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Purpose Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). While the ocular surface is considered one of the major SARS-CoV2 transmission routes, the specific cellular tropism of SARS-CoV2 is not fully understood. In the current study, we evaluated the expression and regulation of two SARS-CoV2 viral entry proteins, TMPRSS2 and ACE2, in human ocular epithelial cells and stem cells. Methods TMPRSS2 and ACE2 expression in ABCB5-positive limbal stem cells (LSCs) were assessed by RNAseq, flow cytometry and immunohistochemistry. PAX6, TMPRSS2, and ACE2 mRNA expression values were obtained from the GSE135455 and DRA002960 RNA-seq datasets. siRNA-mediated PAX6 knockdown (KD) was performed in limbal and conjunctival epithelial cells. TMPRSS2 and ACE2 expression in the PAX6 KD cells was analyzed by qRT-PCR and Western blot. Results We found that ABCB5-positive LSCs express high levels of TMPRSS2 and ACE2 compared to ABCB5-negative limbal epithelial cells. Mechanistically, gene knockout and overexpression models revealed that the eye transcription factor PAX6 negatively regulates TMPRSS2 expression. Therefore, low levels of PAX6 in ABCB5-positive LSCs promote TMPRSS2 expression, and high levels of TMPRSS2 and ACE2 expression by LSCs indicate enhanced susceptibility to SARS-CoV2 infection in this stem cell population. Conclusions Our study points to a need for COVID-19 testing of LSCs derived from donor corneas before transplantation to patients with limbal stem cell deficiency. Furthermore, our findings suggest that expandable human ABCB5+ LSC cultures might represent a relevant novel model system for studying cellular SARS-CoV2 viral entry mechanisms and evaluating related targeting strategies.

Maternal immune activation (MIA) in midpregnancy is a risk factor for neurodevelopmental disorders. Improper brain development may cause malformations of the brain; maldevelopment induced by MIA may lead to a pathology-related phenotype. In this study, a single intraperitoneal injection of 20 mg/kg polyriboinosinic–polyribocytidylic acid [poly(I:C)] was administered to C57BL/6J mice on embryonic day (E) 12.5 to mimic maternal viral infection. Histopathological analysis of neurogenesis was performed using markers for Pax6, Tbr2, and Tbr1. In these fetuses, significant increases were observed in the proportion of Pax6-positive neural progenitor cells and Pax6/Tbr2 double-positive cells 24 h after poly(I:C) injection. There were no differences in the proportion of Tbr1-positive postmitotic neurons 48 h after poly(I:C) injection. At E18.5, there were more Pax6-positive and Tbr2-positive neural progenitor cells in the poly(I:C)-injected group than in the saline-injected group. Gene ontology enrichment analysis of poly(I:C)-induced differentially expressed genes in the fetal brain at E12.5 demonstrated that these genes were enriched in terms including response to cytokine, response to decreased oxygen levels in the category of biological process. At E13.5, activating transcription factor 4 (Atf4), which is an effector of integrated stress response, was significantly upregulated in the fetal brain. Our results show that poly(I:C)-induced MIA at E12.5 leads to dysregulated neurogenesis and upregulates Atf4 in the fetal brain. These findings provide a new insight in the mechanism of MIA causing improper brain development and subsequent neurodevelopmental disorders., Highlights • MIA increases Pax6-positive and Tbr2-positive neural progenitor cells. • MIA impaired the process of neurogenesis from as early as the acute stage. • MIA upregulated Atf4, an effector of integrated stress response, in the fetal brain.

Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.

Aniridia is usually an autosomal dominant, rare disorder characterized by a variable degree of hypoplasia or the absence of iris tissue, with additional ocular abnormalities. Pathogenic variants in the PAX6 gene are associated with aniridia in most patients. However, in up to 30% of individuals, disease results from 11p13 chromosomal rearrangements. Here we present a patient with a clinical diagnosis of partial aniridia born to consanguineous Polish parents. The parents were asymptomatic and ophthalmologically normal. We performed PAX6 sequencing, array comparative genomic hybridization, quantitative real-time PCR, and whole genome sequencing. aCGH revealed a homozygous deletion of the DCDC1 gene fragment in the patient. The same, but heterozygous deletion, was detected in each of the patient's asymptomatic parents and brother. In the presented family, the signs and symptoms of aniridia are observed only in the homozygous proband. Whole genome sequencing analysis was performed to determine other possible causes of the disease and did not detect any additional or alternative potentially pathogenic variant. We report a novel homozygous deletion located in the 11p13 region, which does not include the PAX6 gene or any known PAX6 enhancers. To our best knowledge, this is the first reported case of a patient presented with isolated aniridia carrying a homozygous microdeletion downstream of the PAX6 gene.

