Positive feedback regulation of frizzled-7 expression robustly shapes a steep Wnt gradient in Xenopus heart development, together with sFRP1 and heparan sulfate.

Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when that patterning concerns differentiation of thin tissues. Wnt is a morphogen that organizes cardiac development. Wnt6 patterns cardiogenic mesoderm to induce differentiation of a thin tissue, the pericardium, in Xenopus. In this study, we revealed that a Wnt receptor, frizzled-7, is expressed in a Wnt-dependent manner. With a combination of experiments and mathematical modeling, this receptor-feedback appears essential to shape a steep gradient of Wnt signaling. In addition, computer simulation revealed that this feedback imparts robustness against variations of Wnt ligand production and allows the system to reach a steady state quickly. We also found that a Wnt antagonist sFRP1, which is expressed on the opposite side of the Wnt source, accumulates on N-acetyl-rich heparan sulfate (HS). N-acetyl-rich HS concentration is high between the sources of Wnt and sFRP1, achieving local inhibition of Wnt signaling via restriction of sFRP1 spreading. These integrated regulatory systems restrict the Wnt signaling range and ensure reproducible patterning of the thin pericardium.

A mathematical modelling portrait of Wnt signalling in early vertebrate embryogenesis.

There are two phases of Wnt signalling in early vertebrate embryogenesis: very early, maternal Wnt signalling promotes dorsal development, and slightly later, zygotic Wnt signalling promotes ventral and lateral mesoderm induction. However, recent molecular biology analysis has revealed more complexity among the direct Wnt target genes, with at least five classes. Here in order to test the logic and the dynamics of a new Gene Regulatory Network model suggested by these discoveries we use mathematical modelling based on ordinary differential equations (ODEs). Our mathematical modelling of this Gene Regulatory Network reveals that a simplified model, with one "super-gene" for each class is sufficient to a large extent to describe the regulatory behaviour previously observed experimentally.

Evolutionary diversification of the canonical Wnt signaling effector TCF/LEF in chordates.

Wnt signaling is essential during animal development and regeneration, but also plays an important role in diseases such as cancer and diabetes. The canonical Wnt signaling pathway is one of the most conserved signaling cascades in the animal kingdom, with the T-cell factor/lymphoid enhancer factor (TCF/LEF) proteins being the major mediators of Wnt/ß-catenin-regulated gene expression. In comparison with invertebrates, vertebrates possess a high diversity of TCF/LEF family genes, implicating this as a possible key change to Wnt signaling at the evolutionary origin of vertebrates. However, the precise nature of this diversification is only poorly understood. The aim of this study is to clarify orthology, paralogy, and isoform relationships within the TCF/LEF gene family within chordates via in silico comparative study of TCF/LEF gene structure, molecular phylogeny, and gene synteny. Our results support the notion that the four TCF/LEF paralog subfamilies in jawed vertebrates (gnathostomes) evolved via the two rounds of whole-genome duplications that occurred during early vertebrate evolution. Importantly, gene structure comparisons and synteny analysis of jawless vertebrate (cyclostome) TCFs suggest that a TCF7L2-like form of gene structure is a close proxy for the ancestral vertebrate structure. In conclusion, we propose a detailed evolutionary path based on a new pre-whole-genome duplication vertebrate TCF gene model. This ancestor gene model highlights the chordate and vertebrate innovations of TCF/LEF gene structure, providing the foundation for understanding the role of Wnt/ß-catenin signaling in vertebrate evolution.

Foxh1/Nodal Defines Context-Specific Direct Maternal Wnt/ß-Catenin Target Gene Regulation in Early Development.

Although Wnt/ß-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide ß-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of ß-catenin association. We discover that genomic ß-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfß signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease.

Diverse LEF/TCF Expression in Human Colorectal Cancer Correlates with Altered Wnt-Regulated Transcriptome in a Meta-Analysis of Patient Biopsies.

