Functional regulation of tyrosinase and LAMP gene family of melanogenesis and cell death in immortal murine melanocytes after repeated exposure to ultraviolet B

The aim of the present study was to evaluate the effect of human follicle stimulating hormone (hFSH) on cellular proliferation in the chick embryo ovary. Chick embryos (Babcock B300) were injected on chorioallantoic membrane with a single dose of hFSH (2.0 IU/ embryo) at Days 7, 9, or 13 of incubation or with hCG (2.0 IU/embryo) at Day 13 of incubation. At 17 days of incubation and within 24 h after hatching, left ovaries were dissected and completely dissociated. Cells from the whole ovary were classified into germ cells (primary oocytes), typical steroidogenic cells, and poorly differentiated somatic cells and counted with the aid of a hemocytometer. Aliquots of the cell suspension from the whole left ovary were analyzed by flow cytometry, in order to determine the percentage of cells at each phase of the cell cycle. In addition, samples of the suspension (1.0 x 10(6 )cells) were incubated for 2 h in basal and stimulated conditions measuring 17beta-estradiol secretion in the medium. The ovarian cell number at 17 days of incubation showed that hFSH treatment at Day 7 did not modify the cell number in any of the subpopulations evaluated; treatment at Day 9 resulted in an increase in poorly differentiated somatic cell number, without changes in steroidogenic and germ cells, whereas hFSH treatment at Day 13 augmented the number of poorly differentiated, steroidogenic, and germ cells. The percentage of cells in S-phase was increased 12 and 15 h after hFSH treatment (Day 13). Secretion of 17beta-estradiol was increased in the hFSH-treated group (Day 13) measured at 17 days of incubation. The increase in cell number of the three subpopulations was still observed in the left ovary of the newly hatched chicken. Treatment with hCG at Day 13 of incubation did not change the number of poorly differentiated, steroidogenic, and germ cells in the left ovary, neither in the 17-day-old chick embryo nor in the newly hatched chicken. The 17beta-estradiol secretion in hCG-treated embryos was similar to controls. The present study is the first evidence of an effect of FSH on somatic and germ cell number, together with an increase in 17beta-estradiol production during chick embryo ovary development.     

Growth response to lectins in chick embryo cells at different stages of development

We report here the effect of Robinia lectin and Concanavalin A on fibroblasts and liver cells from chick embryos between the 8th and the 20th day of development. This was observed in vitro after different times of cultivation. There is evidence that these lectins decrease cell number in cultures from young embryo cells but that they stimulate the proliferation of older embryo cells. The optimum concentration of either lectin was 3 mug/ml. No effect was observed on 12- and 14-day cells at the different concentrations of lectin used. The agglutination of fibroblasts by these lectins regularly decreased from the 8th to the 16th day of development. Liver cells however were agglutinated at no stage. These results could perhaps be explained in terms of cell surface changes either during the course of ontogeny or as a result of lectin treatment.     

Postneurulation rapid brain growth represents a critical time for encephalocele formation: a chick model

Current theory regarding the pathogenesis of encephaloceles suggests that the defect occurs after neurulation is completed in which brain tissue herniates through defective mesodermal elements necessary for skull modeling. To better delineate the mechanisms of encephalocele development, we performed a variety of microsurgical manipulations in the developing cranium of the chick embryo during the postneurulation period of early rapid brain growth. The results of the study revealed that encephaloceles could be induced in 78% of chick embryos manipulated at stage 26 that had evidence of marked decompression of the primitive ventricle. On the other hand, control embryos or embryos manipulated without ventricular decompression at stage 26 did not develop encephaloceles (0 of 32). Embryos with decompression of the primitive ventricle treated at earlier stages (20-24) showed only a 5% incidence of encephaloceles. These findings suggest that there is both a critical time point during postneurulation rapid brain growth when encephaloceles are prone to occur and a mesenchymal defect associated with decompression of the primitive ventricle that must be present to induce encephaloceles.     

