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Bloom's syndrome is a genetic disorder characterized by increased incidence of cancer and an immunodeficiency of unknown origin. The BLM gene mutated in Bloom's syndrome encodes a DNA helicase involved in the maintenance of genomic integrity. To explore the role of BLM in the immune system, we ablated murine Blm in the T-cell lineage. In the absence of Blm, thymocytes were severely reduced in numbers and displayed a developmental block at the beta-selection checkpoint that was partially p53 dependent. Blm-deficient thymocytes rearranged their T-cell receptor (TCR) beta genes normally yet failed to survive and proliferate in response to pre-TCR signaling. Furthermore, peripheral T cells were reduced in numbers, manifested defective homeostatic and TCR-induced proliferation, and produced extensive chromosomal damage. Finally, CD4(+) and CD8(+) T-cell responses were impaired upon antigen challenge. Thus, by ensuring genomic stability, Blm serves a vital role for development, maintenance, and function of T lymphocytes, suggesting a basis for the immune deficiency in Bloom's syndrome.

The Friend leukemia virus (FLV)-infected cell line, T-3-Cl-2, undergoes a form of erythroid differentiation in culture when treated with an appropriate inducer, such as dimethylsulfoxide ((CH3)2SO). Thus, whereas untreated cells contain no detectable hemoglobin, treated cells accumulate hemoglobin in quantities comparable to those in the mature mouse red blood cell. We have investigated the mechanism of hemoglobin induction by quantitating the number of globin genes and the amount of globin mRNA in cells before and during the period of hemoglobin accumulation. The results indicate the number of globin genes does not change as the cells accumulate hemogtobin: There are less than 5 globin genes per haploid genome. On the other hand, whereas cells lacking hemoglobin contain little, if any, globin mRNA, hemoglobin-containing cells accumulate, on the average, 8,000 molecules of globin mRNA per cell. The most direct, although, by no means, the only interpretation of these results is that the induction of hemoglobin synthesis involves transcriptional activation of the globin genes. Using this same cell line, we show that mouse globin mRNA sequences are also present in viral particles purified from the culture medium of globin-producing cells. These globin mRNA sequences are absent from viral particles derived from T-3-Cl-2 cells which are not producing globin mRNA. Virus-associated globin mRNA sequences sediment in association with 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60 S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin mRNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin mRNA does not become associated with the 60S viral RNA complex; and that globin mRNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin mRNA. The presence of globin mRNA sequences in FLV particles has important implications in terms of our ability to distinguish between host and viral RNAs in viral particles and in terms of the possible role RNA tumor viruses might play in transduction of genetic information.

One of the hallmarks of cancer cells is their increased resistance to apoptosis induction. Alterations in many apoptosis regulators belonging to the intrinsic pathway confer emerging neoplastic cells with a selective growth advantage in the hostile tumor microenvironment. The realization that those same defects contribute to resistance to radiation and chemotherapeutic agents have prompted the unrelenting search for mitochondria-targeted compounds for the treatment of cancer. Mitochondria play a central role in the process of cell death. They serve as integrators of upstream effector mechanisms. Most importantly, mitochondrial outer membrane permeabilization becomes a commitment point during cell death. Thus, strategies aimed at directly triggering this event by either blocking the activity of antiapoptotic factors or by interfering with vital mitochondrial functions may help to overcome resistance to standard cancer therapy.

The developmental origin of type I interferon (IFN)-producing plasmacytoid dendritic cells (PDCs) is controversial. In particular, the rearrangement of immunoglobulin heavy chain (IgH) genes in murine PDCs and the expression of pre-T cell receptor alpha (pTalpha) gene by human PDCs were proposed as evidence for their "lymphoid" origin. Here we demonstrate that PDCs capable of IFN production develop efficiently from both myeloid- and lymphoid-committed progenitors. Rearranged IgH genes as well as RAG transcripts were found in both myeloid- and lymphoid-derived PDCs. The human pTalpha transgenic reporter was activated in both myeloid- and lymphoid-derived PDCs at a level comparable to pre-T cells. PDCs were the only cell population that activated murine RAG1 knockin and human pTalpha transgenic reporters outside the lymphoid lineage. These results highlight a unique developmental program of PDCs that distinguishes them from other cell types including conventional dendritic cells.

Mutations that lead to the emergence of resistance to apoptosis are commonly observed among tumor cells. Some of the proteins affected are integral parts of the apoptotic cascade such as pro- and antiapoptotic members of the Bcl-2 family. F16 is a small molecule that accumulates in mitochondria of a variety of tumor cells and interferes with their physiological function. Because this interference ultimately triggers apoptosis in many affected cell lines, we examined the effect of antiapoptotic Bcl-2 overexpression on the response of cells to F16. Our results showed that high levels of Bcl-2 did not block the ability of F16 to induce cell death. However, unlike the apoptotic response that followed F16 treatment of cells with moderate Bcl-2 levels, cells resistant to a variety of apoptotic stimuli by virtue of Bcl-2 overexpression succumbed to F16 by necrosis. Thus, this dual ability of the mitochondriotoxic compound F16 to induce apoptosis and necrosis may represent an added advantage by expanding its spectrum of action toward genetically altered tumor cells incapable of apoptosis.

Independent mouse models for Bloom syndrome (BS) exist, each thought to disrupt Blm gene function. However, animals bearing these alleles exhibit distinct phenotypes. Blm tm1Ches and Blm tm1Grdn homozygous mutant animals exhibit embryonic lethality while in another, Blm tm3Brd , homozygosity yields viable, fertile animals with a cancer predisposition. Further characterization reveals the Blm tm3Brd allele to be a hypomorph, producing a diminished quantity of normal mRNA and protein. The Blm tm3Brd allele produces sufficient normal protein to rescue Blm tm1Ches lethality. Evaluation of viable animals reveals an inverse correlation between the quantity of Blm protein and the level of chromosome instability and a similar genotypic relationship for tumor predisposition indicating that Blm protein is rate limiting for maintaining genomic stability and the avoidance of tumors.

