Congenital hydrocephalus in genetically engineered mice.

There is evidence that genetic factors play a role in the complex multifactorial pathogenesis of hydrocephalus. Identification of the genes involved in the development of this neurologic disorder in animal models may elucidate factors responsible for the excessive accumulation of cerebrospinal fluid in hydrocephalic humans. The authors report here a brief summary of findings from 12 lines of genetically engineered mice that presented with autosomal recessive congenital hydrocephalus. This study illustrates the value of knockout mice in identifying genetic factors involved in the development of congenital hydrocephalus. Findings suggest that dysfunctional motile cilia represent the underlying pathogenetic mechanism in 8 of the 12 lines (Ulk4, Nme5, Nme7, Kif27, Stk36, Dpcd, Ak7, and Ak8). The likely underlying cause in the remaining 4 lines (RIKEN 4930444A02, Celsr2, Mboat7, and transgenic FZD3) was not determined, but it is possible that some of these could also have ciliary defects. For example, the cerebellar malformations observed in RIKEN 4930444A02 knockout mice show similarities to a number of developmental disorders, such as Joubert, Meckel-Gruber, and Bardet-Biedl syndromes, which involve mutations in cilia-related genes. Even though the direct relevance of mouse models to hydrocephalus in humans remains uncertain, the high prevalence of familial patterns of inheritance for congenital hydrocephalus in humans suggests that identification of genes responsible for development of hydrocephalus in mice may lead to the identification of homologous modifier genes and susceptibility alleles in humans. Also, characterization of mouse models can enhance understanding of important cell signaling and developmental pathways involved in the pathogenesis of hydrocephalus.

Situs inversus in Dpcd/Poll-/-, Nme7-/- , and Pkd1l1-/- mice.

Situs inversus (SI) is a congenital condition characterized by left-right transposition of thoracic and visceral organs and associated vasculature. The usual asymmetrical positioning of organs is established early in development in a transient structure called the embryonic node. The 2-cilia hypothesis proposes that 2 kinds of primary cilia in the embryonic node determine left-right asymmetry: motile cilia that generate a leftward fluid flow, and immotile mechanosensory cilia that respond to the flow. Here, we describe 3 mouse SI models that provide support for the 2-cilia hypothesis. In addition to having SI, Dpcd/Poll(-/-) mice (for: deleted in a mouse model of primary ciliary dyskinesia) and Nme7(-/-) mice (for: nonmetastatic cells 7) had lesions consistent with deficient ciliary motility: Hydrocephalus, sinusitis, and male infertility developed in Dpcd/Poll(-/-) mice, whereas hydrocephalus and excessive nasal exudates were seen in Nme7(-/-) mice. In contrast, the absence of respiratory tract lesions, hydrocephalus, and male infertility in Pkd1l1(-/-) mice (for: polycystic kidney disease 1 like 1) suggested that dysfunction of motile cilia was not involved in the development of SI in this line. Moreover, the gene Pkd1l1 has considerable sequence similarity with Pkd1 (for: polycystic kidney disease 1), which encodes a protein (polycystin-1) that is essential for the mechanosensory function of immotile primary cilia in the kidney. The markedly reduced viability of Pkd1l1(-/-) mice is somewhat surprising given the absence of any detected abnormalities (other than SI) in surviving Pkd1l1(-/-) mice subjected to a comprehensive battery of phenotype-screening exams. However, the heart and great vessels of Pkd1l1(-/-) mice were not examined, and it is possible that the decreased viability of Pkd1l1(-/-) mice is due to undiagnosed cardiovascular defects associated with heterotaxy.

Disruption of Ini1 leads to peri-implantation lethality and tumorigenesis in mice.

SNF5/INI1 is a component of the ATP-dependent chromatin remodeling enzyme family SWI/SNF. Germ line mutations of INI1 have been identified in children with brain and renal rhabdoid tumors, indicating that INI1 is a tumor suppressor. Here we report that disruption of Ini1 expression in mice results in early embryonic lethality. Ini1-null embryos die between 3.5 and 5.5 days postcoitum, and Ini1-null blastocysts fail to hatch, form the trophectoderm, or expand the inner cell mass when cultured in vitro. Furthermore, we report that approximately 15% of Ini1-heterozygous mice present with tumors, mostly undifferentiated or poorly differentiated sarcomas. Tumor formation is associated with a loss of heterozygocity at the Ini1 locus, characterizing Ini1 as a tumor suppressor in mice. Thus, Ini1 is essential for embryo viability and for repression of oncogenesis in the adult organism.

Analysis of ku80-mutant mice and cells with deficient levels of p53.

