Neural plate- and neural tube-forming potential of isolated epiblast areas in avian embryos

Summary Formation, shaping, and bending of the neural plate and closure of the neural groove are complex processes resulting in formation of the neural tube. Two experiments were performed using avian embryos as model systems to examine these events. First, we transected blastoderms near the level of Hensen's node to determine the potential of prenodal neural plate to form neural tube in isolation from primitive streak regression. Our results demonstrate that shaping and bending of the prenodal neural plate occur under these conditions, but neural groove closure is inhibited. Second, we isolated various areas of postnodal epiblasts to determine their potential to form neural plate. Our results suggest that the area of the postnodal epiblast that can form neural plate consists of paired tracts lying adjacent to the definitive primitive streak and extending caudally at least 1 mm from its cranial end.     

Origin and early migration of primordial germ cells in the chick embryo: A study of the stages definitive primitive streak through eight somites

The germinal crescent in the chick embryo is characterized by small, PAS-positive, nonglycogen granules from 1.5 to 5 μ in diameter. The primordial germ cells (PGCs) were found to originate in and separate from the germinal crescent endoderm through stage 7 (2 somites). Shortly after separation most of the granules in the PGCs lost their organization and the PAS-positive material was distributed irregularly throughout the cytoplasm. A few of these granules remained within the cells indefinitely. Glycogen of an agranular nature which had shifted to one pole of the cell was observed at stage four. Granular glycogen which was distributed throughout the cytoplasm was not observed prior to stage 7 or 8. Cell counts on individual embryos showed noticeable variations as to the number of germ cells between embryos of the same stage. For example, in stage 4 embryos the minimum number of cells counted, including attached and free, was 78 and the maimum 169, while in stage 9 the minimum was 83 and the maximum 469 cells. After separation the germ cells were observed almost anywhere between the ectoderm and the endoderm although the majority remained in the area where they originated.     

The migration of myogenic cells from the somites into the leg region of avian embryos

The migration of myogenic stem cells into the leg anlagen of chick embryos between stages 16--20 of Hamburger and Hamilton was examined. SEM and TEM studies reveal that cell migration starts at stage 16 from the just-formed somites 26-28. The migrating myogenic cells are elongated and oriented in a medio-lateral direction. The leading ends branch into filopodia which contact a fibrillar network. At first, single cells migrate; later on the cells leaving the ventro-lateral edge of the dermatome migrate in strands and have specialized contacts between them. After reaction with ruthenium red and concanavalin A the migrating cells show a thick surface coat to which ruthenium red-positive particles are attached. The surface coat may be important in the interactions among the migrating cells as well as between the cells and the substrate. The migration of myogenic stem cells was found to take place in a matrix of collagenous fibrils and ruthenium red-positive particles, probably containing glycosaminoglycans. At the onset of migration the fibrillar network exhibits a preferred medio-lateral orientation. Therefore, it may be concluded that this alignment of the fibrils influences the direction of cell migration.     

The primitive streak, the caudal eminence and related structures in staged human embryos

The caudal region of the trunk was reassessed in 52 serially sectioned human embryos of stages 8-23, 42 of which were controlled by precise graphic reconstructions. The following observations, new for the human, are presented. (1) The neurenteric canal is an important landmark because rostral to it the neural plate of stages 8, 9, and the main part of the notochord develop, whereas caudal to it the neural plate of stages 10-12 and the caudal portion of the notochord are formed. All somites at stages 9-11 and probably also at stage 12 arise rostral to the site of the neurenteric canal. (2) A 'chordoneural hinge' can be detected in stages 10 and 11, where the caudal part of the neural plate gives off cells that probably participate in the production of mesenchyme. (3) When apparent disappearance of the epiblast is used as a criterion, then the primitive streak seems to end during stage 9. (4) The caudal eminence, derived from the primitive streak and covered by ectoderm, forms at stage 10 caudal to the site of the former neurenteric canal and persists as a terminal cap to at least stage 14, although formation of mesenchyme continues in stages 15 to 17 or 18. (5) As the region rostral to the site of the neurenteric canal grows because of the development of somites, the caudal eminence is shifted caudally. (6) The caudal eminence is most active developmentally during stage 13, when most of the required (ca 6 out of 9) pairs of somites appear. (7) The eminence produces the caudal part of the notochord and, after closure of the caudal neuropore, all caudal structures, but it does not produce even a temporary 'tail' in the human. (8) A temporal overlap results between primary and secondary development in the caudal part of the notochord. (9) Primary development begins very early with the formation of the inner cell mass at stage 3, and includes the development of the somites rostral to the neurenteric canal, whereas secondary development, with the exception of the notochord caudally, commences at stage 12. (10) Primary neurulation lasts from stage 8 to stage 12, secondary from stage 12 to stages 17 or 18. (11) Secondary development and secondary neurulation are characterized morphologically by direct formation of structures (notochord, postcloacal gut, neural cord/neural tube) from mesenchyme.     

