Boni A. Afouda

GATA4, 5 and 6 mediate TGFβ maintenance of endodermal gene expression in Xenopus embryos

The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. Here we detail the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3β by all three GATA factors was abolished when protein synthesis was inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17α and HNF1β by GATA4 and 5 was substantially reduced when protein synthesis was inhibited, induction by GATA6 was minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induced GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, blocked induction of Sox17α and HNF1β by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors were induced by activin, although GATA4 and 6 required lower concentrations. GATA MOs inhibited Sox17α and HNF1β induction by activin at low and high concentrations in the order: GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFβ signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, revealed GATA5 and 6 as the most critical for the development of the gut and the liver.

GATA transcription factors integrate Wnt signalling during heart development.

Cardiogenesis is inhibited by canonical Wnt/β-catenin signalling and stimulated by non-canonical Wnt11/JNK signalling, but how these two signalling pathways crosstalk is currently unknown. Here, we show that Wnt/β-catenin signalling restricts cardiogenesis via inhibition of GATA gene expression, as experimentally reinstating GATA function overrides β-catenin-mediated inhibition and restores cardiogenesis. Furthermore, we show that GATA transcription factors in turn directly regulate Wnt11 gene expression, and that Wnt11 is required to a significant degree for mediating the cardiogenesis-promoting function of GATA transcription factors. These results demonstrate that GATA factors occupy a central position between canonical and non-canonical Wnt signalling in regulating heart muscle formation.

Wnt/β-catenin signalling regulates cardiomyogenesis via GATA transcription factors

A functioning heart muscle is required continuously throughout life. During embryonic development the heart muscle tissue differentiates from mesoderm that has heart‐forming potential. Heart‐forming potential in the embryonic mesoderm is regulated by pro‐cardiogenic transcription factors, such as members of the GATA and NK‐2 transcription factor families. Subsequent heart muscle differentiation involves the expression of cytoskeletal proteins, including myosins and troponins. Different Wnt signalling pathways have various functions in heart development. So‐called ‘canonical’ (Wnt/β‐catenin‐mediated) signalling has a conserved role in vertebrate heart development, regulating and restricting heart development and subsequent heart muscle differentiation. Here we investigated the way in which Wnt/β‐catenin signalling functionally interacts with the GATA family of pro‐cardiogenic transcription factors to regulate subsequent heart muscle differentiation. We used whole Xenopus embryos as an accessible experimental model system for vertebrate heart development. Our experiments confirmed that activation of Wnt signalling results in reduced gata gene expression, as well as reduced gene expression of other pro‐cardiogenic transcription factors and heart muscle differentiation markers. Remarkably, we discovered that when GATA function is experimentally restored, the expression of other pro‐cardiogenic transcription factors and heart muscle differentiation markers is rescued. These findings, obtained from whole‐embryo experiments, show that Wnt signalling regulates heart development at the level of GATA factors, confirming earlier results from tissue‐culture experiments. Furthermore, our rescue experiments in Xenopus embryos revealed differences in functional activity between the various GATA transcription factors involved in heart development. We discovered that GATA4 is more efficient at reinstating the gene expression of other pro‐cardiogenic transcription factors, whereas GATA6 is more potent at promoting the expression of genes associated with terminal heart muscle differentiation. In conclusion, our findings show that the inhibition of heart development by Wnt/β‐catenin signalling during organogenesis is mediated by the loss of expression of GATA pro‐cardiogenic transcription factors and reveal functional differences between those GATA factors in heart development.

Xenopus Explants as an Experimental Model System for Studying Heart Development

Many developmental processes are highly conserved in all vertebrate organisms. This conservation has allowed developmental biologists to use numerous animal models to further our understanding of the molecular mechanisms that govern heart development and congenital heart disease. Amphibian embryos represent a useful model for such studies because their relatively large embryos are available in large numbers and survive simple microsurgery. In addition, until swimming tadpole stages, an amphibian embryo develops using nutrients stored in each of its many cells. This feature has the advantage that explants isolated from embryonic tissue will continue to survive in isolation and differentiate in culture. Furthermore, cells from the ectodermal layer of the blastula or gastrula embryos are stem cell like in that they are pluripotent and can be induced to form various tissues in vitro. Here, we will review work from recent studies in which explants from the amphibian embryos were used to further our understanding of vertebrate heart development. We will bring together the key facts needed for using Xenopus explants as experimental approaches for studying molecular pathways and gene regulatory networks in vertebrate cardiogenesis. The knowledge generated with these approaches supports the usefulness of amphibian explants, and the relevance of the findings strongly validates the conservation of molecular pathways that underlie heart development in all vertebrates.

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. Developmental Dynamics 240:649–662, 2011.

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 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.

Cardiac MHCα expression in Xenopus

We agree that the discrepancy between our results on Xenopus cardiac MHCα expression reported in Afouda et al. ([Afouda et al., 2008][1]) and those of Samuel and Latinkic based on their Xenopus MHCα expression experiments ([Samuel and Latinkic, 2010][2]) might indeed be due to technical details.

13-P107 GATA transcription factors integrate Wnt signaling during heart development

The face is a reflection of our genome. Facial deformities are oftentimes harbingers of an underlying disease states. For example, decreased Hedgehog activity in the developing craniofacial region causes holoprosencephaly and close-set eyes (hypotelorism). We found that excessive Hedgehog activity, caused by truncating the primary cilia on cranial neural crest cells, led to hypertelorism and frontonasal dysplasias (Brugrnann et al., 2009). Here, we show that this loss of the intraflagellar transport protein Kif3a also affects Wnt activity in the face. Using transgenic models to ‘‘map” Wnt activity in the developing face, we found that areas of strong Wnt responsiveness coincided with elevated cell proliferation, which resulted in regionalized outgrowth of the facial prominences. Reducing Wnt signaling, through genetic elimination of Lefl/Tcf4, decreases facial outgrowth but in sharp contrast, deletion of Kif3a in craniofacial mesenchyme resulted in expansion of Wnt responsiveness. The boundaries that form between Hedgehog and Wnt responsiveness were disrupted in the developing face. During Drosophila segmentation Interactions between Hedgehog/Wnt pathways provide instructional cues that establish directionality within an epithelium. Here, we provide evidence that a similar mechanism operates in the embryonic face: our data support a model whereby neural crest cells sense and integrate gradients of Hedgehog and Wnt activity. Disruptions to this Hedgehog/Wnt boundary specification lead to abnormal proliferation in the facial prominences. The resulting phenotype of hypertelorism is a hallmark of numerous craniofacial syndromes, as well as some of the known ciliopathies. These data demonstrate a conserved mechanism whereby patterning and growth is regulated during embryogenesis.