Expression of <i>Th, NurrI</i>, <i>C130021L20Rik</i> and <i>Rspo2</i> is affected in the adult SNc.

<p>(A–G′′) <i>Th</i> expression in the adult midbrain from rostral to caudal, marking the SNc and the VTA respectively. A comparison between wild-type (wt), heterozygous and homozygous <i>Dreher</i> material demonstrates almost identical expression patterns between <i>wt/wt</i> and <i>wt/dr</i> coronal brain sections, whilst obvious defects can be seen in the rostral-lateral part of the SNc in <i>dr/dr</i> midbrains (arrowheads). The medial VTA displays some reduction, although more subtle than in rostral-lateral mdDA domains. (H–K′) Expression of <i>NurrI</i> (H,J,H′,J′), and <i>C130021L20Rik</i> (I,K,I′,K′), shows similar defects as was seen for <i>Th</i>; rostral-lateral expression is diminished (arrowheads). (L–Q′) A complete loss of <i>Rspo2</i> expression in the SNc was observed. In the wild-type, only a subset of cells in the SNc domain express <i>Rspo2</i>, and all <i>Rspo2</i> expression is lost in the affected SNc of the <i>Lmx1a</i> knock-out, as can be observed in higher magnifications (O,Q,O′,Q′).</p

<i>Lmx1a</i> Encodes a Rostral Set of Mesodiencephalic Dopaminergic Neurons Marked by the <i>Wnt</i>/B-Catenin Signaling Activator <i>R-spondin 2</i>

<div><p>Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor <i>Lmx1a</i> is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in <i>Lmx1a</i>-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of <i>Lmx1a</i>. Subsequent expression analysis on brain sections, confirmed that <i>Nurr1</i> is regulated by <i>Lmx1a,</i> and additional downstream targets were identified, like <i>Pou4f1, Pbx1, Pitx2</i>, <i>C130021l20Rik</i>, <i>Calb2</i> and <i>Rspo2</i>. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, <i>Nurr1</i> and <i>C130021l20Rik</i> were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the <i>Wnt</i>/b-catenin signaling activator <i>Rspo2</i> in this domain. Subsequent analysis of <i>Rspo2−/−</i> embryos revealed affected mdDA neurons, partially phenocopying the <i>Lmx1a</i> mutant. To conclude, our study revealed that <i>Lmx1a</i> is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the <i>Wnt</i> activator <i>Rspo2</i>.</p></div

A model integrating several identified targets of <i>Lmx1a</i>.

<p><i>Pou4f1</i> (<i>Brn3a</i>) was strongly up-regulated in the <i>Lmx1a−/−</i> expression array, in line with a suggested role of <i>Lmx1a</i> in repressing an alternative, red nucleus, fate during development (dashed line). <i>Lmx1a</i> targets <i>C130021l20Rik</i> and <i>Calb2</i> require <i>Lmx1a</i> directly or indirectly for correct expression in subsets of the mdDA system. The <i>Nurr1</i> transcriptional pathway induces differentiating mdDA neurons, and loss of <i>Lmx1a</i> resulted in affected <i>Nurr1</i> expression, but also in affected expression of <i>Nurr1</i> transcriptional targets, mainly in rostral-lateral mdDA neurons. <i>Rspo2</i> is involved in the <i>Wnt1</i>/b-catenin signaling pathways, which is implicated in proliferation and cell-cycle exit. Loss of <i>Lmx1a</i>, resulted in decreased <i>Rspo2</i> expression and loss of mdDA neuronal markers as Th, Ahd2 and Pitx3. Interestingly, loss of <i>Rspo2</i> partially phenocopied the defects observed in the <i>Lmx1a-dr/dr</i> mdDA system.</p

Genes regulated by <i>Lmx1a</i> in a microarray analysis of E12.5 <i>Lmx1a-dr/dr</i> embryos.

<p>(A) RNA was collected from E12.5 micro-dissected <i>Lmx1a-dr/dr</i> and <i>wt/wt</i> brains. <i>Lmx1a-dr/dr</i> samples were hybridized against <i>wt/wt</i> pooled RNA control. The heatmap represents up- (red) and down- (green) regulated genes based on log2-ratios of four individual microarray samples. Only the top 20 significantly up- and down-regulated genes are shown. (B) Relative expression levels of the 20 most up- and (C) down-regulated genes (microarray FWER ANOVA, p<0.05). With <i>Rspo2</i> as most down-regulated gene, and <i>Usf1</i> as highest up-regulated gene. (D) Venn-diagrams showing a number of genes that are also regulated in <i>Pitx3</i> and <i>NurrI</i> microarrays derived from previous studies. (E) qPCR validation of significant down-regulation of <i>Rspo2</i>, <i>Nurr1</i>, <i>Calb2</i>, <i>C130021l20Rik</i>, <i>Lmx1a</i>, <i>Cbln1</i> and <i>Pitx2</i>, and of significant up-regulation of <i>Pbx1</i> and <i>Pou4f1</i>, in <i>Lmx1a</i>-deficient embryonic midbrains. <i>18s</i> was used for normalization. Mean expression values in wt are set at 1 (red line) and are indicated with standard error bars (s.e.m.). Grey bars represent mean expression changes in <i>Lmx1a-dr/dr</i> samples compared to wt samples. Statistical analysis was performed with Student’s t-test. *P<0.05 is considered significant; **P<0.01. N = 4 for all analyzed genes and for each phenotype (each experimental sample (n) represents a pool of five micro-dissected midbrains). <i>M, midbrain; MHB, mid-hindbrain border; R, rostral; C, caudal; wt, wild-type.</i></p

