Specification Of Anteroposterior Axis Research Articles

Rspo2 inhibits TCF3 phosphorylation to antagonize Wnt signaling during vertebrate anteroposterior axis specification

The Wnt pathway activates target genes by controlling the β-catenin-T-cell factor (TCF) transcriptional complex during embryonic development and cancer. This pathway can be potentiated by R-spondins, a family of proteins that bind RNF43/ZNRF3 E3 ubiquitin ligases and LGR4/5 receptors to prevent Frizzled degradation. Here we demonstrate that, during Xenopus anteroposterior axis specification, Rspo2 functions as a Wnt antagonist, both morphologically and at the level of gene targets and pathway mediators. Unexpectedly, the binding to RNF43/ZNRF3 and LGR4/5 was not required for the Wnt inhibitory activity. Moreover, Rspo2 did not influence Dishevelled phosphorylation in response to Wnt ligands, suggesting that Frizzled activity is not affected. Further analysis indicated that the Wnt antagonism is due to the inhibitory effect of Rspo2 on TCF3/TCF7L1 phosphorylation that normally leads to target gene activation. Consistent with this mechanism, Rspo2 anteriorizing activity has been rescued in TCF3-depleted embryos. These observations suggest that Rspo2 is a context-specific regulator of TCF3 phosphorylation and Wnt signaling.

The dynamics of morphogenesis in stem cell-based embryology: Novel insights for symmetry breaking

Breaking embryonic symmetry is an essential prerequisite to shape the initially symmetric embryo into a highly organized body plan that serves as the blueprint of the adult organism. This critical process is driven by morphogen signaling gradients that instruct anteroposterior axis specification. Despite its fundamental importance, what triggers symmetry breaking and how the signaling gradients are established in time and space in the mammalian embryo remain largely unknown. Stem cell-based in vitro models of embryogenesis offer an unprecedented opportunity to quantitatively dissect the multiple physical and molecular processes that shape the mammalian embryo. Here we review biochemical mechanisms governing early mammalian patterning in vivo and highlight recent advances to recreate this in vitro using stem cells. We discuss how the novel insights from these model systems extend previously proposed concepts to illuminate the extent to which embryonic cells have the intrinsic capability to generate specific, reproducible patterns during embryogenesis.

Specification of anteroposterior axis by combinatorial signaling during Xenopus development.

The specification of anteroposterior (AP) axis is a fundamental and complex patterning process that sets up the embryonic polarity and shapes a multicellular organism. This process involves the integration of distinct signaling pathways to coordinate temporal-spatial gene expression and morphogenetic movements. In the frog Xenopus, extensive embryological and molecular studies have provided major advance in understanding the mechanism implicated in AP patterning. Following fertilization, cortical rotation leads to the transport of maternal determinants to the dorsal region and creates the primary dorsoventral (DV) asymmetry. The activation of maternal Wnt/ß-catenin signaling and a high Nodal signal induces the formation of the Nieuwkoop center in the dorsal-vegetal cells, which then triggers the formation of the Spemann organizer in the overlying dorsal marginal zone. It is now well established that the Spemann organizer plays a central role in building the vertebrate body axes because it provides patterning information for both DV and AP polarities. The antagonistic interactions between signals secreted in the Spemann organizer and the opposite ventral region pattern the mesoderm along the DV axis, and this DV information is translated into AP positional values during gastrulation. The formation of anterior neural tissue requires simultaneous inhibition of zygotic Wnt and bone morphogenetic protein (BMP) signals, while an endogenous gradient of Wnt, fibroblast growth factors (FGFs), retinoic acid (RA) signaling, and collinearly expressed Hox genes patterns the trunk and posterior regions. Collectively, DV asymmetry is mostly coupled to AP polarity, and cell-cell interactions mediated essentially by the same regulatory networks operate in DV and AP patterning. For further resources related to this article, please visit the WIREs website.

