Seminal fluid values of the 20 patients treated with mDRV/C compared with the results of 21 patients treated initially with ART and then with mDRV/r [6].

<p>Seminal fluid values of the 20 patients treated with mDRV/C compared with the results of 21 patients treated initially with ART and then with mDRV/r [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196257#pone.0196257.ref006" target="_blank">6</a>].</p

Patients included in different procedures of the study according to the seminal viral load.

<p>Patients included in different procedures of the study according to the seminal viral load.</p

Semen quality in patients receiving mDRV/r [6] and mDRV/C.

<p>Semen quality in patients receiving mDRV/r [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196257#pone.0196257.ref006" target="_blank">6</a>] and mDRV/C.</p

HCNR sequence analysis suggests novel <i>meis1</i> upstream regulators.

<p>(A) <i>meis1</i> transcription, detected by <i>in situ hybridization</i> (upper panel) compared to GFP expression, driven by the HHc2:066650-GFP transgenic line (lower panel) at 12 (left), 24 (middle) and 30 hpf (right). Side views are shown. HHc2:066650 drives GFP expression in the eye primordium, the early hindbrain, the retina, the optic tectum and the olfactory bulb, which are <i>meis1</i> expression domains. B) The analysis of the potential binding sites predicted by Jaspar highlights the presence of three potential Pax6 sites. Two of these sites contain a high degree of similarity with the consensus. (C) Pax6_Binding_Site1 also mapped to a microisland of ultraconservation when human and zebrafish orthologous regions were aligned.</p

Pax6 regulates the enhancer activity of HHc2:066650.

<p>A) Representative pictures of transgenic zebrafish embryos at 24 and 48 hpf, corresponding to HHc2:066650 F2 offspring injected with MOpaxA, MOpaxB, or a mixture of both. The red arrow points to the retina, where a strong decrease in the GFP levels is observed after morpholino treatment. A control mopholino-injected individual is shown for comparison. B) Representative mosaic embryos injected with HHc2:066650 construct with/without the two candidate Pax6 binding sites. Both the GFP and RFP channels are included. The boxed region is magnified on the right. The dashed circle delineates the eye. (C) GFP expression was measured in the eye (area marked by the dashed circle in (B)) of wild type and Δpax6_BS version of HHc2:066650 mosaic embryos (n = 9 and 11 embryos, respectively), and normalized with their respective muscle RFP expression. Enhancer signal significantly decreased in the mutant version of HHc2:066650 (p = 0.018, Mann Whitney test). The box plots contains a central rectangle, which spans from the first quartile to the third quartile. A segment inside the rectangle shows the median and whiskers above and below the box show the locations of the minimum and maximum.</p

Chromatin conformation assays measuring <i>in vivo</i> the relative interaction frequency between zHHc2:066650 and <i>meis1</i> promoter.

<p>Relative interaction of the zebrafish HHc2:066650 orthologous region and the <i>meis1</i> promoter, 70.8 Kb upstream of zHHc2:066650. Interaction frequency was measured relative to two control regions: the first control maps 62 Kb upstream zHHc2:066650, while the second one lies 47.8 Kb downstream the enhancer. Interaction between <i>meis1</i> promoter and zHHc2:066650 shows a 3-fold increase when compared to the controls. Error bars represent the standard deviation of three independent measurements.</p

Distribution of the human conserved non-coding regions showing enhancer activity in transgenic Zebrafish.

<p>Vista browser representation of the genomic region surrounding the human <i>MEIS1</i> gene (chr2:065.893.545–067.353.996), using Hg18 human genome as reference. Conservation is represented as pink peaks. Conserved non-coding elements (CNEs) with confirmed enhancer activity in stable transgenic zebrafish lines are marked as vertical bars.</p

Enhancer activities from the HCNRs recapitulate endogenous <i>meis1</i> expression pattern.

<p>A) Dorsal and lateral views of a representative founder illustrate the expression pattern of each HCNR. B) Picture of a <i>meis1 in situ</i> hybridization of a 30 hpf embryo illustrates the different expression territories. A color-based schema of a zebrafish embryo highlights the different domains where GFP expression was found during the study. C) Diagrams of patterns of expressions of highly penetrant enhancers (boxed in red) and of non-tissue specific enhancers (boxed in blue). In each diagram, the number of founder lines showing expression in each particular body structure is represented by the red lines. For example, of three lines of HHc2:066543, all three showed forebrain expression, and additionally, two of them drove expression in the hindbrain while the remaining one in the neural tube. <i>In situ</i> hybridization picture is from ZFIN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033617#pone.0033617-Thisse1" target="_blank">[16]</a>.</p

Enhancer activity displayed by the HCNRs in stable transgenesis at 24–48 hpf.

<p>Table summarizing the GFP expression patterns and the enhancer activity found in the study. The “+” symbol in the enhancer activity column symbolizes the strength of the expression patterns according to GFP expression levels regardless of the similarity among founders. The “−” symbol represents the absence of activity. Columns 4–11 refer to the number of founders showing GFP expression on each territory. A CNE was classified as “negative” if at least two founder lines did not show any GFP expression. To annotate the expression pattern of any positive CNE, at least three stable transgenic lines were analysed. “Booster” refers to HCNRs that, when in different stable lines, drive expression in very different patterns, as if boosting the position effects.</p

Human highly conserved non-coding regions assayed for enhancer activity.

<p>Annotation of the regions corresponding to the human Hg18, that were amplified by PCR and assayed for enhancer activity using the ZED vector. (n.d: not detected).</p