Src acts with WNT/FGFRL signaling to pattern the planarian anteroposterior axis.

Tissue identity determination is crucial for regeneration, and the planarian anteroposterior (AP) axis uses positional control genes expressed from body wall muscle to determine body regionalization. Canonical Wnt signaling establishes anterior versus posterior pole identities through notum and wnt1 signaling, and two Wnt/FGFRL signaling pathways control head and trunk domains, but their downstream signaling mechanisms are not fully understood. Here, we identify a planarian Src homolog that restricts head and trunk identities to anterior positions. src-1(RNAi) animals formed enlarged brains and ectopic eyes and also duplicated trunk tissue, similar to a combination of Wnt/FGFRL RNAi phenotypes. src-1 was required for establishing territories of positional control gene expression in Schmidtea mediterranea, indicating that it acts at an upstream step in patterning the AP axis. Double RNAi experiments and eye regeneration assays suggest src-1 can act in parallel to at least some Wnt and FGFRL factors. Co-inhibition of src-1 with other posterior-promoting factors led to dramatic patterning changes and a reprogramming of Wnt/FGFRLs into controlling new positional outputs. These results identify src-1 as a factor that promotes robustness of the AP positional system that instructs appropriate regeneration.

Chimeric 3D gastruloids - a versatile tool for studies of mammalian peri-gastrulation development.

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 Hippo pathway regulates axis formation and morphogenesis in Hydra.

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.

Injury-induced MAPK activation triggers body axis formation in Hydra by default Wnt signaling.

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 ß-catenin signaling or the application of recombinant Wnts. We propose a model in which a ß-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.

Somite development and regionalisation of the vertebral axial skeleton.

A critical stage in the development of all vertebrate embryos is the generation of the body plan and its subsequent patterning and regionalisation along the main anterior-posterior axis. This includes the formation of the vertebral axial skeleton. Its organisation begins during early embryonic development with the periodic formation of paired blocks of mesoderm tissue called somites. Here, we review axial patterning of somites, with a focus on studies using amniote model systems - avian and mouse. We summarise the molecular and cellular mechanisms that generate paraxial mesoderm and review how the different anatomical regions of the vertebral column acquire their specific identity and thus shape the body plan. We also discuss the generation of organoids and embryo-like structures from embryonic stem cells, which provide insights regarding axis formation and promise to be useful for disease modelling.

Diversity and robustness of bone morphogenetic protein pattern formation.

Pattern formation by bone morphogenetic proteins (BMPs) demonstrates remarkable plasticity and utility in several contexts, such as early embryonic development, tissue patterning and the maintenance of stem cell niches. BMPs pattern tissues over many temporal and spatial scales: BMP gradients as short as 1-2 cell diameters maintain the stem cell niche of the Drosophila germarium over a 24-h cycle, and BMP gradients of several hundred microns establish dorsal-ventral tissue specification in Drosophila, zebrafish and Xenopus embryos in timescales between 30 min and several hours. The mechanisms that shape BMP signaling gradients are also incredibly diverse. Although ligand diffusion plays a dominant role in forming the gradient, a cast of diffusible and non-diffusible regulators modulate gradient formation and confer robustness, including scale invariance and adaptability to perturbations in gene expression and growth. In this Review, we document the diverse ways that BMP gradients are formed and refined, and we identify the core principles that they share to achieve reliable performance.

Eukaryotic initiation factor 4A3 inhibits Wnt/ß-catenin signaling and regulates axis formation in zebrafish embryos.

A key step in the activation of canonical Wnt signaling is the interaction between ß-catenin and Tcf/Lefs that forms the transcription activation complex and facilitates the expression of target genes. Eukaryotic initiation factor 4A3 (EIF4A3) is an ATP-dependent DEAD box-family RNA helicase and acts as a core subunit of the exon junction complex (EJC) to control a series of RNA post-transcriptional processes. In this study, we uncover that EIF4A3 functions as a Wnt inhibitor by interfering with the formation of ß-catenin/Tcf transcription activation complex. As Wnt stimulation increases, accumulated ß-catenin displaces EIF4A3 from a transcriptional complex with Tcf/Lef, allowing the active complex to facilitate the expression of target genes. In zebrafish embryos, eif4a3 depletion inhibited the development of the dorsal organizer and pattern formation of the anterior neuroectoderm by increasing Wnt/ß-catenin signaling. Conversely, overexpression of eif4a3 decreased Wnt/ß-catenin signaling and inhibited the formation of the dorsal organizer before gastrulation. Our results reveal previously unreported roles of EIF4A3 in the inhibition of Wnt signaling and the regulation of embryonic development in zebrafish.

The role of Xenopus developmental biology in unraveling Wnt signalling and antero-posterior axis formation.