Ezh2 regulates anteroposterior axis specification and proximodistal axis elongation in the developing limb

Specification and determination (commitment) of positional identities precedes overt pattern formation during development. In the limb bud, it is clear that the anteroposterior axis is specified at a very early stage and is prepatterned by the mutually antagonistic interaction between Gli3 and Hand2. There is also evidence that the proximodistal axis is specified early and determined progressively. Little is known about upstream regulators of these processes or how epigenetic modifiers influence axis formation. Using conditional mutagenesis at different time points, we show that the histone methyltransferase Ezh2 is an upstream regulator of anteroposterior prepattern at an early stage. Mutants exhibit posteriorised limb bud identity. During later limb bud stages, Ezh2 is essential for cell survival and proximodistal segment elongation. Ezh2 maintains the late phase of Hox gene expression and cell transposition experiments suggest that it regulates the plasticity with which cells respond to instructive positional cues.

Organizing the DV axis during planarian regeneration

During regeneration, lost structures are rebuilt and perfectly integrated within the remaining non-injured tissues. This fascinating process captured the attention of one of the founders of modern genetics, T. H. Morgan. He was particularly interested in understanding regeneration in freshwater planarians, which can regenerate a whole animal from a small piece of their bodies. He performed numerous experiments to understand how polarity is re-established such that an anterior-facing wound regenerates a head whereas a posterior-facing wound regenerates a tail. However, it has not been until more than 100 years later that the molecules required to determine axial polarity have been identified. Several studies have now shown that the Wnt/β-catenin and Hedgehog pathways are required for anteroposterior axis specification, whereas the establishment of the planarian dorsoventral (DV) axis relies on the Bone Morphogenetic Protein (BMP) pathway. Two recent papers have now uncovered additional conserved (anti-dorsalizing morphogenetic protein) and novel (noggin-like genes) elements that regulate planarian DV axis regeneration. Here, we summarize those results and present new data and hypotheses to explain the role that noggin-like genes might play.

Organizing the DV axis during planarian regeneration.

During regeneration, lost structures are rebuilt and perfectly integrated within the remaining non-injured tissues. This fascinating process captured the attention of one of the founders of modern genetics, T.H. Morgan. He was particularly interested in understanding regeneration in freshwater planarians, which can regenerate a whole animal from a small piece of their bodies. He performed numerous experiments to understand how polarity is re-established such that an anterior-facing wound regenerates a head whereas a posterior-facing wound regenerates a tail. However, it has not been until more than 100 years later that the molecules required to determine axial polarity have been identified. Several studies have now shown that the Wnt/β-catenin and Hedgehog pathways are required for anteroposterior axis specification, whereas the establishment of the planarian dorsoventral (DV) axis relies on the Bone Morphogenetic Protein (BMP) pathway. Two recent papers have now uncovered additional conserved (anti-dorsalizing morphogenetic protein) and novel (noggin-like genes) elements that regulate planarian DV axis regeneration. Here, we summarize those results and present new data and hypotheses to explain the role that noggin-like genes might play.

Dishevelled is essential for neural connectivity and planar cell polarity in planarians

The Wingless/Integrated (Wnt) signaling pathway controls multiple events during development and homeostasis. It comprises multiple branches, mainly classified according to their dependence on β-catenin activation. The Wnt/β-catenin branch is essential for the establishment of the embryonic anteroposterior (AP) body axis throughout the phylogenetic tree. It is also required for AP axis establishment during planarian regeneration. Wnt/β-catenin-independent signaling encompasses several different pathways, of which the most extensively studied is the planar cell polarity (PCP) pathway, which is responsible for planar polarization of cell structures within an epithelial sheet. Dishevelled (Dvl) is the hub of Wnt signaling because it regulates and channels the Wnt signal into every branch. Here, we analyze the role of Schmidtea mediterranea Dvl homologs (Smed-dvl-1 and Smed-dvl-2) using gene silencing. We demonstrate that in addition to a role in AP axis specification, planarian Dvls are involved in at least two different β-catenin-independent processes. First, they are essential for neural connectivity through Smed-wnt5 signaling. Second, Smed-dvl-2, together with the S. mediterranea homologs of Van-Gogh (Vang) and Diversin (Div), is required for apical positioning of the basal bodies of epithelial cells. These data represent evidence not only of the function of the PCP network in lophotrocozoans but of the involvement of the PCP core elements Vang and Div in apical positioning of the cilia.

