Effects of colchicine- and cytochalasin B-treatment on the intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells

Treatment with colchicine (antimicrotubular agent) and cytochalasin B (antimicrofilamentous agent) has been used to investigate the possible role played by the cytoskeleton in the maintenance of intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells. On the one hand, embryos treated with colchicine showed modifications in their distribution patterns of yolk droplets and lipid bodies, which suggests the involvement of the microtubular integrity of neuroepithelial cells in the maintenance of normal distribution patterns. On the other hand, the close relationships between vitelline and lipid inclusions and Golgi apparatus observed in untreated embryos seems to be kept in the embryos treated with colchicine and cytochalasin B. Moreover, from the effects of colchicine on Golgi apparatus position a possible functional role for the microtubular system in the maintenance of Golgi apparatus polarity in the chick neuroepithelial cells can be proposed. The results provided here constitute new information about the cellular mechanisms involved in chick neurulation.     

Loss of Occludin and Functional Tight Junctions, but Not ZO-1, during Neural Tube Closure—Remodeling of the Neuroepithelium Prior to Neurogenesis

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.     

Neural tube closure defects caused by papaverine in explanted early chick embryos.

Papaverine (50 micrograms/ml) preferentially inhibited uplifting of neural folds in explanted stage 8 chick embryos. Affected neuroepithelial cells often lost their wedge-shaped and elongated appearance. Also, luminal surfaces of most affected cells were smoother than usual as evidenced by the marked decrease in the number of cytoplasmic extensions, but the integrity of other structures (including cytoskeletal components) was not noticeably affected. The observed changes in cell surface topography were due, at least in part, to the imparied ability of apical microfilaments to contract and their eventual relaxation. The "relaxing" effect of papaverine on neural folds could be reversed by subsequent treatment with ionophore A23187. Since papaverine and ionophore A23187 are known to alter the normal distribution of intracellular Ca2+ and changes in cell surface topography are correlated with contractile activities of apical microfilaments, papaverine elicits neural tube closure defects by lowering intracellular free Ca2+ levels, thereby relaxing contracted apical microfilaments in neuroepithelial cells.     

Embryonic expression of beta-actin-lacZ hybrid gene injected into the fertilized ovum of the domestic fowl.

An experiment was carried out to investigate the expression of cloned DNA injected into the germinal disc of the chick fertilized ovum. The beta-actin-lacZ hybrid gene, MiwZ, was injected, in the closed circular form, into the cytoplasm of the germinal disc at the single-cell stage. The embryos were cultured in vitro, then in recipient eggshells up to day 4 of incubation. The survival rate of the embryos at day 4 was 42% (55/130), and the rate of embryos expressing MiwZ was 64% (35/55). Twenty-two embryos expressed the MiwZ in both embryonic and extraembryonic tissues, while the remainder expressed the MiwZ in only extraembryonic tissues. Mosaic expression was observed in most of the embryos expressing MiwZ in embryonic tissues. Expression throughout all tissues of the embryo including blood cells occurred in one case. In this case, the injected DNA was assumed to have integrated at an earlier stage. The results indicate that it is now possible to investigate the promoter activities of introduced exogenous genes as well as the effect of introduced genes on embryogenesis in early chick embryos. This technique may also facilitate the production of transgenic chicks.     

FGF2 plays a key role in embryonic cerebrospinal fluid trophic properties over chick embryo neuroepithelial stem cells

During early stages of brain development, neuroepithelial stem cells undergo intense proliferation as neurogenesis begins. Fibroblast growth factor 2 (FGF2) has been involved in the regulation of these processes, and although it has been suggested that they work in an autocrine-paracrine mode, there is no general agreement on this because the behavior of neuroepithelial cells is not self-sufficient in explants cultured in vitro. In this work, we show that during early stages of development in chick embryos there is another source of FGF2, besides that of the neuroepithelium, which affects the brain primordium, since the cerebrospinal fluid (E-CSF) contains several isoforms of this factor. We also demonstrate, both in vitro and in vivo, that the FGF2 from the E-CSF has an effect on the regulation of neuroepithelial cell behavior, including cell proliferation and neurogenesis. In order to clarify putative sources of FGF2 in embryonic tissues, we detected by in situ hybridization high levels of mRNA expression in notochord, mesonephros and hepatic primordia, and low levels in brain neuroectoderm, corroborated by semiquantitative PCR analysis. Furthermore, we show that the notochord segregates several FGF2 isoforms which modify the behavior of the neuroepithelial cells in vitro. In addition, we show that the FGF2 ligand is present in the embryonic serum; and, by means of labeled FGF2, we prove that this factor passes via the neuroepithelium from the embryonic serum to the E-CSF in vivo. Considering all these results, we propose that, in chick embryos, the behavior of brain neuroepithelial stem cells at the earliest stages of development is influenced by the action of the FGF2 contained within the E-CSF which could have an extraneural origin, thus suggesting a new and complementary way of regulating brain development.     

