Functional analysis of the preBötC oscillator in the <i>Lp</i> mutant.

<p>A: Photomicrograph of a transverse medullary slice through the preBötC obtained from an E16.5 <i>+/+</i> embryo loaded with calcium green 1-AM observed in direct fluorescence (left panel) and as relative changes in fluorescence (ΔF/F, right panel). The red circles indicate the position of the bilaterally distributed preBötC oscillators. Traces below (ΔF/F preBötC) indicate calcium transients measured in the preBötC region in control conditions (top trace), 10⁻⁷ M Substance P (SP, second trace), 10 µM Riluzole (third trace) and 10 µM CNQX (fourth trace). B and C: Same legend as in A for a <i>Lp/+</i> embryo (B) and a <i>Lp/Lp</i> embryo (C). D: Graph representing the mean frequency of calcium transients measured in the preBötC region in different experimental conditions (indicated below and color coded) for <i>+/+</i> (left part), <i>Lp/+</i> (middle part) and <i>Lp/Lp</i> (right part). Numbers in brackets indicate the number of preparations tested. Asterisks indicate statistically different means. Frequency is increased in the presence of SP, unchanged in the presence of riluzole and blocked in the presence of CNQX for all genotypes. Hence, the preBötC oscillator is functionally preserved in <i>Lp/Lp</i> mutants. D: dorsal, L: lateral.</p

Rostro-caudal distribution of facial motor neurons and respiratory neurons in the hindbrain of wild-type and <i>Lp</i> mutant embryos.

<p>Half slice drawings were obtained from images of brainstem slices labeled with anti-NK1R and Islet1,2 antibodies (dorsal up). The red dots indicate individual e-pF neurons, the gray areas indicate the FMN and the blue ovals indicate the preBötC. The rostro-caudal extents of the preBötC (blue), the e-pF (red) and the FMN (gray) are indicated by vertical colored bars. The numbers on the left of each drawing indicate the rostro-caudal position (in µm) of the slice relative to the preBötC position, which is defined as zero (see the full scale on the left of +/+). Note that the e-pF is more rostrally and loosely distributed in the <i>Lp/Lp</i> preparation compared to the +/+ and <i>Lp/+</i> preparations. Similar distributions were observed in a second preparation for each genotype. XIIn: hypoglossal nucleus, nA: nucleus ambiguus, FMN: facial motor nucleus.</p

Active cells form a functional e-pF oscillator in <i>Lp</i> mutant embryos.

<p>A: Ventral view of the hindbrain over the area encompassing the FMN and the active region. Preparations were obtained from an E15.5 <i>+/+</i> embryo, loaded with calcium Green 1-AM and observed in direct fluorescence (left panel) or as relative changes in fluorescence (ΔF/F, right panel). The yellow oval indicates the position of the FMN, the e-pF network is encircled in red. Traces below indicate calcium transients (ΔF/F) measured in the e-pF region in control conditions (top trace), pH 7.2 (second trace), 10 µM CNQX (third trace), 10 µM Riluzole (fourth trace) and 10⁻⁷ M Substance P (SP, fifth trace). B and C: Same legend as in A for a <i>Lp/+</i> embryo (B) and a <i>Lp/Lp</i> embryo (C). D: Graphs representing the mean frequency of calcium transients measured in the e-pF region in different experimental conditions (indicated below and color coded) for <i>+/+</i> (left part), <i>Lp/+</i> (middle part) and <i>Lp/Lp</i> (right part). Numbers in brackets indicate the number of preparations tested. Asterisks indicate statistically different means. Frequency is increased in the presence of CNQX, SP and in pH 7.2 for all genotypes, while riluzole blocks the rhythmic activity in the active region. The active network found in the mutant shares pharmacological characteristics of the e-pF recorded in the wild type littermates. L: lateral, R: rostral.</p

<i>Lp</i> mice display a spectrum of outflow tract abnormalities.

