Biomedical subjects

R Jaenisch

Publications and source records attributed to R Jaenisch.

At least 91 records · Page 5Linked to original sources

PubMed1997

Myogenin can substitute for Myf5 in promoting myogenesis but less efficiently.

The myogenic basic Helix-Loop-Helix transcription factors, including Myf5, MyoD, myogenin (myg) and MRF4, play important roles in skeletal muscle development. The phenotypes of mutant mice deficient in either gene are different, suggesting that each gene may have a unique function in vivo. We previously showed that targeting myogenin into the Myf5 locus (Myf5(myg-ki)) rescued the rib cage truncation in the Myf5-null mutant, hence demonstrating functional redundancy between Myf5 and myogenin in skeletal morphogenesis. Here we present the results of crossing myogenin knock-in (myg-ki) mice with either MyoD-null or myogenin-null mutants. The Myf5(myg-ki) allele rescued early myogenesis, but Myf5(myg-ki/myg-ki);MyoD(-/-) mutant mice died immediately after birth owing to reduced muscle formation. Therefore, myogenin, expressed from the Myf5 locus, is not able to completely replace the function of Myf5 in muscle development although it is capable of determining and/or maintaining myogenic lineage. Myf5(myg-ki/myg-ki);myg(-/-) mutant mice displayed the same phenotype as myg(-/-) mutants. This indicates that the earlier expression of myogenin cannot promote myogenic terminal differentiation, which is normally initiated by the endogenous myogenin. Thus, our results are consistent with the notion that Myf5 and myogenin are functionally interchangeable in determining myogenic lineage and assuring normal rib formation. Our experiment revealed, however, that some aspects of myogenesis may be unique to a given myogenic factor and are due to either different regulatory sequences that control their temporal and spatial expression or different functional protein domains.

Alleles↗

PubMed1996

A method for high efficiency YAC lipofection into murine embryonic stem cells.

We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection. With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert. These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment.

Animals↗

PubMed1996

Targeted mutagenesis of Timp-1 reveals that lung tumor invasion is influenced by Timp-1 genotype of the tumor but not by that of the host.

Timp-1 has been implicated as a suppressor of tumor metastasis. To study the relative importance of Timp-1 expression by tumor vs host during tumor invasion, three pairs of co-isogenic, tumorigenic cells containing wild-type or mutant Timp-1 alleles were generated. These were used in experimental metastasis assays in wild-type or Timp-1-deficient mice. Timp-1 expression in tumorigenic cells could either increase or decrease tumor invasion of lungs in a tumor cell-specific manner. This suggests that depending on the tumor, Timp-1 can either suppress or potentiate metastasis. Mice deficient for Timp-1 were indistinguishable from wild-type mice in metastasis assays with all tumorigenic cells tested. Although Timp-1 is a secreted protein, and lung produces other Timps, the influence of Timp-1 on lung invasion by the tumorigenic cells tested is cell-autonomous, depending only on Timp-1 genotype of tumor and not that of host.

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PubMed1996

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene.

Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.

Amino Acid Sequence↗

PubMed1996

Different collagenase gene products have different roles in degradation of type I collagen.

Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus. We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus. Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli. Assays utilized collagen from wild type (+/+) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in alpha1(I) chains that prevent collagenase cleavage at the helical locus. MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (+/+) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (beta components converted to alpha chains). Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences amino-terminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA. Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.

Amino Acid Sequence↗

PubMed1996

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Alleles↗

PubMed1996

DNA hypomethylation can activate Xist expression and silence X-linked genes.

