Biomedical subjects

R R Behringer

Publications and source records attributed to R R Behringer.

At least 91 records · Page 5Linked to original sources

PubMed1994

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.

Animals↗

PubMed1994

Visualization and ablation of phenylethanolamine N-methyltransferase producing cells in transgenic mice.

We cloned and sequenced the mouse phenylethanolamine N-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5' flanking sequences from the cloned mouse PNMT gene can direct expression of the Escherichia coli beta-galactosidase (lacZ) gene to predicted regions of the adrenal, eye and brain in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT8 kb-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.

Adrenal Medulla↗

PubMed1994

The in vivo roles of müllerian-inhibiting substance.

The fetal testis functions as the sex differentiator by imposing a masculine pattern of development upon a genetic program that is inherently female. Two hormones produced by the fetal testis mediate the differentiation of the müllerian and Wolffian ducts (Figs. 1 and 4). MIS actively inhibits the development of the müllerian ducts, and testosterone induces the differentiation of the Wolffian ducts. The absence of these two hormones during fetal development in the female (the hormonal equivalent of no testes) permits müllerian duct differentiation and does not induce Wolffian duct development. The in vivo outcomes of ectopic MIS exposure or MIS deficiency illustrate the balance required to coordinately differentiate and cause regression of the respective male and female genital ducts. The observations made in the MIS-deficient mice demonstrate that codevelopment of both genital duct systems interferes with normal development of both systems and ultimately interferes with reproduction and fertility. Thus, reproduction and fertility in mammals appear to be most efficient if only one type of genital duct system develops. The phenotypes of the MIS-overexpressing transgenic mice and the MIS-deficient mice are similar yet different. Some of the explanations that might reconcile these differences probably lie with the receptor for MIS. Since the MIS-overexpressing transgenic mice are exposed to pharmacological levels of MIS during development, it seems possible that this may lead to productive interactions with other related receptors. Candidate genes have been isolated for the MIS receptor that are membrane-bound serine/threonine kinases (Baarends et al., 1994; di Clemente et al., 1994) similar to those cloned for the TGF-beta (Lin et al., 1992) and activin (Mathews and Vale, 1991) type II receptors. Interestingly, expression of these putative MIS receptor genes is localized by in situ hybridization to the mesenchymal cells adjacent to the müllerian ducts, suggesting that MIS most likely alters the surrounding mesenchyme to elicit müllerian duct regression. Experiments are underway to isolate the mouse MIS receptor gene to thereby generate MIS receptor-deficient mice and to compare the phenotype with the MIS gain-of-function and loss-of-function animals. Isolation of the human MIS receptor gene will facilitate the identification of human PMDS patients with normal levels of MIS that have mutations in the MIS receptor gene. Finally, studies of the MIS receptor gene will open up avenues for the molecular characterization of signal transduction pathways that mediate müllerian duct regression and Leydig cell proliferation control.

Androgen-Insensitivity Syndrome↗

PubMed1994

Normal long bone growth and development in type X collagen-null mice.

To investigate the role of type X collagen in skeletal development, we have generated type X collagen-null mice. Surprisingly, mice without type X collagen were viable and fertile and had no gross abnormalities in long bone growth or development. No differences were detected between the type X collagen-null mice and controls when growth plates of both newborn and 3-week old mice were examined by histology and by immunostaining for extracellular matrix components of bone including osteopontin, osteocalcin and type II collagen. Our results suggest that type X collagen is not required for long bone development. However, mice and humans with dominant acting type X collagen mutations have bone abnormalities, suggesting that only the presence of abnormal type X collagen can modify bone growth and development.

Animals↗

PubMed1994

Hypomyelinating peripheral neuropathies and schwannomas in transgenic mice expressing SV40 T-antigen.

We have prepared transgenic mice carrying a temperature-sensitive mutant of the SV40 oncogene (tsA-1609) under the control of 5' flanking sequences from the Schwann cell-specific P0 gene. Four of six founder mice showed moderate to severe hypomyelination in peripheral nerves of tail biopsies, with only rare myelinated fibers. Offspring were obtained from three of these founders. Northern blot and immunohistochemical analyses showed that expression of T-antigen was restricted to the PNS. Mice expressing the highest levels of T-antigen exhibited the most severe hypomyelination. Mice expressing lower levels developed transient mild hypomyelination, but after long latencies developed sporadic schwannomas. An immortalized cell line exhibiting properties of Schwann cells at an arrested stage of differentiation, termed "SCT-1," was derived from one of these tumors.