This review updates the knowledge about the morphological assessment of the foveal hypoplasia in congenital aniridia and resumes the reported genotype-phenotype correlations known to date. Congenital aniridia is a pan ocular disease. Although iris absence is considered the hallmark of this entity, foveal hypoplasia is present in 94.7%-84% of patients. A foveal morphology assessed by optical coherence tomography in which external retina structures can be identified, with presence of the lengthening of photoreceptors outer segment and a greater external retinal thickness, is associated with a better visual outcome, regardless a foveal pit is identified or not. This analysis can be performed once the external retina has completed its differentiation, by 6 years old. PAX6 mutations that introduce premature termination codon, C terminal extension or PAX6 involving deletions have been related to lesser foveal differentiation. Better foveal differentiation has been associated to non-coding PAX6 mutations.

Congenital aniridia is a rare genetic eye disorder with total or partial absence of the iris from birth. In most cases the genetic origin of aniridia is a mutation in the PAX6 gene, lead-ing to involvement of most eye structures. Hypoplasia of the fovea is usually present and is associated with reduced visual acuity and nystagmus. Aniridia-associated keratopathy, glaucoma, and cataract are serious and progressive complications that can further reduce visual function. Treatment of the ocular complications of aniridia is challenging and has a high risk of side effects. New approaches such as stem cell therapy may, however, offer better prognoses. We describe the various ocular manifestations of aniridia, with a special focus on conditions that commonly require treatment. We also review the growing literature reporting systemic manifestations of the disease. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Funding Agencies|European Union COST Action [CA-18116 ANIRIDIA-NET]

The distribution patterns of a set of conserved brain developmental regulatory transcription factors were analyzed in the forebrain of the basal actinopterygian fish Acipenser ruthenus, consistent with the prosomeric model. In the telencephalon, the pallium was characterized by ventricular expression of Pax6. In the subpallium, the combined expression of Nkx2.1/Islet-1 (Isl1) allowed to propose ventral and dorsal areas, as the septo-pallidal (Nkx2.1/Isl1+) and striatal derivatives (Isl1+), respectively, and a dorsal portion of the striatal derivatives, ventricularly rich in Pax6 and devoid of Isl1 expression. Dispersed Orthopedia (Otp) cells were found in the supracommissural and posterior nuclei of the ventral telencephalon, related to the medial portion of the amygdaloid complex. The preoptic area was identified by the Nkx2.1/Isl1 expression. In the alar hypothalamus, an Otp-expressing territory, lacking Nkx2.1/Isl1, was identified as the paraventricular domain. The adjacent subparaventricular domain (Spa) was subdivided in a rostral territory expressing Nkx2.1 and an Isl1+ caudal one. In the basal hypothalamus, the tuberal region was defined by the Nkx2.1/Isl1 expression and a rostral Otp-expressing domain was identified. Moreover, the Otp/Nkx2.1 combination showed an additional zone lacking Isl1, tentatively identified as the mamillary area. In the diencephalon, both Pax6 and Isl1 defined the prethalamic domain, and within the basal prosomere 3, scattered Pax6- and Isl1-expressing cells were observed in the posterior tubercle. Finally, a small group of Pax6 cells was observed in the pretectal area. These results improve the understanding of the forebrain evolution and demonstrate that its basic bauplan is present very early in the vertebrate lineage.

