上表达量的差异均无显著性。结论 Sprouty2基因阻断FGF信号后,促进了早期鸡胚胎神经嵴细胞的迁移,但是FGF信号对此过程的影响可能不是由神经钙黏分子N-Cadherin介导的。     

Migration and distribution of circumpharyngeal crest cells in the chick embryo. Formation of the circumpharyngeal ridge and E/C8+ crest cells in the vertebrate head region.

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8+ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8+ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudal-most pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. DiI labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.     

Differentiation of the chick embryo floor plate

Summary In a number of species, the floor plate of the developing neural tube and spinal cord has been ascribed specialized functions associated with the patterning of neuronal differentiation. The differentiation of the floor plate itself is believed to be closely related to the presence of the underlying notochord. Grafting experiments have previously shown that in the chick embryo an implanted segment of notochord is capable of inducing the adjacent host neural plate or neural tube to produce an additional floor plate, although the inductive effect diminishes with increasing age of the host. We have examined the potential of notochord to promote the appearance of floor plate-like structures from neural tube tissue in culture. To facilitate this, it was necessary initially to examine the immunoreactivity of the early neural tube and floor plate in situ and in vitro with a panel of antibodies to identify a suitable marker for floor plate differentiation in vitro. In situ, the differentiation of the floor plate was characterized by a lack of immunoperoxidase staining with antibody to neurofilaments and the monoclonal antibody HNK-1 throughout the period examined. This distinguished the floor plate from other regions of the neural tube, and was in contrast to its conspicuous affinity for antibodies to N-CAM and highly sialylated N-CAM, which also stained several closely adjacent regions of the neural tube over the period examined. We also found that oligodendrocytes occurred both in the floor plate and in the flanking ventral neural tube, and that astrocytes were too poorly represented throughout the neural tube at the stages examined to be useful markers of floor plate differentiation. We therefore concluded that only the anti-neurofilament and the HNK-1 antibodies were potentially useful markers for floor plate differentiation. When these antibodies were tested on cells in culture, neural tube tissue showed the presence of neurofilament and HNK-1-positive neurites, while floor plate cultures showed few of these. These distributions were consistent with those demonstrated in situ. However, cells staining positively for N-CAM, sialylated N-CAM and the glial cell markers were relatively sparse in floor plate cultures, suggesting that these epitopes were not retained or were masked in cultured cells. As a result of these experiments, we selected the absence of neurofilament-positive cells as a marker for floor plate differentiation in culture. Co-culture of pieces of neural tube from 3-day embryos with notochord segments resulted in the suppression of neurofilament-positive neurite outgrowth from the former, and the consequent production of tissue with floor plate-like characteristics. The absence of neurites was most marked on the side of the neural tube tissue that was apposed to the notochord. Co-culture of neural tube with other tissues did not produce this effect. We suggest that the neurite suppression by notochord in vitro is analogous to its activity in situ, and that neural tubes from 3-day embryos are still competent to respond to notochordal tissue.     

Cardiac neural crest contribution to the pulmonary artery and sixth aortic arch artery complex in chick embryos aged 6 to 18 days

Previous studies of cardiac neural crest (CNC) migration in early chick embryos demonstrated CNC cells in the media of pharyngeal arch arteries three, four, and six, and in the most proximal part of the developing pulmonary arteries. The objectives of this study were to learn (1) to what extent the CNC is involved in the later development of the pulmonary arteries, (2) how the CNC cells are distributed in the sixth aortic arch artery including the wall of the ductus arteriosus in the older embryo, and (3) what happens to the CNC as the pulmonary artery/sixth arch complex grows into its adult configuration. Quail-to-chick chimeras were used to study CNC distribution in embryos aged 6 to 18 days. Controls (undisturbed chick embryos) were collected with chimeras. Each was fixed, processed, sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. The results demonstrated that CNC disappeared from the proximal pulmonary arteries by embryonic day 9 and played no further role in pulmonary artery development. With the exception of the endothelium, CNC completely filled the wall of the sixth aortic arch artery as far distally as its junction with the dorsal aorta in younger embryos and with the aorta in older embryos, thus suggesting the possibility of proximodistal migration of CNC along the sixth aortic arch. The ductus wall, filled with CNC, was intimately associated with the recurrent laryngeal nerve, also filled with CNC, thereby strongly suggesting a role for CNC in ductal closure. © 1993 Wiley-Liss, Inc.     

