Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel 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 identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts.

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.The luxoid (lu) mutation arose spontaneously in 1950 and was initially characterized for its semidominant limb abnormalities and recessive skeletal and male infertility phenotypes 1,2 . A subsequent report suggested that luxoid mutant males had limited short-term production of normal-looking sperm followed by a rapid loss of all Plzf is required in adult male germ cells for stem cell self-renewal

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell death. 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.

Kisspeptins are products of the Kiss1 gene, which bind to GPR54, a G protein-coupled receptor. Kisspeptins and GPR54 have been implicated in the neuroendocrine regulation of GnRH secretion. To test the hypothesis that testosterone regulates Kiss1 gene expression, we compared the expression of KiSS-1 mRNA among groups of intact, castrated, and castrated/testosterone (T)-treated male mice. In the arcuate nucleus (Arc), castration resulted in a significant increase in KiSS-1 mRNA, which was completely reversed with T replacement, whereas in the anteroventral periventricular nucleus, the results were the opposite, i.e. castration decreased and T increased KiSS-1 mRNA expression. In the Arc, the effects of T on KiSS-1 mRNA were completely mimicked by estrogen but only partially mimicked by dihydrotestosterone, a nonaromatizable androgen, suggesting that both estrogen receptor (ER) and androgen receptor (AR) play a role in T-mediated regulation of KiSS-1. Studies of the effects of T on KiSS-1 expression in mice with either a deletion of the ERalpha or a hypomorphic allele to the AR revealed that the effects of T are mediated by both ERalpha and AR pathways, which was confirmed by the presence of either ERalpha or AR coexpression in most KiSS-1 neurons in the Arc. These observations suggest that KiSS-1 neurons in the Arc, whose transcriptional activity is inhibited by T, are targets for the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the anteroventral periventricular nucleus, whose activity is stimulated by T, may mediate other T-dependent processes.

Stem cells support tissue maintenance by balancing self-renewal and differentiation. In mice, it is believed that a homogeneous stem cell population of single spermatogonia supports spermatogenesis, and that differentiation, which is accompanied by the formation of connected cells (cysts) of increasing length, is linear and nonreversible. We evaluated this model using lineage-analysis and live-imaging and found that this putative stem cell population is not homogeneous. Instead, the stem cell pool that supports steady-state spermatogenesis is contained within a subpopulation of single spermatogonia. Also, cysts are not committed to differentiation and appear to recover stem cell potential by fragmentation. Lastly, the fate of individual spermatogonial populations was dramatically altered during regeneration following damage. Thus, there are multiple and reversible paths from stem cell to differentiation, which may also occur in other systems.Maintenance of adult tissues is supported by a small number of undifferentiated stem cells that self-renew to maintain their population and produce differentiating progeny for normal tissue function. It has generally been accepted that differentiating daughter cells progress unidirectionally towards terminal differentiation. This view has been recently challenged by data suggesting that under some circumstances differentiating cells can revert to the self-renewing stem cell pool (1-8). This apparent plasticity may add robustness to maintenance of the stem cell population during normal tissue maintenance and may play a crucial role in tissue regeneration following injury. However, the nature of the self-renewing stem cells and the plasticity of differentiating cells in the maintenance of tissue homeostasis and regeneration are mostly unknown, particularly in mammals.Germ cells share a characteristic feature across all animal species. While the most primitive cells in adult gonads are singly isolated, their differentiating progeny remain connected by intercellular bridges to form syncytial cysts of 2 n cells (9,10). Thus, the length of the cysts reflects their cell division history or lineage. This unique feature has made the germline one of the most tractable systems to study adult stem cell self-renewal and differentiation (2,3).* To whom correspondence should be addressed. shosei@nibb.ac.jp. The study of the spermatogenic stem cell compartment in mammals also relies on the heterogeneity in the cyst length (9,11,12 (Fig. S1).The prevailing rodent stem cell model (14,15) (Fig. 1A) assumes that the stem cell population resides in the A s population and that cyst length reflects the extent of differentiation in a linear manner (9,11). A corollary of this 'A s model' is that A s spermatogonia are functionally homogeneous, that all A s cells are stem cells, and that all cells are equivalent in each morphological category (9,10). This model, proposed in 1971, has provided the framework for years of germline stem cell research in mice and other animals. Despite its simplici...

