Cephalic neurulation and optic vesicle formation in the early mouse embryo

The overall pattern of cephalic neurulation and the concomitant early development of the optic vesicles in mouse embryos were examined by scanning electron microscopy. Paraffin-sectioned specimens were also examined. The overall pattern of closure of the cephalic neural folds accords well with earlier observations of this process. The earliest indication of optic placode formation was seen in histological sections of embryos at the 4-ysomite stage, while optic pit formation was first observed at the 5- to 6-somite stage. The upper halves of the optic vesicles were formed in 10- to 15-hsomite embryos by the fusion of the neural folds at the junction between the mesencephalon and prosencephalon, while closure of the lower halves was associated with the closure of the rostral neuropore, and was usually completed by about the 20-somite stage. By the 25- to 30-somite stage, a rapid increase in the volume of the forebrain was observed, so that the optic vesicles were displaced laterally. An overall increase in the volume of the optic vesicles and decrease in the diameter of the optic stalks were also observed at this time. This account of cephalic neurulation and optic organogenesis provides useful baseline data relevant to the study of the normal early development of the mouse. A comparison is made between similar events in the rat, the hamster, and the human embryo.     

Ultrastructure of secondary neurulation in the chick embryo

Formation of the future lumbosacral level of the spinal cord was studied in two-day-old chick embryos by light and electron (transmission and scanning) microscopy. A neurulation overlap zone occupied this level. The dorsal portion of the neural tube formed by bending of the neural plate and approximation and fusion of neural folds (i.e., by primary neurulation), and the ventral part formed during secondary neurulation by cavitation of an initially solid, compact mass of cells, the medullary cord, derived from the tail bud. Secondary neurulation involved four morphogenetic processes: (1) segregation of the cells of the prospective medullary cord from cells of adjacent regions, (2) formation of a precisely delimited medullary cord, (3) cavitation of the central portion of this cord, and (4) coalescence of all lumina into a single, central cavity. Cell segregation was associated with the formation of a layer of primarily extracellular materials between adjacent organ rudiments. The source and composition of these materials are unknown. Formation of the medullary cord entailed considerable elongation of the peripheral cells of this developing structure and the fabrication of small intercellular junctions, first at the basal (outer) ends of the elongating peripheral cells, and then at their apical (inner) ends. These events resulted in the formation of an outer pseudostratified layer of radially arranged, columnar cells, having characteristics similar to those of the neural plate, and an inner cluster of irregularly shaped and arranged cells. Cavitation always occurred first at the junction between these two cellular populations. The central cells of the medullary cord also eventually elongated, like the peripheral cells, and may have been intercalated into the lateral walls of the developing neural tube as lumina coalesced.     

Endoderm regeneration in the chick embryo studied by SEM

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

Placodes of the chick embryo studied by SEM

Summary The otic, the lens and the nasal placodes have been examined in chick embryos between stages 10 and 18 of Hamburger and Hamilton. At the stage when each placode first becomes visible conspicuous differences have been seen in the surface morphology between those cells which will invaginate and form the placode and those which will remain on the surface of the head, forming the epidermis. The differences become more pronounced with increasing development. The placode cells possess many surface projections whilst the epidermal cells do not. These differences in surface morphology are related to other differences which are visible in TEM sections, the placode cells being highly columnar and extending the full depth of the placode, whilst the epidermal cells are cuboidal or even squamous. This modification in cell shape of the placode cells is correlated with the presence of longitudinally orientated microtubules.The mechanism of invagination is discussed and evidence is presented which supports the idea that there is a migration of cells into the placode from one side. Such a phenomenon would help to explain the asymmetrical structure of the placode, including the presence of the overhanging lip.     

