Scanning-electron-microscopic studies on the pathogenesis of exencephaly and cranioschisis induced in the rat after neural tube closure

Following a single-dose (15 mg/kg) administration of cyclophosphamide on day 12 of gestation to Wistar rats, fetuses were collected at various intervals of time and studied under the scanning electron microscope. Comparisons were made with age-matched controls. Disorganization of the neuro-epithelium (NE) was characterized by expansion of the intercellular space (ICS), appearance of abundant cellular debris, extravascular red blood cells and formation of haemorrhagic cysts in the subependymal zone. Proliferation of the choroid plexus and its surface modifications parallelled the changes in the NE. The ventricular system dilated progressively. Supra-ependymal elements appeared earlier and in greater abundance in the experimental than in the control embryos. The mesenchymal cells of the cyclophosphamide-treated embryos were scanty, poorly organized, had less numerous cytoplasmic processes than the corresponding controls and failed to develop into the primordium of the skull vault. Degenerative changes in the NE, ventricular dilatation and the absence of a supporting skull vault primordium appeared to underlie the reopening of the closed neural tube.     

Electron-microscopic studies on the pathogenesis of exencephaly and cranioschisis induced in the rat after neural tube closure: role of the neuroepithelium and choroid plexus

Exencephaly was induced in Wistar rat fetuses by the administration of a single dose of cyclophosphamide (15 mg/kg) in saline, after neural tube closure. The neuroepithelium (NE) and the choroid plexus were studied electron-microscopically in sections taken from a few hours after treatment to day 19 of gestation. The reduction in polyribosomes and condensation of the nucleus and cytoplasm were followed by cell death and fragmentation in the NE. Such cellular debris were phagocytosed and digested by the apparently normal neuroblasts. Cell proliferation was inhibited. The progressive loss of cells and lack of neuropil arborisation resulted in the expansion of the extracellular space and reduced intercellular contacts. The internal and external limiting membranes became weak. The vascular endothelium was attenuated. There were no obvious discontinuities of endothelium, but clusters of extravascular red blood cells, particularly in the vicinity of capillaries, in the cavitations in the NE and in the ventricular lumen were prominent by day 15. Subsequently, the cavities in the NE frankly communicated with the ventricle internally and subcutaneous blebs externally. The choroid plexus of exencephalic embryos was more extensive than that of the age-matched controls. Hydropic vacuoles, dense bodies, distended mitochondria, clusters of vesicles in basal cytoplasm and lakes of monoparticulate glycogen progressively increased in the plexus cells. Pericapillary oedema was obvious in the core of the plexus. These observations suggest that, in addition to cell death and reduced cell proliferation, haemorrhage, oedema and enhanced cerebrospinal fluid production contribute to reopening of the closed neural tube in this model.     

Retinoic acid inhibits migration of cranial neural crest cells in the cultured mouse embryo

Clinical observations have demonstrated that isotretinoin (13-cis-retinoic acid; cis-RA) is a human teratogen causing primarily heart and craniofacial malformations. Isotretinoin exposure to the early postimplantation mouse embryo in culture results in specific defects in craniofacial development that may be due to an interference in the early migration of cranial neural crest (CNC) cells [Goulding and Pratt, 1986]. The present study was designed to test this hypothesis by examining the migration of these cells in whole embryo culture. Day 8 CD-1 mouse embryos were cultured for 6-48 hr in the presence or absence of cis-RA at 2 X 10(-6) to 2 X 10(-5) M. Embryos either were fixed for light microscopy using Nichols' method for localization of CNC cells or were processed for scanning and transmission electron microscopy. At the light microscopic level, CNC cells in the mid-brain region of control embryos had migrated to the region of the first and second visceral arches after 6 hr in culture. Cis-RA interfered with this migration; CNC cells in treated embryos either did not leave the neuroepithelium (NE) or were aggregated near the NE. Autoradiographic studies indicated that cis-RA did not affect the overall viability or DNA synthesis of the CNC cells. However, at the TEM level, there was a dramatic increase in the number of cellular blebs in the CNC cells. Our results demonstrate a direct effect of 13-cis-RA on the CNC cells and suggest that this effect is due to alterations in the cell surface.     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

