Biomedical subjects

T Doetschman

Publications and source records attributed to T Doetschman.

At least 55 records · Page 3Linked to original sources

PubMed1997

TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes.

The growth and differentiation factor transforming growth factor-beta2 (TGFbeta2) is thought to play important roles in multiple developmental processes. Targeted disruption of the TGFbeta2 gene was undertaken to determine its essential role in vivo. TGFbeta2-null mice exhibit perinatal mortality and a wide range of developmental defects for a single gene disruption. These include cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital defects. The developmental processes most commonly involved in the affected tissues include epithelial-mesenchymal interactions, cell growth, extracellular matrix production and tissue remodeling. In addition, many affected tissues have neural crest-derived components and simulate neural crest deficiencies. There is no phenotypic overlap with TGFbeta1- and TGFbeta3-null mice indicating numerous non-compensated functions between the TGFbeta isoforms.

Abnormalities, Multiple↗

PubMed1996

Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

Alleles↗

PubMed1996

Liver and intestinal fatty acid binding proteins in control and TGF beta 1 gene targeted deficient mice.

The effect of transforming growth factor beta-1 (TGF beta 1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF beta 1-deficient mice. Homozygous TGF beta 1-deficient 129 x CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF beta 1-deficient/immunodeficient C3H mice on a SCID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (I-FABP) which decreased 3-fold in the TGF beta 1-deficient/immunodeficient C3H mice only. Thus, TGF beta 1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.

Animals↗

PubMed1996

Phospholamban gene dosage effects in the mammalian heart.

Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.

Adenosine Triphosphatases↗

PubMed1996

Gastric lesions in transforming growth factor beta-1 heterozygous mice.

Transforming growth factor beta-1 (TGF beta 1) is known to inhibit the growth of many epithelial cell types in culture. Consequently, it is important to determine whether it has any tumor suppressor activity in vitro. Fifteen heterozygous and eight wild type TGF beta 1-deficient mice were examined to determine if there was a difference in lifespan or lesion development due to the loss of one TGF beta 1 allele. Mice were killed when there was evidence of neoplasia or severe illness. There was no significant difference in the lifespan of the two groups. Hyperplastic lesions in the glandular mucosa were seen in 10 TGF beta 1 (+/-) mice. These lesions were localized to the lesser curvature of the stomach, extending from the limiting ridge to the pylorus. Seven of the 10 glandular hyperplastic lesions in the TGF beta 1 (+/-) mice had features similar to human gastritis cystica profunda. Associated with the glandular invasion of the muscularis were a mixed inflammatory infiltration of the surrounding muscular wall and mucosa with chronic vasculitis in the tissues adjacent to these lesions. In contrast to the distinct genotypic differences in lesion incidence observed in the glandular stomach, there was no significant difference in lesion incidence in other organs. The increased incidence of the hyperplastic lesions in the TGF beta 1 (+/-) mice is highly suggestive that allelic loss of TGF beta 1 plays an important role in the genesis of these lesions. However, allelic loss of TGF beta 1 does not cause alterations in the incidence of neoplasia.

Alleles↗

PubMed1996

Keratin 12-deficient mice have fragile corneal epithelia.

PURPOSE: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS: One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS: The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS: The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.

Animals↗

PubMed1995

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Animals↗

PubMed1995

Effect of transforming growth factor-beta 1 and -beta 2 on Schwann cell proliferation on neurites.

Mechanisms regulating Schwann cell proliferation during development are unclear. Schwann cell division is known to be driven by an unidentified mitogen present on the surface of axons, but it is not known whether other molecules play a role in regulating this proliferation. Transforming growth factor-beta (TGF-beta) which is found in the developing peripheral nervous system (PNS) and is mitogenic for neuron-free Schwann cells in vitro could be involved. We have investigated the effects of TGF-beta 1, TGF-beta 2 and antibodies to TGF-beta 1 and TGF-beta 2 on axon driven Schwann cell proliferation. Rat embryonic dorsal root ganglion neurons (DRG) neurons and Schwann cells from the sciatic nerve were isolated, purified and recombined in vitro. Confirming earlier reports by others, we observed that TGF-beta 1 and TGF-beta 2 added to the culture medium stimulated the proliferation of Schwann cells in the absence of neurons. However, when added to neuron-Schwann cell co-cultures, TGF beta caused a variable response ranging from no effect to moderate inhibition of Schwann cell proliferation in different experiments. A stimulation of Schwann cell proliferation by TGF beta was never observed in neuron-Schwann cell co-cultures. Antibodies to TGF-beta 1 and TGF-beta 2 did not influence axon driven Schwann cell proliferation. To further determine the role of TGF-beta in Schwann cell proliferation and myelination, we studied Schwann cell proliferation in cultures from mice in which the TGF-beta 1 gene was delected by homologous recombination. Neuron-Schwann cell cultures from wild-type, heterozygous and homozygous mice were used. No differences were observed in either Schwann cell proliferation or myelination between cultures obtained from homozygous mutants and their heterozygous and wild-type controls. These findings suggest that TGF-beta does not function as a part of the mitogenic mechanism presented by neurons to Schwann cells, but that the presence of active TGF beta in the cellular environment might regulate the degree of proliferation induced by neuronal contact.