Homeodomain-interacting protein kinase 1 (HIPK1) is one of three closely related serine/threonine protein kinases that regulate the activity of a broad range of transcription factors (5, 10, 15, 22, 23). The HIPKs were originally identified as nuclear protein kinases that function as corepressors for various homeodomain-containing transcription factors. Subsequently, HIPK2 was shown to provide a regulatory role for a large transcriptional repressor complex, as it interacts with the repressive elements Groucho and histone deacetylase (HDAC1) (4). HIPK2 also associates with high-mobility group I (y) proteins, a family of nuclear architectural proteins that influence transcription regulation (26). The HIPKs are related to a group of kinases that includes Yak1 (Saccharomyces cerevisiae), YakA (Dictyostelium discoideum), and Minibrain (Drosophila melanogaster, rodents, and humans) (15, 23). This group represents a family of protein kinases that regulate the transition from growth to differentiation in eukaryotic cells. The assignment of HIPKs to this group suggests a common function in growth regulation. In fact, growth-regulatory and tumor suppressor functions have been attributed to the HIPKs (5, 10, 26, 28). For example, HIPK2 transfection into cells results in a dramatic decrease in colony formation (5, 10). This outcome occurs through the cooperative activities of HIPK2 and the tumor suppressor protein p53 (5, 10). HIPK2 phosphorylates p53 on Ser 46, resulting in activation of p53-dependent transcription, cell growth regulation, and apoptosis initiation (5, 10, 31). A potential apoptotic function was also assigned to HIPK3, as it interacts with the cell surface death receptor Fas and phosphorylates the Fas-associated death domain (FADD), a transducer of Fas-mediated apoptotic signaling (28). The association of HIPK3, a nuclear protein, with a cell surface receptor was reconciled by demonstrating diffuse cytoplasmic HIPK3 localization. Daxx is also a nuclear protein that was shown to associate with cytoplasmic and cell surface molecules, including transforming growth factor beta and Fas (25, 32). Overexpressed Daxx was shown to bind the Fas death domain and to mediate apoptotic signaling by activating ASK-1 and subsequently the Jun-N-terminal kinase (JNK) pathway, independent of the FADD/procaspase-8 pathway (3, 32). Within the nucleus, Daxx interacts with the promyelocytic leukemia protein (PML) and localizes to PML oncogenic domains (PODs) (17, 30, 33). Localization of Daxx to PODs correlates with Daxx's proapoptotic activity. For example, Daxx mutants that fail to localize to PODs do not facilitate Fas-induced cell death (30). Furthermore, in the absence of PML, Daxx is dispersed throughout the nucleus, and activated cell death is diminished (33). These findings indicate that Daxx and PML may cooperate in PODs in mediating apoptotic signals. This may be important in understanding the biological nature of acute promyelocytic leukemia, which is characterized by reciprocal chromosomal translocation between the pml and retinoic acid receptor α genes, resulting in the oncogenic fusion product PML-retinoic acid receptor α. In acute promyelocytic leukemia cells, Daxx does not localize to PODs. However, upon treatment with retinoic acid, which induces disease remission, Daxx relocalizes to PODs (33). Despite evidence supporting a proapoptotic function for Daxx, other studies have demonstrated that Daxx is essential to cell survival or is antiapoptotic. Targeted disruption of Daxx in mice results in embryonic lethality accompanied by extensive apoptosis (21). Elevated apoptosis was also observed in Daxx-null embryonic stem cells (21) as well as in fibroblasts in which endogenous Daxx was depleted by RNA interference treatment (J. S. Michaelson and P. Leder, unpublished data). In myeloid precursor cells, Daxx overexpression inhibited activated cell death, indicating an antiapoptotic role for Daxx (2). Furthermore, an antiapoptotic role for Daxx in acute promyelocytic leukemia cells was proposed, as Daxx expression decreased after apoptotic induction with HDAC1 inhibitors (1). It is possible that Daxx provides bipartite functions. Under certain circumstances, Daxx may be essential for cell survival, and under other circumstances, Daxx may propagate apoptotic signals. Similar to the HIPKs, Daxx functions as a transcriptional regulator. For example, Daxx repressed the transcriptional activities of the Pax3 and ETS1 transcription factors (11, 18). Interestingly, Daxx was not able to repress the transcriptional activities of the oncogenic fusion protein Pax3-FKHR present in an alveolar rhabdomyosarcoma. This suggests that Pax3-FKHR circumvents the transcriptional controls normally applied to Pax3 (11). Daxx also either repressed or activated Pax5-mediated transcription (6). The specific effect of Daxx on Pax5 activity varied in different B-cell lines. Activation of Pax5-mediated transcription by Daxx depended on recruitment of the histone acetyltransferase CREB binding protein. To date, the regulation of Daxx transcriptional activity is poorly understood. It was suggested that in the absence of PML, Daxx localized to chromatin, where it recruited HDAC1 and repressed transcription (12, 17). Overexpression of PML but not the oncogenic fusion PML-retinoic acid receptor α recruits Daxx to PODs, thereby inhibiting Daxx repression (16, 17). The recruitment of Daxx from chromatin to PODs requires the secondary modification of PML by the ubiquitin-like molecule SUMO-1 (12, 16, 17). These results demonstrate that Daxx transcriptional regulatory activity is controlled in part by PML, which sequesters Daxx from condensed chromatin to PODs. In this study, we characterized the expression and localization of HIPK1. In addition, we propose two novel mechanisms for regulating Daxx behavior, both mediated by HIPK1. HIPK1 relocates Daxx from PODs, presumably to chromatin, where Daxx participates in transcriptional regulation. In addition, HIPK1 phosphorylates Daxx on Ser 669, a site important in modulating Daxx activity.

A transgenic reporter mouse strain, which expressed the human CD25 (hCD25) surface marker as a reporter under the control of the pre-T cell receptor alpha(pTalpha) promoter, was used to identify lymphoid precursors that expressed pTalpha intracellularly. The hCD25 reporter marked intra- and extrathymic precursors of lymphocytes but not myeloid cells. The earliest intrathymic precursors were CD4(lo)CD8(-)CD25(-)CD44(+)c-Kit(+) cells that expressed elevated levels of Notch-1 mRNA. Clonogenic assays showed that the extrathymic precursors were common lymphoid progenitors (CLPs) that included CD19(-), B220(+), Thy1(+) and CD4(+) cells. Thus, the pTalpha reporter can be used to trace lymphopoiesis between CLPs and alphabeta T cells. The slower extinction of the hCD25 reporter compared to pTalpha enabled us to define points at which pTalpha(-) lineages branched off.

