Prdm14 initiates lymphoblastic leukemia after expanding a population of cells resembling common lymphoid progenitors.

Understanding the heterogeneous genetic mechanisms of tumor initiation in lymphoid leukemias (LL) will lead to improvements in prognostic classification and treatment regimens. In previous studies of mouse leukemias, we showed that retroviral insertion at the ecotropic viral insertion site 32 locus leads to increased expression of Prdm14, a pluripotency gene implicated in the self-renewal capacity of embryonic stem cells and the early stages of breast cancer. Here, we show that PRDM14 is also overexpressed in ∼25% of human lymphoid neoplasms, with increased frequencies in T-cell acute LL and hyperdiploid precursor B-cell acute LL. To test if Prdm14 overexpression could initiate leukemia, mice were transduced with bone marrow cells transfected with a Prdm14 expression vector. LLs developed in 96% of female mice and 42% of male mice. Before the onset of leukemia, differentiation of transduced cells was biased up to 1000-fold toward cells with features of common lymphoid progenitors (CLPs), and lymphoid differentiation showed a relative block at the pro-B stage. Microarray gene expression analysis of expanded CLP-like cells before the onset of leukemia demonstrated upregulation of genes involved in pluripotency, tumor initiation, early B-lineage commitment, Wnt/Ras signaling and the epithelial-to-mesenchymal transition. Among the dysregulated genes were imprinted genes and non-coding RNAs including Dlk1 and Meg3, which are also key pluripotency mediators. Heightened expression of the estrogen-dependent oncogene, Myb, in tumors suggests a basis for the increased frequency of cancer in female mice. These data provide the first direct evidence for the association of Prdm14 with cancer initiation in an in vivo mouse model and in human lymphoid malignancies, while suggesting mechanisms for Prdm14's mode of action.

Isolation and characterization of Vsx1, a novel mouse CVC paired-like homeobox gene expressed during embryogenesis and in the retina.

Gastrula stage mouse embryo RNA was screened by degenerate RT-PCR to yield a novel paired-like homeobox gene. The open reading frame encoded by the cDNA was most similar to human VSX1. Mouse Vsx1 encodes a protein of 363 amino acid residues that contains a CVC domain that was originally identified as a conserved motif among mouse CHX10, goldfish VSX-1 and C. elegans CEH-10. Linkage analysis showed that mouse Vsx1 mapped to the distal region of chromosome 2. RT-PCR analysis detected mouse Vsx1 transcripts from gastrulation and post-gastrulation stage mouse embryos, suggesting a role for Vsx1 during mouse embryogenesis. Analysis of the eyes of mouse chimeras generated with embryonic stem cells in which a lacZ reporter was targeted to the Vsx1 locus suggested that Vsx1 is expressed in the inner nuclear layer of the retina.

Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail.

Neural tube defects (NTDs) such as spina bifida and anencephaly are common congenital malformations in humans (1/1,000 births) that result from failure of the neural tube to close during embryogenesis. The etiology of NTDs is complex, with both genetic and environmental contributions; the genetic component has been extensively studied with mouse models. Loop-tail (Lp) is a semidominant mutation on mouse chromosome 1 (ref. 4). In the two known Lp alleles (Lp, Lpm1Jus), heterozygous mice exhibit a characteristic looped tail, and homozygous embryos show a completely open neural tube in the hindbrain and spinal region, a condition similar to the severe craniorachischisis defect in humans. Morphological and neural patterning studies indicate a role for the Lp gene product in controlling early morphogenesis and patterning of both axial midline structures and the developing neural plate. The 0.6-cM/0.7-megabase (Mb) Lp interval is delineated proximally by D1Mit113/Apoa2/Fcer1g and distally by Fcer1a/D1Mit149/Spna1 and contains a minimum of 17 transcription units. One of these genes, Ltap, encodes a homolog of Drosophila Strabismus/Van Gogh (Stbm/Vang), a component of the frizzled/dishevelled tissue polarity pathway. Ltap is expressed broadly in the neuroectoderm throughout early neurogenesis and is altered in two independent Lp alleles, identifying this gene as a strong candidate for Lp.