Chick embryos can form teratomas from microinjected mouse embryonic stem cells

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra‐embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon‐like or dark‐red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad‐range potential as an experimental host model.     

An early developmental phase of pp60c-src expression in the neural ectoderm

The expression of the normal cellular src protein (pp60c-src) was investigated in the early chick embryo during gastrulation and neurulation by immunoperoxidase staining using antisera, raised against bacterially expressed pp60v-src, that recognizes pp60c-src specifically in normal cells. During gastrulation pp60c-src immunoreactivity appeared primarily in the neural ectoderm and was much less prominent in the mesoderm, endoderm, and nonneural ectoderm. During neurulation pp60c-src immunoreactivity began to disappear from the wall of the closing neural tube so that by the completion of neural tube closure no specific pp60c-src immunoreactivity appeared in any of the neuroepithelial cells composing the neural tube. These studies reveal a developmental phase of pp60c-src expression even earlier than reported previously, when neuroepithelial cells of later embryos undergo terminal neuronal differentiation. These findings raise the possibility that pp60c-src may mediate two different differentiation signals in the neuronal lineage.     

The cellular environment controls the expression of engrailed-like protein in the cranial neuroepithelium of quail-chick chimeric embryos.

We have previously shown that one of two chicken engrailed-like genes, chick En-2, is expressed in a restricted region of the early chick embryo brain: the mes/metencephalon (Gardner et al. 1988). In this study, we examine the role of the cellular environment in regulation of engrailed-like (En) protein expression in quail-chick chimeric embryos. Two types of transplant surgery were performed at the 9-15 somite stage to produce chimeric embryos. In the first, the mid-mesencephalic vesicle or caudal mesencephalic vesicle alar plate (which is En protein-positive) was transplanted from a quail embryo into an En protein-negative region of chick neuroepithelium, the prosencephalon (mMP and cMP grafts, respectively). In the second reciprocal surgery, prosencephalic alar plate which is En protein-negative, was transplanted into the En protein-positive mesencephalic vesicle (PM grafts). A polyclonal antiserum, alpha Enhb-1, which recognizes chick En proteins (Davis et al. 1991) was used to identify En-positive cells 48 h after surgery. In mMP embryos, 71% of integrated grafts had lost En expression (n = 17). In contrast, in cMP grafts, 93% of integrated grafts continued to stain with the antiserum (n = 14). In addition, in 86% of these embryos, the graft induced adjacent chick host diencephalic cells to become En protein-positive as well. All PM grafts contained aEnhb-1-positive cells; such cells never expressed this protein in their normal environment. These early changes in En protein expression correlate well with the morphological changes observed in similar graft surgeries assayed later in development. Thus, our results are consistent with the hypothesis that En genes play a role in the regionalization of the early cranial neuroepithelium.     

Toxic and teratologic effects of verapamil on early chick embryos: evidence for the involvement of calcium in neural tube closure