Animals↗

PubMed1995

Transforming growth factor-beta 3 is required for secondary palate fusion.

Mice lacking TGF-beta 3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-beta 3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.

Animals↗

PubMed1995

Onset and progression of pathological lesions in transforming growth factor-beta 1-deficient mice.

Null-mutant (knockout) mice were obtained through disruption of the sixth exon of the endogenous transforming growth factor-beta 1 allele in murine embryonic stem cells via homologous recombination. Mice lacking transforming growth factor-beta 1 (mutants) were born grossly indistinguishable from wild-type littermates. With time, mutant mice exhibited a wasting phenotype that manifested itself in severe weight loss and dishevelled appearance (between 15 and 36 days of age). Examination of these moribund mice histologically revealed that transforming growth factor-beta 1-deficient mice exhibit a moderate to severe, multifocal, organ-dependent, mixed inflammatory cell response adversely affecting the heart, stomach, diaphragm, liver, lung, salivary gland, and pancreas. Because of the known multifunctional nature of transforming growth factor-beta 1 on the control of growth and differentiation of many different cell types, it is important to determine the degree to which the inflammatory response interacts with or masks other deficiencies that are present. To this end, we examined the extent and nature of the inflammatory lesions in different ages of neonatal knockout mice (5, 7, 10, and 14 days of age) and older moribund mice (> 15 days of age) and compared them with the histology seen in wild-type normal animals. Mild inflammatory infiltrates were first observed in 5-day mutant mice in the heart, by day 7 in the lung, salivary gland, and pancreas, and by day 14 inflammatory lesions were found in almost all organs examined. Moderate to severe inflammation was not present until the mice were 10 to 14 days old. In the older animals, there was a slight increase in the severity of the inflammatory lesions as the mice aged.

Animals↗

PubMed1994

Transforming growth factor-beta 1 in reproduction and development.

Expression patterns of TGF-beta s during embryogenesis and in adult reproductive organs, as well as the activities of these molecules in in vitro assays of biological processes relating to reproduction and development, have suggested that TGF-beta s may play a role in both reproductive function and embryonic development. To investigate the function of TGF-beta 1 in vivo, the murine TGF-beta 1 gene was disrupted by gene targeting, and animals that lacked TGF-beta 1 activity were generated. Homozygous mutant animals were obtained which exhibited a multifocal inflammatory disease. However, the observed numbers of homozygous mutant offspring were less than expected, suggesting the occurrence of some type of prenatal lethality. This paper reviews the proposed role of the TGF-beta s in reproductive and developmental processes and discusses observations obtained from the TGF-beta 1 gene-targeting experiments as they relate to these processes.

Animals↗

PubMed1994

Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to beta-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (+/- dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca(2+)-ATPase for Ca2+ in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the beta-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the heart's contractile responses to beta-adrenergic agonists.

Animals↗

PubMed1993

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

Amino Acid Sequence↗

PubMed1993

Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.

Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders.

Amino Acid Sequence↗

PubMed1993

Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders.

To better understand the formation of the cardiovascular system and its disease states, models amenable to manipulation must be developed. In this article we present two models. One is a small animal model for an inflammatory disorder that can lead to heart failure. Production of this model is based on the ability of blastocyst-derived embryonic stem cells, which can be genetically altered in vitro by a technique called gene targeting, to reconstitute an entire animal when reintroduced into a blastocyst and allowed to colonize the germ line of the resulting chimeric embryo. The other model is based on the capacity of embryonic stem cells to differentiate in culture into embryo-like structures called embryoid bodies. Embryoid bodies contain angioblasts, or prevascular endothelial cells, which can be induced to undergo aspects of vascular development by manipulation of culture conditions.