Aberrantly activated Wnt signaling causes cellular transformation that can lead to human colorectal cancer. Wnt signaling is mediated by Lymphoid Enhancer Factor/T-Cell Factor (LEF/TCF) DNA-binding factors. Here we investigate whether altered LEF/TCF expression is conserved in human colorectal tumor sample and may potentially be correlated with indicators of cancer progression. We carried out a meta-analysis of carefully selected publicly available gene expression data sets with paired tumor biopsy and adjacent matched normal tissues from colorectal cancer patients. Our meta-analysis confirms that among the four human LEF/TCF genes, LEF1 and TCF7 are preferentially expressed in tumor biopsies, while TCF7L2 and TCF7L1 in normal control tissue. We also confirm positive correlation of LEF1 and TCF7 expression with hallmarks of active Wnt signaling (i.e., AXIN2 and LGR5). We are able to correlate differential LEF/TCF gene expression with distinct transcriptomes associated with cell adhesion, extracellular matrix organization, and Wnt receptor feedback regulation. We demonstrate here in human colorectal tumor sample correlation of altered LEF/TCF gene expression with quantitatively and qualitatively different transcriptomes, suggesting LEF/TCF-specific transcriptional regulation of Wnt target genes relevant for cancer progression and survival. This bioinformatics analysis provides a foundation for future more detailed, functional, and molecular analyses aimed at dissecting such functional differences.

Xenopus: Experimental Access to Cardiovascular Development, Regeneration Discovery, and Cardiovascular Heart-Defect Modeling.

Xenopus has been used to study a wide array of developmental processes, benefiting from vast quantities of relatively large, externally developing eggs. Xenopus is particularly amenable to examining the cardiac system because many of the developmental processes and genes involved in cardiac specification, differentiation, and growth are conserved between Xenopus and human and have been characterized in detail. Furthermore, compared with other higher vertebrate models, Xenopus embryos can survive longer without a properly functioning heart or circulatory system, enabling investigation of later consequences of early embryological manipulations. This biology is complemented by experimental technology, such as embryonic explants to study the heart, microinjection of overexpression constructs, and, most recently, the generation of genetic mutations through gene-editing technologies. Recent investigations highlight Xenopus as a powerful experimental system for studying injury/repair and regeneration and for congenital heart disease (CHD) modeling, which reinforces why this model system remains ideal for studying heart development.

Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.

In vitro generated mammalian cardiomyocytes provide experimental models for studying normal mammalian cardiomyocyte development, for disease modeling and for drug development. They also promise to inform future therapeutic strategies for repair of injured or diseased myocardium. Here we provide reliable protocols for differentiation of mouse embryonic stem cells into functional cardiomyocytes, together with Notes about trouble shooting and optimizing such protocols for specific cell lines.

Cardiomyocyte Differentiation from Human Embryonic Stem Cells.

In vitro generated human cardiomyocytes hold the ultimate promise for heart patients for repair of injured or diseased myocardium, but they also provide experimental models for studying normal cardiomyocyte development, for disease modeling and for drug development. Here we provide reliable protocols for differentiation of human embryonic stem cells into functional cardiomyocytes, together with Notes about troubleshooting and optimizing such protocols for specific cell lines. This chapter also briefly discusses other published protocols and those further adapted for differentiation of induced pluripotent stem cells into cardiomyocytes.

Genome-wide transcriptomics analysis of genes regulated by GATA4, 5 and 6 during cardiomyogenesis in Xenopus laevis.

The transcription factors GATA4, GATA5 and GATA6 play important roles in heart muscle differentiation. The data presented in this article are related to the research article entitled "Genome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis" (Afouda et al., 2017) [1]. The present study identifies genes regulated by these individual cardiogenic GATA factors using genome-wide transcriptomics analysis. We have presented genes that are specifically regulated by each of them, as well those regulated by either of them. The gene ontology terms (GO) associated with the genes differentially affected are also presented. The data set will allow further investigations on the gene regulatory network downstream of individual cardiogenic GATA factors during cardiac muscle formation.

Genome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis.