Human cerebral cortical malformations are associated with progenitor proliferation and neuronal migration abnormalities. Progenitor cells include apical radial glia, intermediate progenitors and basal (or outer) radial glia (bRGs or oRGs). bRGs are few in number in lissencephalic species (e.g. the mouse) but abundant in gyrencephalic brains. The LIS1 gene coding for a dynein regulator, is mutated in human lissencephaly, associated also in some cases with microcephaly. LIS1 was shown to be important during cell division and neuronal migration. Here, we generated bRG-like cells in the mouse embryonic brain, investigating the role of Lis1 in their formation. This was achieved by in utero electroporation of a hominoid-specific gene TBC1D3 (coding for a RAB-GAP protein) at mouse embryonic day (E) 14.5. We first confirmed that TBC1D3 expression in wild-type (WT) brain generates numerous Pax6+ bRG-like cells that are basally localized. Second, using the same approach, we assessed the formation of these cells in heterozygote Lis1 mutant brains. Our novel results show that Lis1 depletion in the forebrain from E9.5 prevented subsequent TBC1D3-induced bRG-like cell amplification. Indeed, we observe perturbation of the ventricular zone (VZ) in the mutant. Lis1 depletion altered adhesion proteins and mitotic spindle orientations at the ventricular surface and increased the proportion of abventricular mitoses. Progenitor outcome could not be further altered by TBC1D3. We conclude that disruption of Lis1/LIS1 dosage is likely to be detrimental for appropriate progenitor number and position, contributing to lissencephaly pathogenesis.

Background Lens development is orchestrated by transcription factors. Disease-causing variants in transcription factors and their developmental target genes are associated with congenital cataracts and other eye anomalies. Methods Using whole exome sequencing, we identified disease-causing variants in two large British families and one isolated case with autosomal dominant congenital cataract. Bioinformatics analysis confirmed these disease-causing mutations as rare or novel variants, with a moderate to damaging pathogenicity score, with testing for segregation within the families using direct Sanger sequencing. Results Family A had a missense variant (c.184 G>A; p.V62M) in PAX6 and affected individuals presented with nuclear cataract. Family B had a frameshift variant (c.470–477dup; p.A160R*) in PITX3 that was also associated with nuclear cataract. A recurrent missense variant in HSF4 (c.341 T>C; p.L114P) was associated with congenital cataract in a single isolated case. Conclusions We have therefore identified novel variants in PAX6 and PITX3 that cause autosomal dominant congenital cataract.

AimsAn ependymoma shows divergent morphological and molecular features depending on their location. The paired box 6 (PAX6) transcription factor is a putative tumour suppressor and drives cancer cells towards a stem cell-like state. A transcriptome study reported highPAX6expression in ependymal tumours, but data on protein expression are lacking.MethodsWe, therefore, analysedPAX6expression by immunohistochemistry in 172 ependymoma samples and correlated its expression to histology, WHO grade, anatomical location and molecular subgroups.ResultsMeanPAX6nuclear expression in ependymoma was 27.5% (95% CI 23.3 to 31.7).PAX6expression in subependymoma (mean: 5%) was significantly lower compared with myxopapillary (30%), WHO grade II (26%) and anaplastic ependymoma (35%). Supratentorial ependymomas also displayed significant lowerPAX6levels (15%) compared with spinal cord tumours (30%). Expression levels in YAP1-fused ependymoma (41%) were higher compared with REL-associated protein (RELA)-fusion positive tumours (17%), whilePAX6expression was similar in posterior fossa group A (33%) and B (29%) ependymomas. Kaplan-Meier analysis in RELA-fusion positive ependymomas and posterior fossa group B showed a significant better outcome forPAX6at or above the cut-off of 19.45% compared with tumours withPAX6below the cut-off.ConclusionsWe demonstrate thatPAX6is frequently expressed in human ependymal tumours and immunohistochemistry may be helpful in determining prognostic relevant subgroups.

