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Prevention of gastrulation but not neurulation by antibodies to fibronectin in amphibian embryos.

Gastrulation and formation of the neural plate are major steps in early vertebrate embryogenesis. Although morphogenetic movements leading to the formation of the primary germ layers have been extensively described, the mechanisms governing migration of mesodermal cells and their interactions with ectoderm remain ill-defined. A large body of evidence indicates that fibronectin (FN), a high molecular weight cell-surface-associated glycoprotein, promotes cell adhesion and cell migration throughout embryogenesis. FN has been detected at an early blastula stage in Pleurodeles waltlii. We now show that FN is a component of a dense fibrillar matrix underlying the blastocoel roof; in contrast, the exterior surface of the embryo is devoid of FN. Microsurgical inversion of part of the blastocoel roof does not prevent mesodermal cell migration except at the site of inversion where no FN matrix is available. Perturbation experiments using antibodies to FN demonstrate that the invagination of presumptive mesodermal cells does not occur when the monovalent antibodies are injected before or at the onset of gastrulation; on the other hand, the formation of a neural plate is not prevented when late gastrula stage embryos are treated with antibodies to FN. We conclude that the presence of FN is required for cell migration during gastrulation.

Animals↗

The essential role of Cited2, a negative regulator for HIF-1alpha, in heart development and neurulation.

Cited2 is a cAMP-responsive element-binding protein (CBP)/p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1alpha through its competitive binding with HIF-1alpha to CBP/p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2(-/-) embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1alpha-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2(-/-) hearts. The increase of VEGF levels is significant, because defects in the Cited2(-/-) embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2(-/-) embryos demonstrate an enhanced expression of HIF-1alpha-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.

Animals↗

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo.

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.

Animals↗

Phenytoin-induced teratogenesis: a molecular basis for the observed developmental delay during neurulation.

PURPOSE: We wished to determine whether chronic phenytoin (PHT) exposure could impair neural development and if any morphological alterations could be linked to changes in gene expression. METHODS: Pregnant SWV mice were chronically administered PHT 40 mg/kg/day from gestational day (GD) 0:12 (day:h) until they were killed at various timepoints throughout neural tube closure (NTC). At each timepoint, embryos from both treated and control dams were collected and scored for their progression through NTC. The neural tubes were then isolated and subjected to in situ transcription (IST) and antisense RNA amplification procedures. Using these techniques, we examined the expression of 10 genes: N-cadherin (Ncad), collagen type IV (col-IV), bcl-2, c-jun, PAX-3, collular retinol binding protein-2 (CRBP-2), retinoic acid receptor alpha (RAR alpha), transforming growth factor(beta2) (TGF(beta2)), wee-1, and EMX-2. RESULTS: Chronic PHT exposure not only caused a delay in NTC whereby exposed embryos lagged behind the controls at each collection timepoint, but also significantly altered the expression of specific genes at distinct times during NTC. Early in NTC, PHT induced a significant reduction in the expression of N-cad, col-IV, and c-jun in exposed embryos as compared with controls. In contrast, during the midstages of NTC, the only significant molecular alterations observed in the PHT-exposed embryos was the continued decreased expression of col-IV and an increase in CRBP-2 expression. Finally, in the latter stages of NTC, PHT caused a significant reduction in the expression of bcl-2, RAR alpha, TGF(beta2), EMX-2, and PAX-3. CONCLUSIONS: These results show that although the effects of PHT are morphologically subtle, causing a delay in the development of the neural tube, this delay is accompanied by alterations in critical genes at crucial times of neural development that may account for the observed neurological deficits often associated with PHT exposure.

Abnormalities, Drug-Induced↗

Transient modulation of gene expression in the neurulation staged mouse embryo.

Transient modulation of gene expression in the embryo during early organogenesis will allow studies to be conducted that determine tissue- and stage-specific function(s) of genes. To achieve this goal, viral vectors and antisense oligodeoxynucleotides have been used to produce gain-of-function and loss-of-function models. Adenoviral transduction of whole embryos, embryonic heart and vasculature, and primary neural crest cell culture has been reported. The morphological consequences of overexpression or decreasing expression of selected genes have been evaluated using these tools. Gene-teratogen interaction studies have also been performed. The viral vectors appear to be important tools for modulating gene expression and hold great promise for future research.

Adenoviridae↗

Actin distribution patterns in the mouse neural tube during neurulation.

With the use of antibodies to actin and indirect immunofluorescent techniques regions of increased actin concentration were demonstrated first in basal and later in apical areas of mouse neuroepithelial cells. These patterns of staining corresponded to shape changes observed in cranial neural folds as they initially elevated from the neural plate and later moved toward the midline.

Actins↗

Fuel-mediated teratogenesis: biochemical effects of hypoglycemia during neurulation in mouse embryos in vitro.

Hypoglycemia has been reported to induce congenital malformations and growth retardation in rodent embryos during the period of neural tube closure in vitro. However, the biochemical alterations responsible for the production of the dysmorphogenic effects have not been evaluated. Therefore, the rates of glucose metabolism by glycolysis, citric acid cycle, oxidative pentose phosphate pathway (PPP), and anabolic utilization were evaluated in mouse embryos and extraembryonic membranes using the whole embryo culture technique. Altered glucose metabolism by glycolysis and oxidative PPP, as well as altered anabolic synthesis, were produced by exposure to hypoglycemia. In embryos exposed to mild hypoglycemia (80 mg/dl) altered metabolism by the PPP and an associated effect on nucleic acid synthesis were in part responsible for the dysmorphogenic effects of this treatment. In contrast, severe hypoglycemia (40 mg/dl) appeared to have an immediate effect on glycolytic metabolism in addition to effects on the PPP and nucleic acid synthesis. Therefore, a multifactorial biochemical mechanism contributes to the induction of malformations by severe hypoglycemia in mouse embryos in vitro. Furthermore, the differential effects of moderate vs. severe hypoglycemia on glycolytic metabolism, and possibly energy production, may account for the differences in the severity of these treatments on embryonic growth and the incidence of malformations.

Animals↗

Single cell lineage and regionalization of cell populations during Medaka neurulation.

To study the movement of individual cells and development of cell grouping during neurogenesis, we labeled single cells in early Medaka gastrula at stage 13 [13 hours post-fertilization (hpf)] with a fluorescent vital dye, and analyzed cells and their descendants using time-lapse live recording up to stage 24 (44 hpf). At stage 13, all future neural cells were located in a dorsal 140 degrees sector of the embryo, and migrated toward the vegetal pole; but during stage 15 to 16, they converged towards the midline. Cells that contributed to later neural subdivisions initially formed overlapping populations, but after stage 16+ they formed non-overlapping cell groups having characteristics of tissue 'compartments', preceding development of morphologically distinct neural subdivisions. In early retinal development, a single compartment for future retinal cells was formed superficial to telencephalic and diencephalic compartments, but it was split into left and right eye components at stage 17 in parallel with anterodorsal movement of the diencephalic compartment. At stage 16+, when these compartments were established, Pax6 expression initiated, but only in the laterally located subpopulation of the retina precursor. These observations revise the current view of bilateral retinal development. Continuous live recording of labeled single precursor cells and computer graphics-assisted data analysis, which are presented for the first time in this study, provide excellent means with which to analyze essential cellular processes in organogenesis.

Animals↗

Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.

Embryonic stem cells differentiate into neuroectodermal cells under specific culture conditions. In primates, these cells are organized into rosettes expressing Pax6 and Sox1 and are responsive to inductive signals such as Sonic hedgehog (Shh) and retinoic acid. However, direct derivation of organized neuroectoderm in vitro from preimplantation mammalian embryos has never been reported. Here, we show that bovine inner cell masses from nuclear transfer and fertilized embryos, grown on feeders in serum-free medium, form polarized rosette structures expressing nestin, Pax6, Pax7, Sox1, and Otx2 and exhibiting interkinetic nuclear migration activity and cell junction distribution as in the developing neural tube. After in vitro expansion, neural rosettes give rise to p75-positive neural crest precursor cell lines capable of long-term proliferation and differentiation in autonomic and sensory peripheral neurons, glial cells, melanocytes, smooth muscle cells, and chondrocytes, recapitulating in vitro the unique plasticity of the neural crest lineage. Challenging the rosette dorsal fate by early exposure to Shh induces the expression of ventral markers Isl1, Nkx2.2, and Nkx6.1 and differentiation of mature astrocytes and neurons of central nervous system ventral identity, demonstrating appropriate response to inductive signals. All together, these findings indicate that neural rosettes directly derived from cloned and fertilized bovine embryos represent an in vitro model of early neural specification and differentiation events. Moreover, this study provides a source of highly proliferative neural crest precursor cell lines of wide differentiation potential for cell therapy and tissue engineering applications.