The transcription factors GATA4, GATA5 and GATA6 are important regulators of heart muscle differentiation (cardiomyogenesis), which function in a partially redundant manner. We identified genes specifically regulated by individual cardiogenic GATA factors in a genome-wide transcriptomics analysis. The genes regulated by gata4 are particularly interesting because GATA4 is able to induce differentiation of beating cardiomyocytes in Xenopus and in mammalian systems. Among the specifically gata4-regulated transcripts we identified two SoxF family members, sox7 and sox18. Experimental reinstatement of gata4 restores sox7 and sox18 expression, and loss of cardiomyocyte differentiation due to gata4 knockdown is partially restored by reinstating sox7 or sox18 expression, while (as previously reported) knockdown of sox7 or sox18 interferes with heart muscle formation. In order to test for conservation in mammalian cardiomyogenesis, we confirmed in mouse embryonic stem cells (ESCs) undergoing cardiomyogenesis that knockdown of Gata4 leads to reduced Sox7 (and Sox18) expression and that Gata4 is also uniquely capable of promptly inducing Sox7 expression. Taken together, we identify an important and conserved gene regulatory axis from gata4 to the SoxF paralogs sox7 and sox18 and further to heart muscle cell differentiation.

Genome-wide analysis of canonical Wnt target gene regulation in Xenopus tropicalis challenges ß-catenin paradigm.

Wnt/ß-catenin signaling is an important cell-to-cell signaling mechanism that controls gene expression during embryonic development and is critically implicated in human diseases. Developmental, cellular, and transcriptional responses to Wnt signaling are remarkably context-specific in different biological processes. While nuclear localization of ß-catenin is the key to activation of the Wnt/ß-catenin pathway and target gene expression, the molecular mechanisms of how the same Wnt/ß-catenin signaling pathway induces specific responses remain undetermined. Recent advances in high-throughput sequencing technologies and the availability of genome information for Xenopus tropicalis have enabled us to uncover a genome-wide view of Wnt/ß-catenin signaling in early vertebrate embryos, which challenges previous concepts about molecular mechanisms of Wnt target gene regulation. In this review, we summarize our experimental approaches, introduce the technologies we employed and focus on recent findings about Wnt target gene regulation from Xenopus research. We will also discuss potential functions of widespread ß-catenin binding in the genome that we discovered in this species.

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development.

Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

Tissue- and stage-specific Wnt target gene expression is controlled subsequent to ß-catenin recruitment to cis-regulatory modules.

Key signalling pathways, such as canonical Wnt/ß-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear ß-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of ß-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of ß-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates ß-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence ß-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/ß-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of ß-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/ß-catenin target genes subsequent to ß-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated ß-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts.

Wnt signaling in the heart fields: Variations on a common theme.

Wnt signaling plays an essential role in development and differentiation. Heart development is initiated with the induction of precardiac mesoderm requiring the tightly and spatially controlled regulation of canonical and noncanonical Wnt signaling pathways. The role of Wnt signaling in subsequent development of the heart fields is to a large extent unclear. We will discuss the role of Wnt signaling in the development of the arterial and venous pole of the heart, highlighting the dual roles of Wnt signaling with respect to its time- and dosage-dependent effects and the balance between the canonical and noncanonical signaling. Canonical signaling appears to be involved in retaining the cardiac precursors in a proliferative and precursor state, whereas noncanonical signaling promotes their differentiation. Thereafter, both canonical and noncanonical signaling regulate specific steps in differentiation of the cardiac compartments. Because heart development is a contiguous, rather than a sequential, process, analyses tend only to show a single timeframe of development. The repetitive alternating and reciprocal effect of canonical and noncanonical signaling is lost when studied in homogenates. Without the simultaneous in vivo visualization of the different Wnt signaling pathways, the mechanism of Wnt signaling in heart development remains elusive.

The Wnt signaling mediator tcf1 is required for expression of foxd3 during Xenopus gastrulation.

TCF1 belongs to the family of LEF1/TCF transcription factors that regulate gene expression downstream of Wnt/ß-catenin signaling, which is crucial for embryonic development and is involved in adult stem cell regulation and tumor growth. In early Xenopus embryos, tcf1 plays an important role in mesoderm induction and patterning. Foxd3 emerged as a potential tcf1 target gene in a microarray analysis of gastrula stage embryos. Because foxd3 and tcf1 are coexpressed during gastrulation, we investigated whether foxd3 is regulated by tcf1. By using morpholino-mediated knockdown, we show that during gastrulation foxd3 expression is dependent on tcf1. By chromatin immunoprecipitation, we also demonstrate direct interaction of ß-catenin/tcf complexes with the foxd3 gene locus. Hence, our results indicate that tcf1 acts as an essential activator of foxd3, which is critical for dorsal mesoderm formation in early embryos.

sfrp1 promotes cardiomyocyte differentiation in Xenopus via negative-feedback regulation of Wnt signalling.

