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Biomedical subjects

P A Overbeek

Publications and source records attributed to P A Overbeek.

At least 37 records · Page 2Linked to original sources
PubMed1999

Insertional mutation of the collagen genes Col4a3 and Col4a4 in a mouse model of Alport syndrome.

Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.

Animals↗
PubMed1999

Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression.

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+ channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+ channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+ channel gene. Paradoxically, we find that addition of the extra K+ channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+ channel gene dosage leads to dysregulation of normal K+ channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.

Animals↗
PubMed1999

Anomalous development of the hepatobiliary system in the Inv mouse.

Extrahepatic biliary atresia (BA) is a devastating disease of the neonate in which the hepatic and/or common bile duct is obliterated or interrupted. Infants and children with this diagnosis constitute 50% to 60% of the pediatric population that undergoes orthotopic liver transplantation. However, there is still very little known about the etiology and pathogenesis of BA. Several recent studies have demonstrated that anomalies of situs determination are more commonly associated with BA than previously recognized. In this study, we examined the pathogenesis of jaundice in the inv mouse, a transgenic mouse in which a recessive deletion of the inversin gene results in situs inversus and jaundice. The results show that these mice have cholestasis with conjugated hyperbilirubinemia, failure to excrete technetium-labeled mebrofenin from the liver into the small intestine, lack of continuity between the extrahepatic biliary tree and the small intestine as demonstrated by Trypan blue cholangiography, and a liver histological picture indicative of extrahepatic biliary obstruction with negligible inflammation/necrosis within the hepatic parenchyma. Lectin histochemical staining of biliary epithelial cells in serial sections suggests the presence of several different anomalies in the architecture of the extrahepatic biliary system. These results suggest that the inversin gene plays an essential role in the morphogenesis of the hepatobiliary system and raise the possibility that alterations in the human orthologue of inversin account for some of the cases of BA in which there are also anomalies of situs determination.

Animals↗
PubMed1999

Ectopic gland induction by lens-specific expression of keratinocyte growth factor (FGF-7) in transgenic mice.

During mammalian embryogenesis, epithelial-mesenchymal interactions play a determining role in normal tissue patterning and development. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a mesenchymally-derived mitogen for epithelial cells. As the KGF receptor is expressed by epithelial cells of numerous tissues and KGF is produced in adjacent stromal cells, KGF is thought to play a role in mediating epithelial cell behaviour. To further investigate the role of this molecule in the development of ocular epithelia we employed transgenic mice engineered to overexpress human KGF in the eye. The most striking phenotypic development was the hyperproliferation of embryonic corneal epithelial cells and their subsequent differentiation into functional lacrimal gland-like tissues. This indicates that stimulation of the KGF receptor early in development, in surface ectoderm normally destined to form corneal epithelium, is sufficient to alter the fate of these cells. Furthermore, this suggests that the correct spatial and temporal expression of FGFs plays a critical role in normal lacrimal gland induction. These transgenic mice provide a valuable model system to study the mechanisms underlying cell fate decisions during ocular morphogenesis.

Animals↗
PubMed1999

Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia results in abnormal morphogenesis of teeth, hair and eccrine sweat glands. The gene (ED1) responsible for the disorder has been identified, as well as the analogous X-linked gene (Ta) in the mouse. Autosomal recessive disorders, phenotypically indistinguishable from the X-linked forms, exist in humans and at two separate loci (crinkled, cr, and downless, dl) in mice. Dominant disorders, possibly allelic to the recessive loci, are seen in both species (ED3, Dlslk). A candidate gene has recently been identified at the dl locus that is mutated in both dl and Dlslk mutant alleles. We isolated and characterized its human DL homologue, and identified mutations in three families displaying recessive inheritance and two with dominant inheritance. The disorder does not map to the candidate gene locus in all autosomal recessive families, implying the existence of at least one additional human locus. The putative protein is predicted to have a single transmembrane domain, and shows similarity to two separate domains of the tumour necrosis factor receptor (TNFR) family.