Visual demonstration of the limb-forming zone in the chick embryo lateral plate

The present study was undertaken to determine whether a visible Wolffian ridge, distinct from the lateral fold, can be identified in chick embryos. Ectoderm thickness was measured in stage 11-17 chick embryos. There was a general trend, from thin ectoderm in the midline, to an ectodermal thickening over the somites, intermediate mesoderm, and lateral plate. Other embryos were cut from the yolk, pinned out, and photographed. The lateral fold was then eliminated, and the embryo was rephotographed. The photographs reveal a definite opaic zone, distinct from the lateral fold, in stage 11-18 chick embryos. Furthermore, there is a direct correlation between the opacity of this cellular band and the limb-forming potential of grafted wing, flank, and leg regions (see Stephens et al., '89). At stages 11-14, the wing, flank, and leg exhibit a uniform opacity, and a uniform capacity for limb formation when grafted to a host celom. From stage 15 to stage 18, the opacity in the flank diminishes, and its limb-forming capability disappears. This study demonstrates the presence of an opaic zone, which we have called the limb-forming zone (LFZ) along the lateral side of early chick embryos, which is independent of the lateral fold, is not as extensive as the lateral plate, and is not simply associated with ectodermal thickening, but which is directly correlated with limb-forming potential in the lateral plate.     

Developing visual function in the red jungle fowl embryo.

Measured the onset of visual system function in 35 wild red jungle fowl ( Gallus gallus ) embryos by the pupillary reflex technique and compared it with that of its domesticated descendant, the domestic chicks. The 1st neurally mediated pupillary reflex in the embryos was found at Day 15 of incubation after 77% of incubation was completed. This point did not differ significantly from the onset of this reflex in the domestic chick embryo (after approximately 83% of incubation). Thus, it is concluded that the relatively late onset of overt visual system function in the chick, as compared with several other precocial avian species, was not a result of its history of intense domestication but was most likely a normal characteristic of this and other closely related species. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

In vitro expression of n-cadherin adhesion molecule during early cardiac morphogenesis

The aim of the present study was to investigate, "in vitro", the degree of organogenetic potentiality of the cells of the cardiogenic area during the early developmental stages of the chick embryo. Embryos from between the end of the presomitic stage to the 8 somite stage were studied. The subcephalic fold was cultured in liquid medium for up to 7 days. After 24 hs of culturing, an extended migration ring was observed. In the explants, from 3 somite stage, onwards, beating masses were noted, the shape and size of which suggested a vascular-like structure. Sections of the cultures were processed for the detection of the N-Cadherin adhesion molecule. The observations stated that the diffusion and intensity of expression of this receptor is related to the stage od development of the embryo. Cultures from the presomitic stage to 3 somite stage did not express the molecule. Instead, expression took place in those cultures of embryos at the 3 somite stage, onwards. In the cultures to which the antiserum against N-Cadherin had been to the medium, the formation of vascular-like structures was affected. The changes depended on the age of the embryos. These observations suggest that the expression of the N-Cadherin is related to the potentiality of the presumptive myocardic cells to organize themselves, at least "in vitro", to form a well-defined tridimensional structure. The expression of the adhesion molecule and the potentiality of the cells to build tubular structures were transient features, "in vitro" in our cultures. This suggests that "in vivo" the expression of the N-Cadherin must be aided by factors which, at present, are unidentified.     

No Mycobacterium paratuberculosis detected in intestinal tissue, including Peyer''s patches and lymph follicles, of Crohn''s disease

1. The present study evaluated developmental characteristics in the chick embryo throughout incubation following cell removal from the freshly laid egg. 2. Between 10 and a few hundred cells of the blastoderm were removed for sex diagnosis. Incubation of the treated embryos was then continued in an open egg shell culture system. 3. Cell recovery from different regions within the blastoderm was performed. 4. The experiments presented here demonstrate the persistence of the developmental potential of chicken embryos in an in ovo culture system after removal of different numbers of cells from the germinal disc prior to incubation. 5. No deviations in developmental characteristics were recorded when compared to untreated control cultures. 6. No detrimental effects of double cell biopsies could be observed. 7. A similar number of chicks developed to hatch regardless of the location of manipulation within the blastoderm.     

