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PubMed2003

Patterning the limb before and after SHH signalling.

The vertebrate limb is one of the most relevant experimental models for analysing cell-cell signalling during patterning of embryonic fields and organogenesis. Recently, the combination of molecular and genetic studies with experimental manipulation of developing limb buds has significantly advanced our understanding of the complex molecular interactions co-ordinating limb bud outgrowth and patterning. Some of these studies have shown that there is a need to revise some of the textbook views of vertebrate limb development. In this review, we discuss how signalling by the polarizing region is established and how limb bud morphogenesis is controlled by both long-range and signal relay mechanisms. We also discuss recent results showing that differential mesenchymal responsiveness to SHH signalling is established prior to its expression by the polarizing region.

Animals↗
PubMed1996

A contact-dependent animal-to-vegetal signal biases neural lineages during Xenopus cleavage stages.

The central nervous system (CNS) of Xenopus is derived from three of four tiers of blastomeres of the 32-cell embryo, and each blastomere in these tiers produces a characteristic number of primary spinal neurons. The C-tier blastomeres constitute the boundary between those that contribute to the CNS (A-, B-, and C-tiers) and those that do not (D-tier). To test whether the neural lineages descended from the C-tier are established by intrinsic information or by cell-cell interactions, single B-tier blastomeres were deleted and the lineage of their C-tier neighbors mapped. The contributions of C-tier blastomeres to subdivisions of the CNS and to specific spinal neurons were significantly reduced. Contributions of these blastomeres to other tissues were mostly normal, indicating that those C-tier progeny that no longer contribute to CNS are distributed in small numbers throughout the rest of the clone. To test whether the changes in neural lineages after B-tier deletions were the result of the C-tier blastomere changing position, contacting new neighbors, or losing contact with inductive B-tier neighbors, intact embryos were transiently dissociated within their vitelline membranes at different time points prior to the midblastula transition. This treatment disrupted cell-cell contact, but not gap junction-mediated dye coupling or the positions of neighboring cells. C-tier CNS lineages were reduced as after deletion of the B-tier neighbor, suggesting that the neural fate of C-tier cells depends upon specific B-tier interactions. To determine whether these interactions occurred specifically between B-tier and C-tier neighbors, barriers were inserted transiently between individual B/C pairs; similar reductions in C-tier CNS lineages were observed. These data demonstrate that an animal-to-vegetal, contact-dependent signal passes from B-tier to C-tier blastomeres and is required for the normal C-tier contribution to the CNS. This cell-cell interaction occurs many hours before the onset of zygotic transcription or neural induction and may bias the field of cells that can respond to neural induction.

Animals↗
PubMed2005

Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction.

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, we have analyzed the role of the transcription factors Msx1 and Pax3. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, we show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction.

Animals↗
PubMed2003

A novel inducible element, activated by contact with Rathke's pouch, is present in the regulatory region of the Rpx/Hesx1 homeobox gene.

Reciprocal inductive interactions are postulated to play a role in the determination and differentiation of the pituitary gland and the ventral hypothalamus. The homeobox gene Rpx/Hesxl is expressed during gastrulation in the anterior endoderm, prechordal plate, and the prospective cephalic neural plate, and at later stages of development in Rathke's pouch, the primordium of the pituitary. We have defined the regulatory elements necessary for proper spatial and temporal expression during development in transgenic mice using lacZ reporter genes. Proper spatial and temporal expression in the anterior endoderm prechordal plate and anterior neural plate can be recapitulated with as little as 568 bp of upstream sequence and intragenic sequence containing the first exon and intron. Late-stage expression in Rathke's pouch requires additional negative and positive regulatory elements. Interestingly, deletion analysis uncovered an element that directs transgene expression to a region of the hypothalamus that lies in direct contact with Rathke's pouch. In vitro tissue recombination experiments have established that this expression is induced by contact with the pouch. We propose that this element may be present in other genes that normally respond to signals emanating from the pouch during the development of the hypothalamic-pituitary axis. The Rpx-lacZ transgenic mice provide a novel model system for the molecular dissection of inductive cell signaling during pituitary development.

Animals↗
PubMed2002

Interaction of the IP(3)-Ca(2+) and the FGF-MAPK signaling pathways in the Xenopus laevis embryo: a qualitative approach to the mesodermal induction problem.