Wnt signalling plays an eminent role in development, stem cell growth, and tissue homeostasis. Much of what we know about Wnt signalling, we owe to research in developmental biology. Here I review some salient discoveries in the older literature, beginning with the Lithium experiments in sea urchin by Curt Herbst in the 1890ies, when unknown to him he observed the gradual effects of Wnt overactivation upon embryonic axis formation. After revisiting key discoveries into Wingless signalling in Drosophila, I examine the role that the Xenopus embryo has played as model system in this regard. Not only were components of the Wnt cascade dissected and secreted Wnt antagonists discovered in Xenopus, but it also played a key role in unveiling the evolutionary conserved role of Wnt signalling in primary body axis formation. I conclude that Xenopus developmental biology has played a major role in elucidating the mechanisms of embryonic Wnt signalling.

The role of cnidarian developmental biology in unraveling axis formation and Wnt signaling.

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.

Differential gene regulation in DAPT-treated Hydra reveals candidate direct Notch signalling targets.

In Hydra, Notch inhibition causes defects in head patterning and prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. To understand the molecular mechanisms by which the Notch pathway regulates these processes, we performed RNA-seq and identified genes that are differentially regulated in response to 48 h of treating the animals with the Notch inhibitor DAPT. To identify candidate direct regulators of Notch signalling, we profiled gene expression changes that occur during subsequent restoration of Notch activity and performed promoter analyses to identify RBPJ transcription factor-binding sites in the regulatory regions of Notch-responsive genes. Interrogating the available single-cell sequencing data set revealed the gene expression patterns of Notch-regulated Hydra genes. Through these analyses, a comprehensive picture of the molecular pathways regulated by Notch signalling in head patterning and in interstitial cell differentiation in Hydra emerged. As prime candidates for direct Notch target genes, in addition to Hydra (Hy)Hes, we suggest Sp5 and HyAlx. They rapidly recovered their expression levels after DAPT removal and possess Notch-responsive RBPJ transcription factor-binding sites in their regulatory regions.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Multifaceted Functions of TWSG1: From Embryogenesis to Cancer Development.

Bone morphogenetic proteins (BMPs) play an important role in development. Twisted gastrulation BMP signaling modulator 1 (TWSG1) was initially identified as a regulator of the dorsoventral axis formation in Drosophila. The mechanism of BMP signaling modulation by TWSG1 is complex. TWSG1 inhibits BMP signaling by binding to BMP ligands including BMP4, whereas it enhances signaling by interacting with Chordin, a BMP antagonist. Therefore, TWSG1 can act as both a BMP agonist and antagonist. TWSG1 has various functions ranging from embryogenesis to cancer progression. TWSG1 knockout mice showed neural, craniofacial, and mammary defects. TWSG1 also regulated erythropoiesis and thymocyte development. Furthermore, the relationship between TWSG1 and cancer has been elucidated. Allelic loss of TWSG1 was detected in colorectal cancer. TWSG1 expression was upregulated in papillary thyroid carcinoma and glioblastoma but downregulated in gastric and endometrial cancers. TWSG1 suppressed BMP7-enhanced sphere formation and migration in endometrial cancer cells, indicating its tumor-suppressive role. Further studies are required to clarify the TWSG1 function and its association with BMP signaling in cancer development. Finally, TWSG1 is abundantly expressed in human and mouse ovaries and sustains follicular growth in rodent ovaries. Thus, TWSG1 has various functions ranging from fertility to cancer. Therefore, TWSG1 signaling modulation may be beneficial in treating specific diseases such as cancer.

The multiple roles of caudal in early development of the milkweed bug Oncopeltus fasciatus.

The homeobox transcription factor Caudal has conserved roles in all Bilateria in defining the posterior pole and in controlling posterior elongation. These roles are seemingly similar and are difficult to disentangle. We have carried out a detailed analysis of the expression, function and interactions of the caudal ortholog of the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect with a conservative early development process, in order to understand its different functions throughout development. In Oncopeltus, caudal is not maternally deposited, but has a sequence of roles in the posterior of the embryos throughout early development. It defines and maintains a posterior-anterior gradient in the blastoderm and modulates the activity of segmentation genes in simultaneous segmentation during the blastoderm stage. It later defines the invagination site and the posterior segment addition zone (SAZ) in the germband. It maintains the posterior SAZ cells in an undifferentiated proliferative state, while promoting dynamic expression of segmentation genes in the anterior SAZ. We show that rather than being a simple posterior determinant, Caudal is involved in several distinct regulatory networks, each with a distinct developmental role.

Double abdomen in a short-germ insect: Zygotic control of axis formation revealed in the beetle Tribolium castaneum.

The distinction of anterior versus posterior is a crucial first step in animal embryogenesis. In the fly Drosophila, this axis is established by morphogenetic gradients contributed by the mother that regulate zygotic target genes. This principle has been considered to hold true for insects in general but is fundamentally different from vertebrates, where zygotic genes and Wnt signaling are required. We investigated symmetry breaking in the beetle Tribolium castaneum, which among insects represents the more ancestral short-germ embryogenesis. We found that maternal Tc-germ cell-less is required for anterior localization of maternal Tc-axin, which represses Wnt signaling and promotes expression of anterior zygotic genes. Both RNAi targeting Tc-germ cell-less or double RNAi knocking down the zygotic genes Tc-homeobrain and Tc-zen1 led to the formation of a second growth zone at the anterior, which resulted in double-abdomen phenotypes. Conversely, interfering with two posterior factors, Tc-caudal and Wnt, caused double-anterior phenotypes. These findings reveal that maternal and zygotic mechanisms, including Wnt signaling, are required for establishing embryo polarity and induce the segmentation clock in a short-germ insect.