Classification scheme for genes expressed during formation and progression of the avian primitive streak

We have systematically examined the expression patterns of thirteen genes by in situ hybridization during the formation and progression of the avian primitive streak. Based on common patterns of expression, we classify these genes into three distinct groups. Group 1 genes, subdivided into group 1A (Wnt8c, Slug, Vg1, and Nodal) and group 1B (Fgf8, Brachyury, and Cripto), were expressed first in the epiblast and then, throughout most of the length of the primitive streak. Group 2 genes, namely, cNot1, Sonic hedgehog (Shh), Hnf3 beta and Chordin, were confined to the rostral end of the primitive streak, and then, to Hensen's node. In contrast, Group 3 genes, comprising Goosecoid (GSC) and Crescent, were expressed in the hypoblast. This classification scheme provides a rational basis for categorizing genes expressed during avian gastrulation, and such systematization is likely to provide insight into the relationships among different genes and their potential roles in key events of gastrulation.     

Contribution of single somites to the skeleton and muscles of the occipital and cervical regions in avian embryos

Controversy has surrounded the process of resegmentation of cervico-occipital somites. We have reinvestigated this topic by grafting single somites of quail embryos homotopically into chick embryos. Somites one to five contribute to the skull. Somites one and two contribute to the parasphenoid, which develops by direct ossification in a non-segmental fashion. All cartilaginous derivatives of the somites are segmental. Somite two forms a stripe of cells in the basioccipital, exoccipital and supraoccipital. Somites three to five give rise to the subsequent caudal parts of the basioccipital and exoccipital. Somite five forms the first motion segment including the occipital condyle, the cranial part of the atlas and the tip of the dens axis. Therefore, the border between head and neck is in the centre of somite five, and corresponds to the expression boundary of Choxb-3. Somite six forms the caudal part of the atlas and the cranial part of the axis. Somites two to eight all contribute to the cranio-cervical muscles with the exception of the Mm. rectus capitis dorsalis and ventralis and the M. biventer cervicis, which do not receive contributions from somite two. In contrast, the M. cucullaris capitis is exclusively formed by myogenic cells from somite two, which parallels its exclusive innervation by the accessory nerve. Our data confirm the segmental nature of the occiput, and show that resegmentation is a very regular process involving all except the four cranialmost somites. Except for somites one and two, all of the somites contribute to the muscles located at the appropriate levels. Accepted: 5 July 2000     

Mitotic cells and their microappendages in the primitive streak of the chick embryo

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscroscopy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.     

Blisters in the area pellucida, area opaca, and segmental plate of avian embryos

This is a special communication in an area of special interest to all researchers using avian material. Avian embryos in the Northeast, representing four species (chicken, quail, duck, guinea hen), have been found to be drastically deficient in presomitic tissue (segmental plate tissue) between 45 and 60 h of incubation. These deficiencies first appear in the embryo as blisters, then, through tissue repair, they disappear and the embryos continue seemingly normal development. Similar blisters and excrescences appear in the area pellucida and area opaca between 20 and 30 h of incubation. Associated with these blisters and excrescences in very young embryos and blisters in segmental plates, but not necessarily the result of them, is a high incidence of congenital malformation during later development. These anomalies may be affecting the results obtained in avian research.     