Xist and other X-linked gene expression was examined by fluorescence in situ hybridization in cells of wild type and DNA methyltranferase (Dnmt) mutant embryos and embryonic stem (ES) cells to determine whether demethylation-induced Xist expression leads to inappropriate X chromosome inactivation. In undifferentiated ES cells low-level Xist expression was detected from the single active X chromosome (Xa) in male cells and on both Xa's in female cells. Upon differentiation Xist expression was detected only in female cells, in which Xist RNA colocalized with the entire inactive X chromosome (Xi). Differentiated Dnmt mutant ES cells or cells of mutant postgastrulation embryos showed aberrant patterns of Xist expression: Xist transcripts colocalized with the single X chromosome in male cells and with both X chromosomes in female cells. X-linked gene expression was not detected from chromosomes coated with Xist RNA. These results suggest that ectopic Xist expression, induced by DNA hypomethylation, may lead to the inactivation of X-linked genes. We conclude that Xist-mediated X chromosome inactivation can occur in the absence of DNA methylation, arguing that DNA methylation may be required to repress Xist expression for the maintenance of a transcriptionally active Xa. In differentiated Dnmt mutant ES cells the activation of Xist expression correlated with a dramatic increase in apoptotic bodies, suggesting that Xist-mediated X chromosome inactivation may result in cell death and contribute to the embryonic lethality of the Dnmt mutation.

Animals↗

PubMed1996

A 450 kb transgene displays properties of the mammalian X-inactivation center.

X inactivation results in inactivation of one X chromosome to compensate for gene dosage differences between mammalian females and males. It requires the X-inactivation center (Xic) and Xist in cis. We report that introducing 450 kb of murine Xic/Xist sequences onto autosomes activates female dosage compensation in male ES cells. Xist is induced upon differentiation and can be expressed from both endogenous and ectopic loci, suggesting that elements for counting and choosing Xs are present in the transgene. Differentiating transgenic ES cells undergo excessive cell death. Postnatally, Xist is expressed only from the transgene. Ectopic Xist RNA structurally associates with the autosome and may inactivate a marker gene in cis. These results argue that the Xic is contained within 450 kb and that these sequences are sufficient for chromosome counting, choosing, and initiation of X inactivation.

Animals↗

PubMed1996

Transcription factor AP-2 essential for cranial closure and craniofacial development.

During closure of the neural tube in the mouse, transcription factor AP-2 is expressed in ectoderm and in neural-crest cells migrating from the cranial neural folds. Cranial neural crest cells provide patterning information for craniofacial morphogenesis, generate most of the skull bones, and together with placodal ectoderm, form the cranial ganglia. To study the role of AP-2 during embryogenesis, we undertook a targeted mutagenesis of the AP-2 gene in the mouse. Here we report that AP-2(-/-) mice died perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face, skull, sensory organs and cranial ganglia. Failure of cranial closure between 9 and 9.5 days postcoitum coincided with increased apoptosis in the midbrain, anterior hindbrain and proximal mesenchyme of the first branchial arch, but did not involve loss of expression of twist or Pax-3, two other regulatory genes known to be required for cranial closure.

Animals↗

PubMed1996

Deletion of integrin alpha 1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion.

Integrin alpha 1 is a receptor for laminin and collagen which is expressed widely and dynamically in embryogenesis and has been implicated in various developmental processes including establishment of the placenta and formation of the central and peripheral nervous system. In the adult it is the sole collagen receptor in smooth muscle and liver and is thought to be important for the stability of these tissues. We have generated a null allele of the alpha 1 gene in the germline of mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutation are viable and fertile and have no overt phenotype, demonstrating that the molecule is not required for development. Embryonic fibroblasts derived from mutant animals are unable to spread on or migrate into substrata of collagen IV and are deficient in spreading on and migrating into laminin. Further in vitro analysis of cell spreading and migration suggests that alpha 1 beta 1 is not required for binding to collagen I and implicates a third receptor, possibly integrin alpha 3 beta 1, in collagen I binding.

Alleles↗

PubMed1996

Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes.

