Ectopic expression of Ptf1a induces spinal defects, urogenital defects, and anorectal malformations in Danforth's short tail mice.

Danforth's short tail (Sd) is a semidominant mutation on mouse chromosome 2, characterized by spinal defects, urogenital defects, and anorectal malformations. However, the gene responsible for the Sd phenotype was unknown. In this study, we identified the molecular basis of the Sd mutation. By positional cloning, we identified the insertion of an early transposon in the Sd candidate locus approximately 12-kb upstream of Ptf1a. We found that insertion of the transposon caused overexpression of three neighboring genes, Gm13344, Gm13336, and Ptf1a, in Sd mutant embryos and that the Sd phenotype was not caused by disruption of an as-yet-unknown gene in the candidate locus. Using multiple knockout and knock-in mouse models, we demonstrated that misexpression of Ptf1a, but not of Gm13344 or Gm13336, in the notochord, hindgut, cloaca, and mesonephros was sufficient to replicate the Sd phenotype. The ectopic expression of Ptf1a in the caudal embryo resulted in attenuated expression of Cdx2 and its downstream target genes T, Wnt3a, and Cyp26a1; we conclude that this is the molecular basis of the Sd phenotype. Analysis of Sd mutant mice will provide insight into the development of the spinal column, anus, and kidney.

A Cre knock-in mouse line on the Sickle tail locus induces recombination in the notochord and intervertebral disks.

Sickle tail (Skt) was originally identified by gene trap mutagenesis in mice, and the trapped gene is highly expressed in the notochord, intervertebral discs (IVD), and mesonephros. Here, we report the generation of Skt(cre) mice expressing Cre recombinase in the IVD due to target insertion of the cre gene into the Skt locus by recombinase-mediated cassette exchange. Crossing a conditional lacZ Reporter (R26R), Cre expression from the Skt(cre) allele specifically activates ß-galactosidase expression in the whole notochord from E9.5 onwards. In E15.5 Skt(cre);R26R embryos, reporter activity was detected in the nucleus pulposus and in a portion of the annulus fibrosus, resulting in expansion of Cre-expressing cells in the adult IVD. Reporter activity was also seen in the Skt(cre);R26R mesonephros at E15.5. These results suggest that Skt(cre) mice are useful for exploring the fate specification of notochordal cells and creating models for IVD-related skeletal diseases.

The floor plate is sufficient for development of the sclerotome and spine without the notochord.

Danforth'sshort-tail (Sd) mouse is a semi-dominant mutation affecting the development of the vertebral column. Although the notochord degenerates completely by embryonic day 9.5, the vertebral column exists up to the lumber region, suggesting that the floor plate can substitute for notochord function. We previously established the mutant mouse line, Skt(Gt), through gene trap mutagenesis and identified the novel gene, Skt, which was mapped 0.95cM distal to the Sd locus. Taking advantage of the fact that monitoring notochordal development and genotyping of the Sd locus can be performed using the Skt(Gt) allele, we assessed the development of the vertebra, notochord, somite, floor plate and sclerotome in +-+/+-Skt(Gt), Sd-+/+-+, Sd-Skt(Gt)/+-+, Sd-Skt(Gt)/+-Skt(Gt), Sd-+/Sd-+ and Sd-Skt(Gt)/Sd-Skt(Gt) embryos. In Sd homozygous mutants with a C57BL/6 genetic background, the vertebral column was truncated in the 6th thoracic vertebra, which was more severe than previously reported. The floor plate and sclerotome developed to the level of somite before notochord degeneration and the number of remaining vertebrae corresponded well with the level of development of the floor plate and sclerotome. Defects to the sclerotome and subsequent vertebral development were not due to failure of somitogenesis. Taken together, these results suggest that the notochord induced floor plate development before degeneration, and that the remaining floor plate is sufficient for maintenance of differentiation of the somite into the sclerotome and vertebra in the absence of the notochord.

The Skt gene, required for anorectal development, is a candidate for a molecular marker of the cloacal plate.

