Biomedical subjects

P A Overbeek

Publications and source records attributed to P A Overbeek.

At least 19 recordsLinked to original sources

PubMed2001

Regulation of choroid development by the retinal pigment epithelium.

PURPOSE: The choroidal vasculature is essential for normal retinal function. However, mechanisms that control choroid development are unknown. In the present study, we provide evidence that the retinal pigment epithelium (RPE) plays an essential role in regulation of the choroid development in the mouse eye. METHODS: Transgenic mice that transiently express FGF9 in the embryonic RPE were generated. Postnatal eyes were analyzed by histology and in situ hybridizations. RESULTS: In the transgenic mice, most of the RPE was converted to neural retina. The choroid formed only in regions where patches of RPE were present. The choroid failed to develop in the absence of the RPE. CONCLUSIONS: The presence of the RPE appears to be required for choroid development, suggesting that molecular interactions between RPE and periocular mesenchyme are essential for melanocyte differentiation and vascular development in the choroid.

Animals↗

PubMed2001

Elevated TGFbeta signaling inhibits ocular vascular development.

Alterations in the ocular vasculature are associated with retinal diseases such as retinopathy of prematurity and diabetic retinopathy. Vascular endothelial growth factor (VEGF) as a potent stimulator for normal and abnormal vascular growth has been extensively studied. However, little is known about secreted factors that negatively regulate vascular growth in ocular tissues. We now report that expression of a self-activating TGFbeta1 in the ocular lens of transgenic mice results in inhibition of retinal angiogenesis followed by retinal degeneration. Transgenic TGFbeta1 can rescue the hyperplasic hyaloid tissue and reverse the corneal deficiency in TGFbeta2-null embryos. These results demonstrate that TGFbeta signaling modulates development of ocular vasculature and cornea in a dosage-dependent manner and that TGFbeta1 can substitute for TGFbeta2 in ocular tissues.

Animals↗

PubMed2001

Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival.

Cardiac muscle regeneration after injury is limited by "irreversible" cell cycle exit. Telomere shortening is one postulated basis for replicative senescence, via down-regulation of telomerase reverse transcriptase (TERT); telomere dysfunction also is associated with greater sensitivity to apoptosis. Forced expression of TERT in cardiac muscle in mice was sufficient to rescue telomerase activity and telomere length. Initially, the ventricle was hypercellular, with increased myocyte density and DNA synthesis. By 12 wk, cell cycling subsided; instead, cell enlargement (hypertrophy) was seen, without fibrosis or impaired function. Likewise, viral delivery of TERT was sufficient for hypertrophy in cultured cardiac myocytes. The TERT virus and transgene also conferred protection from apoptosis, in vitro and in vivo. Hyperplasia, hypertrophy, and survival all required active TERT and were not seen with a catalytically inactive mutation. Thus, TERT can delay cell cycle exit in cardiac muscle, induce hypertrophy in postmitotic cells, and promote cardiac myocyte survival.

Animals↗

PubMed2001

A transgenic insertion causing cryptorchidism in mice.

A distinctive feature of gonadal maturation in mammals is the movement to an extraabdominal location. Testicular descent is a complex, multistage process whereby the embryonic gonads migrate from their initial abdominal position to the scrotum. Failure in this process results in cryptorchidism, a frequent congenital birth defect in humans. We report here a new mouse transgenic insertional mutation, cryptorchidism with white spotting (crsp). Males homozygous for crsp exhibit a high intraabdominal position of the testes, associated with complete sterility. Heterozygous males have a wild-type phenotype, and homozygous females are fertile. Surgically descended testes in crsp/crsp males show normal spermatogenesis. Using FISH and genetic analyses, the transgenic insert causing the crsp mutation has been mapped to the distal part of mouse chromosome 5. Transgene integration resulted in a 550-kb deletion located upstream of the Brca2 gene. A candidate gene encoding a novel G protein-coupled receptor (Great) with an expression pattern suggesting involvement in testicular descent has been identified.

Amino Acid Sequence↗

PubMed2001

Misexpression of IGF-I in the mouse lens expands the transitional zone and perturbs lens polarization.

Insulin-like growth factor-I (IGF-I) has been implicated as a regulator of lens development. Experiments performed in the chick have indicated that IGF-I can stimulate lens fiber cell differentiation and may be involved in controlling lens polarization. To assess IGF-I activity on mammalian lens cells in vivo, we generated transgenic mice in which this factor was overexpressed from the alphaA-crystallin promoter. Interestingly, we observed no premature differentiation of lens epithelial cells. The pattern of lens polarization was perturbed, with an apparent expansion of the epithelial compartment towards the posterior lens pole. The distribution of immunoreactivity for MIP26 and p57(KIP2) and a modified pattern of proliferation suggested that this morphological change was best described as an expansion of the germinative and transitional zones. The expression of IGF-I signaling components in the normal transitional zone and expansion of the transitional zone in the transgenic lens both suggest that endogenous IGF-I may provide a spatial cue that helps to control the normal location of this domain.

Animals↗

PubMed2001

X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

Adolescent↗

PubMed2001

Requirement for TGFbeta receptor signaling during terminal lens fiber differentiation.

