Chr. Vermeij-Keers

Limb body wall malformation complex: An embryologic etiology?

Four human fetuses with the limb body wall malformation complex, also known as the amniotic band syndrome, were examined. Besides malformations of the body wall and the limb(s), anomalies of the internal organs were found in three fetuses, suggesting a disturbance of normal morphogenesis before the fifth week of development. When analyzing the observed data in relation to their etiology, no support was found for either the amniotic band or the vascular disruption theory. New insights into morphogenesis suggest that the limb body wall malformation complex results from a malfunction in the ectodermal placodes and that it can be considered to be an embryonic dysplasia.

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker

SummaryThe formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectincoated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.

The Neural Crest: a Study On Cell Degeneration and the Improbability of Cell Migration in Mouse Embryos

Mouse embryos (CPB-S strain) aged 6.0-8.3 days post coitum (p.c.) were investigated in 10-μm and 1-μm series by light microscopy with respect to: (a) the occurrence of degenerating cells in the neural crest, and (b) the stage of development in which the neural crest cells start to lose their epithelial arrangement. A high frequency of cell degeneration was found in all embryos examined, starting with the 6.6-day-p.c. stage, i.e., prior to the ectodermal disruption of the neural crest in the 7.3-day-p.c. stage, and continuing through all of the later stages included in the study. In the 7.3-day-p.c. stage the neural plate has not developed yet and the neural crest is present between the neurectoderm and the future surface ectoderm situated just lateral to the notochordal plate. During the transformation of the neurectoderm, via the neural plate and the neural groove, into the neural tube, the neural crest is shifted first laterally and then dorsally and medially, and drops cells. These cells proliferate immediately and eventually differentiate. In this developmental model there is no need for migration of cells to explain the outgrowth of e.g. facial swellings.

Separation of neural and surface ectoderm after closure of the rostral neuropore

SummarySeparation of neural and surface ectoderm after closure of the rostral neuropore in the head region has been described by investigating the integrity of the basement membranes of these epithelia in 11- to 27-somite rat embryos. The basement membranes were visualized with polyclonal antibodies against laminin. Furthermore, cell degeneration has been investigated in relation to neural crest activity, and discontinuities of the basement membrane in 9- to 30-somite mouse embryos.The separation of the basement membranes of neural and surface ectoderm in the midline is a final phase during the fusion of the neural folds, which takes place from the closure of the rostral neuropore, at the 19-somite stage, until the 27-somite stage (rat embryos), and which occurs focally with variation in the midsagittal and the transverse planes. In the prosencephalon, neural crest activity is absent during the separation phase of both epithelia, but cell degeneration may contribute to the separation of the initially connected basement membranes. A disturbance in the separation of the neural and surface ectoderm may be the pathogenetic basis of midline skull defects, and of the fronto-ethmoidal encephalocele in particular.

Wheat germ agglutinin-gold as a novel marker for mesectoderm formation in mouse embryos cultured in vitro.

The routes of movement of mesectoderm cells in mammalian embryos have not yet been investigated experimentally due to technical problems. However, the recent development of in vitro culture methods have made an experimental approach to this problem in mouse and rat embryos possible. We have used combined lectin and colloidal-gold (WGA-Au) probe as a nontraumatic, easily detectable mesectoderm marker. The probe is introduced into the amniotic cavity by microinjection. All of the cells lining the cavity, including the mesectoderm precursors, phagocytose the colloidal gold, which is then stored in membrane-bound vesicles. The probe remains inside the target mesectoderm cells after their migration into the mesoderm compartment. Vesicles containing gold are detectable in both ultrathin and semithin sections. The applicability of WGA-HRP as a probe was also assessed because of the many properties it shares with WGA-Au, but it proved to be unsatisfactory for this purpose because it is transferred between cells and also to the extracellular spaces.

The cell surface coat in neurulating mouse and rat embryos, studied with lectins

SummaryCarbohydrates in the surface coat of cells are thought to have a function in cell adhesion. The surface coat of cells, located in the fusion zone of the neural walls is investigated during neural tube closure in mammalian embryos. The presence of α-D-mannose, α-D-glucose and N-acetyl-D-glucosamine is quantified with the help of the lectins concanavalin A and wheat germ agglutinin in absence or after enzymic treatment. A two-step incubation is used, in which the second step consists of a protein-gold conjugate. A high incidence of these sugar residues was found in the fusion zone, indicating a relation to the specific capacity of these cells in establishing cell contacts.

Cell degeneration and mitosis in the buccopharyngeal and branchial membranes in the mouse embryo.

SummaryThe frequencies of cell degeneration and mitosis were investigated in the rupturing buccopharyngeal membrane (BPM) and in the persistent first branchial membrane (BM). In the BPM, cell degeneration starts many hours before rupture is visible, but mitotic figures are absent. In the BM this situation is reversed: mitotic figures are regularly observed, but a degenerating cell only occasionally. It is concluded that the ratio between the numbers of degenerating and dividing cells regulates the fate of both the BPM and the BM.

Hypertelorism and the median cleft face syndrome. An embryological analysis.

A microscopical study of the early and late development of the face was performed in 77 human embryos and fetuses. After the transformation of both nasal placodes, via nasal grooves, into the nasal tubes the ectoderm of the face is closed superficially and the early development of the face (less than or equal to 17 mm crown-rump length (C-RL)) is terminated. Between the nasal tubes the internasal groove is present. Furthermore these embryos show physiologically a flat nose and hypertelorism. During the late development (greater than or equal to 17 mm C-RL) of the face the internasal groove disappears due to the outgrowth and differentiation of the nasal septum in the frontocaudal direction. Simultaneously (17-27 mm C-RL) the distance between the eyes decreases relatively, because of a relative lag in transverse growth. The differentiation of the facial mesenchyme into bone centres starts in the same period. From this embryological point of view the major anomalies of the median cleft face syndrome (hypertelorism--orbital as well as interorbital--and cranium bifidum occultum, median cleft nose, median cleft prolabium and median cleft premaxilla), can be classified as secondary or late, i.e., differentiation, defects.

The oro-ocular clefts, an embryological subdivision

Analysing data from an embryological study of the early and late development of the face, performed in 76 human embryos and fetuses, and from the inspection of 2,300 human skulls we recognized two types of oro-ocular malformations, early transformation (primary) and late differentiation (secondary) defects.The early development (≤ 17 mm C-RL) is characterized by transformations and fusions of the facial swellings oriented round each nasal placode. During this developmental period only one type of the oro-ocular defects (Morian I) can be explained, situated between the non-fused lateral nasal and maxillary processes. In this defect the naso-lacrimal duct is always involved.All the other types, e.g., Morian 11 and III, are late (secondary) defects, as they develop later (≥ 17 mm C-RL) during the differentiation of the mesenchyme of the fused facial swellings into bone, cartilage and musculature.