La Vigie marocaine

14 août 19381938/08/14 (A29,N10107)-1938/08/14

Additional file 6: of Conservation analysis of sequences flanking the testis-determining gene Sry in 17 mammalian species

CentriMo input sequences. The 600 bp 5’ flanking sequences used in the CentriMo analysis. (TXT 10 kb

Lymphatic vessels are limited to the tunica albuginea in adult testis.

<p><b>A–C)</b> EGFP-positive vessels are readily observed across the surface of the adult testis emanating from the spermatic cord (asterisk). Strong EGFP expression is also observed within the seminiferous tubules. <b>D–F)</b> Sectioned <i>Prox1</i>-EGFP transgenic adult testis co-stained with the Leydig cell marker HSD3B1 and counterstained with DAPI revealed no lymphatic vessels inside the testis, but within the tunica albuginea (arrow). Additional EGFP expression was verified within the seminiferous tubules (encircled) and localised to spermatids closer to the lumen. <b>G–I)</b> Sectioned <i>Prox1</i>-EGFP adult epididymis co-stained with the smooth muscle cell marker ACTA2 to demarcate the vas deferens and counterstained with DAPI showed prominent EGFP-positive lymphatic vessels, but also EGFP-positive sperm cells. BV = blood vessel; scale bars C = 600 µm, D = 100 µm, G = 300 µm.</p

Lymphatic vessels sprout across, but not beyond, the testis cap at 17.5 dpc.

<p>Representative images captured from optical projection tomograph, also represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052620#pone.0052620.s002" target="_blank">Vid S1</a>. <b>A)</b> During late gestation, EGFP-positive lymphatic vessels are seen growing from pre-existing vessels in the spermatic cord before sprouting across the testis surface. B<b>)</b> The fetal testis also contains a rich network of blood vessels visualised by ENG staining, but <b>C)</b> the EGFP-positive vessels do not overlap with the more extensive blood vasculature. Yellow areas correspond to lymphatic vessels (green) and blood vessels (red) in different planes. <b>D)</b> 3-D model of <i>Prox1</i>-EGFP positive lymphatic network during initial development. <b>E)</b> Magnified region showing two EGFP-positive lymphatic vessels running parallel to the coelomic (arterial) vessel and <b>F)</b> magnified region of the rete testis. T = testis; E1 = head of epididymis; E2 = tail of epididymis; CV = coelomic vessel; RT = rete testis; scale bars: A = 500 µm, B = 250 µm.</p

The adult ovary possesses a rich lymphatic network largely overlapping with the blood vasculature.

<p>Representative images captured from optical projection tomograph, also represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052620#pone.0052620.s003" target="_blank">Vid S2</a>. <b>A)</b> The mature ovary contain a dense network of lymphatic vessels that emanate from the rete ovarii, and <b>B)</b> a rich network of ENG-positive blood vessels. <b>C)</b> Lymphatic vessels also expressing LYVE1 are generally localised to the ovarian and extraovarian rete. <b>D)</b> 3-D model of <i>Prox1</i>-EGFP positive lymphatic network as compared to <b>E)</b> LYVE1-positive lymphatic vessels and ENG-positive blood vessels <b>F)</b> Merged image of ENG, EGFP and LYVE1 expression in the adult ovary reveals distinct patterning of the blood and lymphatic network. Oc = ovarian cortex; Om = ovarian medulla; RO = rete ovarii; F = follicle; scale bar = 1 mm.</p

Lymphatic vessels develop in the postnatal ovary from around 10 dpn. A)

<p>At 7 dpn, EGFP-positive vessels are observed in along one side of the uterine horn and in the ovarian ligaments, but the ovary (encircled) is still devoid of lymphatics. <b>B)</b> At 10 dpn, the ovary possesses a lymphatic network. Lymphatic vessels sprouting laterally at distinct regional distances from a pre-existing vasculature along the length of the uterus have almost encircled the entire uterine horn. <b>C)</b> At 14 dpn, the ovary possesses a distinct lymphatic network and the uterine horn has developed a strikingly segmented lymphatic network encircling the entire tissue <b>D)</b> The adult ovary maintain a high Prox1-RGFP expression and the uterine horn has developed a extensive mesh of lymphatic vessels. <b>E)</b> Prominent <i>Prox1</i>-EGFP positive vessels of the 9 week uterine horn also express endogenous PROX1, and <b>F)</b> the lymphatic marker NRP2, <b>G)</b> both overlapping with <i>Prox1</i>-EGFP expression. OL = ovarian ligament; U = uterine horn; scale bar = 200 µm.</p

Partial phenocopy of known gene knockouts in gonad and pancreas.