Embryogenesis in Najas marina L.: Structural and histochemical approach

In Najas marina L. few polysaccharide grains are observed in zygote, basal cell and embryonal cells until the initiation of embryonic shoot-apex. With the formation of the shoot-apex, numerous polysaccharide grains engorge in the embryonal cells. The basal cell wall, subjacent to the nucellus, stains intensely with PAS (Peiodic Schiff's)-reaction. The concentration of proteins and RNA increases in the basal cell.Interestingly, the embryo shows intraseminal germination. The cells of embryonic shoot-apex, embryonic leaves, root primordium and procambial cells show a few polysaccharide grains while the cells of hypocotyledonary and cotyledonary regions are engorged with polysaccharide (starch) grains. Uniform distribution of proteins and RNA is observed in the embryonic shoot-apex, embryonic-leaves, root primordium and procambium, but the cells of hypocotyledonary and cotyledonary zones exhibit a low profile for these metabolites. The initial root-primordium remains quiescent. Three or 4 epidermal cells, subjacent to this quiescent primordium, differentiate; show densely stained, polarised, protein bands; and act as the future root primordium.The nucleus of the basal cell becomes polyploid and densely stains for proteins, RNA and DNA. At the globular proembryo stage, numerous nucleolar bodies migrate towards the periphery of the nucleus and at the 3-leaf embryo stage, these nucleolar bodies, rich in proteins and RNA, are located in the cytoplasm revealing nucleo-cytoplasmic interaction. The basal cell that never divides, but only enlarges, is persistent in the mature seed.     

Cranial neural crest contributes to the bony skull vault in adult Xenopus laevis: insights from cell labeling studies

As a step toward resolving the developmental origin of the ossified skull in adult anurans, we performed a series of cell labeling and grafting studies of the cranial neural crest (CNC) in the clawed frog, Xenopus laevis. We employ an indelible, fixative-stable fluorescent dextran as a cell marker to follow migration of the three embryonic streams of cranial neural crest and to directly assess their contributions to the bony skull vault, which forms weeks after hatching. The three streams maintain distinct boundaries in the developing embryo. Their cells proliferate widely through subsequent larval (tadpole) development, albeit in regionally distinct portions of the head. At metamorphosis, each stream contributes to the large frontoparietal bone, which is the primary constituent of the skull vault in adult anurans. The streams give rise to regionally distinct portions of the bone, thereby preserving their earlier relative position anteroposteriorly within the embryonic neural ridge. These data, when combined with comparable experimental observations from other model species, provide insights into the ancestral pattern of cranial development in tetrapod vertebrates as well as the origin of differences reported between birds and mammals.     

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.     

The vasculature of the rat embryo: an electron microscope study

The ultrastructure of portions of the arterial and venous systems of the 11.5 day old Wistar rat embryos has been studied by scanning and transmission electron microscopy. The vessels at this stage of development are in the form of capillaries, and the arterial and venous types can be distinguished by the morphology of the endothelial cells by SEM. The endothelial cells of the arterial vessels gave prominent nuclear bulges and numerous microvilli apart from their spindle shape, whilst those of the veins appear flattened, are polygonal in shape, and have few microvilli. Transmission electron microscopy shows that the endothelial cells of the arteries and veins are identical in structure. The ultrastructure of these cells resembles that of endothelial cells at later stages of development including the adult type in that mature forms of cytoplasmic organelles are obtained. In studies on the intercellular junctions and fenestrations with lanthanum nitrate, the impression is formed that the vessels at this stage are impermeable to small molecular size particles, compared with adult capillaries. This suggests that cytoplasmic vesicles must play a major role in the transport of macromolecules in the 11.5 day embryonic vessels.     

Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.     

Hypothalamic sites of catecholamine inhibition of luteinizing hormone in the anestrous ewe.

Norepinephrine (NE) and dopamine (DA) actively inhibit the release of LH in anestrous ewes. This can be detected as an increase in LH pulse frequency following i.v. injection of NE and DA antagonists. The objective of this study was to determine the sites of these inhibitory actions in the ovine hypothalamus by using local administrations of the NE antagonist, phenoxybenzamine (PBZ), or the DA antagonist, pimozide (PIM), into specific hypothalamic areas. Each neurotransmitter antagonist was administered via a chronically implanted steel guide tube into either the preoptic area (POA), retrochiasmatic area (RCh), or the median eminence region (ME). Blood samples were taken every 15 min for 2 h before and 4 h during implantation of these drugs and were analyzed for LH and prolactin by RIA. Control (no treatment) samples were obtained similarly from the same animals on another day. Placement of PBZ into the POA significantly increased LH pulse frequency and mean LH concentrations over control values whereas PIM did not. In contrast, PIM significantly increased LH pulse frequency and mean LH concentrations when placed in the ME or in the RCh, but PBZ did not. No effects of PIM on prolactin concentrations were detected. These results suggest that an NE neural system operates in the POA and that a DA system acts in the medial basal hypothalamus (RCh or ME) to suppress GnRH pulse frequency in the ovary-intact anestrous ewe.     

