Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog(Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants,ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range,cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.
Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.
Partial trisomy distal 4q (denoted 4q+) is a human chromosomal disorder caused by duplication of the distal end of the long arm of chromosome 4 (Chr4). This disorder manifests typical phenotypes, including craniofacial, renal, heart and thumb developmental defects. Although these clinical features are likely caused by a dosage imbalance in the gene network involving the trisomic region, the causative gene or genes and the molecular bases are largely unknown. Here, we report mouse Recombination-induced mutation 4 (Rim4) as a model animal of 4q+. The Rim4 genome contains an insertion of a 6.5 Mb fragment from mouse chromosome 8 into chromosome 6. This insertion fragment contains 17 genes, including Hand2, that encode the basic helix-loop-helix transcription factor and is syntenic to the distal end of human Chr4, 4q32.3 to 4q34.1, which is responsible for 4q+. A comparison of phenotypes between patients with Rim4 and 4q+ revealed that Rim4 shows direct parallels with many phenotypes of 4q+ such as craniofacial, heart, cervical vertebra and limb deformities. Rebalancing the gene dosage by a genetic cross with Hand2 knockout mice ameliorated symptoms of the heart and limb deformities of Rim4. Conversely, an increase in copy number of Hand2 in wild-type mice recaptures the heart and limb deformities of Rim4. Our results collectively demonstrate that overdosage of Hand2 is a major cause for at least the limb and heart phenotypes of 4q+ and that mouse Rim4 provides a unique animal model for understanding the molecular bases underlying the complex phenotypes of 4q+.
BackgroundAlveolar soft part sarcoma (ASPS) is an extremely rare metastatic soft tissue tumor with a poor prognosis for which no effective systemic therapies have yet been established. Therefore, the development of novel effective treatment approaches is required. Tyrosine kinases (TKs) are being increasingly used as therapeutic targets in a variety of cancers. The purpose of this study was to identify novel therapeutic target TKs and to clarify the efficacy of TK inhibitors (TKIs) in the treatment of ASPS.Experimental designTo identify novel therapeutic target TKs in ASPS, we evaluated the antitumor effects and kinase activity of three TKIs (pazopanib, dasatinib, and cabozantinib) against ASPS cells using an in vitro assay. Based on these results, we then investigated the phosphorylation activities of the identified targets using western blotting, in addition to examining antitumor activity through in vivo assays of several TKIs to determine both the efficacy of these substances and accurate targets.ResultsIn cell proliferation and invasion assays using pazopanib, cabozantinib, and dasatinib, all three TKIs inhibited the cell growth in ASPS cells. Statistical analyses of the cell proliferation and invasion assays revealed that dasatinib had a significant inhibitory effect in cell proliferation assays, and cabozantinib exhibited marked inhibitory effects on cellular functions in both assays. Through western blotting, we also confirmed that cabozantinib inhibited c-MET phosphorylation and dasatinib inhibited SRC phosphorylation in dose-dependent fashion. Mice that received cabozantinib and dasatinib had significantly smaller tumor volumes than control animals, demonstrating the in vivo antitumor activity of, these substances.ConclusionsOur findings suggest that cabozantinib and dasatinib may be more effective than pazopanib against ASPS cells. These in vitro and in vivo data suggest that c-MET may be a potential therapeutic target in ASPS, and cabozantinib may be a particularly useful therapeutic option for patients with ASPS, including those with pazopanib-resistant ASPS.
The current study clarified the accuracy of a circulating tumor cell (CTC) detection system to diagnose colorectal cancer using blood samples. The system uses the 'polymeric CTC-chip,' (CTC-chip), which is a microfluidic device that is used for CTC isolation. CTCs are considered sensitive diagnostic biomarkers. However, their concentration in the peripheral blood is low and requires highly sensitive and specific capturing techniques. The capture efficiency of the polymeric CTC-chip was first assessed using cell suspensions of the colorectal cancer cell line HCT-116, which was reported as 90.9% in a phosphate-buffered saline suspension and 65.0% in the blood. The CTC-chip was then used to detect CTCs in blood samples obtained from 13 patients with stage II-IV colorectal cancer. On average, the CTCs/ml was lower in patients with stages II and III colorectal cancer (3.3±2.3) than in those with stage IV (7.0±6.2). In patients with stages II-IV, 92% had ≥1 CTC per ml, which was significantly higher than the positive rate (15%) detected using the carbohydrate antigen 19-9 test (CA19-9). Furthermore, CTCs were detected in all patients with stage II and III colorectal cancer, including a number of patients with negative results for the carcinoembryonic antigen (CEA) and CA19-9 tests. With the polymeric CTC-chip detection system, CTCs can be effective cancer markers, particularly for patients with stage II and III colorectal cancer who often exhibit negative conventional serum marker test results. The CTC-chip system may also facilitate the detection of cancer progression based on CTC concentration.
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