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

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

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 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

Pitx2-Mediated Myogenic Cell Gene Network During Filling Limb Muscle Anlagen.

<p>The network displays ten genes with the greatest fold change from each of the three groups; actin (diamonds), microtubule (octagons) and adhesion (squares) related genes. Color intensity is representative of reported fold change based on their gene expression analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035822#pone-0035822-t001" target="_blank">Table 1</a>), with darker color representing larger fold change. Connections between the nodes are displayed as solid lines for direct interactions and dashed lines for possible interactions.</p

Regulation of Shape and Size of Limb Muscle Anlagen by Pitx2.

<p>(<b>A–H</b>) Whole mount X-gal staining of Pitx2^LacZ/+ and Pitx2^LacZ/LacZ knock-in mice from E10.5–E14.5. Pitx2 was expressed throughout muscle anlagen but not in epidermis, mesenchyme or bone anlagen. Most distal limb muscle anlagen were mildly deformed. Muscle groups are outlined. (<b>I</b>) Percentage of EGFP⁺ cells collected from Pax3^Cre|ROSA^EGFP|Pitx2^LacZ/+ (HET) and Pax3^Cre|ROSA^EGFP|Pitx2^LacZ/LacZ (MUT) embryos at E12.5 forelimb tissue dissociated into single cell suspension. The percentage of EGFP⁺ cells had a mean ± standard error of the mean (SEM) of 17±0.6% for HET (n = 8) and 11±1% for MUT (n = 7). This 26% reduction in EGFP⁺ cells in the MUT forelimb was considered to be significant using unpaired t-test with a p-value = 0.0001. (<b>J</b>) Example histograms of propidium iodide (PI) staining of HET and MUT EGFP⁺ cells isolated from forelimb tissue at E12.5. (<b>K</b>) Results of cell cycle analysis using PI staining showing distribution (mean ± SEM) of the EGFP⁺ cell population between HET (n = 5) and MUT (n = 4) during G1 (69±0.007% HET, 74±0.009% MUT); S (18±0.005% HET, 17±0.006% MUT); and G2 (13±0.003% HET, 9±0.015%MUT) phases. The increase in MUT cells during G1 phase was determined to be significant using unpaired t-test with a p-value  = 0.0102.</p

Pitx2 Target Genes in Abdominal Wall.

1<p>Based on Student’s t-test.</p>2<p>Hoxc11 is not represented on the Mouse Genome 2.0 Array.</p

Somitic MRF Expression Precedes the Onset of Somitic Pitx2 Expression.

<p>(<b>A–D</b>) A lateral stripe of X-gal staining the dorsal somatopleure (smp^d; black dots), forms from the X-gal stained ventral somaopleure at the posterior forelimb level prior to the 28 somite (E10.5) stage shown here and extends in both the rostral and caudal direction. Underlying somites become X-gal positive as the smp^d forms over them. This happens equivalently in mutants and heterozygotes. The second somite behind the left forelimb (asterisk) is enlarged and outlined. Note that somitic X-gal staining is not concentrated at the hypaxial tip. Scale bar, 200 µm. (<b>E, </b><b>F</b>) X-gal staining of late E10.5 embryos is similar in the epaxial myotomes (black dots), but large deformities already exist in the body walls of mutants. Scale bar, 1 mm. (<b>G–J</b>) Expression domains of Myod1 in hypaxial myotomes of interlimb somites are not altered. The second somite behind the left forelimb (asterisk) is enlarged and outlined, for direct comparison with A–D panels. Note the modest deformity imposed by the outward turn of the body wall. Scale bar, 200 µm. (<b>K–N</b>) Pax3^CRE|ROSA^EGFP was used to visualize the entire dermomyotome lineage on the left (<b>K, </b><b>L</b>) and right (<b>M, </b><b>N</b>) side of E10.5 mice. Note the distortion of somite structure behind the left, but not right, forelimb. Scale bar, 400 µm. (<b>O, </b><b>O1, </b><b>P, </b><b>P1</b>) Myf5 expression domains in mutant somites show no apparent difference. The fourth somite behind the forelimb is identified by an asterisk. Black arrows point to the ventral edge of the hypaxial compartment, white arrow and swooshes indicate dorsal to ventral trajectories. Scale bar, 200 µm.</p

Body Wall Muscle Defects in <i>Pitx2</i> Mutants.