<p>(A, B) STRA8 knockdown: IF showed knockdown of STRA8 (A) in Stra8MO-treated XX gonads. Nuclear localisation of meiosis markers (γH2AX (A) and SCP3 (B); indicated by white arrows; see inserts) was absent but germ cells were present (POU5F1 (B); see inserts) in XX Stra8MO-treated gonads. (C–E) Knockdown of SOX9 in the gonad: Western blot for SOX9 (relative to α-TUBULIN or β-ACTIN) showed a downregulation of SOX9 (C) after Sox9MO treatment in XY gonads (<i>n</i> = 3). Downregulation of expression of SOX9 target gene <i>Amh</i> (D) expression was observed by qRT-PCR (<i>n</i> = 8, 15, 11, 4). IF for AMH and HSD3β (E) showed that AMH staining was weaker in XY Sox9MO samples compared to XY controls and that HSD3β-positive FLCs were present but staining was weaker in XY Sox9MO-treated gonads. (F–I) Knockdown of SOX9 in the pancreas: qRT-PCR of Sox9Mo treated pancreata showed <i>Ins1</i> (F) was downregulated but <i>Pax6</i> (G) was unchanged (<i>n</i> = 5, 5, 5, 5). Quantification of PAX6/INS-positive cells revealed that PAX6-positive (H) and INS-positive (I) cell number was unaltered by Sox9MO treatment (<i>n</i> = 3, 4, 2, 2). Scale bars = 100 μM; cMO = control morpholino; xMO = morpholino targeting gene x. For Western blots SOX9 levels were normalised to α-TUBULIN or β-ACTIN loading controls and Sox9MO-treated XY gonads measured relative to cMO treated XY gonads with expression for each blot set to 1. Rel. Ab./control = Relative Abundance of SOX9 to α-TUBULIN or β-ACTIN. For all qRT-PCR levels are shown relative to <i>Tbp</i>, error = S.E.M. For cell quantification error = S.E.M. with individual counts plotted. * = p = 0.05, ** = p = 0.001, *** = p = 0.0001, ns = not statistically significant.</p

Overview of method: MO delivery by heart injection.

<p>(A) Experimental pipeline from harvest of embryos through to injection, culture and downstream analyses. Visualisation of heart injection protocol can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114932#pone.0114932.s001" target="_blank">S1 Video</a> and images B–E. The cocktail of dye and MO in PBS is delivered via injection into the left ventricle of the beating heart at 11.5 dpc (B). Dye can be visualised going around the embryonic vasculature (indicated by white arrows) (C, D) and into the head vasculature (D) before the whole embryo is coloured (E). Schematic of ventricle injection (F) and the embryonic gonad which is highly vascularised (G). Delivery of India ink and F-MO (indicated by white arrows) shows the compounds reaching the mesonephric plexus at 5 min post-injection (H; <i>n</i> = 3); after 30 min F-MO positive cells were observed in the gonad proper (I; <i>n</i> = 3). s = seconds; min = minutes; g = gonad; m = mesonephros; F-MO = carboxyfluorescein-labelled standard control morpholino oligonucleotide. Scale bars: E = 1 mm, H = 0.5 mm.</p

Double knockdown of <i>Gli1/Gli2</i> in XY gonads.

<p>(A–D) Knockdown of GLI1/GLI2 in the gonad: qRT-PCR showed that treatment with Gli1/Gli2MO (<i>n</i> = 6, 5, 5, 8) resulted in no significant downregulation in steroidogenic regulator <i>Sf1/Nr5a1</i> (A) but a significant downregulation in expression of steroidogenic pathway enzymes <i>Hsd3β</i> (B), <i>Cyp11a1</i> (C) and <i>Star</i> (D). No change was observed in <i>Nr5a1</i> expression in Gli1MO or Gli2MO knockdown (E, I). Similarly, there were no changes in expression of steroidogenic pathway enzymes <i>Hsd3β</i> (F, J), <i>Cyp11a1</i> (G, K) and <i>Star</i> (H, L) in Gli1MO (E–H; <i>n</i> = 6, 6, 7, 5) or Gli2MO (I–L; <i>n</i> = 8, 7, 4, 3) single knockdowns. IF showed Sertoli cells (AMH (M) and SOX9 (N)) and germ cells (POU5F1 (M)) were present in XY Gli1/Gli2MO treated gonads and no FOXL2-positive cells were observed (N). Steroidogenic <i>Hsd3β</i>-positive (M) and <i>Nr5a1</i>-positive (N) cells were still present in Gli1/Gli2MO treated XY gonads. Quantification (<i>n</i> = 2) of steroidogenic cells revealed no change in the number of HSD3β-positive Leydig cells (O; green) or SF1-positive/SOX9-negative pre-Leydig cells (O; red). There was a decrease in the number of SOX9-positve Sertoli cells in the Gli1/2MO treated XY gonads (O; yellow). Scale bars = 100 μM; cMO = control morpholino; xMO = morpholino targeting gene x. For all qRT-PCR levels are shown relative to <i>Tbp</i>, error = S.E.M. For cell quantification error = S.E.M. with individual counts plotted. * = p = 0.05, ** = p = 0.001, ns = not statistically significant.</p

Immunofluorescence analysis of C57BL/6J TES deleted XY gonads.

<p>(A) Bright field images of 8-week old wild type, TES^+/- and TES^-/- testes. (B) Immunostaining of 13.5 dpc XY testis of wild type and TES^-/- embryos. (C) Immunostaining of 8-week old XY testes of wild type and TES^-/- mice. Testes were stained for SOX9 (green), FOXL2 (red) and DAPI (blue).</p