Animals↗

PubMed1993

Structural abnormalities associated with congenital megacolon in transgenic mice that overexpress the Hoxa-4 gene.

Congenital megacolon develops in transgenic mice that overexpress the homeobox-containing gene, Hoxa-4. The current study was done to identify abnormalities of the terminal colon that might account for the phenotype. The terminal bowel of transgenic mice was compared with that of control and lethal spotted (ls/ls) mice, a strain in which megacolon also develops. The terminal colon of the transgenic mice contained fewer ganglia than that of controls, but was hypoganglionic, rather than aganglionic like that of ls/ls mice. The neurons present in the adult transgenic colon were significantly increased in size and a subset of very large neurons (> 40 microns in maximum diameter) were observed. Electron microscopic studies of young adult transgenic mice revealed that the ganglia and nerves of the myenteric plexus had the ultrastructure of extraenteric peripheral nerve rather than that of the enteric nervous system (ENS). The myenteric ganglia in the transgenic animals contained Schwann cells associated with a basal lamina that enveloped axons completely and individually, instead of glia. Although collagen is excluded from the ganglia and thin nerve fibers of the normal ENS, a collagen-containing endoneurium surrounded each of the axon-Schwann cell units of the abnormal nerve fibers of the transgenic colon. Some of the neurons of the transgenic mice were located in these nerve bundles rather than in ganglia. There were also smooth muscle abnormalities in the terminal bowel of the transgenic mice. Wide gaps were present in the longitudinal muscle of the transgenic mice; these gaps contained ganglia that were in contact with the adventitia. These longitudinal smooth muscle cells were more irregular than those of controls and they contained fewer puncta adherens; moreover, a larger proportion of the volume of the cytoplasm of transgenic smooth muscle cells was occupied by organelles. Finally, an extensive thickening and reduplication of the basal lamina surrounding the smooth muscle cells of the muscularis mucosa was observed in the transgenic colon and resembled that found in ls/ls mice. These data suggest that both smooth muscle and the innervation of the terminal bowel of neonatal Hoxa-4 transgenic mice are structurally abnormal. Although some of the abnormalities seen in Hoxa-4 transgenic mice are similar to those which arise in ls/ls mice, the two conditions are not identical. In both animals, the data are consistent with the hypothesis that the defects arise as a result of a defective interaction between the precursors of enteric neurons and smooth muscle.

Acetylcholinesterase↗

PubMed1993

Oncogene expression in retinal horizontal cells of transgenic mice results in a cascade of neurodegeneration.

The phenylethanolamine N-methyltransferase promoter directs the expression of the SV40 T antigen to subsets of amacrine and horizontal neurons of the retina in a line of transgenic mice. T antigen expression begins in these cells during the first postnatal week. The horizontal cells appear to develop normally for another week but then begin to die. Subsequently, most of the horizontal cells disappear from the central and mid retina, resulting in loss of the outer plexiform layer and absence of ribbon synapses between the photoreceptors and bipolar cells. Neuronal transformation occurs only in the peripheral retina. These experiments indicate that horizontal neurons are heterogeneous with respect to susceptibility to transformation and that T antigen expression in a subset of horizontal neurons can be a direct cause of neuronal cell death. Furthermore, critical interdependencies exist between horizontal neurons after retinal neurogenesis is complete.

Animals↗

PubMed1993

Insulin-like growth factor I increases brain growth and central nervous system myelination in transgenic mice.

Insulin-like growth factor I (IGF-I) is a potent regulator of oligodendrocyte development and myelination in vitro, but its effect on myelination in vivo has never been tested directly. Therefore, we examined brain growth and myelination in a transgenic mouse line that overexpresses IGF-I. By postnatal day 55, when brain growth and myelination are essentially complete in normal mice, the brains of transgenic mice were 55% larger than those of controls owing to an increase in cell size and apparently in cell number. Most or all brain structures appeared to be affected. At the same time, total myelin content of the transgenic mice was 130% greater than that of controls. Oligodendrocyte number as a percentage of total cell number was not increased in the transgenic mouse brains; the increase in myelin content was primarily the result of an increase in myelin production per oligodendrocyte. These findings indicate that IGF-I is a potent inducer of brain growth and myelination in vivo.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1993

A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice.

