Ample Pairs

We show that the ample degree of a stable theory with trivial forking is preserved when we consider the corresponding theory of belles paires, if it exists. This result also applies to the theory of $H$-structures of a trivial theory of rank $1$.Comment: Research partially supported by the program MTM2014-59178-P. The second author conducted research with support of the programme ANR-13-BS01-0006 Valcomo. The third author would like to thank the European Research Council grant 33882

Loss of <i>mp29</i> resulted in aberrant embryonic development.

<p>The following results were repeated with three independent experiments. (A) Morphology of embryos cultured <i>in vitro</i>. Embryos were collected from uterus at E3.5 and cultured <i>in vitro</i> to E6.5. Embryos were hatched at E6.5 in <i>mp29^+/+</i> and <i>mp29^GT/+</i> embryos. (B) Embryos were isolated from E7.5 to E9.5 for hematoxylin–eosin staining. <i>mp29^+/+</i> and <i>mp29^GT/+</i> embryos at E7.5 had normal gastrulation. ee: embryonic ectoderm. m: mesoderm. ve: visceral endoderm. (C) Whole embryo extracts at E7.5 were lysed in RIPA buffer for Western blotting analysis. Hsp90 was used as a loading control.</p

<i>mp29</i> transgene complemented the deficiency of mp29.

<p><i>mp29^GT/+</i> mice were mated with <i>mp29^Tg/+</i> mice to generate <i>mp29^GT/+mp29^Tg/+</i> littermates. Inbreed of <i>mp29^GT/+mp29^Tg/+</i> male mice with <i>mp29^GT/+mp29^+/+</i> female mice gave birth to <i>mp29^GT/GTmp29^Tg/+</i> mice. (A) Genotyping of complemented mice by PCR analysis. NSPF1/NeoES PCR products indicated the presence of U3NeoSV1 vector. NSPF1/NSPR1 primers were used to determine whether both of alleles were inserted with U3NeoSV1 vector. mPGKF1/mp29R1 PCR products indicated the presence of <i>mp29</i> transgene with a product of 500 bp. Tg refers to the hemizygous <i>mp29</i> transgene. (B) The genotyping of 76 complemented offsprings was examined. Eight pups exhibited homozygously interrupted genotype but with mp29 transgene (<i>mp29^GT/GTmp29^Tg/+</i>). There were no <i>mp29^GT/GT</i> mice that could survive without the presence of <i>mp29</i> transgene. (C) Tissue extracts of the livers from complemented mice were prepared for Western blot analysis. Note that normal expression levels of α-tubulin, Chk1, and Chk2 were restored in <i>mp29^GT/GTmp29^Tg/+</i> mice.</p

Decreased α-tubulin and Chk1 expression in <i>mp29^GT/GT</i> embryo.

<p>Unless otherwise stated, the following results were repeated with three independent experiments. (A) Whole embryo extracts at E7.5 were prepared for Western blotting analysis. (B) RT-PCR for embryos prepared from E7.5. Note lower levels of α-tubulin, Chk1, and Chk2, but not β-tubulin, in <i>mp29^GT/GT</i> embryos. GAPDH was used as an input control. (C) Total RNAs from embryos at E7.5 were extracted and first strand cDNAs were transcribed for quantitative real-time PCR analysis to determine the relative pre-mRNA and mRNA levels of α-tubulin and Chk1. GAPDH was used as a normalized control. (D) NIH3T3 cells were co-transfected with siRNAs and E1A splicing reporter for <i>in vivo</i> alternative splicing assay. Total RNAs were isolated and subjected to RT-PCR. The splicing products were quantitatively analyzed with three independent experiments. GAPDH was used as a normalized control. * indicated <i>p</i><0.05 in one-way ANOVA F-test.</p

Interruption of mouse <i>mp29</i> gene by a gene trap vector.

<p>(A) Schematic of the targeting vector U3NeoSV1 within exon 2 and 3 of <i>mp29</i> gene. (B) Genotyping of mouse tail DNAs by PCR. NSPF1/NeoES primers were used to examine the presence of U3NeoSV1 vector in <i>mp29</i> gene with a product of 250 bp. NSPF1/R1 primers were used to determine if both of alleles were inserted with U3NeoSV1 vectors with a product of 300 bp. 18s RNA was used as a control. +/+: Wild type mice; GT/+: Heterozygous mice with U3NeoSV1 inserted in one allele. (C) Of 86 offspring mice, 28 pups were identified as wild type (+/+) and 58 pups were heterozygous (GT/+).</p

Analysis of embryos from intercrosses of <i>mp29^GT/+</i> mice.

