RARG/RXRA heterodimers bind to <i>Sall4</i> in testis chromatin, on an IR1 motif located in the first intron.

<p>(A) UCSC Genome Browser snapshot of the <i>Sall4</i> locus in NCBI37/mm9 assembly, including tracks for anti-RAR ChIP-seq [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref034" target="_blank">34</a>], RefSeq genes and mammalian conservation (from top to bottom). RARE points to the RAR-binding region identified according to Moutier et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref034" target="_blank">34</a>]. (B) Alignments of the DNA sequences from the indicated species and corresponding to the region containing the RAR-binding region in mouse <i>Sall4</i>. The dotted arrows indicate orientations of the core motifs. Stars and grey boxes highlight the DNA residues and the RAR binding sequences that are conserved in all 6 species, respectively. (C) Left panel: relative expression of <i>Sall4a</i> mRNA quantified by RT-qPCR in whole testes from control (white bars) and <i>Rarg</i>^{<i>−/−</i>} (grey bars) mice at PN5. Error bars represent s.e.m. (n = 9); * <i>p</i> < 0.05. Right panel: schematic representation of <i>Sall4</i> locus and analysis of DNA recovered from testis chromatin immunoprecipitated using antibodies directed against RNApol2, all RAR or all RXR isotypes (RAR or RXR, respectively). The untranslated exon and the transcription start site (TSS) are depicted by an open box and a broken arrow, respectively. The locations of primers used are indicated at −11 kb and in the RAR-binding region (RARE). Mean fold enrichment of three experiments at RARE binding site (grey bars) is relative to the amount of DNA recovered at −11 kb (set at 1, white bars). Error bars represent s.e.m. (n = 4 to 5); * <i>p</i> < 0.05. (D) EMSA showing that RARG/RXRA heterodimers (Het) bind to the ³²P-labelled IR1 of <i>Sall4</i> (lane 4). Binding is competed when increasing amounts of unlabeled IR1 are added to reaction (lanes 5–7), but not when a mutated form IR1 is added (IR1m, lanes 8–10). ³²P-IR1 probe indicates unbound DNA. (E) EMSA showing that RARG/RXRA heterodimers (Het) bound to the DR5 of <i>Rarb</i> (lane 3) are competed both when unlabeled DR5 (lane 4) or increasing amounts of IR1 (lanes 5–7) are added to reaction. ³²P-DR5 probe indicates unbound DNA.</p

Ablation of either RXR or RAR in spermatogonia does not alter the first round of spermatogenesis.

<p>(A) Percentages of seminiferous tubule cross-sections in which preleptotene/leptotene (PR+L), zygotene and early pachytene (Z+eP), late pachytene (P) and diplotene (D) spermatocytes or round spermatids (RS) represent the most advanced germ cell-types in control (white bars), <i>Rxra/b/g</i>^{<i>Spg–/–</i>}(black bars) and <i>Rara/b/g</i>^{<i>Spg–/–</i>}(grey bars) testes at post-natal day 20 (PN20). The bars represent mean ± s.e.m. (n = 4–5). (B-D) Histological sections of seminiferous from post-natal day 25 (PN25) control, <i>Rara/b/g</i>^{<i>Spg–/–</i>} and <i>Rara</i>^{<i>Ser–/–</i>}/<i>Rarg</i>^{<i>–/–</i>} mice stained with hematoxylin and eosin. Note the absence of spermatocytes (C) or of all meiotic and post meiotic germ cells (D) in the mutant testes. (E-J) Detection of spermatogonia expressing KIT (red signal in E, F, H and J) and RARG (red signal in G and I) in control and <i>Rara/b/g</i>^{<i>Spg–/–</i>} testes at 6 weeks of age. ZBTB16 (green nuclear signal in E, F, H and J) identifies spermatogonia. Alexa Fluor 488-conjugated peanut agglutinin (in E and F) labels the acrosomal system of spermatids, allowing precise identification of the stage of the seminiferous epithelium cycle. (G and H) and (I and J) represent consecutive histological sections. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue signal). A₁, A₁ spermatogonia, based on their presence in seminiferous tubule sections that contain both preleptotene spermatocytes (KIT-positive and ZBTB16-negative) and step8 spermatids. Int, intermediate spermatogonia, based on cell density co-expression of KIT and ZBTB16, and peanut hemagglutinin staining of acrosomes on consecutive sections. PR, P, preleptotene and pachytene spermatocytes, respectively; SG, spermatogonia, St7 and St8, step 7 and step8 round spermatids. Scale bars: 30 μm (B-D), 20 μm (E and F) and 55 μm (G-J).</p

Primers used for ChIP and EMSA assays.

<p>Gene, forward (upper line) and reverse (lower line) primers and their use are indicated. IR0, inverted repeats separated by 0 nucleotide; DR0, DR1, DR2, DR4 and DR5, direct repeats separated by 0, 1, 2, 4 and 5 nucleotides, respectively.</p><p>Primers used for ChIP and EMSA assays.</p

RXRA and RXRB are both instrumental to spermatogonia differentiation.