Regulation of TCF3 by Wnt-Dependent Phosphorylation during Vertebrate Axis Specification

A commonly accepted model of Wnt/β-catenin signaling involves target gene activation bya complex of β-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves β-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative invivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.

Evidence for a composite anterior determinant in the hover flyEpisyrphus balteatus(Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

Most insect embryos develop from a monolayer of cells around the yolk, but only part of this blastoderm forms the embryonic rudiment. Another part forms extra-embryonic serosa. Size and position of the serosa anlage vary between species, and previous work raises the issue of whether such differences co-evolve with the mechanisms that establish anteroposterior (AP) polarity of the embryo. AP polarity of the Drosophila embryo depends on bicoid, which is necessary and sufficient to determine the anterior body plan. Orthologs of bicoid have been identified in various cyclorrhaphan flies and their occurrence seems to correlate with a mid-dorsal serosa or amnioserosa anlage. Here, we introduce with Episyrphus balteatus (Syrphidae) a cyclorrhaphan model for embryonic AP axis specification that features an anterodorsal serosa anlage. Current phylogenies place Episyrphus within the clade that uses bicoid mRNA as anterior determinant, but no bicoid-like sequence could be identified in this species. Using RNA interference (RNAi) and ectopic mRNA injection, we obtained evidence that pattern formation along the entire AP axis of the Episyrphus embryo relies heavily on the precise regulation of caudal, and that anterior pattern formation in particular depends on two localized factors rather than one. Early zygotic activation of orthodenticle is separated from anterior repression of caudal, two distinct functions which in Drosophila are performed jointly by bicoid, whereas hunchback appears to be regulated by both factors. Furthermore, we found that overexpression of orthodenticle is sufficient to confine the serosa anlage of Episyrphus to dorsal blastoderm. We discuss our findings in a phylogenetic context and propose that Episyrphus employs a primitive cyclorrhaphan mechanism of AP axis specification.

Silencing ofSmed-βcatenin1generates radial-like hypercephalized planarians

Little is known about the molecular mechanisms responsible for axis establishment during non-embryonic processes such as regeneration and homeostasis. To address this issue, we set out to analyze the role of the canonical Wnt pathway in planarians, flatworms renowned for their extraordinary morphological plasticity. Canonical Wnt signalling is an evolutionarily conserved mechanism to confer polarity during embryonic development, specifying the anteroposterior (AP) axis in most bilaterians and the dorsoventral (DV) axis in early vertebrate embryos. beta-Catenin is a key element in this pathway, although it is a bifunctional protein that is also involved in cell-cell adhesion. Here, we report the characterization of two beta-catenin homologs from Schmidtea mediterranea (Smed-betacatenin1/2). Loss of function of Smed-betacatenin1, but not Smed-betacatenin2, in both regenerating and intact planarians, generates radial-like hypercephalized planarians in which the AP axis disappears but the DV axis remains unaffected, representing a unique example of a striking body symmetry transformation. The radial-like hypercephalized phenotype demonstrates the requirement for Smed-betacatenin1 in AP axis re-establishment and maintenance, and supports a conserved role for canonical Wnt signalling in AP axis specification, whereas the role of beta-catenin in DV axis establishment would be a vertebrate innovation. When considered alongside the protein domains present in each S. mediterranea beta-catenin and the results of functional assays in Xenopus embryos demonstrating nuclear accumulation and axis induction with Smed-betacatenin1, but not Smed-betacatenin2, these data suggest that S. mediterranea beta-catenins could be functionally specialized and that only Smed-betacatenin1 is involved in Wnt signalling.

Fushi tarazu: a Hox gene changes its role.