Differential dysmorphogenesis induced by microinjection of an alkylating agent into rat conceptuses cultured in vitro

A technique of microinjection of small quantities of teratogens into extraembryonic compartments or specific organ primordium of rat conceptuses of pregnancy day 11 is described. Conceptuses microinjected with 50 nl tissue culture medium developed normally for 44-45 hr when cultured in homologous rat serum, indicating that the microinjection procedure itself did not produce any deleterious effects on growth and differentiation of embryos. Microinjection of an alkylating agent, phosphoramide mustard dissolved in tissue culture medium, into the exocoelom produced anomalous embryogenesis, consisting of retarded embryonic growth, anomalies of the neural tube, and general necrosis of various organ primordia. In contrast, the embryonic development remained relatively unaffected by microinjection of identical amounts of this alkylating agent into the amniotic cavity. However, neural-tube differentiation was markedly affected when phosphoramide mustard was injected into anterior neural-tube fluid, producing anencephalic or microcephalic embryos without significant effect on postcephalic organ differentiation. The morphogenesis of the anterior limb was unaffected by local injection of the agent into somitic tissues adjacent to the presumptive limb-bud region. Therefore, it appears that differential dysmorphogenesis could be induced by microinjection of an alkylating agent into different conceptus compartments. These results indicate that even during early embryogenesis various cell types are not equally susceptible to a given teratogen, and that the differential cytotoxicity of the teratogen toward specific embryonic or extraembryonic cells and tissues may account for embryonic anomalies characteristically produced by that agent.     

Early development of the primitive cranial vault in the chick embryo.

The calcified tissues involved in the early morphogenesis of the cranial vault were studied by microradiographic analysis and histological techniques in 12 chick embryos on the 9th, 12th, and 14th days of incubation. On the 9th day, the frontal, parietal, and squamosal bones are comprised of a thin lamina of chondroid tissue deposited at a short distance from the fibers of the dura mater. Woven bone formation takes place in the calvarial mesenchyme only after the 12th day of incubation and occurs mainly on the external side of the chondroid primordium. The present data obviously indicate that the primitive desmocranium of the chick embryo, which is usually known to be formed by intramembranous ossification, consists first of chondroid tissue. This tissue represents thus the initial modality of skeletogenic differentiation within the cephalic mesenchyme of the cranial vault.     

Ultrastructural demonstration of neurohaemal contacts in the internal zone of the median eminence of the Mongolian gerbil (Meriones unguiculatus): correlation with synaptophysin immunohistochemistry

Summary Electron microscopy of the median eminence (ME) of the Mongolan gerbil (Meriones unguiculatus) revealed that, unlike most other mammalian species, abundant neurohaemal contacts were present not only in the external zone (EZ), but also in the internal zone (IZ) up to the subependymal layer. In the IZ, nerve terminals with dense core vesicles and/or small clear vesicles abutted on the outer basal lamina of the perivascular space of portal capillaries, alternating with tanycyte processes. In addition to these neurohaemal contacts, several layers of vesicle-filled varicosities surrounded the portal vasculature. An analysis of serial thin sections showed that the latter varicosities could also reach the perivascular basal lamina or contact it through small extensions in other planes of section. Apparently at least some of the nerve terminals making neurohaemal contacts were en passant in nature. A correlative investigation of synaptophysin (a major integral membrane protein of small synaptic vesicles) immunoreactivity at the light microscopical level demonstrated a conspicuously dense immunostaining around portal capillaries in both EZ and IZ of the proximal and distal ME (neural stalk). Since this perivascular accumulation of immunoreactivity coincides precisely with the ultrastructural accumulation of vesicle-filled axons which establish numerous neurohaemal contacts, it is concluded that this pattern of synaptophysin immunostaining indicates sites of neurohaemal contacts at the light microscopical level. During postnatal development, the perivascular concentration of synaptophysin immunoreactivity in the IZ appeared concomitantly with the early postnatal expansion of long portal capillary loops into the IZ. By direct electron-microscopical demonstration and indirect immunohistochemical evidence, the present study of the gerbil ME reveals that the whole extent of portal capillaries up to the subependymal layer constitutes an area for numerous neurohaemal contacts. Hence, the common view that neurohaemal contacts are restricted to the EZ of the mammalian ME is not generally valid.     

Origin of the brushborder in the differentiating midgut of Melasoma saliceti (Chrysomelidae, Coleoptera) embryos

The embryonic development of Melasoma saliceti takes eight days at room temperature. At the beginning of the 5th day the endoderm cells have already formed a unilayered epithelium of the midgut primordium. The midgut epithelium is formed by flat cells that are not connected by specialized intercellular junctions. Large vesicles can be seen in dilated intercellular spaces of the epithelium. Cytoplasmic projections, similar to microvilli, appear in the vesicles. During the 5th day ofdevelopment, the vesicles grow and become enclosed by the intercellular junctions of a zonula adherens type. During the 6th day of development the cell junctions surrounding the vesicles become transformed into a septate type. On the 8th day of development the vesicles come close to the apical sides of the midgut cells and open towards the yolk. At the same time the microvilli spread over the apical surface of the midgut primordium to form the regular brushborder of the larval midgut. In the species studied the vesicles appear to "prefabricate" the apical surfaces of the future midgut epithelium.     