Several families of growth factors have been identified as regulators of cell fate in the developing lens. Members of the fibroblast growth factor family are potent inducers of lens fiber differentiation. Members of the transforming growth factor beta (TGFbeta) family, particularly bone morphogenetic proteins, have also been implicated in various stages of lens and ocular development, including lens induction and lens placode formation. However, at later stages of lens development, TGFbeta family members have been shown to induce pathological changes in lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFbeta receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in lens fiber differentiation. In this study we have investigated the consequences of disrupting TGFbeta signaling during lens fiber differentiation by using the mouse alphaA-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFbeta receptors in the lens fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus. Inhibition of TGFbeta signaling resulted in altered expression patterns of the fiber-specific proteins, alpha-crystallin, filensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFbeta signaling is a key event during fiber differentiation and is required for completion of terminal differentiation.

Actins↗

PubMed2001

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.

Animals↗

PubMed2001

Secreted FGFR3, but not FGFR1, inhibits lens fiber differentiation.

The vertebrate lens has a distinct polarity with cuboidal epithelial cells on the anterior side and differentiated fiber cells on the posterior side. It has been proposed that the anterior-posterior polarity of the lens is imposed by factors present in the ocular media surrounding the lens (aqueous and vitreous humor). The differentiation factors have been hypothesized to be members of the fibroblast growth factor (FGF) family. Though FGFs have been shown to be sufficient for induction of lens differentiation both in vivo and in vitro, they have not been demonstrated to be necessary for endogenous initiation of fiber cell differentiation. To test this possibility, we have generated transgenic mice with ocular expression of secreted self-dimerizing versions of FGFR1 (FR1) and FGFR3 (FR3). Expression of FR3, but not FR1, leads to an expansion of proliferating epithelial cells from the anterior to the posterior side of the lens due to a delay in the initiation of fiber cell differentiation. This delay is most apparent postnatally and correlates with appropriate changes in expression of marker genes including p57(KIP2), Maf and Prox1. Phosphorylation of Erk1 and Erk2 was reduced in the lenses of FR3 mice compared with nontransgenic mice. Though differentiation was delayed in FR3 mice, the lens epithelial cells still retained their intrinsic ability to respond to FGF stimulation. Based on these results we propose that the initiation of lens fiber cell differentiation in mice requires FGF receptor signaling and that one of the lens differentiation signals in the vitreous humor is a ligand for FR3, and is therefore likely to be an FGF or FGF-like factor.

Animals↗

PubMed2000

Endogenous and ectopic gland induction by FGF-10.

FGF-10, a member of the fibroblast growth factor family, is expressed in mesodermally derived cell populations during embryogenesis. During normal ocular development, FGF-10 is expressed in the perioptic mesenchyme adjacent to the Harderian and lacrimal gland primordia. In this report, we provide evidence that FGF-10 is both necessary and sufficient to initiate glandular morphogenesis. Lens-specific expression of FGF-10 was sufficient to induce ectopic ocular glands within the cornea. In addition, lacrimal and Harderian glands were not seen in FGF-10 null fetuses. Based on these results we propose that FGF-10 is an inductive signal that initiates ocular gland morphogenesis.

Animals↗

PubMed2000

Lens-specific VEGF-A expression induces angioblast migration and proliferation and stimulates angiogenic remodeling.

Vascular endothelial growth factor (VEGF) is a secreted mitogen which specifically stimulates proliferation of vascular endothelial cells in vitro and in vivo. Its expression pattern is consistent with it being an important regulator of vasculogenesis and angiogenesis, and targeted disruption of VEGF-A has demonstrated that it is essential for vascular development. To determine if VEGF-A was sufficient to alter vascularization in the eye we generated transgenic mice which express human VEGF-A(165) specifically in the lens. Expression of transgenic VEGF-A led to excessive proliferation and accumulation of disorganized angioblasts and endothelial cells around the lens. The results support the hypothesis that VEGF-A can initiate the process of vascularization by stimulating chemoattraction and proliferation of angioblasts and endothelial cells and that VEGF-A expression can stimulate angiogenic remodeling. However, VEGF-A alone was not sufficient to direct blood vessel organization or maturation.

Animals↗

PubMed2000

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Activating Transcription Factor 6↗

PubMed2000

A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse.

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.

Animals↗

PubMed2000

FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.

We investigated the mechanism of tissue induction and specification using the lacrimal gland as a model system. This structure begins its morphogenesis as a bud-like outgrowth of the conjunctival epithelium and ultimately forms a branched structure with secretory function. Using a reporter transgene as a specific marker for gland epithelium, we show that the transcription factor Pax6 is required for normal development of the gland and is probably an important competence factor. In investigating the cell-cell signaling required, we show that fibroblast growth factor (FGF) 10 is sufficient to stimulate ectopic lacrimal bud formation in ocular explants. Expression of FGF10 in the mesenchyme adjacent to the presumptive lacrimal bud and absence of lacrimal gland development in FGF10-null mice strongly suggest that it is an endogenous inducer. This was supported by the observation that inhibition of signaling by a receptor for FGF10 (receptor 2 IIIb) suppressed development of the endogenous lacrimal bud. In explants of mesenchyme-free gland epithelium, FGF10 stimulated growth but not branching morphogenesis. This suggested that its role in induction is to stimulate proliferation and, in turn, that FGF10 combines with other factors to provide the instructive signals required for lacrimal gland development.

