Pax6 Gene Expression Research Articles

Heparin-Binding Epidermal-like Growth Factor (HB-EGF) Reduces Cell Death in an Organoid Model of Retinal Damage

In zebrafish and various mammalian species, HB-EGF has been shown to promote Müller glia proliferation and activation of repair mechanisms that have not been fully investigated in human retina. In the current study, 70- to 90-day-old human retinal organoids were treated with 20 μM 4-hydroxytamoxifen (4-OHT), and CRX, REC, NRL, PAX6, VIM, GFAP, and VSX2 gene and protein expression were assessed at various times points after treatment. Organoids with or without 4-OHT-induced damage were then cultured with HB-EGF for 7 days. We showed that 20 μM 4-OHT caused a reduction in the number of recoverin-positive cells; an increase in the number of TUNEL-positive cells; and downregulation of the photoreceptor gene markers CRX, NRL, and REC. Culture of organoids with HB-EGF for 7 days after 4-OHT-induced damage caused a marked reduction in the number of TUNEL-positive cells and small increases in the number of Ki67-positive cells and PAX6 and NOTCH1 gene expression. The current results suggest that treatment of human ESC-derived retinal organoids with 4-OHT may be used as a model of retinal degeneration in vitro. Furthermore, HB-EGF treatment of human retinal organoids increases proliferating Müller cells, but only after 4-OHT induced damage, and may be an indication of Muller reactivity in response to photoreceptor damage. Further studies will aim to identify factors that may induce Müller cell-mediated regeneration of the human retina, aiding in the development of therapies for retinal degeneration.

Genetic analysis of medaka fish illuminates conserved and divergent roles of Pax6 in vertebrate eye development.

Landmark discovery of eye defects caused by Pax6 gene mutations in humans, rodents, and even fruit flies combined with Pax6 gene expression studies in various phyla, led to the master control gene hypothesis postulating that the gene is required almost universally for animal visual system development. However, this assumption has not been broadly tested in genetically trackable organisms such as vertebrates. Here, to determine the functional role of the fish orthologue of mammalian Pax6 in eye development we analyzed mutants in medaka Pax6.1 gene generated by genome editing. We found that transcription factors implicated in vertebrate lens development (Prox1a, MafB, c-Maf, FoxE3) failed to initiate expression in the presumptive lens tissue of Pax6.1 mutant fish resulting in aphakia, a phenotype observed previously in Pax6 mutant mice. Surprisingly, the overall differentiation potential of Pax6.1-deficient retinal progenitor cells (RPCs) is not severely compromised, and the only cell types affected by the absence of Pax6.1 transcription factor are retinal ganglion cells. This is in stark contrast to the situation in mice where the Pax6 gene is required cell-autonomously for the expansion of RPCs, and the differentiation of all retina cell types. Our results provide novel insight into the conserved and divergent roles of Pax6 gene orthologues in vertebrate eye development indicating that the lens-specific role is more evolutionarily conserved than the role in retina differentiation.

Effects of Artificial Light at Night on Fitness-Related Traits of Sea Urchin (Heliocidaris crassispina).

Limited data are available regarding the effects of elevated coastal artificial light at night (ALAN) on intertidal echinoderms. In this study, we investigated the behavioral, morphological, and physiological responses of the sea urchin (Heliocidaris crassispina) after continuous exposure to ALAN at light intensities of 0.1, 300, and 600 Lux for 6 weeks. Our findings revealed that ALAN at 300 Lux substantially reduced food consumption, Lantern weight, and gonadosomatic index (GSI). On the other hand, ALAN at 600 Lux notably prolonged the righting and covering response times and elevated the 5-HIAA/5-HT ratio, while concurrently decreasing food consumption, body weight, Lantern weight, GSI, and Pax6 gene expression. These results indicated that continuous exposure to ALAN could cause an adverse effect on fitness-related traits, including behavioral responses, growth, reproductive performance, and photoreception of sea urchins. The present study provides new insights on the impact of light pollution on echinoderms.

Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6.

Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action. NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20μM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment. Our data suggest that treatment with 10μM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10μM) caused a significant increase in NSC differentiation towards immature (Olig2+) and mature (MBP+) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin+cells) increased in all treatment groups, with the highest and most significant increase obtained at 15μM concentration. It is worth noting that pregnenolone at the highest concentration of 15μM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis. Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.

Optimization of Cdx Transcription Factors Characteristics

This study presents a new application of TOPSIS for the optimization of transcription factors characteristics. This application is essential as it can help compare the characteristics of these proteins and determine the optimized output of their comparison with this decision-making method. The hypothesis in this article was that according to the previous study of the Cdx transcription factors, as the Cdx2 transcription factor showed more robust characteristics than Cdx1 and Cdx4, the TOPSIS method would show a better rank position of these first proteins in comparison with the two other ones. Moreover, the engrailed repressor domain EnRCdx1 used in the plasmid showed the reduction of the pax3 gene expression in comparison with the induced regulation of the gene expression with the production of the Cdx1, Cdx2, and Cdx4 transcription factors using the corresponding plasmids, the worst rank position with TOPSIS was expected for this repressor domain. The results obtained with this ranking method showed that the rank positions of the transcription factors and the repressor domain corresponded to their compared properties. Moreover, the change in the weight values of the candidates showed the modification of their distances from the best and worst alternatives and closeness coefficients. However, as expected, the candidates’ rank positions were unchanged, and the Cdx2 transcription factor was still the best candidate. The results of this article can be used in computer engineering to improve biological applications of these proteins.

Effects of high-molecular-weight polyvinyl chloride on Xenopus laevis adults and embryos: the mRNA expression profiles of Myf5, Esr1, Bmp4, Pax6, and Hsp70 genes during early embryonic development.

Microplastics and associated adverse effects have been on the global agenda in recent years. Because of its importance as a model organism for studies on developmental biology, Xenopus laevis has been chosen as the study animal in in vitro teratogenesis studies. FETAX test uses early-stage embryos of X. laevis to measure the potential of substances to cause mortality, malformation, and growth inhibition in developing embryos. The aim of this study was to examine the effects of high molecular weight polyvinyl chloride (HMW-PVC) on parental X. laevis frogs and their embryos using the FETAX test. To this purpose, a HMW-PVC dose of 1% of body weight/twice each week was provided to frogs by oral gavage throughout 6 weeks. After the procedure, oocytes and sperms of HMW-PVC-exposed frogs were fertilized and FETAX was applied to selected embryos. After the completion of a 96-h incubation period, tadpoles were examined, their live/dead status were determined, their lengths were measured, and their anomalies were photographed. Besides, excised organs of the parental frogs were referred to histopathology examination. On the other hand, the mRNA expression levels of Hsp70, Myf5, Bmp4, Pax6, and Esr1 genes were determined by applying real-time quantitative PCR method to cDNA which was synthesized from the total RNA of embryos. The results showed that treatment with HMW-PVC dose of 1% of body weight/twice each week caused malformations and decreased viability. Hsp70 and Pax6 gene expression levels significantly decreased in all assay groups, as compared with controls. Lung and intestine tissues showed normal appearance in histopatological examination. Further research is required to explain the whole effects of HMW-PVC exposure on X. laevis embryos.

Mechanotransduction Regulates Reprogramming Enhancement in Adherent 3D Keratocyte Cultures.

Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (OCT4, NANOG, SOX2, KLF4, CMYC, ABCG2 and PAX6) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors (OCT4, NANOG, SOX2, KLF4 and CMYC) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4, NANOG, SOX2, KLF4, HNK1, ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2, ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFβ/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.

Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

Aberrant expression of PAX6 gene associated with classical aniridia: identification and functional characterization of novel noncoding mutations.

