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Biomedical subjects

E N Olson

Publications and source records attributed to E N Olson.

At least 127 records · Page 7Linked to original sources
PubMed1997

Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.

Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.

Alleles↗
PubMed1997

Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal, and cardiac muscle cells.

SM22alpha is an adult smooth muscle-specific protein that is expressed in the smooth, cardiac, and skeletal muscle lineages during early embryogenesis before becoming restricted specifically to all vascular and visceral smooth muscle cells (SMC) in late fetal development and adulthood. We have used the SM22alpha gene as a marker to define the regulatory mechanisms that control muscle-specific gene expression in SMCs. Previously, we reported that the 445-base-pair promoter of SM22alpha was sufficient to direct transcription of a lacZ reporter gene in early cardiac and skeletal muscle cell lineages and in a subset of arterial SMCs, but not in venous nor visceral SMCs in transgenic mice. Here we describe two evolutionarily conserved CArG (CC(A/T)6GG) boxes in the SM22alpha promoter, both of which are essential for full promoter activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, skeletal, and cardiac muscle lineages in transgenic mice. Both CArG boxes bind serum response factor (SRF), but SRF binding is not sufficient for SM22alpha promoter activity, since overexpression of SRF in the embryonal teratocarcinoma cell line F9, which normally expresses low levels of SRF, fails to activate the promoter. However, a chimeric protein in which SRF was fused to the transcription activation domain of the viral coactivator VP16 is able to activate the SM22alpha promoter in F9 cells. These results demonstrate the SM22alpha promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to the SM22alpha promoter and they suggest that SRF activates SM22alpha transcription in conjunction with additional regulatory factors that are cell type-restricted.

Animals↗
PubMed1997

Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.

Members of the myocyte enhancer factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, serum response factor)-box transcription factors bind an A-T-rich DNA sequence associated with muscle-specific genes. The murine MEF2C gene is expressed in heart precursor cells before formation of the linear heart tube. In mice homozygous for a null mutation of MEF2C, the heart tube did not undergo looping morphogenesis, the future right ventricle did not form, and a subset of cardiac muscle genes was not expressed. The absence of the right ventricular region of the mutant heart correlated with down-regulation of the dHAND gene, which encodes a basic helix-loop-helix transcription factor required for cardiac morphogenesis. Thus, MEF2C is an essential regulator of cardiac myogenesis and right ventricular development.

Acyltransferases↗
PubMed1997

Regulation of paraxis expression and somite formation by ectoderm- and neural tube-derived signals.

During vertebrate embryogenesis, the paraxial mesoderm becomes segmented into somites, which form as paired epithelial spheres with a periodicity that reflects the segmental organization of the embryo. As a somite matures, the ventral region gives rise to a mesenchymal cell population, the sclerotome, that forms the axial skeleton. The dorsal region of the somite remains epithelial and is called dermomyotome. The dermomyotome gives rise to the trunk and limb muscle and to the dermis of the back. Epaxial and hypaxial muscle precursors can be attributed to distinct somitic compartments which are laid down prior to overt somite differentiation. Inductive signals from the neural tube, notochord, and overlying ectoderm have been shown to be required for patterning of the somites into these different compartments. Paraxis is a basic helix-loop-helix transcription factor expressed in the unsegmented paraxial mesoderm and throughout epithelial somites before becoming restricted to epithelial cells of the dermomyotome. To determine whether paraxis might be a target for inductive signals that influence somite patterning, we examined the influence of axial structures and surface ectoderm on paraxis expression by performing microsurgical operations on chick embryos. These studies revealed two distinct phases of paraxis expression, an early phase in the paraxial mesoderm that is dependent on signals from the ectoderm and independent of the neural tube, and a later phase that is supported by redundant signals from the ectoderm and neural tube. Under experimental conditions in which paraxis failed to be expressed, cells from the paraxial mesoderm failed to epithelialize and somites were not formed. We also performed an RT-PCR analysis of combined tissue explants in vitro and confirmed that surface ectoderm is sufficient to induce paraxis expression in segmental plate mesoderm. These results demonstrate that somite formation requires signals from adjacent cell types and that the paraxis gene is a target for the signal transduction pathways that regulate somitogenesis.

