PTEN基因对早期胚胎神经嵴细胞迁移的作用研究

目的 初步探讨PTEN基因在早期神经嵴细胞迁移中的作用.方法 首先胚胎整体的原位杂交和免疫荧光方法检测鸡胚胎内源性的PTEN基因及蛋白水平的表达情况;其次,利用鸡胚胎体内半侧神经管转染的方法,使神经管一侧PTEN基因过表达,对侧神经管为正常对照侧;最后,通过Pax7的整体胚胎免疫荧光表达观察PTEN基因对其标记的部分神经嵴细胞迁移的影响.结果 内源性PTEN基因在mRNA和蛋白水平表达显示,其在早期胚胎HH4期的神经板即开始明显的表达;通过半侧过表达PTEN基因后观察到过表达PTEN基因侧的头部神经嵴细胞迁移与对照侧相比明显受到抑制,但对躯干部的影响并不明显.结论 PTEN基因可能抑制早期胚胎头部神经嵴细胞的迁移.     

In ovo time-lapse analysis after dorsal neural tube ablation shows rerouting of chick hindbrain neural crest

Previous analyses of single neural crest cell trajectories have suggested important roles for interactions between neural crest cells and the environment, and amongst neural crest cells. To test the relative contribution of intrinsic versus extrinsic information in guiding cells to their appropriate sites, we ablated subpopulations of premigratory chick hindbrain neural crest and followed the remaining neural crest cells over time using a new in ovo imaging technique. Neural crest cell migratory behaviors are dramatically different in ablated compared with unoperated embryos. Deviations from normal migration appear either shortly after cells emerge from the neural tube or en route to the branchial arches, areas where cell-cell interactions typically occur between neural crest cells in normal embryos. Unlike the persistent, directed trajectories in normal embryos, neural crest cells frequently change direction and move somewhat chaotically after ablation. In addition, the migration of neural crest cells in collective chains, commonly observed in normal embryos, was severely disrupted. Hindbrain neural crest cells have the capacity to reroute their migratory pathways and thus compensate for missing neural crest cells after ablation of neighboring populations. Because the alterations in neural crest cell migration are most dramatic in regions that would normally foster cell-cell interactions, the trajectories reported here argue that cell-cell interactions have a key role in the shaping of the neural crest migration.     

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

Effect of hyaluronidase treatment on the distribution of cranial neural crest cells in the chick embryo

The cranial paraxial mesoblast is patterned into segmental units termed somitomeres. Recently we demonstrated the morphological relationship between the migratory pathways of cranial neural crest cells and the patterned primary mesenchyme of chick embryos (Anderson and Meier, '81). Since extracellular matrix, particularly hyaluronate, is also distributed in cranial crest pathways, embryos were given sub-blastodisc injections of hyaluronidase just prior to neural tube fusion and neural crest migration to remove matrix. Histological sections of enzyme-treated embryos showed that Alcian blue staining of hyaluronate was significantly reduced. Surface ectoderm appeared collapsed on the subjacent mesoderm as well. Examination of embryos with the scanning electron microscope (SEM) revealed that paraxial mesoderm remained segmentally patterned even though it appeared more condensed because of a reduction in intercellular space between mesenchymal cells. In enzyme-treated embryos, the rostral crest cells spread over the dorsal surfaces of the first four somitomeres, as they would do normally. This distribution of neural crest cells occurs even when enzyme treatment interferes with neural tube fusion at that level. We conclude that 1) neural tube fusion is not a prerequisite for the timely release of cranial crest in the chick embryo and 2) that much of the organized hyaluronate-rich matrix that lies in the path of cranial crest is not essential for crest emigration or patterned distribution.     

Nodose placode provides ectomesenchyme to the developing chick heart in the absence of cardiac neural crest

Summary The cardiac neural crest provides both ectomesenchyme and parasympathetic postganglionic neurons to the developing heart. Ablation of cardiac neural crest results in persistent truncus arteriosus, a condition in which the conotruncal and aorticopulmonary septa do not form in the developing heart. Parasympathetic postganglionic neurons are abundantly present in hearts with persistent truncus arteriosus, which indicates a regeneration of the neural component of the cardiac neural crest without comparable restitution of the ectomesenchymal component. The neural component has been shown to be provided by cells from the nodose placode following ablation of the cardiac neural crest. This investigation has shown that ectomesenchymal cells are also supplied to a limited extent by the nodose placode which normally has no ectomesenchymal derivatives. Although placode-derived ectomesenchyme helps to strengthen the wall of the cardiac outflow vessel, it is not competent to induce conotruncal and aorticopulmonary septal closure.     