Embryonic stem (ES) cells homozygous for a disruption of the DNA (cytosine-5)-methyltransferase gene (Dnmt) proliferate normally with their DNA highly demethylated but die upon differentiation. Expression of the wild-type Dnmt cDNA in mutant male ES cells caused an increase in methylation of bulk DNA and of the Xist and Igf2 genes to normal levels, but did not restore the methylation of the imprinted genes H19 and Igf2r. These cells differentiated normally in vitro and contributed substantially to adult chimeras. While the Xist gene was not expressed in the remethylated male ES cells, no restoration of the normal expression profile was seen for H19, Igf2r, or Igf2. This indicates that ES cells can faithfully reestablish normal methylation and expression patterns of nonimprinted genes but lack the ability to restore those of imprinted genes. Full restoration of monoallelic methylation and expression was imposed on H19, Igf2, and Igf2r upon germ-line transmission. These results are consistent with the presence of distinct de novo DNA methyltransferase activities during oogenesis and spermatogenesis, which specifically recognize imprinted genes but are absent in the postimplantation embryo and in ES cells.

Alleles↗

PubMed1996

Functional redundancy of the muscle-specific transcription factors Myf5 and myogenin.

The myogenic basic helix-loop-helix transcription factors, Myf5, MyoD, myogenin and MRF4, play key roles in skeletal muscle development. All of them induce myogenic differentiation in cultured non-muscle cells, suggesting that they might be functionally redundant. But the genes are expressed at different times during embryogenesis and mice carrying a mutation in any of the genes have different phenotypes. A rib cage defect was observed in Myf5-deficient mice, which die perinatally. We investigated whether the rib cage defect was due to the failure of the early activation of the gene or to the unique interactions of Myf5 with specific downstream targets. For this we inserted a myogenin complementary DNA into the Myf5 locus by homologous recombination which simultaneously disrupted Myf5 function. We report here that mice homozygous for this myogenin gene knock-in (ki) developed a normal rib cage and were viable, therefore demonstrating functional redundancy of Myf5 and myogenin for rib formation.

Animals↗

PubMed1996

Type I collagen-deficient Mov-13 mice do not retain SPARC in the extracellular matrix: implications for fibroblast function.

The Mov-13 strain of mice was created by the insertion of the murine Moloney leukemia virus into the first intron of the alpha 1 (I) collagen gene. Consequently, Mov-13 embryos do not transcribe alpha 1 (I) collagen mRNA and lack type I collagen protein in the extracellular matrix (ECM). Homozygotes die within 12-14 days of embryonic development, in part from the rupture of large blood vessels, and also exhibit deficiencies in hematopoesis and assembly of the ECM (Lohler et al. [1984] Cell 38:597-607). Several matricellular proteins, proteoglycans, and growth factors bind to type I collagen, e.g., fibronectin, secreted protein acidic and rich in cysteine (SPARC), decorin, and transforming growth factor-beta. Here we investigate the expression and function of SPARC in the absence of type I collagen. We show that fibroblasts isolated from Mov-13 homozygous, heterozygous, and wild-type embryos transcribed and translated SPARC mRNA in vitro. However, accumulation of extracellular SPARC was severely affected in the tissues of Mov-13 homozygotes, whereas extracellular deposition of the secreted glycoproteins fibronectin and type III collagen was not altered. Since SPARC has been shown to be a regulator of cell shape, the functional consequences of the absence of extracellular SPARC were evaluated in collagen gel contraction assays. Fibroblasts isolated from homozygous Mov-13 mice did not contract native type I collagen gels as efficiently as fibroblasts from heterozygous littermates; however, addition of exogenous SPARC enhanced the contraction of collagen by homozygous Mov-13 fibroblasts. The stimulatory effect of SPARC was blocked by antibodies specific for the amino terminus of the protein. These results provide evidence that type I collagen is one of the major extracellular proteins that binds SPARC in vivo. Furthermore, the capacity of fibroblasts to contract ECM in vitro is enhanced by extracellular SPARC. We therefore propose that the remodeling of ECM by cells in vivo is regulated in part by a specific interaction between SPARC and type I collagen.

