The formation of embryonic axes is a critical step during animal development, which contributes to establishing the basic body plan in each particular organism. Wnt signaling pathways play pivotal roles in this fundamental process. Canonical Wnt signaling that is dependent on β-catenin regulates the patterning of dorsoventral, anteroposterior, and left-right axes. Non-canonical Wnt signaling that is independent of β-catenin modulates cytoskeletal organization to coordinate cell polarity changes and asymmetric cell movements. It is now well documented that components of these Wnt pathways biochemically and functionally interact to mediate cell-cell communications and instruct cellular polarization in breaking the embryonic symmetry. The dysfunction of Wnt signaling disrupts embryonic axis specification and proper tissue morphogenesis, and mutations of Wnt pathway genes are associated with birth defects in humans. This review discusses the regulatory roles of Wnt pathway components in embryonic axis formation by focusing on vertebrate models. It highlights current progress in decoding conserved mechanisms underlying the establishment of asymmetry along the three primary body axes. By providing an in-depth analysis of canonical and non-canonical pathways in regulating cell fates and cellular behaviors, this work offers insights into the intricate processes that contribute to setting up the basic body plan in vertebrate embryos.

In vertebrates, the three orthogonal body axes, anteroposterior (AP), dorsoventral (DV) and left-right (LR) are determined at gastrula and neurula stages by the Spemann-Mangold organizer and its equivalents. A common feature of AP and DV axis formation is that an evolutionary conserved interplay between growth factors (Wnt, BMP) and their extracellular antagonists (e.g. Dkk1, Chordin) creates signaling gradients for axial patterning. Recent work showed that LR patterning in Xenopus follows the same principle, with R-spondin 2 (Rspo2) as an extracellular FGF antagonist, which creates a signaling gradient that determines the LR vector. That a triad of anti-FGF, anti-BMP, and anti-Wnt governs LR, DV, and AP axis formation reveals a unifying principle in animal development. We discuss how cross-talk between these three signals confers integrated AP-DV-LR body axis patterning underlying developmental robustness, size scaling, and harmonious regulation. We propose that Urbilateria featured three orthogonal body axes that were governed by a Cartesian coordinate system of orthogonal Wnt/AP, BMP/DV, and FGF/LR signaling gradients.

Hydra has a regenerative capacity that is not limited to individual organs but encompasses the entire body. Various global and integrative genome, transcriptome and proteome approaches have shown that many of the signaling pathways and transcription factors present in vertebrates are already present in Cnidaria, the sister group of Bilateria, and are also activated in regeneration. It is now possible to investigate one of the central questions of regeneration biology, i.e., how does the patterning system become activated by the injury signals that initiate regeneration. This review will present the current data obtained in Hydra and draw parallels with regeneration in Bilateria. Important findings of this global analysis are that the Wnt signaling pathway has a dual function in the regeneration process. In the early phase Wnt is activated generically and in a second phase of pattern formation it is activated in a position specific manner. Thus, Wnt signaling is part of the generic injury response, in which mitogen-activated protein kinases (MAPKs) are initially activated via calcium and reactive oxygen species (ROS). The MAPKs, p38, c-Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERK) are essential for Wnt activation in Hydra head and foot regenerates. Furthermore, the antagonism between the ERK signaling pathway and stress-induced MAPKs results in a balanced induction of apoptosis and mitosis. However, the early Wnt genes are activated by MAPK signaling rather than apoptosis. Early Wnt gene activity is differentially integrated with a stable, β-Catenin-based gradient along the primary body axis maintaining axial polarity and activating further Wnts in the regenerating head. Because MAPKs and Wnts are highly evolutionarily conserved, we hypothesize that this mechanism is also present in vertebrates but may be activated to different degrees at the level of early Wnt gene integration.

Regeneration abilities are widespread among animals and select species can restore any body parts removed by wounds that sever the major body axes. This capability of whole-body regeneration as exemplified in flatworm planarians, Acoels, and Cnidarians involves initial responses to injury, the assessment of wound site polarization, determination of missing tissue and programming of blastema fate, and patterned outgrowth to restore axis content and proportionality. Wnt signaling drives many shared and conserved aspects of the biology of whole-body regeneration in the planarian species Schmidtea mediterranea and Dugesia japonica, in the Acoel Hofstenia miamia, and in Cnidarians Hydra and Nematostella. These overlapping mechanisms suggest whole-body regeneration might be an ancestral property across diverse animal taxa.

Stem cell-derived three-dimensional (3D) gastruloids show a remarkable capacity of self-organisation and recapitulate many aspects of gastrulation stage mammalian development. Gastruloids can be rapidly generated and offer several experimental advantages, such as scalability, observability and accessibility for manipulation. Here, we present approaches to further expand the experimental potency of murine 3D gastruloids by using functional genetics in mouse embryonic stem cells (mESCs) to generate chimeric gastruloids. In chimeric gastruloids, fluorescently labelled cells of different genotypes harbouring inducible gene expression or loss-of-function alleles are combined with wild-type cells. We showcase this experimental approach in chimeric gastruloids of mESCs carrying homozygous deletions of the Tbx transcription factor brachyury or inducible expression of Eomes. Resulting chimeric gastruloids recapitulate reported Eomes and brachyury functions, such as instructing cardiac fate and promoting posterior axial extension, respectively. Additionally, chimeric gastruloids revealed previously unrecognised phenotypes, such as the tissue sorting preference of brachyury deficient cells to endoderm and the cell non-autonomous effects of brachyury deficiency on Wnt3a patterning along the embryonic axis, demonstrating some of the advantages of chimeric gastruloids as an efficient tool for studies of mammalian gastrulation.