<p>Whole-mount X-gal staining to compare nascent abdominal and limb suspension musculature at E14.5 in heterozygote, Pitx2^+/LacZ (<b>A, </b><b>C, </b><b>E, </b><b>G</b>) and mutant, Pitx2^LacZ/LacZ (<b>B, </b><b>D, </b><b>F, </b><b>H</b>) mouse fetuses. Mutant fetus has a sharp rightward kink in the body axis that was straightened somewhat to allow visualization. Right sides are shown because they are less malformed than left sides. Colored dots indicate approximate location of the attachment points from which were used to identify muscle anlagen. (<b>A, </b><b>B</b>) Skinned fetuses show the most superficial muscles beneath the skin. Spinotrapezius (red dots) and latissimus dorsi (orange dots) were truncated at the abdominal attachment end and had faulty texture. Acromiotrapezius (white dots) and levator scapulae ventralis (blue dots) were of normal length but faulty texture. Scale bar, 1 mm. (<b>C, </b><b>D</b>) Muscles identified in outer layer have been dissected away. Serratus dorsalis (black dots) was not detected in the mutant or was displaced (white dots). Two straps of sacrospinalis (red dots) appeared to reach the vertebral column, but were abnormally shortened and of incorrect texture. External oblique (white dots) muscle anlagen in heterozygotes are either absent or severely truncated on their abdominal end. Scale bar, 1 mm. (<b>E, </b><b>H</b>) Ventral view of the abdominal wall shows complete collapse of musculature (white dots). Scale bar, 1 mm. (<b>G, </b><b>H</b>) Frontal sections through the abdomen of X-gal stained and paraffin embedded fetuses, counterstained with eosin/hematoxylin. The left side of the embryo is shown in both panels. The residual muscle fragment cannot be definitively identified. Scale bar, 200 µm. <b>de</b>, dermis; <b>pc</b>, panniculus carnosus; <b>spp</b>, spachopleural mesenchyme; <b>eo</b>, external oblique; <b>io</b>, internal oblique; <b>tv</b>, transversus.</p

Pitx2 Protein Occupancy at MRF Loci in E10.5 Body Wall Chromatin.

<p>The Myf5/Myf6 (<b>A–D</b>), Myod1 (<b>E–H</b>), Myogenin (<b>I–L</b>), and Tcfap2 (<b>M, </b><b>N</b>) loci were examined for Pitx2 (<b>B, </b><b>F, </b><b>J, </b><b>N</b>), HDAC1 (<b>C, </b><b>G, </b><b>K</b>), and HDAC3 (<b>D, </b><b>H, </b><b>L</b>) occupancy in sonically sheared chromatin isolated from E10.5 body wall biopsies. PCR amplicons of 70–150 bp (red boxes) were designed around evolutionarily conserved bicoid core motifs. Each red diamond indicates a separate vertebrate species, which contains the bicoid core motif. Bar graphs show the average amount of signal precipitated from wild type (black) and mutant (white) biopsies, normalized by the ratio of input chromatin between wild type and mutant (Amount_norm). Error bars indicate the standard deviation in triplicate measurements made from individual biopsy pools. All comparisons are between pairs (WT, MUT) of biopsy pools processed in parallel on the same day. If a particular amplicon was examined in several pairs of biopsies, data from the pair with the highest significance of occupancy is shown. <i>Pitx2-ChIP Analysis.</i> (<b>A–B; </b><b>E–F; </b><b>I–J; </b><b>M–N</b>). The probability that Pitx2 occupies the site in wild type tissue is indicated by asterisk code above the site and the bar graphs of the measurement; ****(>99.99%), ***(>99%), **(>95%), *(>90%), ± (>70%), ne (no evidence), nd (not detected). One code is shown for each extract pair measured. The significance of occupancy indicated by the code was established by the Student’s t-test between triplicate measures taken on mutant and wild type samples. P-values were looked up from computed t-values for a two-tailed test, and indicate the probability that the observed difference (in either direction) is due to measurement error. Note that the size of the bars does not directly indicate occupancy levels for the simple reason that mutants lack Pitx2 protein and therefore <i>de facto</i> have zero occupancy. The size of white bars therefore only indicates total noise of measurement, using exactly the same antibody preparation as the parallel wild type measurement, and must be subtracted from that measurement to obtain a direct measure of occupancy (Fig. 8). <i>HDAC-ChIP Analysis</i> (<b>C–D; </b><b>G–H; </b><b>K–L</b>) Significance of difference was measured and is encoded as for Pitx2 ChIP analysis. In these experiments, the significance code indicates the chance that the HDAC occupancy differs between WT and MUT.</p