The PAX6 is essential for ocular morphogenesis and is known to be highly sensitive to changes in gene expression, where neither over- nor under-expression ensures normal ocular development. Two unrelated probands with classical aniridia who were previously considered "PAX6-negative", were studied by whole-genome sequencing. Through the use of multiple in silico deep learning-based algorithms, we identified two novel putative causal mutations, c.-133_-132del in the 5' untranslated region (5'-UTR) and c.-52 + 5G>A in an intron upstream of the PAX6 gene. The luciferase activity was significantly increased and VAX2 binding was disrupted with the former 5'-UTR variant compared with wild-type sequence, which resulted in a striking overexpression of PAX6. The minigene assay showed that the c.-52 + 5G>A mutation caused defective splicing, which resulted in the formation of truncated transcripts.

Structure and Variability of the Pax3 and EDNRB Genes in American Mink (Neovison vison)

The Pax3 protein is a transcription factor responsible for process of differentiation neural crest stem cells during the embryonic period, in individual life Pax3 gene expression is an element of skin cells response to ultraviolet radiation (UV). Mutations of Pax3 gene are possible cause of some color patterns Quarter Horse breed, additionally some variations of Pax3 gene can disturb the process of embryonic or fetal development. Endothelin Receptor Type B (EDNRB) is another candidate gene that can affect on the distribution of the dye in skin or hairs, exons 1, 3, 4 and 5 were sequenced, any nucleotide variations were not observed. Exons 2 and 3 of the Pax3 gene along with the flanking regions were sequenced, in which a few variations were found, some of them were genotyped by means of PCR-RFLP and ASA-PCR. Statistically significant differences between the frequency of examined alleles in the various coat color groups were observed.

Structure and Variability of 5'UTR, Exon 1 and Fragment of Intron 1 of the Pax3 Gene in American Mink (Neovison vison)

The expression product of the Pax3 gene is a transcription factor involved in the process of neural crest’s stem cell differentiation into melanocytes during embryonic development. In individual life, the Pax3 gene expression is an element of the complex process of regulation the degree of melanoblasts differentiation. The state of melanoblasts (being a reservoir of renewable cells) differentiation depends on UV radiation, which stimulates the Pax3 gene expression. In the absence of radiation the expression of the Pax3 gene is inhibited, what stops the melanoblasts at the current level of diversity. Pax3 gene is one of the candidate genes responsible for animals coat color and its mutations are considered a possible cause for the occurrence of the color patterns: “splashed white” and “white spotting” in Quarter Horse breed. The 5'UTR exon 1 and the intron fragment 1 of the Pax3 gene in the American mink were sequenced. Two variations were found: substitution c.-17G>A in the 5′ UTR and substitution c.+142C>G in intron 1. Investigations were carried out on 93 individuals, which were genotyped for polymorphisms in the 5'UTR region using the MTPA-PCR technique. It was established that the study group was in a state of genetic equilibrium, because there were no statistically significant differences between observed genotypes frequency and theoretically calculated according to the Hardy–Weinberg principle and it was found that there were no statistically significant relationship between the Pax3 gene polymorphism and the American mink coat color.

The effects of electromagnetic fields on cultured human retinal pigment epithelial cells.

A great deal of evidence has confirmed that electromagnetic fields (EMFs) can affect the central nervous system. In this study, cultured neonatal human retinal pigment epithelial (hRPE) cells were exposed to pulsed EMF of 1 mT intensity and 50 Hz frequency 8 h daily for 3 days. In addition to cell proliferation and cell death assays, immunocytochemistry for RPE65, PAX6, nestin, and cytokeratin 8/18 proteins were performed. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for NES, PAX6, RPE65, and ACTA2 gene expression. Exposed hRPE cells did not demonstrate significant change in terms of cytomorphology, cell proliferation, or cell death. Protein expression of PAX6 was decreased in treated cells compared to controls and remained unchanged for RPE65, cytokeratin 8/18, and nestin. Gene expressions of NES, RPE65, and PAX6 were decreased in treated cells as compared to controls. Gene expression of ACTA2 did not significantly change. In conclusion, viability of cultivated neonatal hRPE cells did not change after short exposure to a safe dose of pulsed EMF albeit that both gene and protein expressions of retinal progenitor cell markers were reduced. Whether longer exposure durations that are being constantly produced by widely-used electronic devices may induce significant changes in these cells, needs further investigation. Bioelectromagnetics. 39:585-594, 2018. © 2018 Wiley Periodicals, Inc.

