MALFORMATIONS OF THE CENTRAL NERVOUS SYSTEM INDUCED BY NEUROTROPIC DRUGS IN MOUSE EMBRYOS

Out of a sample of fifteen neurotropic drugs consisting of seven antidepressants and anti-psychotics, two antianxiety drugs, one anticonvulsant, three opiates and two synthetic analgesics, twelve were found to be teratogenic for mouse embryos, causing malformations of the central nervous system. After single injections of the teratogenic dose administered at the very beginning of the ninth day of gestation, four days later, i.e. in 13-day-old embryos, the induced defects appeared to make up a recurring syndrome of malformations which consists of several abnormalities present in various frequencies either individually or in combination in the same embryos. These malformations are: exencephaly, craniorachischisis, cervical and thoraco-lumbar myeloschisis, hydrocephalic dilatation of the fourth brain ventricle, Z-shaped kinking of the spinal cord and lumbar hydromyelia. In addition, after administration of some of the drugs, branchyury or anury with or without lumbar myeloaplasia were recorded.
In general the results reported here seem to suggest that because of their possible affinity neurotropic drugs are potentially teratogenic for the embryonic central nervous system if applied at the time of the neural tube closure although it is known that there are drugs in this group which do not cause any malformations of the central nervous system and that many non-neurotropic agents do cause such malformations. Secondly, the results seem to suggest also that the position of the malformations along the cerebro-spinal axis may be depending to some extent on the pharmacological properties of the drugs tested. These conjectures are treated here as entirely provisional pending further investigations.     

Cyclic GMP is not involved in neural induction inXenopus laevis

Summary Recent evidence indicates an important role for cell-surface mediated signal transduction in embryonic induction. We, therefore, started a systematic search to identify signal transduction pathways which are activated during embryonic induction and specifically during neural induction. We showed previously that the protein kinase C and cAMP pathways mediate neural induction inXenopus laevis. Here, we investigated whether cGMP is also involved in the early development of the nervous system. We measured the cGMP content of whole embryos at embryonic stages which mark important events in the early development of the nervous system, as well as in the developing neural tissue itself, after this was induced from ectoderm by dorsal mesoderm. No changes in cGMP content were found, either in whole embryos at different developmental stages, or in developing neural tissue from these stages. We also found no evidence for the presence of nitroprusside stimulatable guanylate cyclase in these developmental stages. A cGMP analogue, 8-Br-cGMP, was not able to induce neural tissue, either alone or in combination with known neural inducers, the phorbol ester TPA and 8-Br-cAMP. 8-Br-cGMP also had no negative influence on the neural inducing ability of dorsal mesoderm or TPA, alone or in combination with 8-Br-cAMP. We conclude that cGMP has no role in the early development of the central nervous system inXenopus laevis. This conclusion underlines the specificity of the signal transduction pathways (PKC and cAMP pathways) that do mediate neural induction.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Influenza-B-virus-induced eye and brain malformations during early chick embryogenesis and localization of the viral RNA in specific areas

Influenza is prevalent worldwide, and the teratogenic effects of influenza infection have been suspected to occur within the developing central nervous system. We herein report the sequelae of influenza B viral infection during early chick embryogenesis. Chick embryos at Hamburger-Hamilton stage 9 were infected by an in ovo injection under the blastoderm of influenza B virus (B/Taiwan/25/99). At 48 h after infection, gross malformations of the eye and brain, ranging from 25 to 58% of 168 infected embryos, were observed, in contrast to 3–6% among 71 mock-infected controls (p < 0.0001 for both eye and brain malformations). Histological analyses showed extensive tissue degeneration and aggregates of cells in the head mesenchyme, suggesting cell death and heterotopia. Influenza B viral RNA was directly localized by in situ hybridization with probes specific for the HA segment. Viral RNA was extensively detected in the head surface ectoderm and in the lung bud. In the developing brain, viral RNA was specifically located in the anterior neural retina, habenular area, mid-thalamus, and rhombencephalon. Our data show that influenza B virus can be a teratogenic agent in neural and nonneural embryonic tissues, raising concern for transplacental infection during early pregnancy.     