Alleles↗
PubMed1999

Involvement of a novel Tnf receptor homologue in hair follicle induction.

Although inductive interactions are known to be essential for specification of cell fate in many vertebrate tissues, the signals and receptors responsible for transmitting this information remain largely unidentified. Mice with mutations in the downless (dl) gene have defects in hair follicle induction, lack sweat glands and have malformed teeth. These structures originate as ectodermal placodes, which invaginate into the underlying mesenchyme and differentiate to form specific organs. Positional cloning of the dl gene began with identification of the transgenic family OVE1. One branch of the family, dl(OVE1B), carries an approximately 600-kb deletion at the dl locus caused by transgene integration. The mutated locus has been physically mapped in this family, and a 200-kb mouse YAC clone, YAC D9, has been identified and shown to rescue the dl phenotype in the spontaneous dl(Jackson) (dl(J), recessive) and Dl(sleek) (Dl(slk), dominant negative) mutants. Here we report the positional cloning of the dl gene, which encodes a novel member of the tumour necrosis factor (Tnf) receptor (Tnfr) family. The mutant phenotype and dl expression pattern suggests that this gene encodes a receptor that specifies hair follicle fate. Its ligand is likely to be the product of the tabby (Ta) gene, as Ta mutants have a phenotype identical to that of dl mutants and Ta encodes a Tnf-like protein.

Amino Acid Sequence↗
PubMed1999

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.

Animals↗
PubMed1998

Cloning of inv, a gene that controls left/right asymmetry and kidney development.

Most vertebrate internal organs show a distinctive left/right asymmetry. The inv (inversion of embryonic turning) mutation in mice was created previously by random insertional mutagenesis; it produces both a constant reversal of left/right polarity (situs inversus) and cyst formation in the kidneys. Asymmetric expression patterns of the genes nodal and lefty are reversed in the inv mutant, indicating that inv may act early in left/right determination. Here we identify a new gene located at the inv locus. The encoded protein contains 15 consecutive repeats of an Ank/Swi6 motif at its amino terminus. Expression of the gene is the highest in the kidneys and liver among adult tissues, and is seen in presomite-stage embryos. Analysis of the transgenic genome and the structure of the candidate gene indicate that the candidate gene is the only gene that is disrupted in inv mutants. Transgenic introduction of a minigene encoding the candidate protein restores normal left/right asymmetry and kidney development in the inv mutant, confirming the identity of the candidate gene.

Amino Acid Sequence↗
PubMed1998

Disregulation of ocular morphogenesis by lens-specific expression of FGF-3/int-2 in transgenic mice.

FGF-3, originally named int-2, was discovered as an oncogene frequently activated in mammary carcinomas resulting from the chromosomal integration of the mouse mammary tumor virus (MMTV). Int-2 was later designated FGF-3 based on sequence homology with other members of the fibroblast growth factor (FGF) family. FGF-1 is the prototypical member of the FGF family, and is the only family member which activates all known FGF receptor isoforms. Transgenic mice expressing in the lens a form of FGF-1 engineered to be secreted show premature differentiation of the entire lens epithelium. In contrast, transgenic mice engineered to secrete FGF-2 in the lens do not undergo premature differentiation of the lens epithelium (C. M. Stolen et al., 1997, Development 124, 4009-4017). To further assess the roles of FGFs and FGF receptors in lens development, the alpha A-crystallin promoter was used to target expression of FGF-3 to the developing lens of transgenic mice. The expression of FGF-3 in the lens rapidly induced epithelial cells throughout the lens to elongate and to express fiber cell-specific proteins including MIP and beta-crystallins. This premature differentiation of the lens epithelium was followed by the degeneration of the entire lens. Since FGF-1 and FGF-3 can both activate one FGF receptor isoform (FGFR2 IIIb) that is not activated by FGF-2, these results suggest that activation of FGFR2 IIIb is sufficient to induce fiber cell differentiation throughout the lens epithelium in vivo. Furthermore, transgenic lens cells expressing FGF-3 were able to induce the differentiation of neighboring nontransgenic lens epithelial cells in chimeric mice. Expression of FGF-3 in the lens also resulted in developmental alterations of the eyelids, cornea, and retina, and in the most severely affected transgenic lines, the postnatal appearance of intraocular glandular structures.

