Biomedical subjects

M L Breitman

Publications and source records attributed to M L Breitman.

At least 37 records · Page 2Linked to original sources

PubMed1993

An everted repeat mediates retinoic acid induction of the gamma F-crystallin gene: evidence of a direct role for retinoids in lens development.

The vertebrate lens is a classical system for examining mechanisms of tissue determination and differentiation, yet little is known about the signaling molecules controlling its development. Here, we report that retinoic acid (RA), a substance known for its teratogenic effects on the eye and as a natural endogenous morphogenetic agent, acts as a regulator of gene expression in the lens. We have identified a novel type of RA response element (RARE) within the lens-specific mouse gamma F-crystallin promoter, consisting of two (A/G)GGTCA motifs in an everted arrangement spaced by 8 nucleotides. This element (gamma F-RARE) mediates activation of the gamma F-crystallin promoter by ligand-activated endogenous lens cell RA receptors (RARs) and confers RA responsiveness when linked to a heterologous promoter. gamma F-RARE is bound in vitro by RAR/RXR heterodimers, and both receptors cooperate in vivo to trans-activate this element. These observations demonstrate a direct effect of RA on lens-specific gene expression and reveal a novel role for retinoids in the development and homeostasis of the mammalian eye.

Amino Acid Sequence↗

PubMed1993

The proximal promoter of the mouse arrestin gene directs gene expression in photoreceptor cells and contains an evolutionarily conserved retinal factor-binding site.

Regulatory sequences and nuclear factors governing tissue-restricted expression of the mouse arrestin gene were investigated. The results showed that while proximal promoter sequence positions -38 to +304 are sufficient to direct low levels of retina-specific gene expression, sequences extending upstream to position -209 support higher levels of expression in the retina, as well as detectable expression in the lens, pineal gland, and brain. Within the interval between positions -209 and -38, a broadly expressed nuclear factor, Bd, binds to sequences centered between positions -205 and -185, a region which contains two direct repeats of the hexamer, TGACCT. The proximal promoter binds three apparently retina-specific nuclear factors, Bp1, Bp2, and Bp3, through overlapping sequences centered between positions -25 and -15. Bp1 and Bp3 also recognize a closely related sequence found in the promoter regions of several other vertebrate photoreceptor-specific genes. Moreover, the consensus binding site for Bp1, designated PCE I, is identical to RCS I, an element known to play a critical role eliciting photoreceptor-specific gene expression in Drosophila melanogaster. The results suggest that PCE I and RCS I are functionally as well as structurally similar and that, despite marked differences in the fly and vertebrate visual systems, the transcriptional machinery involved in photoreceptor-specific gene expression has been strongly evolutionarily conserved.

Animals↗

PubMed1993

flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors.

We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximal-lateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.

Amino Acid Sequence↗

PubMed1993

The endothelial-specific receptor tyrosine kinase, tek, is a member of a new subfamily of receptors.

We have cloned a 4.2-kb murine cDNA encoding the Tek receptor tyrosine kinase (RTK), which is expressed in endothelial cells and their progenitors. The 1122-residue protein contains an extracellular domain comprising three fibronectin type III repeats fused to two immunoglobulin-like loops that are in turn separated by three epidermal growth factor-like repeats. The association of these different structural motifs and their characteristic arrangement in the Tek extracellular domain has been reported for only one other RTK, Tie, an endothelial-specific RTK of human origin. We show here that Tek and Tie are encoded by distinct genes and that, together, these receptors define a new subfamily of RTKs. In addition, we demonstrate that the tek cDNA, when introduced into COS cells, encodes a product of 140 kDa and that this protein and/or tek transcripts are detectable in highly vascularized embryonic tissues and in some, but not all, cell lines of endothelial origin.

Amino Acid Sequence↗

PubMed1993

Progressive and regressive fate of lens tumors correlates with subtle differences in transgene expression in gamma F-crystallin-SV40 T antigen transgenic mice.