Brain, eye, and face defects as a result of ectopic localization of Sonic hedgehog protein in the developing rostral neural tube.

BACKGROUND: Normal development of the face, eyes, and brain requires the coordinated expression of many genes. One gene that has been implicated in the development of each of these structures encodes the secreted protein, Sonic hedgehog (Shh). During central nervous system development, Shh is required for ventral specification along the entire neural axis. To further explore the role of Shh in chick brain and craniofacial development, we overexpressed Shh in the developing rostral neural tube METHODS: In order to determine if Shh is sufficient to ventralize the forebrain, we localized ectopically recombinant Shh protein to the rostral neural tube of chick embryos. The resulting embryos were evaluated morphologically and by assaying gene expression. RESULTS: Disruption in normal gene expression patterns was observed with a reduction or loss in expression of genes normally expressed in the dorsal forebrain (wnt-3a, wnt-4, and Pax-6) and expansion of ventrally expressed genes dorsally (HNF-3beta, Ptc). In addition to the genetic alterations observed in the neural tube, a craniofacial phenotype characterized by a reduction in many cranial neural crest-derived structures was observed. The eyes of Shh-treated embryos were also malformed. They were small with expansion of the retinal pigmented epithelium, enlarged optic stalks, and a reduction of neural retina. DISCUSSION: The ectopic localization of recombinant Shh protein in the rostral neural tube resulted in severe craniofacial anomalies and alterations of gene expression predicted by other studies. The system employed appears to be a model for studying the embryogenesis of malformations that involve the brain, eyes, and face.     

Influence of L.5-hydroxytryptophan (L.5-HTP) on the development of chick embryo]

L.5-hydroxytryptophan (L.5-HTP) injections provoke, in the chick embryo, some malformations of the nervous system, when treated at 24 hours of incubation. The same treatement after 48 hours of incubation does not lead to malformations, but to a reduction in size which is as much obvious as the embryos are treated at a later stage. It seems that there could be some relation between the serotonin metabolism and the growth hormon secretion.     

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chick^GFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. Chick^GFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.     

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage^1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.     

Exposure of neural crest cells to elevated glucose leads to congenital heart defects, an effect that can be prevented by N-acetylcysteine

BACKGROUND: Diabetes mellitus during pregnancy increases the risk for congenital heart disease in the offspring. The majority of the cardiovascular malformations occur in the outflow tract and pharyngeal arch arteries, where neural crest cells are essential for normal development. We studied the effects of specific exposure of neural crest cells to elevated glucose on heart development. Antioxidants reduce the damaging effect of glucose on neural crest cells in vitro; therefore, we investigated the effect of supplementing N-acetylcysteine in vivo. METHODS: Cardiac neural crest of HH 8-12 chicken embryos was directly exposed by a single injection in the neural tube with 30 mM D-glucose (or 30 mM L-glucose as a control). To examine the effect of a reduction in oxidative stress, we added 2 mM N-acetylcysteine to the injected D-glucose. RESULTS: Exposure of neural crest cells to elevated D-glucose-induced congenital heart malformations in 82% of the embryos. In the embryos injected with L-glucose, only 9% developed a heart malformation. As expected, all malformations were located in the outflow tract and pharyngeal arch arteries. The frequency of heart malformations decreased from 82% to 27% when 2 mM N-acetylcysteine was added to the injected D-glucose. CONCLUSIONS: These data are the first to confirm that the vulnerability of neural crest cells to elevated glucose induces congenital heart malformations. The fact that N-acetylcysteine limits the teratogenicity of glucose implies that its damaging effect is mediated by an increase of oxidative stress in the neural crest cells.     

