Ectopic lingual expression of ameloblast markers and signaling molecules in <i>Bcl11b^ep−/−</i> incisor.

<p>(A-B) BCL11B immunostaining in sections of <i>Bcl11b^L2/L2</i> and <i>Bcl11b^ep−/−</i> mice at E16.5. The epithelium is outlined by white dots. (C-L) RNA ISH using the indicated probes in sections of <i>Bcl11b^L2/L2</i> and <i>Bcl11b^ep−/−</i> incisors at E16.5. The epithelium is outlined by red dots. Black and red arrowheads denote lingual mesenchymal and epithelial staining, respectively. Scale bar, 500 µm.</p

BCL11B expression during incisor development.

<p>BCL11B immunostaining in sections of wild-type mice at indicated developmental stages. The epithelium is outlined by white dots. Scale bars: (A-B) 100 µm; (C) 200 µm; (D-E) 500 µm. a, ameloblasts; an, anterior; cl, cervical loop; de, dental epithelium; df, dental follicle; dm, dental mesenchyme; iee, inner enamel epithelium; lab, labial; lin, lingual; oee, outer enamel epithelium; pm, papillary mesenchyme; po, posterior; sr, stellate reticulum; vl, vestibular lamina.</p

Altered ameloblast development in <i>Bcl11b^−/−</i> incisors.

<p>RNA ISH using the indicated probes in sections of wild-type and <i>Bcl11b^−/−</i> mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B, E-F) 500 µm; other panels, 200 µm.</p

Epithelial invagination defect in <i>Bcl11b^−/−</i> developing incisors between initiation and early bud stage.

<p>(A-D) H&E staining in sections of wild-type and <i>Bcl11b^−/−</i> mice at indicated developmental stages. The epithelium is outlined in black. (E-H) BrdU immunostaining (green) in sections of wild-type and <i>Bcl11b^−/−</i> mice. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (I-J) BrdU index of wild-type and <i>Bcl11b^−/−</i> dental epithelium; *** denotes statistical significance at p ≤ 0.001, n = 3. (K-N) RNA ISH using a <i>Bmp4</i> probe in sections of wild-type and <i>Bcl11b^−/−</i> mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows denote epithelial staining. Scale bar, 100 µm.</p

Pitx2 Target Genes In Forelimb Migratory Muscle Progenitor Cell Lineage.

1<p>indicates one of the three array sets was inconsistent with the other two, expression means and standard deviation was calculated using only the two consistent arrays.</p>2<p>members of the adhesome.</p

Alterations in <i>Bcl11b^−/−</i> incisor development at cap stage.

<p>(A-B) H&E staining in sections of wild-type and <i>Bcl11b^−/−</i> mice at E14.5. The epithelium is outlined in black. (C-D) BrdU immunostaining (green) in sections of wild-type and <i>Bcl11b^−/−</i> mice at E14.5. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (E) BrdU index of wild-type and <i>Bcl11b^−/−</i> CLs; *** denotes statistical significance at p ≤ 0.001, n = 3. (F-G) RNA ISH using an <i>Fgf10</i> probe in sections of wild-type and <i>Bcl11b^−/−</i> mice at E14. Black arrows indicate mesenchymal staining; black asterisks denote the staining between dental epithelium and vestibular lamina. Scale bar, 200 µm. cl, cervical loop; ek, enamel knot; vl, vestibular lamina.</p

Flow-Sorting EGFP⁺ MMP Cells from Forelimbs.

<p>(<b>A</b>) EGFP indicating Lbx1 expression in Lbx1^EGFP/+ E12.5 mouse. (<b>B</b>) Immunohistochemistry of cross-sectioned Lbx1^EGFP/+|Pitx2^LacZ/+ E12.5 mouse forelimb. beta-Gal(Pitx2) and Lbx1(EGFP) are co-localized in the flexor and extensor muscle groups. (<b>C</b>) FACS analysis of sorted Lbx1^EGFP/+ cells. The automated multiwell plating function on the MoFlo was used to test a variety of substrate and media at systematically controlled plating densities. Cells were sorted at a rate of 10,000 cells/sec with a purity of 95–99+%, depending on the stringency of gating. Cell number (Y axis, log scale) vs. florescence intensity (X axis, FL1) plot. The “a” peak represented EGFP⁻ cell population, and the “b” peak represented the GFP⁺ cell population. The GFP⁺ population represents 5–7% of the total limb bud cellular pool. (<b>D</b>) RNA samples were quantified and ran on an Agilent Bioanalyser 2100 to assess RNA quality prior to microarray analysis. (<b>E</b>) Comparison of expression of total RNA from HET (y axis) vs. WT (y axis). Each dot in both axes represents relative RNA expression levels for an individual gene in WT vs. HET respectively. If a dot is perfectly located in the diagonal line, then the relative gene expression level for the representing gene exhibits no difference within HET and WT. (<b>F</b>) Comparison of expression of total RNA from MUT (y axis) vs. WT (x axis). Each dot in both axes represents relative RNA expression levels for an individual gene in MUT vs. WT respectively. (<b>G</b>) Comparison of expression of total RNA from MUT (y axis) vs. HET (x axis). Each dot in both axes represents relative RNA expression levels for an individual gene in MUT vs. HET respectively. Pitx2 expression levels were indicated by arrow. Pitx2 was strongly down regulated in the Pitx2 mutants. Comparison of expression of total RNA of genes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035822#pone-0035822-t001" target="_blank">Table 1</a> of HET (y axis) vs. WT (x axis) (<b>H</b>), MUT (y axis) vs. WT (x axis) (<b>I</b>), MUT (y axis) vs. HET (x axis) (<b>J</b>).</p