Absence of Ku80 results in increased sensitivity to ionizing radiation, defective lymphocyte development, early onset of an age-related phenotype, and premature replicative senescence. Here we investigate the role of p53 on the phenotype of ku80-mutant mice and cells. Reducing levels of p53 increased the cancer incidence for ku80(-/-) mice. About 20% of ku80(-/-) p53(+/-) mice developed a broad spectrum of cancer by 40 weeks and all ku80(-/-) p53(-/-) mice developed pro-B-cell lymphoma by 16 weeks. Reducing levels of p53 rescued populations of ku80(-/-) cells from replicative senescence by enabling spontaneous immortalization. The double-mutant cells are impaired for the G(1)/S checkpoint due to the p53 mutation and are hypersensitive to gamma-radiation and reactive oxygen species due to the Ku80 mutation. These data show that replicative senescence is caused by a p53-dependent cell cycle response to damaged DNA in ku80(-/-) cells and that p53 is essential for preventing very early onset of pro-B-cell lymphoma in ku80(-/-) mice.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.

Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells.

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs) and genomic sequence has created a 'gene function gap' with the identification of new genes far outpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.

Defective global genome repair in XPC mice is associated with skin cancer susceptibility but not with sensitivity to UVB induced erythema and edema.

It is generally presumed that xeroderma pigmentosum (XP) patients are extremely sensitive to developing UV erythema, and that they have a more than 1000-fold increased skin cancer risk. Recently established mouse models for XP can be employed to investigate the mechanism of these increased susceptibilities. In line with human data, both XPA and XPC knockout mice have been shown to have an increased susceptibility to UVB induced squamous cell carcinomas. In XPA knockouts, nucleotide excision repair of UV induced DNA photolesions is completely defective (i.e., both global genome repair and transcription coupled repair are defective). We determined the strand specific removal of cyclobutane pyrimidine dimers and pyrimidine [6-4] pyrimidone photoproducts from the p53 gene in cells from XPC knockout mice and wild-type littermates. Analogous to human XPC cells, embryonic fibroblasts from XPC knockout mice are only capable of performing transcription coupled repair of DNA photolesions. We show that these XPC knockout mice, in striking contrast to XPA knockout mice, do not have a lower minimal erythema/edema dose than their wild-type littermates. Hence, defective global genome repair appears to lead to skin cancer susceptibility, but does not influence the sensitivity to acute effects of UVB radiation, such as erythema and edema. The latter phenomena thus relate to the capacity to perform transcription coupled repair, which suggests that blockage of RNA synthesis is a key event in the development of UV erythema and edema.

The tumorigenic potential and cell growth characteristics of p53-deficient cells are equivalent in the presence or absence of Mdm2.

The Mdm2 oncoprotein forms a complex with the p53 tumor suppressor protein and inhibits p53-mediated regulation of heterologous gene expression. Recently, Mdm2 has been found to bind several other proteins that function to regulate cell cycle progression, including the E2F-1/DP1 transcription factor complex and the retinoblastoma tumor-suppressor protein. To determine whether Mdm2 plays a role in cell cycle control or tumorigenesis that is distinct from its ability to modulate p53 function, we have examined and compared both the in vitro growth characteristics of p53-deficient and Mdm2/p53-deficient fibroblasts, and the rate and spectrum of tumor formation in p53-deficient and Mdm2/p53-deficient mice. We find no difference between p53-deficient fibroblasts and Mdm2/p53-deficient fibroblasts either in their rate of proliferation in culture or in their survival frequency when treated with various genotoxic agents. Cell cycle studies indicate no difference in the ability of the two cell populations to enter S phase when treated with DNA-damaging agents or nucleotide antimetabolites, and p53-deficient fibroblasts and Mdm2/p53-deficient fibroblasts exhibit the same rate of spontaneous immortalization following long-term passage in culture. Finally, p53-deficient mice and Mdm2/p53-deficient mice display the same incidence and spectrum of spontaneous tumor formation in vivo. These results demonstrate that deletion of Mdm2 has no additional effect on cell proliferation, cell cycle control, or tumorigenesis when p53 is absent.

High susceptibility to ultraviolet-induced carcinogenesis in mice lacking XPC.

Compromise of genetic information by mutation may result in the dysregulation of cellular growth control and subsequent tumour formation. Xeroderma pigmentosum (XP) is a rare autosomal disease characterized by hypersensitivity of the skin to sunlight and > 1,000-fold increased risk of skin cancers in sun-exposed parts of the body. Cell fusion studies have revealed eight complementation groups in XP (A-G, and an XP-variant form); group C is one of the most common forms of the disease. We have isolated a mouse homologue of the human gene for XP group C and generated XPC-deficient mice by using embryonic stem cell technology. Mice homozygous for the XPC mutant allele (xpcm1/xpcm1) are viable and do not exhibit an increased susceptibility to spontaneous tumour generation at one year of age. However, xpcm1/xpcm1 mice were found to be highly susceptible to ultraviolet-induced carcinogenesis compared with mice heterozygous for the mutant allele (xpcm1/+) and wild-type controls. Homozygous xpcm1 mutant mice also display a spectrum of ultraviolet-exposure-related pathological skin and eye changes consistent with the human disease xeroderma pigmentosum group C.

p53 deficiency does not affect the accumulation of point mutations in a transgene target.