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of thepre-T cell receptor α(pTa) gene, a T-cell-specific transcriptional target of Notch. ThepTaenhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation ofpTatransgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway.

The evolutionarily conserved Hus1 proteins function in DNA damage response pathways that serve to maintain genomic stability [1, 2]. Cells lacking mouse Hus1 are hypersensitive to certain genotoxins [3], and we have explored the molecular basis for this defect by examining how Hus1 inactivation affects genotoxin-induced signaling events. p53 accumulation and activation in response to DNA damage appeared normal in Hus1 null cells. Likewise, Hus1 was dispensable for genotoxin-induced Chk2 phosphorylation. In contrast, Chk1 phosphorylation after genotoxic stress was greatly reduced in the absence of Hus1, but was restored in Hus1 null fibroblasts complemented by infection with a Hus1 -expressing retrovirus. These results demonstrate that mouse Hus1 is required for a specific subset of DNA damage signaling events and functions to promote genotoxin-induced Chk1 phosphorylation.

The neurogenic gene brainiac was first isolated in Drosophila melanogaster, where it interacts genetically with members of the Notch signaling cascade. We have isolated a murine homologue of the Drosophila brainiac gene and delineated its highly specific expression pattern during development and adult life. We find particularly strong expression in the developing central nervous system, in the developing retina, and in the adult hippocampus. Targeted deletion of mouse Brainiac 1 expression leads to embryonic lethality prior to implantation. Null embryos can be recovered as blastocysts but do not appear to implant, indicating that mouse Brainiac 1, likely a glycosyltransferase, is crucial for very early development of the mouse embryo.

The expression of the pre-T cell receptor α (pTa) gene occurs exclusively in immature T lymphocytes and is regulated by poorly defined mechanisms. We have analyzed the role of the upstream enhancer in pTa expression using conventional and bacterial artificial chromosome (BAC) reporter transgenes. The deletion of the enhancer completely abolished the expression of pTa BAC reporter in transgenic mice. Conversely, the combination of pTa enhancer and promoter targeted transgenes specifically to immature thymocytes, recapitulating the expression pattern of pTa. The core enhancer is conserved between mice and humans and contains a critical binding site for the transcription factor c-Myb. We also show that pTa promoter contains a conserved tandem E box site activated by E protein, HEB. These data establish the enhancer as a critical element regulating pTa gene expression and identify additional targets for c-Myb and E proteins in T cell development.

Cell cycle checkpoints are surveillance mechanisms that monitor the cell cycle and protect genome integrity by inducing cell cycle arrest or programmed cell death in response to DNA damage, mitotic spindle defects, or errors in the ordering of cell cycle events (Hartwell and Weinert 1989; Elledge 1996). Among these regulatory pathways are the DNA replication checkpoint, which couples mitosis to the completion of DNA replication, and the DNA damage checkpoint, which promotes the repair of DNA lesions before DNA replication or cell division. In addition to overseeing progression of the mitotic cell cycle, checkpoint proteins also act to maintain telomeres (Dahlen et al. 1998; Ahmed and Hodgkin 2000), to monitor meiosis (for review, see Page and Orr-Weaver 1997), and to regulate dNTP biosynthesis and maintain the cellular capacity for DNA synthesis (Desany et al. 1998; Zhao et al. 1998). Defects in checkpoint pathways can result in cell death or genomic instability. In mammals, genomic instability is thought to promote tumorigenesis (Hartwell and Kastan 1994; Lengauer et al. 1998), and mutations in the checkpoint genes p53 (for review, see Levine 1997), ATM (for review, see Rotman and Shiloh 1999), and hCHK2 (Bell et al. 1999) are associated with familial cancer syndromes. In addition, cellular responses to anticancer drugs, many of which act by damaging DNA or inhibiting DNA replication, may depend largely on the activity of cell cycle checkpoints (Hartwell and Kastan 1994). Recent studies indicate that several features of cell cycle checkpoints are conserved throughout evolution. As a result, knowledge of cell cycle checkpoints in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe provides a framework for developing a further understanding of checkpoint pathways in higher eukaryotes. In fission yeast six non-essential genes (hus1+, rad1+, rad3+, rad9+, rad17+, and rad26+), known as the checkpoint rad genes, are required for both the DNA replication and DNA damage checkpoints (Al-Khodairy and Carr 1992; Enoch et al. 1992; Rowley et al. 1992; Al-Khodairy et al. 1994). With the possible exception of rad26+, each of the checkpoint rad genes is represented in mammals by a closely related homolog (for review, see Caspari and Carr 1999; Dasika et al. 1999). Evidence that this sequence homology reflects conserved protein function is provided by the rad3+-related gene ATM, which encodes a bona fide mammalian checkpoint protein. In addition to the checkpoint rad genes, other fission yeast genes that function in checkpoint responses, such as chk1+ and cds1+, also appear to be retained in higher eukaryotes (for review, see Caspari and Carr 1999; Dasika et al. 1999). Taken together, these evolutionarily-conserved checkpoint genes may represent a core checkpoint apparatus, analysis of which will likely provide considerable insight into mammalian DNA damage responses. Fission yeast hus1+ (hydroxyurea sensitive) was originally identified in a screen for genes that, when inactivated, conferred sensitivity to the DNA replication inhibitor hydroxyurea (HU) (Enoch et al. 1992). Fission yeast lacking hus1+ appear normal under standard growth conditions, but fail to arrest the cell cycle after DNA damage or blockage of DNA synthesis (Enoch et al. 1992; Kostrub et al. 1997). Instead, hus1− strains proceed into mitosis with damaged or incompletely replicated genomes. In addition, hus1+ plays a role in the poorly understood process of recovery from S-phase arrest, as HU-treated hus1− yeast become irreversibly damaged during interphase, before undergoing premature cell division (Enoch et al. 1992). Although the Hus1p polypeptide lacks domains that might indicate its biochemical function, sequence analyses suggest that Hus1p may be structurally similar to the DNA sliding clamp protein PCNA, which forms a trimer that encircles DNA (Aravind et al. 1999; Caspari et al. 2000). Hus1p is phosphorylated after DNA damage, and this modification requires the other checkpoint rad proteins, including the ATM-related kinase Rad3p (Kostrub et al. 1998). In addition, Hus1p physically associates with two other checkpoint polypeptides, Rad1p and Rad9p (Kostrub et al. 1998; Caspari et al. 2000). Homologs of hus1+ have been identified in a number of higher eukaryotes, including Mus musculus and Homo sapiens (Dean et al. 1998; Kostrub et al. 1998). The mammalian Hus1 proteins are similar in length to Hus1p and share ∼30% amino acid identity with the S. pombe protein. The notion that these homologs are functionally equivalent to their fission yeast counterpart is strengthened by the finding that the human HUS1 protein interacts with human RAD1 and RAD9 polypeptides (St. Onge et al. 1999; Volkmer and Karnitz 1999). Mouse Hus1 is a single copy gene located at the proximal end of chromosome 11 (Weiss et al. 1999). It is expressed throughout embryonic development and displays a ubiquitous expression pattern in adult tissues. To determine the role of Hus1 in murine development and cell cycle checkpoint function, we produced a targeted disruption of Hus1. Here, we report that Hus1 inactivation causes mid-gestational embryonic lethality. Hus1-null embryos show transcriptional induction of DNA damage-responsive genes and yield cells that contain increased spontaneous chromosomal abnormalities, suggesting that Hus1 is required for the maintenance of genomic stability. In addition, analysis of Hus1-deficient fibroblast cultures indicates that mouse Hus1, like S. pombe hus1+, has a role in cellular responses to genotoxic stress.

