Liver Specification in the Absence of Cardiac Differentiation Revealed by Differential Sensitivity to Wnt/ß Catenin Pathway Activation.

Embryonic precursors of liver and heart, whilst not sharing cellular origin, develop in close proximity through a dynamic series of inductive signaling events. During gastrulation anterior endoderm (AE) provides cardiogenic signals that act on adjacent mesoderm, resulting in induction of cardiac precursors. Subsequently cardiogenic mesoderm generates a FGF signal that acts on adjacent AE to induce foregut organ specification. Additional signals such as BMP and Wnt provide further information required for liver specification. Most findings on liver specification were derived from mouse explant studies as well as experiments with Xenopus and zebrafish embryos. To address some of the limitations of these models, here we used two complementary ex vivo models based on Xenopus embryos: pluripotent animal cap explants expressing Gata4 transcription factor and conjugates of gastrula-stage AE with animal caps (AC). We show that in these models liver specification is not sensitive to Wnt signaling manipulation, in contrast to the requirement for Wnt antagonism shown in vivo. FGF pathway is not necessary for Gata4-induced liver specification in animal cap explants but is required for prolonged period in sandwiches of AE and AC. In contrast, BMP signaling is shown to be essential for Gata4-induced liver specification. Our findings may have implications for research on liver differentiation from embryonic stem cells.

Ventricular cell fate can be specified until the onset of myocardial differentiation.

The mechanisms that govern specification of various cell types that constitute vertebrate heart are not fully understood. Whilst most studies of heart development have utilised the mouse embryo, we have used an alternative model, embryos of the frog Xenopus laevis, which permits direct experimental manipulation of a non-essential heart. We show that in this model pluripotent animal cap explants injected with cardiogenic factor GATA4 mRNA express pan-myocardial as well as ventricular and proepicardial markers. We found that cardiac cell fate diversification, as assessed by ventricular and proepicardial markers, critically depends on tissue integrity, as it is disrupted by dissociation but can be fully restored by inhibition of the BMP pathway and partially by Dkk-1. Ventricular and proepicardial cell fates can also be restored in reaggregated GATA4-expressing cells upon transplantation into a host embryo. The competence of the host embryo to induce ventricular and proepicardial markers gradually decreases with the age of the transplant and is lost by the onset of myocardial differentiation at the late tailbud stage (st. 28). The influence of the host on the transplant was not limited to diversification of cardiac cell fates, but also included induction of growth and rhythmic beating, resulting in generation of a secondary heart-like structure. Our results additionally show that efficient generation of secondary heart requires normal axial patterning of the host embryo. Furthermore, secondary hearts can be induced in a wide range of locations within the host, arguing that the host embryo provides a permissive environment for development of cardiac patterning, growth and physiological maturation. Our results have implications for a major goal of cardiac regenerative medicine, differentiation of ventricular myocardium.

A functional connectome: regulation of Wnt/TCF-dependent transcription by pairs of pathway activators.

BACKGROUND: Wnt/ß-catenin signaling is often portrayed as a simple pathway that is initiated by Wnt ligand at the cell surface leading, via linear series of interactions between 'core pathway' members, to the induction of nuclear transcription from genes flanked by ß-catenin/TCF transcription factor binding sites. Wnt/ß-catenin signaling is also regulated by a much larger set of 'non-core regulators'. However the relationship between 'non-core regulators' is currently not well understood. Aberrant activation of the pathway has been shown to drive tumorgenesis in a number of different tissues. METHODS: Mammalian cells engineered to have a partially-active level of Wnt/ß-catenin signaling were screened by transfection for proteins that up or down-regulated a mid-level of TCF-dependent transcription induced by transient expression of an activated LRP6 Wnt co-receptor (∆NLRP). RESULTS: 141 novel regulators of TCF-dependent transcription were identified. Surprisingly, when tested without ∆NLRP activation, most up-regulators failed to alter TCF-dependent transcription. However, when expressed in pairs, 27 % (466/1170) functionally interacted to alter levels of TCF-dependent transcription. When proteins were displayed as nodes connected by their ability to co-operate in the regulation of TCF-dependent transcription, a network of functional interactions was revealed. In this network, 'core pathway' components (Eg. ß-catenin, GSK-3, Dsh) were found to be the most highly connected nodes. Activation of different nodes in this network impacted on the sensitivity to Wnt pathway small molecule antagonists. CONCLUSIONS: The 'functional connectome' identified here strongly supports an alternative model of the Wnt pathway as a complex context-dependent network. The network further suggests that mutational activation of highly connected Wnt signaling nodes predisposed cells to further context-dependent alterations in levels of TCF-dependent transcription that may be important during tumor progression and treatment.