Regulatory elements of the mouse gamma F-crystallin gene were used to derive transgenic mice expressing SV40 large T antigen in terminally differentiating fiber cells of the ocular lens. The resulting gamma F-crystallin-T antigen mice developed either malignant or regressive lens tumors in a strain-dependent fashion. Developmental and RNA analyses revealed that in both 'tumor-progressing' and 'tumor-regressing' mouse strains expression of the transgene blocked morphological differentiation of lens fibers without appreciably affecting gamma-crystallin gene expression, a marker of terminal lens fiber cell differentiation. Strain-dependent differences in tumorigenic outcome could be correlated with both subtle differences in transgene expression and the ability of tumor cells to escape from the normal confines of the lens. The results implicate the importance of cellular environment to malignant tumor development and provide insight into those features of normal lens ontogeny that may render the lens refractory to the development of spontaneous tumors.

Animals↗

PubMed1993

Altered growth and spontaneous transformation of cells cultured from v-jun transgenic mice recapitulate wound-induced multistage tumorigenesis.

H-2K/v-jun transgenic mice develop sarcomas at sites of wounding via a multistep process characterized by discrete pathological stages. To study this progression in vitro, cells from different stages of tumorigenesis were cultured and examined for their growth properties. The results show that whereas transgenic fibroblasts do not manifest enhanced proliferative potential in vivo in the absence of wounding, they do show obvious proliferative advantage relative to nontransgenic fibroblasts in vitro, including the capacity for indefinite growth. In addition, relative to nontransgenic fibroblasts, transgenic cells show altered sensitivity to platelet-derived growth factor and tumor necrosis factor alpha, both of which are known to be mobilized during wounding. No obvious differences in growth potential are observed between transgenic fibroblasts and cells cultured from wound-induced premalignant lesions, and confluent cultures of both cell populations give rise to spontaneous foci of transformed myogenic and nonmyogenic cells that resemble those of late-stage malignant wound sarcomas. Relative to transgenic fibroblast cultures, however, premalignant lesion cultures segregate transformed cells at a greater frequency and after shorter intervals of in vitro growth. The results suggest that wound-induced multistage tumorigenesis can be recapitulated in vitro and that cells cultured from different stages of tumorigenesis retain biological properties that reflect the pathological stage from which they are derived.

Animals↗

PubMed1992

Temporal regulation of six crystallin transcripts during mouse lens development.

Using the polymerase chain reaction (PCR) and RNA blot analysis, we have examined the differential expression patterns of the gamma-crystallins during lens development. Since only four of these genes had been previously characterized, the cDNAs for the remaining two genes, gamma C and gamma F, were isolated and sequenced. The steady-state mRNA profiles were then determined by RNA blot analysis of samples from embryonic stages to 180 days after birth, with gene-specific probes for gamma A, gamma B, gamma C, and gamma D, and a common probe for gamma E and gamma F. Due to the paucity of mismatches between the gamma E and gamma F-crystallin genes, the PCR technique was exploited to determine their relative abundance. The data showed that while all six gamma-crystallin genes were expressed in the embryonic lens, they were differentially regulated during development. At early stages, the levels of gamma B and gamma C mRNAs were found to be relatively low in comparison to those for gamma A, gamma D, gamma E and gamma F. After 30-40 days, however, the levels of gamma A, gamma E, and gamma F mRNAs declined rapidly, and the gamma B, gamma C and gamma D transcripts became the major gamma-crystallin mRNA species. The utility of the PCR technique in studying the relative abundance of steady-state gamma-crystallin mRNAs was also investigated.

Animals↗

PubMed1992

A frameshift mutation in the gamma E-crystallin gene of the Elo mouse.

