Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells
Raymond M. AnchanSalil A. LachkeBehzad Gerami‐NainiJ. Suzanne LindseyNicholas NgCatherine NaberMichael D. NickersonResy CavallescoSheldon RowanJennifer L. EatonQiongchao XiRichard L. Maas
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Abstract:
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.Keywords:
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A paired homeodomain transcription factor, PAX6, is a well-known regulator of eye development, and its heterozygous mutations in humans cause congenital eye anomalies such as aniridia. Because it was recently shown that PAX6 also plays an indispensable role in islet cell development, a PAX6 gene mutation in humans may lead to a defect of the endocrine pancreas. Whereas heterozygous mutations in islet-cell transcription factors such as IPF1/IDX-1/STF-1/PDX-1 and NEUROD1/BETA2 serve as a genetic cause of diabetes or glucose intolerance, we investigated the possibility of PAX6 gene mutations being a genetic factor common to aniridia and diabetes. In five aniridia and one Peters’ anomaly patients, all of the coding exons and their flanking exon-intron junctions of the PAX6 gene were surveyed for mutations. The results of direct DNA sequencing revealed three different mutations in four aniridia patients: one previously reported type of mutation and two unreported types. In agreement with polypeptide truncation and a lack of the carboxyl-terminal transactivation domain in all of the mutated PAX6 proteins, no transcriptional activity was found in the reporter gene analyses. Oral glucose tolerance tests revealed that all of the patients with a PAX6 gene mutation had glucose intolerance characterized by impaired insulin secretion. Although we did not detect a mutation within the characterized portion of the PAX6 gene in one of the five aniridia patients, diabetes was cosegregated with aniridia in her family, and a single nucleotide polymorphism in intron 9 of the PAX6 gene was correlated with the disorders, suggesting that a mutation, possibly located in an uncharacterized portion of the PAX6 gene, can explain both diabetes and aniridia in this family. In contrast, the patient with Peters’ anomaly, for which a PAX6 gene mutation is a relatively rare cause, showed normal glucose tolerance (NGT) and did not show a Pax6 gene mutation. Taken together, our present observations suggest that heterozygous mutations in the PAX6 gene can induce eye anomaly and glucose intolerance in individuals harboring these mutations.
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Abstract Background Congenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells. Results Pax6 +/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6 +/+ and Pax6 +/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6 +/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6 +/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels. Conclusion The study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6 +/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.
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Background
Paired box gene 6 (Pax6) is a master regulator for eye and brain development. Pax6 mutations or changes in its expression cause a series of ocular diseases including absence of iris, corneal opacity, cataract, glaucoma, abnormal fovea, retinoblastoma, and Wilm's tumor-aniridia-qenital ahormalies-retardation (WAGR). As a transcription factor, it is expressed in the region of anterior surface ectoderm corresponding to the future adenohypophyseal, olfactory and lens placodes, optic vesicle and other parts of the future brain and thus control the development of eye, brain, pituitary grand, nose and pancreas. Pax6 exists in 4 different isoforms, whose functions are subjected to regulation by different post-translation modifications. A complete understanding of the structure and functions of Pax6 and its associations with relevant diseases is helpful for ophthalmologists to investigate the pathogenesis and treatment of implicated ocular diseases caused by Pax6 gene mutation or changing in its expression.
Key words:
Paired box gene 6; Transcription factor; Humans; Eye abnormalities-causing gene; Mutation; Regulation of gene expression; Brain development; Eye development
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Abstract The PAX6 gene on chromosome 11p13 was isolated by positional cloning (Ton et al., 1991) as a strong candidate gene for the human eye anomaly aniridia (OMIM 106210) (Fig. 86–1). The gene was identi(ed within the aniridia subregion of the Wilms’ tumor, aniridia, genitourinary abnormalities, and mental retardation (WAGR, OMIM 194072) contiguous deletion site. Its expression pattern, assessed by RNA in situ hybridization in human and mouse development, is consistent with a role for PAX6 in developmental eye disease, although it is broader than the spectrum of tissues affected in aniridia.
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Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.
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The transparency of the mammalian lens is primarily maintained by short range order among the major pro- teins of the lens fiber cells, the crystallins. Although these proteins are highly conserved at the amino acid sequence level, it has proven difficult to establish that they possess other than structural functions. We find that when non-lens proteins are added to concentrated solutions of a-crystallin, aggregation is induced, pre- sumably through excluded volume effects. In contrast, the monomeric y-crystallins and the low molecular weight form of &crystallin (pL) cause a decrease in the size of cr-crystallin. When the naturally aggregated form of cY-crystallin is examined, y- and &-crystallin, as well as a reducing agent, also cause partial dissocia- tion as detected by dynamic light scattering and size exclusion chromatography, while no effect is seen with non-crystallin proteins. Furthermore, the chemical cross-linking of cY-crystallin is inhibited by y- and &- crystallin but not by other proteins. The ability of y- crystallin to inhibit the association of a-crystallin is primarily localized to the r-11 form which contains a high degree of exposed thiols. Only small amounts of y- and fiL-crystallin, however, can be cross-linked to cw-crystallin in mixtures of the three proteins even at very high protein concentrations. These results suggest that one possible role for the lower molecular weight crystallins may be to minimize through a reductive effect the intrinsic tendency of cr-crystallin to aggre- gate, an association reaction implicated in the loss of lens transparency. The transparency of the vertebrate ocular lens appears to be derived largely through short range order among the con- centrated proteins of the lens fiber cells, the crystallins (1,2). The crystallins are a group of soluble globular proteins which are expressed at extremely high concentrations in the lens and whose primary function appears to be to establish an essential refractive index gradient (3). It is now generally accepted that any significant disruption of the crystallin’s short range order, regardless of the mechanism or etiologic pathway, will result in increased light scattering by the lens tissue and, consequently, lens opacification or cataract for- * This work was partially supported by the National Eye Institute, National Institutes of Health Grant EY06727, and National Institute of Health Research Career Development Award AI00663 (to C. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked
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Mutations in the paired box 6 (PAX6)gene cause a wide variety of eye anomalies, including aniridia. PAX6 mutations are not well described in the Chinese population so this study is aimed at exploring the role of PAX6 mutations in Taiwanese patients with congenital eye anomalies.Seventeen patients with single or multiple congenital eye anomalies were enrolled. Genomic DNA was prepared from venous blood leukocytes, and the coding regions of PAX6 were analyzed by PCR and direct sequencing. Clinical manifestations of the patients were then correlated to PAX6 mutations.Five PAX6 mutations were identified in one case each. Three mutations c.317T>A (p.L106X), c.142-1G>T, and c.656del10 (p.Q219QfsX20) were novel and the other two, c.331delG (p.V111SfsX13) and c.949C>T (p.R317X), have been reported. All five cases had aniridia; three had other eye anomalies; and four had developmental delay. Only one case had other affected family members. In the ten cases that had no PAX6 mutation, only one had aniridia.Both novel and known PAX6 mutations were identified in the current study, and PAX6 mutations were closely associated with aniridia. Absence of a positive family history does not exclude PAX6 mutation. The frequent occurrence of developmental delay in patients with PAX6 mutation argues for a prompt diagnosis of the disease.
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