Animals↗
PubMed1998

The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness.

dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. The HAND genes have a complementary left-right cardiac asymmetry of expression with dHAND predominantly on the right side and eHAND on the left side of the looped heart tube. Here we show that although eHAND is asymmetrically expressed along the anterior-posterior and dorsal-ventral embryonic axes, it is symmetrically expressed along the left-right axis at early stages of embryonic and cardiac development. After cardiac looping, dHAND and eHAND are expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left-right (LR) sidedness of dHAND and eHAND expression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however, dHAND expression persists in the pulmonary ventricle and eHAND in the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. Previously we showed that dHAND-null mice fail to form a right-sided pulmonary ventricle. Here mice homozygous for the dHAND and inv mutations are demonstrated to have only a right-sided ventricle which is morphologically a left (systemic) ventricle. These data suggest that the HAND genes are involved in development of segments of the heart tube which give rise to specific chambers of the heart during cardiogenesis, rather than controlling the direction of cardiac looping by interpreting the cascade of LR embryonic signals.

Animals↗
PubMed1998

Lens-specific expression of transforming growth factor beta1 in transgenic mice causes anterior subcapsular cataracts.

Transgenic mice were generated by microinjection of a construct containing a self-activating human TGF-beta1 cDNA driven by the lens-specific alphaA-crystallin promoter. Seven transgenic founder mice were generated, and four transgenic lines expressing TGF-beta1 were characterized. By postnatal day 21, mice from the four families exhibited anterior subcapsular cataracts. The lenses in these mice developed focal plaques of spindle-shaped cells that expressed alpha-smooth muscle actin, and that resembled the plaques seen in human anterior subcapsular cataracts. Transgenic mice showed additional ocular defects, including corneal opacification and structural changes in the iris and ciliary body. The corneal opacities were associated with increased exfoliation of the squamous layer of the corneal epithelium and increased DNA replication in the basal epithelium.

Animals↗
PubMed1998

YAC rescue of downless locus mutations in mice.

Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10. To determine which of the YACs contain the dl gene, we generated YAC transgenic mice by mouse embryo microinjections. The 200-kb YAC B25.D9 was found to rescue all of the downless defects. In addition, the transgenic YAC rescued the dominant Sleek (Dlslk) allele. Since the sequences within the YAC are entirely deleted in one of the transgenic mutants, our results establish that Sleek encodes a dominant-negative protein whose effects can be reversed by expression of extra copies of the wild-type locus.

Alleles↗
PubMed1998

Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice.

Fibroblast growth factors (FGFs), such as FGF-1, have been shown to induce differentiation of lens epithelial cells both in tissue culture and in transgenic mice. In the present study, using the alpha A-crystallin promoter, we generated transgenic mice that express different FGFs (FGF-4, FGF-7, FGF-8, FGF-9) specifically in the lens. All four FGFs induced changes in ocular development. Microphthalmic eyes were evident in transgenic mice expressing FGF-8, FGF-9 and some lines expressing FGF-4. A developmental study of the microphthalmic eyes revealed that, by embryonic day 15, expression of these FGFs induced lens epithelial cells to undergo premature fiber differentiation. In less severely affected lines expressing FGF-4 or FGF-7, the lens epithelial cells exhibited a premature exit from the cell cycle and underwent a fiber differentiation response later in development, leading to cataract formation. The responsiveness of lens cells to different FGFs indicates that these proteins stimulate the same or overlapping downstream signalling pathway(s). These overlapping effects of different FGFs on a common cell type indicate that the normal developmental roles for these genes are determined by the temporal and spatial regulation of their expression patterns. The fact that any of these FGFs can induce ocular defects and loss of lens transparency implies that it is essential for the normal eye to maintain very specific spatial control over FGF expression in order to prevent cataract induction.