A reexamination of the role of microfilaments in neurulation in the chick embryo

Formation of wedge-shaped neuroepithelial cells, owing to the constriction of apical bands of microfilaments, is widely believed to play a major part in bending of the neural plate. Although cell "wedging" occurs during neurulation, its exact role in bending is unknown. Likewise, although microfilament bands occupy the apices of neuroepithelial cells, whether these structures are required for cell wedging is unknown. Finally, although it is known that cytochalasins interfere with neurulation, it is unknown whether they block shaping or furrowing of the neural plate, or elevation, convergence, or fusion of the neural folds. The purpose of this study was to reexamine the role of microfilaments in neurulation in the chick embryo. Embryos were treated with cytochalasin D (CD) to depolymerize microfilaments and were analyzed 4-24 hr later. CD did not prevent neural plate shaping, median neural plate furrowing, wedging of median neuroepithelial cells, or neural fold elevation. However, dorsolateral neural plate furrowing, wedging of dorsolateral neuroepithelial cells, and convergence of the neural folds were blocked frequently by CD. In addition, neural folds always failed to fuse across the midline in embryos treated with CD, and neural crest cell migration was prevented. These data indicate that only the later aspects of neurulation may require microfilaments, and that certain neuroepithelial cells, particularly those that normally wedge with median furrowing and elevation of the neural folds, become (and remain) wedge-shaped in the absence of apical microfilament bands. Thus, microfilament-mediated constriction of neuroepithelial cell apices is not the major force for median neuroepithelial cell wedging and elevation of the chick neural plate. Further studies are needed to localize the motor(s) for these processes.     

Distribution of cell surface glycoconjugates during secondary neurulation in the chick embryo

Lectin histochemistry was used to examine the expression of cell surface glycoconjugates during secondary neurulation in chick embryos. Fourteen lectins were applied to serial sections of the caudal region of embryos at the various stages of tail bud development. The lectins Bandeiraea simplicifolia, Dolichos biflorus agglutinin, Phaseolus vulgaris leukoagglutinin, soybean agglutinin, Sophora japonica agglutinin, Ulex europaeus agglutinin and succinylated wheat germ agglutinin (sWGA) showed very light or no binding to the developing medullary cord of the tail bud. With the other lectins, staining occurred throughout the early tail bud and solid medullary cord. During cavitation, however, differential expression of cell surface glycoconjugates by different cell populations was observed. The lectins concanavalin A, Lens culinaris agglutinin, Pisum sativum agglutinin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin and WGA showed basic similarities in the distribution of lectin binding. Of these, the binding pattern of WGA was the most striking. As the medullary cord cells were separating into central mesenchymal and peripheral epithelial populations, WGA bound preferentially to the epithelial cells and the notochord. The lectin PNA, however, became preferentially bound to the mesenchymal cells. Heavy staining by WGA (specific for N-acetylglucosamine and sialic acid) where sWGA staining (specific for N-acetylglucosamine only) was faint suggested that WGA binding was due to the presence of sialic acid containing glycoconjugates.     

Deceleration and acceleration in the rate of posterior neuropore closure during neurulation in the curly tail (ct) mouse embryo

Summary Curly tail (ct) is a mouse mutant producing spinal neural tube defects as a result of delayed closure of the posterior neuropore (PNP). The purpose of the present study was to determine in ct/ct embryos the time of onset of the delay in PNP closure, and the pattern of this closure, as well as to study the possibility that reopening of the neural tube occurs. Normal spinal neurulation was studied in non-mutant Swiss (Sw) embryos. In the latter, the average PNP length diminished steadily between the 7- and 25-somite stages, and then decreased more rapidly, indicating an acceleration of closure rate, until the 30- to 32-somite stage, when all PNPs closed. PNP width decreased steadily between the stages of 7 and 30 somites. In ct/ct embryos the average PNP length showed a slight increase between the stage of 23 to 28 somites, indicating a temporary deceleration of closure rate, and the range of PNP sizes increased markedly. This was followed by a decrease in PNP length until the 37-somite stage, indicating an acceleration of closure rate. From the stage of 32 somites onwards, the proportion of embryos with closed PNPs gradually increased to 90%. The population of ct/ct embryos was subdivided. Embryos with large PNPs showed a marked deceleration of closure rate during a period of 11 somite stages, followed by a brief but very high acceleration of closure rate. This resulted in closure of the PNP in a proportion of these embryos, while in the remainder of the embryos the deceleration phase had been too enhanced to allow complete catch up of closure during the acceleration phase; these embryos would develop spina bifida. Embryos with relative small PNPs also showed a deceleration of closure rate, but only during a period of four somite stages. This was followed by an acceleration, resulting in closure of all PNPs at the stage of 32 to 33 somites. The enlargement of the PNP in ct/ct embryos was not due to re-opening of a closed neural tube, but resulted from a sharp decline in the rate of PNP closure combined with a normal rate of caudal elongation of the embryo. It is concluded that the ct strain forms a homogeneous population, with a large variation of its specific phenotype: deceleration of PNP closure during a restricted period. The disturbance of spinal neurulation in ct/ct embryos takes the form of a deceleration/acceleration pattern, resulting in a net delay of closure. It is suggested that, due to the ct mutation, forces are generated in the embryonic axis which oppose a normal neurulation process at a specific stage of development.     