Synthesis of delta-aminolaevulinate synthase by isolated liver polyribosomes

1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta-aminolaevulinate synthase in vitro. These results suggest that haemin represses the synthesis of delta-aminolaevulinate synthase by decreasing the amount of mRNA for the enzyme available for translation.     

Endotoxic activity of cell-free rumen fluid from cattle fed hay or grain

The cell-free rumen fluid from cattle fed hay or grain exhibited the following biological characteristics which strongly suggest the presence of endotoxin or a toxic principle similar to endotoxin of gram-negative bacteria: proved lethal to mice when injected with actinomycin D; proved extremely lethal to chick embryo; induced biphasic pyogenic response in rabbits; enhanced susceptibility to bacterial infection in mice; evoked positive epinephrine skin reaction in rabbits and phenol-water or aqueous ether proved lethal to mice and chick embryos. A quantitative difference in concentrations of endotoxin was observed on LD50 in mice and chick embryos and response to the epinephrine skin test in rabbits. Cell-free rumen fluid of grain-fed cattle contained at least twice as much endotoxin as that of hay-fed cattle. Endotoxin in cell-free rumen fluid and in higher concentration in cattle fed grain than in those fed hay support the hypothesis that rumen bacterial endotoxins may participate in the pathogenesis of diseases associated with high grain feeding such as lactic acidosis and the sudden-death syndrome.     

Activities of enzymes involved in the metabolism of platelet-activating factor in neural cell cultures during proliferation and differentiation

Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways. The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A2 activity, and acetyl-CoA. The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator. We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA). In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1-3). During this period no significant changes of lyso-PAF AcT activity was observed. Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions. The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures. PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day. RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate. These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced.     

Ontogeny of behavioral responsiveness to sound in the chick embryo as indicated by electrical recordings of motility.

Gathered normative behavioral data regarding the ontogeny of responsiveness to sound in the chicken embryo. As a prerequisite, a sensitive and accurate method for recording embryonic motility was developed (Exp I). By means of platinum electrodes inserted just beneath the shell membrane, potentials resulting from heartbeat and movement of 300 White Leghorn embryos were recorded on a polygraph. The technique was found to be effective when applied to chick embryos 6 days and older. Correlations between visual observations of activity and the records produced by the electronic technique substantiated its accuracy. Behavioral responses of 300 chick embryos (Stages 39–43) to acoustic stimulation (Exp II) were then recorded. High-intensity (115-db) tones of 400, 700, and 1,400 Hz were used as stimuli. The earliest consistent responses were recorded from Stage 40 (Days 14–25) Ss; the 700 and 1,400 Hz tones produced statistically reliable inhibition of movement during the stimulus period compared with the post-stimulus period. Reliable increases in movement during the stimulus period were first recorded at Stage 42 (Days 16–27) in response to 700 and 1,400 Hz and at Stage 43 (Days 17–28) in response to 400 Hz. (42 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The mouse homeobox gene, Gbx2: genomic organization and expression in pluripotent cells in vitro and in vivo