The susceptibility of the mammary gland to carcinogenesis is influenced by its normal development, particularly during developmental stages such as puberty and pregnancy that are characterized by marked changes in proliferation and differentiation. Protein kinases are important regulators of proliferation and differentiation, as well as of neoplastic transformation, in a wide array of tissues, including the breast. Using a RT-PCR-based cloning strategy, we have identified 41 protein kinases that are expressed in breast cancer cell lines and in the murine mammary gland during development. The expression of each of these kinases was analyzed throughout postnatal mammary gland development as well as in a panel of mammary epithelial cell lines derived from distinct transgenic models of breast cancer. Although the majority of protein kinases isolated in this screen have no currently recognized role in mammary development, most kinases examined were found to exhibit developmental regulation. After kinases were clustered on the basis of similarities in their temporal expression profiles during mammary development, multiple distinct patterns of expression were observed. Analysis of these patterns revealed an ordered set of expression profiles in which successive waves of kinase expression occur during development. Interestingly, several protein kinases whose expression has previously been reported to be restricted to tissues other than the mammary gland were isolated in this screen and found to be expressed in the mammary gland. In aggregate, these findings suggest that the array of kinases participating in the regulation of normal mammary development is considerably broader than currently appreciated.

We recently characterized a genomic region located upstream of the mouse pre-T cell receptor α ( pTa ) gene, which controls pTa expression in pre-T cells. We now report an unexpected homology between this region and a region in the mouse X chromosome inactivation center between the 3′ end of the Xist gene and the start of an antisense transcript Tsix. The homology is extended over 4 kb of genomic sequence split by an expanded repeat region and is observed only in the mouse, not in the rat. Despite high sequence similarity to the pTa transcriptional enhancer, the homologous X chromosome fragment appears to have lost its enhancer activity. These data underscore the complex organization of the mouse genome and, in particular, of the X chromosome inactivation center.

ErbB-2 is a critical oncogenic marker in human breast cancer. Its appearance correlates with poor prognosis and it is, therefore, an important target for physiologic investigation and therapeutic intervention. With this in mind, we have created and characterized two mouse breast cancer models that express rat wild type neu, the homologue of ErbB-2, and rat mutant neu under the control of the normal mouse neu promoter. These models in which the copy number of the neu gene is moderately amplified should more closely parallel the expression pattern of ErbB-2 seen in some cases of human breast cancer. Transgenic mouse models were constructed by injecting one of the two pronuclei of a fertilized FVB/n egg and implanting it into a pseudopregnant Swiss/Webster mouse. Tissue expression was analyzed through the use of reverse transcription polymerase chain reaction and mammary histopathology examined by fixing, staining and mounting of the entire gland. In the former wild type model, we show that low level, long term expression of neu leads to abnormal lobuloalveolar development in virginal glands and incomplete regression in multiparous glands. Malignant foci form following multiple rounds of pregnancy and regression. In the latter model, a similarly directed transgene carrying the constitutively activated, mutant form of the rat neu gene, a stronger but similar phenotype is displayed. Evidently minor perturbations in amplified neu expression are sufficient to alter mammary development and induce malignant transformation.

The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments. Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx's physiologic function remains unknown. To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice. Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo.

Cell cycle checkpoints are regulatory mechanisms that arrest the cell cycle or initiate programmed cell death when critical events such as DNA replication fail to be completed or when DNA or spindle damage occurs. In fission yeast, cell cycle checkpoint responses to DNA replication blocks and DNA damage require the hus1 + gene. Mammalian homologs of hus1 + were recently identified, and here we report a detailed analysis of mouse Hus1. An approximately 4.2-kb full-length cDNA encoding the 32-kDa mouse Hus1 protein was isolated. The genomic structure and exon–intron boundary sequences of the gene were determined, and mouse Hus1 was found to consist of nine exons. Mouse Hus1 was mapped to the proximal end of chromosome 11 and is therefore a candidate gene for the mouse mutation germ cell deficient, which maps to the same genomic region. Finally, mouse Hus1 was found to be expressed in a variety of adult tissues and at several stages of embryonic development.

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn -deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.

Neu differentiation factor (NDF, also called neuregulin) is a potent inducer of epithelial cell proliferation and has been shown to induce mammary carcinomas in transgenic mice. Notwithstanding this proliferative effect, we have shown that a novel isoform of NDF can induce apoptosis when overexpressed. Here we report that this property also extends to other NDF isoforms and that the cytoplasmic portion of NDF is largely responsible for the apoptotic effect, whereas the proliferative activity is likely to depend upon the secreted version of NDF. In accordance with these contradictory properties, we find that tumors induced by NDF display extensive apoptosis in vivo. NDF is therefore an oncogene whose deregulation can induce transformation as well as apoptosis.