Identification of a new chemically induced allele (Lp(m1Jus)) at the loop-tail locus: morphology, histology, and genetic mapping.

Loop-tail (Lp) is a semidominant mutation that affects neurulation in mice. Heterozygous animals are characterized by a looped-tail appearance (pig tail) and wobbly head movements while homozygous embryos exhibit a neural tube closure defect that extends from the caudal midbrain to the tip of the tail. The Lp gene has been finely mapped to the distal part of chromosome 1, and a positional cloning strategy has been initiated to isolate the defective gene. This study represents the characterization of a new Lp allele (Lp(m1Jus)) induced by N-ethyl-N-nitrosurea mutagenesis. Lp(m1Jus)/+ mice have a looped-tail appearance, and both Lp(m1Jus)/Lp(m1Jus) homozygotes and Lp/Lp(m1Jus) compound heterozygotes fail to initiate neural tube closure along most of the embryonic axis. These data indicate that the Lp(m1Jus) allele causes a neural tube defect and overall phenotype similar to that of the original Lp allele. Segregation analysis of 90 (Lp(m1Jus)/+ x C57BL/6J)F(1) x C57BL/6J looped-tail mice with seven markers that define the Lp genetic map (D1Mit455/D1Mit146/D1Mit148/D1Mit270-1 cM-D1Mit113-0.4 cM-Lp-0.2 cM-D1Mit149-0.8 cM-D1Mit115) showed significant linkage between Lp(m1Jus) and all loci analyzed (P < 0.0001). Eight crossovers were detected with the proximal cluster of D1Mit455, D1Mit146, D1Mit148, and D1Mit270, indicating a recombination rate higher than expected in this region, and a single recombinant was encountered with the distal markers D1Mit149 and D1Mit115. Based on these phenotypic and genetic data, Lp(m1Jus) is most likely allelic to Lp, thereby representing a valuable additional tool for the positional cloning of the Lp gene and its subsequent molecular characterization.

The winged helix/forkhead transcription factor Foxq1 regulates differentiation of hair in satin mice.

Satin (sa) homozygous mice have a silky coat with high sheen arising from structurally abnormal medulla cells and defects in differentiation of the hair shaft. We demonstrate that the winged helix/forkhead transcription factor, Foxq1 (Forkhead box, subclass q, member 1) is mutant in sa mice. An intragenic deletion was identified in the radiation-induced satin mutant of the SB/Le inbred strain; a second allele, identified by an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, has a missense mutation in the conserved winged helix DNA-binding domain. Homozygous mutants of the two alleles are indistinguishable. We show that Foxq1 is expressed during embryogenesis and exhibits a tissue-restricted expression pattern in adult tissues. The hair defects appear to be restricted to the inner structures of the hair; consequently, Foxq1 has a unique and distinct function involved in differentiation and development of the hair shaft. Despite an otherwise healthy appearance, satin mice have been reported to exhibit suppressed NK-cell function and alloimmune cytotoxic T-cell function. We show instead that the immune defects are attributable to genetic background differences.

Gene defect in ectodermal dysplasia implicates a death domain adapter in development.

Members of the tumour-necrosis factor receptor (TNFR) family that contain an intracellular death domain initiate signalling by recruiting cytoplasmic death domain adapter proteins. Edar is a death domain protein of the TNFR family that is required for the development of hair, teeth and other ectodermal derivatives. Mutations in Edar-or its ligand, Eda-cause hypohidrotic ectodermal dysplasia in humans and mice. This disorder is characterized by sparse hair, a lack of sweat glands and malformation of teeth. Here we report the identification of a death domain adapter encoded by the mouse crinkled locus. The crinkled mutant has an hypohidrotic ectodermal dysplasia phenotype identical to that of the edar (downless) and eda (Tabby) mutants. This adapter, which we have called Edaradd (for Edar-associated death domain), interacts with the death domain of Edar and links the receptor to downstream signalling pathways. We also identify a missense mutation in its human orthologue, EDARADD, that is present in a family affected with hypohidrotic ectodermal dysplasia. Our findings show that the death receptor/adapter signalling mechanism is conserved in developmental, as well as apoptotic, signalling.