Toxic and teratologic effects of verapamil, a calcium antagonist, on chick embryos explanted at stage 8 (four-somite stage) and cultured for 6-8 hours were investigated. In general, embryos responded to verapamil in a dose-related manner. Concentrations lower than 2 micrograms/ml had no apparent effect on the development of embryos. A concentration of 15 micrograms/ml significantly increased the incidence of embryos (approximately 80% of viable embryos) with neural tube closure defects and less numerous somites. Higher concentrations (e.g., 30 micrograms/ml) were embryotoxic and over 90% of the embryos were either severely malformed or dead after 8 hours of incubation. Compared to controls, verapamil-treated neuroepithelial cells had smoother apical surfaces and less conspicuous microfilament bundles. The deleterious effects of verapamil (15 micrograms/ml) could be reversed by subculturing the affected embryos, within 3 hours of treatment, on nutrient medium alone or on nutrient medium containing 25 micrograms/ml chlorotetracycline (CTC), a calcium agonist, the latter being more effective provided that treatment did not exceed 4 hours. Exposure of the developing neuroepithelium to 15 micrograms/ml verapamil for 3-4 hours resulted in a significant reduction in free Ca2+ levels, as revealed by the pyroantimonate precipitation method, throughout neuroepithelial cells. Overall results suggest that verapamil causes neural tube closure defects by reducing intracellular free Ca2+ levels, thereby relaxing apical microfilament bundles of developing neuroepithelial cells.     

Alternate expression of the HNK-1 epitope in rhombomeres of the chick embryo.

Rhombomeres are regarded as the manifestation of innate segmentation within the vertebrate CNS. To investigate developmental changes occurring in the CNS and PNS, a series of chick embryos were immunostained with several monoclonal antibodies. The HNK-1-immunoreactivity (IR) appeared in rhombomeres (r) 3 and r5 around stage 15, when r2 and r4 were not stained. This alternate pattern is similar to the Krox-20 gene expression in the mouse embryo. At levels of r2 and r4, HNK-1+ neural crest cell masses were attached to the CNS forming cranial sensory ganglia. In these rhombomeres, an accumulation of neuroepithelial cells near the cranial nerve root and early development of neuroblasts in the basal plate were observed. The above observations seem to suggest that the alternate HNK-1-IR in rhombomeres might be related to the expression of cell adhesion molecules, and therefore also to the adhesion of the cranial ganglion precursors to the CNS, which takes place every other rhombomere in the preotic region. Thus, the alternate pattern of the HNK-1-IR seems to be related to the morphogenesis of preotic branchial nerves.     

Role of cell-cycle in regulating neuroepithelial cell shape during bending of the chick neural plate

Summary Neuroepithelial cells transform from spindle-shaped to wedge-shaped within the median and paired dorsolateral hinge points of the bending neural plate, but the mechanisms underlying these localized changes are unclear. This study was designed to evaluate further the hypothesis that localized wedging of neuroepithelial cells during bending involves basal cellular expansion resulting from alteration of the cell-cycle. Neurulating chick embryos were treated with tritiated thymidine, and transverse sections through the midbrain were examined autoradiographically. Parameters of the cell-cycle as well as nuclear position and size were assessed in the median hinge point, which contains predominantly wedge-shaped cells, and in adjacent lateral areas of the neural plate, which contain predominantly spindle-shaped cells. Both the DNA-synthetic phase and non-DNA synthetic portion of the cell-cycle were significantly longer in the median hinge point than in lateral neuroepithelial areas, some nuclei in both regions were located basally during these phases, and virtually all basal nuclei in the median hinge point were large. Additionally, the mitotic phase was significantly shorter in the median hinge point than in lateral areas. We present a model to explain how alteration of the cell-cycle in the median hinge point could generate wedging of cells in this region.     

Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos

We have used the vital dye, DiI, to analyze the contribution of sacral neural crest cells to the enteric nervous system in chick and mouse embryos. In order to label premigratory sacral neural crest cells selectively, DiI was injected into the lumen of the neural tube at the level of the hindlimb. In chick embryos, DiI injections made prior to stage 19 resulted in labelled cells in the gut, which had emerged from the neural tube adjacent to somites 29-37. In mouse embryos, neural crest cells emigrated from the sacral neural tube between E9 and E9.5. In both chick and mouse embryos, DiI-labelled cells were observed in the rostral half of the somitic sclerotome, around the dorsal aorta, in the mesentery surrounding the gut, as well as within the epithelium of the gut. Mouse embryos, however, contained consistently fewer labelled cells than chick embryos. DiI-labelled cells first were observed in the rostral and dorsal portion of the gut. Paralleling the maturation of the embryo, there was a rostral-to-caudal sequence in which neural crest cells populated the gut at the sacral level. In addition, neural crest cells appeared within the gut in a dorsal-to-ventral sequence, suggesting that the cells entered the gut dorsally and moved progressively ventrally. The present results resolve a long-standing discrepancy in the literature by demonstrating that sacral neural crest cells in both the chick and mouse contribute to the enteric nervous system in the postumbilical gut.     