In this work we propose that the animal-vegetal gradient spatial distribution of the IP(3) receptors observed in the Xenopus embryo can effect a uniform FGF inducting input signal, allowing for different modes of transcription of the Xbra gene, producing the differentiation of the cells of the marginal zone. We analyze this hypothesis with a model for the interaction of the calcium signaling system with the MAPK cascade during the FGF mesodermal induction process, consisting of five non-linear coupled differential equations. A numerical treatment of a one- and two-cell system shows that the calcium flux between cells enhances the Raf activity levels, leading to oscillatory behavior. This qualitative result may be of consequence for the expression of the ventralizing characteristics of the FGF inducting signal.

Animals↗
PubMed1998

Neural crest induction in Xenopus: evidence for a two-signal model.

We have investigated the molecular interactions underlying neural crest formation in Xenopus. Using chordin overexpression to antagonize endogenous BMP signaling in whole embryos and explants, we demonstrate that such inhibition alone is insufficient to account for neural crest induction in vivo. We find, however, that chordin-induced neural plate tissue can be induced to adopt neural crest fates by members of the FGF and Wnt families, growth factors that have previously been shown to posteriorize induced neural tissue. Overexpression of a dominant negative XWnt-8 inhibits the expression of neural crest markers, demonstrating the necessity for a Wnt signal during neural crest induction in vivo. The requirement for Wnt signaling during neural crest induction is shown to be direct, whereas FGF-mediated neural crest induction may be mediated by Wnt signals. Overexpression of the zinc finger transcription factor Slug, one of the earliest markers of neural crest formation, is insufficient for neural crest induction. Slug-expressing ectoderm will generate neural crest in the presence of Wnt or FGF-like signals, however, bypassing the need for BMP inhibition in this process. A two-step model for neural crest induction is proposed.

Animals↗
PubMed1999

Amphibian embryos as a model system for organ engineering: in vitro induction and rescue of the heart anlage.

Beating hearts can be induced under in vitro conditions when the dorsal blastopore lip (including the zone of Spemann organizer) is treated with Suramin. In contrast, untreated organizer forms dorsal mesodermal derivatives as notochord and somites. When those in vitro produced heart precursor tissues are transplanted ectopically in the posterior trunk area of early larvae, secondary beating heart structures will be formed. Furthermore, the replacement of the heart primordium of the host embryo by heart tissue induced under in vitro conditions will result in the rescue of the heart anlage. This model could be a valuable tool for the study of the multi-step molecular mechanisms of heart structure induction under in vitro conditions and vasculogenesis after transplantation into the host embryo.

Animals↗
PubMed1985

Inductive interactions in early amphibian development and their general nature.

After a short discussion on cell interactions in general and inductive interactions in particular, the almost completely epigenetic nature of amphibian development is emphasized. In the symmetrized egg undergoing cleavage a large-scale inductive interaction occurs which leads to the formation of the meso-endoderm. Meso-endoderm formation gives rise to the morphogenic process of gastrulation. In the ensuing triple-layered embryo inductive interactions are strongly enhanced. The following large-scale inductive interaction leads to the formation of the neural anlage. This is again followed by the morphogenetic process of neurulation or neural tube formation. Subsequent interactions between the germ layers of the triple-layered embryo give rise to the formation of the regional pattern of organ anlagen. Finally, the most promising approaches to the nature of inductive interactions for mesoderm and endoderm formation are discussed.

Amphibians↗
PubMed1978

Metabolic inhibitors and kidney tubule induction.

The induction of kidney tubules in metanephric mesenchyme has previously been shown to require close contact between the interacting tissues. In our study we show that low concentrations of inhibitors of RNA, DNA and protein synthesis inhibit tubule induction, although they do not seem to prevent the formation of contacts between the interacting tissues. The effective concentrations were about the same as those which inhibited the synthesis of macromolecules. Cycloheximide caused an increased synthesis of RNA. Low concentrations of Mitomycin C inhibited DNA synthesis but not tubule formation. A concentration of the inhibitors which caused weakened induction also caused a marked decrease in leucine incorporation. We concluded therefore, that a decrease in protein synthesis in the inducing tissue is responsible for the inhibition of induction.

Animals↗
PubMed1983

Nature of the hypoblastic influence on the chick embryo epiblast.

