Sumihare Noji

Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly

Preaxial polydactyly (PPD) is a common limb malformation in human. A number of polydactylous mouse mutants indicate that misexpression of Shh is a common requirement for generating extra digits. Here we identify a translocation breakpoint in a PPD patient and a transgenic insertion site in the polydactylous mouse mutant sasquatch (Ssq). The genetic lesions in both lie within the same respective intron of the LMBR1/Lmbr1 gene, which resides ≈1 Mb away from Shh. Genetic analysis of Ssq reveals that the Lmbr1 gene is incidental to the phenotype and that the mutation directly interrupts a cis-acting regulator of Shh. This regulator is most likely the target for generating PPD mutations in human.

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Pitx2. a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

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.

Role of Pax-5 in the regulation of a mid-hindbrain organizer’s activity

The mes‐metencephalic boundary (isthmus) has been suggested to act as an organizer in the development of the optic tectum. Pax‐5 was cloned as a candidate for regulator of the organizing center. Isthmus‐specific expression of Pax‐5 and analogy with the genetic cascade in Drosophila suggest that Pax‐5 may be at a higher hierarchical position in the gene regulation cascade of tectum development. To examine this possibility, a gain‐of‐function experiment on Pax‐5 was carried out. In ovo electroporation on E2 chick brain with the eucaryotic expression vector that encodes chick Pax‐5 cDNA was used. Not only was a considerable amount of Pax‐5 expressed ectopically in the transfected brain, but irregular bulging of the neuroepithelium was induced in the diencephalon and mesencephalon. At Pax‐5 misexpressing sites, uptake of BrdU was increased. Histological examination of E7 transfected brain revealed that Pax‐5 caused transdifferentiation of diencephalon into the tectum‐like structure. In the bulges of the E7 mesencephalon, differentiation of laminar structure was repressed when compared to the normal side. In transfected embryos, En‐2, Wnt‐1 and Fgf8 were up‐regulated ectopically, and Otx2 was down‐regulated in the diencephalon to mesencephalon. Moreover, Ephrin‐A2, which is expressed specifically in the tectum with a gradient highest at the caudal end, is suggested to be involved in pathfinding of the retinal fibers, and was induced in the bulges. When the mouse Fgf8 expression vector was electroporated, Pax‐5 and chick Fgf8 were also induced ectopically. These results suggest that Pax‐5, together with Fgf8, hold a higher position in the genetic hierarchy of the isthmus organizing center and regulate its activity.

Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice

Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin‐related proteins, and myostatin antibodies. Although follistatin shows potent myostatin‐inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (Kd) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (Kd) of a follistatin‐derived myostatin inhibitor, designated FS I‐I, to activin and myostatin are 64.3 μM and 46.8 nM, respectively. Transgenic mice expressing FS I‐I, under the control of a skeletal muscle‐specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I‐I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I‐I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I‐I mice. These results indicate that myostatin blockade by FS I‐I has a therapeutic potential for muscular dystrophy.—Nakatani, M., Takehara, Y., Sugino, H., Matsumoto, M., Hashimoto, O., Hasegawa, Y., Murakami, T., Uezumi, A., Takeda, S., Noji, S., Sunada, Y., Tsuchida, K. Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice. FASEB J. 22, 477–487 (2008)

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.

Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation.

© 1997 Federation of European Biochemical Societies.

Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.

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.