Histological and ultrastructural studies of secondary neurulation in mouse embryos

The histological and ultrastructural features of secondary neurulation in C57BL/6 mouse embryos were examined as a first step in the analysis of how this process occurs in mammalian embryos. Secondary neurulation involves two major events in mouse embryos: (1) formation of the medullary rosette (9.5- to 10-day embryos) or plate (11- to 12-day embryos), and (2) cavitation. These two events occur simultaneously. The medullary rosette consists of elongated tail bud cells, radially arranged around a central lumen formed by cavitation. The secondary portion of the neural tube forms in 9.5- to 10-day embryos by progressive enlargement of the central lumen and addition (by cell recruitment or mitosis) of tail bud cells to the rosette. The medullary plate likewise consists of elongated tail bud cells, but these cells do not surround a central cavity. Instead, cells of the medullary plate extend ventrad from the basal aspect of the dorsal surface ectoderm to a slit-like cavity formed by cavitation. Formation of the secondary neural tube occurs in 11- to 12-day embryos, principally by the recruitment of more lateral and ventral tail bud cells into the medullary plate. Free cells and cellular debris are frequently encountered in the forming lumen of the secondary neural tube, but cells exhibiting signs of necrosis were absent in cavitating regions. Numerous small intercellular junctions form at the inner (juxtaluminal) ends of tail bud cells as the medullary rosette or plate is forming and cavitation is occurring. These observations suggest that cavitation per se (i.e., formation of a lumen) during secondary neurulation is a relatively passive phenomenon, which results principally from neighboring cells becoming polarized apicobasally and incorporated into a primitive neuroepithelium. The latter constitutes the walls of the forming secondary neural tube.     

Topographical changes along the neural fold associated with neurulation in the hamster and mouse

The topography of the ectoderm was examined by scanning electron microscopy during neurulation in hamster and mouse embryos. Stages from the appearance of the neural folds to closure of the posterior neuropore were studied. Progressive development of a zone of altered cellular morphology was observed along the crests of the neural folds. This zone evolved from and abrupt transition between surface and neural regions of the ectoderm to a narrow band of flattened cells which exhibited numerous membranous “ruffles” in the mouse, or blebs and presumably degenerating cells in the hamster, immediately prior to contact between the folds. These alterations were more prominent along the anterior than the posterior portions of the folds. Contact of the folds occurred first between the flattened cells with subsequent union of the surface cells. Stages of neural crest cell formation were observed subjacent to the zone of alterations in histological sections. It is suggested that the observed surface alterations may reflect changes in the membrane properties of the altered cells which are correlated with both neural crest formation and initial adhesion between the folds.     

Regional differences in morphogenesis of the neuroepithelium suggest multiple mechanisms of spinal neurulation in the mouse