The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo.

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

The forkhead box containing transcription factor FoxB is a potential component of dorsal-ventral body axis formation in the spider Parasteatoda tepidariorum.

In the spider, determination of the dorsal-ventral body (DV) axis depends on the interplay of the dorsal morphogen encoding gene decapentaplegic (Dpp) and its antagonist, short gastrulation (sog), a gene that is involved in the correct establishment of ventral tissues. Recent work demonstrated that the forkhead domain encoding gene FoxB is involved in dorsal-ventral axis formation in spider limbs. Here, Dpp likely acts as a dorsal morphogen, and FoxB is likely in control of ventral tissues as RNAi-mediated knockdown of FoxB causes dorsalization of the limbs. In this study, we present phenotypes of FoxB knockdown that demonstrate a function in the establishment of the DV body axis. Knockdown of FoxB function leads to embryos with partially duplicated median germ bands (Duplicitas media) that are possibly the result of ectopic activation of Dpp signalling. Another class of phenotypes is characterized by unnaturally slim (dorsal-ventrally compressed) germ bands in which ventral tissue is either not formed, or is specified incorrectly, likely a result of Dpp over-activity. These results suggest that FoxB functions as an antagonist of Dpp signalling during body axis patterning, similarly as it is the case in limb development. FoxB thus represents a general player in the establishment of dorsal-ventral structures during spider ontogeny.

PLETHORA transcription factors orchestrate de novo organ patterning during Arabidopsis lateral root outgrowth.

Plant development is characterized by repeated initiation of meristems, regions of dividing cells that give rise to new organs. During lateral root (LR) formation, new LR meristems are specified to support the outgrowth of LRs along a new axis. The determination of the sequential events required to form this new growth axis has been hampered by redundant activities of key transcription factors. Here, we characterize the effects of three PLETHORA (PLT) transcription factors, PLT3, PLT5, and PLT7, during LR outgrowth. In plt3plt5plt7 triple mutants, the morphology of lateral root primordia (LRP), the auxin response gradient, and the expression of meristem/tissue identity markers are impaired from the "symmetry-breaking" periclinal cell divisions during the transition between stage I and stage II, wherein cells first acquire different identities in the proximodistal and radial axes. Particularly, PLT1, PLT2, and PLT4 genes that are typically expressed later than PLT3, PLT5, and PLT7 during LR outgrowth are not induced in the mutant primordia, rendering "PLT-null" LRP. Reintroduction of any PLT clade member in the mutant primordia completely restores layer identities at stage II and rescues mutant defects in meristem and tissue establishment. Therefore, all PLT genes can activate the formative cell divisions that lead to de novo meristem establishment and tissue patterning associated with a new growth axis.

Roles of maternal wnt8a transcripts in axis formation in zebrafish.

In early zebrafish development, the program for dorsal axis formation begins soon after fertilization. Previous studies suggested that dorsal determinants (DDs) localize to the vegetal pole, and are transported to the dorsal blastomeres in a microtubule-dependent manner. The DDs activate the canonical Wnt pathway and induce dorsal-specific genes that are required for dorsal axis formation. Among wnt-family genes, only the wnt8a mRNA is reported to localize to the vegetal pole in oocytes and to induce the dorsal axis, suggesting that Wnt8a is a candidate DD. Here, to reveal the roles of maternal wnt8a, we generated wnt8a mutants by transcription activator-like effector nucleases (TALENs), and established zygotic, maternal, and maternal zygotic wnt8a mutants by germ-line replacement. Zebrafish wnt8a has two open reading frames (ORF1 and ORF2) that are tandemly located in the genome. Although the zygotic ORF1 or ORF2 wnt8a mutants showed little or no axis-formation defects, the ORF1/2 compound mutants showed antero-dorsalized phenotypes, indicating that ORF1 and ORF2 have redundant roles in ventrolateral and posterior tissue formation. Unexpectedly, the maternal wnt8a ORF1/2 mutants showed no axis-formation defects. The maternal-zygotic wnt8a ORF1/2 mutants showed more severe antero-dorsalized phenotypes than the zygotic mutants. These results indicated that maternal wnt8a is dispensable for the initial dorsal determination, but cooperates with zygotic wnt8a for ventrolateral and posterior tissue formation. Finally, we re-examined the maternal wnt genes and found that Wnt6a is an alternative candidate DD.

Development of the aboral domain in Nematostella requires ß-catenin and the opposing activities of Six3/6 and Frizzled5/8.

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvß-catenin regulates both oral and aboral development. In the oral hemisphere, Nvß-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvß-catenin knockdown. In addition, Nvß-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvß-catenin in the patterning of the entire oral-aboral axis of Nematostella.

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos.

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.