Oocytes and sperm are some of the most differentiated cells in our bodies, yet they generate all cell types after fertilization. Accumulating evidence suggests that this extraordinary potential is conferred to germ cells from the time of their formation during embryogenesis. In this Review, we describe common themes emerging from the study of germ cells in vertebrates and invertebrates. Transcriptional repression, chromatin remodeling, and an emphasis on posttranscriptional gene regulation preserve the totipotent genome of germ cells through generations.

Within the mammalian testis, specialized tight junctions between somatic Sertoli cells create basal and apical polarity within the cells, restrict movement of molecules between cells, and separate the seminiferous epithelium into basal and adluminal compartments. These tight junctions form the basis of the blood-testis barrier, a structure whose function and dynamic regulation is poorly understood. In this study, we used microarray gene expression profiling to identify genes with altered transcript levels in a mouse model for conditional androgen insensitivity. We show that testosterone, acting through its receptor expressed in Sertoli cells, regulates the expression of claudin 3, which encodes a transient component of newly formed tight junctions. Sertoli cell-specific ablation of androgen receptor results in increased permeability of the bloodtestis barrier to biotin, suggesting claudin 3 regulates the movement of small molecules across the Sertoli cell tight junctions. These results suggest that androgen action in Sertoli cells regulates germ cell differentiation, in part by controlling the microenvironment of the seminiferous epithelium. Our studies also indicate that hormonal strategies for male contraception may interfere with the blood-testis barrier.androgen receptor ͉ testosterone ͉ tight junctions

SummaryWe have generated a transgenic mouse line, Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia, and is detected through pre-leptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/ GFP)21Lbe (Z/EG) reporter females and compared the expression of Enhanced Green Fluorescent Protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF -a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development. KeywordsCre recombinase; Stra8 promoter; spermatogonia; spermatocytes; Z/EG These experiments were initiated to produce a transgenic mouse line that expresses cre in undifferentiated spermatogonia. To date, the use of cre-mediated recombination to inactivate genes in developing germ cells at specific stages has been limited by the restricted expression patterns of available cre drivers. Recombination in primordial germ cells is possible using the alkaline phosphatase, liver/bone/kidney Alpl tm1(cre)Nagy mouse line (Lomeli et al., 2000). However, significant developmental events occur between the onset of cre at embryonic day (E)9.5 and the appearance of spermatogonia in the postnatal testis. Additional cre activity is detected in the placenta, intestine, and neural tube. Excision of floxed DNA in primary spermatocytes and elongating spermatids is achievable using synaptonemal complex protein 1 Tg(Sycp1-cre)4Min (Vidal et al., 1998) and protamine 1 Tg(Prm-cre)58Og (O'Gorman et al., 1997), respectively, but the temporal expression of these transgenes is too late to be of use in undifferentiated spermatogonia. Meanwhile, the expression of cre driven by the growth differentiation factor 9 (Gdf9) and zona pellucida 3 (Lan et al., 2004;Lewandoski et al., 1997).We therefore selected the promoter of the premeiotic male and female germ cell-specific gene Stra8 to drive expression of improved Cre recombinase. Endogenous Stra8 is first expressed in ovarian germ cells at E12.5 and continues until E16.5, while in males it is first transcribed in early-stage spermatogonia in the postnatal testis and persists in premeiotic germ cells throughout adulthood (Menke et al., 2003;Oulad-Abdelghani et al., 1996). A 1.4 Kb Stra8 promoter fragment was recently fused to EGF...