Hyeyoon Lee

Cholesterol selectively activates canonical Wnt signalling over non-canonical Wnt signalling

Wnt proteins control diverse biological processes through β-catenin-dependent canonical signaling and β-catenin-independent non-canonical signaling. The mechanisms by which these signaling pathways are differentially triggered and controlled are not fully understood. Dishevelled (Dvl) is a scaffold protein that serves as the branch point of these pathways. Here, we show that cholesterol selectively activates canonical Wnt signaling over non-canonical signaling under physiological conditions by specifically facilitating the membrane recruitment of the PDZ domain of Dvl and its interaction with other proteins. Single molecule imaging analysis shows that cholesterol is enriched around the Wnt-activated Frizzled and low-density lipoprotein receptor-related protein 5/6 receptors and plays an essential role for Dvl-mediated formation and maintenance of the canonical Wnt signaling complex. Collectively, our results suggest a new regulatory role of cholesterol in Wnt signaling and a potential link between cellular cholesterol levels and the balance between canonical and non-canonical Wnt signaling activities.

β-Arrestin 1 mediates non-canonical Wnt pathway to regulate convergent extension movements.

β-Arrestins are multifaceted proteins that play critical roles in termination of G protein-coupled receptor (GPCR) signaling by inducing its desensitization and internalization as well as in facilitation of many intracellular signaling pathways. Here, we examine using Xenopus embryos whether β-arrestin 1 might act as a mediator of β-catenin-independent Wnt (non-canonical) signaling. Xenopus β-arrestin 1 (xβarr1) is expressed in the tissues undergoing extensive cell rearrangements in early development. Gain- and loss-of-function analyses of xβarr1 revealed that it regulates convergent extension (CE) movements of mesodermal tissue with no effect on cell fate specification. In addition, rescue experiments showed that xβarr1 controls CE movements downstream of Wnt11/Fz7 signal and via activation of RhoA and JNK. In line with this, xβarr1 associated with key Wnt components including Ryk, Fz, and Dishevelled. Furthermore, we found that xβarr1 could recover CE movements inhibited by xβarr2 knockdown or its endocytosis defective mutant. Overall, these results suggest that β-arrestin 1 and 2 share interchangeable endocytic activity to regulate CE movements downstream of the non-canonical Wnt pathway.

Ubiquitin C-terminal hydrolase37 regulates Tcf7 DNA binding for the activation of Wnt signalling.

The Tcf/Lef family of transcription factors mediates the Wnt/β-catenin pathway that is involved in a wide range of biological processes, including vertebrate embryogenesis and diverse pathogenesis. Post-translational modifications, including phosphorylation, sumoylation and acetylation, are known to be important for the regulation of Tcf/Lef proteins. However, the importance of ubiquitination and ubiquitin-mediated regulatory mechanisms for Tcf/Lef activity are still unclear. Here, we newly show that ubiquitin C-terminal hydrolase 37 (Uch37), a deubiquitinase, interacts with Tcf7 (formerly named Tcf1) to activate Wnt signalling. Biochemical analyses demonstrated that deubiquitinating activity of Uch37 is not involved in Tcf7 protein stability but is required for the association of Tcf7 to target gene promoter in both Xenopus embryo and human liver cancer cells. In vivo analyses further revealed that Uch37 functions as a positive regulator of the Wnt/β-catenin pathway downstream of β-catenin stabilization that is required for the expression of ventrolateral mesoderm genes during Xenopus gastrulation. Our study provides a new mechanism for chromatin occupancy of Tcf7 and uncovers the physiological significance of Uch37 during early vertebrate development by regulating the Wnt/β-catenin pathway.

Analysis of the expression of microtubule plus-end tracking proteins (+TIPs) during Xenopus laevis embryogenesis.

Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracellular transport in diverse cellular processes. Microtubule plus-end tracking proteins (+TIPs), a structurally and functionally diverse group of proteins, are specifically accumulated in the microtubule plus end and regulate dynamic microtubule behavior. We characterized the +TIPs, Clip1, p150(glued), Clasp1, Lis1 and Stim1, in Xenopus laevis and report their expression patterns during embryogenesis in this paper. All the five +TIP genes are maternally expressed and have similar expression patterns during Xenopus embryo development. The expression of +TIPs is localized in the animal hemisphere and ectoderm region at early stages of embryonic development. As development progresses to later stages, the ectodermal expression of +TIPs persists in head and neural tube structures. Clasp1, p150(glued) and Lis1 in particular are specifically expressed in the cranial nerves. Importantly, +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs, and our analysis provides insight that could serve as the basis for future research of microtubules in vertebrate development, cell movements during gastrulation and neurogenesis.

Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1

ABSTRACT Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of Xenopus. We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of Xenopus embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development. Summary: The E3 ubiquitin ligase March2 and the Dsh-interacting protein Dapper1 cooperatively regulate turnover of Dsh via lysosomal degradation to antagonize Wnt signaling and contribute to Xenopus head formation.

Involvement of Tmem150b during Xenopus convergent extension

zipper forward. Behind the zipper, junctional exchange (Ne/Epi+Ne/ Epi-NNe/Ne+Epi/Epi), cell detachment, and local inhibition of Myosin II on new junctions, reduce posterior resistance to zipper progression. Here, we show that differential expression of Cadherin2 (Cad-2), a classic cadherin, controls these asymmetries for zipper progression. Cad-2 is normally expressed only in neural cells. When GFP-tagged Cad-2 transgene is expressed in all neural cells, Cad2::GFP enriched at homotypic cell junctions between neural cells, but absent from heterotypic junctions between neural and epidermal cells. Equalizing Cad-2 expression across the Ne/Epi boundary results in local accumulation of Cad-2 and local inhibition of myosin activation along that boundary and prevents zippering. In contrast, creating ectopic boundaries between Cad-2-expressing and nonexpressing cells causes ectopic myosin activation at those boundaries. These and other results suggest homophilic Cad-2 interactions inhibit myosin activation on homotypic Ne/Ne boundaries, and directs myosin activation to heterotypic Ne/Epi boundaries. Thus, by promoting local activation of Myosin II along Ne/Epi junctions ahead of the zipper and local inhibition of Myosin II at homotypic Ne/Ne contacts behind the zipper, Cad-2 creates a global contractile asymmetry that is required for unidirectional zipper progression.

Uch37 is required for Tcf1-mediated mesoderm development during Xenopus gastrulation

laryngeal and syringeal muscles are Isl1 derived in birds, implying a pharyngeal mesoderm origin. We’re currently completing this study with transcriptomic analyses and quail/chick transplantation in order to document the origin and molecular requirements of these muscles. Our study focuses on establishing a comprehensive view of the emergence and formation of the intricate pharyngeal musculature acting as a functional unit for feeding and vocalizing in mammals.

March2 is required for Xenopus head organizer formation by antagonizing Wnt signaling pathway

The development of an organism largely depends on the orchestration between cellular growth and the timing of developmental cell cycles, which is fine tuned by several genes. The transcript stability and abundance of these genes during development and throughout adulthood regulates animal form and function while mutation in them causes the cells to lose control over the timely progression of developmental cell cycles and may trigger tumorigenesis. mRNA decapping is a key step of mRNA degradation involving the removal of the 5’m7G cap of mRNAs. In Drosophila melanogaster, the mRNA decapping enzyme 2-DCP2, performs this step in association with other proteins. DCP2 has been studied extensively in various model organisms in the light of maintenance of transcript abundance and stability. In Drosophila melanogaster, loss-of-function alleles of DCP2, viz., DCP2BG01766 and DCP2e00034 cause absolute embryonic lethality in homozygotes, whereas another allele, DCP2l(3)tb causes larval brain tumors and absolute pupal lethality in the homozygotes. DCP2BG01766 and DCP2l(3)tb show defects in cuticular denticles and elevated expression of activated JNK in the cells of the lateral epidermis. Parallely, the DCP2BG01766 mutant embryos show accumulation of Rab11, a vesicular trafficking protein in the lateral epidermis as well as elevated expression of activated JNK in the cells of the neuro-ectoderm. The tumorous allele, DCP2l(3)tb exhibits hyperplastic nature, with increased number and size of cells without loss of epithelial polarity along with impaired fasciculation of axons, implying DCP2 to be an essential candidate for development of the nervous system in Drosophila. Taken together, our results shed light on hitherto undescribed roles of DCP2 during Drosophila development, besides its cognate function.