PAX3: A Molecule with Oncogenic or Tumor Suppressor Function Is Involved in Cancer

Metastasis is the most deadly aspect of cancer and results from acquired gene regulation abnormalities in tumor cells. Transcriptional regulation is an essential component of controlling of gene function and its failure could contribute to tumor progression and metastasis. During cancer progression, deregulation of oncogenic or tumor suppressive transcription factors, as well as master cell fate regulators, collectively influences multiple steps of the metastasis cascade, including local invasion and dissemination of the tumor to distant organs. Transcription factor PAX3/Pax3, which contributes to diverse cell lineages during embryonic development, plays a major role in tumorigenesis. Mutations in this gene can cause neurodevelopmental disease and the existing literature supports that there is a potential link between aberrant expression of PAX3 genes in adult tissues and a wide variety of cancers. PAX3 function is tissue-specific and could contribute to tumorigenesis either directly as oncogene or as a tumor suppressor by losing its function. In this review, we discuss comprehensively the differential role played by PAX3 in various tissues and how its aberrant expression is implicated in disease development. This review particularly highlights the oncogenic and tumor suppressor role played by PAX3 in different cancers and underlines the importance of precisely identifying tissue-specific role of PAX3 in order to determine its exact role in development of cancer.

Functional characteristics of novel pancreatic Pax6 regulatory elements.

Complex diseases, such as diabetes, are influenced by comprehensive transcriptional networks. Genome-wide association studies have revealed that variants located in regulatory elements for pancreatic transcription factors are linked to diabetes, including those functionally linked to the paired box transcription factor Pax6. Pax6 deletions in adult mice cause rapid onset of classic diabetes, but the full spectrum of pancreatic Pax6 regulators is unknown. Using a regulatory element discovery approach, we identified two novel Pax6 pancreatic cis-regulatory elements in a poorly characterized regulatory desert. Both new elements, Pax6 pancreas cis-regulatory element 3 (PE3) and PE4, are located 50 and 100 kb upstream and interact with different parts of the Pax6 promoter and nearby non-coding RNAs. They drive expression in the developing pancreas and brain and code for multiple pancreas-related transcription factor-binding sites. PE3 binds CCCTC-binding factor (CTCF) and is marked by stem cell identity markers in embryonic stem cells, whilst a common variant located in the PE4 element affects binding of Pax4, a known pancreatic regulator, altering Pax6 gene expression. To determine the ability of these elements to regulate gene expression, synthetic transcriptional activators and repressors were targeted to PE3 and PE4, modulating Pax6 gene expression, as well as influencing neighbouring genes and long non-coding RNAs, implicating the Pax6 locus in pancreas function and diabetes.

First report of Klein-Waardenburg Syndrome in Iran and a novel pathogenic splice site variant in PAX3 gene

ObjectivesWaardenburg Syndrome (WS) as a congenital auditory-pigmentary syndrome is a clinically and genetically heterogeneous disorder. Based upon clinical manifestations, it can be classified into four types. Loss of function mutations in PAX3 gene cause WS1 and WS3 (Klein-Waardenburg syndrome). While WS2 and WS4 have locus heterogeneity with multiple causative genes. Here we report a novel splice site variant in a pedigree with multiple affected members. Based on diagnostic criteria, three of them are associated with WS3. The remained patients classified as type 1. MethodsPCR amplification and Sanger sequencing were performed for all exons and all exon-intron boundaries of PAX3 (NM_181,459) gene of the proband. Then available symptomatic and asymptomatic members were screened for the detected variant. Interpretation and classification of the variant were done based on the current guidelines. ResultsWe identified a novel heterozygous splice site variant (c.586+2T > C) in donor site of intron 4 of PAX3 gene in our proband. Moreover, this variant was co-segregated with the disease in other available five affected members. Also, the detected variant was not detected in any of the investigated asymptomatic members. This variant was classified as a pathogenic variant. ConclusionsThis study shows significant intra-familial clinical heterogeneity and absence of phenotype-genotype correlation in a pedigree with Waardenburg Syndrome. However, severity of phenotypes and additional symptoms in the patients can be related to alternative splicing and different levels of PAX3 gene expression. Detailed evaluation of more cases can shed light on this and case-reports are valuable traffic sign in the road. This article is the first report of Waardenburg syndrome type 3 in Iran.

SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-β1) signalling in renal cell carcinoma.

Canonical TGF-β1 signalling promotes tumor progression by facilitating invasion and metastasis, whereby release of TGF-β1, by (for example) infiltrating immune cells, induces epithelial to mesenchymal transition (EMT). PAX2, a member of the Paired box family of transcriptional regulators, is normally expressed during embryonic development, including in the kidney, where it promotes mesenchymal to epithelial transition (MET). PAX2 expression is silenced in many normal adult tissues. However, in contrast, PAX2 is expressed in several cancer types, including kidney, prostate, breast, and ovarian cancer. While multiple studies have implicated TGF-β superfamily members in modulating expression of Pax genes during embryonic development, few have investigated direct regulation of Pax gene expression by TGF-β1. Here we have investigated direct regulation of PAX2 expression by TGF-β1 in clear cell renal cell carcinoma (CC-RCC) cell lines. Treatment of PAX2-expressing 786-O and A498 CC-RCC cell lines with TGF-β1 resulted in inhibition of endogenous PAX2 mRNA and protein expression, as well as expression from transiently transfected PAX2 promoter constructs; this inhibition was abolished in the presence of expression of the inhibitory SMAD, SMAD7. Using ChIP-PCR we showed TGF-β1 treatment induced SMAD3 protein phosphorylation in 786-O cells, and direct SMAD3 binding to the human PAX2 promoter, which was inhibited by SMAD7 over-expression. Overall, these data suggest that canonical TGF-β signalling suppresses PAX2 transcription in CC-RCC cells due to the direct binding of SMAD proteins to the PAX2 promoter. These studies improve our understanding of tumor progression and epithelial to mesenchyme transition (EMT) in CC-RCC and in other PAX2-expressing cancer types.

PAX6 Promoter Methylation Correlates with MDA-MB-231 Cell Migration, and Expression of MMP2 and MMP9

Objective:Breast cancer is a heterogeneous disease characterized by an accumulation of genetic and epigenetic alterations that lead tumor cells to acquire characteristics like the capacity for invasion and metastasis. Metastasis remains a major challenge in cancer management and understanding of its molecular basis should result in improved prevention, diagnosis, and treatment of breast cancer patients. The aim of this study was to investigate how promoter DNA methylation regulates PAX6 gene expression and influences breast carcinoma cell migration.Methods:PAX6 promoter methylation was detected by Methyl Specific-Multiplex Ligation Probe Amplification (MS-MLPA). Gene expression was evaluated using qRT-PCR, while the effect of PAX6 on migration was ssessed by wound healing assay. In addition, MMP2 and MMP9 genes were studied using different bioinformatic tools.Results:The PAX6 promoter is methylated in breast cancer cell lines and methylation in this region impacts on its expression. Migration assays revealed that PAX6 overexpression promotes cell migration, while PAX6 inhibition decreases it. More importantly, we found that migration is affected by PAX6 methylation status. Employing bioinformatic analysis, binding sites for PAX6 on the regulatory regions of the MMP2 and MMP9 genes were established, PAX6 overexpression increasing MMP2 and MMP9 expression at the mRNA level.Conclusion:Our study provides novel insights into epigenetic events that regulate PAX6 expression and molecular mechanisms by which PAX6 modifies the migration capacity of breast cancer cells.