Animals↗

PubMed2000

Regulation of mouse lens fiber cell development and differentiation by the Maf gene.

Maf is a basic domain/leucine zipper domain protein originally identified as a proto-oncogene whose consensus target site in vitro, the T-MARE, is an extended version of an AP-1 site normally recognized by Fos and Jun. Maf and the closely related family members Neural retina leucine zipper (Nrl), L-Maf, and Krml1/MafB have been implicated in a wide variety of developmental and physiologic roles; however, mutations in vivo have been described only for Krml1/MafB, in which a loss-of-function causes abnormalities in hindbrain development due to failure to activate the Hoxa3 and Hoxb3 genes. We have used gene targeting to replace Maf coding sequences with those of lacZ, and have carried out a comprehensive analysis of embryonic expression and the homozygous mutant phenotype in the eye. Maf is expressed in the lens vesicle after invagination, and becomes highly upregulated in the equatorial zone, the site at which self-renewing anterior epithelial cells withdraw from the cell cycle and terminally differentiate into posterior fiber cells. Posterior lens cells in Maf(lacZ) mutant mice exhibit failure of elongation at embryonic day 11.5, do not express (&agr;)A- and all of the (beta)-crystallin genes, and display inappropriately high levels of DNA synthesis. This phenotype partially overlaps with those reported for gene targeting of Prox1 and Sox1; however, expression of these genes is grossly normal, as is expression of Eya1, Eya2, Pax6, and Sox2. Recombinant Maf protein binds to T-MARE sites in the (alpha)A-, (beta)B2-, and (beta)A4-crystallin promoters but fails to bind to a point mutation in the (alpha)A-crystallin promoter that has been shown previously to be required for promoter function. Our results indicate that Maf directly activates many if not all of the (beta)-crystallin genes, and suggest a model for coordinating cell cycle withdrawal with terminal differentiation.

Animals↗

PubMed2000

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis.

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.

Animals↗

PubMed2000

Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2.

PURPOSE: Previous studies have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause lens fiber cell proliferation and apoptosis. Because pRb is thought to block cell cycle progression by inhibition of E2F transcription factors, experiments were conducted to test whether overexpression of different E2F family members would be sufficient to induce fiber cell proliferation and subsequent apoptosis. The in vivo functions of the transcription factor E2F2 have not previously been analyzed or described in transgenic mice. METHODS: Human E2F1 and E2F2 cDNAs were linked to the alphaA-crystallin promoter. Transgenic mice were generated by microinjection. Changes in cell cycle regulation were assayed by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation and by in situ hybridization. Cell death was assayed using the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS: At embryonic day (E)15.5, strong expression of the E2F1 and E2F2 transgenes was detected in lens fiber cells with little or no expression in epithelial cells. BrdU incorporation and TUNEL assays showed that overexpression of either E2F1 or E2F2 in lens fiber cells was sufficient to cause cell cycle entry and subsequent apoptosis. Expression of either E2F1 or E2F2 was sufficient to induce the transcription of cyclins (A2, B1, and E), as well as p53 and Bax in the lens fibercells. CONCLUSIONS: Expression of either E2F1 or E2F2 can induce postmitotic lens fiber cells to re-enter the cell cycle. Inappropriate cell cycle entry is recognized by p53 in each case, and programmed cell death ensues.

Animals↗

PubMed1999

Tyrosinase-related protein 2 promoter targets transgene expression to ocular and neural crest-derived tissues.

In an effort to identify a promoter suitable for studying early ocular development, we generated transgenic mice carrying the lacZ reporter gene linked to the tyrosinase-related protein 2 (TRP2) promoter. TRP2-lacZ was expressed in early retinal pigment epithelium (RPE) and early neural crest cells in embryos. The promoter activity was robust and consistent in independent transgenic lines. The transgene was also expressed in the optic nerve and neural crest-derived neuronal cells in which the endogenous TRP2 gene is not expressed. This suggests that repressor elements may be missing in the promoter used in this study. To test whether this promoter can be used to study melanocyte development, we cross-mated TRP2-lacZ transgenic mice with mice heterozygous for the Patch (Ph) mutation. The pattern of beta-galactosidase activity in the embryos correlates well with the pigmentation phenotype in postnatal and adult Ph/+ mice. We also generated transgenic mice expressing fibroblast growth factor 9 (FGF9) directed by the TRP2 promoter and examined the effect on ocular development. Ectopic expression of FGF9 in the early embryonic RPE switched its differentiation pathway to a neuronal fate, resulting in formation of a duplicated neural retina in transgenic mice. These studies demonstrate that the TRP2 promoter is valuable for transgenic studies of ocular differentiation and development of neural crest cells.

Animals↗