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

Determination of Absolute Pitx2 Occupancy levels in Embryonic Biopsies.

<p>(<b>A</b>) Normalized WT chromatin immunoprecipitate signals (WTPPTnorm) were obtained by dividing signals from wild type precipitates by the ratio (WT/MUT) of input signals. Mutant chromatin immunoprecipitate signals (MUTPPT) represent collective measurement noise, with the identical antibody preparation, and were therefore subtracted to obtain the absolute amount of signal due to Pitx2 occupancy, which is plotted on the Y-axis. Error bars were calculated, by standard error propagation techniques, from the standard deviations in the measurements of both the wild type and mutant signals. The primer pair and extract pair used for each measurement is indicated on the X-axis. Data was sorted by significance of occupancy computed as described in legend to Fig. 6. Significance coding thresholds are indicated by vertical dashed lines, with associated P-values. Note that the Pitx2-specific signal approaches zero as the significance of occupancy approaches accepted scientific standards (P<0.05 or P<0.1). (<b>B</b>) The percent of genomes in the biopsy that were occupied by Pitx2 was calculated, for each particular primer-pair/extract combination, by dividing the Pitx2-specific signal by the average input signal (wild type input measurements were normalized by the overall input ratio before being averaged together with the mutant input measurements). Error bars were calculated directly from measurement errors by standard error propagation techniques executed at each step in the calculation. Data is sorted in the same order as in panel A. Gaps on the x-axis indicate a primer pair that was tested in more than one extract pair; only the measurement of highest significance is shown. Note that there is an approximate correspondence between the amount of Pitx2-specific signal (Panel A) and the percentage of genomes occupied (Panel B) at low P-values. This correspondence breaks down as P-values rise above accepted scientific norms (P<0.05 or P<0.1). Within the significant zone, the correspondence between absolute Pitx2-specific signal and calculated percent occupancy appears to be determined by the efficiency of particular probe sets in generating signal.</p

Developmental and Draft Transcriptional Network Models.

<p>(<b>A</b>) Hypaxial Pitx2^- somites (yellow) become embedded in the Pitx2⁺ SMP (blue) prior to the onset of Pitx2 expression in the somites. The SMP expresses an abdominal lateral plate mesoderm <i>Hox</i> code (bright pink). Loss of Pitx2 alters the <i>Hox</i> code in the abdominal wall (dull pink), but does not affect expression levels of MRFs in somites (still yellow). The altered surroundings stunt the somites and ultimately prevent muscle development in the abdominal wall. (<b>B</b>) Draft network model shows that Pitx2 normally activates <i>Hox9-11</i> paralogs in their abdominal domains and represses MRFs in somite-derived structures such as the myotome, and dermomyotome in the abdominal wall cells. Interactions of Pitx2 in somatic cells are not depicted in this model.</p

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

<p>The Hox 9-11 paralog regions of the Hoxa (<b>A</b>), Hoxb (<b>B</b>), Hoxc (<b>C</b>), and Hoxd (<b>D</b>) clusters were examined for Pitx2 occupancy in sonically sheared chromatin isolated from E10.5 embryonic body wall biopsies. 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). 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). Error analysis was done as described in the legend to Fig. 6.</p

Population-Specific Regulation of Chmp2b by <em>Lbx1</em> during Onset of Synaptogenesis in Lateral Association Interneurons

<div><p>Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene <em>Lbx1</em> plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of <em>Lbx1</em> in the embryonic mouse neural tube. Chmp2b was expressed and depended on <em>Lbx1</em> in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.</p> </div

Lbx1-Independent Domain Absent at Cervical Levels.

<p>(A–D) Dorsolateral neural tube at cervical levels. A lateral column (bracketed by arrowheads) shows cells that are colabeled by Lbx1(GFP) and Lhx1/5. A subset of these cells are also labeled by Chmp2b in heterozygotes but not in mutants. The size of the lateral column is also reduced, possibly reflecting the loss of dI4 cells, as observed at thoracic levels. The <i>Lbx1</i>-independent expression domain in the circumferential trajectory, representing dI1B cells, is absent in both genotypes. (E–H). Dorsolateral neural tube at thoracic levels. A lateral column (bracketed by arrowheads) shows dI4 cells that are colabeled by Lbx1(GFP) and Lhx1/5. Almost all of the cells in this column are labeled by Chmp2b in heterozygotes. The column is absent in mutants. A large <i>Lbx1</i>-independent expression domain in the circumferential trajectory, representing dI1B cells, is present in both genotypes. It is adjacent to the dI4 column and obscured the loss of Chmp2b RNA in the dI4 column at thoracic levels (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048573#s3" target="_blank">results</a>). (I, J) Quantification of cells in GFP/Lhx1/5/Chmp2b labeled heterozygote sections at cervical (n = 4) and thoracic (n = 4) levels, respectively. Primary antibodies against GFP, Lhx1/5, and Chmp2b were detected using appropriate Cy2 (green), Cy3 (red), and Cy5 (infrared)secondary antibodies, respectively. Colors were electronically switched, for clarity, to those indicated by the labels.</p

Lbx1-Dependent and Lbx1-Independent Chmp2b Expression Domains.

<p>(A,B) Chmp2b RNA at mid-forelimb levels in the dorsal and ventral portions of the mantle zone is <i>Lbx1</i>-dependent and <i>Lbx1</i>-independent, respectively. RNA expression in the motor columns (black arrow) varied considerably in adjacent sections, but was not generally affected when series of sections along corresponding parts of the body axis were compared. Arrowheads indicate the region of the intermediate zone (IZ), where Chmp2b RNA signal remains intense in mutants. The IZ lies lateral and adjacent to the ventricular zone (VZ). Chmp2b signal can be compared in the circumferential trajectory at the level of the sulcus limitans (SL), in the circled region (white arrow). (C, D) Chmp2b protein at E12.5 at mid-forelimb levels in the dorsal and ventral portions of the mantle zone (MZ) is Lbx1-dependent and <i>Lbx1</i>-independent, respectively. The dorsolateral funiculus (DLF; *) and ventrolateral funiculus (VLF; **) are indicated. (E) Only a subset of the Lbx(GFP)⁺ cells within the lateral dorsal horn express Chmp2b (arrowheads). Green cells lacking Chmp2b protein are intermingled (arrows). (F) Quantitative evaluation of the fraction of Chmp2b⁺ area in the neural tube that was colabeled by GFP. All images of the neural tube in this report will be oriented medial to lateral (left to right) and dorsal to ventral (top to bottom).</p