<i>Foxp1</i> knockdown does not affect neuronal differentiation.

<p>E14.5 mouse embryos were electroporated with <i>Foxp1</i> shRNA-b or shRNA-Scr, and brains were fixed at P2. Coronal sections were immunostained for Satb2 (A) and Cux1 (B). a, b, c, High-magnification images of the boxed regions in the left panels. a, b, c in (A) and (B) represent layer II-IV, layer V and white matter, respectively. (C), (D), Immunofluorescence staining of Ctip2 and Tbr1 in brain sections from control and <i>Foxp1</i> shRNA-b. (C)-a, (D)-a, High-magnification images of the boxed regions in the left panels. Scale bar, 50μm in (A), (B), (C) and (D).</p

<i>Foxp1</i> is expressed at CP in mouse embryonic brain and repressed by shRNA constructs.

<p>Brain sections (14 μm) obtained from E16.5 (A) and E18.5 (B) mice were immunostained with antibodies against FOXP1 (green) and Satb2 (red). a, High-magnification images of the boxed regions in the left panels. Scale bar, 20μm in A, 50μm in B. (C) and (D), Relative mRNA expression of <i>Foxp1</i> in N2a cells (C) and in cultured cortical cells (D) transfected with shRNA-Scr or two <i>Foxp1</i> shRNAs (shRNA-a and shRNA-b). ***p<0.001, one-way ANOVA. (E), GFP-pCAGGS was cotransfected into N2a cells with shRNA-Scr, <i>Foxp1</i> shRNA-a or shRNA-b. After 48h, cells were collected and analyzed for the expression of FOXP1 and GAPDH by western blot. The expressions of FOXP1 were quantitated as rations of the background-subtracted intensities for FOXP1 to GAPDH. (F), GFP-pCAGGS was electroporated with shRNA-Scr, <i>Foxp1</i> shRNA-a or shRNA-b into cerebral cortices at E14.5. Coronal sections were obtained from the cerebral cortices at E17.5 and immunostained with anti-GFP (green) and anti-FOXP1 (red). a, b, c, High-magnification images of the boxed regions in the left panels. Arrows indicate GFP-positive neurons. Scale bar, 50μm in F, 20μm in c.</p

Effects of <i>Foxp1</i> knockdown on the placement of cortical neurons.

<p>(A), (B) E14.5 embryos were electroporated with the control or <i>Foxp1</i> shRNA-b, together with GFP-pCAGGS, and analyzed at P2, P4, P7, and P14. Scale bar, 50μm. (C), (D), (E), (F) Histograms showed the percentage of transfected cells in different regions of the cerebral cortex at P2, P4, P7, and P14. *p<0.05 and ***p<0.001 for comparisons between shRNA-b and the corresponding control, Student’s <i>t</i>-test.</p

<i>Foxp1</i> regulates neuronal morphogenesis <i>in vitro</i>.

<p>(A), Cortical neurons were transfected <i>in utero</i> at E14.5 and isolated at E15.5 for primary cultures <i>in vitro</i>. At day four <i>in vitro</i>, the neurons transfected with GFP-pCAGGS together with shRNA-Scr (n = 39) or <i>Foxp1</i> shRNA-b (n = 53) were analyzed by Neurolucida. Scale bar, 50μm. (B), Quantitative analysis of total lengths of the axon and dendrites from images in (A). ***p<0.001, Student’s <i>t</i>-test.</p

Knockdown of <i>Foxp1</i> in pyramidal neurons leads to abnormal dendritic development at P30.

<p>(A), Coronal sections (80 μm) from <i>Foxp1</i> knockdown and the control mice. Scale bar: 50μm. (B), Representative images and Neurolucida tracings of GFP-positive, layer II-III neurons from the control (n = 18) and <i>Foxp1</i> knockdown (n = 17) mice at E15.5. Scale bar: 50μm. (C), Representative images and Neurolucida tracing of GFP-positive neurons stalled in layer V (n = 15) from shRNA-b transfected mice at E15.5 or from the control (n = 11) transfected mice at E13.5. (D), (E), (F), Quantitative and statistical analysis of dendritic parameters in layer II-III. (G), (H), (I), Quantitative and statistical analysis of dendritic parameters in layer V at P30. *p<0.05, **p<0.01, Student’s <i>t</i>-test.</p

Suppression of <i>Foxp1</i> inhibits neuronal migration.