Cultured chick yolk sac epithelium: structure and IgG surface binding

The chick yolk sac endoderm transports maternal immunoglobulin G (IgG) from the yolk into the embryo during development, providing the newly hatched chick with passive immunity until it becomes immunocompetent. To study this transport process, chick yolk sac endodermal cells isolated from embryos of 6 to 18 days of incubation were grown in vitro on a collagen substrate. The cultured cells possessed a remarkable structural similarity to the in vivo tissue and reformed a polarized confluent epithelium with tight junctions and desmosomes joining the cells at their apical margins. In addition, the cells exhibited apical microvilli, numerous phagolysosomes in the cytoplasm and retained the expression of the yolk sac endoderm-specific enzyme marker, cysteine lyase. Importantly, the cultured cells retained the ability to specifically bind IgG as demonstrated by indirect immunofluorescence. Chicken IgG bound to the cultured cells at 4 degrees C in a diffuse pattern that clustered into a punctate pattern when a second antibody was used. Cultures from yolk sacs of day 6 through day 18 of development all demonstrated this immunofluorescent labeling for at least 14 days in culture. These results demonstrate that cultured yolk sac endoderm maintains its differentiated morphology and ability to bind IgG.     

Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route.

The behavior of quail primordial germ cells (PGC) after injection into chick embryos by the intravascular route was examined. The quail (donor) PGC, taken from the bloodstream of quail embryos (recipient) at stage 13-14, were injected into the vitelline vessels of chick embryos (recipient) at stage 15. In the recipient embryos, the PGC of the quail and the chick were histochemically distinguished by a double-staining technique involving a lectin, from Wistaria floribunda (WFA) and the PAS reaction. One day after injection, quail PGC appeared in the prospective gonadal region of recipient chick embryos, being localized among the recipient chick PGC. This result indicates that a staining technique specific for WFA lectin is useful for identification of quail PGC and that quail PGC can be transferred by a vascular route for the production of germline chimeras.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.     

Pathogenesis of persistent truncus arteriosus induced by nimustine hydrochloride in chick embryos

Nimustine hydrochloride (ACNU) is a nitrosourea derivative anticancer agent which has been shown to cause persistent truncus arteriosus in chick embryos. The objective of this study was to confirm the teratogenic effects of ACNU on the cardiovascular system of chick embryos and to determine whether ACNU induces persistent truncus arteriosus by interfering with neural crest cells. Various doses of ACNU ranging from 10 to 200 micrograms were injected under the chorioallantoic membrane of chick embryos on the third day of incubation. Saline solution was used as the control. After 10 to 11 days of incubation, 242 (46%) survivors of the 524 treated eggs were obtained. The survival rates of the embryos and the frequencies of cardiovascular anomalies were dose dependent. Of 146 embryos with cardiovascular anomalies, 104 (71%) had persistent truncus arteriosus. Ventricular septal defect and double-outlet right ventricle were seen in 37 (25%) and one (1%), respectively. Aortic arch anomalies were seen in 116 embryos (79%). Quail-chick chimeras (chick embryos with quail cardiac neural crest) were treated with 50 micrograms of ACNU and examined histologically 24 hours later. These chimeras showed dying neural crest cells in the pharyngeal arches. Dying cells were also noted in the neural tube, cranial ganglia, retina, and otocyst. These results suggest that persistent truncus arteriosus in chick embryos treated with ACNU is induced by neural crest cell death.     