Developmental eye and neural tube defects in the eye blebs mouse.

In the mouse, eye blebs (eb) is a spontaneous mutation that presents a useful model for the study of abnormal eye development. Since its initial description three decades ago, little information has been generated regarding the developmental course of eb eyes. Although the gene for eb has not been identified, much can be learned from the developmental defects present in the eb mouse. First detected in the eye at embryonic day 11.5 (E11.5), the eb defect is observed as an increased vascularization throughout the developing eye and head region. As development proceeds, the embryonic eye fills with blood, and the resulting hematoma distorts the shape of the iris. The eyelids fail to close, and animals are born with open eyes. Lens degeneration and retinal folding are characteristic of eb, as are microphthalmia and thick, disorganized irises. A second presentation of the eb defect is disruption of neural tube closure in the anterior and hindbrain neuropores. These eb animals are born with open neural tubes but with apparently normal eyes.

Effects of ENU dosage on mouse strains.

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F(1) hybrids to ENU. The results show that most F(1) hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

Mouse as the measure of man?

The humble house mouse's cohabitation with humans has been noted since the birth of agriculture, about 10 000 years ago, in the fertile flood plains of the Middle East. In recent times, however, the mouse has been elevated from pest to model for the study of human health and disease. Recent genomics and genetics initiatives will ensure the continued growth of the house mouse as a disease model.

Optimal N-ethyl-N-nitrosourea (ENU) doses for inbred mouse strains.

ENU is a powerful germline mutagen in the mouse, providing the opportunity to analyze the functions of large numbers of genes in the mammalian genome. In many mutagenesis experiments, it would be beneficial to exploit the advantages of inbred mouse strains. To perform an effective ENU mutagenesis screen using inbred mice, a dosage regimen is required to determine the optimal dose of ENU for that inbred strain, a time-consuming preliminary process. We have carried out dosage regimens for mutagenizing doses of ENU in ten inbred strains of mouse: 129X1/SvJ, 129S6/SvEv, A/J, BALB/cJ, BTBR/N, C3He/J, C3HeB/FeJ, C57BL/6J, C57BR/cdJ, and CBA/CaJ, and determined an optimal dose for each strain, defined by length of sterile period and number of males to survive treatment. Three strains: A/J, BALB/cJ and C57BL/6J, are able to tolerate high doses, up to 300 mg/kg body weight, and are highly recommended for mutagenesis studies.

Genetic profile of insertion mutations in mouse leukemias and lymphomas.

Murine leukemia retroviruses (MuLVs) cause leukemia and lymphoma in susceptible strains of mice as a result of insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Using a novel approach to amplify and sequence viral insertion sites, we have sequenced >200 viral insertion sites from which we identify >35 genes altered by viral insertion in four AKXD mouse strains. The class of genes most frequently altered are transcription factors, however, insertions are found near genes involved in signal transduction, cell cycle control, DNA repair, cell division, hematopoietic differentiation, and near many ESTs and novel loci. Many of these mutations identify genes that have not been implicated in cancer. By isolating nearly all the somatic viral insertion mutations contributing to disease in these strains we show that each AKXD strain displays a unique mutation profile, suggesting strain-specific susceptibility to mutations in particular genetic pathways.

A comparative transcript map and candidates for mutant phenotypes in the Tyrp1 (brown) deletion complex homologous to human 9p21-23.