Cholinergic traits in the neural crest: Acetylcholinesterase in crest cells of the chick embryo

Previous work by our group has demonstrated that mesencephalic neural crest cells at an early stage of migration are able to synthesize acetylcholine (ACh). Acetylcholinesterase (AChE), the enzyme responsible for ACh degradation, was examined in neural crest cells of the chick embryo, using cytochemical and biochemical methods. Observations at the light microscope level showed that cholinesterase activity, identified as true AChE, was present at all axial levels in presumptive crest cells of the neural folds, soon after closure of the neural tube. Subsequently, AChE activity was found in cells of the individualized neural crest and in crest cells migrating at cephalic and trunk levels. Cell counts revealed that 88–94% of the total crest population was AChE-positive. Electron microscope observations indicated that the enzyme was confined to perinuclear and endoplasmic reticulum cisternae. The AChE of migrating mesencephalic neural crest cells was identified as the dimeric form (sedimentation coefficient 6.9 S) of the catalytic subunit. These results indicate that the specific AChE is present in the majority of neural crest cells all along the neural axis. Thus the ability to synthesize and degrade ACh is expressed at least in some neural crest cells at an early stage of development.     

Epigenetic regulation in neural crest development

The neural crest (NC) is a multipotent, migratory cell population that arises from the developing dorsal neural fold of vertebrate embryos. Once their fates are specified, neural crest cells (NCCs) migrate along defined routes and differentiate into a variety of tissues, including bone and cartilage of the craniofacial skeleton, peripheral neurons, glia, pigment cells, endocrine cells, and mesenchymal precursor cells (Santagati and Rijli,2003; Dupin et al.,2006; Hall,2009). Abnormal development of NCCs causes a number of human diseases, including ear abnormalities (including deafness), heart anomalies, neuroblastomas, and mandibulofacial dysostosis (Hall,2009). For more than a century, NCCs have attracted the attention of geneticists and developmental biologists for their stem cell-like properties, including self-renewal and multipotent differentiation potential. However, we have only begun to understand the underlying mechanisms responsible for their formation and behavior. Recent studies have demonstrated that epigenetic regulation plays important roles in NC development. In this review, we focused on some of the most recent findings on chromatin-mediated mechanisms for vertebrate NCC development.     

Acetylcholinesterase activity in neural crest cells of the early chick embryo

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.     

Adrenergic innervation of the developing chick heart: neural crest ablations to produce sympathetically aneural hearts

Ablation of various regions of premigratory trunk neural crest which gives rise to the sympathetic trunks was used to remove sympathetic cardiac innervation. Neuronal uptake of [3H]-norepinephrine was used as an index of neuronal development in the chick atrium. Following ablation of neural crest over somites 10-15 or 15-20, uptake was significantly decreased in the atrium at 16 and 17 days of development. Ablation of neural crest over somites 5-10 and 20-25 caused no decrease in [3H]-norepinephrine uptake. Removal of neural crest over somites 5-25 or 10-20 caused approximately equal depletions of [3H]-norepinephrine uptake in the atrium. Cardiac norepinephrine concentration was significantly depressed following ablation of neural crest over somites 5-25 but not over somites 10-20. Light-microscopic and histofluorescent preparations confirmed the absence of sympathetic trunks in the region of the normal origin of the sympathetic cardiac nerves following neural crest ablation over somites 10-20. The neural tube and dorsal root ganglia were damaged in the area of the neural-crest ablation; however, all of these structures were normal cranial and caudal to the lesioned area. Development of most of the embryos as well as the morphology of all of the hearts was normal following the lesion. These results indicate that it is possible to produce sympathetically aneural hearts by neural-crest ablation; however, sympathetic cardiac nerves account for an insignificant amount of cardiac norepinephrine. The adrenal medulla is the most likely source of cardiac norepinephrine in sympathetically aneural hearts.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells

Although neural crest cells are known to be very responsive to environmental cues during their development, recent evidence indicates that at least some subpopulations may be committed to a specific differentiation program prior to migration. Because the neural crest is composed of a heterogeneous mixture of cells that contributes to many vertebrate cell lineages, assessing the properties of specific subpopulations and the effect of the environment on their development has been difficult. To address this problem, we have isolated a pure subpopulation of chick mesencephalic neural crest cells by fluorescence no-flow cytometry after labeling them with monoclonal antibodies (Mabs) to a 75-kDa cell surface antigen that is associated with high affinity choline uptake. When cultures of chick mesencephalic neural crest cells are labeled with these Mabs and a fluorescent second step antibody, approximately 5% of the cells are antigen-positive (A+). After sorting, 100% of the resulting cultured mesencephalic neural crest cells are A+. The Mabs we used also label all of the neurons of the embryonic chick and quail ciliary ganglion in vivo and in vitro. We have compared the effect of various cell culture media on the isolated neural crest subpopulation and the heterogeneous chick mesencephalic neural crest from which it was derived. A+ cells were passaged and grown in a variety of media, each of which differently affected its characteristics and development. A+ cells proliferated in the presence of 15% fetal bovine serum (FBS) and high concentrations (10-15%) of chick embryo extract, but did not differentiate, although they retained basal levels of choline acetyltransferase (ChAT) activity. However, in chick serum and high (25 mM as opposed to 7 mM) K+, and heart-, iris-, or lung-conditioned medium, all of which are known to promote survival and/or cholinergic development of ciliary ganglion neurons, the cells ceased to proliferate and all of the cells in the culture became "neuron-like" within 10 days. No neuron-like cells were found in liver-, notocord-, or neural tube-conditioned media if FBS was used. When A+ cells were eliminated either by complement-mediated cytotoxicity or by laser-ablating A+ cells during no-flow cytometry, all ChAT activity was also eliminated, and no neuron-like cells or ChAT activity was found in cultures during a subsequent 3-week culture period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular regulation of neural crest development

The neural crest is a transient embryonic structure that gives rise to a multitude of different cell types in the vertebrate. As such, it is an iideal model to study the processes of vertebrate differentiation and development. This review focuses on two major questionsrelated to neural crest development. The first question concerns the degree and time of commitment of the neural crest cellsto differntt cell lineages and the emerging role of the homebox containing genes in regulating this process. Evidence from the cephalic crest suggests that the commitment process does start before the neural crest cells migrate away from the neural tube and gene ablation experiments suggest that different homeobox genes are required for the development of neural and mesenchymal tissue derivatives. However, clonal analysis of neural crest cell before migration suggests that many of the cells remain multi-potential indicating that the final determinative steps occur progressively during migration and in association with environmental influences. The second question concerns the nature of the environmental factors that determine the differentiation of neural crest cells into discrete lineages. Evidence is provided, mainly from in vitro experiments, that purified growth factors selectively promote the differentiation of neural crest cells down either sympathetic, adrenal, sensory, or melanocytic cell lineages.     

Epiblast origin and early migration of neural crest cells in the chick embryo

Chick embryos carrying transplants labeled with tritiated thymidine demonstrate that the neural crest originates in the anterior epiblast, at the junction of areas destined for epidermis and neural tube. As the neural tube begins to fold and the axis lengthens, cells along this junction are drawn dorsomedially; at the seven-somite stage they begin to separate from the epithelium of the head, and migrate into the angle between the epidermis and the neural tube. The paraxial mesoderm already populating this angle originates in more posterior and medial portions of the epiblast than do the neural crest cells; after invagination at the primitive streak, it migrates anterolaterally, ventral to the ectoderm layer, until it too is folded dorsomedially into the angle between the epidermis and the neural tube.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.     

Gene-regulatory interactions in neural crest evolution and development

In this review, we outline the gene-regulatory interactions driving neural crest development and compare these to a hypothetical network operating in the embryonic ectoderm of the cephalochordate amphioxus. While the early stages of ectodermal patterning appear conserved between amphioxus and vertebrates, later activation of neural crest-specific factors at the neural plate border appears to be a vertebrate novelty. This difference may reflect co-option of genetic pathways which conferred novel properties upon the evolving vertebrate neural plate border, potentiating the evolution of definitive neural crest.