Carboxy terminus of GATA4 transcription factor is required for its cardiogenic activity and interaction with CDK4.

GATA4-6 transcription factors regulate numerous aspects of development and homeostasis in multiple tissues of mesodermal and endodermal origin. In the heart, the best studied of these factors, GATA4, has multiple distinct roles in cardiac specification, differentiation, morphogenesis, hypertrophy and survival. To improve understanding of how GATA4 achieves its numerous roles in the heart, here we have focused on the carboxy-terminal domain and the residues required for interaction with cofactors FOG2 and Tbx5. We present evidence that the carboxy terminal region composed of amino acids 362-400 is essential for mediating cardiogenesis in Xenopus pluripotent explants and embryos. In contrast, the same region is not required for endoderm-inducing activity of GATA4. Further evidence is presented that the carboxy terminal cardiogenic region of GATA4 does not operate as a generic transcriptional activator. Potential mechanism of action of the carboxy terminal end of GATA4 is provided by the results showing physical and functional interaction with CDK4, including the enhancement of cardiogenic activity of GATA4 by CDK4. These results establish CDK4 as a GATA4 partner in cardiogenesis. The interactions of GATA4 with its other well described cofactors Tbx5 and FOG2 are known to be involved in heart morphogenesis, but their requirement for cardiac differentiation is unknown. We report that the mutations that disrupt interactions of GATA4 with Tbx5 and FOG2, G295S and V217G, respectively, do not impair cardiogenic activity of GATA4. These findings add support to the view that distinct roles of GATA4 in the heart are mediated by different determinants of the protein. Finally, we show that the rat GATA4 likely induces cardiogenesis cell autonomously or directly as it does not require activity of endodermal transcription factor Sox17, a GATA4 target gene that induces cardiogenesis non-cell autonomously.

Cyclin D2 is a GATA4 cofactor in cardiogenesis.

The G1 cyclins play a pivotal role in regulation of cell differentiation and proliferation. The mechanisms underlying their cell-specific roles are incompletely understood. Here, we show that a G1 cyclin, cyclin D2 (CycD2), enhances the activity of transcription factor GATA4, a key regulator of cardiomyocyte growth and differentiation. GATA4 recruits CycD2 to its target promoters, and their interaction results in synergistic activation of GATA-dependent transcription. This effect is specific to CycD2 because CycD1 is unable to potentiate activity of GATA4 and is CDK-independent. GATA4 physically interacts with CycD2 through a discreet N-terminal activation domain that is essential for the cardiogenic activity of GATA4. Human mutations in this domain that are linked to congenital heart disease interfere with CycD2-GATA4 synergy. Cardiogenesis assays in Xenopus embryos indicate that CycD2 enhances the cardiogenic function of GATA4. Together, our data uncover a role for CycD2 as a cardiogenic coactivator of GATA4 and suggest a paradigm for cell-specific effects of cyclin Ds.

Dissociation of cardiogenic and postnatal myocardial activities of GATA4.

Transcription factor GATA4 is a critical regulator of the embryonic and postnatal heart, but the mechanisms and cofactors required for its diverse functions are not fully understood. Here, we show that whereas the N-terminal domain of GATA4 is required for inducing cardiogenesis and for promoting postnatal cardiomyocyte survival, distinct residues and domains therein are necessary to mediate these effects. Cardiogenic activity of GATA4 requires a 24-amino-acid (aa) region (aa 129 to 152) which is needed for transcriptional synergy and physical interaction with BAF60c. The same region is not essential for induction of endoderm or blood cell markers by GATA4, suggesting that it acts as a cell-type-specific transcriptional activation domain. On the other hand, a serine residue at position 105, which is a known target for mitogen-activated protein kinase (MAPK) phosphorylation, is necessary for GATA4-dependent cardiac myocyte survival and hypertrophy but is entirely dispensable for GATA4-induced cardiogenesis. We find that S105 is differentially required for transcriptional synergy between GATA4 and serum response factor (SRF) but not other cardiac cofactors such as TBX5 and NKX2.5. The findings provide new insight into GATA4 mechanisms of action and suggest that distinct regulatory pathways regulate activities of GATA4 in embryonic development and postnatal hearts.

A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription.

The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, beta-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of beta-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors.

Early activation of FGF and nodal pathways mediates cardiac specification independently of Wnt/beta-catenin signaling.

