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ABSTRACT: Mammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF) signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas.
Ras transgenic lenses and corneal epithelial cells showed increased proliferation with concomitant increases in cyclin D1 and D2 expression. This initial increase in proliferation is sustained in the cornea but not in the lens epithelial cells. Coincidentally, cdk inhibitors p27Kip1 and p57Kip2 were upregulated in the Ras transgenic lenses but not in the corneas. Phospho-Erk1 and Erk2 levels were elevated in the lens but not in the cornea and Spry 1 and Spry 2, negative regulators of Ras-Raf-Erk signaling, were upregulated more in the corneal than in the lens epithelial cells. Both lens and corneal differentiation programs were sensitive to Ras activation. Ras transgenic embryos showed a distinctive alteration in the architecture of the lens pit. Ras activation, though sufficient for upregulation of Prox1, a transcription factor critical for cell cycle exit and initiation of fiber differentiation, is not sufficient for induction of terminal fiber differentiation. Expression of Keratin 12, a marker of corneal epithelial differentiation, was reduced in the Ras transgenic corneas.
Collectively, these results suggest that Ras activation a) induces distinct sets of downstream targets in the lens and cornea resulting in distinct cellular responses and b) is sufficient for initiation but not completion of lens fiber differentiation.
BMC Developmental Biology 01/2010; 10:13. · 2.79 Impact Factor
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ABSTRACT: There is evidence that isoprostanes (IsoPs) can regulate exogenously applied excitatory amino acid neurotransmitters in bovine retina in vitro. However, the regulation of retinal morphology and endogenous neurotransmitter levels by IsoPs is unknown. We examined the effects of intravitreally injected 8-iso-PGE(2) on retinal tissue integrity and viability and amino acid neurotransmitters in bovine eye organ culture ex vivo. Exposure of bovine eyeballs to simulated experimental conditions revealed no retinal apoptosis and necrosis in TUNEL and DAPI staining and hematoxylin and eosin staining assays, respectively, and no changes in basal levels of amino acids in retina and vitreous humor. Furthermore, intravitreal injection of 8-iso-PGE(2) into bovine eyeballs had no effect on retinal apoptosis and integrity. Interestingly, 8-iso-PGE(2) caused a concentration-dependent attenuation of retinal glutamate and its metabolite glutamine and glycine levels, while GABA was unaffected. 8-Iso-PGE(2) (1 and 100 microM) significantly (P < 0.001) attenuated glutamate levels by 33.9% and 48.0%, respectively. 8-Iso-PGE(2) (100 microM) inhibited (P < 0.01) retinal glutamine and glycine levels by 37.7% and 35.5%, respectively. The IsoP exhibited no effect on vitreous humor glutamine and glycine levels, while glutamate and GABA were not detected. Thus, 8-iso-PGE(2) can regulate retinal amino acids without inducing cell death in bovine retina ex vivo.
Methods and Findings in Experimental and Clinical Pharmacology 10/2008; 30(8):615-26. · 0.93 Impact Factor
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ABSTRACT: The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and alphaA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.
Developmental Biology 09/2008; 323(1):53-63. · 4.07 Impact Factor
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ABSTRACT: The purpose of this study was to reassess the role of the lens as an "embryonic organizer" of ocular tissues.
We ablated the lens in mice by lens-specific expression of an attenuated version of diphtheria toxin A subunit(Tox176) driven by a modified crystallin promoter. Alterations in the differentiation programs of ocular tissues were examined by hematoxylin and eosin staining, in situ hybridization, and immunohistochemistry.
Transgenic mice in the family OVE1757 exhibited severe microphakia. Apoptotic lens fibers were seen by embryonic day 15 (E15) and the lenses were completely ablated by post natal day 8. Multiple defects were seen in the anterior chamber. Corneal endothelial cells did not differentiate properly. The mesenchymal cells that would normally give rise to the endothelial layer were found to express N-cadherin, but they failed to form tight junctions and undergo a mesenchymal-to-epithelial transition. Although early specification of the presumptive ciliary body and iris was detected, subsequent differentiation of the iris was blocked. No dramatic changes were seen in the development of the retina.
These results support the hypothesis that an intact lens is essential for proper differentiation of both the corneal endothelium and the iris and that the lens "organizes" the development of tissues in the anterior chamber.
Molecular vision 02/2007; 13:2289-300. · 2.20 Impact Factor
