Robert J. Munroe

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. Mcm4Chaos3 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, McmChaos3/− embryos died late in gestation, indicating that Mcm4Chaos3 is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4Chaos3/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.

Mouse mutants from chemically mutagenized embryonic stem cells

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

The Mouse Genomic Instability Mutation chaos1 Is an Allele of Polq That Exhibits Genetic Interaction with Atm

ABSTRACT 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 θ. 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 (∼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.

Toward the Genetics of Mammalian Reproduction: Induction and Mapping of Gametogenesis Mutants in Mice

Abstract The genetic control of mammalian gametogenesis is inadequately characterized because of a lack of mutations causing infertility. To further the discovery of genes required for mammalian gametogenesis, phenotype-driven screens were performed in mice using random chemical mutagenesis of whole animals and embryonic stem cells. Eleven initial mutations are reported here that affect proliferation of germ cells, meiosis, spermiogenesis, and spermiation. Nine of the mutations have been mapped genetically. These preliminary studies provide baselines for estimating the number of genes required for gametogenesis and offer guidance in conducting new genetic screens that will accelerate and optimize mutant discovery. This report demonstrates the efficacy and expediency of mutagenesis to identify new genes required for mammalian gamete development.

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.

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

BackgroundThe 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.ResultsDumbo (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.ConclusionThe 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.

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells

BackgroundThe 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.ResultsWe 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.ConclusionsTogether, these data suggest that Sel1l is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development.

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.

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

BackgroundForward 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.ResultsWe 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.ConclusionThis 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.

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.