DNA repair is required by organisms to prevent the accumulation of mutations and to maintain the integrity of genetic information. Mammalian cells that have been treated with agents that damage DNA have an increase in p53 levels, a p53-dependent arrest at G1 in the cell cycle, and a p53-dependent apoptotic response. It has been hypothesized that this block in cell cycle progression is necessary to allow time for DNA repair or to direct the damaged cell to an apoptotic pathway. This hypothesis predicts that p53-deficient cells would have an abnormal apoptotic response and exhibit a "mutator" phenotype. Using a sensitive assay for the accumulation of point mutations, small deletions, and insertions, we have directly tested whether p53-deficient cells exhibit an increased frequency of mutation before and after exposure to DNA-damaging agents. We report that wild-type and p53-deficient fibroblasts, thymocytes, and tumor tissue have indistinguishable rates of point mutation accumulation in a transgenic lacI target gene. These results suggest that the role of p53 in G1 checkpoint control and tumor suppression does not affect the accumulation of point mutations.

p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells.

Terminal differentiation is coupled to withdrawal from the cell cycle. The cyclin-dependent kinase inhibitor (CKI) p21Cip1 is transcriptionally regulated by p53 and can induce growth arrest. CKIs are therefore potential mediators of developmental control of cell proliferation. The expression pattern of mouse p21 correlated with terminal differentiation of multiple cell lineages including skeletal muscle, cartilage, skin, and nasal epithelium in a p53-independent manner. Although the muscle-specific transcription factor MyoD is sufficient to activate p21 expression in 10T1/2 cells, p21 was expressed in myogenic cells of mice lacking the genes encoding MyoD and myogenin, demonstrating that p21 expression does not require these transcription factors. The p21 protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation.

In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice.

Fibroblast cultures were derived from mouse embryos containing either one (p53+/-) or two (p53-/-) inactivated p53 alleles and compared to normal embryo fibroblasts for a number of growth parameters. Early passage p53-deficient embryo fibroblasts (p53-/-) divided faster than normal embryo fibroblasts, achieved higher confluent densities, and had a higher fraction of division-competent cells under conditions of low cell density. Flow cytometry studies of early passage embryo fibroblasts showed that the percent of p53-deficient cells in G0/G1 was lower than in normal cells, consistent with the argument that p53 mediates a G1 block. When p53-deficient and normal cells were passaged for long periods of time, the homozygote (p53-/-) fibroblasts grew at a high rate for over 50 passages and never entered a non-growing senescent phase characteristic of the heterozygote (p53+/-) and normal (p53+/+) cells. The p53-deficient fibroblasts were genetically unstable during passaging, with the p53-/- cells showing a high degree of aneuploidy and the p53+/- cells displaying a moderate level of chromosomal abnormalities by passage 25. Surprisingly, the heterozygote cells lost their single wild type allele very early during culturing and in spite of this loss most heterozygote lines entered into senescence. We conclude that the loss of p53 by itself is insufficient to confer immortality on a cell, but does confer a growth advantage. Taken together, the findings confirm that the absence of p53 promotes genomic instability, which in turn may result in genetic alterations which directly produce immortality.

Cytoplasmic beta-actin promoter produces germ cell and preimplantation embryonic transgene expression.

The cytoplasmic beta-actin promoter, commonly used as strong promoter in many gene regulation studies, produces a pattern of male germ cell and preimplantation, embryonic gene expression in transgenic mice. In seven of ten expressing transgenic lines, a chicken beta-actin-lacZ fusion gene was expressed in adult testes. In addition, five of the ten lines demonstrated transgene expression in the preimplantation mouse embryo. This is the first example of transgene expression at the stages of both gamete and early embryo. Overall, the site or transgene integration appeared to influence transgene expression in adult tissues.

Targeting of gene expression to skeletal and cardiac muscle of trangenic animals.

The tissue restricted and developmental potentiation of transcription by chicken alpha-skeletal actin promoter regions fused to the reporter gene chloramphenicol acetyl transferase (CAT) were characterized in transgenic mice. Six of eight expressing transgenic mouse lines containing the chicken alpha-skeletal actin promoter fused to CAT resulted in preferential transgene transcription in skeletal muscle tissue, similar to the endogenous mouse alpha-skeletal actin gene. Two of the eight lines departed from the preferred pattern of skeletal muscle expression with primary expression of the transgene in the heart, a tissue containing primarily cardiac actin isoforms. Developmentally, a transition from embryonic heart to fetal and neonatal skeletal muscle expression was produced by the transgene promoter, a pattern of regulation similar to that of the endogenous alpha-skeletal actin gene. Instances of departure of transgene expression from the endogenous gene implied the existance of higher order muscle gene regulatory mechanisms.