PMC

(a) Heatmap showing the results of deep sequencing analyses. Only microRNAs representing more than 0.1% of total microRNAs in at least one tissue are shown. Number of reads for a microRNA in a given tissue was obtained by averaging the different sample repetitions. Columns describe the ratios between tissues selected for comparison. Proximity of vertical position indicates the similarity of expression profile of detected microRNAs and was determined using the MeV application. (b) Close-up of specific regions of the heat map highlighting the miR-200-family a group of microRNAs preferentially expressed in the OB and the miR-34 family that is induced during ciliogenesis. miR-34a does not cluster in the heatmap due to strong expression in OB glia. (c) Histogram representing the absolute number of reads per tissue obtained in the deep sequencing analyzis for each member of the miR-200 family. All miR-200 family members are exclusively expressed in the OB but not in the stem cell or migratory compartments.

(a) Sagittal section of a GAD67-GFP knock-in mouse forebrain. Diagrams are dot plots of the FACsorting experiment performed on wild-type (left) and GAD67-GFP knock-in mice (right). Three cell populations were sorted from GAD67-GFP knock-in mouse brain and used for subsequent qRT-PCR analyses. (b) qRT-PCR characterization of the three populations. GluR2 is expressed on both, GABAergic neuronal progenitors and fully differentiated neurons. Doublecortin is exclusively expressed in neuronal progenitors. GFP negative cells do not significantly express these neuronal markers. (c) qRT-PCR analysis of the expression of miR-200b and miR-141 in the three sorted populations showing a preferential expression in the GFP-low fraction. (d) qRT-PCR characterization of the two purified cell fractions issued from the MACS experiment designed to discriminate neuronal vs glial fraction from the OB based. Neuronal (NeuN, GluR2, DCX) and glial (GFAP, Olig1, Olig2) markers validate the expected neuronal and glial identities. (e) qRT-PCR analysis demonstrates that miR-200b and miR-141 are enriched in the neuronal fraction. For b-e the qPCR values shown in the histograms result from 2 (b,d) or 3 (c,e) qPCR experiments (4 wells per condition in each experiment) (f) Electroporation of an expression construct driving GFP with regulatory sequences of the human miR-429/miR-200a/miR-200b cluster leads to GFP-labeled cells in the OB. Scale bar: 70 μm. Error bars: sem.

(a) Representation of the two vectors designed to over-express (miR-200-gof) or down-regulate (miR-200-sponge) the expression of all miR-200 family members in parallel (b) Luciferase assay performed on HeLa cells transfected with the Zeb2-UTR vector together with control vectors (control condition), with the miR-200 expression vector alone (miR-200-gof) or with the miR-200 expression vector and the miR-200 sponge plasmid (miR-200-gof + miR-200 sponge condition). Data represent the mean ± s.e.m of values from 4 wells. miR-200 sponge partially rescues the inhibitory activity of the miR-200 expression vector. (c) Fluorescent images showing OB neurons 15 days after lateral co-electroporation of pCX-GFP and pCX-mcs2 control vector (left column) or pCX-GFP and miR-200 sponge vector (right column). Arrows indicate cells expressing both GFP and NeuN, asterisk shows a cell positive for GFP but negative for the late pan-neuronal marker NeuN. (d) Mean of GFP + cells that do not express NeuN (n = number of animals analyzed). Difference between groups were analyzed using Man and Whitney test (P = 0.009023). (e) Ratios of GFP + cells showing BrdU integration 2 days after lateral electroporation of a GFP vector. Difference between groups were analyzed using Man and Whitney test. (f) Fluorescent images showing neuroblasts in the RMS 7 days after lateral co-electroporation of pCX-GFP and pCX-mcs2 control vector or pCX-GFP and miR-200-gof stained for calretinin. Only in the miR-200 over-expression condition GFP + cells expressing calretinin are detected. (g) Ratios of GFP + cells co-expressing calretinin at 4 and 7 dpe (n = number of animals analyzed). Differences between groups were analyzed using Man and Whitney test. Scale bars: 30 μm in (c,e).

(a) Zeb2 mRNA (red) is widely expressed in the forebrain with particularly prominent presence in the SVZ and RMS. (b) Images showing GFP cells in the RMS stained with Zeb2 antibody 4 days after in vivo electroporation in control or miR-200-gof conditions. (c) Quantification of mean Zeb2 staining intensity 4 days after in vivo electroporation cells at 4 dpe in control or miR-200-gof conditions. This showed a significant reduction in Zeb2 protein expression in neuronal precursors, regardless of their calretinin expression status. Differences between groups of cells were analyzed pairwise with a t-test (control vs miR-200 calretinin positive P < 2.2e-16; control vs miR-200 calretinin negative P < 2.2e-16); n = number of cells used for analysis; an = number of animals from which analyzed cells were issued. (d) Zeb2 expression normalizes the miR-200-gof mediated induction in calretinin expression. Differences between groups were analyzed pairwise with the Man and Whitney test (control (n = 5 animals) vs miR-200 (n = 5 animals) P = 0.008816, miR-200 (n = 5 animals) vs miR-200 + Zeb2 (n = 7 animals) P = 0.04236). (e) Calretinin immunostaining of coronal forebrain section through the SVZ of Gsh2-Cre; Zeb2^+/+ (wt) or Gsh2-Cre; Zeb^Fl/Fl knockout (Zeb2 −/−) animals at P5 at the level indicated in the schema. (f) The number of calretinin immunoreactive cells in the aSVZ. is much higher in knockout (Zeb2 −/−) than in control (wt) animals. (g) qRT-PCR analysis in FACS sorted SVZ cells from P2 animals reveals a massive increase in calretinin mRNA expression in knockout (Zeb2 −/−) compared to control (wt) animals. In (f,g) difference between groups was analyzed using t- test. Scale bars: 1 mm in a, 20 μm in (b) 200 μm in (e).