Amino Acid Sequence↗
PubMed1997

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Animals↗
PubMed1997

Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.

The zinc finger transcription factor GATA4 has been implicated in heart development based on its early expression in precardiogenic splanchnic mesoderm and its ability to activate the expression of a number of cardiac-specific genes. To determine the role of GATA4 in embryogenesis, we generated mice homozygous for a GATA4 null allele. Homozygous GATA4 null mice arrested in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lacked a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia failed to migrate ventrally but instead remained lateral and generated two independent heart tubes that contained differentiated cardiomyocytes. We show that these deformities resulted from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. We propose that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the AIP.

Animals↗
PubMed1997

D-mef2 is a target for Tinman activation during Drosophila heart development.

The NK-type homeobox gene tinman and the MADS box gene D-mef2 encode transcription factors required for the development and differentiation of the Drosophila heart, and closely related genes regulate cardiogenesis in vertebrates. Genetic analyses indicate that tinman and D-mef2 act at early and late steps, respectively, in the cardiogenic lineage. However, it is unknown whether regulatory interactions exist between these developmental control genes. We show that D-mef2 expression in the developing Drosophila heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D-mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila.

Animals↗
PubMed1997

State of commitment of prospective neural plate and prospective mesoderm in late gastrula/early neurula stages of avian embryos.

We examined the ability of epiblast regions of known prospective fate from the late gastrula/early neurula stage of avian embryos to self-differentiate when placed heterotopically, testing their state of commitment. Three sites were examined: paranodal prospective neural plate ectoderm, containing cells fated to form a portion of the lateral wall of the neural tube at essentially all rostrocaudal levels of the neuraxis; prospective mesoderm from the caudolateral epiblast, containing cells fated to ingress through the primitive streak and to form lateral plate mesoderm; and prospective mesoderm from one level of the primitive streak, containing cells fated to continue ingressing and form paraxial mesoderm. Grafts from all sites exhibited plasticity. Grafts from the prospective neural plate ectoderm could readily substitute for regions of prospective mesoderm, when transplanted to either the epiblast or primitive streak, undergoing an epithelial-mesenchymal transition and, where appropriate, expressing paraxis, a gene expressed in paraxial mesoderm. Similarly, grafts containing prospective mesoderm from the epiblast could readily substitute for regions of the prospective neural plate ectoderm, undergoing convergent-extension movements characteristic of neuroectodermal cells and expressing appropriate genes such as Engrailed-2 and Hoxb-1. Grafts containing prospective mesoderm from the primitive streak could also incorporate into the neural plate and undergo convergence-extension movements of neurulation, although their principal contribution was to mesodermal and endodermal structures. Collectively, our results demonstrate that at the late gastrula/early neurula stage, germ layer-specific properties are not irrevocably fixed for prospective ectodermal and mesodermal regions of the blastoderm. Moreover, the signals responsible for the induction of these two tissue types must still be present and available at these late stages.

Animals↗
PubMed1997

A novel A/T-rich element mediates ANF gene expression during cardiac myocyte hypertrophy.

The induction of the atrial natriuretic factor (ANF) gene during alpha 1-adrenergic stimulation of neonatal rat ventricular myocytes has served as a model for gene expression during cardiac muscle cell hypertrophy. This study describes and identifies a single regulatory element that mediates expression of the ANF gene. Deletional mutations were generated in a 639-bp fragment of the ANF promoter that confers alpha 1-adrenergic inducibility to a luciferase reporter gene in transient transfection assays in ventricular myocytes. The results of gel mobility shift and diethylpyrocarbonate (DEPC) interference studies with nuclear cardiac cell extracts identified the nucleotide contract points for a novel A/T-rich element (ANF-AT) at positions -582/-575 that partially mediates alpha 1-adrenergic inducibility. Mutations in the ANF-AT element reduced alpha-adrenergic inducibility of an ANF-TK-luciferase fusion gene in cardiac cells by 35% but had no effect on expression in other muscle and non-muscle cells tested. Gel mobility supershift assays with antibodies directed against the MEF-2 protein, the homeobox protein MHox, or the zinc finger protein HF-1b, document that these factors are not major components of the endogenous ANF-AT binding activity in cardiac muscle cells. The current study provides evidence for a role for a novel A/T-rich element in the regulation of ANF gene expression in cardiac ventricular myocytes.