The gene for the iron-binding protein transferrin is transcribed at a high level in liver hepatocytes but is also active in several other cell types, including oligodendrocytes in the brain. Enhancer elements between bp -560 and -44 of the transferrin gene promoter specifically activated transcription from a heterologous promoter in transgenic mouse liver and brain. Within this region, a potent cis-acting element between bp -98 and -83 was found to be essential for gene activity in both cultured hepatocytes and transgenic mouse liver. The -98 to -83 element contains a CCAAT sequence and is specifically bound by a nuclear factor from mouse liver that is homologous to rat liver C/EBP (CAAT enhancer-binding protein). Point mutations within this binding site inhibit factor binding and abolish transcription in transfected hepatoma cells. When placed in the context of the 3,000-bp transferrin promoter, the C/EBP binding site mutation causes a complete loss of transcription in transgenic mouse liver; however, transgene expression in the brain of the same animals was unaffected. These results suggest a modular structure for the transferrin promoter and demonstrate that deletions or specific point mutations can be used to generate transgene promoters with an activity more restricted than that of their endogenous counterparts.

Animals↗

PubMed1993

Sequences 5' of the homeobox of the Hox-1.4 gene direct tissue-specific expression of lacZ during mouse development.

The murine homeobox-containing gene Hox-1.4 is expressed in restricted patterns during embryogenesis and in male germ cells. To begin identification of the cis-acting elements regulating this expression, transgenic mice were generated carrying a chimeric construct that contained approx. 4 kb of 5' flanking sequence and approx. 1 kb of structural gene, fused in frame to the E. coli lacZ gene. This construct directed expression of the resulting Hox-1.4,beta-galactosidase fusion protein in a pattern that reproduced virtually the complete embryonic and adult sites of expression of the endogenous gene. Embryonic expression of the fusion protein was first detected in mesoderm at day 8.0 of gestation (E 8.0). Between gestational ages E 8.5 to E 12.5, beta-gal expression was observed in the somites, the lateral walls of the posterior myelencephalon, the dorsal region and ventral wall of the spinal cord, spinal ganglia and prevertebrae and their surrounding mesenchyme, between presumptive ribs, as well as in mesenchymal layers in the lung, kidney and portions of the gut. Expression was also noted in the pancreas and in the supporting cells and sheath around subsets of peripheral nerves, sites that had not been detected previously. Adult expression was observed in testes, specifically in meiotic and post-meiotic male germ cells. In contrast, transgenic mice carrying 5' deletions of the construct which leave approx. 1.2 kb or approx. 2.0 kb of Hox-1.4 sequence 5' to the embryonic promoter, did not exhibit beta-gal staining. These deletion experiments defined at least one cis-acting control element necessary for the expression of the Hox-1.4 gene to a 2 kb region located 2 to 4 kb 5' of the embryonic transcription start site.

Animals↗

PubMed1992

P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice.

We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.

Age Factors↗

PubMed1992

Atrial natriuretic peptide synthesis in atrial tumors of transgenic mice.

Transgenic mice harboring a chimeric gene linking mouse protamine 1 5'-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atrial natriuretic peptide (ANP) immunoreactivity (1,698 +/- 993 vs. 60 +/- 18 fmol/ml for controls) and hematocrit (56 +/- 8 vs. 51 +/- 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5'-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atrial cell line.

Amino Acid Sequence↗

PubMed1992

Directed expression of an oncogene to Sertoli cells in transgenic mice using mullerian inhibiting substance regulatory sequences.

Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.

Animals↗

PubMed1991

Human beta-globin locus control region: analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice.

The human beta-globin locus control region (LCR) is essential for high-level expression of human epsilon-, gamma-, and beta-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human beta-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human beta-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletions of both 5' and 3' sequences, a 373-base-pair (bp) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 18-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained.

Animals↗

PubMed1991

Mapping anti-müllerian hormone (Amh) and related sequences in the mouse: identification of a new region of homology between MMU10 and HSA19p.