BACKGROUND AND AIMS: It has been reported that a dorsal cloacal plate defect is associated with anorectal malformations (ARMs); however, there has been very little information reported about the developmental mechanisms involved with cloacal plate formation. Danforth's short tail (Sd) mutant mice show ARMs. In our previous study, the co-presence of Skt ( Gt ) mutation, in which Skt gene is disrupted by the gene-trap vector (p-U8), increased the incidence of ARMs in Sd mutant to 100%. Our aims in this study are determining the Skt expression around the cloaca during the anorectal development and demonstrating the role of Skt gene in ARMs. METHODS: Embryos, normal controls [+Skt ( Gt )/+Skt ( Gt )] and ARMs models [Sd Skt ( Gt )/+Skt ( Gt )], from embryonic day (E) 9.5 to E12.5, were evaluated with X-gal staining. RESULTS: In control embryos, Skt expression was detected both in the endoderm and ectoderm of the cloacal plate from E9.5 onward. At E12.5, Skt expression was also detected in the mesenchyme neighboring the dorsal cloacal plates. In [Sd Skt ( Gt )/+Skt ( Gt )] mutant embryos, the cloacal plates failed to extend proximodistally and, consequently, the dorsal part of cloacal plate was defective at E11.5. Skt expressing cells were detected in the shortened cloacal plate and in the thickened mesenchyme dorsal to it. CONCLUSIONS: We showed the spatial and temporal expression of Skt gene in the cloacal plate formation. This gene could be a marker for the cloacal plate during the anorectal development. Furthermore, Skt was considered to be associated with the embryogenesis of ARMs.

Synoviocyte-derived angiopoietin-like protein 2 contributes to synovial chronic inflammation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis of the diarthrodial joints. Several proinflammatory cytokines derived from both infiltrating inflammatory cells and activated resident cells within the RA joint play a fundamental role in the processes that cause inflammation. However, anticytokine treatment is beneficial but not curative, the effects are only partial, and nonresponses are common. Therefore, an effort has been made to identify other key regulators of inflammation in articular structures to develop new therapies to suppress synovial inflammation and joint destruction in RA. Adipose tissue-derived angiopoietin-like protein 2 (Angptl2) activates an inflammatory cascade in endothelial cells and induces chemotaxis of monocytes/macrophages in obesity, resulting in initiation and propagation of inflammation within adipose tissues and obesity-related metabolic diseases. Angptl2 mRNA and protein are abundantly expressed in hyperplastic rheumatoid synovium of RA patients, especially in fibroblast-like and macrophage-like synoviocytes, but not in B and T lymphocytes. Angptl2 concentration in joints of RA patients was also significantly increased in comparison with patients with osteoarthritis, which in comparison with RA represents a significantly lower inflammatory grade form of arthritis. Notably, Angptl2 promoted increased chemotactic activities of CD14+CD16- monocytes from synovial fluid of RA patients. Therefore, Angptl2 acts as an important rheumatoid synovium-derived inflammatory mediator in RA pathogenesis.

Association of the tag SNPs in the human SKT gene (KIAA1217) with lumbar disc herniation.

Lumbar disc herniation (LDH) is one of the most common musculo-skeletal diseases. Recent studies have indicated that LDH has strong genetic determinants, and several susceptibility genes have been reported to associate with LDH; however, its etiology and pathogenesis still remain unclear. KIAA1217 (alias SKT, the human homolog of murine Skt [Sickle tail]) is a good candidate for an LDH susceptibility gene because SKT is specifically expressed in nucleus pulposa of intervertebral discs (IVDs) in humans and mice, and Skt(Gt) mice, which are established through a large-scale gene-trap mutagenesis, exhibit progressive, postnatal onset abnormality of the IVDs. Here, we report the association of SKT with LDH. Using tag SNPs, we examined the association in two independent Japanese case-control populations and found a significant association with SKT rs16924573 in the allele frequency model (p = 0.0015). The association was replicated in a Finnish case-control population (p = 0.026). The combined p value of the two population by meta-analysis is 0.00040 (OR, 1.34; 95% CI, 1.14-1.58). Our data indicate that SKT is involved in the etiology of LDH.

Early pre-implantation lethality in mice carrying truncated mutation in the RNA polymerase 1-2 gene.

Ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) is an important initial step for the production of ribosomes. The RNA polymerase 1-2 (Rpo1-2) gene is comprised of 15 exons and encodes 1135 amino acids (aa) of the second largest subunit in Pol I. In a gene trap screen, we have identified an insertional mutation (Rpo1-2(Gt)) in the 14th exon of Rpo1-2, resulting in a truncation of 312aa from the C-terminal. In Rpo1-2(Gt/Gt) embryos, the synthesis of rRNA was severely impaired. Rpo1-2(Gt/Gt) embryos could develop to the morula stage, and thereafter displayed nucleolus disruption and apoptotic cell death. These results indicate that the loss of rDNA transcription induced nucleolar structure disorganization and apoptosis in preimplantation embryos.

A novel murine gene, Sickle tail, linked to the Danforth's short tail locus, is required for normal development of the intervertebral disc.

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforth's short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.