Fibronectin distribution in the chick embryo during formation of the blastula

Summary The distribution of the fibronectin-rich extracellular matrix (ECM) in the chick embryo during formation of the blástula has been evaluated semiquantitatively using an electron microscopical immunogold staining technique. During the first 10 h of postlaying development, fibronectin was found in both embryonic area pellucida and extra-embryonic area opaca of the blastoderm. In the area pellucida, the fibronectin was (1) associated with the basal lamina of the epiblast, (2) present between epiblastic and hypoblastic cells and (3) occasionally internalized in hypoblastic cells. Along the embryonic axis, a transient and high density of ECM was associated with the front of the anteriorly and rapidly expanding hypoblast. Very high density of fibronectin was observed in the marginal zone of the area pellucida, where the epiblastic and deeper cell layers show contacts and intense re-arrangements. In the area opaca, fibronectin was at first found only sporadically between contacting cells, but its density increased steadily and markedly during the first day of development. These rapid and significant changes in the regional distribution of fibronectin-rich ECM are discussed with respect to the early morphogenesis of the chick embryo.     

Onset of elastogenesis and downregulation of smooth muscle actin as distinguishing phenomena in artery differentiation in the chick embryo

During development, the arterial system is grossly divided into elastic and muscular vessel types. Apart from local environmental factors, it has been suggested that vascular smooth muscle cell origin (mesoderm or neural crest) is involved in this, as yet poorly understood, arterial differentiation. We describe differentiation of the thoracic arterial system in the chick embryo, using immunohistochemical techniques staining for muscle-specific actin, vinculin and desmin and histological staining to visualise elastin. The initial developmental stages of the vessel wall in all arteries appeared to be highly similar, with all arteries showing peri-endothelial actin and vinculin staining. Major alterations did not occur until the start of elastogenesis, which coincided with complete loss of actin staining from the proximal part of the great arteries. Later in development, however, actin was re-expressed in a subpopulation of medial cells, which also expressed vinculin and desmin. Concomitantly another, nonmuscular, cell type became evident in the great arteries. Transient loss of actin expression and segregation of very distinct cell populations occurred only in vessels prone to elastic development and known to receive a neural crest contribution. In contrast, arteries that developed a muscular phenotype never lost the initially acquired peri-endothelial actin expression. We also show a significant difference in the organisation of elastic fibres between elastic vessels that contain neural crest derivatives and those that do not. The ductus arteriosus still presents as an enigma in the sense that it is the only part of the pharyngeal arch complex that develops a muscular phenotype.     

Pathway formation and the terminal distribution pattern of the spinocerebellar projection in the chick embryo

Summary Pathway formation and the terminal distribution pattern of spinocerebellar fibers in the chick embryo were examined by means of an anterograde labelling technique with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP).Spinocerebellar fibers, which originate in the lumbar spinal cord and are located in the marginal layer of the spinal cord, reach the corsal part of the cerebellar plate on embryonic day (E)8. On the way to the cerebellum the fibers form one distinct bundle, that suggests that gross projection errors probably do not occur during the formation of the spinocerebellar pathway.On E10, labelled fibers are located mostly in the medullary zone of the anterior lobe. By E12, the number of labelled fibers increases greatly in the inner granular and molecular layers. In transverse sections labelling was distributed throughout the mediolateral extent of the medullary zone. By E14, sagittal strips of labelling were clearly recognized in lobules II–IV; however, labelled terminals were present throughout lobule I. Although the adult pattern of terminal distribution is attained by E14, the mossy fiber terminals are still quite immature. The density of labelling decreased greatly by E16, and small terminal varicosities were first recognized. Structural differentiation of mossy fiber terminals continues to the end of the embryonic or the newly posthatched period.     

On the differentiation and migration of some non-neuronal neural crest derived cell types

Summary Neural tubes containing premigratory neural crest cells from head and trunk levels as well as somites containing neural crest cells that have migrated away from the neural crest were grafted orthotopically and heterotopically from quail embryos to chicken embryos. Schwann cells and melanocytes of donor origin developed after all grafting procedures. Cartilage developed only from neural crest cells of head levels. No skeletal muscle was ever observed to develop from the neural crest. The development of these different cell types from heterotopically grafted premigratory neural crest cells indicates that the neural crest is not a population of pluripotent undeterminated cells, but that at least some determinated cells are present within it before the onset of emigration of neural crest cells from the neural crest. Different neural-crest-derived cell populations exhibit different migratory behaviour: After heterotopically grafting quail neural crest cells to the wing buds of chicken embryos. Schwann cells and non-epidermal melanocytes were found to have migrated proximally and distally away from the grafts. Epidermal melanocytes of donor origin were found to have migrated in a distal direction essentially.This work was supported by the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung (P 4680)     

Effect of the notochord on the differentiation of a floor plate area in the neural tube of the chick embryo

