Sun-Cheol Choi

Wnt3a-Mediated Formation of Phosphatidylinositol 4,5-Bisphosphate Regulates LRP6 Phosphorylation

The canonical Wnt–β-catenin signaling pathway is initiated by inducing phosphorylation of one of the Wnt receptors, low-density lipoprotein receptor-related protein 6 (LRP6), at threonine residue 1479 (Thr1479) and serine residue 1490 (Ser1490). By screening a human kinase small interfering RNA library, we identified phosphatidylinositol 4-kinase type II α and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled and dishevelled, the latter of which directly interacted with and activated PIP5KI. In turn, PtdIns (4,5)P2 regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a signaling mechanism for Wnt to regulate LRP6 phosphorylation.

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements.

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph-ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.

The involvement of lethal giant larvae and Wnt signaling in bottle cell formation in Xenopus embryos

Lethal giant larvae (Lgl) plays a critical role in establishment of cell polarity in epithelial cells. While Frizzled/Dsh signaling has been implicated in the regulation of the localization and activity of Lgl, it remains unclear whether specific Wnt ligands are involved. Here we show that Wnt5a triggers the release of Lgl from the cell cortex into the cytoplasm with the concomitant decrease in Lgl stability. The observed changes in Lgl localization were independent of atypical PKC (aPKC), which is known to influence Lgl distribution. In ectodermal cells, both Wnt5a and Lgl triggered morphological and molecular changes characteristic of apical constriction, whereas depletion of their functions prevented endogenous and ectopic bottle cell formation. Furthermore, Lgl RNA partially rescued bottle cell formation in embryos injected with a dominant negative Wnt5a construct. These results suggest a molecular link between Wnt5a and Lgl that is essential for apical constriction during vertebrate gastrulation.

Rap2 is required for Wnt/β‐catenin signaling pathway in Xenopus early development

The Wnt/β‐catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/β‐catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer‐specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits β‐catenin stabilization and the induction of ectopic dorsal axis and Wnt‐responsive genes caused by XWnt8, Dsh or β‐catenin, but has no effect on the signaling activities of a stabilized β‐catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh‐mediated β‐catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/β‐catenin signaling as a modulator of the subcellular localization of Dsh.

Regulation of Activin/Nodal Signaling by Rap2-Directed Receptor Trafficking

We show that Rap2, a member of the Ras GTPase family, positively regulates Activin/Nodal signaling activity by controlling the trafficking of its receptors. In the absence of ligand activation, Rap2 directs internalized Activin/Nodal receptors into a recycling pathway, thereby preventing their degradation and maintaining their levels on the cell surface. Upon ligand activation, Rap2 no longer promotes receptor recycling but delays its turnover. In both cases, Rap2 contributes to upregulation of signaling activity by antagonizing Smad7. In addition, we found that the efficiency of Activin/Nodal receptor recycling is different between dorsal and ventral halves of Xenopus early embryo, which results from the asymmetric expression of Rap2 and Smad7. Consequently, they regulate cell responsiveness to ligands and the spatiotemporally dynamic activation of Smad2 along the dorsoventral axis of the embryo. Therefore, these findings suggest a molecular basis for the regulation of signaling activity and embryonic patterning by Activin/Nodal receptor trafficking.

Role of Tbx2 in defining the territory of the pronephric nephron

Despite extensive study of the development of the nephron, which is the functional unit of the kidney, the molecular mechanisms underlying the determination of nephron size remain largely unknown. Using the Xenopus pronephros, we demonstrate here that Tbx2, a T-box transcriptional repressor, functions to demarcate the territory of the pronephric nephron. Tbx2 is specifically expressed around three distinct components of the pronephric nephron: the tubule, duct and glomus. Gain of function of Tbx2 inhibits nephric mesoderm formation. Conversely, Tbx2 loss of function expands the boundary of each component of the pronephric nephron, resulting in an enlarged pronephros. BMP signals induce Tbx2 in the non-nephric mesoderm, which inhibits the expression of the nephric markers Hey1 and Gremlin. Importantly, these pronephric molecules repress Tbx2 expression by antagonizing BMP signals in the nephric mesoderm. These results suggest that the negative regulatory loops between BMP/Tbx2 and Gremlin or Hey1 are responsible for defining the territory of the pronephric nephron.

