A Dominant, Recombination-Defective Allele of Dmc1 Causing Male-Specific Sterility

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1(Mei11), encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1(Mei11) exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells

<p>Abstract</p> <p>Background</p> <p>The vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. <it>Suppressor enhancer lin12/Notch 1 like (Sel1l</it>) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by <it>Sel1l </it>remain elusive.</p> <p>Results</p> <p>We have characterized the pancreatic phenotype of mice carrying a gene trap mutation in <it>Sel1l</it>. We show that <it>Sel1l </it>expression in the developing pancreas coincides with differentiation of the endocrine and exocrine lineages. Mice homozygous for the gene trap mutation die prenatally and display an impaired pancreatic epithelial morphology and cell differentiation. The pancreatic epithelial cells of <it>Sel1l </it>mutant embryos are confined to the progenitor cell state throughout the secondary transition. Pharmacological inhibition of Notch signaling partially rescues the pancreatic phenotype of <it>Sel1l </it>mutant embryos.</p> <p>Conclusions</p> <p>Together, these data suggest that <it>Sel1l </it>is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development.</p

Mutation in Mouse Hei10, an E3 Ubiquitin Ligase, Disrupts Meiotic Crossing Over

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5′ splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I

Mouse H6 Homeobox 1 (Hmx1) mutations cause cranial abnormalities and reduced body mass

<p>Abstract</p> <p>Background</p> <p>The H6 homeobox genes <it>Hmx1</it>, <it>Hmx2</it>, and <it>Hmx3 </it>(also known as <it>Nkx5-3</it>; <it>Nkx5-2 </it>and <it>Nkx5-1</it>, respectively), compose a family within the NKL subclass of the ANTP class of homeobox genes. Hmx gene family expression is mostly limited to sensory organs, branchial (pharyngeal) arches, and the rostral part of the central nervous system. Targeted mutation of either <it>Hmx2 </it>or <it>Hmx3 </it>in mice disrupts the vestibular system. These tandemly duplicated genes have functional overlap as indicated by the loss of the entire vestibular system in double mutants. Mutants have not been described for <it>Hmx1</it>, the most divergent of the family.</p> <p>Results</p> <p>Dumbo (<it>dmbo</it>) is a semi-lethal mouse mutation that was recovered in a forward genetic mutagenesis screen. Mutants exhibit enlarged ear pinnae with a distinctive ventrolateral shift. Here, we report on the basis of this phenotype and other abnormalities in the mutant, and identify the causative mutation as being an allele of <it>Hmx1</it>. Examination of dumbo skulls revealed only subtle changes in cranial bone morphology, namely hyperplasia of the gonial bone and irregularities along the caudal border of the squamous temporal bone. Other nearby otic structures were unaffected. The semilethality of <it>dmbo/dmbo </it>mice was found to be ~40%, occured perinatally, and was associated with exencephaly. Surviving mutants of both sexes exhibited reduced body mass from ~3 days postpartum onwards. Most dumbo adults were microphthalmic. Recombinant animals and specific deletion-bearing mice were used to map the <it>dumbo </it>mutation to a 1.8 Mb region on Chromosome 5. DNA sequencing of genes in this region revealed a nonsense mutation in the first exon of H6 Homeobox 1 (<it>Hmx1</it>; also <it>Nkx5-3</it>). An independent spontaneous allele called misplaced ears (<it>mpe</it>) was also identified, confirming <it>Hmx1 </it>as the responsible mutant gene.</p> <p>Conclusion</p> <p>The divergence of <it>Hmx1 </it>from its paralogs is reflected by different and diverse developmental roles exclusive of vestibular involvement. Additionally, these mutant <it>Hmx1 </it>alleles represent the first mouse models of a recently-discovered Oculo-Auricular syndrome caused by mutation of the orthologous human gene.</p

High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5

<p>Abstract</p> <p>Background</p> <p>Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals.</p> <p>Results</p> <p>We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (<it>Tbc1d14</it>, <it>Nol14</it>, <it>Tyms</it>, <it>Cad</it>, <it>Fbxl5</it>, <it>Haus3</it>), and mutations in genes we or others previously reported (<it>Tapt1</it>, <it>Rest</it>, <it>Ugdh</it>, <it>Paxip1</it>, <it>Hmx1, Otoe, Nsun7</it>). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in <it>Tbc1d14 </it>provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.</p> <p>Conclusion</p> <p>This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.</p

