Contribution of small and large Leydig cell aggregates to the total Leydig cell aggregate area per testis in e21.5 rat testes after <i>in utero</i> exposure to vehicle (control) or dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e20.5 (full treatment window), e15.5–e18.5 (MPW window) or e19.5–e20.5 (late window).

<p>Values are Means ± SEM for 8–15 animals from 3–5 litters per treatment group. ***p<0.001, in comparison with controls; ^ap<0.001 in comparison with Dex group (except p<0.05 when Dex is compared with DBP-500 late window treatment); ^bp<0.05 in comparison with DBP-500 late window group; ^cp<0.001 in comparison with DBP-750 late window group; ^dp<0.01 in comparison with DBP-750 MPW window group; ^ep<0.05 in comparison with DBP-750 full treatment window group.</p

Anogenital distance (AGD) and intratesticular testosterone (ITT) in rats at e21.5 after <i>in utero</i> exposure to vehicle (control), dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e20.5 (full treatment window), e15.5–e18.5 (MPW window) or e19.5–e20.5 (late window).

<p>Only treatments which included the masculinization programming window (MPW) resulted in a significant reduction in AGD in animals (A), whereas ITT was maximally reduced when treatment included the late (e19.5–e20.5) window (B). Values are Means ± SEM for 18–39 animals from 3–7 litters per group. ***p<0.001, in comparison with controls; ^ap<0.001 in comparison with Dex group; ^bp<0.001 in comparison with DBP-500 late window group; ^cp<0.001 in comparison with DBP-750 late window group; ^dp<0.01 in comparison with DBP-750 MPW window group; ^ep<0.05 in comparison with DBP-500 full treatment window group.</p

Immunohistological analysis of focal dysgenetic areas in rat testes exposed to vehicle (control) or dibutyl phthalate (DBP).

<p>(A–B) Double immunofluorescence for 3β-HSD (blue) and Sox-9 (red) on e21.5 testis sections from (A) vehicle (control) and (B) DBP-exposed (750 mg/kg/) animals, illustrating focal dysgenesis in which Leydig cell aggregates contain ectopically localized Sertoli cells. Green depicts DAPI nuclear counterstain. SC = seminiferous cords. Scale bar = 20 µm. (C1–C6) Example of sections stained for 3β-HSD (brown) used for Leydig cell aggregate analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030111#pone-0030111-g003" target="_blank">Figure 3</a>). Arrows indicate large Leydig cell aggregates, asterisks indicate seminiferous cords. Scale bar = 200 µm. (D–E) Double immunohistochemistry for 3β-HSD (blue) and SMA (brown) on postnatal day (pnd) 8 testis sections from (D) vehicle (control) and (E) DBP-exposed (500 mg/kg/) animals, illustrating focal dysgenesis after DBP-exposure, with large Leydig cell aggregates and malformed seminiferous cords and intratubular Leydig cells (arrows). Scale bar = 50 µm.</p

Relationship between Leydig cell (LC) aggregation ( = focal dysgenesis) and anogenital distance (AGD) (A, B) or intratesticular testosterone (ITT) at e21.5 (C, D) or between AGD and ITT at e21.5 (E, F) in animals exposed <i>in utero</i> to vehicle (control), dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex during all treatment windows (A, C, E), or during the full treatment window (e13.5–e20.5) only (B, D, F).

<p>Dysgenesis is negatively correlated with AGD irrespective of treatment period (A, B) whereas all other correlations were affected by the treatment period (full details in text).</p

Relationship between Leydig cell (LC) aggregation ( = focal dysgenesis) and anogenital distance (AGD) at postnatal day (pnd) 8 after <i>in utero</i> exposure to vehicle (control), dibutyl phthalate (DBP-500 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e21.5.

<p>(A) Average size of the 3 largest Leydig cell aggregates is shown as Means ± SEM for 8–17 animals from 3–6 litters. ***p<0.001, in comparison with controls; other comparisons are indicated by capped lines. (B) Correlation between dysgenesis and anogenital distance (AGD) in pnd8 animals.</p

Proximity of V_κ genes to E2a binding sites correlates with frequencies of long-range interactions.

<p>(A) Schematic representation of the <i>Igκ</i> locus, showing the location of all functional V_κ (grey, top), J_κ and C_κ gene segments, and the κ regulatory elements Sis, iEκ, and 3′Eκ. MAR, matrix attachment region. V_κ genes within close proximity (as defined by colocalization on the same 3C-Seq restriction fragment) to the indicated TFs or H3K4 hypermethylation (as detected by previous ChIP-seq studies; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#s4" target="_blank">Materials and Methods</a> for references) are shown. At the bottom, highly used (>1.0% used) V_κ gene segments are depicted (orange), which cluster within two large high-usage domains (yellow shading). Primary V_κ gene usage data was taken from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#pbio.1001791-AokiOta1" target="_blank">[54]</a>. (B) Average usage of V_κ genes marked only by an Ikaros binding site or those marked by binding sites of both Ikaros and E2a. (C) Comparison of average interaction frequencies (for the three κ regulatory elements indicated) between V_κ⁻ fragments (no V_κ), V_κ⁺ fragments containing an Ikaros binding site only, and V_κ⁺ fragments containing both an Ikaros and E2a binding site. Bars represent average frequencies for <i>Btk</i>^−/−<i>Slp65</i>^−/− pre-B cells (yellow) and WT pre-B cells (grey). (D) Classification of V_κ⁺ fragments, containing an Ikaros binding site only (top) or containing both an Ikaros and E2a binding site (bottom), based on the effect of pre-BCR signaling on their interactions with the three κ regulatory elements indicated. Increase and decrease were defined as >1.5-fold change of interaction frequencies detected in WT pre-B cells versus <i>Btk</i>^−/−<i>Slp65</i>^−/− pre-B cells. (E) Proposed model of pre-BCR signaling-mediated changes in κ enhancer action. In pro-B cells (left) the enhancers show minimal coordination and their interactions are not yet (fully) focused on the V_κ genes. Upon pre-BCR signaling and differentiation to pre-B cells (right), TFs bind the locus to coordinate enhancer action and focus their interactions to the V_κ genes, inducing germline transcription (GLT) and accessibility to the V(D)J recombinase. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#s3" target="_blank">Discussion</a> for more details. Statistical significance was determined using a Mann–Whitney U test (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s., not significant, <i>p</i>≥0.05).</p