硫代硫酸钠干扰斑马鱼胚胎发育并致畸

硫的衍生物潜在的威胁着胚胎的发育过程.斑马鱼被用于研究不同浓度（1×10-6～1 mol/L）的硫代硫酸钠（sodium thiosulfate, STS）对胚胎发育的影响,在解剖显微镜下实时观察斑马鱼胚胎发育的全过程.采用Western 印迹法检测乙酰化的微管蛋白——α-微管蛋白（acetylated tubulin, α-tubulin）和神经元增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达,分别检测STS暴露后胚胎的运动神经元功能,神经元的增殖状态.发育中的斑马鱼胚胎暴露于0.1～1 mol/L STS,呈现出严重的发育迟缓,并且伴随多脏器畸形;暴露于10 μmol/L～10 mmol/L STS,胚胎呈现循环系统,神经系统以及颌面部畸形.胚胎在48 hpf (hours post fertilization)时,对STS的暴露敏感高于24 hpf和96 hpf.STS可能干扰细胞的增殖及运动神经元的正常分化.STS可能干扰正常的细胞骨架结构,并在胚胎发育晚期影响细胞增殖,对胚胎神经系统、循环系统及颌面部有致畸作用.     

Mesoderm induction in the future tail region of Xenopus

Summary We have used interspecific grafts between Xenopus borealis and Xenopus laevis to study the signalling system that produces tail mesoderm. Early gastrula ectoderm grafted into the posterior neural plate region of neurulae responds to a mesodermal inducing signal in this region and forms mainly tail somites; this signal persists until at least the early tail bud stage. Ventral ectoderm grafted into the posterior neural plate loses its competence to respond to this signal after stage 10 1/2. We have established the specification of anterior and posterior neural plate ectoderm. In ectodermal sandwiches or when grafted into unusual positions, anterior regions gave rise to mainly nervous system and posterior regions to large amounts of muscle, together with some nervous system. Thus it was impossible to assess the competence of posterior neural plate ectoderm to form further mesoderm and hence to establish if mesodermal induction continues during neurulation in unmanipulated embryos.     

Development of the enteric nervous system in chick embryonic duodenum in terms of the distribution of tubulin

An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.     

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction.

The development of the vertebrate nervous system is initiated in amphibia by inductive interactions between ectoderm and a region of the embryo called the organizer. The organizer tissue in the dorsal lip of the blastopore of Xenopus and Hensen's node in chick embryos have similar neural inducing properties when transplanted into ectopic sites in their respective embryos. To begin to determine the nature of the inducing signals of the organizer and whether they are conserved across species we have examined the ability of Hensen's node to induce neural tissue in Xenopus ectoderm. We show that Hensen's node induces large amounts of neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may reflect notoplate induction. The competence of the ectoderm to respond to Hensen's node extends much later in development than that to activin-A or to induction by vegetal cells, and parallels the extended competence to neural induction by axial mesoderm. The actions of activin-A and Hensen's node are further distinguished by their effects on lithium-treated ectoderm. These results suggest that neural induction can occur efficiently in response to inducing signals from organizer tissue arrested at a stage prior to gastrulation, and that such early interactions in the blastula may be an important component of neural induction in vertebrate embryos.     

The interference of naloxone hydrochloride in the teratogenic activity of opiates

Diamorphine hydrochloride, methadone hydrochloride, and the synthetic enkephalin analogue FK 33-824 are potent teratogens for the central nervous system in mouse embryos. They induce the "neurotropic syndrome of malformations," which is restricted to the central nervous system if administered during the critical period of neural tube closure. Pretreatment with corresponding equimolecular doses of the antagonist naloxone hydrochloride applied 30 minutes before treatment with the opiate agonists abolishes the major severe malformations, i.e., exencephaly, craniorachischisis, and brachyury, and reduces the number of cases of kinking of the spinal cord. Dilation of the fourth brain ventricle remains unaffected. It is suggested that the mechanism of interference in the teratogenicity of the opiates by naloxone hydrochloride reported here is based on competition for opiate receptors. In general, these observations are regarded as evidence that the pharmacological affinity of opiate agonists to receptors in the central nervous system is responsible for the malformations caused by them in this system.     

FGF signaling is essential for the early events in the development of the chick nervous system and mesoderm.