<p><b>A,B</b>) <i>In situ</i> hybridisation on E10.5 <i>Lp/+</i> and <i>Lp/Lp</i> embryos reveals normal expression of <i>Tbx20</i> in the mutant embryo, but illustrates the abnormal heart loop (the outline of the outflow tract and ventricular chambers is indicated by the dotted lines). <b>C,D</b>) H&E sections of E14.5 <i>Lp/+</i> and <i>Lp/Lp</i> embryos show the double outlet right ventricle in the mutant embryo (the arrows indicate the communication between and the aorta and the ventricle). <b>E–H</b>) β-gal staining (blue) of wholemount stained <i>Lp/+</i> and <i>Lp/Lp</i> E10.5 embryos shows that NCC migration (labelled by <i>Wnt1-Cre</i> based lineage tracing) appears normal in the mutants. Transverse sections (G,H) show that although the OFT is reduced in length, there is normal migration of NCC into the outflow vessel (arrow). The bars in G,H indicate the characteristic shortened outflow tract seen in the mutant. <b>I–L</b>) β-gal staining of wholemount stained <i>Lp/+</i> and <i>Lp/Lp</i> E9.5 embryos shows that the SHF, labelled by <i>Isl1-Cre</i> based lineage tracing, appears normal in the mutants, however the cells appear disorganised (arrows). <b>M,N</b>) Isl1 antibody labels SHF cells in the distal outflow tract (brown staining – arrows). These cells appear disorganised in the <i>Lp/Lp</i> embryo at E9.5 (N′ arrow, compare to M′). Ao – aorta, LV - left ventricle, OFT - outflow tract, RV - right ventricle.</p

SHF-specific loss of <i>Vangl2</i> results in outflow tract defects.

<p><b>A,B,E,F</b>) Targeted deletion of Vangl2 by <i>Wnt1-Cre</i>, in NCC, does not result in neural tube (A,E) or outflow tract defects (B,F). <b>C,D,G,H</b>) In contrast, although there are no neural tube defects when <i>Vangl2</i> is deleted in the <i>Isl1-Cre</i> expressing SHF (G), the resultant embryos do have double outlet right ventricle (H – compare with D). <b>I–P</b>) No defects were seen when <i>Vangl2</i> was deleted in either <i>Nkx2.5-Cre</i> expressing cardiac progenitors or <i>Mlc2v-Cre</i> expressing differentiated cardiomyocytes. In each case the arrows show the communication between the ventricle and the aorta. All embryos are E14.5. Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004871#pgen.1004871.s004" target="_blank">S4 Fig</a>. Ao – aorta, LV - left ventricle, RV - right ventricle, <i>Vangl2^f</i> – <i>Vangl2^flox</i>. Scale bar  = 2 mm (white), 500 µm (black).</p

Bilateral e-pF oscillators exhibit synchronized activity in <i>Lp</i> mutant embryos.

<p>A–C: Left: Ventral view of whole hindbrain preparations from E15.5 <i>+/+</i> (A), <i>Lp/+</i> (B) and <i>Lp/Lp</i> (C) embryos loaded with Calcium Green 1-AM and observed in direct fluorescence. The red lines indicate the position of the left (L) and the right (R) e-pF oscillators. The two traces (middle panels) correspond to the calcium transients illustrated as relative fluorescent changes (ΔF/F) recorded simultaneously in the corresponding e-pF oscillators in one preparation. The dashed lines in blue highlight the synchronicity between the activities of both e-pF oscillators. Right: corresponding superimposed left-right e-pF cross-correlograms illustrated for 4 different preparations. Bilateral synchronous activity in the two e-pF oscillators is preserved the <i>Lp</i>/+ and <i>Lp</i>/<i>Lp</i> mutants. R: rostral.</p

Altered distribution of active cells at the pial surface of <i>Lp</i> mutant hindbrain preparations.

<p>A–C: Ventral view of whole hindbrain preparations from E15.5 <i>+/+</i> (A), <i>Lp/+</i> (B) and <i>Lp/Lp</i> (C) embryos loaded with Calcium Green 1-AM and observed in direct fluorescence. Yellow ovals indicate the position of the facial motor nucleus (FMN) that is clearly visible in direct light (see right side of preparations in A and B). D–F: Maps for rhythmic active cells (red circles) detected at a high magnification for corresponding genotypes in the region delimited by the white rectangles in A–C. Active cells located at the ventral surface of the preparations are found for all genotypes. G–I: Histograms of the rostro-caudal distribution of active cells relative to the constant preBötC position for 2 wild-type (G), 3 <i>Lp/+</i> (H), and 3 <i>Lp/Lp</i> (I) embryos. The red arrows indicate the rostral and the caudal extremity of the FMN. The distribution of active cells shows a significant rostral displacement in <i>Lp/Lp</i> embryos. J–L: Calcium transients illustrated as relative fluorescent changes (ΔF/F) recorded in the region encompassing the active cells (delimited by the blue rectangles in A–C). Relative fluorescent changes intensity are color-coded, white corresponding to the strongest activity (see the color scale at the bottom). The traces below show the spontaneous calcium changes recorded over time in the entire active region for each genotype. R: rostral.</p

Location of the facial motor nucleus and Phox2b-positive cells in the hindbrain of <i>Lp</i> mutant embryos.