3C-Seq analysis of long-range chromatin interactions within the Igκ locus and flanking regions.

<p>(A) Overview of long-range interactions revealed by 3C-Seq experiments performed on the indicated cell fractions, representing a gradient of pre-BCR signaling, whereby the iEκ element (top), the 3′Eκ element (center), or the Sis element (bottom) was used as a viewpoint. Shown are the relative interaction frequencies (average of two replicate experiments) for the indicated genomic locations. The ∼8.4 Mb region containing the Ig<i>κ</i> locus (yellow shading) and flanking regions (cyan shading) is shown and genes and genomic coordinates are given (bottom). The locations of the two BAC probes used for 3D DNA-FISH are indicated by a green (distal V_κ probe) and red (proximal C_κ/enhancer probe) rectangle (<i>bottom</i>). Pre-B cell fractions were FACS-purified from the indicated mice on a VH81x transgenic <i>Rag1</i>^−/− background (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#pbio-1001791-g001" target="_blank">Figure 1</a> for gating strategy). Erythroid progenitor cells were used as a nonlymphoid control. (B) 3D DNA-FISH analysis comparing locus contraction in cultured bone-marrow–derived <i>E2a</i>^−/− pre-pro-B, <i>Rag1</i>^−/− pro-B, and VH81x <i>Rag1</i>^−/− pre-B cells (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#pbio.1001791.s006" target="_blank">Figure S6</a> for phenotype of IL-7 cultured B-lineage cells). Locations of the BAC probes used are indicated at the bottom of panel A. Representative images for all three cell types are shown on the left, quantifications (>100 nuclei counted per cell type) on the right. The red lines indicate the median distance between the two probes. Statistical significance was determined using a Mann–Whitney U test (***<i>p</i><0.001; n.s., not significant, <i>p</i>≥0.05).</p

Modulation of long-range chromatin interactions within the Igκ locus by pre-BCR signaling.

<p>Quantitative analysis of 3C-Seq datasets using the three indicated κ regulatory elements as viewpoints. (A) Average long-range chromatin interaction frequencies (from two replicate 3C-seq experiments) with upstream (∼2.0 Mb), V_κ (∼3.2 Mb), and downstream (∼3.2 Mb) regions, as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#pbio-1001791-g003" target="_blank">Figure 3A</a>, for the five B-cell precursor fractions representing a pre-BCR signaling gradient. Average interaction frequencies per region were calculated as the average number of 3C-Seq reads per restriction fragment within that region. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#s4" target="_blank">Materials and Methods</a> section for more details. (B) Average interaction frequencies within the V_κ region were determined for fragments that do not (−) contain a functional V_κ gene and for those that do contain a functional V_κ gene (+). (C) Correlation plots of average interaction frequencies of the two enhancer elements with the 101 functional V_κ genes for WT pre-B cells (left) versus <i>Btk</i>^−/−<i>Slp65</i>^−/− pre-B cells (right). On the log scale, frequencies <1 were set to 10⁰. Statistical significance was determined using a Mann–Whitney U test (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s., not significant, <i>p</i>≥0.05).</p

Long-range chromatin interactions of κ regulatory elements correlate with TF binding and histone modifications.

<p>(A-D) For fragments within the V_κ region, average 3C-seq interaction frequencies were calculated for fragments that did (+) or did not (−) contain binding sites for TFs or H3K4 histone modifications (as determined by previous ChIP-Seq studies; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001791#s4" target="_blank">Materials and Methods</a> for references). Data for the three viewpoint and the five B-cell precursor fractions representing a pre-BCR signaling gradient are shown for Ctcf (A), Ikaros (B), E2a (C), and H3K4 di- and tri-methylation (Me2/3). Statistical significance was determined using a Mann–Whitney U test (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s., not significant, <i>p</i>≥0.05).</p

Genes differentially expressed between WT, Btk, or Slp65 single or double mutant V_H81X Tg <i>Rag1</i>^−/− pre-B cells or <i>Rag1</i>^−/− pro-B cells.

a<p><i>p</i> value in ANOVA analysis.</p>b<p>Fold change times up-regulated or down-regulated (−) when compared with WT (V_H81X Tg <i>Rag1</i>^−/−) pre-B cells.</p><p>Groups are Rag1 KO <i>Rag1</i>^−/− pro-B cells; Btk KO <i>Btk</i>^−/− V_H81X Tg <i>Rag1</i>^−/− pre-B cells; Slp65 KO <i>Slp65</i>^−/− V_H81X Tg <i>Rag1</i>^−/− pre-B cells; BtkSlp65 KO <i>Btk</i>^−/−<i>Slp65</i>^−/− V_H81X Tg <i>Rag1^−/−</i> pre-B cells.</p>c<p>n.s., <i>p</i>>0.01.</p