Wnt signalling is a key regulator of vertebrate heart development, yet it is unclear which specific Wnt signalling components are required to regulate which aspect of cardiogenesis. Previously, we identified Wnt6 as an endogenous Wnt ligand required for controlling heart muscle differentiation via canonical Wnt/ß-catenin signalling. Here we show for the first time a requirement for an endogenous Wnt signalling inhibitor for normal heart muscle differentiation. Expression of sfrp1 is strongly induced in differentiating heart muscle. We show that sfrp1 is not only able to promote heart muscle differentiation but is also required for the formation of normal size heart muscle in the embryo. sfrp1 is functionally able to inhibit Wnt6 signalling and its requirement during heart development relates to relieving the cardiogenesis-restricting function of endogenous wnt6. In turn, we discover that sfrp1 expression in the heart is regulated by Wnt6 signalling, which for the first time indicates that sfrp genes can function as part of a Wnt negative-feedback regulatory loop. Our experiments indicate that sfrp1 controls the size of the differentiating heart muscle primarily by regulating cell fate within the cardiac mesoderm between muscular and non-muscular cell lineages. The cardiac mesoderm is therefore not passively patterned by signals from the surrounding tissue, but regulates its differentiation into muscular and non-muscular tissue using positional information from the surrounding tissue. This regulatory network might ensure that Wnt activation enables expansion and migration of cardiac progenitors, followed by Wnt inhibition permitting cardiomyocyte differentiation.

Different requirements for GATA factors in cardiogenesis are mediated by non-canonical Wnt signaling.

GATA factors and Wnt signals are key regulators of vertebrate cardiogenesis, but specific roles for individual GATA factors and how they interact with Wnt signaling remain unknown. We use loss of function and overexpression approaches to elucidate how these molecules regulate early cardiogenesis in Xenopus. In order to minimize indirect effects due to abnormal early embryogenesis, we use pluripotent embryonic tissues as cardiogenic assays. We confirm central roles for GATA4, 5, and 6 in cardiogenesis, but also discover individual and different requirements. We show that GATA4 or 6 regulate both cardiogenic potential and subsequent cardiomyocyte differentiation but that GATA5 is involved in regulating cardiomyocyte differentiation. We also show that Wnt11b signaling can rescue reduced cardiac differentiation resulting from loss of function of GATA4 and 6 but not GATA5. We conclude that Wnt11b mediates the differential requirements for GATA factors during vertebrate cardiogenesis.

Glucocorticoid-dependent transdifferentiation of pancreatic progenitor cells into hepatocytes is dependent on transient suppression of WNT signalling.

Developmentally, the pancreas and liver are closely related and pathological conditions - including elevated glucocorticoid levels - result in the appearance of hepatocytes in the pancreas. The role of the WNT signalling pathway in this process has been examined in the model transdifferentiating pancreatic acinar AR42J-B-13 (B-13) cell. Glucocorticoid treatment resulted in a transient loss of constitutive WNT3a expression, phosphorylation and depletion of beta-catenin, loss of beta-catenin nuclear localisation, and significant reductions in T-cell factor/lymphoid enhancer factor (Tcf/Lef) transcriptional activity before overt changes in phenotype into hepatocyte-like (B-13/H) cells. A return to higher Tcf/Lef transcriptional activity correlated with the re-expression of WNT3a in B-13/H cells. beta-catenin knock down alone substituted for and enhanced glucocorticoid-dependent transdifferentiation. Overexpression of a mutant beta-catenin (pt-Xbeta-cat) protein that blocked glucocorticoid-dependent suppression of Tcf/Lef activity resulted in inhibition of transdifferentiation. A small-molecule activator of Tcf/Lef transcription factors blocked glucocorticoid-dependent effects, as observed with pt-Xbeta-cat expression. Quercetin - a Tcf/Lef inhibitor - did not promote transdifferentiation into B-13/H cells, but did potentiate glucocorticoid-mediated transdifferentiation. These data demonstrate that the transdifferentiation of B-13 cells into hepatocyte-like cells in response to glucocorticoid was dependent on the repression of constitutively active WNT signalling.