Animals↗

PubMed1992

Mouse phospholamban gene expression during development in vivo and in vitro.

To establish a murine model that may allow for definition of the precise role of phospholamban in myocardial contractility through selective perturbations in the phospholamban gene, we initiated studies on the role of phospholamban in the murine heart. Intact beating hearts were perfused in the absence or presence of isoproterenol, and quantitative measurements of cardiac performance were obtained. Isoproterenol stimulation was associated with increases in the affinity of the sarcoplasmic reticulum Ca2+ pump for Ca2+ that were due to phospholamban phosphorylation. To assess the regulation of phospholamban gene expression during murine development, Northern blot and polymerase chain reaction analyses were used. Phospholamban mRNA was first detected in murine embryos on the ninth day of development (the time when the cardiac tube begins to contract). In murine embryoid bodies, which have been shown to recapitulate several aspects of cardiogenesis, phospholamban mRNA was detected on the seventh day (the time when spontaneous contractions are first observed). Only those embryoid bodies that exhibited contractions expressed phospholamban transcripts, and these were accompanied by expression of the protein, as revealed by immunofluorescence microscopy. Sequence analysis of the cDNA encoding phospholamban in embryoid bodies indicated complete homology to that in adult hearts. The deduced amino acid sequence of murine phospholamban was identical to rabbit cardiac phospholamban but different from dog cardiac and human cardiac phospholamban by one amino acid. These data suggest that phospholamban, the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, is present very early in murine cardiogenesis in utero and in vitro, and this may constitute an important determinant for proper development of myocardial contractility.

Amino Acid Sequence↗

PubMed1991

Myosin heavy chain gene expression in mouse embryoid bodies. An in vitro developmental study.

Embryoid bodies (EBs) are obtained when mouse pluripotential embryonic stem cells are grown in the absence of an embryonic fibroblast feeder layer. Seven- to 9-day-old EBs undergo rhythmical, spontaneous contractions and express the appropriate tissue- and developmental stage-specific cardiac and skeletal myosin heavy chain (MHC) genes. To study the expression patterns of these MHC genes in vitro we isolated and partially sequenced the cDNAs expressed in EBs such that specific oligonucleotides suitable for polymerase chain reaction analyses and appropriate riboprobes for in situ hybridizations could be made. The data show the beta-cardiac gene is expressed first during EB development (days 3 and 4), and alpha-cardiac gene expression begins at approximately day 8. A similar pattern of expression is also detected during mouse embryogenesis in utero. Only those EBs that expressed both the alpha- and beta-cardiac transcripts contracted. In situ hybridization of EBs using riboprobes shows that the spatial distribution of the cardiac MHC transcripts differs. No expression of the genes was detected in day 8 or older nonbeating EBs. These data suggest that developing EBs closely mimic the pre- and early postsomitic patterns of in vivo expression of the cardiac MHC genes and thus provide a useful system in which to study early aspects of mammalian cardiogenesis.

Amino Acid Sequence↗

PubMed1990

Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro.

In the mouse embryo, early organogenesis is characterized by the formation of a functional cardiac muscle, such that 9-day embryos exhibit beating, although not fully developed hearts. In light of this observation, we found it intriguing that mouse embryoid bodies (EB), which can develop in vitro from totipotential embryonic stem cells, undergo spontaneous contractile activity. To determine if these cells are capable of recapitulating aspects of cardiogenesis, a cDNA library was prepared from beating EB and screened with a chicken skeletal myosin heavy chain cDNA. We found that the predominant myosin transcripts in the library encode the alpha- and beta-cardiac isoforms. In addition, an embryonic skeletal myosin cDNA was isolated. The myosin heavy chain transcripts in both EB and 9-day embryonic hearts were found to be the same. Transcript-specific primers were prepared, and polymerase chain reaction analyses on single EB were carried out. The data show that a single EB is capable of expressing both the alpha- and beta-isoforms as well as very low amounts of the embryonic skeletal transcript. These data indicate that EB transcribe the appropriate tissue- and developmental stage-specific myosin heavy chain genes and therefore serve as a model system for studying early cardiogenic processes at the molecular level.

Animals↗