The establishment of anterior-posterior (AP) regional identity is an essential step in the appropriate development of the vertebrate central nervous system. An important aspect of AP neural axis formation is the inherent plasticity that allows developing cells to respond to and recover from the various perturbations that embryos continually face during the course of development. While the mechanisms governing the regionalization of the nervous system have been extensively studied, relatively less is known about the nature and limits of early neural plasticity of the anterior-posterior neural axis. This study aims to characterize the degree of neural axis plasticity in Xenopus laevis by investigating the response of embryos to a 180-degree rotation of their AP neural axis during gastrula stages by assessing the expression of regional marker genes using in situ hybridization. Our results reveal the presence of a narrow window of time between the mid- and late gastrula stage, during which embryos are able undergo significant recovery following a 180-degree rotation of their neural axis and eventually express appropriate regional marker genes including Otx, Engrailed, and Krox. By the late gastrula stage, embryos show misregulation of regional marker genes following neural axis rotation, suggesting that this profound axial plasticity is a transient phenomenon that is lost by late gastrula stages.

Hydra\'s almost unlimited regenerative potential is based on Wnt signaling, but so far it is unknown how the injury stimulus is transmitted to discrete patterning fates in head and foot regenerates. We previously identified mitogen-activated protein kinases (MAPKs) among the earliest injury response molecules in Hydra head regeneration. Here, we show that three MAPKs-p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs)-are essential to initiate regeneration in Hydra, independent of the wound position. Their activation occurs in response to any injury and requires calcium and reactive oxygen species (ROS) signaling. Phosphorylated MAPKs hereby exhibit cross talk with mutual antagonism between the ERK pathway and stress-induced MAPKs, orchestrating a balance between cell survival and apoptosis. Importantly, Wnt3 and Wnt9/10c, which are induced by MAPK signaling, can partially rescue regeneration in tissues treated with MAPK inhibitors. Also, foot regenerates can be reverted to form head tissue by a pharmacological increase of β-catenin signaling or the application of recombinant Wnts. We propose a model in which a β-catenin-based stable gradient of head-forming capacity along the primary body axis, by differentially integrating an indiscriminate injury response, determines the fate of the regenerating tissue. Hereby, Wnt signaling acquires sustained activation in the head regenerate, while it is transient in the presumptive foot tissue. Given the high level of evolutionary conservation of MAPKs and Wnts, we assume that this mechanism is deeply embedded in our genome.

Patterning along an axis of polarity is a fundamental step in the development of a multicellular animal embryo. In the cellular field of an early spider embryo, Hedgehog signaling operates to specify a \"fuzzy\" French-flag-like pattern along the primary axis, which is related to the future anterior-posterior (A-P) axis. However, details regarding the generation and development of a diversity of cell states based on the embryo polarity are not known. To address this issue, we applied single-cell RNA sequencing to the early spider embryo consisting of approximately 2,000 cells. Our results confirmed that this technique successfully detected 3 cell populations corresponding to the germ layers and some transient cell states. We showed that the data from dissociated cells had sufficient information for reconstruction of a correct global A-P polarity of the presumptive ectoderm, without clear segregation of specific cell states. This outcome is explained by the varied but differentially overlapping expression of Hedgehog-signal target genes and newly identified marker genes. We also showed that the data resources generated by the transcriptome analysis are applicable to a genome-wide search for genes whose expression is spatially regulated, based on the detection of pattern similarity. Furthermore, we performed single-nucleus RNA sequencing, which was more powerful in detecting emerging cell states. The single-cell and single-nucleus transcriptome techniques will help investigate the pattern-forming processes in the spider model system in an unbiased, comprehensive manner. We provided web-based resources of these transcriptome datasets for future studies of pattern formation and cell differentiation.

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution.

Cnidarians are fascinating creatures at the base of metazoan evolution possessing an almost unlimited regeneration capacity that has attracted the interest of researchers, from Abraham Trembley\'s discovery of regeneration to the present. They share a simple body plan and a high morphogenetic plasticity that has led to a broad spectrum of life cycles. With molecular genomics it became unequivocally clear that Cnidaria are the sister group of the Bilateria and how similar their molecular toolkit is to that of more complex animals. This has renewed interest in these simple animals, which have had an important role in the establishment of fundamental concepts for developmental biologists from the beginning. This review focuses on our current understanding of signaling centers (organizers) and morphogenetic gradients in cnidarians and how they relate to the emergence of the bilaterian body axes. The data are largely based on the cnidarian models Hydra and Nematostella and are supported by new studies on forms with a complete cnidarian life cycle, such as the medusozoans Aurelia and Clytia. Molecular studies on cnidarian development have revealed the existence of an ancient Wnt signaling center at the site of gastrulation, which was instrumental for the formation of a primary body axis and can be traced back to the common ancestor of bilaterian and non-bilaterian animals. New molecular data also suggest that the molecular vectors for the dorso-ventral and left-right body axis in bilaterians, Bmp and Nodal signaling, respectively, were already present but had different fates in the two clades. The close link of developmental processes in bilaterians and cnidarians but also their distinct differences make cnidarians an indispensable model for tackling fundamental questions in developmental biology from patterning, regeneration and other recent molecular approaches to theoretical concepts.