Fibroblast growth factor (FGF) belongs to a family of polypeptides with diverse biological functions. In the present study we have assessed the role of FGF signaling in the development of nervous system and mesodermal tissues in chick embryo. Treatment of in vitro cultured embryos with exogenous, human recombinant FGF led to abnormalities in neural induction and development, notochord formation and somitogenesis as studied by gross morphology and histology. Overall growth and development was also adversely affected as seen from the measurement of body axis length. Further, treatment of embryos with FGF resulted in differential modulation of expression of two genes important in normal development as studied by whole mount in situ hybridization using DIG-labeled riboprobes. The expression of Brachyury, which is necessary for mesoderm formation, was down-regulated in FGF-treated embryos. The expression of noggin, the product which participates in the patterning of the chick neural tube was, on the other hand, up-regulated within 2 h. We also studied development of neural and mesodermal tissues in conditions where FGF signaling was defective. This was achieved by culturing the embryos in the presence of suramin. In the presence of low doses of suramin (100-150 nmole/culture), abnormalities were detected mainly in the mesodermal structures while at higher doses (200-400 nmole/culture), the nervous system too was found to be abnormal in a large proportion of embryos. Treatment of chick embryos with suramin (200 nmole/culture) also modulated the expression of Brachyuryand noggin within a 2 h period. The results showthat FGF signaling plays an important role in the molecular events leading to the development of nervous system and mesodermal tissues in the chick embryo.     

Spontaneous cyclic embryonic movements in humans and guinea pigs

Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cyclic. This study explores the distribution of SB-to-SB intervals in human and guinea pig embryos before the appearance of more complex movements such as general movements. We hypothesized that the activity in both species is cyclic. We made 15-min sonographic recordings of SBs between 5 weeks and 0 days (5wk0d) and 7wk0d conceptional age (CA) in 18 human embryos of uncomplicated IVF pregnancies (term 38 weeks) and in 20 guinea pig embryos between 3wk4d and 4wk0d CA (term 9 weeks). SB-to-SB interval durations were categorized as long (≥10 s) or short (<10 s) intervals. For human embryos, the median values for long and short intervals were 61 s (range, 10-165 s) and 3 s (range, 1-9 s) respectively; for guinea pigs 38 s (range, 10-288 s) and 5 s (range, 1-9 s), respectively. During development, the duration of long intervals decreased while the number of short intervals increased for both species. The earliest embryonic motility in the human and guinea pig is performed cyclically with distinct developmental milestones. The resemblance of their interval development offers promising possibilities to use the guinea pig as a noninvasive animal model of external influences on motor and neural development.     

Spatial aspects of neural induction in Xenopus laevis

A monoclonal antibody, 2G9, has been identified and characterised as a marker of neural differentiation in Xenopus. The epitope is present throughout the adult central nervous system and in peripheral nerves. Staining is first detected in embryos at stage 21 in the thoracic region. By stage 29 it stains the whole central nervous system, except the tail tip. The epitope is present in a 65K Mr protein, and includes sialic acid. The antibody also reacts with neural tissue in mice and axolotls and newts. 2G9 was used to show that both notochord and somites are capable of neural induction, and the stimulus is present as late as stage 22. Attempts to demonstrate the induction of nervous system by developing nervous system (homoiogenetic induction) were unsuccessful. The view that the lateral extent of the nervous system might be determined by that of the inductive stimulus is discussed. Neural induction was detected as early as stage 10 and occurs in embryos without gastrulation and without cell division from stage 7 1/2.     

Distribution of laminin in the developing peripheral nervous system of the chick

During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and on neighboring cell surfaces may provide "choices" of pathways for growth cone migration. The extracellular matrix glycoprotein laminin appears in early embryos and mediates neuronal adhesion and neurite extension in vitro. In this study, we have examined the distribution of laminin at early periods of peripheral nervous system development. The distribution of laminin, demonstrated by immunostaining frozen sections of chick embryos, was compared to the distribution of fibronectin and of early peripheral neurites as revealed with an antibody to a neurofilament-associated protein. Laminin is present in the neural tube basement membrane, in early ganglia, and in developing dorsal and ventral roots, where the laminin staining pattern parallels that of neurofilaments. In early ganglia and nerve roots, laminin immunostaining defines loose "meshworks" rather than basement membranes, which seem to form slightly later in these structures. In contrast, fibronectin is absent in neural tube basement membrane, ganglia, and nerve roots, although it is present along neural crest migratory pathways and in intersomitic spaces. Our observations of laminin distribution are consistent with the possibility that laminin provides an adhesive surface for neurite extension at some stages of early peripheral nervous system development.