Akira Yukita

Molecular links among the causative genes for ocular malformation: Otx2 and Sox2 coregulate Rax expression

The neural-related genes Sox2, Pax6, Otx2, and Rax have been associated with severe ocular malformations such as anophthalmia and microphthalmia, but it remains unclear as to how these genes are linked functionally. We analyzed the upstream signaling of Xenopus Rax (also known as Rx1) and identified the Otx2 and Sox2 proteins as direct upstream regulators of Rax. We revealed that endogenous Otx2 and Sox2 proteins bound to the conserved noncoding sequence (CNS1) located ≈2 kb upstream of the Rax promoter. This sequence is conserved among vertebrates and is required for potent transcriptional activity. Reporter assays showed that Otx2 and Sox2 synergistically activated transcription via CNS1. Furthermore, the Otx2 and Sox2 proteins physically interacted with each other, and this interaction was affected by the Sox2-missense mutations identified in these ocular disorders. These results demonstrate that the direct interaction and interdependence between the Otx2 and Sox2 proteins coordinate Rax expression in eye development, providing molecular linkages among the genes responsible for ocular malformation.

Desumoylation Activity of Axam, a Novel Axin-Binding Protein, Is Involved in Downregulation of β-Catenin

ABSTRACT Axam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of β-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of β-catenin. An Axam fragment which contains both domains was able to decrease the level of β-catenin. On substitution of Ser for Cys547 in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate β-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopus embryos, AxamC547S showed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate β-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.

XIdax, an inhibitor of the canonical Wnt pathway, is required for anterior neural structure formation in Xenopus

Wnt signaling pathways are involved during various stages in the development of many species. In Xenopus, the accumulation of β‐catenin on the dorsal side of embryo is required for induction of the organizer, while the head structure formation requires inhibition of Wnt signaling. Here, we report a role for xIdax, a negative regulator of Wnt signaling. XIdax is expressed in neural tissues at the neurula stage, and in the restricted region of the tadpole brain. Ectopic expression of xIdax inhibits the target gene expression, suggesting that xIdax can inhibit canonical Wnt signaling. To examine the function of xIdax, a morpholino oligo for xIdax (xIdaxMO) was designed. An injection into an animal pole cell caused a loss of forebrain. The anterior neural marker expression is decreased in xIdaxMO‐injected embryo, suggesting that xIdax is required for anterior neural development. Moreover, a negative regulator that acts downstream of xIdax rescued this defect. We propose that Idax functions are dependent on the canonical Wnt pathway and are crucial for the anterior neural development. Developmental Dynamics 230:79–90, 2004.

Ripply2 is essential for precise somite formation during mouse early development

The regions of expression of Ripply1 and Ripply2, presumptive transcriptional corepressors, overlap at the presomitic mesoderm during somitogenesis in mouse and zebrafish. Ripply1 is required for somite segmentation in zebrafish, but the developmental role of Ripply2 remains unclear in both species. Here, we generated Ripply2 knock‐out mice to investigate the role of Ripply2. Defects in segmentation of the axial skeleton were observed in the homozygous mutant mice. Moreover, somite segmentation and expression of Notch2 and Uncx4.1 were disrupted. These findings indicate that Ripply2 is involved in somite segmentation and establishment of rostrocaudal polarity.

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1high and Thy-1low) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1high cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1low cells. Furthermore, subcutaneous implantation of Thy-1high cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.

Nucleoredoxin regulates the Wnt/planar cell polarity pathway in Xenopus

The Wnt signaling pathway is conserved across species, and is essential for early development. We previously identified nucleoredoxin (NRX) as a protein that interacts with dishevelled (Dvl) in vivo to negatively regulate the Wnt/β‐catenin pathway. However, whether NRX affects another branch of the Wnt pathway, the Wnt/planar cell polarity (PCP) pathway, remains unclear. Here we show that NRX regulates the Wnt/PCP pathway. In Xenopus laevis, over‐expression or depletion of NRX by injection of NRX mRNA or antisense morpholino oligonucleotide, respectively, yields the bent‐axis phenotype that is typically observed in embryos with abnormal PCP pathway activity. In co‐injection experiments of Dvl and NRX mRNA, NRX suppresses the Dvl‐induced bent‐axis phenotype. Over‐expression or depletion of NRX also suppresses the convergent extension movements that are believed to underlie normal gastrulation. We also found that NRX can inhibit Dvl‐induced up‐regulation of c‐Jun phosphorylation. These results indicate that NRX plays crucial roles in the Wnt/PCP pathway through Dvl and regulates Xenopus gastrulation movements.

Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis.

Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.

Novel Daple-like protein positively regulates both the Wnt/β-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.

Localization of Thy-1–positive Cells in the Perichondrium During Endochondral Ossification

We elucidated the localization of Thy-1–positive cells in the perichondrium of fetal rat limb bones to clarify the distribution of osteogenic cells in the process of endochondral ossification. We also examined the formation of calcified bone-like matrices by isolated perichondrial cells in vitro. At embryonic day (E) 15.5, when the cartilage primodia were formed, immunoreactivity for Thy-1 was detected in cells of the perichondrium adjacent to the zone of hypertrophic chondrocytes. At E17.5, when the bone collar formation and the vascular invasion were initiated, fibroblast-like cells at the sites of vascular invasion, as well as in the perichondrium, showed Thy-1 labeling. Double immunostaining for Thy-1 and osterix revealed that Thy-1 was not expressed in the osterix-positive osteoblasts. Electron microscopic analysis revealed that Thy-1–positive cells in the zone of hypertrophic chondrocytes came in contact with blood vessels. Perichondrial cells isolated from limb bones showed alkaline phosphatase activity and formed calcified bone-like matrices after 4 weeks in osteogenic medium. RT-PCR demonstrated that Thy-1 expression decreased as calcified nodules formed. Conversely, the expression of osteogenic marker genes Runx2, osterix, and osteocalcin increased. These results indicate that Thy-1 is a good marker for characterizing osteoprogenitor cells.

XSUMO-1 is required for normal mesoderm induction and axis elongation during early Xenopus development

The small ubiquitin‐related modifier (SUMO) is a member of the ubiquitin‐like protein family, and SUMO conjugation (SUMOylation) resembles ubiquitination. Despite many SUMOylation target proteins being reported, the role of this system in vertebrate development remains unclear. We inhibited the function of Xenopus SUMO‐1 (XSUMO‐1) using a morpholino antisense oligo against XSUMO‐1 (XSUMO‐1‐MO) to clarify the role of SUMOylation. XSUMO‐1‐MO inhibited normal axis formation in embryos and elongation of activin‐treated animal caps. The expression of several mesoderm markers was reduced by XSUMO‐1‐MO. We measured activin‐like activity by using a reporter construct containing a multimer of activin‐responsive elements from the Goosecoid promoter, [DE(6x)Luc]. This assay showed that XSUMO‐1‐MO directly inhibited activin/nodal signaling. Furthermore, XSUMO‐1‐MO inhibited ectopic axis formation induced by XSmad2, and XSmad2/4 mRNA could not rescue the axis elongation defect induced by XSUMO‐1‐MO. These results suggested that XSUMO‐1 is required for normal axis elongation, at least partly mediating activin/nodal signaling. Developmental Dynamics 236:2757–2766, 2007.