Mutations in the mouse formin (Fmn) gene result in limb deformities and incompletely penetrant renal aplasia. A molecular genetic approach was taken to characterize novel circular RNAs from the Fmn gene and to understand the developmental effects of gene-targeted mutations. RT-PCR and ribonuclease protection analyses were done to characterize the circular RNA transcripts arising from the Fmn gene. Two lines of mice with gene-targeted deletions of specific Fmn exons, namely exon 4 or exon 5, were generated and analyzed. In our analysis of formin cDNAs, we discovered a class of transcripts in which the exon order is reversed such that downstream exons are joined to the acceptor end of a specific exon that lies 5′ to them in the genome. RT-PCR and ribonuclease protection analyses indicate that these transcripts are circular and are the major transcripts arising from this locus in adult brain and kidney. To gain insight into the biological function of these transcripts, we have systematically deleted the relevant exons using gene-targeted homologous recombination. The resulting mice fail to produce circular transcripts, but appear to produce normal amounts of the linear RNA isoforms from this locus. While these deficient mice have normal limbs, they display variably penetrant renal aplasia characteristic of other mutant formin alleles. Our results demonstrate novel circular transcripts arising from the Fmn gene. Moreover, their high levels of expression suggest that they are not products of aberrant splicing events, but instead, may play important biological roles. Mice with gene-targeted deletions of Fmn exons 4 or 5 lack these circular transcripts and have an incompletely penetrant renal agenesis phenotype. While the biologic function of circular Fmn RNA transcripts is not entirely known, our work suggests their possible involvement in kidney development.

The death domain serine/threonine kinase RIP interacts with the death receptors Fas and tumor necrosis receptor 1 (TNFR1). In vitro, RIP stimulates apoptosis, SAPK/JNK, and NF-kappaB activation. To define the physiologic role(s) that RIP plays in regulating apoptosis in vivo, we introduced a rip null mutation in mice through homologous recombination. RIP-deficient mice appear normal at birth but fail to thrive, displaying extensive apoptosis in both the lymphoid and adipose tissue and dying at 1-3 days of age. In contrast to a normal thymic anti-Fas response, rip-/- cells are highly sensitive to TNFalpha-induced cell death. Sensitivity to TNFalpha-mediated cell death in rip-/- cells is accompanied by a failure to activate the transcription factor NF-kappaB.

We have assessed five signal transduction pathways to determine the role each might play in the malignant transformation of mammary epithelium initiated by neu, heregulin/NDF, TGFalpha, v-Ha-ras and c-myc in transgenic mice. The study involves a molecular and pharmacologic assessment of Erk/MAP kinase, Jnk/SAP kinase, PI 3-kinase, protein kinase C, and the Src-related kinases Lck and Fyn. Our results indicate that oncogenes capable of transforming mammary gland epithelium activate and require specific signal transduction pathways. For example, mammary tumors initiated by neu, v-Ha-ras and c-myc have high levels of active Erk/MAP kinase and their anchorage independent growth is strongly inhibited by PD098059, an inhibitor of Mek/ MAP kinase kinase. By contrast, Erk/MAP kinase activity is weak in tumors initiated by TFGalpha and heregulin/NDF and the corresponding cell lines are not growth inhibited by PD098059. Similarly, PI 3-kinase is strongly activated in neu, TGFalpha and heregulin/NDF initiated tumor cell lines, but not in c-myc or v-Ha-ras initiated tumor cell lines. The anchorage independent growth of all these tumor cell lines are, however, inhibited by the specific PI 3-kinase inhibitor LY294001. Further illustrating this oncogene-based specificity, PP1, a specific inhibitor of the Src-like kinases, Lck and Fyn, blocks anchorage-independent cell growth only in the TGFalpha initiated mammary tumor cell line. Taken together with additional observations, we conclude that certain oncogenes reliably require the recruitment/activation of specific signal transduction pathways. Such specific relationships between the initiating oncogene and a required pathway may reflect a direct activating effect or the parallel activation of a pathway that is a necessary oncogenic collaborator for transformation in the mammary gland. The work points to a molecular basis for targeting therapy when an initiating oncogene can be implicated; for example, because of amplification, increased expression, genetic alteration, or heritable characteristics.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm -deficient mice recapitulate most of the AT phenotype. p21 cip1/waf1 (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm -deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21 . Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.

Wnt-10b is expressed during the formation of the mammary rudiment in mouse embryos and its expression continues through puberty when the mammary ductal pattern is established under control of ovarian steroids. Recently, viral activation of the Wnt-10b locus has linked its overexpression to mammary tumor formation, suggesting a role for Wnt-10b in patterning and growth-regulation of the mammary gland. To test this notion, we created lines of transgenic mice that express elevated levels of Wnt-10b under the control of the MMTV promoter/enhancer. Overexpression of this gene resulted in profound developmental alterations in the mammary gland, including expanded glandular development and the precocious appearance of alveoli in virgin females. Moreover, transgenic male mice also exhibited dramatic mammary development involving highly branched mammary ducts and gynecomastia. Aberrant expression of Wnt-10b in the mammary rudiments of males evidently bypasses the normal requirement for ovarian hormonal control in stimulating mammary ductal growth and the repressive effects of androgens. In addition to these developmental effects, transgenic mice of both sexes were highly susceptible to the development of mammary adenocarcinomas. Such tumors arose in a solitary manner indicating that Wnt-10b is a proto-oncogene which provides a necessary, but insufficient signal for oncogenesis. Relevant to this, there was no evidence of amplified expression of FGF mRNAs in these tumors though the Fgf's are a class of genes often implicated as collaborators in Wnt-mediated tumor formation. Indeed, co-expression of MMTV-Wnt-10b and MMTV-FGF-3/int-2 resulted in sterile offspring with highly disorganized mammary epithelium, demonstrating a potent interaction between their respective developmental pathways. These results suggest that Wnt-10b, or other Wnt genes expressed early in mammary development, play a role in regulating sexual dimorphism and show potent transforming activity when overexpressed.