Animals↗

[Morphometric analysis of neural tube cells in neurulating chick embryos].

Preliminary report on a morphometric study of the neuroepithelium of 1 S and 7 S -- stage chick embryos. We show first that the nucleo-cytoplasmic ratio and then, that the volume fraction of intercellular spaces, nuclei and cytoplasm as they relate to the neural tube, differ significatively from cephalic to caudal portion of the neural tube. At stage 7 S only the volume fraction of intercellular spaces, nuclei and cytoplasm in the neural tube change along the cephalo-caudal axis of the embryos. Finally, between the two stages, at the back of the head, 1) volume fraction of intercellular spaces in the neural tube remains unchanged 2) nucleo-cytoplasmic ratio increase 3) volumetric density and size of mitochondria do not change, while surface density of endoplasmic reticulum in cytoplasm increases.

Animals↗

[Forskolin and cyclic adenosine monophosphate inhibit the neurulation action of concanavalin A on explants of the gastrula ectoderm of amphibians].

We studied effects of forskolin, an activator of adenylate cyclase activity, and dioctanoyl-cyclic adenosine monophosphate (do-cAMP) on neutralizing (N) activity of concanavalin A (Con A). Biological testing was performed using explanted animal pole ectoderm of the Rana temporaria early gastrula. Con A treatment (200 micrograms/ml, 2 h) resulted in neutralization of 70-90% explants. If the explants were previously treated with forskolin (100 microM, 1 h), Con A effect decreased to 10%. When Con A and forskolin were applied simultaneously, no N-effect was observed. The same results were obtained with simultaneous treatment of the explants with Con A and do-cAMP (10(-5) M). Moreover, treatment with forskolin of the explants previously treated with Con A inhibited their neural differentiation. We suggest that N-effect of Con A is calcium dependent; the increase in intracellular cAMP after treatment of explants with forskolin or do-cAMP interferes with intracellular Ca2+ release and this results in the inhibited N-effect of Con A.

Animals↗

Organelle distribution in the wedge-, spindle- and inverted wedge-shaped neuroepithelial cells during chick embryo neurulation.

The formation of the neural tube is a morphogenetic process involving cell-shape changes. The activity of the cytoskeletal elements of the neuroepithelial cells and the interkinetic nuclear migration affect the neuroepithelial cell shape in a way that these cells acquire three distinct conformations of wedge-shaped, spindle-shaped and inverted wedge-shaped cells. Each of them is ultrastructurally characterized in this paper with regard to the intracellular distribution of mitochondria, rough endoplasmic reticulum, yolk droplets, lipid bodies and Golgi apparatus. These distribution patterns are analyzed in relation to the role played by the neuroepithelial cells during the successive phases of neural tube formation.

Animals↗

Development of neural tube basal lamina during neurulation and neural crest cell emigration in the trunk of the mouse embryo.

In the trunk of higher vertebrates, the neural crest (NC) cells remain temporarily within the dorsal portion of the neural tube after fusion of the neural folds; shortly thereafter they emigrate, invading surrounding spaces and tissues. One of the factors postulated to be important in the initiation of migration of NC cells is the disruption of the basal lamina (BL) over the dorsal portion of the neural tube. It has been assumed by many that the BL must be discontinuous in order that the NC cells can leave the neural tube; and indeed, experiments performed in our laboratory, and by others, have shown that NC cells cannot penetrate an intact BL. Therefore, we have undertaken a systematic ultrastructural study to evaluate the condition of the BL during neural fold elevation and NC cell emigration. Our results show that: (i) BL surrounding the neural epithelium (NE) becomes progressively more extensive from neural fold to migratory stages. It first forms on the lateral portion of the neuroepithelium of the neural folds and then extends ventrally into the region adjacent to the notochord; (ii) BL becomes continuous beneath the epidermal ectoderm (EE) that overlies the NC cell region only during the terminal stages of NC cell emigration; (iii) BL does not form over the dorsal portion of the neural tube until NC emigration is terminated; and (iv) the morphology of the BL changes as development proceeds. We conclude that absence of a BL over the premigratory NC cell population in the trunk of mouse embryos is a necessary but not a sufficient condition for emigration to take place.

Animals↗