A panel of 78 backcross progeny, BALB/cJ x (BALB/cJ x CAST/Ei)F1, was used to map the gene encoding anti-Müllerian hormone (Amh), also called Müllerian inhibiting substance, to mouse Chromosome 10 (MMU10). This analysis identified a new region of linkage homology between human Chromosome 19p (HSA 19p) and MMU10 and localized an apparent recombinational hot spot in (C57BL/6J x Mus spretus)F1 females [compared with (BALB/cJ x CAST/Ei)F1 males] to the interval between phenylalanine hydroxylase (Pah) and mast cell growth factor (Mgf). In addition, eight unlinked polymorphic sequences, provisionally designated Amh-related sequences (Amh-rs1 through Amh-rs8), were identified by Southern blot analysis using Amh probes. Amh-rs1, -rs2, -rs4, and -rs7 were mapped to MMU1, 13, 12, and 15, respectively, by recombinant inbred (RI) strain and intraspecific backcross analyses. The NXSM RI strain distribution patterns for the four unmapped loci are also presented.

Animals↗

PubMed1991

Transferrin- and albumin-directed expression of growth-related peptides in transgenic sheep.

Chimeric genes containing either the mouse transferrin (Trf) enhancer/promoter fused to the structural sequences encoding bovine growth hormone (GH) or the mouse albumin (Alb) enhancer/promoter fused to the gene for human growth hormone-releasing factor (GRF) were microinjected into sheep zygotes. A low percentage of resulting transgenic sheep chronically expressed the respective genes, resulting in elevated plasma concentrations of circulating GH or GRF, respectively. Growth hormone-releasing factor expression induced elevated plasma levels of endogenous GH production. In addition, elevated levels of circulating insulin-like growth factor-I were observed in the bovine GH-expressing Trf transgenic sheep. Growth of these founder transgenic sheep relative to controls were not enhanced. In part, this may be due to the development of the diabetic condition exhibited by both transgenic groups. These results demonstrate that the mouse Trf and Alb enhancer/promoters are active in sheep and suggest that alternate strategies for expressing growth-related genes may be required to modulate growth in sheep.

Albumins↗

PubMed1990

Abnormal sexual development in transgenic mice chronically expressing müllerian inhibiting substance.

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein normally secreted by the Sertoli cells of the fetal and adult testis and by granulosa cells of the postnatal ovary. The production of MIS in the male fetus brings about the regression of the Müllerian ducts, the anlagen of the uterus, oviducts, and upper vagina. In addition, purified MIS induces the formation of seminiferous cord-like structures in fetal rat ovaries cultured in vitro, suggesting that MIS may influence testicular differentiation. We have produced transgenic mice chronically expressing human MIS under the control of the mouse metallothionein-1 promoter to investigate its role during sexual development. In females, chronic expression led to the inhibition of Müllerian duct differentiation, resulting in a blind vagina and no uterus or oviducts. At birth the ovaries had fewer germ cells than normal; during the next two weeks germ cells were lost and the somatic cells became organized into structures resembling seminiferous tubules. Apparently, these structures degenerate as they are undetectable in adult females. The majority of transgenic males developed normally. But in two lines with the highest levels of MIS expression, some males showed feminization of the external genitalia, impairment of Wolffian duct development, and undescended testes. These results suggest that MIS has several distinct roles in mammalian sexual development.

Androgens↗

PubMed1990

Human sickle hemoglobin in transgenic mice.

DNA molecules that contain the human alpha- and beta s-globin genes inserted downstream of erythroid-specific, deoxyribonuclease I super-hypersensitive sites were coinjected into fertilized mouse eggs and a transgenic mouse line was established that synthesizes human sickle hemoglobin (Hb S). These animals were bred to beta-thalassemic mice to reduce endogenous mouse globin levels. When erythrocytes from these mice were deoxygenated, greater than 90 percent of the cells displayed the same characteristic sickled shapes as erythrocytes from humans with sickle cell disease. Compared to controls the mice have decreased hematocrits, elevated reticulocyte counts, lower hemoglobin concentrations, and splenomegaly, which are all indications of the anemia associated with human sickle cell disease.

Anemia, Sickle Cell↗