A demonstration of cellular interactions during the formation of mesoderm and primordial germ cells in Pleurodeles waltlii

Animal, vegetal, dorsal and ventral blastomeres of eight-cell embryos of the urodele Pleurodeles waltlii were isolated and cultured for 15 days. The four animal blastomeres produced vesicles delimited by an irregularly shaped epidermis. In all other explants, the formation of mesodermal structures occurred, which can be interpreted as the result of inductive interaction, occurring during segmentation, between the ectodermal animal cap and vegetal yolk mass. Primordial germ cells (PGCs), which formed in 78% of cases when the presumptive ventral half to the embryo was cultured, occurred in only 48% of cases when the two ventral vegetal blastomeres were cultured alone. The absence of PGCs in the explants emanating from the four vegetal blastomeres is thought to have been due to inhibition of differentiation by notochord. This hypothesis has been confirmed by culture experiments in which the addition of presumptive chordomesoderm of young gastrulae prevented the differentiation of PGCs under conditions in which they are normally formed. These observations suggest that, in urodeles, PGCs do not arise from cells segregated as early as the eight-cell stage, but are the product of later inductive interaction between ectoderm and endoderm.     

A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl.

We have determined the fate of presumptive mesodermal cells in the early Pleurodeles waltl gastrula. We labeled all cells in a gastrula with RLDx cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the presumptive regions are generally distributed according to classical fate maps for urodeles but that the boundaries between presumptive regions are indistinct, due to extensive intermingling between cells at the edges of grafted regions. We have shown that there is a high dorsal to low ventral gradient of mixing between superficial and deep cells.     

A suppressor/enhancer screen in Drosophila reveals a role for wnt-mediated lipid metabolism in primordial germ cell migration

Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration.     

Detection, using fluorescent lectins, of specific carbohydrate chains in primordial germ cells of anuran amphibians

A study of primordial germ cells (PGC) of Amphibia Anura was carried out after treatment of sections by different fluorescein isothiocyanate conjugated lectins (FITC-lectins). Specific labelling on the PGC is obtained with lectins, the activity of which is inhibited by D-galactose or N-acetyl-galactosamine. These osidic groups appear to be located more specifically on the PGC. The same labelling pattern is not obtained with lectins possessing major affinity for mannose, glucose, fucose and N-acetyl-glucosamine. Furthermore, changes in labelling pattern are observed during migration of PGC. It is suggested that D-galactose and N-acetyl-galactosamine might be related to membrane activity of PGC during migration. Ultrastructural study of the visualization of cell surface carbohydrates supplies some information on the localisation of these lectins receptors.     

Control of neural crest cell dispersion in the trunk of the avian embryo

Many hypotheses have been advanced to explain the orientation and directional migration of neural crest cells. These include positive and negative chemotaxis, haptotaxis, galvanotaxis, and contact inhibition. To test directly the factors that may control the directional dispersion of the neural crest, I have employed a variety of grafting techniques in living embryos. In addition, time-lapse video microscopy has been used to study neural crest cells in tissue culture. Trunk neural crest cells normally disperse from their origin at the dorsal neural tube along two extracellular pathways. One pathway extends laterally between the ectoderm and somites. When either pigmented neural crest cells or neural crest cells isolated from 24-hr cultures are grafted into the space lateral to the somites, they migrate: (1) medially toward the neural tube in the space between the ectoderm and somites and (2) ventrally along intersomitic blood vessels. Once the grafted cells contact the posterior cardinal vein and dorsal aorta they migrate along both blood vessels for several somite lengths in the anterior-posterior axis. Neural crest cells grafted lateral to the somites do not immediately move laterally into the somatic mesoderm of the body wall or the limb. Dispersion of neural crest cells into the mesoderm occurs only after blood vessels and nerves have first invaded, which the grafted cells then follow. The other neural crest pathway extends ventrally alongside the neural tube in the intersomitic space. When neural crest cells were grafted to a ventral position, between the notochord and dorsal aorta, in this intersomitic pathway at the axial level of the last somite, the grafted cells migrate rapidly within 2 hr in two directions: (1) dorsally, in the intersomitic space, until the grafted cells contact the ventrally moving stream of the host neural crest and (2) laterally, along the dorsal aorta and endoderm. All of the above experiments indicate that neither a preestablished chemotactic nor adhesive (haptotactic) gradient exists in the embryo since the grafted neural crest cells will move in the reverse direction along these pathways toward the dorsal neural tube. For the same reason, these experiments also show that dispersal of the neural crest is not directed passively by other environmental controls, since the cells can clearly move counter to their usual pathway and against such putative passive mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Demonstration of neural induction using nuclear markers inXenopus

