Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

Quantitative analysis of mid-gestation mouse aggregation chimaeras: non-random composition of the placenta

Mouse chimaeras were produced by aggregating eight-cell embryos from two different F₂ matings, abbreviated to AF₂ and BF₂ respectively: (C57BL/ OIa.AKR-Gpi-1s ^a, c/Ws female × BALB/c male)F₂ and (C57BL/Ws female × CBA/Ca male)F₂. Quantitative electrophoresis of glucose phosphate isomerase (GPI-1) was used to estimate the proportions of the two cell populations in different tissues of the 12 day chimàeric conceptuses, with the % GPI-1A indicating the percentage of cells derived from the AF₂ embryos. The % GPI-1A was found to be highly positively correlated within the primitive ectoderm lineage (between the fetus, amnion and yolk sac mesoderm) and within the primitive endoderm lineage (between the yolk sac endoderm and the parietal endoderm) but no correlation (either positive or negative) was seen between the two lineages. This confirms the results of a previous,study of chimaeras made between partially congenic strains and suggests the original conclusions have general validity. The % GPI-1A in the placenta was corrected for the expected contribution of maternal GPI-1, based on control experiments involving transfer of homozygous Gpi-1s ^b /Gpi-1s ^b embryos to the uteri of Gpi-1s ^a /Gpi-1s ^a pseudopregnant females. The corrected % GPI- lA in the placenta was positively correlated with that in each of the three primitive ectoderm derivatives. This suggests either (1) exchange of cells between the polar trophectoderm and the underlying part of the inner cell mass that forms the primitive ectoderm or (2) cells are incompletely mixed in the chimaeric blastocyst and patches of AF₂ and BF₂ cells straddle the boundary between the polar trophectoderm and the underlying primitive ectoderm. The second explanation does not imply the existence of shared developmental lineages between trophectoderm and primitive ectoderm in non-chimaeric embryos. Unlike that of any other tissue, the distribution of placental GPI-1A was U-shaped; in 17/28 placenta samples the proportion of the minor component was 10% or less. This suggests that the placental trophoblast is derived from a small number of coherenct clones of polar trophectoderm cells (either a small number of polar trophectoderm cells or a larger number if the two cell populations are not finely intermingled). Thus, although as a population the placentas of chimaeric conceptuses are balanced with respect to the % GPI-1A (mean close to 50%), individually most placentas are extremely unbalanced in their chimaeric composition (< 10% or > 90% GPI-IA). This non-random composition of the chimaeric placentas is in contrast to the widely held assumption that the distribution of cells in chimaeric conceptuses is normally random. Correspondence to: J.D. West     

Embryonic development and metamorphosis of the scyphozoan <Emphasis Type="Italic">Aurelia</Emphasis>

We investigated the development of Aurelia (Cnidaria, Scyphozoa) during embryogenesis and metamorphosis into a polyp, using antibody markers combined with confocal and transmission electron microscopy. Early embryos form actively proliferating coeloblastulae. Invagination is observed during gastrulation. In the planula, (1) the ectoderm is pseudostratified with densely packed nuclei arranged in a superficial and a deep stratum, (2) the aboral pole consists of elongated ectodermal cells with basally located nuclei forming an apical organ, which is previously only known from anthozoan planulae, (3) endodermal cells are large and highly vacuolated, and (4) FMRFamide-immunoreactive nerve cells are found exclusively in the ectoderm of the aboral region. During metamorphosis into a polyp, cells in the planula endoderm, but not in the ectoderm, become strongly caspase 3 immunoreactive, suggesting that the planula endoderm, in part or in its entirety, undergoes apoptosis during metamorphosis. The polyp endoderm seems to be derived from the planula ectoderm in Aurelia, implicating the occurrence of “secondary” gastrulation during early metamorphosis.     

Fine structural changes associated with the onset of calcium,sodium and water transport by the chick chorioallantoic membrane

Summary An attempt is made to correlate structure and transport function in the embryonic chorioallantoic membrane. The fine structure of the endoderm and ectoderm in the membrane was examined with particular attention given to the morphological changes that occur when transport is established,in vivo. Two distinctive cells, a granule-rich cell and a mitochondria-rich cell, appear in the endoderm at the time allantoic fluid sodium, chloride and water reabsorption commences. These are indistinguishable from the cells described in toad bladder epithelium. It is suggested that the granule-rich cell is responsible for bulk water movement and the mitochondria-rich cell is specifically engaged in active sodium transport. In the ectoderm, two distinctive cell types are also found to be associated with the onset of active calcium transport. These are referred to as the capillary-covering cell and the villus-cavity cell. The preponderate capillary-covering cell is most likely responsible for transcellular calcium transport. It is postulated that the function of the villus-cavity cell is to secrete hydrogen ions which are necessary, along with carbonic anhydrase, to mobilize Ca⁺⁺ from the insoluble calcium carbonate of the eggshell.     