The observed rostral-lateral phenotype is retained in E14.5 <i>Lmx1a-dr/dr</i>.

<p>(AA–JJ′) ISH analysis of several mdDA markers in wild-type and <i>Lmx1a-dr/dr</i> tissue. Small arrows indicate the fasciculus retroflexus, as extra anatomical marker for comparing wild-type and mutant sections. (A–F′′) <i>Lmx1a</i> transcript levels are lowered in <i>Lmx1a-dr/dr</i> (large arrowheads), while between the heterozygous mutant and wild-type control no differences are observed. (G–L′) <i>Aadc</i> expression is laterally down-regulated, except for a clear rostral expansion of a group of cells in the diencephalon (G′K′,L′). Medially, <i>Aadc</i> expression is expanded rostrally and dorsally. (M–R′) <i>Pitx3</i> expression defects are found in the rostral-lateral parts were transcript levels are lower (arrowhead). (S–X′) A more pronounced defect is observed for <i>Th</i>, which is clearly reduced in lateral sections (arrowheads). (Y–DD′) <i>En1</i> expression is slightly decreased in most lateral domains. (EE–JJ′) For <i>Ahd2,</i> the most lateral expression domains are lost while more medial expression domains are unchanged. (KK–KK′) IHC analysis of E12.5 sagittal <i>Lmx1a</i> wild-type and knock-out tissue. The initial TH fiber outgrowth (green) appears unaffected in the mutant, since growth patterns towards LMX1A+ cells (red) are almost identical, despite a smaller number of TH+ cells. (LL–OO′) Medial to lateral analysis of TH fiber outgrowth and innervation within the striatum, in wild-type and <i>Lmx1a-dr/dr</i> tissue at E14.5, displays slightly fewer TH+ fibers in the knock-out. However, no clear phenotype is observed in direction or organization of the fibers, and they arrive at the expected site in the striatum (OO,OO′, green arrowheads).</p

mdDA markers TH, AHD2 and PITX3 display loss of expression in <i>Rspo2-LacZ/LacZ</i> embryonic midbrain.

<p>Sagittal sections of <i>Rspo2</i> control and knock-out (<i>LacZ/LacZ</i>) littermate mouse brains, at E14.5. (A–G′) TH protein expression analysis reveals a decrease in TH+ cells in the <i>Rspo2</i>-ablated mdDA neuronal field. Lines indicating the mid-hindbrain border (mhb) and the fasciculus retroflexus (fr) are added for clarity. (A′′–G′′) More detailed images of (A′–G′). BGAL protein staining can be observed in <i>Rspo2-LacZ/LacZ</i> mutant cells. Most of these cells do not express TH (anymore). (H–N′) A decrease in PITX3-expressing mdDA neurons in <i>Rspo2-LacZ/LacZ</i> tissue is shown. (O–U′) From lateral to medial to lateral, AHD2 analysis reveals a decrease in expression in the paramedian and lateral mdDA neuronal subset, in the absence of <i>Rspo2</i>. (V–X′) Higher magnifications of (C,C′), (J,J′) and (Q,Q′), showing a mild but clear decrease of mdDA neurons expressing TH, PITX3 and AHD2 in the paramedian midbrain. (Y–AA′) Higher magnifications of (F,F′), (M,M′) and (O,O′), showing a clear decrease in the number of TH-, PITX3-, and AHD2-positive neurons in the lateral mdDA neuronal field.</p

PARG forms complexes with Smad proteins and de-ADP-ribosylates Smad3.

<p>(<b>a</b>) Immunoprecipitation of Flag-Smad2, Flag-Smad3 or Flag-Smad4 followed by immunoblotting for myc-PARG in cell lysates of transiently transfected 293T cells with the indicated plasmids and after stimulation with vehicle (-TGFβ, left panel) or 5 ng/ml TGFβ1 for 30 min (right panel). Expression levels of all transfected proteins are shown in the TCL immunoblot of the 293T cells. (<b>b</b>) Immunoprecipitation of Flag-Smad2/3/4 followed by immunoblotting for myc-PARG in cell lysates of transiently transfected 293T cells with the indicated plasmids and in the absence of stimulation with TGFβ. Expression levels of all transfected proteins are shown in the TCL immunoblot of the 293T cells. α-Tubulin immunoblot serves as protein loading control. Stars mark non-specific protein bands. (<b>c</b>) Immunoprecipitation of endogenous Smad2/3 followed by immunoblotting for transfected myc-PARG in 293T cells stimulated with vehicle (-TGFβ) or with 5 ng/ml TGFβ1 for 30 min. Negative control immunoprecipitation using non-specific IgG is shown. TCL shows the levels of endogenous Smad2/3 proteins and transfected myc-PARG before immunoprecipitation. Smad2/3 immunoblot also serves as protein loading control. (<b>d</b>) In vitro de-ADP-ribosylation assay of Smad3 using PARG. GST-Smad3 was first ADP-ribosylated using recombinant PARP-1. The proteins were pulled-down and washed, prior to reconstitution with PARG reaction buffer and increasing amounts of recombinant PARG (shown as milli-units (mU) of enzymatic activity). The ADP-ribosylated proteins are shown in the autoradiogram along with the CBB-stained input GST-Smad3 levels. Panels a–c show results from representative experiments that were repeated at least twice and panel d shows results from representative experiments that were repeated at least three times.</p

PLA of endogenous Smad3 and PARP-1 complexes in HaCaT cells.

<p>(<b>a</b>) HaCaT cells were analyzed with PLA using antibodies against Smad3 and PARP-1 after transfection with control or the indicated specific siRNAs and stimulation with vehicle (-TGFβ) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. Cells stimulated with 10 mM hydrogen peroxide for 10 min served as positive control. PLA with single antibodies against Smad3 or PARP-1 are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b</b>) Quantification of the experiment shown in panel (a) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p

TGFβ induces formation of endogenous complexes between Smads and PARP-1/2 in HaCaT cells.

<p>(<b>a</b>) Immunoprecipitation of Flag-Smad2/3/4 followed by immunoblotting for PARP-1 and PARP-2 in cell lysates of transiently transfected HEK-293T cells with the indicated plasmids and after stimulation with vehicle (-TGFβ) or 5 ng/ml TGFβ1 for 30 min. Expression levels of all transfected proteins and endogenous PARP-1 and PARP-2 are shown in the total cell lysate (TCL) immunoblot of the HEK 293T cells. PARP-1 immunoblot also serves as protein loading control. Stars mark non-specific protein bands. (<b>b</b>) Immunoprecipitation of Smad2/3 followed by immunoblotting for PARP-1, PARP-2, Smad2/3 and Smad4 in HaCaT cells stimulated with vehicle (-TGFβ) or with 5 ng/ml TGFβ1 for 30 min. Negative control immunoprecipitation using non-specific IgG is shown. TCL shows the levels of endogenous proteins before immunoprecipitation. PARP-1 immunoblot also serves as protein loading control and C-terminal phospho-Smad2 (p-Smad2) serves as control for the efficiency of stimulation of TGFβ signaling. (<b>c</b>) Immunoprecipitation of Smad2/3 followed by immunoblotting for PARP-1, PARP-2, Smad2/3 and Smad4 in HaCaT cells transfected with the indicated siRNAs and stimulated with 5 ng/ml TGFβ1 for 30 min or not (-TGFβ). Efficiency of knockdown of PARP-1 and PARP-2, total Smad levels, phospho-Smad2 levels and protein loading (α-tubulin) controls can be seen in the TCL. (<b>d</b>) In vitro PARylation assay after glutathion-pulldown of control GST protein or GST-Smad3, truncated mutant of GST-Smad3 (ΔMH2) and GST-Smad4 in the presence of recombinant PARP-1 and/or recombinant PARP-2 as indicated. A star (weak signal) indicates the position of PARP-2 in addition to the arrow. A longer exposure of the autoradiogram around the migrating position of PARP-2 is shown at the bottom. Note the position of ADP-ribosylated Smad proteins that migrate at the size of the core non-ADP-ribosylated proteins. The input amounts of recombinant proteins were calculated based on staining of test SDS-PAGE with CBB as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone.0103651.s001" target="_blank">Fig. S1</a>. The figure shows results from representative experiments that were repeated at least twice.</p

PLA of endogenous PARP-1 and PARP-2 ADP-ribosylation after TGFβ stimulation in HaCaT cells.

<p>(<b>a, c</b>) HaCaT cells were analyzed with PLA using antibodies against PARP-1 and PAR chains (<b>a</b>) or antibodies against PARP-2 and PAR (<b>c</b>) after stimulation with vehicle (0 min) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. PLA with single antibodies against PARP-1 or PAR are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b, d</b>) Quantification of the experiments shown in panels (a, c) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p