Functional Redundancy of DICER Cofactors TARBP2 and PRKRA During Murine Embryogenesis Does Not Involve miRNA Biogenesis.

Several in vitro studies have suggested that canonical microRNA (miRNA) biogenesis requires the DICER cofactors TARBP2 and PRKRA for processing of pre-miRNAs to mature miRNAs. To investigate the roles of TARBP2 and PRKRA in miRNA biogenesis in vivo, and to determine possible functional redundancy, we first compared the phenotypes of Tarbp2 and Prkra single and double mutants. In contrast to Dicer -/- embryos, which die by embryonic day 7.5 (E7.5), single Tarbp2 -/- and Prkra -/- mice survive beyond E7.5 and either die perinatally or survive and exhibit cranial/facial abnormalities, respectively. In contrast, only a few Tarbp2 -/- ; Prkra -/- double mutants survived beyond E12.5, suggesting genetic redundancy between Tarbp2 and Prkra during embryonic development. Sequencing of miRNAs from single-mutant embryos at E15.5 revealed changes in abundance and isomiR type in Tarbp2 -/- , but not Prkra -/- , embryos, demonstrating that TARBP2, but not PRKRA, functions in miRNA biogenesis of a subclass of miRNAs, and suggesting that functional redundancy between TARBP2 and PRKRA does not involve miRNA biogenesis. Genetics 2018 Apr; 208(4):1513-22

A large CRISPR-induced bystander mutation causes immune dysregulation.

A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic bystander mutations that escape detection by routine targeted genotyping assays

LIN28A Marks the Spermatogonial Progenitor Population and Regulates Its Cyclic Expansion

One of the hallmarks of highly proliferative adult tissues is the presence of a stem cell population that produces progenitor cells bound for differentiation. Progenitor cells undergo multiple transit amplifying (TA) divisions before initiating terminal differentiation. In the adult male germline, daughter cells arising from the spermatogonial stem cells undergo multiple rounds of TA divisions to produce undifferentiated clones of interconnected 2, 4, 8, and 16 cells, collectively termed Aundifferentiated (Aundiff ) spermatogonia, before entering a stereotypic differentiation cascade. Although the number of TA divisions markedly affects the tissue output both at steady state and during regeneration, mechanisms regulating the expansion of the TA cell population are poorly understood in mammals. Here, we show that mice with a conditional deletion of Lin28a in the adult male germline, display impaired clonal expansion of the progenitor TA Aundiff spermatogonia. The in vivo proliferative activity of Aundiff spermatogonial cells as indicated by BrdU incorporation during S-phase was reduced in the absence of LIN28A. Thus, contrary to the role of LIN28A as a key determinant of cell fate signals in multiple stem cell lineages, in the adult male germline it functions as an intrinsic regulator of proliferation in the population of Aundiff TA spermatogonia. In addition, neither precocious differentiation nor diminished capacity for self-renewal potential as assessed by transplantation was observed, suggesting that neither LIN28A itself nor the pool of Aal progenitor cells substantially contribute to the functional stem cell compartment. Stem Cells 2014;32:860-87

Androgen-dependent sertoli cell tight junction remodeling is mediated by multiple tight junction components.

Sertoli cell tight junctions (SCTJs) of the seminiferous epithelium create a specialized microenvironment in the testis to aid differentiation of spermatocytes and spermatids from spermatogonial stem cells. SCTJs must be chronically broken and rebuilt with high fidelity to allow the transmigration of preleptotene spermatocytes from the basal to adluminal epithelial compartment. Impairment of androgen signaling in Sertoli cells perturbs SCTJ remodeling. Claudin (CLDN) 3, a tight junction component under androgen regulation, localizes to newly forming SCTJs and is absent in Sertoli cell androgen receptor knockout (SCARKO) mice. We show here that Cldn3-null mice do not phenocopy SCARKO mice: Cldn3(-/-) mice are fertile, show uninterrupted spermatogenesis, and exhibit fully functional SCTJs based on imaging and small molecule tracer analyses, suggesting that other androgen-regulated genes must contribute to the SCARKO phenotype. To further investigate the SCTJ phenotype observed in SCARKO mutants, we generated a new SCARKO model and extensively analyzed the expression of other tight junction components. In addition to Cldn3, we identified altered expression of several other SCTJ molecules, including down-regulation of Cldn13 and a noncanonical tight junction protein 2 isoform (Tjp2iso3). Chromatin immunoprecipitation was used to demonstrate direct androgen receptor binding to regions of these target genes. Furthermore, we demonstrated that CLDN13 is a constituent of SCTJs and that TJP2iso3 colocalizes with tricellulin, a constituent of tricellular junctions, underscoring the importance of androgen signaling in the regulation of both bicellular and tricellular Sertoli cell tight junctions. Mol Endocrinol 2014 Jul; 28(7):1055-72

