Shin-ichiro Nishimatsu

Genes for bone morphogenetic proteins are differentially transcribed in early amphibian embryos

We have previously demonstrated that activin, a member of the TGF-beta family, has a potent mesoderm-inducing activity in Xenopus embryos. In the course of screening for activin-related genes from Xenopus, we have cloned cDNAs for Xenopus homologue of BMP-2, -4 and -7. Northern blot analysis revealed that these BMP genes are maternally encoded and differentially regulated after fertilization. Alkaline phosphatase-inducing assay using the recombinant BMP proteins has shown that at least BMP-2 and -4 have similar activity to mammalian counterparts.

Involvement of Frizzled-10 in Wnt-7a signaling during chick limb development

The dorsal ectoderm of the limb bud is known to regulate anterior–posterior patterning as well as dorsal– ventral patterning during vertebrate limb morphogenesis. Wnt‐7a, expressed in the dorsal ectoderm, encodes a key molecule implicated in these events. In the present study, chicken frizzled‐10 (Fz‐10) encoding a Wnt receptor was used to study mechanisms of Wnt‐7a signaling during chick limb patterning, because its expression is restricted to the posterior‐distal region of the dorsal limb bud. Fz‐10 transcripts colocalize with Sonic hedgehog (Shh) in the dorsal side of stages 18–23 chick limb buds. It was demonstrated that Fz‐10 interacts with Wnt‐7a to induce synergistically the expression of Wnt‐responsive genes, such as Siamois and Xnr3, in Xenopus animal cap assays. In the chick limb bud, Fz‐10 expression is regulated by Shh and a signal from the dorsal ectoderm, presumably Wnt‐7a, but not by signals from the apical ectodermal ridge. These results suggest that Fz‐10 acts as a receptor for Wnt‐7a and has a positive effect on Shh expression in the chick limb bud.

Wnt10a Is Involved in AER Formation During Chick Limb Development

The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/β‐catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of β‐catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/β‐catenin signaling pathway. Developmental Dynamics 233:282–287, 2005.

Activin as a cell differentiation factor

Activin, originally discovered as a polypeptide hormone that is capable of stimulating follicle-stimulating hormone secretion from pituitary cells in vitro, has recently been found to have a much wider range of biological activities. There are a number of reports of activin action as a cell differentiation factor on various types of cells rather than as a modulator of hormone secretion, as predicted initially, based on its structural similarity to transforming growth factor-beta. Studies of the distribution of activin and its receptor in a variety of tissues and its wide-ranging actions clearly illustrates its multifunctional properties. In particular, activin has been shown to be a potential regulator of early development of Xenopus laevis. Observation of activin effect in embryogenesis is of general importance to our understanding of the role of the family of growth factors in developmental processes.

Bone morphogenetic protein-3 family members and their biological functions.

Bone morphogenetic protein-3 and 3b (BMP-3 and BMP-3b) together represent a unique subgroup of the BMP family. BMP-3b shares 82% amino acid identity with BMP-3 in the mature region (ligand domain), but only 37% in the pro-region (pro-domain). In osteoblasts, BMP-3 and 3b have similar antagonistic activity against BMP-2, but they are differentially regulated. In developing embryos, BMP-3 and 3b have different dorsalizing activities. BMP-3b triggers secondary head formation in an autonomous manner, whereas BMP-3 induces aberrant tail formation. Loss-of-function analysis demonstrates that coordinated activity of xBMP-3b and cerberus, a head inducer, are required for head formation in Xenopus embryos. At the molecular level, BMP-3b antagonizes both nodal-like proteins (Xnr1 and derriere) and ventralizing BMPs (BMP-2 and ADMP), whereas BMP-3 only antagonizes ventralizing BMPs. Moreover, BMP-3b, but not BMP-3, associates with the monomeric form of Xnr1, a nodal-like protein. These molecular features of BMP-3 and 3b are due to their distinct pro-regions. These findings suggest that the processing of precursor regions and assembly of BMP-3 and 3b are important in various developmental processes and organogenesis.

Expression of tbx20 RNA during chick heart development

The T‐box gene family encodes a set of transcription factors that are involved in various developmental processes. We isolated tbx20 gene from chick embryos and examined in detail its expression patterns during heart development. In situ hybridization showed that tbx20 was expressed in the lateral plate mesoderm and subsequently in the primitive heart tube. At stages of looped heart, tbx20 was localized in the outflow tract (OT) and atrioventricular (AV) canal, in which valvuloseptal endocardial cushion develops. At later stages, although tbx20 was expressed predominantly in the nascent right ventricle, transcripts of tbx20 were down‐regulated in the left ventricle. These results suggest that tbx20 may play important roles in a variety of developmental processes in cardiogenesis, such as chamber‐specification and septation. Developmental Dynamics 230:576–580, 2004.

Coordination of BMP-3b and cerberus is required for head formation of Xenopus embryos

Bone morphogenetic proteins (BMPs) and their antagonists are involved in the axial patterning of vertebrate embryos. We report that both BMP-3b and BMP-3 dorsalize Xenopus embryos, but act as dissimilar antagonists within the BMP family. BMP-3b injected into Xenopus embryos triggered secondary head formation in an autonomous manner, whereas BMP-3 induced aberrant tail formation. At the molecular level, BMP-3b antagonized nodal-like proteins and ventralizing BMPs, whereas BMP-3 antagonized only the latter. These differences are due to divergence of their pro-domains. Less BMP-3b than BMP-3 precursor is proteolytically processed in embryos. BMP-3b protein associated with a monomeric form of Xnrl, a nodal-like protein, whereas BMP-3 did not. These molecular features are consistent with their expression profiles during Xenopus development. XBMP-3b is expressed in the prechordal plate, while xBMP-3 is expressed in the notochord. Using antisense morpholino oligonucleotides, we found that the depletion of both xBMP-3b and cerberus, a head inducer, caused headless Xenopus embryos, whereas the depletion of both xBMP-3 and cerberus affected the size of the somite. These results revealed that xBMP-3b and cerberus are essential for head formation regulated by the Spemann organizer, and that xBMP-3b and perhaps xBMP-3 are involved in the axial patterning of Xenopus embryos.

Identification of chick frizzled-10 expressed in the developing limb and the central nervous system.

We have identified chick frizzled (Fz)-10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensens node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior-dorsal region of the distal mesenchyme, and gradually expands to the anterior-distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog (Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.

Multiple genes for Xenopus activin receptor expressed during early embryogenesis

Four distinct cDNAs for activin receptor designated as XSTK2, 3, 8 and 9 have been cloned from a Xenopus laevis cDNA library. The protein structures deduced from the cDNAs have shown that they all have a putative extracellular ligand‐binding domain, a single transmembrane domain and cytoplasmic Ser/Thr kinase domain, except that XSTK2 is extremely similar to the XSTK3 gene but lacks a carboxyl‐terminal part of the kinase motif. Northern blot analysis showed that all transcripts are maternally inherited. The levels or transcript for XSTK2, 3 and 8 appeared to fluctuate during early development while those for XSTK9 maintain constant.

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3P104L) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-β) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-β type I receptor (TβRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-β1, -β2, and -β3 signaling. Here, we show that a TβRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-β1, as well as CAV3P104L. Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3P104L mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-β family members in muscle. These data indicate that both TGF-β-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TβRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-β signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.