Animals↗
PubMed1997

Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo.

Mouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations. However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology. Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells. Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alphaMyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alphaMyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein. Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells. Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.

Adenoviridae↗
PubMed1997

Inhibition of cell death by lens-specific overexpression of bcl-2 in transgenic mice.

Previous studies on cell cycle regulation in the ocular lens using transgenic mice have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause postmitotic lens fiber cells to enter the cell cycle. However, when the p53 gene and protein are intact, inactivation of pRb in this terminally differentiated cell type results in cell death, rather than continued proliferation. Since bcl-2 has been shown to act as a cell death repressor, the ability of this gene to block p53-dependent apoptosis in lenses was examined. Transgenic mice were generated that overexpress bcl-2 in a lens-specific fashion. Surprisingly, overexpression of bcl-2 was sufficient to interfere with normal fiber cell differentiation, inducing cataracts, microphakia, vacuolization, fiber cell disorganization, and inhibition of fiber cell denucleation. The bcl-2 mice were mated to mice exhibiting lens-specific expression of the N-terminal region of simian virus 40 large T antigen (termed truncT). The resulting double transgenic mice showed a marked reduction in the truncT-induced fiber cell death. Apoptosis in the truncT mice could also be suppressed by crossing these mice into a p53-deficient background. Either overexpression of bcl-2 or loss of p53 in truncT mice resulted in proliferation of fiber cells around the cortex of the lens. These proliferating fiber cells continue to express beta- and gamma-crystallin proteins, which are normally only expressed following withdrawal from the cell cycle. The p53 protein is known to upregulate expression of certain target genes, including p21, a protein that can block cell cycle progression by inhibition of cyclin-dependent kinases. In order to assess whether bcl-2 interferes with the transcriptional activation activity of p53, transgenic lenses were assayed by in situ hybridization for levels of p21 expression. Lenses that expressed both truncT and bcl-2 showed elevated p21, implying that bcl-2 does not inhibit apoptosis by directly inhibiting p53, but instead may block a later step in the apoptosis pathway. In addition, overexpression of p21 is not sufficient to cause apoptosis. These experiments show that the lenses of transgenic mice represent a valuable in vivo setting for studies of both induction and inhibition of programmed cell death.

Animals↗
PubMed1997

Expression of testis angiotensin-converting enzyme is mediated by a cyclic AMP responsive element.

Testis angiotensin-converting enzyme (testis ACE), an ACE isozyme that plays an important role in male fertility, is transcribed from a unique promotor active only in developing spermatids. In vitro analysis suggests the importance of a cyclic AMP response element (CRE)-like region within the testis ACE promoter, and similar DNA motifs are important in the expression of a variety of testis-specific genes. In the present study, we examined the effects of mutations in the CRE-like element on testis ACE promoter activity in vivo using transgenic mice. Disruption of this element reduced reporter gene expression to near background levels. In contrast, conversion of the CRE-like element to a consensus CRE-binding site resulted in high level expression of the reporter gene specifically in the testis. These experiments prove that the CRE-like element is essential for testis ACE promoter activity, although it does not appear to be responsible for its tissue specificity. These data provide insight into how a phenotypically differentiated tissue, ie, male gem cells, regulate tissue-specific gene expression.

Animals↗
PubMed1996

Lens-specific expression of PDGF-A alters lens growth and development.

The vertebrate lens provides an in vivo model to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development by in situ hybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-alpha receptor (PDGF-alphaR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-alphaR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens development in vivo, we generated transgenic mice that express human PDGF-A in the lens under the control of the alphaA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific beta-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cells in vivo. Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.

Aging↗