Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.     

BMP regulates vegetal pole induction centres in early xenopus development

BACKGROUND: Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. RESULTS: The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. CONCLUSIONS: These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

FGF is a prospective competence factor for early activin-type signals in Xenopus mesoderm induction

Normal pattern formation during embryonic development requires the regulation of cellular competence to respond to inductive signals. In the Xenopus blastula, vegetal cells release mesoderm-inducing factors but themselves become endoderm, suggesting that vegetal cells may be prevented from expressing mesodermal genes in response to the signals that they secrete. We show here that addition of low levels of basic fibroblast growth factor (bFGF) induces the ectopic expression of the mesodermal markers Xbra, MyoD and muscle actin in vegetal explants, even though vegetal cells express low levels of the FGF receptor. Activin, a potent mesoderm-inducing agent in explanted ectoderm (animal explants), does not induce ectopic expression of these markers in vegetal explants. However, activin-type signaling is present in vegetal cells, since the vegetal expression of Mix.1 and goosecoid is inhibited by the truncated activin receptor. These results, together with the observation that FGF is required for mesoderm induction by activin, support our proposal that a maternal FGF acts at the equator as a competence factor, permitting equatorial cells to express mesoderm in response to an activin-type signal. The overlap of FGF and activin-type signaling is proposed to restrict mesoderm to the equatorial region.     

Continuing organizer function during chick tail development

Development of the posterior body (lumbosacral region and tail) in vertebrates is delayed relative to gastrulation. In amniotes, it proceeds with the replacement of the regressed node and primitive streak by a caudal blastema-like mass of mesenchyme known as the tail bud. Despite apparent morphological dissimilarities, recent results suggest that tail development in amniotes is in essence a continuation of gastrulation, as is the case in Xenopus. However, this has been inferred primarily from the outcome of fate mapping studies demonstrating discrete, regionalized cell populations in the tail bud, like those present at gastrulation. Our analysis of the tail bud distribution of several molecular markers that are expressed in specific spatial domains during chick gastrulation confirms these results. Furthermore, we present evidence that gastrulation-like ingression movements from the surface continue in the early chick tail bud and that the established tail bud retains organizer activity. This 'tail organizer' has the expected properties of being able to recruit uncommitted host cells into a new embryonic axis and induce host neural tissue with posteriorly regionalized gene expression when grafted to competent host cells that are otherwise destined to form only extra-embryonic tissue. Together, these results indicate that chick tail development is mechanistically continuous with gastrulation and that the developing tail in chick may serve as a useful experimental adjunct to investigate the molecular basis of inductive interactions operating during gastrulation, considering that residual tail organizing activity is still present at a surprisingly late stage.     

Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos

The cadherin-catenin complex has an important role in cell-cell adhesion and may also function in signaling pathways. We report that overexpression of three cadherin types in Xenopus embryos causes them to develop with reduced dorsal axial structures. The same phenotype is produced in embryos that have been depleted of maternal beta-catenin protein by an antisense oligodeoxynucleotide complementary to beta-catenin mRNA. They show an inhibition in the expression of dorsal mesodermal markers MyoD and goosecoid, but not of ventral and general mesodermal markers. They lack notochords, somites, and neural tubes and are defective in dorsal mesodermal signaling in Nieuwkoop assays. The phenotype can be rescued by the injection of beta-catenin mRNA and not by the injection of Xwnt-8 mRNA. These results show that beta-catenin has an important role in dorsal mesoderm induction. They directly demonstrate the activity of a maternal mRNA in axis specification.     

A posteriorising factor, retinoic acid, reveals that anteroposterior patterning controls the timing of neuronal differentiation in Xenopus neuroectoderm

During early development of the Xenopus central nervous system (CNS), neuronal differentiation can be detected posteriorly at neural plate stages but is delayed anteriorly until after neural tube closure. A similar delay in neuronal differentiation also occurs in the anterior neural tissue that forms in vitro when isolated ectoderm is treated with the neural inducer noggin. Here we examine the factors that control the timing of neuronal differentiation both in embryos and in neural tissue induced by noggin (noggin caps). We show that the delay in neuronal differentiation that occurs in noggin caps cannot be overcome by inhibiting the activity of the neurogenic gene, X-Delta-1, which normally inhibits neuronal differentiation, suggesting that it represents a novel level of regulation. Conversely, we show that the timing of neuronal differentiation can be changed from late to early after treating noggin caps or embryos with retinoic acid (RA), a putative posteriorising agent. Concommittal with changes in the timing of neuronal differentiation, RA suppresses the expression of anterior neural genes and promotes the expression of posterior neural genes. The level of early neuronal differentiation induced by RA alone is greatly increased by the additional expression of the proneural gene, XASH3. These results indicate that early neuronal differentiation in neuralised ectoderm requires posteriorising signals, as well as signals that promote the activity of proneural genes such as XASH3. In addition, these result suggest that neuronal differentiation is controlled by anteroposterior (A-P) patterning, which exerts a temporal control on the onset of neuronal differentiation.     