<p>(A), E14.5 mouse embryos electroporated with a GFP expression plasmid along with shRNA-Scr, <i>Foxp1</i> shRNA-a or shRNA-b together with pCMV-mFoxp1 were allowed to develop until E17.5. Coronal sections (14 μm) of E17.5 brains were immunostained with an antibody against GFP (green). Nuclei were stained with DAPI (blue). Short black lines indicate the borders between CP, IZ, SVZ/VZ. Scale bar, 50μm. (B), Statistical analysis of the percentages of GFP-positive cells in the indicated regions of the cerebral cortex as showed in (A). *p<0.05, ***p<0.001, one-way ANOVA. (C), E14.5 mice receiving mir30-ScrRNA or mir30-shRNA-b were examined at E17.5. Scale bar, 50μm. (C)-a and (C)-b, Representative images of migrating neurons (Green) stained with FOXP1 antibody (Red) showed FOXP1 expression in control neurons but not in mir30-shRNA-b expressing neurons. (D), Statistical analysis of the percentages of electroporated cells in the indicated regions of the cerebral cortex as showed in (C). ***p<0.001, Student’s <i>t</i>-test. (E), GFP-pCAGGS was coelectroporated with pX330 or pX330-gFoxp1 into E14.5 mouse brains. Sections were examined at E17.5. Scale bar, 50μm. Immunostaining for FOXP1 (Red) indicated a lack of FOXP1 expression in pX330-gFoxp1 transfected neurons (E-b). Vector pX330 transfected neurons expressed FOXP1 in the CP normally (E-a). (F), Quantitative analysis of the distribution of electroporated cells in cortical layers as showed in (E). **p<0.01 and ***p<0.001, Student’s <i>t</i>-test.</p

Regulated genes in transgenic mice and validation of <i>Ctgf</i> as a target.

<p>(A): qRT-PCR using limb RNA (E12.5-E14.5) from wildtype (Wt) and transgenic littermates (Tg) (N = 8–10 for each stage). Measurements were carried out individually, in duplicates, and normalized to <i>Adam9</i> and <i>Sdha</i>. Relative normalized values are presented on the y-axis. Significances are indicated in each diagram by asterisks (*: <i>p</i>≤0.05, **: <i>p</i>≤0.01, ***: <i>p</i>≤0.001). Variations are indicated by the standard deviation (SD). In 7/8 candidates an upregulation was confirmed as significant in at least one embryonic stage. (B): nCounter analysis of <i>CTGF</i> and <i>SHOX</i> expression in NHDF and U2OS cells after transient transfections of <i>SHOX</i> and <i>p.Y141D</i>. <i>CTGF</i> is significantly downregulated in NHDF cells, whereas it is significantly upregulated in U2OS cells. Values on y-axis represent absolute counts of mRNA, normalized to <i>ADAM9</i>, <i>HPRT1</i> and <i>SDHA</i>. Significancies are indicated by asterisks. (C): <i>In situ</i> hybridization using a <i>Ctgf</i> antisense riboprobe on embryonic limbs from wildtype and <i>SHOX</i>-transgenic littermates (N = 8) at stage E12.5. In transgenic embryos, enhanced and distalized expression of <i>Ctgf</i> was detected in the middle part of the developing limbs.</p

Analysis of postnatal bone parameters of <i>Col2a1-SHOX</i>-transgenic mice.

<p>(A): Alcian Blue/Alizarin Red S staining at different developmental (E14.5, E18.5) and postnatal (P28) stages does not reveal apparent differences between transgenic and wildtype skeletal elements. (B): Postnatal <i>in vivo</i> time-course analysis of bone growth in 65 animals of two transgenic lines by μ-CT analysis. Tibiae and femora of wildtype and <i>Tg(Col2a1-SHOX)</i> littermates at the age of 4, 12 and 24 weeks were scanned, female and male individuals were evaluated separately. Total bone length, cortical bone thickness and bone volume do not show significant differences between wildtype and transgenic females or males. Some transgenic animals presented longer bones and weaker structures of the cortical bone in the subcartilaginous region (indicated in the μ-CT images). Other micromorphological parameters (bone mineral density (BMD), trabecular volume and thickness) showed no significant differences. Statistical analyses were performed using student's t-test. (C): hematoxilin and eosin (H&E) stainings of the growth plate in wildtype and transgenic tibiae. Consistent differences between wildtype and <i>Tg(Col2a1-SHOX)</i> adult growth plates (24 weeks of age) did not exist (N = 8), but some transgenic tibiae showed a buckling, and the columns of chondrocytes became shorter and were not strictly oriented in a parallel assembly compared to the wildtype (right image).</p

Analysis of <i>CTGF</i> as a direct transcriptional target of SHOX.