The Hox genes play a role in anteroposterior axis specification of bilaterian animals that has been conserved for more than 600 million years. However, some of these genes have occasionally changed their roles in evolution. For example, the insect gene fushi tarazu (ftz), although localised in the Hox cluster, no longer acts as a Hox gene, but is involved in segmentation and nervous system development. Recent data of Mouchel-Vielh et al., and Hughes and Kaufman on ftz homologues in a crustacean and a myriapod, respectively, shed new light onto the evolution of this gene.

The initial phase of embryonic patterning in mammals

Although specification of the antero-posterior axis is a critical intial step in development of the fetus, it is not known either how, or at what stage in development, this process begins. Such information is vital for understanding not only normal development in mammals but also monozygotic twinning, which, at least in man, is associated with a significantly increased incidence of birth defects. According to recent studies in the mouse, specification of the fetal anteroposterior axis begins well before gastrulation, and probably even before the conceptus implants. Moreover, evidence is accruing that the origin of relevant asymmetries depends on information that is already present in the zygote before it embarks on cleavage. Hence, early development in mammals does not differ as markedly from that in other animals as has generally been assumed. Consequently, at present, the possibility of adverse effects of techniques used to assist human reproduction cannot be disregarded.

Role of the anterior visceral endoderm in restricting posterior signals in the mouse embryo.

Recent genetic and embryological experiments have demonstrated that head formation in the mouse embryo is dependent on signals provided by two organising centers during gastrulation, the anterior visceral endoderm (AVE) and the anterior primitive streak (also called the Early Gastrula Organiser, EGO). However the molecular nature of the signals triggering anterior neural formation from the epiblast is not clearly understood. The analysis of mouse mutants has allowed the identification of some of the molecular players involved in the process of head formation. In this review, we describe different mutant embryos in which impairment of visceral endoderm function leads to similar defects in antero-posterior axis specification. These phenotypes are consistent with a role of the AVE in protecting anterior embryonic regions from signals that promote posterior development. We propose that a genetic cascade in the AVE, involving HNF3beta, Lim1, Otx2, Smad2 and ActRIB, leads to the production of secreted TGFbeta antagonists that protect the anterior epiblast region from Nodal signalling.

The Early Expression Control of Xepsin by Nonaxial and Planar Posteriorizing Signals in Xenopus Epidermis

The control mechanism of the anteroposterior axis specification in Xenopus epidermis was studied by comparing the expression of a novel anterior marker, Xepsin, with that of a panepidermal marker, type I keratin. Xepsin mRNA, which encodes a novel Xenopus serine protease, is transcribed zygotically with the expression peak in neurula stages. In normal development, its expression is limited to the anterior and anterior–dorsal portions within epidermis during neurula and tailbud stages, respectively. In UV-irradiated ventralized embryos (dorsoanterior index, DAI 0 and 1), an expression boundary for Xepsin is apparently formed within the epidermis. In contrast, Xepsin expression was observed throughout the epidermis in LiCl-treated dorsalized embryos (DAI 10), as seen from an expression pattern indistinguishable from that of type I keratin. These data suggest that posteriorizing signals which suppress the transcription of Xepsin are present in nonaxial regions and absent in the anterior dorsal mesoderm. That posteriorizing signals were present in nonaxial regions was also supported by a conjugation experiment in which Xepsin expression was suppressed in ectodermal explants conjugated with lateral or ventral marginal zone. Moreover, the partly suppressed expression of Xepsin in the epidermal region of exogastrulae indicates that the signals may travel horizontally within the plane of the epidermis. We also present data showing that both treatment with retinoic acid and the overexpression of a constitutively active form of a retinoic acid receptor caused the suppression of Xepsin mRNA transcription, suggesting that anterior–posterior patterning in the central nervous system and in the epidermis may share common endogenous factors, i.e., retinoids, in the Xenopus embryo.