BACKGROUND: Cardiac induction, the first step in heart development in vertebrate embryos, is thought to be initiated by anterior endoderm during gastrulation, but what the signals are and how they act is unknown. Several signaling pathways, including FGF, Nodal, BMP and Wnt have been implicated in cardiac specification, in both gain- and loss-of-function experiments. However, as these pathways regulate germ layer formation and patterning, their specific roles in cardiac induction have been difficult to define. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanisms of cardiac induction directly we devised an assay based on conjugates of anterior endoderm from early gastrula stage Xenopus embryos as the inducing tissue and pluripotent ectodermal explants as the responding tissue. We show that the anterior endoderm produces a specific signal, as skeletal muscle is not induced. Cardiac inducing signal needs up to two hours of interaction with the responding tissue to produce an effect. While we found that the BMP pathway was not necessary, our results demonstrate that the FGF and Nodal pathways are essential for cardiogenesis. They were required only during the first hour of cardiogenesis, while sustained activation of ERK was required for at least four hours. Our results also show that transient early activation of the Wnt/beta-catenin pathway has no effect on cardiogenesis, while later activation of the pathway antagonizes cardiac differentiation. CONCLUSIONS/SIGNIFICANCE: We have described an assay for investigating the mechanisms of cardiac induction by anterior endoderm. The assay was used to provide evidence for a direct, early and transient requirement of FGF and Nodal pathways. In addition, we demonstrate that Wnt/beta-catenin pathway plays no direct role in vertebrate cardiac specification, but needs to be suppressed just prior to differentiation.

Expression of Xenopus tropicalis HNF6/Onecut-1.

Onecut genes belong to a family of transcription factors that are known to be important in embryonic development. In the present study, we analyzed the pattern of expression of Onecut-1/HNF6 in Xenopus tropicalis using RT-PCR and whole mount in situ hybridization. Expression of the Xenopus tropicalis Onecut-1/HNF6 gene was found to be conserved in the neural tube, the sensory placodes and in the anterior ventral endoderm in a domain consistent with the developing liver primordium.

NKCC1 (SLC12a2) induces a secondary axis in Xenopus laevis embryos independently of its co-transporter function.

NKCC1 is a broadly expressed Na(+)-K(+)-Cl(-) co-transporter involved in regulation of ion flux across the cell membrane and in regulating cell volume. Whilst much is known about the co-transporter activity of NKCC1 and its regulation by protein kinases and phosphatases, little is known about the activities of NKCC1 that are co-transporter independent. In this report we show that over-expression of NKCC1 in embryos of Xenopus laevis induces secondary axes, independently of its co-transporter activity. In addition, over-expression of NKCC1 results in the formation of neural tissue in ectodermal explants. We also show that NKCC1 is expressed broadly but non-uniformly in embryos of Xenopus laevis and Xenopus tropicalis, with prominent expression in the notochord, nervous system and stomach. These results provide insights into an additional, previously unreported activity of NKCCl.

GATA4 and GATA5 are essential for heart and liver development in Xenopus embryos.

BACKGROUND: GATA factors 4/5/6 have been implicated in the development of the heart and endodermal derivatives in vertebrates. Work in zebrafish has indicated that GATA5 is required for normal development earlier than GATA4/6. However, the GATA5 knockout mouse has no apparent embryonic phenotype, thereby questioning the importance of the gene for vertebrate development. RESULTS: In this study we show that in Xenopus embryos GATA5 is essential for early development of heart and liver precursors. In addition, we have found that in Xenopus embryos GATA4 is important for development of heart and liver primordia following their specification, and that in this role it might interact with GATA6. CONCLUSION: Our results suggest that GATA5 acts earlier than GATA4 to regulate development of heart and liver precursors, and indicate that one early direct target of GATA5 is homeobox gene Hex.

Xenopus laevis transgenesis by sperm nuclear injection.

The stable integration of transgenes into embryos of the frog Xenopus laevis is achieved using the procedure described here. Linear DNA containing the transgene is incorporated randomly into sperm nuclei that have had their membranes disrupted with detergent treatment. Microinjection of these nuclei into unfertilized eggs produces viable embryos that can be screened for activity of the transgene. The proportion of embryos that harbor the transgene varies from 10 to 40% of the total number of surviving embryos. Multiple copies of the transgene can integrate as a concatemer into the sperm genome, and more than one site of DNA integration might occur within resulting animals. Germ cell transmission of the transgene is routine and the procedure is well suited to the production of transgenic reporter frog lines. One day should be allocated for the preparation of the sperm nuclei, which are stored as aliquots for future use. The transgenesis reaction and egg injection take one morning.

Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex.