A classical notion regarding the expression of murine embryonic zeta- and adult alpha-globin genes holds that there is a switch in globin production from the embryonic to the adult form during fetal development. Our previous in situ hybridization studies challenged this view, since both zeta- and alpha-globin mRNAs can be detected simultaneously in the earliest erythrocyte populations. This finding raises the possibility that zeta-globin production might be wholly or partially redundant in embryos in which the adult alpha-globin is also expressed. To test this possibility, we created a null mutation of the zeta-globin gene using homologous recombination in embryonic stem cells. Many outbred mice homozygous for the zeta-null mutation were able to develop normally, undermining the notion that there is an absolute need for zeta-globin and indicating that alpha-globin alone can serve the survival needs of the fetus. Interestingly, insertion of the PGK-Neo cassette (used to create the null mutation) into the zeta-globin gene appears to influence the expression of the nearby alpha-globin genes, giving rise to reduced alpha-globin production and to an alpha-thalassemia-like syndrome. There is also evidence indicating the strong influence of genetic background on the zeta-null and alpha1-null phenotypes, both of which are much more severe in the 129/SvEv inbred genetic background. These quantitative differences can potentially be exploited to identify genes important for erythropoiesis.

A classical notion regarding the expression of murine embryonic ζ- and adult α-globin genes holds that there is a switch in globin production from the embryonic to the adult form during fetal development. Our previous in situ hybridization studies challenged this view, since both ζ- and α-globin mRNAs can be detected simultaneously in the earliest erythrocyte populations. This finding raises the possibility that ζ-globin production might be wholly or partially redundant in embryos in which the adult α-globin is also expressed. To test this possibility, we created a null mutation of the ζ-globin gene using homologous recombination in embryonic stem cells. Many outbred mice homozygous for the ζ-null mutation were able to develop normally, undermining the notion that there is an absolute need for ζ-globin and indicating that α-globin alone can serve the survival needs of the fetus. Interestingly, insertion of the PGK-Neo cassette (used to create the null mutation) into the ζ-globin gene appears to influence the expression of the nearby α-globin genes, giving rise to reduced α-globin production and to an α-thalassemia–like syndrome. There is also evidence indicating the strong influence of genetic background on the ζ-null and α1-null phenotypes, both of which are much more severe in the 129/SvEv inbred genetic background. These quantitative differences can potentially be exploited to identify genes important for erythropoiesis.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase [1]; this generally requires 1–2 months of work for each construct. The single ‘knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector. This approach leads to inactivation of the gene of interest [2]. Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing [3]. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. Our approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs [4–8], simplifying this technically demanding method.

Mutations of the murine limb deformity (ld) locus are responsible for a pleiotropic phenotype of completely penetrant limb malformations and incompletely penetrant renal agenesis and/or dysgenesis. The ld locus encodes a complex family of mRNA and protein isoforms. To examine the role of one of the more prominent of these isoforms, isoform IV, we specifically eliminated it by gene targeting. Unlike other mutant ld mice, homozygous mice bearing this isoform IV disruption display incompletely penetrant renal agenesis, but have perfectly normal limbs. Whole mount in situ hybridization demonstrated that this targeted disruption was specific for isoform IV and did not interfere with the expression of other ld isoforms. The isoform IV-disrupted allele of ld does not complement the renal agenesis phenotype of other ld alleles, in a manner consistent with its penetrance, and like the isoform IV-deficient mice, these compound heterozygotes have normal limbs. Sequence analysis of formin isoform IV in other ld mutant alleles did not detect any amino acid changes relative to the strain of origin of the mutant allele. Thus, the disruption of isoform IV is sufficient for the renal agenesis phenotype, but not the limb phenotype of ld mutant mice. Structural mutations in this isoform are only one of several genetic mechanisms leading to the renal phenotype, since amino acid changes in this isoform were not detected. These results demonstrate that this gene is limb deformity, and that variable isoform expression may play a role in generating the pleiotropic ld phenotype.

Fibroblast growth factor receptor-1 (FGFR-1) is a membrane-spanning tyrosine kinase that serves as a high-affinity receptor for fibroblast growth factors. It has recently been shown that FGFR-1 mutant embryos die during gastrulation displaying severe growth retardation and defective mesodermal structures. This early lethality has obscured functions of FGFR-1 that might occur later in development. To circumvent these embryonic defects, we generated chimeras by injecting FGFR-1-deficient (R1-/-) ES cells into wild-type blastocysts. We found that the fgfr-1 gene plays an important role after gastrulation and that it acts in a cell-autonomous fashion. Embryos with a high contribution of R1-/- cells replicate the FGFR-1 null phenotype and die during gastrulation. In contrast, the majority of embryos with a low contribution of R1-/- cells complete gastrulation and display malformations of posterior structures at later stages of embryogenesis. These abnormalities include truncation of embryonic structures, limb bud malformation, partial duplication of the neural tube, tail distortion, and spina bifida caused by the amplification of neural tissue in the posterior portion of the spinal cord. Thus, FGFR-1 plays a role in neurulation, suggesting that there may be a connection between FGFR-1-mediated signal pathways and neural tube defects, the most common malformations in the human central nervous system.

The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and have recently been shown to inhibit human immunodeficiency virus entry into cells. Eotaxin is a C–C chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders and, unlike all other eosinophil chemoattractants, is eosinophil specific. However, given the large number of chemoattractants that have activities on eosinophils, it is unclear whether eotaxin has an important role in vivo. Furthermore, it remains unclear why there is constitutive expression of eotaxin in healthy states in the absence of eosinophilic inflammation. To begin to determine the significance of eotaxin at baseline and during eosinophil-mediated disease processes, we have used targeted gene disruption to generate mice that are deficient in eotaxin. Such mice demonstrate that eotaxin enhances the magnitude of the early (but not late) eosinophil recruitment after antigen challenge in models of asthma and stromal keratitis. Surprisingly, a role for eotaxin in regulating the constitutive number of eosinophils in the peripheral circulation is also demonstrated. These results indicate a contributory role for eotaxin in the generation of peripheral blood and antigen-induced tissue eosinophilia.

We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm-deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm-deficient mice then exhibit many of the same symptoms found in ataxia-telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.

