Tadayoshi Hayata

OPN deficiency suppresses appearance of odontoclastic cells and resorption of the tooth root induced by experimental force application.

Osteopontin (OPN) is a major non‐collagenous bone matrix protein implicated in the regulation of cell function. Although OPN is rich in the cementum of the tooth, the significance of OPN in this tissue is not understood. Tooth root resorption is the most frequent complication of orthodontic tooth movement (TM). The objective of this study was to examine the pathophysiological role of OPN in cementum of the tooth root. For this purpose, the upper right first molar (M1) in OPN‐deficient and wild‐type (WT) mice was subjected to mechanical force via 10 gf NiTi coil spring while the left side molar was kept intact to serve as an internal control. Micro‐CT section and the level of tartrate resistant acid phosphatase (TRAP)‐positive cells on the tooth root surface defined as odontoclasts were quantified at the end of the force application. In WT mice, force application to the tooth caused appearance of odontoclasts around the mesial surface of the tooth root resulting in tooth root resorption. In contrast, OPN deficiency significantly suppressed the force‐induced increase in the number of odontoclasts and suppressed root resorption. This force application also induced increase in the number of TRAP‐positive cells in the alveolar bone on the pressure side defined as osteoclasts, while the levels of the increase in osteoclastic cell number in such alveolar bone were similar between the OPN‐deficient and WT mice. These observations indicate that OPN deficiency suppresses specifically tooth root resorption in case of experimental force application. J. Cell. Physiol. 214: 614–620, 2008.

JunD Suppresses Bone Formation and Contributes to Low Bone Mass Induced by Estrogen Depletion

JunD is an activator protein‐1 (AP‐1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD‐deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency‐induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD‐deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy‐induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c‐jun, fra‐1, and fra‐2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion‐induced osteopenia via its action to suppress bone formation and to enhance bone resorption. J. Cell. Biochem. 103: 1037–1045, 2008.

EXPRESSION OF BRACHYURY-LIKE T-BOX TRANSCRIPTION FACTOR, XBRA3 IN XENOPUS EMBRYO

Abstract The XenopusBrachyury-like Xbra3 gene is a novel T-box gene that is closely associated with XenopusBrachyury. The expression pattern of Xbra3 during development is similar to that of Xbra. During gastrulation Xbra3 is expressed in the marginal zone, with a gradient of increasing expression from ventral to dorsal. In the early neurula stage Xbra3 is expressed in the notochord and posterior mesoderm, but by the tailbud stage its expression is restricted to the forming tailbud and the posterior portion of the notochord.

EXPRESSION OF XENOPUS T-BOX TRANSCRIPTION FACTOR, TBX2 IN XENOPUS EMBRYO

Abstract We report here the cloning and expression of the Xenopus orthologue of the T-box transcription factor gene Tbx2 (optomotor-blind in Drosophila). Tbx2 is first detected in the ventral mesodermal cells just above the yolk plug at late gastrula. At the neurula stage it is strongly expressed in the cement gland, dorsal root ganglia, and otic vesicle region. At the tailbud stage strong Tbx2 expression is observed in the dorsal part of the optic cup and trigeminal ganglia, and it is also expressed in the branchial arches, heart anlage, nasal pit, proctodeum, and the region around the pronephros.

Xenopus cDNA microarray identification of genes with endodermal organ expression

The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ‐related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior regions. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major organs, revealed 915 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development. Developmental Dynamics 236:1633–1649, 2007.

The Role of XBtg2 in Xenopus Neural Development

In early neural development, active cell proliferation and apoptosis take place concurrent with cell differentiation, but how these processes are coordinated remains unclear. In this study, we characterized the role of XBtg2 in Xenopus neural development. XBtg2 transcripts were detected at the edge of the anterolateral neural plate and the neural crest region at the midneurula stage, and in eyes and in part of the neural tube at the tailbud stage. Translational inhibition of XBtg2 affected anterior neural development and impaired eye formation. XBtg2 depletion altered the expression patterns of the early neural genes, Zic3 and SoxD, at the midneurula stage, but not at the early neurula stage. At the midneurula stage, XBtg2-depleted embryos exhibited a marked decrease in the expression of anterior neural genes, En2, Otx2, and Rx1, withoutany changes in neural crest genes, Slug and Snail, or an epidermal gene, XK81. These results suggest that XBtg2 is required for the differentiation of the anterior neural plate from the midneurula stage, but not for the specification of the fate and patterning of the neural plate. XBtg2-depleted embryos also exhibited an increase in both proliferation and apoptosis in the anterior neural plate; however, the altered expression patterns of neural markers were not reversed by inhibition of either the cell cycle or apoptosis. Based on these data, we propose that XBtg2 plays an essential role in the anterior neural development, by regulating neural cell differentiation, and, independently, cell proliferation and survival.

XBtg2 is required for notochord differentiation during early Xenopus development

The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochord tissue, which did not undergo vacuolation. The XBtg2‐depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5‐D‐4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.

Overexpression of the secreted factor Mig30 expressed in the Spemann organizer impairs morphogenetic movements during Xenopus gastrulation

The Spemann organizer secretes several antagonists of growth factors during gastrulation. We describe a novel secreted protein, Mig30, which is expressed in the anterior endomesoderm of the Spemann organizer. Mixer-inducible gene 30 (Mig30) was isolated as a target of Mixer, a homeobox gene required for endoderm development. The Mig30 gene encodes a secreted protein containing a cysteine-rich domain and an immunoglobulin-like domain that belongs to the insulin-like growth factor-binding protein family. Overexpression of Mig30 in the dorsal region results in the retardation of morphogenetic movements during gastrulation and leads to microcephalic embryos. Overexpression of Mig30 also inhibits activin-induced elongation of ectodermal explants without affecting gene expression patterns in mesoderm and endoderm. These results suggest that Mig30 is involved in the regulation of morphogenetic movements during gastrulation in the extracellular space of the Spemann organizer.

Ciz, a transcription factor with a nucleocytoplasmic shuttling activity, interacts with C-propeptides of type I collagen.

Ciz is a zinc finger transcription factor with nucleocytoplasmic shuttling activity. Ciz-deficient mice show high bone mass phenotype. As a first step to address how Ciz suppresses bone formation, we examined the binding partners of Ciz based on a yeast two-hybrid screening. While Ciz is an intracellular protein, 47% of the positive clones were genes encoding extracellular matrix proteins, including Col1a1, Col1a2, Fbln2, and Rpsa. In vitro coimmunoprecipitation experiments using in vitro translated proteins revealed direct binding of Ciz-DeltaZF (zinc finger) to C-propeptides of Col1a1 and Col1a2. In vivo association of the transfected Ciz and C-propeptide of Col1a1 was observed in COS-7 cells based on immunoprecipitation. In terms of intracellular localization, overexpressed C-propeptides of Col1 and Ciz were co-localized in nuclei. These results revealed that Ciz interacts with C-propeptides of type I collagen and this association takes place in nuclei.