Plzf is required in adult male germ cells for stem cell self-renewal

Adult germline stem cells are capable of self-renewal, tissue regeneration and production of large numbers of differentiated progeny. We show here that the classical mouse mutant luxoid affects adult germline stem cell self-renewal. Young homozygous luxoid mutant mice produce limited numbers of normal spermatozoa and then progressively lose their germ line after birth. Transplantation studies showed that germ cells from mutant mice did not colonize recipient testes, suggesting that the defect is intrinsic to the stem cells. We determined that the luxoid mutant contains a nonsense mutation in the gene encoding Plzf, a transcriptional repressor that regulates the epigenetic state of undifferentiated cells, and showed that Plzf is coexpressed with Oct4 in undifferentiated spermatogonia. This is the first gene shown to be required in germ cells for stem cell self-renewal in mammals

Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation.

Previously we established Zygote Electroporation of Nucleases (ZEN) technology as an efficient and high-throughput way to generate genetically modified mouse models. However, there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol. In this study, we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation. Using this approach, we were able to introduce precise nucleotide substitutions, large segment deletion and short segment insertion into targeted loci with high efficiency. J Genet Genomics 2016 May 20; 43(5):319-2

Deregulated sex chromosome gene expression with male germ cell-specific loss of dicer1.

MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs that mediate post-transcriptional gene silencing by inhibiting mRNA translation and promoting mRNA decay. DICER1, an RNase III endonuclease encoded by Dicer1, is required for processing short 21-22 nucleotide miRNAs from longer double-stranded RNA precursors. Here, we investigate the loss of Dicer1 in mouse postnatal male germ cells to determine how disruptions in the miRNA biogenesis pathway may contribute to infertility. Reduced levels of Dicer1 transcripts and DICER1 were confirmed in germ cell knock-out (GCKO) testes by postnatal day 18 (P18). Compared to wild-type (WT) at 8 weeks, GCKO males had no change in body weight; yet showed significant reductions in testis mass and sperm number. Histology and fertility tests confirmed spermatogenic failure in GCKO males. Array analyses at P18 showed that in comparison to WT testes, 75% of miRNA genes and 37% of protein coding genes were differentially expressed in GCKO testes. Among these, 96% of miRNA genes were significantly down-regulated, while 4% miRNA genes were overexpressed. Interestingly, we observed preferential overexpression of genes encoded on the sex chromosomes in GCKO testes, including more than 80% of previously identified targets of meiotic sex chromosome inactivation (MSCI). Compared to WT, GCKO mice showed higher percentages of germ cells at early meiotic stages (leptotene and zygotene) but lower percentages at later stages (pachytene, diplotene and metaphase I) providing evidence that deletion of Dicer1 leads to disruptions in meiotic progression. Therefore, deleting Dicer1 in early postnatal germ cells resulted in deregulation of transcripts encoded by genes on the sex chromosomes, impaired meiotic progression and led to spermatogenic failure and infertility

Failure of meiotic sex chromosome inactivation (MSCI) in P18 <i>Dicer1</i> GCKO testes.

<p>A) Pie charts show the number of genes that are normally silenced during MSCI that were detected by array overlaid with the percentages of X- and Y-genes in GCKO testes with expression levels that were either significantly up-regulated (83.8% and 100%, respectively) or not changed (16.2 and 0%, respectively. B) Bar graph showing the number of overexpressed (>1.5 RFC) X- and Y-genes with or without binding sites for miRNAs shown to be deregulated in P18 GCKO testes. The majority of overexpressed genes (102/123 = 82.9%) contain recognition sites for deregulated miRNAs.</p