<p>(A): Genomic structure of the human <i>CTGF</i> region. ChIP-Seq analysis in ChMM cultures revealed an accumulation of Shox binding in the <i>Ctgf</i> promoter region (grey peaks), especially in a region 3–4 kb from the transcriptional start site (TSS) where an evolutionary conserved sequence (ECR) of 597 bp (human chr6:132317086-132318077) was identified (green bar). (B): Location of the pGL3 ECR and pGL3 ECR+ reporter constructs (grey bars) within the <i>CTGF</i> upstream region. The ECR+ construct encompasses the ECR and an upstream region including ATTA/TAAT motifs and palindromes. SHOX binding motifs (ATTA/TAAT sites and palindromes) in the <i>CTGF</i> 5′ region around the ECR are indicated by asterisks. Red bars represent the location of the generated oligonucleotides for EMSA. (C): Luciferase reporter gene assays in NHDF and U2OS cells. pcDNA4/TO <i>SHOX</i> was cotransfected with a luciferase reporter vector harbouring either the ECR or the ECR+ sequence. Transfections and measurements were carried out in triplicates. A significant activation in the luciferase activity was observed 24 h after <i>SHOX</i> transfection in NHDF cells using both reporter constructs (1.7-fold/2.5-fold with <i>p</i> = 0.02/0.007 for ECR/ECR+). In U2OS cells, an alteration was not observed for the ECR reporter, but a significant reduction was demonstrated for the ECR+ reporter construct (1.0-fold/2.8-fold with <i>p</i> = 0.1/0.003 for ECR/ECR+). (D): EMSA. The SHOX wildtype (Wt) and the mutant p.R153L proteins bind to oligonucleotides 1 and 2, whereas the defective proteins p.Y141D and p.A170P cannot. All fragments of oligonucleotides 1 and 2 containing an ATTA/TAAT site are sensitive to SHOX binding (1a–c, 2a–b). The fragment lacking this motif does not bind (oligonucleotide 2c). Using the SHOX-3 antibody (Ab), we demonstrate that the binding is SHOX-specific. (E): Immunohistochemistry performed on pubertal tibial growth plates. Staining was performed using preimmune serum as a negative control, SHOX antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098543#pone.0098543-Marchini1" target="_blank">[19]</a> and a CTGF-specific antibody. Both the SHOX and CTGF proteins were detected in growth plate chondrocytes.</p

Generation and expression analysis of <i>SHOX</i>-transgenic mice.

<p>(A): The <i>SHOXa</i> cDNA was tagged with a Lumio and SV40 Poly(A) sequence and cloned under the control of a murine <i>Col2a1</i> promotor/enhancer expression cassette. (B): Genotyping was performed using specific primers spanning the first 409 nucleotides of the <i>SHOXa</i> cDNA. No PCR product was detected in wildtype animals. (C):-Southern Blot analysis of the two transgenic lines (1 and 2) used for our investigations. Genomic DNA was digested with <i>BamHI</i>, <i>EcoRV</i> and <i>Hind III</i>. <i>BamHI</i> digestion results in a 1.3 kb fragment that corresponds to the Lumio/SV40-tagged <i>SHOX</i> cDNA, which was flanked by <i>BamHI</i> sites. The presence of only one signal per lane indicates a single integration site of the transgene. (D): Relative quantitative expression of <i>Col2a1</i> and <i>SHOXa</i> transcripts in limbs of wildtype and transgenic littermates (N = 5–8 per litter) at E12.5, E13.5 and E14.5. The expression of the transgene corresponds to the expression dynamics of <i>Col2a1</i>. <i>SHOX</i> levels are generally low with highest expression at E12.5. Values are variable among individual animals as indicated by the standard deviation (SD). (E): WISH of wildtype (Wt) and transgenic (Tg) embryonic limbs from E11.5-E14.5 (N = 20 for each stage). The transgene is weakly expressed in the developing limb at E11.5 and becomes defined around the cartilaginous anlagen at E12.5. From E13.5 onwards, the expression is mainly seen in the mesenchyme around the developing cartilage and in the perichondrium and decreases during later stages.</p