Cyclin D2 arrests Xenopus early embryonic cell cycles

Xenopus cyclin D2 mRNA is a member of the class of maternal RNAs. It is rare and stable during early embryonic development. To investigate the potential role of cyclin D2 during early embryonic cell cycles, cyclin D2 was injected into one blastomere of a two-cell embryo. This injection induced a cell cycle arrest in the injected blastomere. To analyze more precisely the mechanism of this arrest, we took advantage of cycling egg extracts that recapitulate major events of the cell cycle when supplemented with demembranated sperm heads. When Xenopus cyclin D2 is added to egg extracts, the first round of DNA replication occurs as in control extracts. However, Xenopus cyclin D2 blocks subsequent rounds of DNA replication and the oscillations of histone H1 kinase activity associated with cdc2 kinase, indicating that the cell cycle is arrested after the first S-phase. The block induced by Xenopus cyclin D2 is not due to a lack of the mitotic cyclin B2 that accumulates normally. Radiolabeled Xenopus cyclin D2 enters nuclei after completion of the first S-phase and remains stable over the entire period of the arrest. These features suggest that Xenopus cyclin D2 could play an original role during early development, controlling the G2-phase and/or the G2/M transition.     

Localized maternal proteins in Xenopus revealed by subtractive immunization

It has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in the Xenopus oocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within the Xenopus egg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning.     

The incidence of HIV infection among women using family planning methods in Dar es Salaam, Tanzania

Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH4Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH4Cl, before or after NH4Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers.     

Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on effector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. We present evidence that dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-specific gene induction by eFGF in Xenopus animal cap explants. We also show that dominant-negative mutants of Grb2 and Nck can inhibit eFGF-induced Erk1 activation in Xenopus animal caps, and that targeting the first two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Furthermore, combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibited Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in different cells.     

The role of pulmonary surfactant in obstructive airways disease

The Spemann organizer is largely responsible for organizing and patterning the anteroposterior axis during the development of amphibians. In this report, we examine the degree of anteroposterior pattern in the earliest gastrula organizer of Xenopus using a combination of embryological and molecular techniques. When we divide the earliest gastrula organizer, a region measuring 20 cells high by 25 cells wide, into stereotyped anterior (vegetal) and posterior (animal) halves, each half not only has a distinct fate and state of specification, but also induces a unique set of region-specific neural genes. When wrapped in animal cap ectoderm, the anterior half induces only anterior-specific genes (XAG-1 and otxA), while the posterior half induces anterior (otxA and reduced levels of XAG-1) and posterior (Hox B9) neural genes, revealing early localization of neural posteriorizing activity to posterior mesendoderm. This is the earliest demonstration of regionalized neural induction by the Xenopus organizer. Additionally, based on the expression of gsc, Xbra, and Xnot, we show that the organizer is patterned both at the early gastrula stage and prior to the appearance of bottle cells.     

Functional Rescue of photoreceptors from the damaging effects of constant light by survival-promoting factors in the rat

PURPOSE: To investigate whether and how survival-promoting agents rescue photoreceptor cell function and morphology from constant light damage, the authors recorded electroretinographic (ERG) responses and examined light micrographs of retinas in those rats given intravitreal injection of midkine (MK) and basic fibroblast growth factor (bFGF) before constant exposure. METHODS: Albino Sprague-Dawley rats were injected with MK, bFGF, or phosphate-buffered saline (PBS) 2 days before the onset of 1 week of constant light. ERG responses were recorded using white flash stimuli with the intensity range of 4 log units, followed by histologic examinations of retinas, including quantitative assessment of the outer nuclear layer thickness as an index of photoreceptor cell loss. RESULTS: ERG responses were barely detectable in uninjected eyes after 1 week of constant light. On the other hand, distinct responses were recordable in eyes injected intravitreally with MK and bFGF, and the degree of ERG rescue in terms of the amplitude of b-wave was approximately 40% to 60% compared with normal eyes. Intravitreally injected PBS showed slight, but noticeable, preservation of ERG responses as well. Histologic examination revealed that MK and bFGF protected photoreceptors from light damage. A good correlation was found between anatomic rescue of photoreceptors as assessed by outer nuclear layer thickness and the functional rescue as defined by the magnitude of ERG responses. CONCLUSIONS: Functional and anatomic rescue of photoreceptors in albino rats from constant light damage is achieved by prior intravitreal injection of MK and bFGF.