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Biomedical subjects

M L Breitman

Publications and source records attributed to M L Breitman.

At least 19 recordsLinked to original sources
PubMed1998

A model for spontaneous B-lineage lymphomas in IgHmu-HOX11 transgenic mice.

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR alpha/delta regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHmu-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin's lymphoma of peripheral mature B cell origin.

Animals↗
PubMed1998

Characterization of lpd (lipid defect): a novel mutation on mouse chromosome 16 associated with a defect in triglyceride metabolism.

Recent epidemiological studies have identified plasma triglyceride as a risk factor for atherogenesis. We have generated a mouse transgenic line that carries a recessive mutation designated lpd (lipid defect). Homozygous lpd mice develop as runts and die by age 10-15 days with striking liver pathology characterized by the presence of numerous lipid-containing vacuoles and extensive accumulation of triglycerides. Cloning of the mutant insertion locus and the wild-type lpd locus have revealed a duplication of host genomic sequences at the site of integration. Mapping of the lpd locus with the Jackson Laboratory BSS interspecific backcross panel of (C57BL/6JEi x SPRET/Ei) F1 x SPRET/Ei placed the lpd locus to the distal part of chromosome 16. These observations suggest that the transgene disrupts a putative gene at the lpd locus and that lpd is a novel locus related to triglyceride metabolism. The lpd mutant mice may serve as models for human disorders of fatty livers or hypertriglyceridemia.

Animals↗
PubMed1998

The Tlx-2 homeobox gene is a downstream target of BMP signalling and is required for mouse mesoderm development.

TGFbeta-related factors are critical regulators of vertebrate mesoderm development. However, the signalling cascades required for their function during this developmental process are poorly defined. Tlx-2 is a homeobox gene expressed in the primitive streak of mouse embryos. Exogenous BMP-2 rapidly activates Tlx-2 expression in the epiblast of E6.5 embryos. A Tlx-2 promoter element responds to BMP-2 signals in P19 cells, and this response is mediated by BMP type I receptors and Smad1. These results suggest that Tlx-2 is a downstream target gene for BMP signalling in the primitive streak where BMP-4 and other TGFbeta-related factors are expressed. Furthermore, disruption of Tlx-2 function leads to early embryonic lethality. Similar to BMP4 and ALK3 mutants, the mutant embryos display severe defects in primitive streak and mesoderm formation. These experiments thus define a BMP/Tlx-2 signalling pathway that is required during early mammalian gastrulation.

Activin Receptors↗
PubMed1996

Expression of the bZIP transcription factor gene Nrl in the developing nervous system.

Proteins of the Maf/Nrl subfamily of bZIP transcription factors are involved in the regulation of tissue-specific gene expression. The Nrl gene, initially identified from a subtracted retinal library, is expressed in all cell layers of the adult retina, including photoreceptors. The Nrl protein has high sequence homology with Maf proteins, binds to an AP-1 like sequence element, and in photoreceptors appears to be involved in regulating the expression of rhodopsin. In the present study, we investigated the expression of Nrl in the developing and adult mouse using in situ hybridization and RT-PCR. We demonstrate that beginning at embryonic day 12.5 Nrl is expressed throughout the developing central and peripheral nervous system, with the exception of the nasal epithelium. The spatial pattern of hybridization suggests that Nrl is transcribed in post-mitotic, differentiating neurons, the developing cephalic mesenchyme and lens. Nrl expression is downregulated postnatally in the brain, and becomes restricted to neocortex and brainstem in the adult. High levels of Nrl transcripts, however, persist in the mature photoreceptors and other retinal neurons. Our studies suggest a role for the Nrl protein in neuronal differentiation and in mature neurons of the adult retina.

Animals↗
PubMed1996

Regulation of flt-1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium.

Flt-1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt-1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt-1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt-1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up-regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt-1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt-1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt-1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt-1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt-1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.

Animals↗
PubMed1995

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

Animals↗
PubMed1995

Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.

The vascular endothelial growth factor (VEGF) and its high-affinity binding receptors, the tyrosine kinases Flt-1 and Flk-1, are thought to be important for the development of embryonic vasculature. Here we report that Flt-1 is essential for the organization of embryonic vasculature, but is not essential for endothelial cell differentiation. Mouse embryos homozygous for a targeted mutation in the flt-1 locus, flt-1lcz, formed endothelial cells in both embryonic and extra-embryonic regions, but assembled these cells into abnormal vascular channels and died in utero at mid-somite stages. At earlier stages, the blood islands of flt-1lcz homozygotes were abnormal, with angioblasts in the interior as well as on the periphery. We suggest that the Flt-1 signalling pathway may regulate normal endothelial cell-cell or cell-matrix interactions during vascular development.

Animals↗
PubMed1995

The optimal binding sequence of the Hox11 protein contains a predicted recognition core motif.

HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. We determined the DNA binding specificity of the Hox11 protein by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contained a core TAAGTG motif that is consistent with a prediction based on the residues at specific positions that potentially make DNA base contacts and models of homeodomain-DNA interaction proposed from studies with other homeodomains. The specific interaction between Hox11 and the selected optimal binding sequence was further confirmed by band-shift and DNA competition assays. Given that the Hox11 homeodomain shares low homology with other well studied homeodomains, the presence of a predictable recognition core motif in its optimal binding sequence supports the notion that different homeodomains interact with DNA in a similar manner, through highly conserved residues at specific positions that allow contact with DNA.

Amino Acid Sequence↗
PubMed1995

Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development.

We report the detailed developmental expression profiles of three endothelial specific receptor tyrosine kinases (RTKs) flk-1, tek, tie, as well as vascular endothelial growth factor (VEGF), the flk-1 ligand. We also examined the expression of the other VEGF receptor, flt-1, during placental development. flk-1, tek, and tie transcripts were detected sequentially at one-half day intervals starting at E7.0, suggesting that each of these RTKs play a unique role during vascularization of the mouse embryo. All three RTKs were expressed in the extraembryonic and embryonic mesoderm in regions that eventually give rise to the vasculature. Except for the expression of tek and flk-1 in the mesoderm of the amnion, the expression of these RTKs from E8.5 onwards was virtually indistinguishable. An abundant amount of flt-1 transcripts was found in the spongiotrophoblast cells of the developing placenta from E8.0 onwards. This cellular compartment is located between the maternal and labyrinthine layers of the placenta, which both express VEGF. VEGF transcripts were detected as early as E7.0 in the endoderm juxtaposed to the flk-1 positive mesoderm, and later in development VEGF expression displayed an expression profile both contiguous with that of flk-1, and also in tissues found some distance from the flk-1-expressing endothelium. These results suggest a possible dual role for VEGF which includes a chemotactic and/or a cellular maintenance role for VEGF during vascularization of the mouse embryo.

Animals↗
PubMed1994

Genetic mapping of two mouse homeobox genes Tlx-1 and Tlx-2 to murine chromosomes 19 and 6.

We have previously cloned two mouse homeobox genes Tlx-1 (T-cell leukemia homeobox gene-1) and a related gene Tlx-2 based on their homology to human HOX11, a putative proto-oncogene involved in human T-cell leukemia. We have mapped Tlx-1 to mouse chromosome 19 and Tlx-2 to chromosome 6 by linkage analysis using an interspecific backcross (C57BL/6J x Mus spretus) F1 x M. spretus. The proposed gene orders and genetic distances for Tlx-1 and Tlx-2 are centromere 19-Lpc-1-(25.53 cM)-Pltr-4-(5.32 cM)-Tlx-1- (3.19 cM)-Ins-1-(7.45 cM)-Xmv-18, and centromere 6-Tcrb-(12.90 cM)-Mltr-3-(10.75 cM)-Tlx-2-(18.42 cM)-Xmv-6.

Animals↗
PubMed1994

Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.

The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null allele of the tek gene by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative alleles of Tek or homozygous for a null allele of the tek locus both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had approximately 30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo.

Animals↗
PubMed1994

Novel zinc finger proteins that interact with the mouse gamma F-crystallin promoter and are expressed in the sclerotome during early somitogenesis.

Lens-specific expression of the mouse gamma-F-crystallin gene is determined, at least in part, by a 23-bp DNA element, the gamma F-1-binding motif, located in the promoter region of the gene. To characterize the transcription factors that regulate gamma F-crystallin gene expression through this element, we have isolated three chicken cDNAs that encode proteins capable of binding specifically to the gamma F-1-binding motif. These three cDNAs represent differential splicing products from a single gene, gamma FBP. The protein isoforms encoded by two of these cDNAs differ in their ability to modulate the activity of promoters containing the gamma F-1-binding motif. Among them, gamma FBP-B functions as a transcriptional repressor in lens cells, and it's expression is developmentally regulated during lens development, suggesting a role for this isoform in the spatial regulation of gamma F-crystallin gene expression. We also show that expression of the different mRNA transcripts are differentially regulated in various tissues. Furthermore, gamma FBP transcripts are highly expressed in presomitic mesoderm and then over the entire epithelial somite. During somitic differentiation, gamma FBP expression becomes restricted to the sclerotome. These expression patterns suggest a regulatory role for the gamma FBP isoforms in sclerotome specification and/or differentiation.

Alternative Splicing↗
PubMed1994

Cone cells fail to develop normally in transgenic mice showing ablation of rod photoreceptor cells.

Transgenic mice were derived containing the cytotoxic dt-alpha gene driven by opsin promoter sequences. Mice expressing this construct showed progressive degeneration of rod photoreceptor cells commencing at birth, with obvious depletion of such cells by postnatal day 7. Ablation of rod photoreceptor cells in the transgenic retina was accompanied by the failure of developing cone cells to elaborate outer segments, although all other aspects of cone cell cytodifferentiation appeared normal. The results suggest that the 1.0-kb opsin promoter segment contains rod cell type specificity and that cone cells require maturation of rod cells to complete the late stages of their terminal differentiation and for their maintenance and cellular integrity.

