UPF2 is essential for prospermatogonial development in murine testes.

<p>(A) A schematic diagram showing the critical time points during male germ cell development. Two Cre deletor lines used express Cre in the male germline starting from E15.5 (<i>Ddx4-Cre</i>) and P4 (<i>Stra8-Cre</i>) as indicated. PGC, primordial germ cell; E, embryonic day; spg, spermatogonium; pL, pre-leptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diplotene spermatocyte; rspd, round spermatid; espd, elongating/elongated spermatid. (B) Gross morphology of the testis and the epididymis of WT and Ddx4-KO mice. (C) Testis growth curves of WT and Ddx4-KO mice. Testis weight is presented as means ± SEM (n = 6). (D) Testicular histology of WT and Ddx4-KO mice at P60. Seminiferous tubules of Ddx4-KO mice were devoid of germ cells and suffused with vacuoles (*). Only Sertoli cells (arrows) were present along the basal membrane. Scale bar = 30μm.</p

UPF2 is required for postnatal germ cell development in mice.

<p>(A) Gross morphology of the testis and the epididymis of WT and Stra8-KO mice. Scale bar = 2mm. (B) Testis growth curve during postnatal development between WT and Stra8-KO mice (n = 6 for each genotype). (C) Histology of WT and Stra8-KO testes and epididymides at P60. While abundant mature spermatozoa were observed in the WT cauda epididymis, only a few degenerating, immature male germ cells were present in the Stra8-KO cauda epididymis. (D-E) Percentage of seminiferous tubules containing vacuoles (D) or multinucleated giant cells (E) in WT and Stra8-KO testes (n = 4 for each genotype). (F) Percentage of tubules containing apoptotic cells based on terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; Scale bar = 50μm.</p

Histological analyses of the developing testes in WT and Ddx4-KO mice.

<p>(A) HE staining of paraffin-embedded testicular sections. At postnatal day 5 (P5), the number and morphology of germ cells and Sertoli cells are indistinguishable between WT and Ddx4-KO testes. Upon P10, preleptotene or leptotene spermatocytes (arrows) started to appear in WT testes, but not in Ddx4-KO testes due to germ cell loss and delay in meiotic entry. At P20 and thereafter, numerous vacuoles (*) were present, indicative of massive germ cell depletion in Ddx4-KO testes. Scale bar = 50μm. (B) Loss of prospermatogonia during neonatal testicular development in Ddx4-KO males. Immunostaining of SOHLH1, a marker for prospermatogonia and spermatogonia, in the testes of Ddx4-KO male mice at embryonic day 18 (E18), postnatal day 0 (P0), P2, P3 and P5. Scale bar = 70μm. (C) Quantitative analyses of the average number of germ cells per tubule cross-section in WT and Ddx4-KO testes at E18, P0, P2, P3 and P5. Data are presented as Means ± SD, n = 20. * denotes statistical significance (P<0.05).</p

RNA-Seq analyses reveal no global PTC upregulation, but a drastic increase in the 3’UTR length among upregulated, ubiquitously expressed transcripts in Stra8-KO total testes or purified germ cells.

<p>(A) Heat map showing the clustering of de-regulated transcripts identified through RNA-Seq analyses in Stra8-KO testes. A total of 3,839 transcripts were significantly downregulated, while 1,971 transcripts were significantly upregulated in Stra8-KO total testes (Cutoff: FDR<0.05). (B) PTC analyses of RNA-Seq data demonstrated that among all 23,575 PTC-positive transcripts (17,6% of all transcripts), only 137 were upregulated (~7% of upregulated), suggesting a lack of global PTC upregulation in Stra8-KO testes. All de-regulated genes and transcripts are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005863#pgen.1005863.s006" target="_blank">S1 Table</a>. (C) MA plots of transcript expression showing no global upregulation of PTC-positive transcripts in either <i>Upf2</i>-null spermatocytes (spc) or <i>Upf2</i>-null round spermatids (rspd). Transcripts without annotated start codons (NO ORF), transcripts without PTC (PTC-negative), and transcripts containing PTC (PTC-positive) were analyzed. The blue line indicates the mean log2 fold change along the x-axis. (D) Violin plots showing the length distribution of full-length transcript, ORF, 5’UTRs and 3’UTR among up- (1,559), down- (2,654) and non-regulated (85,910) transcripts in Stra8-KO testes (Cutoff: FDR < 0.05). Only transcripts derived from genes expressing multiple transcripts were analyzed. The three dots in each violin plot indicate the 25^th, 50^th (median) and 75^th percentile of the visualized data. (E) Statistics for the pairwise comparisons of the length distributions shown in D. The most significant length increases were observed in 3’UTRs of the up-regulated transcripts in Stra8-KO testes (p-value = 1.05e-149, between up- and down- groups). (F) The median 3’UTR length of the up-, down- and non-regulated transcripts analyzed in D.</p