Graded amounts of Xenopus dishevelled specify discrete anteroposterior cell fates in prospective ectoderm

Signals emitted from the prospective dorsal marginal zone (the organizer) are thought to specify neuroectodermal cell fates along the anteroposterior (AP) axis, but the mechanisms underlying this signaling event remain to be elucidated. To assess the effect of Xenopus Dishevelled (Xdsh), a proposed component of the Wnt, Notch and Frizzled signal transduction pathways, on AP axis determination, it was supplied in varying doses to presumptive ectodermal cells. Two-fold increments in levels of microinjected Xdsh mRNA revealed a gradual shift in cell fates along the AP axis. Lower doses of Xdsh mRNA activated anterior neuroectodermal markers, XAG1 and Xotx2, whereas the higher doses induced more posterior neural tissue markers such as En2, Krox20 and HoxB9. At the highest dose of Xdsh mRNA, explants contained maximal amount of HoxB9 transcripts and developed notochord and somites. When compared with Xdsh, Xwnt8 mRNA also activated anterior neuroectodermal markers, but failed to elicit mesoderm formation. Analysis of explants overexpressing Xdsh at the gastrula stage revealed activation of several organizer-specific genes which have been implicated in determination of neural tissue (Xotx2, noggin, chordin and follistatin). Whereas Goosecoid, Xlim1 and Xwnt8 were not induced in these explants, another early marginal zone marker, Xbra, was activated at the highest level of Xdsh mRNA. These observations suggest that the effects of Xdsh on AP axis specification may be mediated by combinatorial action of several early patterning genes. Increasing levels of Xdsh mRNA activate posterior markers, whereas increasing amounts of the organizer stimulate the extent of anterior development (Stewart, R.M. and Gerhart, J.C. (1990) Development 109, 363–372). These findings argue against induction of the entire organizer by Xdsh in ectodermal cells and implicate signal transduction pathways involving Xdsh in AP axis determination. Thus, different levels of a single molecule, Xdsh, can specify distinct cell states along the AP axis.

Mechanism of anteroposterior axis specification in vertebrates. Lessons from the amphibians.

Interest in the problem of anteroposterior specification has quickened because of our near understanding of the mechanism in Drosophila and because of the homology of Antennapedia-like homeobox gene expression patterns in Drosophila and vertebrates. But vertebrates differ from Drosophila because of morphogenetic movements and interactions between tissue layers, both intimately associated with anteroposterior specification. The purpose of this article is to review classical findings and to enquire how far these have been confirmed, refuted or extended by modern work. The "pre-molecular" work suggests that there are several steps to the process: (i) Formation of anteroposterior pattern in mesoderm during gastrulation with posterior dominance. (ii) Regional specific induction of ectoderm to form neural plate. (iii) Reciprocal interactions from neural plate to mesoderm. (iv) Interactions within neural plate with posterior dominance. Unfortunately, almost all the observable markers are in the CNS rather than in the mesoderm where the initial specification is thought to occur. This has meant that the specification of the mesoderm has been assayed indirectly by transplantation methods such as the Einsteckung. New molecular markers now supplement morphological ones but they are still mainly in the CNS and not the mesoderm. A particular interest attaches to the genes of the Antp-like HOX clusters since these may not only be markers but actual coding factors for anteroposterior levels. We have a new understanding of mesoderm induction based on the discovery of activins and fibroblast growth factors (FGFs) as candidate inducing factors. These factors have later consequences for anteroposterior pattern with activin tending to induce anterior, and FGF posterior structures. Recent work on neural induction has implicated cAMP and protein kinase C (PKC) as elements of the signal transduction pathway and has provided new evidence for the importance of tangential neural induction. The regional specificity of neural induction has been reinvestigated using molecular markers and provides conclusions rather similar to the classical work. Defects in the axial pattern may be produced by retinoic acid but it remains unclear whether its effects are truly coordinate ones or are concentrated in certain regions of high sensitivity. In general the molecular studies have supported and reinforced the "pre-molecular ones". Important questions still remain: (i) How much pattern is there in the mesoderm (how many states?) (ii) How is this pattern generated by the invaginating organizer? (iii) Is there one-to-one transmission of codings to the neural plate? (iv) What is the nature of the interactions within the neural plate? (v) Are the HOX cluster genes really the anteroposterior codings?