The mouse Tyrp1 deletion complex is a valuable resource for high-resolution mapping of genes and phenotypes to the central region of Chromosome (Chr) 4. The distal part of the complex is homologous to human Chr 9p21-23, and we have used the available radiation hybrid maps to identify human transcripts in the region. We localize seven genes to a human YAC contig that spans the full extent of the distal deletion complex and show that the mouse homologs of four of these, including Cer1, map within the complex. On the basis of location and/or expression, we exclude genes as candidates for several known phenotypes in the region and identify a candidate transcript for the neonatal lethal phenotype l(4)Rn2.

Capitalizing on large-scale mouse mutagenesis screens.

Variation is the crux of genetics. Mutagenesis screens in organisms from bacteria to fish have provided a battery of mutants that define protein functions within complex pathways. Large-scale mutation isolation has been carried out in Caenorhabditis elegans, Drosophila melanogaster and zebrafish, and has been recently reported in the mouse in two screens that have generated many new, clinically relevant mutations to reveal the power of phenotype-driven screens in a mammal.

Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia.

AKXD recombinant inbred mice develop a variety of leukemias and lymphomas due to retrovirally mediated insertional activation of cellular proto-oncogenes. We describe a new retroviral insertion site that is the most frequent genetic alteration in AKXD B-cell leukemias. Multiple genes flank the site of viral insertion, but the expression of just two, Hex and mEg5, is significantly upregulated. Hex is a divergent homeobox gene that is transiently expressed in many hematopoietic lineages, suggesting an involvement in cellular differentiation. mEg5 is a member of the bim-C subfamily of kinesin related proteins that are necessary for spindle formation and stabilization during mitosis. Our data provide the first genetic evidence for the activation of these genes in leukemia, and suggest that unscheduled expression of Hex and mEg5 contributes to the development of B-cell leukemia. In addition, this work highlights the use of genomic approaches for the study of position effect mutations.

Mouse ENU mutagenesis.

The progress of human genome sequencing is driving genetic approaches to define gene function. Strategies such as gene traps and chemical mutagenesis will soon generate a large mutant mouse resource. Point mutations induced by N -ethyl- N -nitrosourea (ENU) provide a unique mutant resource because they: (i) reflect the consequences of single gene change independent of position effects; (ii) provide a fine-structure dissection of protein function; (iii) display a range of mutant effects from complete or partial loss of function to exaggerated function; and (iv) discover gene functions in an unbiased manner. Phenotype-driven ENU screens in the mouse are emphasizing relevance to human clinical disease by targeting cardiology, physiology, neurology, immunity, hematopoiesis and mammalian development. Such approaches are extremely powerful in understanding complex human diseases and traits: the base-pair changes may accurately model base changes found in human diseases, and subtle mutant alleles in a standard genetic background provide the ability to analyze the consequences of compound genotypes. Ongoing mouse ENU mutagenesis experiments are generating a treasure trove of new mutations to allow an in-depth study of a single gene, a chromosomal region or a biological system.

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.

A mouse chromosome 19 genetic map including the Lvis1 viral insertion site.

Somatic insertion mutations, such as those caused by the insertion of a proviral element, can contribute to abnormal cell growth by activating cellular proto-oncogenes or inactivating tumor suppressor genes. Lvis1 is a genomic locus frequently disrupted by viral insertion in AKXD B-cell lymphomas (manuscript submitted for publication). To determine whether insertion mutations at Lvis1 affect a known proto-oncogene or tumor suppressor gene, we mapped Lvis1 to distal mouse chromosome 19. A detailed molecular genetic map of this region was constructed, localizing Lvis1 relative to 20 gene and microsatellite markers, 3 of which have not been mapped in the mouse (Nfkb2, Nlz, and Wnt8b). This analysis revealed that Lvis1 maps between two previously identified viral insertion sites, His2 and Frat1, and does not cosegregate with known gene markers. In addition, our study refines the gene order for distal mouse chromosome 19 and expands the comparative map between mouse chromosome 19 and the human chromosome 10q23-q26 homology region.