Stage XIII chick blastoderms deprived of the marginal zone, the area opaca and the posterior half of the hypoblast, when incubated further developed axes whose orientation in 50% of the cases was according to the original blastoderm's orientation, whilst in 50% of the cases they developed at 90 degrees from the posterior side. Those results illustrate the quantitative differences in inductivity between the anterior and the posterior hypoblastic halves. Normally the posterior region has the highest effect but other regions can also bring about the development of an embryonic axis if allowed to act upon the epiblast for a sufficiently long period of time. The possible ways in which a chick hypoblast influences the epiblast to develop an embryo are examined in the light of recent findings and of new experiments described below.

Animals↗
PubMed2001

Arkadia enhances nodal-related signalling to induce mesendoderm.

Nodal-related members of the transforming growth factor (TGF)-beta family regulate the induction of mesoderm, endoderm, and mesendoderm, a tissue specific to the Spemann organizer. How these different tissues form in response to the same signalling molecules is not completely understood. It has been suggested that concentration-dependent effects, mediated by extracellular cofactors and antagonists, are responsible for the differences. Here we show that the nuclear protein Arkadia specifically potentiates the mesendoderm-inducing activity of a subset of TGF-beta family members. The combined activities of Arkadia and Xenopus nodal-related-1 are sufficient to induce mesendoderm and suppress mesoderm. Arkadia dorsalizes ventral tissues, resulting in the induction of organizer-specific gene expression. Blocking nodal signalling extracellularly inhibits these effects. Arkadia influences nodal activity when co-expressed and can function in cells adjacent to those producing the nodal signal. Our findings, together with the observation that Arkadia mutant mice lack a node and node-derived mesendoderm, identify Arkadia as an essential modulator of the nodal signalling cascade that leads to induction of Spemann's organizer.

Animals↗
PubMed1995

Identification of inducing, responding, and suppressing regions in an experimental model of notochord formation in avian embryos.

The notochord normally arises from committed cells in the rostral tip of the primitive streak. After removal of these cells from the avian gastrula, embryos with notochords nevertheless develop in the majority of cases. A region required for the formation of this reconstituted notochord lies lateral to the primitive streak. In the present study we have determined that this region acts as an inducer for more lateral cells in the epiblast, which actually give rise to the reconstituted notochord. The strongest inducing region lies between 0-250 micrometer lateral to the streak and 500-750 micrometer caudal to the rostral end of the streak and chiefly contains cells normally fated to form lateral plate and somitic mesoderm. The responding region is located 250-500 micrometer lateral to the streak and 0-750 micrometer caudal to the rostral end of the streak. This area chiefly contains cells normally fated to form neural ectoderm, although cells normally fated to form lateral plate and somitic mesoderm are also within this area. The inducing and responding areas interact to form reconstituted notochord either when the primitive streak, including its rostral end (Hensen's node), is removed from the cultured blastoderm or when the inducer and responder are grafted together into an ectopic site. Grafting Hensen's node into isolates containing both inducer and responder blocks formation of reconstituted notochord, suggesting that Hensen's node suppresses formation of lateral notochords during normal development. These findings increase our understanding of the early interactions between mesoderm and ectoderm and provide a novel model system that is well defined and accessible for studying inductive events in higher vertebrates.

Animals↗
PubMed2001

Boundaries and functional domains in the animal/vegetal axis of Xenopus gastrula mesoderm.

Patterning of the Xenopus gastrula marginal zone in the axis running equatorially from the Spemann organizer-the so--called "dorsal/ventral axis"--has been extensively studied. It is now evident that patterning in the animal/vegetal axis also needs to be taken into consideration. We have shown that an animal/vegetal pattern is apparent in the marginal zone by midgastrulation in the polarized expression domains of Xenopus brachyury (Xbra) and Xenopus nodal-related factor 2 (Xnr2). In this report, we have followed cells expressing Xbra in the presumptive trunk and tail at the gastrula stage, and find that they fate to presumptive somite, but not to ventrolateral mesoderm of the tailbud embryo. From this, we speculate that the boundary between the Xbra- and Xnr2-expressing cells at gastrula corresponds to a future tissue boundary. In further experiments, we show that the level of mitogen-activated protein kinase (MAPK) activation is polarized along the animal/vegetal axis, with the Xnr2-expressing cells in the vegetal marginal zone having no detectable activated MAPK. We show that inhibition of MAPK activation in Xenopus animal caps results in the conversion of Xnr2 from a dorsal mesoderm inducer to a ventral mesoderm inducer, supporting a role for Xnr2 in induction of ventral mesoderm.

Actins↗