The murine Elo (eye lens obsolescence) mutation confers a dominant phenotype characterized by malformation of the eye lens. The mutation maps to chromosome 1, in close proximity to the gamma E-crystallin gene which is the 3'-most member of the gamma-crystallin gene cluster. We have analysed the sequence of this gene from the Elo mouse and identified a single nucleotide deletion which destroys the fourth and last "Greek key" motif of the protein. This mutation is tightly associated with the phenotype, as no recombination was detected in 274 meioses. In addition, the mutant mRNA is present in the affected lens, providing further support for our hypothesis that the deletion is responsible for the dominant Elo phenotype.

Animals↗

PubMed1992

Skeletal muscle arises as a late event during development of wound sarcomas in v-jun transgenic mice.

Mice carrying an H-2K-v-jun transgene develop malignant sarcomas by a multistage mechanism following wounding. Here we show that these malignancies are often heterogeneous in composition, containing both undifferentiated mesenchymal cells as well as focal areas of skeletal muscle. Such myogenic areas are not detectable in premalignant precursor lesions, suggesting that cells competent for muscle differentiation arise at a late stage of tumorigenesis. Immunohistochemical staining of transgenic sarcomas reveals that levels of v-Jun correlate inversely with muscle-specific gene expression, suggesting that high levels may be inhibitory to myogenesis. Consistent with this idea, we demonstrate that whereas high levels of v-Jun are able to block MyoD-dependent gene expression in vitro, the levels of v-Jun in sarcoma-derived myogenic cells are below the threshold required to produce this effect. The cell of origin of v-jun wound sarcomas, as well as the relationship between myogenic determination and multistage tumorigenesis, are discussed in the light of these results.

Actins↗

PubMed1992

Embryology and morphology of microphthalmia in transgenic mice expressing a gamma F-crystallin/diphtheria toxin A hybrid gene.

BACKGROUND: Transgenic mice in which elongating lens fiber cells were ablated resulting in microphthalmia have been reported, however, their embryology and detailed morphology have not. EXPERIMENTAL DESIGN: The morphology of homozygous and hemizygous CDI, transgenic mice carrying the gamma FDT-A gene was studied by light microscopy on different days of gestation as well as postpartum. The findings were compared with normal CD-1 wild type controls. RESULTS: The earliest changes in mouse embryos transgenic for the gamma F-crystallin/diphtheria toxin A transgene are seen on day 12, when apoptotic cells appear in the area of elongation. In hemizygous embryos, ocular development is relatively normal until day 17 when the lens and eye are slightly smaller than normal and the lens vesicle is filled with abnormal lens material. At this time, the posterior capsule of the lens may rupture, releasing abnormal lens material which disperses throughout the eye, perturbing growth and other ocular structures. Additional breaks may subsequently occur and the ultimate morphology of the hemizygotes correlates with when the posterior capsule ruptures, how much lens material is released, and where it disperses. In homozygous embryos, due to extensive ablation of lens fiber cells, the "lens" becomes a diminutive mass of abnormal lens material, posteriorly located within the eye, and otherwise unable to fulfill its mechanical or inductive role in the development of the cornea, anterior chamber, iris, ciliary epithelium, and zonules with the result that all of these structures are markedly abnormal or absent. In addition, the lens is necessary for the accumulation of vitreous which in turn is required for the growth of the eye as a whole. In homozygous animals, vitreous does not accumulate and severe microphthalmia results. CONCLUSIONS: This study confirms and extends previous observations and conclusions on the central, orchestrating role of the lens in the development of the eye and illustrates the power of transgenic technology to elucidate the finer points of mammalian ocular development.

Animals↗

PubMed1992

tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors.

A search for protein tyrosine kinases expressed during murine cardiogenesis resulted in the isolation of a novel tyrosine kinase, designated tek, which maps to mouse chromosome 4 between the brown and pmv-23 loci. The deduced amino acid sequence of tek predicts that it encodes a putative receptor tyrosine kinase that contains a 21 amino acid kinase insert and which is most closely related in its catalytic domains to FGFR1 and the product of the ret proto-oncogene. In situ hybridization analysis of adult tissues, as well as sectioned and whole-mount embryos, showed that tek is specifically expressed in the endocardium, the leptomeninges and the endothelial lining of the vasculature from the earliest stages of their development. Moreover, examination of the morphology of tek-expressing cells, and staging of tek expression relative to that of the endothelial cell marker von Willebrand factor, revealed that tek is expressed prior to von Willebrand factor and appears to mark the embryonic progenitors of mature endothelial cells. tek encodes a novel putative receptor tyrosine kinase that may be critically involved in the determination and/or maintenance of cells of the endothelial lineage.