Animals↗
PubMed1997

The A10 cell line: a model for neonatal, neointimal, or differentiated vascular smooth muscle cells?

OBJECTIVES: The A10 cell line was derived from the thoracic aorta of embryonic rat and is a commonly used model of vascular smooth muscle cells (VSMC). Despite its wide use this cell line has not been well characterized. This is especially important in light of recent evidence of phenotypically distinct cell populations isolated from rat vascular tissue. Therefore, the present study was undertaken to confirm the VSMC nature of A10 cells and to investigate whether these cells particularly resemble adult, neonatal, or neointimal rat VSMC. METHODS: A variety of defining characteristics were used that included immunofluorescent analysis for smooth muscle alpha-actin, smooth and non-muscle myosin heavy chains, desmin and vimentin; Western analysis for smooth muscle and non-muscle myosin heavy chains; mRNA analysis for smooth muscle myosin heavy chain, calponin, SM22 alpha, tropoelastin and PDGF-B peptide; and functional assays of cell migration, proliferation and agonist induced intracellular Ca transients. RESULTS: A10 cells expressed smooth muscle alpha-actin, SM22 alpha, smooth muscle calponin and vimentin, characteristic of in vivo rat VSMCs; however they also resembled de-differentiated smooth muscle cells in that they expressed non-muscle myosin rather than smooth muscle myosin heavy chain. A10 cells resembled cultured rat neonatal smooth muscle cells ("pup cells") in that they had an epithelioid shape and lacked functional PDGF-alpha receptors: however they did not express PDGF-B mRNA or proliferate in low serum containing medium as do neonatal cells. A10 cells had several characteristics in common with neointimal cells including the expression of alpha-actin, vimentin, and non-muscle myosin and the lack of expression of PDGF-B mRNA as well as the ability to migrate in response to PDGF-BB. CONCLUSION: In conclusion, A10 cells are nondifferentiated VSMC that differ from neonatal but bear significant resemblance to neointimal cells.

Actins↗
PubMed1997

Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity.

Skeletal, cardiac and smooth muscle cells express overlapping sets of muscle-specific genes, such that some muscle genes are expressed in only a single muscle cell lineages. Recent studies in transgenic mice have revealed that, in many cases, multiple, independent cis-regulatory regions, or modules, are required to direct the complete developmental pattern of expression of individual muscle-specific genes, even within a single muscle cell type. The temporospatial specificity of these myogenic regulatory modules is established by unique combinations of transcription factors and has revealed unanticipated diversity in the regulatory programs that control muscle gene expression. This type of composite regulation of muscle gene expression appears to reflect a general strategy for the control of cell-specific gene expression.

Animals↗
PubMed1997

Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND.

dHAND and eHAND are related basic helix-loop-helix (bHLH) transcription factors that are expressed in mesodermal and neural crest-derived structures of the developing heart. In contrast to their homogeneous expression during avian cardiogenesis, during mouse heart development we show that dHAND and eHAND are expressed in a complementary fashion and are restricted to segments of the heart tube fated to form the right and left ventricles, respectively. dHAND and eHAND represent the earliest cardiac chamber-specific transcription factors yet identified. Targeted gene deletion of dHAND in mouse embryos resulted in embryonic lethality at embryonic day 10.5 from heart failure. Our description of the cardiac phenotype of dHAND mutant embryos is the first demonstration of a single gene controlling the formation of the mesodermally derived right ventricle and the neural crest-derived aortic arches and reveals a novel cardiogenic subprogramme for right ventricular development.

Animals↗
PubMed1997

The MEF2A 3' untranslated region functions as a cis-acting translational repressor.