Animals↗
PubMed1994

Two distinct target cells for v-jun mediated wound tumorigenesis.

Transgenic mice expressing v-jun under the control of the H-2K promoter develop dermal fibrosarcomas and rhabdomyosarcomas via a multistep process following wounding. To assess the relative roles that wounding and the H-2K promoter play in this process, we compared the phenotype of H-2K-v-jun mice with that of animals expressing v-jun under the control of the metallothionein I (MTI) promoter. MT-v-jun animals also develop wound-induced neoplasms by a multistage process. Both early and late features of tumorigenesis in MT-v-jun mice are different, however, from what is observed in H-2K-v-jun animals. First, the acute hyperplastic response that is characteristic of H-2K-v-jun granulation tissue is not observed in MT-v-jun wounds. Second, the myogenic components that are readily detected in the majority of late stage H-2K neoplasms are never observed in their MT counterparts. Moreover, analysis of wound tumours arising in animals expressing both MT-v-jun and H-2K-v-jun reveals that the two transgenes are not expressed in identical malignant cell populations. These results imply that mesenchymal granulation tissue is heterogeneous in composition and that the different cellular phenotypes of MT-v-jun and H-2K-v-jun malignancies result from oncogenic activation of wound-derived cells which differ in their differentiation potential. Thus, whereas the wounding component of multistage tumorigenesis is attributable to the action of v-jun, the transcriptional regulatory elements which drive its expression determine the nature of the target cells which give rise to wound-induced neoplasms.

Animals↗
PubMed1993

Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in v-jun transgenic murine cells.

The oncogene jun encodes a transcription factor of the AP-1 family. In mice carrying viral jun (v-jun) as a transgene, wounding is a prerequisite for tumorigenesis, suggesting collaboration between the transgene and a wound-related event. To define possible candidates for this collaborative process, we examined the effect of several wound-related polypeptide growth factors on cells from transgenic mice. Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in embryo fibroblasts and tumor cell revertants from these mice. This effect was specific for the two cytokines and was restricted to cells from v-jun transgenic mice. Anchorage independence required the continued presence of the cytokines. Transfection of transgenic cells with a v-jun expression plasmid also induced anchorage independence and a tumorigenic phenotype in transgenic tumor cell revertants. However, there was no correlation between anchorage independence, expression of Jun, and AP-1 activity. These results suggest that while increased transgene expression can enhance the growth properties of v-jun transgenic cells, there exist other cytokine-dependent mechanisms that have a similar effect. Retinoic acid, dexamethasone, or forskolin inhibits induction of anchorage independence by tumor necrosis factor alpha, interleukin 1 alpha, and transfected v-jun. Although these agents affect both AP-1 transactivation potential and DNA binding in the transgenic cells, the changes are not correlated with the inhibition of growth.

Animals↗
PubMed1993

Developmental regulation and cell type-specific expression of the murine gamma F-crystallin gene is mediated through a lens-specific element containing the gamma F-1 binding site.

The mouse gamma F-crystallin gene, one of six differentially regulated members of the gamma-crystallin gene family, is expressed exclusively in central nuclear fiber cells of the adult lens. The expression of this gene is controlled through regulatory elements contained in two upstream enhancers and the proximal promoter. Here we show that while the upstream enhancers and the proximal promoter could each direct gene expression in fiber cells formed at early stages of lens growth and development, cooperation between these elements is required to achieve expression in fiber cells formed at later stages. Evidence is provided that cooperative interaction between these elements modulates gene expression by increasing promoter strength. We also show that sequences within the proximal promoter region that bind lens cell nuclear factor gamma F-1 are sufficient to elicit gene expression in central nuclear fiber cells of the adult lens.

Animals↗
PubMed1993

Characterization and developmental expression of Tlx-1, the murine homolog of HOX11.

HOX11, a human homeobox gene with putative oncogenic potential, was originally discovered at the chromosome 10 breakpoint in T-cell acute lymphoblastic leukemias bearing the chromosomal translocation t(10;14)(q24;q11). To provide insight into the possible roles of this gene in development, we isolated and characterized its murine homolog, Tlx-1, and examined its profile of expression. Tlx-1 transcripts are first detected at E8.5 in the surface ectoderm and central mesenchyme of the first branchial arch. This expression subsequently extends to the 2nd, 3rd, and 4th branchial arches, as well as the presumptive pharynx, as these structures develop. Between E12.5 and E15.5, the profile of Tlx-1 expression becomes more complex; expression is observed in the developing pancreas and salivary glands, as well as in several components of the nervous system, including the trigeminal, glossopharyngeal and vestibulocochlear ganglia, the spinal cord, and the curvature of the pons-medulla. In addition, expression is seen in the pinna and external auditory meatus of the outer ear, the tooth primordia, and specific cell populations of the mandible and tongue. These complex patterns of expression are consistent with multiple and varied roles for Tlx-1 in development and suggest that Tlx-1 marks, amongst other cell populations, structures derived from cranial neural crest cells and migratory paraxial mesoderm that arise at corresponding levels along the rostral-caudal axis of the developing embryo.

Amino Acid Sequence↗