Amino Acid Sequence↗

PubMed1991

Expression of S-antigen in retina, pineal gland, lens, and brain is directed by 5'-flanking sequences.

S-antigen (S-Ag) is an abundant protein of the retina and pineal gland that elicits experimental autoimmune uveitis and pinealocytis in several animal species. To study the elements regulating the expression of S-Ag, we generated transgenic mice expressing the chloramphenicol acetyl transferase (CAT) gene under the control of a 1.3-kilobase pair 5'-flanking segment of the mouse S-Ag gene. While all of the transgenic mice expressed CAT activity in the retina, in some animals CAT activity was also detected in the pineal gland, lens, and brain. Immunoblotting, polymerase chain reaction-mediated detection of RNA, and immunocyto-staining of transgenic tissues with antibodies to CAT and S-Ag established that the profile of expression of the transgene corresponded to that of S-Ag; both proteins were detectable in retinal photoreceptor cells, pinealocytes, lens fiber and epithelial cells, the cerebellum, and the cerebral cortex. These results indicate that S-Ag is expressed in a wider spectrum of the cell types than previously recognized and that a 1.3-kilobase pair S-Ag promoter segment contains sufficient information to direct appropriate tissue-specific gene expression in transgenic mice.

Animals↗

PubMed1991

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.

Animals↗

PubMed1991

Interaction of a lens cell transcription factor with the proximal domain of the mouse gamma F-crystallin promoter.

The elements regulating lens-specific expression of the mouse gamma F-crystallin gene were examined. Here we show that mouse gamma F-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different gamma-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse gamma F-crystallin gene.

Animals↗

PubMed1990

Obligatory wounding requirement for tumorigenesis in v-jun transgenic mice.

Avian sarcoma virus 17 induces fibrosarcomas in chickens and can transform a number of avian cell types in vitro by the action of v-jun. This gene and the related cellular genes c-jun, jun B and jun D, encode transactivating (or repressing) DNA-binding proteins that form homo- or heterodimeric (Jun-Jun and Jun-Fos) complexes which recognize the AP-1 consensus sequence TGACTCA, a response element that confers sensitivity to the tumour-promoting phorbol ester TPA. We have produced several lines of transgenic mice carrying the v-jun oncogene, driven by the promoter of the widely expressed H-2KK major histocompatibility complex (MHC) class I antigen gene. Transgenic animals are initially phenotypically normal, but after full-thickness wounding they show abnormal wound repair, characterized by hyperplastic granulation tissue. Many of these lesions are slowly progressive because of continuing fibroblast proliferation, and over 2-5 months some give rise to dermal fibrosarcomas. This reproducible multistep transition through a proliferative but benign intermediate is associated with characteristic increments in v-jun expression. Moreover, hyperplastic wound repair and its progression are both related to transgene dosage, suggesting that there exists a quantitative requirement or threshold for v-jun action. Our results indicate that v-jun is not oncogenic in transgenic mice as a result of a 'single-hit' mechanism, but rather, in addition to an obligatory wound, that secondary genetic or epigenetic events (possibly conscripting normal constituents of wound repair) are necessary for tumour development and progression.

Animals↗

PubMed1990

Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene.