Myocyte enhancer factor 2 (MEF2) proteins serve as important muscle transcription factors. In addition, MEF2 proteins have been shown to potentiate the activity of other cell-type-specific transcription factors found in muscle and brain tissue. While transcripts for MEF2 factors are widely expressed in a variety of cells and tissues, MEF2 proteins and binding activity are largely restricted to skeletal, smooth, and cardiac muscle and to brain. This disparity between MEF2 protein and mRNA expression suggests that translational control may play an important role in regulating MEF2 expression. In an effort to identify sequences within the MEF2A message which control translation, we isolated the mouse MEF2A 3' untranslated region (UTR) and fused it to the chloramphenicol acetyltransferase (CAT) reporter gene. Here, we show by CAT assay that the MEF2A 3' UTR dramatically inhibits CAT gene expression in vivo and that this inhibition is due to an internal region within the highly conserved 3' UTR. RNase protection analyses demonstrated that the steady-state level of CAT mRNA produced in vivo was not affected by fusion of the MEF2A 3' UTR, indicating that the inhibition of CAT activity resulted from translational repression. Furthermore, fusion of the MEF2A 3' UTR to CAT inhibited translation in vitro in rabbit reticulocyte lysates. We also show that the translational repression mediated by the 3' UTR of MEF2A is regulated during muscle cell differentiation. As muscle cells in culture differentiate, the translational inhibition caused by the MEF2A 3' UTR is relaxed. These results demonstrate that the MEF2A 3' UTR functions as a cis-acting translational repressor both in vitro and in vivo and suggest that this repression may contribute to the tissue-restricted expression and binding activity of MEF2A.

Animals↗
PubMed1997

Angiotensin II-induced stimulation of smooth muscle alpha-actin expression by serum response factor and the homeodomain transcription factor MHox.

The objective of the present study was to examine the molecular mechanisms whereby angiotensin II (Ang II) stimulates smooth muscle (SM) alpha-actin expression in rat aortic smooth muscle cells (SMCs). Nuclear run-on analysis and transfection studies indicated that the effects of Ang II on SM alpha-actin were mediated at least in part at the transcriptional level. Transfection of various rat SM alpha-actin promoter/chloramphenicol acetyltransferase (CAT) constructs into SMCs demonstrated that the first 155 bp of the SM alpha-actin promoter was sufficient to confer maximal Ang II responsiveness, conferring an approximately 4-fold increase in reporter activities in these SMCs compared with vehicle-treated SMCs. Mutation of either of two highly conserved CArG elements, designated A (-62) and B (-112), completely abolished Ang II-induced increases in reporter activity, whereas mutation of a homeodomain-like binding sequence at -145 (ATTA) reduced reporter activity by half. Results of EMSAs showed that nuclear extracts from Ang II-treated SMCs exhibited enhanced binding activity of serum response factor (SRF) to the CArG elements and of a homeodomain factor, MHox, to the ATTA element. Northern analyses showed that Ang II also stimulated marked increases in MHox mRNA levels. Western analyses demonstrated that Ang II-induced increases in SRF binding were not due to increased SRF protein expression. Recombinant MHox markedly enhanced binding activity of SRF in EMSAs. Finally, MHox overexpression transactivated a SM alpha-actin promoter/CAT reporter construct by approximately 3.5-fold in transient cotransfection studies. These results provide evidence for involvement of a homeodomain transcription factor, MHox, in Ang II-mediated stimulation of SM alpha-actin via a CArG/SRF-dependent mechanism.

Actins↗
PubMed1996

Requirement of the paraxis gene for somite formation and musculoskeletal patterning.

The segmental organization of the vertebrate embryo is first apparent when somites form in a rostrocaudal progression from the paraxial mesoderm adjacent to the neural tube. Newly formed somites appear as paired epithelial spheres that become patterned to form vertebrae, ribs, skeletal muscle and dermis. Paraxis is a basic helix-loop-helix transcription factor expressed in paraxial mesoderm and somites. Here we show that in mice homozygous for a paraxis null mutation, cells from the paraxial mesoderm are unable to form epithelia and so somite formation is disrupted. In the absence of normal somites, the axial skeleton and skeletal muscle form but are improperly patterned. Unexpectedly, however, we found that formation of epithelial somites was not required for segmentation of the embryo or for the establishment of somitic cell lineages. These results demonstrate that paraxis regulates somite morphogenesis, and that the function of somites is to pattern the axial skeleton and skeletal muscles.

Animals↗