Mammalian Twisted Gastrulation Is Essential for Skeleto-Lymphogenesis

Abstract

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4).However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.Transforming growth factor ␤ (TGF-␤) superfamily members bone morphogenetic proteins (BMPs) are critical developmental regulators. Mutations in TGF-␤ family ligands, receptors, and signal transducers such as SMADs are associated with a number of human diseases. TSG was identified in Drosophila as one of the seven zygotic genes that govern the fate of dorsal cells in Drosophila embryos (37). TSG encodes a secreted, cysteine-rich protein that modulates the activity of the Decapentaplegic (DPP) protein, which corresponds to vertebrate BMP-4, and mutations in TSG result in defects of dorsal midline structures called amnioserosa in Drosophila (20). In searching for essential soluble factors produced from the aorta-gonad-mesonephros (AGM) region where definitive hematopoiesis arises (21), we employed the retrovirus-mediated signal sequence trap method previously developed (16), using mRNA from the AGM region of the 10.5-day-postcoitum (dpc) mouse embryo, and isolated a mouse homologue of Drosophila TSG. In 2000, Xenopus twisted gastrulation (TSG) was found to bind directly to BMP-4 to promote BMP-4 signaling by regulating the extracellular availability of BMP-4 (25). Since the dorsoventral axis is inverted between Drosophila and vertebrates and ventralizing factor BMP-4 is essential for mesoderm formation (33) and hematopoietic stem cell (HSC) survival (4), we speculated that TSG may also be involved in ventralization and mesoderm-derived organogenesis, including hematopoiesis, in mammals. Meanwhile, four groups using fine molecular analyses reported that TSG acts rather as a BMP-4 (DPP) antagonist by forming a ternary complex of TSG, BMP-4, and BMP-4 antagonist Chordin or by collaborating with a protease, Tolloid, to generate a Supersog (truncated stable form of Sog [Chordin]) in fly, fish, and frog (6,28,29,35). Thus, it is controversial whether TSG acts...

CCAAT/Enhancer-binding protein homologous protein (CHOP) decreases bone formation and causes osteopenia

“…It is not unusual for intracellular and extracellular proteins to be necessary for a biological effect, but when expressed in excess to be inhibitory. For example, Hes-1 is necessary for adipogenesis, but its overexpression inhibits adipogenesis, twisted gastrulation and connective tissue growth factor are necessary for BMP effects on osteoblastogenesis, but when in excess they can be inhibitory [48][49][50][51]. Similarly, CHOP is necessary for normal osteoblastic function, but when in excess, and under specific conditions in vivo, it may interact with intracellular proteins necessary for osteoblastic function and become inhibitory.…”

von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

“…Our result indicates that this inhibition must be enhanced by the presence of Tsg. Recent results from Tsg-deficient mice indicated a critical role of Tsg in skeletal development of vertebrates (47)(48)(49)(50). Tsg has also been shown to be an important modulator of BMP-regulated cartilage development and chondrocyte differentiation (51).…”

Disruption of Sept6, a Fusion Partner Gene of MLL, Does Not Affect Ontogeny, Leukemogenesis Induced by MLL-SEPT6, or Phenotype Induced by the Loss of Sept4

“…Mice were euthanized, and their tissue samples were analyzed. Circulating blood cells were counted, and tissue specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin as described previously (37). Cytospin preparations of bone marrow cells and blood smears were stained with Wright-Giemsa or peroxidase stain to assess cell morphology.…”

“…The targeting vector was linearized by KpnI digestion at the 3Ј end of the tk cassette and electroporated into E14-1 male embryonic stem (ES) cells, followed by selection with G418 and ganciclovir, as previously described (37). To detect the correct homologous recombination, screening by a Southern blot analysis of BamHI-or SphI-digested genomic DNA from the ES cells was done as previously described (35) by using a 5Ј probe or a 3Ј probe (Fig.…”

“…A 9.1-kb genomic clone carrying the third exon of Sept6 was obtained by screening the genomic library cloned in phage (Stratagene), which was derived from mouse strain 129/SvJ as described previously (37,56). To construct the targeting vector, a 1.1-kb XhoI-BamHI fragment of pMC1NeoPolyA (Stratagene) harboring the neomycin resistance gene (Neo) flanked in the same orientation by two fragments derived from the genomic clone, a 4.0-kb SacI-BstXI fragment at the 5Ј end and a 1.5-kb BamHI-BamHI fragment at the 3Ј end, was introduced into a region between the SacI and ClaI sites of the modified pBluescript vector lacking the XhoI site.…”