A study of neuroepithelial morphogenesis in the mouse embryo has identified three modes of neural tube formation that occur consecutively as neurulation progresses along the spinal region. The three modes of neurulation differ in the extent to which the neuroepithelium exhibits formation of hinge points, i.e. localised bending owing to reduction in apical surface area. In Mode 1, bending occurs only in the neuroepithelium overlying the notochord, creating a median hinge point. The neural folds remain straight along both apical and basal surfaces, resulting in a neural tube with a slit-shaped lumen. In Mode 2, the neuroepithelium forms paired dorsolateral hinge points, as well as a median hinge point, whereas the remaining portions of the neuroepithelium do not bend. This produces a neural tube with a diamond-shaped lumen. In Mode 3 neurulation, the entire neuroepithelium exhibits bending, so that the cells specific hinge points are not discernible; the resulting neural tube has a circular lumen. The three modes of neurulation are present in all three strains of mice studied: C57BL/6, CBA/Ca and curly tail, a mutant predisposed to neural tube defects. However, curly tail embryos exhibit a delay in transition from Mode 2 to Mode 3, preceding faulty closure of the posterior neuropore. This heterogeneity of neurulation morphogenesis in the mouse embryo indicates that the underlying mechanisms may vary along the body axis. Specifically, we suggest that Mode 1 neurulation is driven largely by forces generated extrinsic to the neuroepithelium, in adjacent tissues, whereas Mode 3 neurulation is dependent primarily on forces generated intrinsic to the neuroepithelium. Down the body axis, there is a gradual decrease in the area of ectoderm involved in neural induction and, as neurulation reaches lower spinal levels, the newly induced neural plate exhibits marked indentation from the time of its first appearance. The transition from primary neurulation (neural folding of Mode 3 type) to secondary neurulation (neural tube formation by cavitation) appears to be a smooth continuation of this trend, with loss of contact between the newly induced neuroepithelium and the outside of the embryo.     

Thymus colonization in the developing mouse embryo

We have directly followed the formation of and the thymus colonization by pro-T lymphocytes in the developing C57BL/6 mouse embryo by using the monoclonal antibody JORO 37-5 specific for pro-T lymphocytes, immunofluorescence staining and flow fluorocytometry or microscopy analysis. The results show that JORO 37-5+ cells begin to appear in the liver at day 9 of gestation. These JORO 37-5+ cells migrate to and colonize the thymus 1 day later, where they expand vigorously during the next 4-5 days and, subsequently, switch off expression of JORO 37-5 as they further differentiate into mature thymocytes.     

Origin and development of the epicardium in the mouse embryo

Summary The formation of the epicardium was investigated in the mouse embryo using scanning electron microscopy (SEM) in order to establish a three-dimensional perspective concerning epicardial development in mammals. The epicardium first appears as aggregates of cells scattered on the caudal surface of the ventricle and atria where these regions face the septum transversum in a 9-day-old embryo. These aggregated cells seem to have originated from the mesothelial projections extending from the surface of the septum transversum. Then, the cells of each aggregate flatten, subsequently fusing with each other to form a continuous sheet of epicardium. The fusion of aggregates proceeds in a cranial direction. Finally, the bulbus cordis and truncus arteriosus become invested by migrating cells at the cranial end of the epicardial sheet about 11 days after fertilization. The present observations are discussed in comparison with those made previously in avian embryos.     

Visceral heterotaxy syndrome induced by retinoids in mouse embryo.

Visceral heterotaxy syndrome causes abnormal arrangement of thoracoabdominal organs and severe complex cardiac anomalies by abnormal laterality. The purpose of the present study is to analyze the incidence and pattern of heterotaxy syndrome in etretinate and all-tran retinoic acid treated pregnant DDY mice. Pregnant DDY mice were intragastrically given a single dose of 15 mg/kg of etretinate at day 6, 7 of gestation, 30 mg/kg of etretinate at day 7 of gestation and 20 mg/kg of all-trans retinoic acid at day 7 of gestation. The incidence of visceral heterotaxy was highest in the etretinate 15 mg/kg treated group on day 7 of gestation (38.5%). The major cardiovascular anomalies in heterotaxy syndrome were common atrium, common atrioventricular valve, atrioventricular septal defect, transposition of great arteries, pulmonary atresia, pulmonary artery hypoplasia and aortic arch anomalies. Atrial situs of heterotaxy syndrome were right isomerism, solitus-like, inversus-like and left atrial aplasia, but right isomerism was observed most frequently. The results suggest that retinoic acid exerts a significant effect on the determination of atrial situs during the development of mouse embryo.     