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using <Emphasis Type="Italic">EGFP</Emphasis> transfection

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.     

Induction and patterning of the primitive streak, an organizing center of gastrulation in the amniote.

The primitive streak is the organizing center for amniote gastrulation. It defines the future embryonic midline and serves as a conduit of cell migration for germ layer formation. The migration patterns of endodermal and mesodermal precursors through the streak have been studied in great detail. Additional new breakthroughs recently have revealed the cell biological and molecular mechanisms that govern streak induction and patterning. These findings include (1) identification of the ontogeny and inductive signals of streak precursors, (2) the potential cellular mechanism of streak extension, and (3) the molecular and functional diversification along the anterior-posterior and mediolateral axes within the primitive streak. These findings indicate that amniote embryos initiate gastrulation by using both evolutionarily conserved and divergent mechanisms. The data also provide a foundation for understanding how the midline axis is defined and maintained during gastrulation of the amniotes.     

Morphology,topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21–S23

Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage‐matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high‐resolution episcopic microscopy (HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21–S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis.     

Axial rotation of murine embryos, a study of asymmetric mitotic activity in the neural tube of somite stages

Summary Axial rotation is an important event during a certain period of development of Amniote embryos. In murine embryos a sharp lordosis changes into a kyphosis. The result is the typical fetal position. In this study a temporal and topological relation is found between an asymmetric mitotic activity in the neural tube and the rotation process. The mitotic asymmetry is lost when rotation is completed. A causal relationship between mitotic activity and rotation is postulated.     

Extracellular matrix couples the convergence movements of mesoderm and neural plate during the early stages of neurulation

Background : During the initial stages zebrafish neurulation, neural plate cells undergo highly coordinated movements before they assemble into a multicellular solid neural rod. We have previously identified that the underlying mesoderm is critical to ensure such coordination and generate correct neural tube organization. However, how intertissue coordination is achieved in vivo during zebrafish neural tube morphogenesis is unknown. Results : In this work, we use quantitative live imaging to study the coordinated movements of neural ectoderm and mesoderm during dorsal tissue convergence. We show the extracellular matrix components laminin and fibronectin that lie between mesoderm and neural plate act to couple the movements of neural plate and mesoderm during early stages of neurulation and to maintain the close apposition of these two tissues. Conclusions : Our study highlights the importance of the extracellular matrix proteins laminin and libronectin in coupling the movements and spatial proximity of mesoderm and neuroectoderm during the morphogenetic movements of neurulation. Developmental Dynamics 245:580–589, 2016. © 2016 Wiley Periodicals, Inc.     

Monoclonal antibodies identifying subsets of ectodermal,mesodermal, and endodermal cells in gastrulating and neurulating avian embryos

The goal of our laboratory research is to elucidate the mechanisms underlying gastrulation and neurulation, using the avian embryo as a model system. In previous studies, we used two approaches to map the morphogenetic movements involved in these processes: (1) we constructed quail/chick transplantation chimeras in which grafted quail cells could be identified within chick host embryos by the presence of nucleolarassociated heterochromatin, and (2) we microinjected exogenous cell markers. However, it would be advantageous to be able to detect endogenous markers to demarcate various subsets of cells within the unmanipulated embryo. To elucidate such a series of natural markers, we have used monoclonal antibodies to identify epitopes found on subsets of ectodermal, mesodermal, and endodermal cells. Antibodies were made by immunizing mice against either homogenized ectoderm (i. e., Prospective neural plate and surface ectoderm) or primitive streak, which had been microdissected from stage 3 chick embryos. Additionally, we screened a panel of antibodies made against soluble protein obtained from isolates of cell nuclei from late embryonic chick brain. Here, we describe the labeling patterns of three monoclonal antibodies, called MAb-GL1, GL2, and GL3 (GL, germ layer), during avian gastrulation and neurulation. Our results show that labeling early avian embryos with monoclonal antibodies can reveal previously undetected distributions of cells bearing shared epitopes, providing new labels for subsets of cells in each of the three primary germ layers. © 1993 Wiley-Liss, Inc.