This paper reviews recent work on a project that uses a computer-aided approach for making 3-D reconstructions of serially sectioned mouse embryos (the digital mouse). The captured images are aligned using a warping program so that almost perfect alignment of adjacent sections is achieved with minimal deformation. The sections that are viewed on the computer screen are in fact computer-generated grey-level images with a resolution of about 10 microm. The reconstructed embryo may then be resectioned in any plane to simulate as near as possible an exact match on the computer screen to the viewer's own material. Individual anatomical domains may then be painted in different colors, and these domains may be selected by querying the textual database containing anatomical and other information. Further, it is now possible to generate 3-D images of individual anatomically-discrete components or related sets of components of a particular system in isolation from the rest of the embryo, or, if required, against a 'ghost-like' image of the intact embryo, or specific parts of an embryo. In the article, examples are given of the use of the system in interpreting the vascular, gut and paraxial mesoderm systems, while both the advantages and disadvantages of this approach are also discussed. The eventual aim will be to provide 3-D reconstructions of mouse embryos from fertilization up to 14 days postcoitum of development. When completed, this project will allow the accurate spatial mapping of gene-expression and cell lineage data onto the digital Atlas of normal mouse embryonic development.     

In vitro cytostatic activity of 1,2,4-triazolo- and 1,2,3,4-tetrazolo pyridazines

PROBLEM: There is considerable controversy concerning the root cause and mechanisms of early embryo loss. It has been suggested that most pregnancy losses occur due to morphogenetic anomalies of the embryo. It has also been suggested that the maternal specific immune system rejects the embryo. METHODS: Existing data on the cell and molecular biology of early embryo loss in murine experimental models is reviewed. RESULTS: Using the CBA(female) x DBA/2(male) model of early embryo loss, it has been established that maternal inflammatory cells infiltrate the decidua basalis of all implantation sites within 48 hr after implantation. For most embryos, the relatively low numbers of macrophages (Mphi) and natural killer-like (NK-like) cells of maternal origin remain relatively constant after day 8, whereas 20-30% of the embryos show a significant increase in inflammatory cells in the maternal decidua, corresponding to the incidence of early embryo resorption visible at day 12. Evidence will be reviewed to suggest that decidual NK-like cells are not cytolytic but may be producing the Mphi-activating cytokine interferon gamma (IFN-gamma), which activates decidual Mphi and other cells. Furthermore, embryo loss is ameliorated by in vivo treatment with anti-IFNgamma or anti-NK antisera, indicating that NK-like cells and/or IFNgamma are required for embryo loss, but not for embryo survival. In resorbing embryos, the inflammatory Mphi show evidence of having been primed during early pregnancy, in that in vitro incubation with lipopolysaccharide induced the production of tumor necrosis factor alpha and nitric oxide. CONCLUSION: These findings support the concept that early embryo loss is a nonspecific event mediated by the triggering of cytotoxin production by primed decidual macrophages.     

Brachially innervated ectopic hindlimbs in the chick embryo. I. Limb motility and motor system anatomy during the development of embryonic behavior

The functional status of brachially innervated hindlimbs, produced by transplanting hindlimb buds of chick embryos in place of forelimb buds, was quantified by analyzing the number and temporal distribution of spontaneous limb movements. Brachially innervated hindlimbs exhibited normal motility until E10 but thereafter became significantly less active than normal limbs and the limb movements were more randomly distributed. Contrary to the findings with axolotls and frogs, functional interaction between brachial motoneurons and hindlimb muscles cannot be sustained in the chick embryo. Dysfunction is first detectable at E10 and progresses to near total immobility by E20 and is associated with joint ankylosis and muscular atrophy. Although brachially innervated hindlimbs were virtually immobile by the time of hatching (E21), they produced strong movements in response to electrical stimulation of their spinal nerves, suggesting a central rather than peripheral defect in the motor system. The extent of motoneuron death in the brachial spinal cord was not significantly altered by the substitution of the forelimb bud with the hindlimb bud, but the timing of motoneuron loss was appropriate for the lumbar rather than brachial spinal cord, indicating that the rate of motoneuron death was dictated by the limb. Measurements of nuclear area indicated that motoneuron size was normal during the motoneuron death period (E6-E10) but the nuclei of motoneurons innervating grafted hindlimbs subsequently became significantly larger than those of normal brachial motoneurons. Although the muscle mass of the grafted hindlimb at E18 was significantly less than that of the normal hindlimb (and similar to that of a normal forelimb), electronmicroscopic examination of the grafted hindlimbs and brachial spinal cords of E20 embryos revealed normal myofiber and neuromuscular junction ultrastructure and a small increase in the number of axosomatic synapses on cross-sections of motoneurons innervating grafted hindlimbs compared to motoneurons innervating normal forelimbs. The anatomical data indicate that, rather than being associated with degenerative changes, the motor system of the brachial hindlimb of late-stage embryos is intact, but inactive.     

