Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or in vitro-fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated Chk2-/- mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide-hydrate ("CHK2iII") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.

Comprehensive models of human primary and metastatic colorectal tumors in immunodeficient and immunocompetent mice by chemokine targeting.

Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired subcutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening.

miR-34 cooperates with p53 in suppression of prostate cancer by joint regulation of stem cell compartment.

The miR-34 family was originally found to be a direct target of p53 and is a group of putative tumor suppressors. Surprisingly, mice lacking all mir-34 genes show no increase in cancer formation by 18 months of age, hence placing the physiological relevance of previous studies in doubt. Here, we report that mice with prostate epithelium-specific inactivation of mir-34 and p53 show expansion of the prostate stem cell compartment and develop early invasive adenocarcinomas and high-grade prostatic intraepithelial neoplasia, whereas no such lesions are observed after inactivation of either the mir-34 or p53 genes alone by 15 months of age. Consistently, combined deficiency of p53 and miR-34 leads to acceleration of MET-dependent growth, self-renewal, and motility of prostate stem/progenitor cells. Our study provides direct genetic evidence that mir-34 genes are bona fide tumor suppressors and identifies joint control of MET expression by p53 and miR-34 as a key component of prostate stem cell compartment regulation, aberrations in which may lead to cancer.

IQ motif-containing G (Iqcg) is required for mouse spermiogenesis.

Spermiogenesis in mammals is the process by which the newly formed products of meiosis, haploid spermatids, undergo a dramatic morphological transformation from round cells into flagellated spermatozoa. The underlying genetic control of spermiogenesis is complicated and not well-characterized. We have used forward genetic screens in mice to illuminate the mechanisms of spermatozoon development. Here, we report that the oligoasthenoteratospermia in a male-specific infertility mutant (esgd12d) is attributable to disruption of a gene called Iqcg (IQ motif-containing G). The causality of the mutation was confirmed with a targeted null allele. Loss of Iqcg disrupts spermiogenesis such that tail formation either occurs incompletely or breaks apart from the sperm heads. Orthologs are present in diverse species as distant as hemichordates, mollusks, and green algae. Consistent with a conserved role in flagellar formation and/or function, the orthologous Chlamydomonas protein is present in that organism's flagella. Because IQ motif-containing genes typically regulate calmodulin (CaM), which in turn can impact the actin cytoskeleton, these findings suggest a potential role for localized calcium signaling in sperm flagellum morphogenesis.

Exposure to open-fire cooking and cognitive performance in children.

We reexamined field data on cognitive performance in light of recent research that shows open-fire cooking--with its emission of harmful substances--to pose a risk to healthy physical development. Tests of three- to nine-year-old children in four communities around the world yielded evidence concerning block-building skills, memory, and the discernment of embedded figures. Naturalistic observations of these children were also undertaken in everyday settings. Open-fire cooking (as opposed to cooking on kerosene stoves) was associated with both lower cognitive performance and less frequent structured play at all ages. Although these correlational results do not reveal causal mechanisms, they are consistent with ideas about negative developmental consequences of exposure to open-fire cooking and suggest that research is needed on the effect on brain development of practices involving production of indoor smoke.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

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

BACKGROUND: 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. RESULTS: 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 (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3), and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7). 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 Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis. CONCLUSION: 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.

PDCD2 is essential for inner cell mass development and embryonic stem cell maintenance.

PDCD2 is a conserved eukaryotic protein implicated in cell cycle regulation by virtue of its interactions with HCFC1 and the NCOR1/SIN3A corepressor complex. Pdcd2 transcripts are enriched in ES cells and other somatic stem cells, and its ortholog is essential for hematopoietic stem cell maintenance in Drosophila. To characterize the physiological role(s) of mammalian PDCD2, we created a disruption allele in mice. Pdcd2(-/-) embryos underwent implantation but did not undergo further development. Inner cell masses (ICMs) from Pdcd2(-/-) blastocysts failed to outgrow in vitro. Furthermore, embryonic stem cells (ESCs) require PDCD2 as demonstrated by the inability to generate Pdcd2(-/-) ESCs in the absence of an ectopic transgene. Upon differentiation of ESCs by retinoic acid treatment or LIF deprivation, PDCD2 levels declined. In conjunction with prior studies, these results indicate that in vivo, PDCD2 is critical for blastomere and ESC maintenance by contributing to the regulation of genes in a manner essential to the undifferentiated state of these cells.

Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development.

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells.

BACKGROUND: 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. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive. RESULTS: We have characterized the pancreatic phenotype of mice carrying a gene trap mutation in Sel1l. We show that Sel1l 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 Sel1l mutant embryos are confined to the progenitor cell state throughout the secondary transition. Pharmacological inhibition of Notch signaling partially rescues the pancreatic phenotype of Sel1l mutant embryos. CONCLUSIONS: Together, these data suggest that Sel1l is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development.

Contributions of societal modernity to cognitive development: a comparison of four cultures.

This study examined how societal changes associated with modernization are related to cognitive development. Data were from 4 cultural communities that represented a broad range of traditional and modern elements: the Garifuna (Belize), Logoli (Kenya), Newars (Nepal), and Samoans (American Samoa). Naturalistic observations and the performances of 3-, 5-, 7-, and 9-year-old children (N = 192) on 7 cognitive measures were examined. Results replicated age-related improvement on all measures. Contributions of modernity were evident in children's play behaviors and cognitive performances, especially in skills related to schooling. Modernization and schooling independently predicted differences on most of the measures. Results are discussed in relation to the Flynn effect, the worldwide increase in cognitive scores across generations, and the ways in which societal modernization may contribute to cognitive development.