<p>A–C: In situ hybridization of <i>Egr2</i> (black) and <i>Hoxb1</i> (red) in E8.5 whole hindbrain preparations (lateral view) obtained from a <i>+/+</i> (A), <i>Lp/+</i> (B) and <i>Lp/Lp</i> (C) embryos. Wt (+/+) embryo processed for <i>Egr2</i> in situ only. Expression of <i>Egr2</i> in rhombomere 3 (r3) and r5, and of <i>Hoxb1</i> in r4 are unaffected in <i>Lp</i>/+ and <i>Lp/Lp</i> embryos. D–F: In situ hybridization of <i>Tbx20</i> expression in E14.5 whole hindbrain preparations (ventral view) obtained from +/+ (D), <i>Lp/+</i> (E) and <i>Lp/Lp</i> (F) embryos. Facial motoneurons failed to migrate properly both in <i>Lp/+</i> and <i>Lp/Lp</i> embryos. The black squares indicate the regions illustrated in G, H and I. G–O: Anti-Phox2b (red) and anti-Islet1,2 (green) immunofluorescence on whole hindbrain preparations (G–I, ventral view) and transverse sections (J–O) through E15.5 hindbrains of <i>+/+</i> (G, J, M), <i>Lp/+</i> (H, K, N) and <i>Lp/Lp</i> (I, L, O) embryos. White arrowheads in J and K point to Phox2b-positive cells located ventral to the facial nucleus. Numbered dashed lines in G–I refer for each genotype to the axial level corresponding to the sections illustrated in J–R. P–R: Anti-NK1R (red) and anti-Islet1,2 (green) immunofluorescence on transverse sections through E15.5 hindbrain preparations obtained at level 1 for a <i>+/+</i> embryo (P), and at level 2 for <i>Lp/+</i> (Q) and <i>Lp/Lp</i> (R) embryos. Slices illustrated in M and P, N and Q, O and R, correspond to adjacent slices obtained from the same animal, thus showing possible co-expression of several markers in the same neuronal population. Immunostainings indicate the presence of Phox2b-positive or NK1R-positive cell groups present at the ventral surface of the hindbrain for all genotypes. D: dorsal, L: lateral, R: rostral.</p

Response to a low pH challenge is unaffected in the <i>Lp/+</i> mutant.

<p>Integrated phrenic nerve discharge (Int C4) at pH 7.4 (upper trace) and pH 7.2 (bottom traces) for control (A) and <i>Lp</i>/+ (B) embryos at E16.5. C: Quantification of burst frequencies for control (left) and heterozygous (right) embryos at pH 7.4 (white bars) and pH 7.2 (gray bars). Numbers of hindbrain preparations analyzed are indicated on the bars. The motor output of the respiratory network recorded from C4 nerve roots is comparable in <i>Lp/+</i> and wild type preparations in control conditions, and the response to low pH is preserved in the <i>Lp/+</i> mutant.</p

Disruption of epithelial organisation in the distal outflow tract of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos.

<p><b>A–H</b>) At E9.0 in control embryos, β-catenin (green; B,D) is localised to the basolateral domain of the cells in the transition zone of the distal outflow wall and laminin (red; C,D) is becoming localised to the basement membrane underlying this. In <i>Vangl2^flox/flox; Isl1-Cre</i> littermates, β-catenin (F,H) and laminin (G,H) are less abundant and the tissue appears disorganised (n = 3). <b>I–P</b>) By E9.5, immunofluorescent staining for β-catenin is localised to the basolateral region of cells in the control embryo and shows the pseudo-stratified epithelium of the transition zone (J,L). In contrast, although β-catenin expression is still abundant in the transition zone of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos, the cells appear disorganised and it is difficult to determine its subcellular distribution (N - arrows). Laminin is found basally to the cells of the transition zone in control embryos (K - arrows), but is lost in some places and surrounds other cells within the transition zone of <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (O – arrows, n = 3). Note that whereas the distal outflow wall is 2-3 cell layers thick in the control embryo (L), in some places it is 4-5 cell layers thick in the mutant (P). <b>Q–T</b>) γ-tubulin staining of MTOCs at E9.5 shows that these are localised to the apical side of the cells in the distal outflow wall in control embryos (Q and rose plot S). In contrast, the position of the MTOC is much more variable in <i>Vangl2^flox/flox; Isl1-Cre</i> embryos (R and rose plot T), frequently localising to the basolateral side of the cell layer (n = 5) Chi-square, p<0.001. Ap =  Apical, Ba =  Basal, Dis  =  distal, Prox  =  proximal, <i>Vangl2^f</i>  =  <i>Vangl2^flox</i>. Quantification of γ-tubulin performed on 10 embryos (5 control, 5 mutant), with a total of 178 and 193 cells from control and mutant embryos respectively. Scale bar  = 20 µm.</p