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-somite 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-somite 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.     

Comparison of staging systems for the gastrulation and early neurulation period in rodents: a proposed new system.

Because there is no standard developmental staging system for the early postimplantation period of rodent embryos, investigators must now choose between a variety of systems that differ significantly. We have reviewed many of these staging systems and have summarized the ambiguities within them and the inconsistencies among them. In order to compare systems, we first obtained a consensus of the order of developmental events from the literature, and then attempted to fit existing systems into this order taking into account inconsistencies in terminology and blurred borderlines between stages. We were able to do this for most systems but not all because some were too divergent. We found that inconsistencies in definition of some terms, such as "primitive streak stage" and those used to describe the early neurulation process (neural plate, neural groove, neural folds, and head fold) cause much confusion. In order to develop an unambiguous system which can be used by all investigators, we propose to modify Theiler's system, which is one of the most commonly used systems but is not defined precisely during the early postimplantation period. We suggest making subdivisions of the original stages as follows: 1) stage 8 into 8a and 8b, by the degree of extension of the proamniotic cavity into the extraembryonic region; 2) stage 10 into 10a and 10b, by the completion of amnion formation; 3) stage 11 into 11a, 11b, and 11c, by the appearance of neural folds and foregut pocket. After Stage 12, the number of somite pairs can be used to precisely stage embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fate mapping the avian epiblast with focal injections of a fluorescent-histochemical marker: ectodermal derivatives

A microinjection technique is described for fate mapping the epiblast of avian embryos. It consists of injecting the epiblast of cultured blastoderms with a fluorescent-histochemical marker, examining rhodamine fluorescence at the time of injection in living blastoderms, and assaying for horseradish peroxidase activity in histological sections obtained from the same embryos collected 24 h postinjection. Our results demonstrate that this procedure routinely marks cells, allowing their fates to be determined and prospective fate maps to be constructed. Two such maps are presented for ectodermal derivatives of the epiblast: one for late stages of Hensen's node progression (stages 3c through 4) and one for early stages of node regression (stages 4 + through 5). These new maps have six significant features. First, they show that regardless of whether the node is progressing or regressing, the flat neural plate extends at least 300 microns cranial to, 300 microns bilateral to and 1 mm caudal to the center of Hensen's node. Second, they confirm our previous fate mapping studies based on quail/chick chimeras. Namely, they show that the prenodal midline region of the epiblast forms the floor of the forebrain and the ventrolateral part of the optic vesicles as well as MHP cells (i.e., mainly wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate); in contrast, paranodal and postnodal regions contribute L cells (i.e., mainly spindle-shaped neurepithelial cells constituting the lateral aspects of the neural plate). Third, they reveal a second source of MHP cells, Hensen's node, verifying previous studies of others based on tritiated thymidine labeling. Fourth, they demonstrate, in contrast to studies of other based on vital staining, carbon marking, and chorioallantoic grafting but in accordance with our previous studies based on quail/chick chimeras, that the cells contributing to the four craniocaudal subdivisions of the neural tube (i.e., forebrain, midbrain, hindbrain, and spinal cord) are not yet spatially segregated from one another at the flat neural plate stage, although more cranial neural plate cells tend to form more cranial subdivision and more caudal cells tend to form more caudal subdivisions. Thus, single injections routinely mark multiple neural tube subdivisions. Probable reasons for the discrepancy between our present results and the previous results of others is discussed. Fifth, they suggest that cells contributing to the surface ectoderm and neural plate are not yet completely spatially segregated from one another at the flat neural plate stage, particularly in caudal postnodal regions. Sixth, they delineate the locations of the otic placodes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific cellular localization of tyrosinase mRNA during Ciona intestinalis larval development

A Ciona intestinalis cDNA clone that encodes a protein highly homologous to other tyrosinases was isolated. Northern blot analysis showed that expression of Ciona tyrosinase starts at the early neurula stage and continues throughout the tail-bud and tadpole larval stages. The earliest tyrosinase expression was detected, by in situ hybridization, at the neural plate stage, in pigment precursor cells located along the two neural folds, in the animal region of the embryo. In the course of embryonic development the strong hybridization signal was always localized, within the rostral part of the developing brain, in the pigment precursor cells and was later detected in the otolith and ocellus. These results are discussed in relation to tyrosinase as an early marker of neural induction.     

Early regulative ability of the neuroepithelium to form cardiac neural crest

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6–8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations.     

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.     

Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy

The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level.     

Ultrastructural studies of amphibian neural fold fusion.