The role of the basal lamina in mouth formation in the embryo of the starfish Pisaster ochraceus

Details of mouth formation in normal and exogastrulated Pisaster ochraceus larvae have been studied by light microscopy and transmission and scanning electron microscopy. As the archenteron begins to bend, the cells in the presumptive mouth region dissociate and migrate into the blastocoele where they become mesenchyme cells. This leaves a defect in the “blind” endodermal tube, which is covered by a basal lamina. Subsequently this exposed basal lamina bulges to form a blister which appears to extend across the blastocoele to make contact with spikelike projections from the future stomodeal region of the ectoderm. Mesenchyme cell processes are associated with both the basal lamina blister and the ectoderm in this region and may provide both motive power and guidance for contact. Shortly after contact is made the blister of basal lamina from the endoderm fuses with the basal lamina of the ectodermal cells and the ectoderm begins to invaginate. At this time the lateral walls of the presumptive oesophagus are largely formed of naked basal lamina with some loosely associated cells on the endodermal side. Eventually the lateral walls of the proximal part of the oesophagus become cellular, giving rise to an epithelium. A cell plug located between the stomodeum and oesophagus persists for some time before finally breaking down to complete the larval digestive tract. Experiments with exogastrulae suggest that many of these developmental patterns are determined before gastrulation.     

Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass

Summary Early amphibian gastrula ectoderm (Triturus alpestris) has been treated with vegetalizing factor. While normal sandwiches (animal caps of two eggs) differentiated mainly into endoderm derived tissues, giant-sandwiches (a combination of 8 animal caps) formed mesodermal and neural tissues in addition. The results support the interpretation that ectoderm will differentiate into endoderm derived tissues when all or nearly all cells are induced (presumably depending on certain threshold concentrations of the inducer). This is the case in the normal sandwich after treatment with high concentrations of vegetalizing factor for 24 h. However, in a giantsandwich it must be assumed that only the cells in the vicinity of the inducer will be triggered to differentiate into endoderm derived tissues. Mesodermal structures will be formed by secondary interactions between the induced ectoderm (endoderm) and non induced ectodermal cells. The induction of neural structures could be explained as a further interaction between mesodermalized and non induced ectodermal cells. This chain of events is compared with the steps of determination in normogenesis.     

Satellite glial cell responses to neuronal firing in the nervous system of Helix pomatia

Patch clamp experiments were conducted on satellite glial cells attached to the cell body of neurons in place within the nervous system of the snail Helix pomatia. The glial cells were studied using cell-attached and whole-cell patch clamp configurations while the underlying neurons were under current or voltage clamp control.The resting potential of the glial cells (–69 mV) was more negative than that of the underlying neurons (–53 mV), due to their high K⁺ selectivity. Densely packed K⁺ channels were present, some of which were active at the cell resting potential. Neuronal firing elicited a cumulative depolarization of the glial cells. Large K⁺ currents flowing from V-clamped neurons depolarized the glial layer by up to 30 mV. The glial depolarization was directly correlated with the size of the neuronal K⁺ current. The glial cells recovered their resting potential within 2–5 sec. The neuronal depolarization induced a delayed (20–30 sec) and persistent (3–4 min) increase in the glial K⁺ channel opening probability. Likewise, pulses of K⁺ (20–50 mM)-rich saline activated the glial channels, unless the underlying neuron was held hyperpolarized. In low Ca²⁺-high Mg²⁺ saline, neuron depolarization and K⁺-rich saline did not activate the glial K⁺ channels.These data indicate that a calcium-dependent signal released from the neuronal cell body was involved in glial channel regulation. Neuron-induced channel opening may help eliminate the K⁺ ions flowing from active neurons.I. Gommerat is recipient of a fellowship from the Ministère de la Recherche et de la Technologie.This work was supported by the CNRS and by a grant from the Fondation pour la Recherche Médicale. We would like to thank Mrs. M. André and Mr. G. Jacquet for technical assistance and Mrs. J. Blanc for improving the English.     

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)⁺ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).     

Inhibition of voltage-dependent Na+ current in cell-fusion hybrids containing activated c-Ha-ras

Summary The electrophysiological properties of EJ (human bladder carcinoma), GM2291 (human fetal lung fibroblast), and of three hybrid cell lines obtained from their cell fusion were investigated using the patch-clamp technique. GM2291 cells, which are nontumorigenic, express voltage-dependent Na⁺ channels. The pharmacology and gating properties of the Na⁺ channels in GM2291 cells are distinct from neuronal and cardiac Na⁺ channels. EJ cells, which are tumorigenic and contain activated c-Ha-ras, express inward rectifier K⁺ channels. The three cell-fusion hybrid lines, named 145 (nontumorigenic), 145L (non-tumorigenic but morphologically altered), and 147TR2 (fully tumorigenic segregant), have been previously shown to express levels of activated c-Ha-ras similar to those of the EJ parental line. Voltage-dependent Na⁺ channels were observed in none of the hybrid cell lines, while inward rectifier K⁺ channels were observed in each of the hybrid cell lines. The possibility that c-Ha-ras inhibits expression of a voltage-dependent Na⁺ channel is discussed.     