<p>Mean percentages of tubule sections showing normal cellular associations (white bars), abnormal associations resembling the VAD situation with either one or two generations of germ cells lacking (grey bars), and degenerated epithelium containing only spermatogonia and Sertoli cells (black bars) in testes of 12 month-old mice (n = 5) with the indicated genotype. Mice lacking <i>Rxrg</i> and either <i>Rxra</i> (<i>Rxra;g</i>^{<i>Sgp–/–</i>} mutants) or <i>Rxrb</i> (<i>Rxrb;g</i>^{<i>Sgp–/–</i>} mutants) are marginally affected. In contrast, mice simultaneously lacking <i>Rxra</i> and <i>Rxrb</i> (<i>Rxra;b</i>^{<i>Sgp–/–</i>} mutant) displayed a high proportion of affected tubule sections. Additional ablation of <i>Rxrg</i> does not worsen the pathological phenotype (<i>Rxra;b</i>^{<i>Sgp–/–</i>} mutant). This indicates that RXRG is dispensable, whereas RXRA and RXRB are both required and exert redundant functions in spermatogonia.</p

Proposed model for the regulation of <i>Kit</i> expression by ATRA during the transition from A_al to A₁ spermatogonia.

<p>(A) Spermatogonia at an undifferentiated state. ATRA is not available to activate RARG/RXRA heterodimer and transcription of <i>Sall4</i> is low. Transcription of <i>Kit</i> is also low because ZBTB16 is bound to its promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref048" target="_blank">48</a>]. In addition, translation of <i>Kit</i> mRNA already present in cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref002" target="_blank">2</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref049" target="_blank">49</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref050" target="_blank">50</a>] is prevented by the <i>Mirc1</i>, <i>Mirc3</i> and <i>miR221/222</i> small interfering RNAs (brown comb) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref050" target="_blank">50</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref051" target="_blank">51</a>]. (B) Spermatogonia at a differentiating state upon ATRA action. (i) At the A_al-A₁ transition, one possible way for ATRA (yellow triangle) to regulate <i>Kit</i> expression is to activate RARG/RXRA heterodimer, which increases <i>Sall4a</i> expression (high, our study). (ii) SALL4A in large amount can then sequesters ZBTB16 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref014" target="_blank">14</a>], clearing <i>Kit</i> promoter and relieving the repression of <i>Kit</i> transcription normally exerted by ZBTB16 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref048" target="_blank">48</a>]. (iii) ATRA is also proposed to increase the level of SOHLH1, which can replace ZBTB16 on regulatory regions to increase <i>Kit</i> expression (high) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref052" target="_blank">52</a>]. <i>Sohlh1</i> is however not a direct target of RARG as RAR-binding sites are not found in this gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref034" target="_blank">34</a>] and its expression is not induced by BMS961 (our study). (iv) Alternatively, SALL4A can also interact with DNMT3A/B to facilitate the epigenetic shift required for A₁ differentiation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref042" target="_blank">42</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref043" target="_blank">43</a>]. (v) In parallel, ATRA can further induce KIT protein through decreasing expression of microRNA such as <i>Mirc1</i>, <i>Mirc3</i> and <i>miR221/222</i> that prevent <i>Kit</i> mRNA translation. How ATRA regulate miRNA expression is however unknown, as RARE have not been identified in the vicinity of their promoters [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref034" target="_blank">34</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref050" target="_blank">50</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref051" target="_blank">51</a>]. ATRA can also function as a rapid, non-genomic, agent by (vi) increasing the loading of <i>Kit</i> mRNA on polysomes (grey ovals) and its translation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref041" target="_blank">41</a>] and (vii) inducing phosphorylation of KIT (orange stars) and of downstream KIT-effectors, reinforcing commitment towards the A₁ spermatogonia fate [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005501#pgen.1005501.ref053" target="_blank">53</a>].</p

Ablation of RXR in spermatogonia blocks their division, but does not affect meiosis.

<p>(A,B) TUNEL assays on histological sections from 8 week-old control and <i>Rxra;b;g</i>^{<i>Spg–/–</i>} testis as indicated. Red signals correspond to apoptotic cells and nuclei are counterstained with DAPI (in blue). (C-F) Immunohistochemical detection of BrdU (red signals). After administration, incorporated BrdU has been similarly transferred to spermatids at 17 days (C,D) or to pachytene spermatocytes at 9 days (E,F) both in control and mutant seminiferous tubules. In contrast, spermatogonia retaining BrdU are observed only in mutants (E,F). PR and P, preleptotene and pachytene spermatocytes, respectively; S, Sertoli cells; SG, spermatogonia; St7 and St16, step 7 and 16 spermatids, respectively; Z, zygotene spermatocytes. Roman numerals refer to the stages of the seminiferous epithelium cycle. In mutant testes, one asterisk and two asterisks indicate tubule sections without pachytene spermatocytes and without round spermatids, respectively. Scale bar: 160 μm (A,B), 40 μm (C,D) and 25 μm (E,F).</p

Ablation of RXR blocks spermatogonia are at the undifferentiated stage.