Animals↗

PubMed1996

Clonal cell lines produced by infection of neocortical neuroblasts using multiple oncogenes transduced by retroviruses.

Toward understanding the early molecular development of neocortical neurons, we report the generation of two clonal murine cell lines derived by sequential oncogenic retroviral infection of neocortical neuronal precursors. The resulting cell lines stably express the telencephalon-specific gene BF-1 and a gene enriched in the neocortical ventricular zone, vzg-1. They also express early neuronal but not glial markers, possess neuron-like processes without chemical synapses by ultrastructure, and appear pyramidal or bipolar with processes resembling apical dendrites, bifurcating and beaded axons, or growth cones. Immortalization appears to have occurred through the expression of oncogene combinations SV40 large T with vras or SV40 large T with both vsrc and vmyc. These cell lines may represent developing neocortical neuroblasts blocked from complete differentiation, and they should be useful in the isolation and analysis of genes involved in early neocortical development.

Animals↗

PubMed1996

Experimental manipulation of genomic methylation.

Cytosine methylation is an important mechanism of gene regulation in mammals. Mouse embryos with reduced DNA methylation due to targeted disruption of the DNA methyltransferase gene show deregulated expression of imprinted genes. Loss of imprinting associated with loss of allele-specific methylation is one example of an epigenetic alteration found in tumor cells. Changes in DNA methylation may also be associated with facilitating protooncogene expression and inactivating tumor suppressor genes. However, cytosine methylation has additional deleterious consequences for the genome as well. CpG dinucleotides, the target of DNA methylation, are five-fold underpresented in the genome due to the high mutability of methylated cytosine. C-T transition mutations resulting from deamination of 5-methylcytosine are involved in both genetic disease and cancer. Lastly, aberrant DNA methylation may promote the genetic instability of a chromosomal locus. We review the genetic and epigenetic roles for DNA methylation during tumorigenesis gleaned from altered methycytosine patterns in tumor cells, and from pharmacologic, dietary or genetic manipulation of DNA methylation levels.

Animals↗

PubMed1996

The role of DNA methylation in cancer genetic and epigenetics.

The past few years have seen a wider acceptance of a role for DNA methylation in cancer. This can be attributed to three developments. First, the documentation of the over-representation of mutations at CpG dinucleotides has convincingly implicated DNA methylation in the generation of oncogenic point mutations. The second important advance has been the demonstration of epigenetic silencing of tumor suppressor genes by DNA methylation. The third development has been the utilization of experimental methods to manipulate DNA methylation levels. These studies demonstrate that DNA methylation changes in cancer cells are not mere by-products of malignant transformation, but can play an instrumental role in the cancer process. It seems clear that DNA methylation plays a variety of roles in different cancer types and probably at different stages of oncogenesis. DNA methylation is intricately involved in a wide diversity of cellular processes. Likewise, it appears to exert its influence on the cancer process through a diverse array of mechanisms. It is our task not only to identify these mechanisms, but to determine their relative importance for each stage and type of cancer. Our hope then will be to translate that knowledge into clinical applications.

Animals↗

PubMed1996

De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells.

It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

Animals↗

PubMed1996

Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis.

A mutation was targeted to the murine alpha3 integrin gene. Homozygous mutant mice survived to birth, but died during the neonatal period. The mutation caused abnormal kidney and lung development. Mutant kidneys displayed decreased branching of the medullary collecting ducts, although the number of nephrons was not altered. Proximal tubules exhibited two distinct subsets of abnormalities, with the epithelial cells either containing excess lysosomes or becoming microcystic. In addition, glomerular development was markedly affected. In mutant kidneys, the extent of branching of glomerular capillary loops was decreased, with capillary lumina being wider than normal. The glomerular basement membrane was disorganized and glomerular podocytes were unable to form mature foot processes. Branching of the bronchi in lungs of mutant mice was also decreased and the large bronchi extended to the periphery. These results indicate a role for integrin receptors in basement membrane organization and branching morphogenesis.

Animals↗