Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Representation of the strategy used for transcriptomic analysis in time and space in the dorsal and lateral OB lineages. pCX-GFP plasmid was introduced into neural stem cells (NSCs) residing within the dorsal or lateral V-SVZ and GFP-positive cells were isolated by FACS at different time points after electroporation (Elpo). The mRNA content was analyzed by micro-array . ( B ) Quantification of Vax1 mRNA expression detected by micro-array analysis in dorsal (brown) and lateral (purple) progenies during neurogenesis. ( C–H ) In situ hybridization revealing Vax1 mRNA (in blue) combined with immuno-histochemistry using antibodies detecting (in brown) PAX6 ( C, C’, G ), ASCL1 ( D, D’ ), DLX2 ( E, E’, H ) or KI67 ( F, F’ ) proteins in the V-SVZ ( C–F ) or RMS ( G, H ) at postnatal day 3 ( P3 ). ( C’–F’ ) High magnification of cellular staining in the V-SVZ (area indicated by the yellow bracket in C-F). Arrows ( C’ ): examples of strong PAX6 only positive cell in the dorso-lateral SVZ; blue staining underneath labels cells from a distinct plane. Arrow heads ( E’, F’ ): double positive cells for DLX2 and KI67, respectively. High magnification of the RMS highlights the differential expression of Vax1 and Pax6 along the dorso-ventral axis ( G’,G” ) and the co-localization with Dlx2 ( H’,H” ). ( I ) Schematic representation of gene expression profile in different cell types of the neurogenic sequence. Circular arrow indicates proliferating cells. LV: lateral ventricle, RG: radial glia, TAP: transit amplified precursor, VZ: ventricular zone, SVZ: sub-ventricular zone. D: dorsal, L: lateral, S: septal, V: ventral. Scale bars: 100 µm ( C–F ), 20 µm ( C’–F’ ), 50 µm ( G–H ).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Plasmid Preparation, Isolation, Electroporation, Microarray, Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Quantitative Proteomics, Gene Expression, Sequencing, Amplification

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: Cells were analyzed 15 days after electroporation of lateral V-SVZ progenitors by Cre mRNA in WT or Vax1cKO mice. Data are shown as means ± SD, dots represent individual animals. WT: n = 12, Vax1cKO: n = 17. Figure 2—figure supplement 2—source data 1. Quantification of tdTomato+ PGC in Vax1 mutant.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Electroporation, Mutagenesis

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to determine the expression of microRNAs in Vax1-overexpressing progenitors. PCAG-Vax1 and pCX-GFP were simultaneously introduced into NSCs by electroporation of the lateral wall of postnatal P1 brains. Lateral V-SVZ was dissected out 2 days after electroporation and GFP+ cells were isolated by flow cytometry (FACS) to perform quantitative RT-PCR analysis. ( B ) Quantification of Vax1 mRNA level by qRT-PCR in control and Vax1OE conditions, normalized to beta-actin and reported in Vax1 condition as relative level to control, validating the overexpression of Vax1 after electroporation. ( C ) Quantification of miR-7 expression in both conditions. Expression level of miR-7 was normalized by invariant expression of microRNA let-7a and reported in Vax1 condition as relative level to control. Experiments in B and C were performed in triplicate, and data were obtained from ( B ) two independent biological replications or ( C ) three technical replications. ( D ) Genome browser images representing the chromosomal portions encoding the three MiR-7 loci (depicted in pink). Mir-7–1 lies within an intronic sequence of the Hnrnpk gene whereas MiR-7–2 and MiR-7b reside within intergenic sequences. Vax1-binding sites found in the upstream regulatory region of the three MiR-7 are represented by red boxes. ( E ) Model of cross-regulatory interaction between Vax1 , miR-7, and Pax6 in the lateral V-SVZ to control the number of dopaminergic neurons generated by the neural stem cells regionalized in this aspect. This model is supported by our present data and previous work where it was shown that miR-7 was required to inhibit PAX6 expression in lateral NSCs to produce the correct number of dopaminergic neurons in the postnatal OB. Here, we propose that Vax1 acts upstream of miR-7 by positively regulating its expression and consequently inhibiting PAX6. However, it is also possible that Vax1 directly represses the expression of Pax6 mRNA (dashed line) by acting on its promoter . Additionally, Vax1 is required to generate Calbindin neurons from the ventral aspect of the lateral V-SVZ. Figure 5—source data 1. Quantification of expression level of Vax1 and miR-7 in V-SVZ cells.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Expressing, Electroporation, Isolation, Flow Cytometry, Quantitative RT-PCR, Control, Over Expression, Sequencing, Binding Assay, Generated

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to target lateral V-SVZ NSCs with Cre -mRNA. Tomato+ recombined cells were isolated 2 days after electroporation. ( B ) Vax1 mRNA level quantified by RT-PCR was normalized to beta-actin and reported in Vax1cKO condition as relative level to control (WT).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Isolation, Electroporation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet:

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Sequencing, Recombinant, Plasmid Preparation, SYBR Green Assay, Software, Imaging

Journal: Antibiotics

Article Title: Phage Cocktails with Daptomycin and Ampicillin Eradicates Biofilm-Embedded Multidrug-Resistant Enterococcus faecium with Preserved Phage Susceptibility

doi: 10.3390/antibiotics11091175

Figure Lengend Snippet: Evaluation of phage resistance in R497 and HOU503 at the end of 24 h time kill analyses. Phages are represented in the table as follows: 1, ATCC 113; 2, NV-497; 3, NV-503-01; 4, NV-503-02.

Article Snippet: Against both R497 and HOU503, DAP in combination with phage NV-497 at a multiplicity of infection (MOI) of 0.01 was additive, with an FIC index of 1 ( A), while DAP in the presence of a 4-phage cocktail (ATCC 113, NV-497, NV-503-01, NV-503-2; each at an MOI of 0.01), was synergistic, with an FIC index of 0.5 ( B).

Techniques: