Ectopic Activation Of Wnt Signaling Research Articles

Epigenomic landscape of enhancer elements during Hydra head organizer formation

BackgroundAxis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra.ResultsTo gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity.ConclusionsThe activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

Wnt Signaling Regulates Ipsilateral Pathfinding in the Zebrafish Forebrain through slit3

The nervous system relies upon correct interconnections to exert its normal function. During vertebrate embryonic development, highly stereotyped scaffolds of axon tracts are formed early in the brain to set the foundation for the neuronal interconnections. During zebrafish early development, anterior dorsal telencephalic (ADt) neurons extend axons along the ipsilateral supraoptic tract (SOT) and the contralateral anterior commissure (AC). Our previous study shows axonal outgrowths along the AC/SOT tracts are coordinated by the guidance molecules Dcc-Netrin and Robo2-Slit, but it is not known how the expressions of these guidance molecules are regulated in the forebrain tissues. Here we show ectopic activation of Wnt signaling abolishes the ipsilateral SOT originating from the ADt neurons. Further mechanistic studies show ectopic activation of Wnt signaling significantly reduces Slits’ expression, whilst mis-expression of Slit3 rescues SOT outgrowth. Together, our findings indicate Wnt signaling controls the ipsilateral axonal outgrowth through the regulation of slits’ expression in the zebrafish forebrain.

Ectopic activation of WNT signaling in human embryonal carcinoma cells and its effects in short- and long-term in vitro culture

Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/β-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4+/SSEA4−/SSEA1+ profile. Cisplatin treatment indicated that the OCT4+/SSEA4−/SSEA1+ NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells.

Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells.

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

Canonical wnt signaling regulates atrioventricular junction programming and electrophysiological properties.

Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. To determine the role of canonical Wnt signaling in the myocardium during AVC development. We used a novel allele of β-catenin that preserves β-catenin's cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that the loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with the loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiological criteria. Aberrant AVC development can lead to ventricular pre-excitation, a characteristic feature of Wolff-Parkinson-White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of β-catenin protein levels can rescue Notch-mediated ventricular pre-excitation and dysregulated ion channel gene expression. Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electric programming upstream of Tbx3. Our data further suggest that ventricular pre-excitation may require both morphological patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue.

The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling

The Wnt1-Cre transgenic mouse line is extensively used in the study of the development of the neural crest and its derivatives and the midbrain. The Wnt1 gene has important developmental roles in formation of the midbrain–hindbrain boundary, regulation of midbrain size, and neurogenesis of ventral midbrain dopaminergic (mDA) neurons. Here, we report that Wnt1-Cre transgenic mice exhibit phenotypes in multiple aspects of midbrain development. Significant expansion of the midbrain and increased proliferation in the developing inferior colliculus is associated with ectopic expression of Wnt1. Marked elevation of Wnt1 expression in the ventral midbrain is correlated with disruption of the differentiation program of ventral mDA neurons. We find that these phenotypes can be attributed to ectopic expression of Wnt1 from the Wnt1-Cre transgene leading to the ectopic activation of canonical Wnt/β-catenin signaling. Since these caveats could complicate the utility of Wnt1-Cre in some developmental circumstances, we report a new Wnt1-Cre2 transgenic mouse line that can serve the same purposes as the original without the associated phenotypic complications. These studies reveal an important caveat to a widely-used reagent, provide an improved version of this reagent, and indicate that the original Wnt1-Cre transgenic mouse line may be useful as a gain of function model for interrogating Wnt signaling mechanisms in multiple aspects of midbrain development.

Organizer formation in Hydra is disrupted by thalidomide treatment

Thalidomide is a drug that is well known for its teratogenic properties in humans. Surprisingly, thalidomide does not have teratogenic effects on mouse development. We investigated the effect of thalidomide on patterning in hydra, an early metazoan with a very simple axial symmetry. Hydra develops asexually via Wnt-dependent organizer formation, leading to the budding of a new organism. We observe both induction and inhibition of organizer formation depending on cellular context. Interestingly, thalidomide treatment altered budding and the developing organizer, but had little effect on the adult. Expression of Hybra1, a marker of the organizer increased upon thalidomide treatment. However when the organizer is induced by ectopic activation of Wnt signaling via GSK3 inhibition, thalidomide suppresses induction. We show that inhibition of Wnt signaling is not mediated by induction of the BMP pathway. We show that thalidomide activity on organizer formation in hydra depends on the activity of casein kinase1 and the abundance of β-catenin. Finally, we find that interstitial cells, multipotent cells which give rise to nemoatocytes, neural, digestive and germline cells, are partially responsible for the inhibitory effect of thalidomide.

Radial Glial Neural Progenitors Regulate Nascent Brain Vascular Network Stabilization Via Inhibition of Wnt Signaling

Author SummaryThe brain is an energy-intensive organ that consumes about 10 times as much energy per unit volume as the rest of the body. It therefore requires a highly efficient vascular network for oxygen and nutrient delivery, and as a result compromises in blood vessel networks influence a wide array of brain diseases. Our current understanding is that brain-specific neural cell types are involved in shaping its vascular network, but unfortunately little is known about the cellular or molecular mechanisms involved. Using a mouse genetic model, we have found that radial glial cells, a stem cell type well known for its fundamental role in neural circuit formation, also play an unexpected role in brain vessel development. We find that radial glial cells are essential for the stabilization of newly formed blood vessels in the late embryonic brain, and do so in large part through down-regulating canonical Wnt signaling in endothelial cells (which line the interior surface of blood vessels). These findings provide new insight into how new vessels in the brain are normally stabilized and how this process may be compromised and contribute to diseases.