Evolution de la capacité morphogénétique de la région stomodéale chez l'embryon dePleurodeles waltlii Michah (amphibien urodéle). etude par transplantation intrablastocélienne et par culture in vitro

Summary Transplantations into the blastocoel of gastrulae and cultures in vitro of the stomodeal region ofPleurodeles waltlii Michah embryos have been carried out. These experiments gave the following results:Neural crest cells reach the stomodeal region at the young tail-bud stage (stage 22), and are able to take part in formation of teeth and cartilage, it is possible to dissociate the phenomena of mouth opening, complete mouth formation and complete head formation, differentiation of a digestive tube from the pharyngeal endoderm and the formation of a mouth opening both depend on the presence of mesentoderm and mesectoderm in the explant or in the environment, during the tail-bud stage a symmetry factor plays a part in mouth formation, bone formation likewise coincides with the presence of neural crest cells.

     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Transfer of Protection to Murine Typhoid Conferred by L-Form Salmonella typhimurium in Dependence of Cooperation between L Form-Adopted Macrophages and L Form-Induced Lyt-2+ T Cells

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L-form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6-week immune mice but not from 24-week immune animals transferred anti-S. typhimurium immunity. Treatment with anti-macrophage antiserum and complement most effectively abolished protective capacity in 6-week immune cells, while anti-T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt-2⁺ T cells selected from 6-week immune spleen cells. These Lyt-2⁺ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6-week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24-week immune spleen cells may be due to the decrease in the L form-colonization. These results suggest that cooperation between the L form-colonized macrophages and L form-induced cytotoxic Lyt-2⁺ T cells contributes to anti-S. typhimurium immunity, and might imply the immunological difference between the 6-week immune phagocytes and the cells at an early stage of infection or immunization.     

SEM analysis of amphibian mesodermal migration

Summary At the end of gastrulation, the lateral mesoderm of amphibian embryos migrates ventrally between the ectoderm and the endoderm. The present study is an examination of the morphology of the leading cells of the mesodermal sheet and of the substratum over which they move (the inner surface of the ectoderm). The cells of the leading edge of the mesoderm are generally round, with very short and narrow flattened projections in the forward direction. These projections do not have a ruffled morphology, regardless of whether fixation is carried out before or after the ectoderm and mesoderm are dissected away from the endoderm. The inner surface of the ectoderm is covered with fine (450–500A) filamentous extracellular material and the ectoderm cells sometimes extend cytoplasmic processes (approx. 0.1 wide) onto the leading surface of the mesoderm or onto adjacent ectoderm cells. These studies indicate that the morphology of cell migration in amphibians is closer to that seen inFundulus than to that characteristic of chick or mammalian cells.This paper is dedicated to the memory of Mac V. Edds, Jr., who warmly encouraged the developmental biologists of the Pioneer Valley     

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17⁺ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17⁺ cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP⁺ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17⁺ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.     

In the FVB/N HER-2/<Emphasis Type="Italic">neu</Emphasis> transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>-based vaccines

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8⁺ T cells and the presence of CD4⁺CD25⁺ regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8⁺ T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8⁺ T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.     

An ultrastructural study of larval settlement in the sea anemone Urticina crassicornis (Cnidaria,Actiniaria)

Laboratory-reared larvae of the sea anemone Urticina (= Tealia) crassicornis have been examined by electron microscopy prior to and following settlement on algal substrata. At 18 days postfertilization, the free-swimming planula larva measures about 600 μm long. A stomodaeal invagination occurs at the narrow end of the larva and connects with a solid mass of endoderm in the core region. The endoderm possesses septa with well-developed myonemes and is situated subjacent to a thin sheet of mesoglea. The uniformly ciliated ectoderm that constitutes the outer layer of the larva contains: (1) spirocysts, (2) nematocysts, (3) mucus, (4) three types of membrane-bound granules, (5) a basiepithelial nerve plexus, and (6) a few nongranular cells that may represent sensory neurons. Within several minutes after the introduction of the algal substratum, the planula characteristically directs its broadened aboral end toward the alga and secretes a refractile sheet of material. As the aboral end attaches to the substratum, the larva becomes noticeably shorter along its oral-aboral axis, presumably owing to the contractions of myonemes that are located within the endodermal septa. All three types of granules and the ectodermal mucoid substances are exocytosed during settlement, but spirocysts and nematocysts characteristically remain undischarged. Ovoid, PAS⁺ granules are believed to be at least partly responsible for adhesion, since these granules are concentrated at the aboral end prior to settlement and are somewhat similar in ultrastructure to putative viscid granules produced by other species. Contrary to a previous report based on light microscopy, no discrete sensory organ is evident in serial sections of the aboral ectoderm. The ability of planulae to detect suitable substrata appears to depend instead on sparsely distributed sensory cells that occur throughout the larval ectoderm.