Altered Focal Adhesion in Pitx2 Mutant Myogenic Cells.

<p>Muscle progenitor cells were isolated from E12.5 forelimb tissue of Pitx2^LacZ/+ (<b>A, C, E</b>) and Pitx2^LacZ/LacZ (<b>B, D, F</b>) mice. Cells were stained with alexa 488 phalloidin, (F-actin), talin (focal adhesions) and beta-Gal(Pitx2) (<b>A–F</b>). (<b>G</b>) Myogenic cells had a mean ± standard error of the mean (SEM) of 26±4 (total = 475) for HET and 18±1 (total = 330) for MUT of focal adhesions per cell (n = 18). This difference in mean focal adhesion number was determined statistically significant using a two-tailed unpaired t-test (p = 0.0464). The distribution of the number focal adhesions between the leading and trailing edges of the muscle progenitor cells was not affected; the leading edges had an mean ± SEM of 14±2 (total  = 255) for HET and 9±1 (total  = 170) for MUT cells, while at the trailing edges had means ± SEM of 12±2 (total  = 220) for HET and 9±1 (total  = 160) for MUT cells. Neither of the differences of focal adhesion number at leading or trailing edges between HET and MUT cells were determined statistically significant using two tailed unpaired t-test (p = 0.05 and p = 0.0848, respectively). While differences between the leading and trailing edges within HET or MUT cells were also determined not statistically significant using two tailed paired t-test (p = 0.219 and p = 0.355). (<b>H</b>) The size of focal adhesions had an mean size ± SEM of 2.14±0.27 micrometer in HET and 3.09±0.3 micrometer in MUT cells. This difference in focal adhesion size was determined to be very statically significant using two-tailed unpaired t-test (p = 0.0013). The mean size ± SEM of focal adhesions at the leading edge was 1.70±0.24 micrometer for HET and 3.03±0.3 micrometer for MUT, while at the trailing edge the mean size ± SEM was 2.57±0.26 micrometer for HET and 3.15±0.3 micrometer for MUT. The mean focal adhesion size at the leading edge between HET and MUT cells were determined to be very statistically significantly different using unpaired two tailed t-test (p-value  = 0.0016). When comparing leading and trailing focal adhesion size within HET cells there is a statistically significant difference in mean size two-tailed paired t-test (p-value  = 0.001). Comparing mean focal adhesion size between leading and trailing edges within MUT cells showed no statistically significant difference using two tailed paired t-test (p  = 0.1325). White bar denotes 50 micrometer.</p

Increased Actin Bundling and Presence of Tau and Stathmin in Pitx2 Mutant Myogenic Cells.

<p>Immunostaining for Phalloidin (F-Actin) and beta-Gal(Pitx2) (<b>A–D</b>), tropomyosin and beta-Gal(Pitx2) (<b>E–H</b>) desmin (Intermediate Filament) and beta-Gal(Pitx2) (<b>I–L</b>) on Pitx2^LacZ/+, Mapt (Tau) and beta-Gal(Pitx2) (<b>M–P</b>), stathmin (stmn) and beta-Gal(Pitx2) (<b>Q–T</b>), and tubulin (tub) and beta-Gal(Pitx2) (<b>U–X</b>) on Pitx2^LacZ/+. The F-actin, tropomyosin and desmin labeled fibers in cells in the MUT forelimbs were not aligned and cluster together as in the HET. MUT myogenic cells failed to develop protrusions, connect and align to each other. Mapt and stmn were highly expressed in forelimb tissue and myogenic primary cultured cells. Mapt, stmn and tub expression levels were increased in both tissue and primary myogenic cells cultures. Arrows denote points of interest between genotypes. White bar denotes 50 micrometer.</p

Altered expression of TGFβ genes and <i>Fst</i> in <i>Bcl11b^−/−</i> incisor.

<p>RNA ISH using the indicated probes in sections of wild-type and <i>Bcl11b^−/−</i> incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Black and red asterisks denote staining in the posterior part of the dental follicle and epithelial tip of the incisor, respectively. Scale bars: (A-B, E-F, I-J) 500 µm; other panels, 200 µm.</p