Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

RNA editing is a process that modifies RNA nucleotides and changes the efficiency and fidelity of the central dogma. Enzymes that catalyze RNA editing are required for life, and defects in RNA editing are associated with many diseases. Recent advances in sequencing have enabled the genome-wide identification of RNA editing sites in mammalian transcriptomes. Here, we demonstrate that canonical RNA editing (A-to-I and C-to-U) occurs in liver, white adipose, and bone tissues of the laboratory mouse, and we show that apparent non-canonical editing (all other possible base substitutions) is an artifact of current high-throughput sequencing technology. Further, we report that high-confidence canonical RNA editing sites can cause non-synonymous amino acid changes and are significantly enriched in 3' UTRs, specifically at microRNA target sites, suggesting both regulatory and functional consequences for RNA editing.

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.

Adenosine to inosine (A-to-I) RNA editing occurs in a wide range of tissues and cell types and can be catalyzed by one of the two adenosine deaminase acting on double-stranded RNA enzymes, ADAR and ADARB1. Editing can impact both coding and noncoding regions of RNA, and in higher organisms has been proposed to function in adaptive evolution. Neither the prevalence of A-to-I editing nor the role of either ADAR or ADARB1 has been examined in the context of germ cell development in mammals. Computational analysis of whole testis and cell-type specific RNA-sequencing data followed by molecular confirmation demonstrated that A-to-I RNA editing occurs in both the germ line and in somatic Sertoli cells in two targets, Cog3 and Rpa1. Expression analysis demonstrated both Adar and Adarb1 were expressed in both Sertoli cells and in a cell-type dependent manner during germ cell development. Conditional ablation of Adar did not impact testicular RNA editing in either germ cells or Sertoli cells. Additionally, Adar ablation in either cell type did not have gross impacts on germ cell development or male fertility. In contrast, global Adarb1 knockout animals demonstrated a complete loss of A-to-I RNA editing in spite of normal germ cell development. Taken together, these observations demonstrate ADARB1 mediates A-to-I RNA editing in the testis and these editing events are dispensable for male fertility in an inbred mouse strain in the lab.

Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.

Glial-cell-derived neurotrophic factor (GDNF) is a well-studied neuroregenerative factor; however, the degree to which it supports hair formation and skin wound repair is not known. By using a Gfra1 (GDNF family receptor alpha 1) knock-in reporter mouse line, GDNF signaling was found to occur within hair bulge stem cells (BSCs) during the initiation of the hair cycle and early stages of hair formation after depilation. Both recombinant and transgene overexpression of GDNF promoted BSC colony growth, hair formation, and skin repair after wounding through enhanced self-renewal of BSCs and commitment of BSC-derived progenitors into becoming epidermal cells at the injury site. Conditional ablation of Gfra1 among BSCs impaired the onset of the hair cycle, while conditional ablation of the GDNF family member signal transducer, Ret, within BSCs prevented the onset of the hair cycle and depilation-induced anagen development of hair follicles. Our findings reveal that GDNF promotes hair formation and wound repair and that bulge stem cells are critical mediators of both.

Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

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.

Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.

The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Androgen deprivation in men leads to increased adiposity, but the mechanisms underlying androgen regulation of fat mass have not been fully defined. Androgen receptor (AR) is expressed in monocytes/macrophages, which are resident in key metabolic tissues and influence energy metabolism in surrounding cells. Male mice bearing a cell-specific knockout of the AR in monocytes/macrophages (M-ARKO) were generated to determine whether selective loss of androgen signaling in these cells would lead to altered body composition. Wild-type (WT) and M-ARKO mice (12-22 weeks of age, n = 12 per group) were maintained on a regular chow diet for 8 weeks and then switched to a high-fat diet for 8 additional weeks. At baseline and on both the regular chow and high-fat diets, no differences in lean mass or fat mass were observed between groups. Consistent with the absence of differential body weight or adiposity, no differences in food intake (3.0 ± 0.5 g per day for WT mice vs 2.8 ± 0.4 g per day for M-ARKO mice) or total energy expenditure (0.6 ± 0.1 Kcal h-1 for WT mice vs 0.5 ± 0.1 Kcal h-1 for M-ARKO mice) were evident between groups during high-fat feeding. Liver weight was greater in M-ARKO than that in WT mice (1.5 ± 0.1 g vs 1.3 ± 0.0 g, respectively, P = 0.02). Finally, M-ARKO mice did not exhibit impairments in glucose tolerance or insulin sensitivity relative to WT mice at any study time point. In aggregate, these findings suggest that AR signaling specifically in monocytes/macrophages does not contribute to the regulation of systemic energy balance, adiposity, or insulin sensitivity in male mice.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.

We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

The ability to track animals accurately is critical for behavioral experiments. For video-based assays, this is often accomplished by manipulating environmental conditions to increase contrast between the animal and the background in order to achieve proper foreground/background detection (segmentation). Modifying environmental conditions for experimental scalability opposes ethological relevance. The biobehavioral research community needs methods to monitor behaviors over long periods of time, under dynamic environmental conditions, and in animals that are genetically and behaviorally heterogeneous. To address this need, we applied a state-of-the-art neural network-based tracker for single mice. We compare three different neural network architectures across visually diverse mice and different environmental conditions. We find that an encoder-decoder segmentation neural network achieves high accuracy and speed with minimal training data. Furthermore, we provide a labeling interface, labeled training data, tuned hyperparameters, and a pretrained network for the behavior and neuroscience communities.

The majority of patients with benign prostate hyperplasia (BPH) exhibit chronic prostate inflammation and the extent of inflammation correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms are not clearly understood. We established a unique mouse model Prostate Ovalbumin Expressing Transgenic 3 (POET3) that mimics chronic non-bacterial prostatitis in men to study the role of inflammation in prostate hyperplasia. After the injection of ovalbumin peptide-specific T cells, POET3 prostates exhibited an influx of inflammatory cells and an increase in pro-inflammatory cytokines that led to epithelial and stromal hyperplasia. We have previously demonstrated with the POET3 model that inflammation expands the basal prostate stem cell (bPSC) population and promotes bPSC differentiation in organoid cultures. In this study, we investigated the mechanisms underlying the impact of inflammation on bPSC. We found that AR activity was enhanced in inflamed bPSC and was essential for bPSC differentiation in organoid cultures. Most importantly, we identified, for the first time, interleukin 1 receptor antagonist (IL-1RA) as a key regulator of AR in basal stem cells. IL-1RA was one of the top genes upregulated by inflammation and inhibition of IL-1RA abrogated the enhanced AR nuclear accumulation and activity in organoids derived from inflamed bPSC. The mirroring effects of IL-1RA recombinant protein and IL-1α neutralizing antibody suggest that IL-1RA may function by antagonizing IL-1α inhibition of AR expression. Furthermore, we established a lineage tracing model to follow bPSC during inflammation and under castrate conditions. We found that inflammation induced bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation in bPSC is sufficient for their proliferation and differentiation under androgen-deprived conditions. However, proliferation of the differentiated bPSC in the luminal layer significantly diminished with castration, suggesting inflammation may not maintain AR activity in stromal cells, as stromal cells deprived of androgen after castration could no longer provide paracrine growth factors essential for luminal proliferation. Taken together, we have discovered novel mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that contributes to prostate hyperplasia.