Connective-tissue growth factor (CTGF) is a member of the CCN family of secreted proteins. CCN family members contain four characteristic domains and exhibit multiple activities: they associate with the extracellular matrix, they can mediate cell adhesion, cell migration and chemotaxis, and they can modulate the activities of peptide growth factors. Many of the effects of CTGF are thought to be mediated by binding to integrins, whereas others may be because of its recently identified ability to interact with BMP4 and TGF beta. We demonstrate, using Xenopus embryos, that CTGF also regulates signalling through the Wnt pathway, in accord with its ability to bind to the Wnt co-receptor LDL receptor-related protein 6 (LRP6). This interaction is likely to occur through the C-terminal (CT) domain of CTGF, which is distinct from the BMP- and TGF beta-interacting domain. Our results define new activities of CTGF and add to the variety of routes through which cells regulate growth factor activity in development, disease and tissue homeostasis.

Transcriptional regulation of the cardiac-specific MLC2 gene during Xenopus embryonic development.

The mechanisms by which transcription factors, which are not themselves tissue restricted, establish cardiomyocyte-specific patterns of transcription in vivo are unknown. Nor do we understand how positional cues are integrated to provide regionally distinct domains of gene expression within the developing heart. We describe regulation of the Xenopus XMLC2 gene, which encodes a regulatory myosin light chain of the contractile apparatus in cardiac muscle. This gene is expressed from the onset of cardiac differentiation in the frog embryo and is expressed throughout all the myocardium, both before and after heart chamber formation. Using transgenesis in frog embryos, we have identified an 82 bp enhancer within the proximal promoter region of the gene that is necessary and sufficient for heart-specific expression of an XMLC2 transgene. This enhancer is composed of two GATA sites and a composite YY1/CArG-like site. We show that the low-affinity SRF site is essential for transgene expression and that cardiac-specific expression also requires the presence of at least one adjacent GATA site. The overlapping YY1 site within the enhancer appears to act primarily as a repressor of ectopic expression, although it may also have a positive role. Finally, we show that the frog MLC2 promoter drives pan myocardial expression of a transgene in mice, despite the more restricted patterns of expression of murine MLC2 genes. We speculate that a common regulatory mechanism may be responsible for pan-myocardial expression of XMLC2 in both the frog and mouse, modulation of which could have given rise to more restricted patterns of expression within the heart of higher vertebrates.

Induction of cardiomyocytes by GATA4 in Xenopus ectodermal explants.

The earliest step in heart formation in vertebrates occurs during gastrulation, when cardiac tissue is specified. Dorsoanterior endoderm is thought to provide a signal that induces adjacent mesodermal cells to adopt a cardiac fate. However, the nature of this signalling and the precise role of endoderm are unknown because of the close proximity and interdependence of mesoderm and endoderm during gastrulation. To better define the molecular events that underlie cardiac induction, we have sought to develop a simple means of inducing cardiac tissue. We show that the transcription factor GATA4, which has been implicated in regulating cardiac gene expression, is sufficient to induce cardiac differentiation in Xenopus embryonic ectoderm (animal pole) explants, frequently resulting in beating tissue. Lineage labelling experiments demonstrate that GATA4 can trigger cardiac differentiation not only in cells in which it is present, but also in neighbouring cells. Surprisingly, cardiac differentiation can occur without any stable differentiation of anterior endoderm and is in fact enhanced under conditions in which endoderm formation is inhibited. Remarkably, cardiac tissue is formed even when GATA4 activity is delayed until long after explants have commenced differentiation into epidermal tissue. These findings provide a simple assay system for cardiac induction that may allow elucidation of pathways leading to cardiac differentiation. Better knowledge of the pathways governing this process may help develop procedures for efficient generation of cardiomyocytes from pluripotent stem cells.

XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus.

Phagocytic myeloid cells provide the principle line of immune defence during early embryogenesis in lower vertebrates. They may also have important functions during normal embryo morphogenesis, not least through the phagocytic clearance of cell corpses arising from apoptosis. We have identified two cDNAs that provide sensitive molecular markers of embryonic leukocytes in the early Xenopus embryo. These encode a peroxidase (XPOX2) and a Ly-6/uPAR-related protein (XLURP-1). We show that myeloid progenitors can first be detected at an antero-ventral site in early tailbud stage embryos (a region previously termed the anterior ventral blood island) and transiently express the haematopoetic transcription factors SCL and AML. Phagocytes migrate from this site along consistent routes and proliferate, becoming widely distributed throughout the tadpole long before the circulatory system is established. This migration can be followed in living embryos using a 5 kb portion of the XLURP-1 promoter to drive expression of EGFP specifically in the myeloid cells. Interestingly, whilst much of this migration occurs by movement of individual cells between embryonic germ layers, the rostral-most myeloid cells apparently migrate in an anterior direction along the ventral midline within the mesodermal layer itself. The transient presence of such cells as a strip bisecting the cardiac mesoderm immediately prior to heart tube formation suggests that embryonic myeloid cells may play a role in early cardiac morphogenesis.

Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos.

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.