PIP2 mediates Dvl-Fz interaction during Xenopus embryogenesis

Programmed cell death (PCD) during Drosophila metamorphosis is controlled by steroid hormone ecdysone. Metamorphosis of Drosophila involves the destruction of most of the larval tissues and differentiation and morphogenesis of the tissues that form the adult fly. Unlike other larval tissues, Malpighian tubules (MTs) are unique in not undergoing ecdysone induced PCD and are carried to adults. We are interested in understanding the genetic mechanism that regulates tissue’s survival and death decision in response to ecdysone signal. To answer this question we compared transcriptomes of MTs, a tissue which evades PCD in response to ecdysone signals and Salivary glands, which undergo PCD during the same circumstances. We found that out of several ecdysone induced early genes, E93 showed differential expression in MTs in comparison to salivary glands. E93 was significantly downregulated (~1000 fold) in MTs at pupal stage in comparison to salivary glands. Over expression of E93 in MTs resulted in cell death, though the expression of apoptotic genes including hid and reaper, effector caspase (drice) were unaltered. However, Forkhead (Fkh), a known competence factor for steroid induced cell death was found to be down regulated in E93 overexpression background, therefore probably sensitizing the Malpighian tubules to death inducing signals. The protective role Fkh in MTs was further confirmed by its increased expression level in pupal stage in comparison to salivary glands of the same stage, where it is downregulated by prepupal ecdysone signal. Thus we propose that E93 appears to be one of the deciding factors in Malpighian tubules fate/survival and it does this by probably regulating the expression of Fkh.

Abstract B12: March E3 ubiquitin ligase is required for head formation by mediating Dishevelled degradation during Xenopus development

Wnt signaling controls numerous biological events including vertebrate development, adult tissue homeostasis and organogenesis. In addition, dysregulation of Wnt itself or its downstream signaling components causes human birth defects and cancer. Dishevelled (Dvl) is a crucial scaffolding protein that exerts activation of Wnt signaling pathway. Recently, several Dvl-interacting proteins have been suggested to mediate the stability of Dvl. However, the molecular mechanisms by which Dvl undergoes degradation remain to be elucidated. Here we identify March, a RING domain-containing ubiquitin ligase, targeting Dvl protein and show that it regulates canonical Wnt signaling in Xenopus. During Xenopus embryogenesis, March transcripts were ubiquitously expressed. We intriguingly found that March depletion by microinjection of morpholino oligonucleotide caused defects in the anterior head formation, including loss of the forebrain, cement gland and eye structures. We also found that these defective phenotypes were caused by the Wnt antagonistic function of March in the head organizer region. Knockdown of March suppressed expression of head organizer genes Gsc, Chd and Otx2 at the dorsal marginal region of the gastrula embryo. Wnt reporter assay, western blot analysis measuring phosphorylation of LRP6 and activation of β-catenin, RT-PCR and axis duplication assay in Xenopus revealed that March inhibits canonical Wnt signaling pathway. Furthermore, we identified that March protein associates with Dvl and mediates its poly-ubiquitination for lysosomal degradation in Xenopus embryos. Taken together, these results suggest that March retains cytosolic pools of Dvl protein and leads subsequent limitation of Wnt signaling for vertebrate head development. Citation Format: Hyeyoon Lee, Seong-Moon Cheong, Jin-Kwan Han. March E3 ubiquitin ligase is required for head formation by mediating Dishevelled degradation during Xenopus development. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B12.