<p>Analysis of embryos from intercrosses of <i>mp29^GT/+</i> mice.</p

Impaired G2/M checkpoint in <i>mp29^GT/GT</i> embryos.

<p>Unless otherwise stated, the following results were repeated with three independent experiments. (A) <i>mp29^+/+</i>, <i>mp29^GT/+</i>, and <i>mp29^GT/GT</i> blastocysts at E3.5 were treated with aphidicolin (1 µM) for 8 h, incubated with nocodazole (0.1 µg/ml) for an additional 16 h, and then fixed and stained with antibody specific to phosphohistone H3 at Ser10. (B) <i>mp29^+/+</i>, <i>mp29^GT/+</i>, and <i>mp29^GT/GT</i> blastocysts at E3.0 were irradiated with UV light (30 J/m²), incubated with nocodazole for 16 h, and then co-immunostained with anti-phosphohistone H3 at Ser10 antibody and TUNEL fluorescein. The genotypes of each embryo were determined by PCR. Images were obtained using Leica DM6000B microscope.</p

Inhibition of CaMKII and MEK1/2 reduces M6Ab early neurite outgrowth in nerve growth factor (NGF)-differentiated PC12 cells.

<p>(A) PC12 cells were transfected with the pcDNA3-M6Ab-GFP or pcDNA3-GFP plasmids. After transfection, PC12 cells were differentiated with 100 ng/ml NGF for 72 h in the presence of 10 µM KN-62 or 10 µM U1026 (Con; 0.1% DMSO). Cells were fixed, and images were taken with a Zeiss LSM510 laser scanning confocal microscope. (B) Quantification of the total number of neurites, total length of neurites, and filopodium-like processes in a 20-µm neurite length. Results are expressed as the mean ± SD of at least 40∼50 neurites. At least three independent experiments were analyzed. *Significant difference compared with the respective control of WT M6Ab overexpression (P<0.05). Scale bars, 10 µm.</p

Overexpression of zM6Ab can induce neurite outgrowth and filopodia in PC12 and COS-1 cells.

<p>(A) PC12 cells were transfected with the control pcDNA3-GFP or pcDNA3-M6Ab-GFP or pcDNA3-M6Ab-T166A-GFP plasmids. Twenty-four hours after transfection, cells were treated with nerve growth factor (NGF) (100 ng/ml) for 2 days. Cells were then fixed, and images were taken with a Zeiss LSM510 laser scanning confocal microscope. The insets are the 2× magnified images of the boxed areas. (B) Quantification of the total number of neurites, total length of neurites, and filopodium-like processes in a 20-µm neurite length. * indicates a significant difference compared with the respective control of GFP (P<0.05). (C) COS-1 cells were transfected with the control pcDNA3-GFP or pcDNA-M6Ab-GFP or pcDNA3-M6Ab-T166A-GFP plasmids. Cells were fixed, and images were taken with a Zeiss LSM510 laser scanning confocal microscope. Scale bars, 10 µm.</p

The Nogo-C2/Nogo Receptor Complex Regulates the Morphogenesis of Zebrafish Lateral Line Primordium through Modulating the Expression of <i>dkk1b</i>, a Wnt Signal Inhibitor

<div><p>The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in <i>p75</i>, <i>TROY</i>, and <i>Nogo-C2/NgRH1a</i> morphants. Notably, the expression levels of <i>pea3</i>, a downstream marker of Fgf signaling, and <i>dkk1b</i>, a Wnt signaling inhibitor, were both decreased in <i>p75</i>, <i>TROY</i>, and <i>Nogo-C2/NgRH1a</i> morphants; moreover, <i>dkk1b</i> mRNA injection could rescue the defects in neuromast formation resulting from knockdown of <i>p75</i> or <i>TROY</i>. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and <i>dkk1b</i> expression, thereby ensuring stable organization of the PLL primordium.</p></div