The role of the transformer genes in the development of genitalia and analia of Drosophila melanogaster

The autosomal mutations transformer (tra) and transformer-2 (tra-2) of Drosophila convert chromosomal females (X/X) into phenotypical males. Our analysis aims at an understanding of the role which the transformer genes play in the development of the sexually dimorphic genital disc. In each Drosophila embryo, this disc starts development with a male and a female genital primordium, and an anal primordium. Our experiments involved the production of cell clones that were made homozygous for tra or tra-2 at different times of development. Homozygous clones were obtained by inducing mitotic recombination in three types of females heterozygous for tra or tra-2. The cells of the homozygous tra/tra or tra-2/tra-2 clones responded by changing from the female into the male pathway. Male genital structures developed if the clones were induced not later than 81 hr into development. In the analia, male clones appeared up to 120 hr. Our results show that the action of the wild-type alleles of tra⁺ and tra-2⁺ is required until late in larval development to repress the male genital primordium and to support development of the female primordium, as well as to maintain the anal primordium in the female pathway. Our data also suggest that the embryonic genital disc consists of two compartments, one containing the precursors for penis and analia, the other those of the male and female genitalia.     

Organogenesis of the Caenorhabditis elegans intestine

The intestine of Caenorhabditis elegans is an epithelial tube consisting of only 20 cells and is derived clonally from a single embryonic blastomere called E. We describe the cellular events that shape the intestine. These events include cytoplasmic polarization of cells in the intestinal primordium, the intercalation of specific sets of cells, the generation of an extracellular cavity within the primordium, and adherens junction formation. The polarization of the intestinal primordium is associated with the generation of an asymmetric microtubule cytoskeleton, and microtubule function plays a role in subsequent cell polarity. We show that an isolated E blastomere is capable of generating polarized intestinal cells, indicating that some of the major events in intestinal organogenesis do not depend upon interactions with surrounding tissues. We compare and contrast intestinal organogenesis with some of the basic steps in development of a second epithelial organ, the pharynx, and suggest how these differences lead to organs with distinct shapes.     

"Chemical surgery" as an approach to study morphogenetic events in embryonic mouse limb

Cytosine arabinoside (Ara-C) or retinoic acid (RA) was injected into pregnant mice in doses which induce a high incidence of limb defects. Within 4 hr of the treatment, extensive cell death was observed in the embryonic limb buds. However, the location of necrotic cells and the eventual limb defects were different for the two chemicals. Ara-C killed cells in those regions of the limb which were undergoing active proliferation. RA, on the other hand, had no effect on actively dividing cells but was lethal to cells of chondrogenic lineage at stages when their proliferation rate had fallen 7- to 10-fold below the original rate. In all cases, an excellent correlation between the location of dead cells (as seen 4 hr after drug treatment) and the eventual bony defects (as seen in the term fetuses) was observed. The unique properties of Ara-C and RA have been exploited in determining the relative levels of cytodifferentiation in the embryonic mouse limb buds. It is concluded that in the limbs of early 11th day mouse embryos (comparable to chick stage 19–20), differentiation of future skeletal elements has not yet begun. However, by the 12th day (comparable to chick stage 23), cell populations destined to form most of the future cartilages (except for digits) have already been established.     

Cyclophosphamide teratogenesis: evidence for compensatory responses to induced cellular toxicity

Cyclophosphamide (CP) administered ip to pregnant mice on day 10 of gestation (day of plug = day 0) is teratogenic (exencephaly, cleft palate, and limb malformations) at 20 mg/kg and embryolethal at higher doses. In the present study, CP was administered at 1, 5, 10, or 20 mg/kg on day 10 of gestation. Embryos were removed at 8 and 28 hr postdosing, and two embryos from each litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death. The remaining embryos were frozen and forelimb buds subsequently removed for flow cytometric (FCM) analysis of the cellular DNA synthetic cycle. Additional litters were examined near term (day 17) for morphological abnormalities; these data were correlated with embryonic toxicity as detected by NBS staining and FCM analysis. Only the highest dose produced malformations. In marked contrast, a dose-related increase in the percentage of limb bud cells in the S (DNA synthetic) phase of the cell cycle was detectable at all doses. Inhibition of DNA synthesis was detected at all doses 8 hr post exposure and persisted through 28 hr for doses greater than or equal to 10 mg/kg. NBS staining indicated increased cell death in the alar plate of the neural tube 28 hr after exposure to 10 mg/kg CP and generally increased cell death in areas of rapid cell proliferation throughout the embryo at 20 mg/kg. The absence of an overt teratogenic response at dose levels that produced significant perturbation of the cell cycle indicates that a measure of embryonic damage can be compensated for or repaired. The implications of these findings for the existence of thresholds in developmental toxicity are discussed.