Tsutomu Nohno

Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybridization

Hepatocyte growth factor (HGF) has been demonstrated to be synthesized and secreted by non-parenchymal liver cells for liver regeneration after hepatic injury. We performed in situ hybridization to identify HGF-producing cell types in rat liver hepatitis induced by administrating carbon tetrachloride as a hepatotoxin. We found that transcripts of the HGF gene are localized in the Kupffer and endothelial cells in normal livers and increased remarkably in the Kupffer cells of the damaged livers. Thus, HGF is concluded to be synthesized in the Kupffer and endothelial cells to repair the liver tissue in paracrine fashion. No significant increase in the transcripts of the HGF gene was observed in livers after partial hepatectomy, indicating that a mechanism on liver regeneration after the hepatectomy differs from that on liver repairs. Since the HGF gene expression was also found in lung and kidney, HGF may be a ubiquitous factor for tissue repairs.

Identification of a Human Type II Receptor for Bone Morphogenetic Protein-4 That Forms Differential Heteromeric Complexes with Bone Morphogenetic Protein Type I Receptors

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-β-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.

Wnt regulation of chondrocyte differentiation

The Wnt family of growth factors are important regulators of several developmental processes including skeletogenesis. To further investigate the role of Wnts we analysed their expression in the developing chick limb and performed functional analyses in vivo and in vitro. We found that Wnt5b and Wnt11 are restricted within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a is found in the joints and perichondrium, while Wnt4 is expressed in the developing joints and, in some bones, a subset of the hypertrophic chondrocytes. These Wnts mediate distinct effects on the initiation of chondrogenesis and differentiation of chondrocytes in vitro and in vivo. Wnt4 blocks the initiation of chondrogenesis and accelerates terminal chondrocyte differentiation in vitro. In contrast, Wnt5a and Wnt5b promote early chondrogenesis in vitro while inhibiting terminal differentiation in vivo. As Wnt5b and Wnt11 expression overlaps with and appears after Indian hedgehog (Ihh), we also compared their effects with Ihh to see if they mediate aspects of Ihh signalling. This showed that Ihh and Wnt5b and Wnt11 control chondrogenesis in parallel pathways.

Wnt signalling regulates myogenic differentiation in the developing avian wing

The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and ΔLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.

Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full‐coding sequences of chicken Wnt‐5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt‐4 and Wnt‐11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt‐5a expression is detected in the limb‐forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal–distal axis at stages 20–28 and also along the anterior–posterior axis during early stages. It disappeared in the cartilage‐forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt‐4 and Wnt‐11, other members of the Wnt‐5a‐subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt‐4 was expressed in the articular structure and Wnt‐11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt‐5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt‐11 expression was down‐regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt‐5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt‐5a is involved in pattern formation along the proximal–distal axis by regulation of chondrogenic differentiation.

Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ)

SummaryThe glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln+ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), an indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.

Expression pattern of acidic and basic fibroblast growth factor genes in adult rat eyes

Although the retinal angiogenic and mitogenic factors have been identified to be acidic and basic fibroblast growth factors (aFGF and bFGF), little information has so far been available about the cells producing them and their function in retinal tissues. We found, by in situ hybridization, that the expression pattern of the aFGF gene differed remarkably from that of the bFGF gene in adult rat eyes. Our results demonstrated that the aFGF gene was produced by photoreceptor visual cells, neuronal cells in the inner nuclear layer and ganglion cells of the retina, in addition to pigment epithelial cells of the choroid, iris and ciliary body, and epithelial cells of the cornea, conjunctiva and lens, while bFGF was synthesized solely by the photoreceptor visual cells.

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.

Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells.

Background Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown. Results Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation. Conclusions These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.

Expression of retinoic acid receptor genes in neural crest-derived cells during mouse facial development

Retinoic acid (RA) is known as a teratogen that induces abnormalities in facial structures which are made up mainly of neural crest‐derived mesenchyme. We investigated expression patterns of RA receptor (RAR) genes (subtypes α, β, γ) during mouse facial development. The expression of the rarβ gene is specific for the mesenchyme around developing eyes and nose, whereas the RARγ gene is expressed in the mesenchyme differentiating to facial cartilages and bones. In contrast, the RARα gene is expressed weakly and uniformly over the facial region. These results suggest that crucial roles of endogenous RA in facial development depend on differential functions of the RAR subtypes.