We have previously generated microphthalmic mice lacking lens fiber cells by targeting the expression of the diphtheria toxin A (DT-A) gene in transgenic mice with regulatory sequences associated with the mouse gamma 2-crystallin gene. Because of the extreme toxicity of DT to animal cells and the potential leakiness of many tissue-specific regulatory regions, we investigated whether there might be an experimental advantage in using a mutant, attenuated form of the DT-A gene (tox-176) fused to gamma 2-crystallin regulatory sequences to ablate fiber cells in the ocular lens. In contrast to the microphthalmia observed in transgenic animals carrying the native DT-A gene, independent lines of mice transgenic for the gamma 2tox176 construct displayed predominantly cataracts or clinical anophthalmia. These contrasting phenotypes were transmitted within each pedigree, although for some lines some phenotypic heterogeneity among offspring was noted. The difference in phenotype between cataractous and clinically anophthalmic transgenic lines could not be ascribed to differences in the transgene copy number. Instead, the results suggest that transgene expression and hence the extent of genetic ablation are modulated by the site of chromosomal integration and, to a lesser extent, by epigenetic events. They also suggest that the attenuated gamma 2tox176 construct can integrate into chromosomal regions that are particularly favorable for expression without compromising embryological development and therefore that the tox-176 gene may be more versatile and effective than the wild-type DT-A gene for achieving genetic ablation with a broad range of cell- or tissue-specific regulatory sequences.

Animals↗

PubMed1990

Homologous and heterologous enhancers modulate spatial expression but not cell-type specificity of the murine gamma F-crystallin promoter.

Previous studies have shown that mouse gamma F-crystallin sequences -759 to +45, which include the core promoter and two upstream enhancer elements, contain sufficient information for directing gene expression to terminally differentiated fiber cells of the ocular lens. To investigate the role that proximal sequences of the mouse gamma F-crystallin promoter play in the developmental regulation of gene expression, we generated transgenic mice containing the lacZ gene driven by either mouse gamma F-crystallin sequences -171 to +45, which lack functional enhancers, or a hybrid hamster alpha A-/mouse gamma F-crystallin promoter, which contains the hamster alpha A-crystallin enhancer instead of operational gamma F-crystallin enhancers. In situ analysis of lacZ expression in these mice revealed that the mouse gamma F-crystallin promoter segment -171 to +45, which shows low activity in vitro, is able to direct gene expression to the fiber cells in the nucleus of the lens. However, animals expressing gamma 171-lacZ show both a lower level of expression of the lacZ gene and a narrower pattern of staining in the lens nucleus than mice expressing gamma 759-lacZ, which contains the two enhancer elements located between -392 and -278 and -226 to -123.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Evidence that beta 1-6 branched Asn-linked oligosaccharides on metastatic tumor cells facilitate invasion of basement membranes.

In previous studies we have shown that the ability of murine tumor cells to metastasize in situ is directly linked to expression of -GlcNAc beta 1-6Man alpha 1-6Man beta 1-branched complex-type Asn-linked oligosaccharides in tumor-cell glycoproteins. Here we demonstrate that cell-surface expression of beta 1-6 branched oligosaccharides in metastatic tumor cells is specifically associated with increased invasion of human amnion basement membranes in vitro. Compared to nonmetastatic SP1 murine mammary carcinoma cells, 2 metastatic sublines expressed higher levels of beta 1-6 branched oligosaccharides and were found to be invasive but poorly adhesive on the amnion basement membrane. Swainsonine, a non-toxic inhibitor of Asn-linked oligosaccharide processing which blocks the pathway prior to initiation of the beta 1-6 linked antenna, blocked metastatic tumor-cell invasion and increased adhesiveness. Swainsonine and the metalloprotease inhibitor O-phenanthroline inhibited invasion, apparently via independent mechanisms. O-phenanthroline did not affect tumor-cell adhesion to the amnion basement membrane and swainsonine did not block secretion of metalloproteases, beta-hexosaminadase or tissue plasminogen activator activity by the tumor cells. These results suggest that tumor-cell invasion of basement membranes requires both secretion of hydrolase activities and expression of beta 1-6 branched complex-type oligosaccharides at the tumor cell surface, such oligosaccharides being associated with reduced tumor-cell adhesion to extracellular matrix.

Alkaloids↗