β-Arrestin2 mediates the initiation and progression of myeloid leukemia

β-Arrestins were initially discovered as negative regulators of G protein-coupled receptor signaling. Although β-arrestins have more recently been implicated as scaffold proteins that interact with various mitogenic and developmental signals, the genetic role of β-arrestins in driving oncogenesis is not known. Here we have investigated the role of β-arrestin in hematologic malignancies and have found that although both β-arrestin1 and -2 are expressed in the hematopoietic system, loss of β-arrestin2 preferentially leads to a severe impairment in the establishment and propagation of the chronic and blast crisis phases of chronic myelogenous leukemia (CML). These defects are linked to a reduced frequency, as well as defective self-renewal capacity of the cancer stem-cell population, in mouse models and in human CML patient samples. At a molecular level, the loss of β-arrestin2 leads to a significant inhibition of β-catenin stabilization, and ectopic activation of Wnt signaling reverses the defects observed in the β-arrestin2 mutant cells. These data cumulatively show that β-arrestin2 is essential for CML disease propagation and indicate that β-arrestins and the Wnt/β-catenin pathway lie in a signaling hierarchy in the context of CML cancer stem cell maintenance.

P58. The biphasic function of Wnt signaling during neural differentiation of mouse embryonic stem cells

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. However, contrary to these results, undifferentiated ES cells shows very low level of endogenous Wnt signaling and ectopic activation of Wnt signaling blocks neural differentiation. Therefore it is not clear whether or when endogenous Wnt/β-catenin signaling is necessary for self-renewal or neural differentiation of ES cells. When we examined the expression profile of Wnt signaling components, expressions of most Wnts are very low in undifferentiated ES cells while some of Wnts are increased during differentiation. We generated stable ES cell lines that probe Wnt signaling activity and these cells showed that Wnt signaling was very low in undifferentiated ES cells while increased during embryonic body formation or neural differentiation. However, conditioned media from differentiated ES cells did not induce higher reporter activity than that of undifferentiated ES cells, which suggests that ES cells may change the sensitivity to Wnt signaling during differentiation whereas the levels of secreted Wnts are same. Changes in the composition of Tcfs or Lef1 in ES cells at different conditions may be a possible mechanism. In addition, we observed that activation of Wnt/β-catenin signaling at different time periods resulted in differential effects on neural differentiation of 46C ES cells that show increased Sox1 promoter driven GFP during neural differentiation; activation of Wnt signaling by the treatment with BIO (a GSK3β inhibitor) in early or late period blocks or enhances neural differentiation, respectively. Consistent with these results, inactivation of Wnt/β-catenin signaling in late period by the treatment with endo-IWR1 (an Axin stabilizer) blocked neural differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays biphasic roles during neural differentiation of mouse embryonic stem cells. Supported by the National Research Foundation of Korea (2006-2004046) and Brain Korea 21 program.

The Wnt Signaling Receptor Lrp5 Is Required for Mammary Ductal Stem Cell Activity and Wnt1-induced Tumorigenesis

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors, which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype, loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds, which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently, the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore, Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally, we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors.

Lhx5 promotes forebrain development and activates transcription of secreted Wnt antagonists

In vertebrate embryos, induction and patterning of the forebrain require the local inhibition of caudalizing signals, such as Wnts, emanating from the mesendoderm and caudal brain. Here, we report that Lhx5, expressed in the rostral neuroectoderm, regulates the local inhibition of Wnts. Activation of Lhx5 expands forebrain structures, whereas inhibition of Lhx5 function compromises forebrain development in zebrafish embryos. Lhx5 can rescue forebrain deficiencies caused by excess Wnt activity, and inhibition of Lhx5 function results in ectopic activation of Wnt signaling. Lhx5 regulates the expression of two secreted Frizzled-related Wnt antagonists, Sfrp1a and Sfrp5. These Sfrps can reduce the ectopic activation of Wnt signaling and rescue the forebrain deficiencies caused by inhibition of Lhx5 function. Our results demonstrate that Lhx5 is a required factor that promotes forebrain development and inhibits Wnt signaling by activating the transcription of secreted Wnt antagonists.

Long-range, nonautonomous effects of activated Notch1 on tissue homeostasis in the nail☆

Using an antibody directed against γ-secretase-generated antigen unique to activated Notch1, we mapped Notch1 activation strictly to suprabasal cells in epidermis, nail matrix, and other skin appendages during normal development. The consequences of Notch1 activation in keratinizing nail cells were investigated in a transgenic mouse model. Ectopic activation of Notch1 in postmitotic cells within the nail keratogenous zone resulted in longer nails. BrdU labeling revealed an increased number of mitotic cells in transgenic nails. The matrix and keratogenous zone expanded distally due to the increase in cell numbers. The mitosis-promoting effects by a gene product expressed exclusively in postmitotic cells indicates a long-range effect of transgenic Notch1 on regulation of nail homeostasis. We demonstrate that activation of Notch1 in the keratogenous zone resulted in ectopic activation of Wnt signaling, the first such evidence in vertebrates. However, we detected little or no β-catenin activation in proliferating matrix cells, indicating that Wnt is at most an indirect mediator of Notch-induced proliferation. These data support the existence of a novel, cell-nonautonomous role for Notch in maintaining homeostasis of stratified squamous epithelia by indirectly promoting mitosis in basally located cells.