<p><i>In situ</i> hybridization with anti-sense probes for <i>Gfra1</i>, <i>Zbtb16</i>, <i>Kit</i> and <i>Stra8</i> on histological sections of 12 month-old control (A-D), <i>Rxra;b;g</i>^{<i>Spg–/–</i>}(E-H) and <i>Rara;b;g</i>^{<i>Spg–/–</i>}(I-L) testes. Undifferentiated spermatogonia expressing <i>Gfra1</i> and <i>Zbtb16</i> are found in both control and mutant testes. In contrast differentiating spermatogonia expressing <i>Kit</i> and <i>Stra8</i> are found in control (C,D) but not in mutant (G,H,K,L) testes. T1, tubule sections showing normal germ cell associations; T4, tubule sections containing only spermatogonia and Sertoli cells; L, Leydig cells. Scale bar: 80 μm.</p

Opposing function of SOX and RSPO1 signaling in the fate of the gonad.

<p>A- In XX gonads, RSPO1 activates WNT/beta-catenin signaling to promote ovarian differentiation. Ablation of <i>Rspo1</i> results in partial sex reversal with ovotestis development, which coincides with <i>Sox9</i> expression. However additional deletion of <i>Sox9</i> in the XX <i>Rspo1</i>^KO (i.e., <i>Rspo1^KOSox9^cKO</i>) still allows ovotestis formation, implying that <i>Sry</i> and <i>Sox9</i> are not required for testicular differentiation in female-to-male sex reversal. B- In XY gonads, whereas <i>Sox9</i> deletion triggers ovarian development, additional deletion of <i>Rspo1</i> in XY <i>Rspo1^KOSox9^cKO</i> gonads restores testis development. This is associated with the expression of other SOX genes like SOX 8 and SOX10, other masculinising factors.</p

Ablation of RXR in spermatogonia induces age-related testis degeneration.

<p>(A,B) Periodic acid-Schiff stains illustrating overviews and (C-H) details of germ cell associations in the seminiferous epithelium of 12 week-old control and <i>Rxra;b;g</i>^{<i>Sgp–/–</i>} testes, as indicated. Normal gem cell associations at epithelial stage VII (C) coexist with abnormal associations mimicking, to some extent, this epithelial stage, but lacking: pachytene spermatocytes (D,H), preleptotene spermatocytes (E,G) and round spermatids (F,H). (I,J) Hematoxylin and eosin stain showing overviews of 12 month-old control and <i>Rxra;b;g</i>^{<i>Sgp–/–</i>} testes: seminiferous tubules containing only spermatogonia and Sertoli cells represent the end-stage of degeneration in the mutant testes. PR and P, preleptotene and pachytene spermatocytes, respectively; St7 and St16, step 7 and 16 spermatids, respectively; T2, tubule sections lacking generation(s) of germ cells around their entire circumference; T3, tubule sections with disorganization of the germ cell layer; T4, tubule sections containing only spermatogonia and Sertoli cells. Germ cell populations present in a given seminiferous tubule cross-section are highlighted by colored bars: red, preleptotene spermatocytes; green, pachytene spermatocytes; blue, step 7 (round) spermatids; purple, step 16 (elongated, mature) spermatids. Roman numerals indicate the stages of the seminiferous epithelium cycle. Scale bar, 80 μm (A,B and I,J) and 30 μm (C-H).</p

<i>Sall4a</i> expression in undifferentiated spermatogonia is controlled by ligand-activated RARG.

<p>(A) Relative expression of <i>Sall4a</i> mRNA quantified by RT-qPCR in <i>Aldh1a1-3</i>^{<i>Ser−/−</i>} testes cultured in the absence (−) or in the presence (+) of cycloheximide (CHX) and treated for 6 hours with vehicle (white bar) and BM961 (grey bars). Error bars represent s.e.m. (n = 5); * <i>p</i> < 0.05. (B) Western blot analysis of protein extracts from testes of mutants as indicated treated with BMS961 (+) or with vehicle (−), using anti-SALL4 or anti-ACTIN antibodies. NS points to an unspecific signal. (C) Relative expression of <i>Sall4a</i>, <i>Sall4b</i> and <i>Zbtb16</i> mRNA quantified by RT-qPCR in whole testes from control (white bars), <i>Rara;b;g</i>^{<i>Spg−/−</i>} (grey bars) and <i>Rxra;b;g</i>^{<i>Spg−/−</i>} (black bars) mice at PN60. Error bars represent s.e.m. (n = 5); * <i>p</i> < 0.05.</p