Selenite restores Pax6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters.

Arsenic is a worldwide environmental pollutant that generates public health concerns. Various types of cancers and other diseases, including neurological disorders, have been associated with human consumption of arsenic in drinking water. At the molecular level, arsenic and its metabolites have the capacity to provoke genome instability, causing altered expression of genes. One such target of arsenic is the Pax6 gene that encodes a transcription factor in neuronal cells. The aim of this study was to evaluate the effect of two antioxidants, α-tocopheryl succinate (α-TOS) and sodium selenite, on Pax6 gene expression levels in the forebrain and cerebellum of Golden Syrian hamsters chronically exposed to arsenic in drinking water. Animals were divided into six groups. Using quantitative real-time reverse transcriptase (RT)-PCR analysis, we confirmed that arsenic downregulates Pax6 expression in nervous tissues by 53 ± 21% and 32 ± 7% in the forebrain and cerebellum, respectively. In the presence of arsenic, treatment with α-TOS did not modify Pax6 expression in nervous tissues; however, sodium selenite completely restored Pax6 expression in the arsenic-exposed hamster forebrain, but not the cerebellum. Although our results suggest the use of selenite to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population require further studies.

Early Developmental Perturbations in a Human Stem Cell Model of MODY5/HNF1B Pancreatic Hypoplasia

SummaryPatients with an HNF1BS148L/+ mutation (MODY5) typically exhibit pancreatic hypoplasia. However, the molecular mechanisms are unknown due to inaccessibility of patient material and because mouse models do not fully recapitulate MODY5. Here, we differentiated MODY5 human-induced pluripotent stem cells (hiPSCs) into pancreatic progenitors, and show that the HNF1BS148L/+ mutation causes a compensatory increase in several pancreatic transcription factors, and surprisingly, a decrease in PAX6 pancreatic gene expression. The lack of suppression of PDX1, PTF1A, GATA4, and GATA6 indicates that MODY5-mediated pancreatic hypoplasia is mechanistically independent. Overexpression studies demonstrate that a compensatory increase in PDX1 gene expression is due to mutant HNF1BS148L/+ but not wild-type HNF1B or HNF1A. Furthermore, HNF1B does not appear to directly regulate PAX6 gene expression necessary for glucose tolerance. Our results demonstrate compensatory mechanisms in the pancreatic transcription factor network due to mutant HNF1BS148L/+ protein. Thus, patients typically develop MODY5 but not neonatal diabetes despite exhibiting pancreatic hypoplasia.

A New Gestational Diabetes Mellitus Model, Hyperglycemia-Induced Eye Malformation via Inhibiting Pax6 in Chick Embryo

ABSTRACTGestational diabetes mellitus (GDM) is one of the leading causes of fetal malformations. However, few models have been developed to study the underlying mechanisms of GDM-induced fetal eye malformation. In this study, a high concentration of glucose (0.2 mmol per egg) was injected into the air sac of chick embryos on embryo development day (EDD) 1 to develop a hyperglycemia model. Results showed that 47.3% of embryonic eye malformation happened on EDD 5. In this model, the key genes regulating eye development, Pax6, Six3 and Otx2, were downregulated by hyperglycemia. Among these genes, the expression of Pax6 was the most vulnerable to hyperglycemia, being suppressed by 70%. A reduction in Pax6 gene expression induced eye malformation in chick embryos. However, increased expression of Pax6 in chick embryos could rescue hyperglycemia-induced eye malformation. Hyperglycemia stimulated O-linked N-acetylglucosaminylation, which caused oxidative stress in chick embryos. Pax6 was found to be vulnerable to free radicals, but the antioxidant edaravone could restore Pax6 expression and reverse eye malformation. These results illustrated a successful establishment of a new chick embryo model to study the molecular mechanism of hyperglycemia-induced eye malformation. The suppression of the Pax6 gene is probably mediated by oxidative stress and could be a crucial target for the therapy of GDM-induced embryonic eye malformation.