Slingshot: a PiggyBac based transposon system for tamoxifen-inducible ‘self-inactivating’ insertional mutagenesis

We have developed a self-inactivating PiggyBac transposon system for tamoxifen inducible insertional mutagenesis from a stably integrated chromosomal donor. This system, which we have named ‘Slingshot’, utilizes a transposon carrying elements for both gain- and loss-of-function screens in vitro. We show that the Slingshot transposon can be efficiently mobilized from a range of chromosomal loci with high inducibility and low background generating insertions that are randomly dispersed throughout the genome. Furthermore, we show that once the Slingshot transposon has been mobilized it is not remobilized producing stable clonal integrants in all daughter cells. To illustrate the efficacy of Slingshot as a screening tool we set out to identify mediators of resistance to puromycin and the chemotherapeutic drug vincristine by performing genetrap screens in mouse embryonic stem cells. From these genome-wide screens we identified multiple independent insertions in the multidrug resistance transporter genes Abcb1a/b and Abcg2 conferring resistance to drug treatment. Importantly, we also show that the Slingshot transposon system is functional in other mammalian cell lines such as human HEK293, OVCAR-3 and PE01 cells suggesting that it may be used in a range of cell culture systems. Slingshot represents a flexible and potent system for genome-wide transposon-mediated mutagenesis with many potential applications

Comparative Oncogenomics Implicates the Neurofibromin 1 Gene (NF1) as a Breast Cancer Driver

Identifying genomic alterations driving breast cancer is complicated by tumor diversity and genetic heterogeneity. Relevant mouse models are powerful for untangling this problem because such heterogeneity can be controlled. Inbred Chaos3 mice exhibit high levels of genomic instability leading to mammary tumors that have tumor gene expression profiles closely resembling mature human mammary luminal cell signatures. We genomically characterized mammary adenocarcinomas from these mice to identify cancer-causing genomic events that overlap common alterations in human breast cancer. Chaos3 tumors underwent recurrent copy number alterations (CNAs), particularly deletion of the RAS inhibitor Neurofibromin 1 (Nf1) in nearly all cases. These overlap with human CNAs including NF1, which is deleted or mutated in 27.7% of all breast carcinomas. Chaos3 mammary tumor cells exhibit RAS hyperactivation and increased sensitivity to RAS pathway inhibitors. These results indicate that spontaneous NF1 loss can drive breast cancer. This should be informative for treatment of the significant fraction of patients whose tumors bear NF1 mutations

Genetically enhanced asynapsis of autosomal chromatin promotes transcriptional dysregulation and meiotic failure

During meiosis, pairing of homologous chromosomes and their synapsis are essential prerequisites for normal male gametogenesis. Even limited autosomal asynapsis often leads to spermatogenic impairment, the mechanism of which is not fully understood. The present study was aimed at deliberately increasing the size of partial autosomal asynapsis and analysis of its impact on male meiosis. For this purpose, we studied the effect of t12 haplotype encompassing four inversions on chromosome 17 on mouse autosomal translocation T(16;17)43H (abbreviated T43H). The T43H/T43H homozygotes were fully fertile in both sexes, while +/T43H heterozygous males, but not females, were sterile with meiotic arrest at late pachynema. Inclusion of the t12 haplotype in trans to the T43H translocation resulted in enhanced asynapsis of the translocated autosome, ectopic phosphorylation of histone H2AX, persistence of RAD51 foci, and increased gene silencing around the translocation break. Increase was also on colocalization of unsynapsed chromatin with sex body. Remarkably, we found that transcriptional silencing of the unsynapsed autosomal chromatin precedes silencing of sex chromosomes. Based on the present knowledge, we conclude that interference of meiotic silencing of unsynapsed autosomes with meiotic sex chromosome inactivation is the most likely cause of asynapsis-related male sterility

Expression of arf tumor suppressor in spermatogonia facilitates meiotic progression in male germ cells

The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16Ink4a and p19Arf) that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to oncogenic stress. Although p19Arf is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19Arf in spermatogonia does not interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell-autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells