Relaxin family peptide receptor 2 (RXFP2) is the cognate receptor of a peptide hormone insulin-like 3 (INSL3). INSL3 is expressed at high levels in both fetal and adult Leydig cells. Deletion of Insl3 or Rxfp2 genes in mice caused cryptorchidism resulting from a failure of gubernaculum development. Using a novel mouse transgenic line with a knock-in LacZ reporter in the Rxfp2 locus, we detected a robust Rxfp2 expression in embryonic and early postnatal gubernaculum in males and in postmeiotic spermatogenic cells in adult testis. To study the role of INSL3/RXFP2 signaling in male reproduction, we produced a floxed Rxfp2 allele and used the Cre/loxP approach to delete Rxfp2 in different tissues. Using Cre transgene driven by retinoic acid receptor beta promoter, conditional gene targeting in gubernacular mesenchymal cells at early embryonic stages caused high intraabdominal cryptorchidism as in males with a global deletion of Rxfp2. However, when the Rxfp2 was deleted in gubernacular smooth or striated muscle cells, no abnormalities of testicular descent or testis development were found. Specific ablation of Rxfp2 in male germ cells using Stra8-icre transgene did not affect testis descent, spermatogenesis, or fertility in adult males. No significant change in germ cell apoptosis was detected in mutant males. In summary, our data indicate that the INSL3/RXFP2 signaling is important for testicular descent but dispensable for spermatogenesis and fertility in adult males.

More than 60% of supratentorial ependymomas harbor a ZFTA–RELA (ZRfus) gene fusion (formerly C11orf95–RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. Significance: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA–RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing. This article is highlighted in the In This Issue feature, p. 2113

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icre transgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63 genes' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.

As a dual function protein, β-catenin affects both cell adhesion and mediates canonical Wnt/β-catenin cell signaling. β-Catenin is prominently expressed in somatic Sertoli cells in the testis and postmeiotic germ cells, suggesting an additional role in spermatogenesis. It was reported previously that Cre/loxP-mediated conditional inactivation of the β-catenin gene (Ctnnb1) in male gonads using a protamine promoter-driven Cre transgene (Prm-cre) resulted in partial infertility, reduced sperm count, and abnormal spermatogenesis. In this report, we demonstrated that the conditional deletion of Ctnnb1 using a germ cell specific Cre transgene (Stra8-icre) had no effect on male fertility. We have shown that the Stra8-icre transgene was highly efficient in generating deletion in early pre-meiotic and post-meiotic cells. No differences in anatomical or histological presentation were found in the mutant testis, the production of viable sperm was similar, and no abnormalities in DNA sperm content were detected. We concluded that β-catenin is fully dispensable in germ cells for spermatogenesis. The conflicting results from the earlier study may have been due to off-target expression of Prm-cre in testicular somatic cells. In future studies, the analysis of conditional mutants using several Cre-transgenes should be encouraged to reduce potential errors.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half‐life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis‐related genes in primary human stellate cells. RNA sequencing analysis of TGF‐β1–activated LX‐2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS‐induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α‐smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.—Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis. FASEB J. 33, 12435–12446 (2019). http://www.fasebj.org

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<p>HA-ZRfus1 bound and over-expressed genes compared to mouse normal brain</p>

<p>DNA motifs detected and shared with mouse embryonic fibroblasts treated with TNF-alpha</p>