Summary Two nuclear markers were used to investigate the origin of cells in secondary embryos ofXenopus induced by dorsal lip transplants, and to determine the ability of the chordomesoderm to direct cells to change their fates.³H-thymidine was used to label cells transplanted between individualX. laevis embryos, and nuclear quinacrine fluorescence was used to distinguishX. borealis tissues transplanted toX. laevis hosts. In the first set of experiments, dorsal lip tissue (also known as the dorsal marginal zone; DMZ) was transplanted to the ventral marginal zone (VMZ) of host embryos. The marginal zone is the toroid of presumptive mesodermal cells which involutes during gastrulation. Examination of the secondary embryos resulting from these grafts revealed that their notochords were derived almost exclusively from transplanted cells whereas their nervous systems and somites were composed almost entirely of host cells. Next, the nuclear markers were used to show the normal fates of the tissue of the ventral equatorial region immediately above the VMZ by orthotopic grafting. This tissue was found to give rise to structures in the ventral posterior portions of the tailbud embryo. Finally, the same ventral tissue was labeled and transplanted to the dorsal equatorial region above the DMZ. As a result, it was induced to change its fate and become neural. These results lend unequivocal support to Spemann's theory of neural induction which has recently been questioned.     

Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route.

The behavior of quail primordial germ cells (PGC) after injection into chick embryos by the intravascular route was examined. The quail (donor) PGC, taken from the bloodstream of quail embryos (recipient) at stage 13-14, were injected into the vitelline vessels of chick embryos (recipient) at stage 15. In the recipient embryos, the PGC of the quail and the chick were histochemically distinguished by a double-staining technique involving a lectin, from Wistaria floribunda (WFA) and the PAS reaction. One day after injection, quail PGC appeared in the prospective gonadal region of recipient chick embryos, being localized among the recipient chick PGC. This result indicates that a staining technique specific for WFA lectin is useful for identification of quail PGC and that quail PGC can be transferred by a vascular route for the production of germline chimeras.     

The involvement of cAMP signaling pathway in axis specification in Xenopus embryos

The cAMP signaling system has been postulated to be involved in embryogenesis of many animal species, however, little is known about its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT(1a)R) which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos, ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induced secondary axes but were lacking anterior structures. At the molecular level, 5-HT(1a)R stimulation induced expression of the dorsal mesoderm marker genes, and downregulated expression of the ventral markers but had no effect on expression of the pan mesodermal marker gene in ventral marginal zone explants. In addition, ventral expression and stimulation of the receptor partially restored dorsal axis of UV-irradiated axis deficient embryo. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in patterning early embryos.     

Identification of RALDH-3, a novel retinaldehyde dehydrogenase, expressed in the ventral region of the retina

In the developing retina, a retinoic acid (RA) gradient along the dorso-ventral axis is believed to be a prerequisite for the establishment of dorso-ventral asymmetry. This RA gradient is thought to result from the asymmetrical distribution of RA-generating aldehyde dehydrogenases along the dorso-ventral axis. Here, we identified a novel aldehyde dehydrogenase specifically expressed in the chick ventral retina, using restriction landmark cDNA scanning (RLCS). Since this molecule showed enzymatic activity to produce RA from retinaldehyde, we designated it retinaldehyde dehydrogenase 3 (RALDH-3). Structural similarity suggested that RALDH-3 is the orthologue of human aldehyde dehydrogenase 6. We also isolated RALDH-1 which is expressed in the chick dorsal retina and implicated in RA formation. Raldh-3 was preferentially expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region and then in the ventral retina, earlier than Raldh-1 in chick and mouse embryos. High level expression of Raldh-3 was also observed in the nasal region. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. These results suggested that Raldh-3 is the key enzyme in the formation of an RA gradient along the dorso-ventral axis during the early eye development, and also in the development of the olfactory system.     

Regulation of axon guidance by slit and netrin signaling in the Drosophila ventral nerve cord

Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.     

Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus)

Blood was collected from Stage 13 to 14 (1) chick embryos. Primordial germ cells (PGCs) were separated from blood cells by Ficoll density gradient centrifugation. One hundred Rhode Island Red PGCs per embryo were transferred to the blood stream of Stage 14 to 15 White Leghorn embryos. Also, one hundred White Leghorn PGCs per embryo were transferred to the blood stream of Stage 14 to 15 Rhode Island Red embryos. Hatched male and female chicks were raised until sexual maturity, and progeny tests were performed by mating these PGC recipients with Rhode Island Red chickens of the opposite sex. Chicks apparently derived from the transferred PGCs, based on the feather color of the chicks, were produced from all 4 possible mating combinations. The present results indicate that the germ line of PGC recipient chickens consists of 2 distinct populations of germ cells.     

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro . Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo , suggesting the presenge of other growth factor(s) in vivo . We previously reported that tumor necrosis factor-α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4-m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo . Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo ; however, it did stop afterwards. Such proliferating PGC continue to express c-kit and Oct-3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES-like EG cells.     

Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse

The primordial germ cells (PGCs) of the mouse are derived from proximal epiblast cells that are adjacent to the extraembryonic ectoderm during gastrulation. Previous studies have demonstrated that extraembryonic ectoderm-derived BMP4 and BMP8B are both required for PGC generation. Here we show that Bmp2, a member of the Dpp class of the Bmp superfamily, also plays a role in PGC generation. PGC number is significantly reduced in Bmp2 heterozygous and homozygous embryos at the N2 generation onto C57BL/6 background. Bmp2 homozygous embryos also have a short allantois and about 50% of them do not undergo normal chorioallantoic fusion. Using whole-mount in situ hybridization, we show that Bmp2 is primarily expressed in the endoderm of mouse pregastrula and gastrula embryos. Using a genetic approach, we further show that Bmp2 and Bmp4, but not Bmp2 and Bmp8b, have an additive effect on PGC generation. These results suggest that PGC generation in the mouse embryo is regulated not only by extraembryonic ectoderm-derived BMP4 and BMP8B, but also by endoderm-derived BMP2.     

The mechanism for primordial germ-cell migration is conserved between Japanese eel and zebrafish

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).     

Presence and roles of calcium gradients along the dorsal-ventral axis in Drosophila embryos

Dorsal-ventral specification of the Drosophila embryo is mediated by signaling pathways which have been very well described in genetic terms. However, little is known about the physiology of Drosophila development. By imaging patterns of free cytosolic calcium in Drosophila embryos, we found that several calcium gradients are generated along the dorsal-ventral axis. The most pronounced gradient is formed during stage 5, in which calcium levels are high dorsally. Manipulation of the stage 5 calcium gradient affects specification of the amnioserosa, the dorsal-most region of the embryo. We further show that this calcium gradient is inhibited in pipe, Toll, and dorsal mutants, but is unaltered in decapentaplegic (dpp) or punt mutants, suggesting that the stage 5 calcium gradient is formed by a suppression of ventral calcium concentrations. We conclude that calcium plays a role in specification of the dorsal embryonic region.     

Effects of the removal of neural crest anlage upon endodermal morphogenesis and primordial germ cells migration in toad embryo

Summary The anlagen of neural tube or neural tube and neural crests were removed from toad embryos at the early neurula stage. The removal of the neural tube anlage does not affects the normal development of embryos. The removal of neural tube plus neural crest anlagen results in major disturbances of both endodermal morphogenesis and primordial germ cell migration. The possible indirect influence of neural crest cells upon the migration of the primordial germ cells is discussed. The neural crests cells could be involved in the formation and/or release of an attractive morphogen from embryonic chordomesoderm responsible for the migration of the primordial germ cells.     

Inductive activity and enduring cellular constitution of a supernumerary apical ectodermal ridge grafted to the limb bud of the chick embryo.

Apical ectodermal ridges (AERs) isolated from 3- to 4-day chick and quail embryos were prepared by means of trypsinization and microdissection and then were grafted to the dorsal or ventral side of a host chick wing bud. They induced supernumerary limb outgrowths from the host bud showing, respectively, a bidorsal or biventral organization, as determined by the patterns of feather germs. The grafted ridge cells persisted, as revealed by histological sections of supernumerary chick limb parts growing under the influence of quail AERs, whose cells are readily distinguished after application of the Feulgen reagent.These results show that the AER induces limb outgrowth regardless of whether it is associated with dorsal or ventral limb ectoderm and that its continued existence is not dependent on contributions of ectodermal cells from the opposed ectodermal faces of the limb bud. The AER is pictured as maintaining the subjacent mesoderm in a condition of developmental plasticity without specifying its differentiation with respect to the proximodistal axis. It remains uncertain whether the positional values of cells that develop under the influence of the AER arise within these cells themselves or appear in response to influences from proximal sources.