Fig 2: Proposed model for the regulation of Kit expression by ATRA during the transition from Aal to A1 spermatogonia.(A) Spermatogonia at an undifferentiated state. ATRA is not available to activate RARG/RXRA heterodimer and transcription of Sall4 is low. Transcription of Kit is also low because ZBTB16 is bound to its promoter [48]. In addition, translation of Kit mRNA already present in cells [2,49,50] is prevented by the Mirc1, Mirc3 and miR221/222 small interfering RNAs (brown comb) [50,51]. (B) Spermatogonia at a differentiating state upon ATRA action. (i) At the Aal-A1 transition, one possible way for ATRA (yellow triangle) to regulate Kit expression is to activate RARG/RXRA heterodimer, which increases Sall4a expression (high, our study). (ii) SALL4A in large amount can then sequesters ZBTB16 [14], clearing Kit promoter and relieving the repression of Kit transcription normally exerted by ZBTB16 [48]. (iii) ATRA is also proposed to increase the level of SOHLH1, which can replace ZBTB16 on regulatory regions to increase Kit expression (high) [52]. Sohlh1 is however not a direct target of RARG as RAR-binding sites are not found in this gene [34] 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 [42,43]. (v) In parallel, ATRA can further induce KIT protein through decreasing expression of microRNA such as Mirc1, Mirc3 and miR221/222 that prevent Kit mRNA translation. How ATRA regulate miRNA expression is however unknown, as RARE have not been identified in the vicinity of their promoters [34,50,51]. ATRA can also function as a rapid, non-genomic, agent by (vi) increasing the loading of Kit mRNA on polysomes (grey ovals) and its translation [41] and (vii) inducing phosphorylation of KIT (orange stars) and of downstream KIT-effectors, reinforcing commitment towards the A1 spermatogonia fate [53].

Fig 3: RARG/RXRA heterodimers bind to Sall4 in testis chromatin, on an IR1 motif located in the first intron.(A) UCSC Genome Browser snapshot of the Sall4 locus in NCBI37/mm9 assembly, including tracks for anti-RAR ChIP-seq [34], RefSeq genes and mammalian conservation (from top to bottom). RARE points to the RAR-binding region identified according to Moutier et al. [34]. (B) Alignments of the DNA sequences from the indicated species and corresponding to the region containing the RAR-binding region in mouse Sall4. 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 Sall4a mRNA quantified by RT-qPCR in whole testes from control (white bars) and Rarg −/− (grey bars) mice at PN5. Error bars represent s.e.m. (n = 9); * p < 0.05. Right panel: schematic representation of Sall4 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); * p < 0.05. (D) EMSA showing that RARG/RXRA heterodimers (Het) bind to the 32P-labelled IR1 of Sall4 (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 Rarb (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.

Fig 4: Sall4a expression in undifferentiated spermatogonia is controlled by ligand-activated RARG.(A) Relative expression of Sall4a mRNA quantified by RT-qPCR in Aldh1a1-3 Ser−/− 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); * p < 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 Sall4a, Sall4b and Zbtb16 mRNA quantified by RT-qPCR in whole testes from control (white bars), Rara;b;g Spg−/− (grey bars) and Rxra;b;g Spg−/− (black bars) mice at PN60. Error bars represent s.e.m. (n = 5); * p < 0.05.

Fig 5: ‘STITCH 5.0’ protein–protein interaction network reinforces the putative involvement of RXR signaling in the regulation of PAI-1-mediated ECM turnover. The stronger associations are represented by thicker lines. Protein–protein interactions are shown in grey, and chemical–protein interactions are depicted in green. The interactions between chemicals are exhibited in red. Pparg: peroxisome proliferator-activated receptor gamma; Rxra, retinoid X receptor alpha; Rxrb, Retinoid X receptor beta; Rarg, retinoic acid receptor gamma; Mmp9, matrix metallopeptidase 9; Mmp13, matrix metallopeptidase 13; Col4a1, collagen, type IV, alpha 1; Col4a2, collagen, type IV, alpha 2; Fn1, fibronectin 1; Lama5, laminin, alpha 5; Eln, elastin; Plau, plasminogen activator, urokinase; Timp1, tissue inhibitor of metalloproteinase 1.