Ectopic activation of the TAL-1 gene in T lymphocytes occurs in the majority of cases of human T cell acute lymphoblastic leukemia (T-ALL), yet experiments to date have failed to demonstrate a direct transforming capability for tal-1. The tal-1 gene product is a serine phosphoprotein and basic helix-loop-helix (bHLH) transcription factor known to regulate embryonic hematopoiesis. We have established a transgenic mouse model in which tal-1 mis-expression in the thymus results in the development of clonal T cell lymphoblastic leukemia/lymphoma. Thus, overexpression of tal-1 alone can be transforming, verifying its pathogenic role in human T-ALL. In addition, leukemogenesis is accelerated dramatically by transgenic co-expression of tal-1 and the catalytic subunit of casein kinase IIalpha (CKIIalpha), a serine/threonine protein kinase known to modulate the activity of other bHLH transcription factors. Although tal-1 is a substrate for CKII, the synergy of the tal-1 and CKIIalpha transgenes appears to be indirect, perhaps mediated through the E protein heterodimeric partners of tal-1. These studies prove that dysregulated tal-1 is oncogenic, providing a direct molecular explanation for the malignancies associated with TAL-1 activation in human T-ALL.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

The murine limb deformity (ld) locus encodes a set of proteins, termed formins, that are required for embryonic limb and kidney development. Previous studies had indicated that these proteins are located in the nucleus and cytoplasm and have biochemical properties consistent with an action within the nucleus. To test the notion that nuclear localization is crucial for formin function, we carried out molecular and biochemical studies on three ld alleles. We show that two transgene-induced alleles, ldTgHd and ldTgBri, generate similar COOH-truncated formins that lack the terminal 110 amino acids, while a third allele, ldIn2, generates a less extensively truncated formin that lacks the terminal 42 amino acids. Using subcellular fractionation analysis, we find that wild-type formin is detected in both nuclear and cytosolic fractions; in contrast, the truncated formins encoded by ldTgHd and ldTgBri are strictly cytosolic. The less extensively truncated ldIn2 formin shows a similar, but less complete, localization defect. Consistent with this weaker cellular phenotype, hind limbs from ldIn2 mice have milder skeletal defects than those of ldTgBri mice. These observations define a small region in the carboxyl terminus that is required for nuclear localization and suggest that nuclear localization plays a role in formin action.

Understanding the processes that control selective eosinophilia is of fundamental importance in a variety of human diseases (e.g., allergies, parasitic infections, malignancy). Interleukin 5, an eosinophil-specific growth and activating factor, and eotaxin appear to collaborate in this process. Eotaxin is a recently described chemotactic factor that belongs to the C-C (or β) chemokine family and has been implicated in animal and human eosinophilic inflammatory states. We have recently reported the molecular characterization of murine eotaxin and now report the biological properties of purified recombinant murine eotaxin in vitro and in vivo in the presence or absence of interleukin 5 (IL-5) in mice. Murine eotaxin was expressed in bacteria and purified by affinity chromatography and HPLC. Activity was tested in vitro by examining chemotactic and calcium flux responses of purified murine leukocytes. Additionally, desensitization of calcium flux responses to other chemokines, eosinophil survival assays, and basophil histamine release were examined. Finally, eotaxin was delivered to wild-type or IL-5 transgenic mice and the host response was examined. Eotaxin had activity only when the recombinant molecule had the native mature amino terminus and contained the first 25 amino acids of the mature protein. It was active in vitro at an effective concentration between 10 and 100 ng/ml in both Chemotaxis and calcium flux assays toward eosinophils, but not macrophages or neutrophils. Furthermore, intranasal or subcutaneous application of eotaxin selectively recruited large numbers of eosinophils into the mouse lung and skin, respectively, only in the presence of interleukin 5. Macrophage inflammatory protein-1α, a related C-C chemokine active on eosinophils, and eotaxin were not able to cross-desensitize. Eotaxin had no affect on the in vitro survival of eosinophils and did not induce basophil histamine release. Mouse eotaxin is an eosinophil specific chemoattractant that has a markedly enhanced effect in vivo in the presence of another eosinophil selective cytokine IL-5, and utilizes a signal transduction receptor pathway that is distinct from that utilized by macrophage inflammatory protein-1α. This data suggests that the development of tissue eosinophilia in vivo involves a two-step mechanism elicited by interleukin 5 and eotaxin.

Eotaxin is an eosinophil–specific chemoattractant that has been recently identified in rodent models of asthma and host response against tumors. To determine whether a similar molecule might play a role in human inflammatory diseases characterized by eosinophilia, we isolated the human eotaxin gene. We demonstrate that human eotaxin is an early response gene of cytokine–stimulated epithelial and endothelial cells, and is induced in peripheral blood eosinophils by interleukin–3. Eotaxin is directly chemotactic for eosinophils, but not mononuclear cells or neutrophils. Eotaxin messenger RNA accumulates markedly in the lesions of patients with inflammatory bowel disease (ulcerative colitis and Crohn's disease), but not in the lesions of patients with diverticulitis. These results now provide a mechanism involving eotaxin to explain the eosinophil infiltration seen in a variety of human diseases; as such, an eotaxin antagonist may be a novel therapy for certain human diseases characterized by tissue eosinophilia.

The formins, proteins involved in murine limb and kidney development, contain a proline-rich region that matches consensus sequences for Src homology 3 (SH3) ligands. To identify proteins that interact with formins, we used this proline-rich region to screen mouse limb bud expression libraries for formin binding proteins (FBPs). As expected, we found one class of FBPs that contains SH3 domains, including two novel members of this class. In addition, however, we also found a novel class of FBPs that contains one or two copies of a 26 amino acid homology region that has been recently termed the WWP or WW motif. We demonstrate that WWP/WW domains as short as 26 amino acids can act as modular protein-binding interfaces that bind with high affinity to proline-rich sequences that are similar and, in some cases, identical to SH3 ligands. Furthermore, we find that the WWP/WW domain can compete with the Abl SH3 domain in binding a proline-rich peptide present in formin. Our results suggest that these novel protein interaction domains can perform functions similar to those of SH3 domains and, thus, might regulate SH3 interactions with target proteins through competitive binding.