The effect of propranolol upon chick embryo cardiogenesis

The teratogenic effects of propranolol HCl on cardiac development were studied in chick embryos of days 3 and 4 of incubation. Propranolol was injected into the yolk sac at doses ranging from 0.05 to 0.6 mg per egg. All the treated and control embryos were examined on day 7. The LD50 for the embryos treated on the 3rd and 4th day was 0.15 and 0.35 mg per embryo, respectively. Cardiac anomalies such as aortic stenosis ventricular septal defects and common truncus arteriosus were observed. Other malformations included atrial septal defects, thin atrial wall and defects of the pulmonic, aortic and atrioventricular valves. The incidence of cardiac anomalies in the controls was very low. Propranolol was observed to slow the heart rate in the experimental embryos. It is suggested that slowing of heart rate at the early stages of heart development caused aberrant bloodstream flow patterns which probably resulted in the genesis of cardiac anomalies. The results of this study indicate that propranolol has teratogenic effects on chick embryo cardiogenesis.     

Pathogenicity of K. ozaenae for mice and chick embryos

The authors tested the virulence of K. ozaenae--its old museum strains, the freshly isolated ones and those passaged on meat-peptone agar; experiments were carried out on mice and chick embryos. To mice the culture was administered intraperitoneally, subcutaneously, intranasally, and to embryos--into the allantoic cavity and on the chorioallantoic membrane. Irrespective of the method of administration, the freshly isolated strains were highly virulent both for mice and for chick embryos. The virulence of such strains decreased in the process of passaging on the nutrient medium. Old museum strains were of low virulence for albino mice and avirulent for chick embryos. In comparing the virulent and avirulent strains there were found no differences in the antigenic structure and toxicity of the Boiven complex, cytoplasm, membrane, capsular polysaccharide or whole virulent and avirulent bacteria killed by heating.     

GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. We have isolated and characterized a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalized the embryo and decreased the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild-type GSK3beta animalized the embryo and caused the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appeared to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes.     

Induction of primitive streak and Hensen's node by the posterior marginal zone in the early chick embryo

In the preprimitive streak chick embryo, the search for a region capable of inducing the organizer, equivalent to the Nieuwkoop Center of the amphibian embryo, has focused on Koller's sickle, the hypoblast and the posterior marginal zone. However, no clear evidence for induction of an organizer without contribution from the inducing tissue has been provided for any of these structures. We have used DiI/DiO labeling to establish the fate of midline cells in and around Koller's sickle in the normal embryo. In the epiblast, the boundary between cells that contribute to the streak and those that do not lies at the posterior edge of Koller's sickle, except at stage X when it lies slightly more posteriorly in the epiblast. Hypoblast and endoblast (a second lower layer formed under the streak) have distinct origins in the lower layer, and goosecoid expression distinguishes between them. We then used anterior halves of chick prestreak embryos as recipients for grafts of quail posterior marginal zone; quail cells can be identified subsequently with a quail-specific antibody. Anterior halves alone usually formed a streak, most often from the posterior edge. Quail posterior marginal zones without Koller's sickle were grafted to the anterior side of anterior halves. These grafts were able to increase significantly the frequency of streaks arising from the anterior pole of stage X-XI anterior halves without contributing to the streak or node. Stage XII anterior halves no longer responded. A goosecoid-expressing hypoblast did not form under the induced streak, indicating that it is not required for streak formation. We conclude that the marginal zone posterior to Koller's sickle can induce a streak and node, without contributing cells to the induced streak.