The fusion of neural folds to form the neural tube is a process in which presumptive contacting surfaces become adhering. An ultrastructural examination of regions of neural folds in the neurulae of three amphibian species (Hyla regilla, Rana pipiens, and Xenopus laevis), using both transmission and scanning electron microscopy, revealed that, prior to fusion, there is formation of vesicles within cells lining the neural groove, development of extracellular vesicles, changes in the surface morphology of the cells forming the fusion area, and extension of projections (filopodia) from cells lining the neural groove. The association of intra- and extracellular vesicles and filopodia with cells of the neural groove and folds suggests that these organelles may be involved in preparing the neural folds for initial contact, adhesion, and fusion. Ultrastructural differences in reaction of neural fold cell surfaces to staining by ruthenium red, colloidal iron, Alcian blue-lanthanum nitrate, and concanavalin A-hemocyanin indicate that the glycosaminoglycan compositions of these cell surfaces differ from those of presumptive epidermal cells.     

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. Here, we demonstrate that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. We found that Doc expression was restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevented the lateral extension and deepening of these folds at the larval stage and delayed wing disc bending in the early pupal stage. Ectopic Doc expression was sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression expressed Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins were depleted. Overexpression of Doc along the blade/hinge folds caused precocious wing disc bending, which could be suppressed by co-expressing MMP2RNAi.     

Compatibility of chick embryo eye anlagen with the ectoderm of the early amphibian gastrula in vitro

Eye vesicles were isolated from the early chick embryos (stage 9+ after Hamburger and Hamilton, 1951) and combined with the Rana temporaria early gastrula ectoderm (EGE) in vitro. The tissues were jointly incubated in medium 199 diluted twice with deionized water at 22 +/- 1 degree for 7-8 days or the eye vesicles were removed from the EGE ectoderm within 16-18 h. At the joint long-term incubation of these tissues, a toxic effect of the chick embryonic tissues on the EGE cells was noted. In none of the experiments, the inducing effect of the eye vesicle on the EGE was found. Similar data were obtained when the EGE was jointly cultivated with the brain (stage 9-10) and retina (stage 15) of chick embryos. The brain of the chick embryos at stage 15 exerted a weak neuralizing effect on the EGE. In the control experiments, the eye vesicles explanted with the chick embryonic ectoderm remained viable till the end of cultivation but no lentoids formed in the ectoderm. The absence of lens-inducing effect at the joint cultivation of the chick embryonic eye vesicles with the EGE is considered as a result of disturbance of the synthesis or secretion of the corresponding agents rather than a sequence of the species "incompatibility" of the inductor and reacting tissue. Hence, the use of "xenogenic" tissue recombinants is not justified when analyzing the lens-inducing activity of the eye vesicles.     

Pattern of structural differentiation in the optic nerve of trout (Salmo gairdneri)

Summary The development of the trout optic nerve is quantitatively described from early ontogenesis into adulthood. The nerve is oval in cross section until stage 34, thereafter the formation of vertically aligned parallel folds can be observed and thus the unique shape of a folded ribbon is gradually attained. Quantitative measurements revealed a linear increase in cross sectional area, caused in part by the formation of new folds and in part by an increase in size of the preexisting ones. We attribute the continuous expansion of individual folds to an increase in fiber size subsequent to myelination rather than to the addition of new fibers. The total number of glial cells increased concomitantly per fold.Myelinogenesis starst at stage 33 with the ensheathement of axons beginning at the dorsal edge of the primary fold and follows a highly ordered pattern throughout development, strictly succeeding neural outgrowth. The functional significance of this pattern is discussed.     

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.     

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.     

Studies on the mechanisms of neurulation in the chick: interrelationship of contractile proteins, microfilaments, and the shape of neuroepithelial cells

Electron microscopy and indirect immunofluorescence were employed to correlate the distribution patterns of major contractile proteins (actin and myosin) with 1) the organizational state of microfilaments, 2) the apical cell surface topography, 3) the shape of the neuroepithelial cells, and 4) the degree of bending of the neuroepithelium during neurulation in chick embryos at Hamburger and Hamilton stages 5-10 of development. Both actin and myosin are present at these developmental stages and colocalize in the neural plate as well as in later phases of neurulation. During elevation of neural folds, actin- and myosin-specific fluorescence is always most intense in regions where the greatest degree of bending of the neuroepithelium takes place [e.g., the midline of the V-shaped neuroepithelium (early neural fold stage) and the midlateral walls of the "C"-shaped neuroepithelium (mid-neural-fold stage)]. This intense fluorescence coincides with 1) a particularly dense packing of microfilaments and 2) highly constricted cell apices. After neural folds make contact, there is an overall reduction in both the intensity of apical fluorescence and the thickness of apical microfilament bundles, especially in the roof and floor of the neural tube. The remaining fluorescence in the contact area is apparently related to cellular movements during fusion of neural folds.     