The study of limb development has provided insight into pattern formation during vertebrate embryogenesis. Genetic approaches offer powerful ways to identify the critical molecules and their pathways of action required to execute a complex morphogenetic program. We have applied genetic analysis to the process of limb development by studying two mouse mutants, limb deformity (ld) and Strong's luxoid (lst). These mutations confer contrasting phenotypic alterations to the anteroposterior limb pattern. The six mutant ld alleles are fully recessive and result in oligosyndactyly of all four limbs. By contrast, the two mutant lst alleles result in a mirror-image polydactylous limb phenotype inherited in a semidominant fashion. Morphological and molecular analysis of embryonic limbs has shown that the ld and lst alleles affect the extent and distribution of two key signaling centers differentially: the apical ectodermal ridge and the zone of polarizing activity. Molecular characterization of the ld gene has defined a new family of evolutionarily conserved proteins termed the formins. The underlying molecular defect in the lst mutation has not been identified; however, both loci are tightly linked on mouse chromosome 2, suggesting the possibility that they may be allelic. In this study, we have used genetic analysis to examine the epistatic and allelic relationships of ld and lst. We observed that in + ld/lst + double heterozygotes, a single mutant ld allele is able to suppress the semi-dominant polydactylous lst limb phenotype. By segregating the lst and ld loci in a backcross, we observed that these loci recombine and are separated by a genetic distance of approximately 6 cM. Therefore, while our observations demonstrate a genetic interaction between ld and lst, it is probable that ld and lst are not allelic. Instead, lst and ld may be operating either in a linear or in a parallel (bypass) genetic pathway to affect the limb signaling centers.

Transgenic mice that overexpress v-Ha-ras, c-myc, c-neu or int-2 proto-oncogenes in the mammary epithelium develop breast tumors with morphologies that are characteristic of each initiating oncogene. Since these morphological differences reflect distinctive patterns of tumor-specific gene expression, the identification of the products of these genes might shed light on the mechanisms of transformation and/or the identity of target cells that are transformed by specific classes of oncogenes. By focusing on the tyrosine phosphorylation pathway, we have found that the transmembranal protein-tyrosine phosphatase epsilon (PTP epsilon) is highly expressed in murine mammary tumors initiated by c-neu and v-Haras, but not in mammary tumors initiated by c-myc or int-2. This difference is striking and occurs both in primary tumors and in epithelial cells cultured from them. Moreover, PTP epsilon overexpression appears to be mammary tumor-specific in that it is not found in other ras-based tumors and cell lines. These observations suggest that PTP epsilon either plays a role in ras- and neu-mediated transformation of mammary epithelium or marks mammary epithelial cells particularly susceptible to transformation by these oncogenes. Because of its distinctive expression in these mammary tumors, we have further characterized murine PTP epsilon, cloning and determining the complete structures of its cDNAs and showing that it is a glycoprotein that is N-glycosylated in a tissue-specific manner.

We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812-amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.

Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects. The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects, usually aplasia, are thought to result from failure of ureteric bud outgrowth. Since the ld locus gives rise to multiple RNA isoforms encoding several different proteins (termed formins), we wished to understand their role in the formation of these organs. Therefore, we first examined the embryonic expression patterns of the four major ld mRNA isoforms. Isoforms I, II and III (all con-taining a basic amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney including the ureteric bud. Isoform IV (containing an acidic amino terminus) is expressed in the notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney including the ureteric bud. Using a lacZ reporter assay in transgenic mice, we show that this differential expression of isoform IV results from distinct regulatory sequences upstream of its first exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud outgrowth, while isoform IV may be involved in AER differentiation. To define further the developmental consequences of the ld limb defect, we analyzed the expression of a number of genes thought to play a role in limb development. Most signifi-cantly, we find that although the AERs of ld limb buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4 (fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus we conclude that one or more formins are necessary to initiate and/or maintain fgf-4 production in the distal limb. Since ld limbs form distal structures such as digits, we further conclude that while fgf-4 is capable of support-ing distal limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development. In addition, ld limbs show a severe decrease in the expression of several mesodermal markers, including sonic hedgehog (shh), a marker for the polarizing region and Hoxd-12, a marker for posterior mesoderm. We propose that incomplete differentiation of the AER in ld limb buds leads to reduction of polarizing activity and defects along the anteroposterior axis.

Parental genomic imprinting refers to the phenomenon by which alleles behave differently depending on the sex of the parent from which they are inherited. In the case of the murine transgene RSVIgmyc, imprinting is manifest in two ways: differential DNA methylation and differential expression. In inbred FVB/N mice, a transgene inherited from a male parent is undermethylated and expressed; a transgene inherited from the female parent is overmethylated and silent. Using a series of RSVIgmyc constructs and transgenic mice, we show that the imprinting of this transgene requires a cis-acting signal that is principally derived from the repeat sequences that make up the 3' portion of the murine immunoglobulin alpha heavy-chain switch region. Such imprinting is relatively independent of the site of transgene insertion but is influenced by the structure of the transgene itself. Imprinting is also modulated by genetic background. Detailed studies indicate that the paternal allele is undermethylated and expressed in inbred FVB/N mice and in heterozygous F1 FVB/N/C57Bl/6J mice but is overmethylated and silent in inbred C57Bl/6J mice. Consequently, the FVB/N genome appears to carry alleles of modulating genes that dominantly block methylation and permit expression of the paternally imprinted transgene. Furthermore, our results suggest that overmethylation is the default status of both parental alleles and that the paternal allele can be marked in trans by polymorphic factors that act in postblastocyst embryos.

Transgenic mice harboring the int-2/Fgf-3 protooncogene under transcriptional control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit a dramatic, benign hyperplasia of the mammary gland. In one int-2 transgenic line (TG.NX), this growth disturbance is evoked by pregnancy and regresses after parturition. Regression of hyperplastic mammary epithelium is less complete after successive pregnancies, and, within 10 months, most TG.NX mice stochastically develop mammary carcinomas that are transplantable in virgin, syngeneic mice. To identify genes that cooperate with int-2 in cell transformation, we infected TG.NX transgenic mice with MMTV. In a cohort of 14 animals, most mammary tumors represented clonal or oligoclonal outgrowths harboring one to five proviral MMTV integrants. Eight of 35 (23%) MMTV+ tumors exhibited proviral insertion at the Wnt-1 locus. No provirus was detected at the int-2, int-3, or Wnt-3 loci. By Southern analysis, two tumors had proviral insertions at the same genomic location, which was mapped to chromosome 15. Cloning of this int locus identified an additional member of the Wnt gene family. The predicted 389-amino acid protein is most closely related to zebrafish Wnt-10a (58% amino acid identity over 362 residues) and, based on homology analysis, was designated Wnt-10b. This newly discovered Wnt family member was expressed in the embryo and mammary gland of virgin but not pregnant mice and represents a candidate collaborating oncogene of int-2/Fgf-3 in the mammary epithelium.