Summary The role of a notochord fragment on the origin of an additional floor plate area in the neural tube is investigated by quantitative morphological methods. In 1.5 to 2 day chick embryos a notochordal fragment was implanted in close apposition to the lateral wall of the neural groove in the region between prospective wing and leg bud. At 4 days, adjacent to the implant a distinct area of the neural wall was present, which resembled the natural floor plate with respect to its thickness, the abluminal location of elongated nuclei and the absence of neuroblasts. The mitotic density of this area was reduced. This additional floor plate was distinct when the experiment was performed at 1.5 days but was hardly recognizable when it was carried out at 2 days.From these results it is concluded that a) the notochord induces floor plate like structures and diminishes proliferation, and b) that the period of floor plate induction by the notochord is very restricted.     

Neural crest patterning and the evolution of the jaw

Here we present ideas connecting the behaviour of the cranial neural crest during development with the venerable, perhaps incorrect, view that gill-supporting cartilages of an ancient agnathan evolved into the skeleton of an early gnathostome's jaw. We discuss the pattern of migration of the cranial neural crest ectomesenchyme in zebrafish, along with the subsequent arrangement of postmigratory crest and head mesoderm in the nascent pharyngeal segments (branchiomeres), in diverse gnathostomes and in lampreys. These characteristics provide for a plausible von Baerian explanation for the problematic inside-outside change in topology of the gills and their supports between these 2 major groups of vertebrates. We consider it likely that the jaw supports did indeed arise from branchiomeric cartilages.     

An in vitro model of gonad differentiation in the chick embryo

Summary Embryonic gonads of 6 1/2 to 12 days old chick embryos were enzymatically dissociated. The cell suspensions were cultured in small gas permeable bags of foil (Biofolie Heraeus) in a roller culture apparatus. The cells formed multiple small aggregates, in which sex specific differences developed within two days. In cell suspensions of embryonic testes smooth spheric aggregates formed with well delineated testicular cords in the center and a tunica albuginea-like mesenchymal layer at the outside. Most of the male germ cells were incorporated in the central cords. A number of germ cells were barred from entering the cords by the tunica albuginea-like mesenchymal layer and populated the outer surface of the aggregates. The aggregates of left ovary were irregular in shape and characterized by clusters of germ cells residing in an outer cortical zone. The aggregates of the right ovary, which regresses in vivo, showed poor growth and did not differentiate, thus, indicating that the suppression of right ovary was not removed in culture. In the roller cultures of dissociated embryonic gonads male and female morphogenesis was mimicked in a reproducible manner, so that the system can be used for further experimental studies of gonadal development.     

Endoderm regeneration in the chick embryo studied by SEM

Summary Regeneration of the area pellucida endoderm of the chick embryo was studied by scanning electron microscopy (SEM).A new endoderm was formed by in situ changes in the shape and relationships of mesoderm cells.Initially the cells flattened and lost their processes except along cell boundaries. Later even these processes were lost and an epithelium was formed. The area of regenerated endoderm coincided with the area of mesoderm at the time of endoderm removal, confirming the mesodermal origin of the new layer. Remnants of the original endoderm did not contribute to the regenerated layer. Contact inhibition was observed at the boundary between original and regenerated endoderms.     

Cationized ferritin and phosvitin uptake by coated vesicles of the early chick embryo

Summary The endocytosis of cationized ferritin and of a phosvitinferritin conjugate by cells of the chick embryo area pellucida has been examined. Cationized ferritin was bound mainly to the free surface of the epiblast but was absent from the region of the primitive streak. The binding was patchy and experiments suggest that the anionic sites which bind cationized ferritin are themselves naturally clustered. Uptake of cationized ferritin was exclusively by coated pits. The resulting coated vesicles delivered the cationized ferritin to membrane-bound sites of accumulation in the cytoplasm and to the close vicinity of Golgi bodies. The cationized ferritin was frequently found to share intracellular vacuoles with yolk granules. The uptake was not affected by the presence of microfilament or microtubule-inhibiting agents. Native ferritin, even at concentrations forty times that of cationized ferritin, was not bound or endocytosed.Coated pits in the epiblast were often associated with overlying extracellular yolk granules. This suggested that the yolk might be inducing the formation of the coated pits. The yolk protein phosvitin was coupled to ferritin and this conjugate was found to be endocytosed by coated pits. This uptake was inhibited in the presence of an excess of free phosvitin but not by albumin, indicating some selectivity for phosvitin over other proteins. The phosvitin conjugate was also found sharing intracellular vacuoles with yolk. We conclude that the cells of the area pellucida, and in particular those of the epiblast, have an active coated vesicle uptake system which may be able to selectively endocytose yolk or yolk protein.