Morphogenesis of myocardial trabeculae in the mouse embryo

Formation of trabeculae in the embryonic heart and the remodelling that occurs prior to birth is a conspicuous, but poorly understood, feature of vertebrate cardiogenesis. Mutations disrupting trabecular development in the mouse are frequently embryonic lethal, testifying to the importance of the trabeculae, and aberrant trabecular structure is associated with several human cardiac pathologies. Here, trabecular architecture in the developing mouse embryo has been analysed using high‐resolution episcopic microscopy (HREM) and three‐dimensional (3D) modelling. This study shows that at all stages from mid‐gestation to birth, the ventricular trabeculae comprise a complex meshwork of myocardial strands. Such an arrangement defies conventional methods of measurement, and an approach based upon fractal algorithms has been used to provide an objective measure of trabecular complexity. The extent of trabeculation as it changes along the length of left and right ventricles has been quantified, and the changes that occur from formation of the four‐chambered heart until shortly before birth have been mapped. This approach not only measures qualitative features evident from visual inspection of 3D models, but also detects subtle, consistent and regionally localised differences that distinguish each ventricle and its developmental stage. Finally, the combination of HREM imaging and fractal analysis has been applied to analyse changes in embryonic heart structure in a genetic mouse model in which trabeculation is deranged. It is shown that myocardial deletion of the Notch pathway component Mib1 (Mib1^flox/flox; cTnT‐cre) results in a complex array of abnormalities affecting trabeculae and other parts of the heart.     

Fusion of nasal swellings in the mouse embryo

Summary Fusion between the epithelial linings of the medial and lateral nasal swellings transforms the nasal groove into a primitive nasal cavity and forms an epithelial seam, the nasal fin, in the line of contact. Epithelial contact occurs between a restricted group of opposing epithelial cells; adjacent eithelial cells do not fuse but form the linings of the nasal and oral cavities. After its formation, the epithelial nasal fin regresses and is replaced by mesenchymal cells, except for a small posterior portion which remains as the bucconasal membrane.DNA synthesis at 3 different periods (20, 10 and 5 h) before contact on day 11 3/4 was examined in the fusing epithelia and adjacent non-fusing epithelia. DNA synthetic activity decreased in both regions at successive stages of development. Howerer, the decrease in the presumptive fusing epithelia at 10 and 5 h before contact was noteworthy in that it was significantly greater than in the non-fusing epithelia. In the fusing epithelia this decrease of DNA synthetic activity occurred not only in prospective degenerating cells, but was a general phenomenon involving viable cells also.To analyze the regression of the nasal fin, it was studied in serial sections. The majority of the cells were viable and only few degenerating cells were seen, suggesting that not all cells of the nasal fin undergo necrosis. Since the epithelial cells of the nasal fin always appeared to be separated from the surrounding mesenchymal cells, the transformation of surviving cells into mesenchymal cells appears unlikely. It is postulated that surviving epithelial cells are incorporated into the adjacent epithelia of the primitive oral and nasal cavities.     

Prostacyclin receptor signaling and early embryo development in the mouse

BACKGROUND: Prostacyclin (PGI(2)) plays an important role in mouse embryo development and implantation. However, it is unclear whether its action is mediated via the I prostaglandin receptor (IP). METHODS: We compared the preimplantation development of IP deleted (IP-/-) embryos and wild-type (WT) embryos. We also evaluated the effect of iloprost, a stable PGI(2) analog, and L-165041, a peroxisome proliferator activated receptor delta (PPARdelta) ligand, on IP-/- versus WT embryos. Finally, we compared the development of heterozygous IP deficient embryos carrying a normal maternal IP allele versus paternal IP allele. RESULTS: Development of IP-/- embryos lagged behind WT embryos and was not enhanced by either the PGI(2) analog or the PPARdelta ligand. WT embryos had slightly higher, although statistically not significant, implantation rates than IP-/- embryos. Heterozygous IP deficient embryos carrying a normal maternal IP allele showed better development and responded to the PGI(2) analog, unlike those carrying the normal paternal IP allele. CONCLUSIONS: IP receptors play an important role in preimplantation embryo development and mediate the embryo's response to exogenous PGI(2). Early embryo development depends on the oocyte IP receptor.