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

BACKGROUND: The H6 homeobox genes Hmx1, Hmx2, and Hmx3 (also known as Nkx5-3; Nkx5-2 and Nkx5-1, 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 Hmx2 or Hmx3 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 Hmx1, the most divergent of the family. RESULTS: Dumbo (dmbo) 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 Hmx1. 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 dmbo/dmbo 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 dumbo 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 (Hmx1; also Nkx5-3). An independent spontaneous allele called misplaced ears (mpe) was also identified, confirming Hmx1 as the responsible mutant gene. CONCLUSION: The divergence of Hmx1 from its paralogs is reflected by different and diverse developmental roles exclusive of vestibular involvement. Additionally, these mutant Hmx1 alleles represent the first mouse models of a recently-discovered Oculo-Auricular syndrome caused by mutation of the orthologous human gene.

Mutagenesis of mouse embryonic stem cells with ethylmethanesulfonate.

Unraveling the function of the mammalian genome relies heavily on analyses of the laboratory mouse. Because of its powerful genetics and available technologies to manipulate the genome, plus its developmental and physiological similarities to humans, it has become a goal to generate mutations in all mouse genes and analyze the phenotypic consequences. Gene targeting in embryonic stem (ES) cells is the method of choice for making null mutations in known genes of interest. However, forward genetics approaches, in which mutations are produced randomly throughout the genome, has the advantage of producing alleles of varying severity both within known genes, in non-coding regulatory elements, or in other unannotated functional elements. Such forward genetic mutation screens in mice have typically involved treating male mice with N-ethyl-N-nitrosourea (ENU), followed by three generations of breeding to render potential recessive mutations homozygous, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of ES cells. This enables the use of multiple alternative chemicals with different mutational spectra, can reduce breeding to two generations, and impart a higher mutational load. Furthermore, ES cell mutagenesis can be used to create banks of clones that can be screened for point mutations in genes of interest, and to conduct forward genetic screens in vitro to detect potential phenotypes prior to generation of mice. In this chapter, we provide a detailed protocol for mutagenizing ES cells with the point mutagen ethylmethanesulfonate (EMS).

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice.

Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.

The mouse gcd2 mutation causes primordial germ cell depletion.

Germ cell depletion 2 (gcd2) is a chemically induced recessive mutation that causes infertility in male and female mice. The infertility is caused by germ cell depletion as early as 11.5 days post-coitum, when primordial germ cells have completed their migration to the embryonic gonads. Thus, the gcd2 mutation affects the proliferation and/or survival of germ cells after they arrive in the embryonic gonad, a developmental time when little is known about the requirements for germ cell proliferation and survival. The sterility phenotype is incompletely penetrant, has variable expressivity, and is modulated by strain background. The penetrance ranges from 37% in strain C57BL/6J to nearly 100% in CAST/EiJ. Genetic mapping localized gcd2 to a approximately 1Mb region on Chr 2. This interval contains a small number of annotated genes, of which none are known to have a role in germ cell development. Sequencing the coding regions of these genes failed to reveal a mutation, and BACs containing two of the candidate genes failed to rescue the phenotype. This raises the possibilities that the gcd2 mutation resides in non-coding sequences, and regulates genes outside the genetically defined critical region.

Transgenic rescue of the mouse t complex haplolethal locus Thl1.

Chromosomal deletions can uncover haploinsufficient or imprinted regions of the genome. Previously, the haploinsufficient locus t haplolethal 1 (Thl1) was identified and localized to a 1.3-Mb region using overlapping deletions around the Sod2 and D17Leh94 loci of the mouse t complex on Chr 17. Germline chimeric mice, produced from embryonic stem (ES) cells containing radiation-induced deletions of the Thl1 locus, never produced viable deletion-bearing progeny when mated to C57BL/6J (B6) females. However, deletion-bearing offspring could be obtained by mating to females of other strains. In this article we describe a transgenic approach to narrow the critical region for Thl1. BAC clones were introduced into a deletion-bearing ES cell line and one was shown to rescue the Thl1 phenotype, reducing the critical region to 140 kb. Analysis of the gene content of this region suggests two strong Thl1 candidates, Pdcd2 and a novel SET domain-containing gene termed Tset1. A more detailed analysis using mice carrying overlapping deletions identified subregions that influence the phenotypic characteristics of Thl1 hemizygotes.

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis.

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genome-wide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)-mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.

The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.

chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase theta;. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority ( approximately 90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.

Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8.

A novel mutation, mei8, was isolated in a forward genetic screen for infertility mutations induced by chemical mutagenesis of ES cells. Homozygous mutant mice are sterile. Mutant females exhibit ovarian dysgenesis and lack ovarian follicles at reproductive maturity. Affected males have small testes due to arrest of spermatogenesis during meiotic prophase I. Genetic mapping and positional cloning of mei8 led to the identification of a mutation in Rec8, a homolog of the yeast meiosis-specific cohesin gene REC8. Analysis of meiosis in Rec8(mei8)/Rec8(mei8) spermatocytes showed that, while initiation of recombination and synapsis occurs, REC8 is required for the completion and/or maintenance of synapsis, cohesion of sister chromatids, and the formation of chiasmata, as it is in other organisms. However, unlike yeast and Caenorhabditis elegans, localization of REC8 on meiotic chromosomes is not required for the assembly of axial elements.

Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase.

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.