Identification and developmental expression of par-6 gene in Xenopus laevis

The par genes (partitioning defective) are required to establish polarity in the Caenorhabditis elegans embryo. We have identified the Xenopus homologue of C. elegans PAR-6 (XPAR-6). XPAR-6 is a protein of 377 amino acids with one PDZ domain which is involved in mediating protein-protein interactions. It shares 59% and 58% amino acid identity with the mouse and Drosophila PAR-6, respectively, and 54% overall identity with C. elegans PAR-6. Xpar-6 is expressed both maternally and zygotically. Xpar-6 is first detected in the animal half of the egg, and this pattern of expression persists into the cleavage and blastula stages. At the gastrula stage, the message is detected in animal pole area and in a broad domain of ventral region, but is excluded from dorsal region. With the onset of neurulation, the localized expression of Xpar-6 becomes more obvious, leading to it being enriched in the dorsolateral region along the lateral edges of neural plate and anterior presumptive head region surrounding the anterior border of neural plate. At late tailbud stage, Xpar-6 transcripts show localized expression throughout the head, labeling the branchial arches, eyes, otic vesicles and brain, while more posteriorly Xpar-6 labels the somites, pronephros, tail tip and proctodeum. Therefore, this analysis suggests that Xpar-6 has a regionalized pattern of expression during Xenopus early embryogenesis.

Xenopus Dab2 is required for embryonic angiogenesis

BackgroundThe molecular mechanisms governing the formation of the embryonic vascular system remain poorly understood. Here, we show that Disabled-2 (Dab2), a cytosolic adaptor protein, has a pivotal role in the blood vessel formation in Xenopus early embryogenesis.ResultsXenopus Disabled-2 (XDab2) is spatially localized to the blood vessels including the intersomitic veins (ISV) in early embryos. Both antisense morpholino oligonucleotide (MO)-mediated knockdown and overexpression of XDab2 inhibit the formation of ISV, which arise from angiogenesis. In addition, we found that activin-like signaling is essential for this angiogenic event. Functional assays in Xenopus animal caps reveal that activin-like signals induce VEGF expression and this induction can be inhibited by XDab2 depletion. However, XDab2 MO has no effects on the induction of other target genes by activin-like signals. Furthermore, we show that the disruption of the sprouting ISV in XDab2-depleted embryos can be rescued by coexpression of VEGF.ConclusionTaking together, we suggest that XDab2 regulates the embryonic angiogenesis by mediating the VEGF induction by activin-like signaling in Xenopus early development.

Negative regulation of Activin/Nodal signaling by SRF during Xenopus gastrulation.

Activin/Nodal signaling is essential for germ-layer formation and axial patterning during embryogenesis. Recent evidence has demonstrated that the intra- or extracellular inhibition of this signaling is crucial for ectoderm specification and correct positioning of mesoderm and endoderm. Here, we analyzed the function of Xenopus serum response factor (XSRF) in establishing germ layers during early development. XSRF transcripts are restricted to the animal pole ectoderm in Xenopus early embryos. Ectopic expression of XSRF RNA suppresses mesoderm induction, both in the marginal zone in vivo and caused by Activin/Nodal signals in animal caps. Dominant-negative mutant or antisense morpholino oligonucleotide-mediated inhibition of XSRF function expands the expression of mesendodermal genes toward the ectodermal territory and enhances the inducing activity of the Activin signal. SRF interacts with Smad2 and FAST-1, and inhibits the formation of the Smad2-FAST-1 complex induced by Activin. These results suggest that XSRF might act to ensure proper mesoderm induction in the appropriate region by inhibiting the mesoderm-inducing signals during early embryogenesis.

XPteg (Xenopus proximal tubules-expressed gene) is essential for pronephric mesoderm specification and tubulogenesis

Retinoic acid (RA) signaling is important for the early steps of nephrogenic cell fate specification. Here, we report a novel target gene of RA signaling named XPteg (Xenopus proximal tubules-expressed gene) which is critical for pronephric development. XPteg starts to be expressed at the earliest stage of embryonic kidney specification and was restricted to the pronephric proximal tubules during kidney development. Anti-sense morpholino (MO)-mediated knockdown of XPteg perturbed formation of pronephros as demonstrated by reduced expression of pronephric tubule markers. Conversely, overexpression of XPteg promoted endogenous and ectopic expression of those markers and expanded pronephric tubules. Treatment of retinoic acid induced the expression of XPteg in the pronephric field without protein synthesis. Furthermore, we found that the pronephric defects caused by a dominant negative RA receptor could be rescued by coexpression of XPteg. Taken together, these results suggest that XPteg functions as a direct transcriptional target of RA signaling to regulate pronephric tubulogenesis in Xenopus early development.