Mapping of the early neural primordium in quail-chick chimeras. II. The prosencephalic neural plate and neural folds: implications for the genesis of cephalic human congenital abnormalities

Mapping of the avian neural primordium was carried out at the early somitic stages by substituting definite regions of the chick embryo by their quail counterpart. The quail nuclear marker made it possible to identify precisely the derivatives of the grafted areas within the chimeric cephalic structures. A fate map of the prosencephalic neural plate and neural folds is presented. Moreover the origin of the forebrain meninges from the pro- and mesencephalic neural crest is demonstrated. In the light of the data resulting from these experiments, we present a rationale for the genesis of malformations of the face and brain and of congenital endocrine abnormalities occurring in man.     

The two sites of fusion of the neural folds and the two neuropores in the human embryo

BACKGROUND: Since reports on a pattern of multiple sites of fusion of the neural folds in the mouse appeared, it has been widely assumed that a similar pattern must be valid for the human. In the absence of embryological evidence, claims have been made that such a pattern can be discerned by classifying neural tube defects. METHODS: The neural folds and tube, as well as the neuropores, were reassessed in 98 human embryos of Stages 8-13; 61 were controlled by precise graphic reconstructions. RESULTS: Careful study of an extensive series of staged human embryos shows that two de novo sites of fusion of the neural folds appear in succession: alpha in the rhombencephalic region and beta in the prosencephalic region, adjacent to the chiasmatic plate. Fusion from Site alpha proceeds bidirectionally (rostrad and caudad), whereas that from beta is unidirectional (caudad only). The fusions terminate in neuropores, of which there are two: rostral and caudal. Highly variable accessory loci of fusion, without positional stability and of unknown frequency, may be encountered in Stage 10 but seemingly not later, and their existence has been known for more than half a century. CONCLUSIONS: Two sites of fusion (a term preferred to closure) of the neural folds and two neuropores are found in the human embryo. No convincing embryological evidence of a pattern of multiple sites of fusion, such as has been described in the mouse, is available for the human. The construction of embryological details from information derived from other species or from the examination of later anomalies is liable to error. Neural tube defects are reviewed and although they have been considered on the basis of five, four, or three sites of fusion, interpretations based on two sites can as readily be envisaged.     

Nutritive phagocytosis in animal cells

Summary Nutritive phagocytosis in the hydroid Clava squamata was studied with the electron microscope, using carbon particles of 0.6 as an indicator.An early step in phagocytosis is the transformation, in many cells, of the free border from a type with cylindrical microvilli to one with a complicated system of cytoplasmic folds.Particles fixed at the actual stage of ingestion are found (a) between two cytoplasmic folds, (b) between a fold and a relatively straight portion of the cell surface, or (c) in a depression of an otherwise straight portion of the cell surface.Ingested carbon particles were always found enclosed by a membrane, with a layer of moderate electron density between the carbon particle and the membrane.The ingested carbon particles are localized apically in small vesicles each containing one particle (interpreted as primary phagocytic vesicles) or at deeper levels of the cell, in larger vesicles containing many carbon particles (interpreted as secondary phagocytic vesicles).Other cytoplasmic changes during phagocytosis relate to the distribution of mitochondria and the occurence and distribution of flattened vesicles of a characteristic appearance.With the technical assistance of Birgitta af Burén.Financial support from Swedish Natural Science Research Foundation is gratefully acknowledged.     

An ultrastructural examination of the role of cell membrane surface coat material during neurulation

Data from neural crest cultures indicate that cell surface coat material (CSM) is directly involved in cellular migration and events surrounding differentiation. To investigate whether the CSM also has a morphogenetic role, embryos of the amphibian Ambystoma maculatum were examined ultrastructurally throughout the stages of neurulation. Segments of the neural axis were fixed in glutaraldehyde-containing Alcian blue 8GX, which reportedly enhances preservation of CSM, and were postfixed in OsO4 containing 1 percent lanthanum nitrate, which stains the CSM. The medial groove formed by the appearance of the neural ridges contains a large amount of CSM and numerous vesicles coated with lanthanum-positive material. In contrast, the lateral ridge surfaces are covered by a small amount of uniformly distributed CSM and a paucity of vesicles. As the ridges begin to fold there is a progressive increase in the amount of CSM within the presumptive neural tube region. Further convergence of the neural folds is accompanied by an increase of CSM at their leading edges. As the folds approximate each other, lanthanum-positive material physically bridges the gap. However, as the apposing tissue actually abuts to form the neural tube, no CSM is observed in the remaining interspace. The specific distribution and sequential accumulation of cell CSM during the events of neurulation strongly suggest its direct participation in the morphogenetic process.