Notochordal induction of cell wedging in the chick neural plate and its role in neural tube formation

Cells in the median hinge point (MHP) of the bending chick neural plate are tightly apposed to the underlying notochord. These cells differ from those in adjacent lateral neuroepithelial areas (L) in that MHP cells are short and mainly wedge-shaped and line a furrow, whereas L cells are tall and mainly spindle-shaped and do not line a furrow. Cell generation time also differs in these regions. These consistent differences are detectable only after the notochord has formed and established contact with the neural plate; it is unclear whether they result from self-differentiation or induction. Two experiments were performed to evaluate the hypothesis that MHP characteristics develop owing to inductive interactions between the notochord and overlying neuroepithelial cells. First, notochordless chick embryos were generated to determine whether midline neuroepithelial cells still developed typical MHP characteristics. In the absence of the notochord, such characteristics did not develop. Second, isolated segments of quail notochord were transplanted subjacent to L of chick hosts to ascertain whether the notochord is capable of inducing MHP characteristics in L cells. When transplanted notochordal segments established apposition with host L cells, the apposing L cells usually developed typical MHP characteristics. Collectively, these results provide strong evidence that the notochord plays an inductive role in the formation of MHP characteristics. This investigation further revealed that bending can occur in the absence of MHP characteristics, forming a neural tube with an abnormal morphology. Thus, the formation of such characteristics, particularly cell wedging, is not required for bending but plays a major role in generating the normal cross-sectional morphology of the neural tube.     

Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain

An immunohistochemical survey was carried out on frozen sections of the early embryonic chick brain between 1 and 6 days of incubation, with antisera to the three neurofilament proteins (NF-L, NF-M, NF-H). Large numbers of replicating neuroepithelial cells were found to express one of these proteins, NF-M, generations before the existence of any postmitotic neuroblasts (Days 1-2 1/2 of incubation). NF-L and NF-H could not be detected. Not all primordial brain regions contained NF-M-positive cells, but in those that did, every cell was positive. These regions included the dorsal forebrain, optic vesicles, and dorsal hindbrain, but not the dorsal midbrain. All cells in all regions of the cephalic neural tube contained vimentin, whether or not they also contained NF-M. This NF-M expression was transient in the sense that later generations of these NF-M-positive neuroepithelial cells became NF-M negative, before finally giving rise to some descendents that ultimately express all three NF proteins. This transient NF-M expression was found in certain other cells of early embryos, including cardiac myoblasts. The identity of the component in these early neural and nonneural tissues, that bound the antibody, was demonstrated to be identical to adult brain NF-M by one- and two-dimensional immunoblots. These findings demonstrate an unusual kind of biochemical heterogeneity among neuroepithelial cells, and they are relevant to considerations regarding lineage analysis and lineage "markers" in the vertebrate central nervous system.     

Continuous delivery of a monoclonal antibody against Reissner’s fiber into CSF reveals CSF-soluble material immunorelated to the subcommissural organ in early chick embryos

The subcommissural organ (SCO) is an ependymal differentiation located in the dorsal midline of the caudal diencephalon under the posterior commissure. SCO cells synthesize and release glycoproteins into the cerebrospinal fluid (CSF) forming a threadlike structure known as Reissner’s fiber (RF), which runs caudally along the ventricular cavities and the central canal of the spinal cord. Numerous monoclonal antibodies have been raised against bovine RF and the secretory material of the SCO. For this study, we selected the 4F7 monoclonal antibody based on its cross-reactivity with chick embryo SCO glycoproteins in vivo. E4 chick embryos were injected with 4F7 hybridoma cells or with the purified monoclonal antibody into the ventricular cavity of the optic tectum. The hybridoma cells survived, synthesized and released antibody into the CSF for at least 13 days after the injection. E5 embryos injected with 4F7 antibody displayed precipitates in the CSF comprising both the monoclonal antibody and anti-RF-positive material. Such aggregates were never observed in control embryos injected with other monoclonal antibodies used as controls. Western blot analysis of CSF from E4-E6 embryos revealed several immunoreactive bands to anti-RF (AFRU) antibody. We also found AFRU-positive material bound to the apical surface of the choroid plexus primordia in E5 embryos. These and other ultrastructural evidence suggest the existence of soluble SCO-related molecules in the CSF of early chick embryos.C. Hoyo-Becerra and M.D. López-ávalos contributed equally to this study and should be considered as first authors. C. Hoyo-Becerra was the recipient of a predoctoral fellowship (PFPI) from the Ministerio de Educacion y Cultura (Spain). This work was supported by grants from DGICYT (BFI2003-03348; Spain) and FIS (01/0948; Spain), FIS (01-0948, PI021517; Spain) and ISCIII (red CIEN, nodo Fundación Carlos Haya).