UPF2 selectively eliminates longer 3’UTR transcripts derived from ubiquitously expressed genes in spermatocytes and round spermatids.

<p>(A-B) Gene ontology (GO) analyses of differentially expressed genes (FDR < 0.05) for up- and down-regulated genes respectively (P<0.01; top 18 GO terms are shown). (C-D) Gene ontology (GO) analyses of genes with transcripts selectively upregulated upon <i>Upf2</i> inactivation in spermatocytes (Spc) and round spermatids (Rspd) respectively (P<0.01; top 18 GO terms are shown). (E) A working model for UPF2-mediated 3’UTR length control in male germ cells. While the testis-specific transcription factor (TF) complex containing the yet-to-be-identified testis-specific APA factors produces testis-specific gene transcripts with shorter 3’UTRs, the ubiquitous TF complex cooperates with the ubiquitous APA complex to generate both shorter and longer 3’UTR transcripts from ubiquitously expressed genes in male germ cells. The transcripts with longer 3’UTRs are then selectively degraded by the UPF2-directed NMD in the chromatoid body, leading to enrichment of shorter 3’UTR transcripts in haploid male germ cells.</p

UPF2 is a novel component of the chromatoid body (CB) and is highly expressed in spermatocytes and round spermatids in murine testes.

<p>(A) qPCR analyses of <i>Upf2</i> mRNA levels in individual testicular cell types purified from adult murine testes, including spermatogonia (spg), spermatocytes (spc), round spermatids (rspd), Sertoli cells (Sertoli) and Leydig cells (Leydig). Biological triplicates (n = 3) were analyzed and relative <i>Upf2</i> mRNA levels are shown as means ± SEM. (B) Immunofluorescent localization of UPF2 in WT murine testes. UPF2 is abundantly expressed in the cytoplasm of spermatocytes and round spermatids in WT testes with the highest expression confined to an intensive “dot-like” structure, resembling the chromatoid body (CB, arrows), in round spermatids. Scale bar = 50μm. (C) Double immunofluorescent staining of UPF2 and MAEL, a CB marker, in adult murine testes. Arrowheads indicate CBs in round spermatids. Scale bar = 50μm. (D) Co-localization of UPF2 and DDX25, a CB marker protein. In both stages VIII and I seminiferous tubules, UPF2 mostly overlaps with DDX25 in CBs in the round spermatids (arrows). However, UPF2 is absent in a few DDX25-positive dots (arrowheads), which may represent other types of cytoplasmic granular structures, e.g. the satellite body. Scale bar = 15μm.</p

Additional file 7: Table S5. of Transcriptional changes induced by bevacizumab combination therapy in responding and non-responding recurrent glioblastoma patients

Gene set enrichment analysis of down-regulated genes (DOCX 18 kb

Introduction: The Law-Medicine Center 50th Anniversary Symposium: The Field of Health Law: It’s Past and Future

Introduction to The Law-Medicine Center 50th Anniversary Symposium: The Field of Health Law: Its Past and Future, Cleveland, Ohio 2004

Additional file 5: Figure S1. of Transcriptional changes induced by bevacizumab combination therapy in responding and non-responding recurrent glioblastoma patients

Subsampling analysis. This analysis subsampled pairs of non-responders (Comparison 3) to random groups of 6 patients 100 times. Comparison 1 and Comparison 3 shows the number of differentially expressed genes in the paired comparison analysis of responders and non-responders, respectively (DOCX 51 kb