Aniridia patients have poor visual acuity and iris malformation. The fovea in these patients is underdeveloped, but the relationship between structure and electrophysiologic function remains incompletely understood. This study correlates electrophysiology, visual acuity and optical coherence tomography (OCT) in patients with aniridia secondary to mutations in the PAX6 gene and compares with age-similar controls. Patients were recruited from clinical practice. The mfERG protocol was a 4-min 103-hexagon protocol covering approximately 40° in diameter of central retina. Diagnosys full-field ERG (ffERG) and VERIS multifocal ERG (mfERG) were obtained using standard International Society for Clinical Electrophysiology of Vision protocols. OCT central thickness was recorded, and an OCT foveal score was calculated. Nonparametric permutation testing was utilized to determine the statistical significance. A total of 6 aniridia patients and 25 control patients were recruited. On mfERG, aniridia patients had significantly lower amplitudes in rings 1–3 (p = 0.0006, 0.0013, 0.0132), shorter latencies in ring 1 (p = 0.0312) and longer latencies in rings 5 and 6 (p = 0.0026, p = 0.0042) than controls. There was a significantly positive relationship in aniridia patients between logMAR visual acuity and mfERG amplitude in ring 4 (p = 0.0392) and ring 5 (p = 0.0489). On ffERG, there was no difference in amplitudes, though photopic 3.0 a- and b-wave latency, 30 Hz flicker latency and scotopic 0.01 b-wave latency were significantly longer in aniridia patients versus control (p = 0.0018, 0.0.0005, 1.00 x $$10^{{ - 4}}$$ , 0.0198). Thicker central macula on OCT correlated with lower mfERG amplitudes in rings 4–6 (p = 0.0369, 0.0292, 0.0255). There was no correlation between visual acuity and central macular thickness or foveal hypoplasia score as determined by OCT. Higher amplitude on mfERG correlated with poorer visual acuity in rings 4 and 5 in patients with PAX6 mutations. The slope of the change in amplitude from central to peripheral rings on the mfERG is significantly different in aniridia patients compared to controls, with a slower drop-off of amplitude from center to periphery. Additionally, mfERG in aniridia showed lower amplitudes than controls in rings 1–3. These changes along with the lack of correlation between visual acuity and central macular thickness/OCT score suggest that changes in electrical topography may be important to visual deficits in patients with PAX6 gene mutations.

BackgroundThe paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants.MethodsBy locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available.ResultsFive out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.ConclusionWe describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.

The development of the retina and the signaling pathways involved in this process are conserved among vertebrates. Therefore, it was hypothesized that active factors stimulating the retinal regeneration in newts can cause transdifferentiation of human retinal pigment epithelial (RPE) cells. To verify this, we studied the effect of conditioned media (CM) obtained from in vitro regenerating retinas of newts on the phenotype, expression of a number of genes, and localization of cell proteins of human RPE cells of the ARPE-19 line. Within 120 h of cultivation, a change in the morphology of human RPE cells was observed, which manifested in a change in shape and an increase in cell size, which was accompanied by a decrease in proliferative activity. 24 hours after exposure to CM, ARPE-19 showed a short-term increase in the expression of TUBB3 (a panneuronal marker) and a decrease in the expression of BMP2 and BMP4 (neural diffe-rentiation inhibitors). Using immunocytochemical (ICC) methods, we observed an increase in the intensity of staining of cells with antibodies to βIII-tubulin protein and a decrease in the intensity of staining with antibodies to the Cx43 protein of gap junctions (compared to the control) during the period between 24 and 72 h after exposure. At the same time, we observed a decrease in the levels of mRNA expression of the RPE markers OTX2 and KRT18, as well as the KLF4 gene, which is responsible for proliferation and differentiation. Additionally, the shift in the distribution of β-catenin to cytoplasmic localization was recorded using the ICC method. To exclude mesenchymal (and osteogenic in particular) differentiation under the effect of CM, we studied expression of the SPP1 and RUNX2 genes by qPCR. No SPP1 transcription was found in ARPE-19 cells in the control and under the effect of CM, and a decrease in the expression level of RUNX2 mRNA under the effect of CM was observed. Therefore, an attempt to shift to neuronal differentiation was observed in ARPE-19 cells during the period between 24 and 72 h after exposure to the CM of newt retinal regenerates, as indicated by an increase in transcription and translation of βIII-tubulin, a weakening of intercellular adhesion, and a decrease in the expression of inhibitors of neural differentiation BMP2 and BMP4, as well as RPE markers OTX2 and KRT18. However, after that, due to the short-term effect of CM, during the period between 72 and 120 h after exposure, the cells returned to the initial differentiation, as evidenced by a decrease in the expression of neuronal differentiation markers (NES, TUBB3, PAX6) and an increase in the expression of RPE markers (OTX2, MITF).

The abnormal neuroectodermal development due to the PAX 6 gene mutation results in aniridia, a rare disorder. We report herein a family that presented with aniridia, myopia, ptosis, glaucoma, cataract, and sensory nystagmus. This family provides a unique resource for the mutational analysis of the CRYBB1, CRYBA4, and PAX-6 gene. In addition, it is important to remember that mutations are often a part of these ocular abnormalities. Patient awareness, education, and involvement in treatment are essential to prevent amblyopia associated with the cataract and nystagmus related to aniridia.

Aim: To investigate the association of the pair box 6 gene (PAX6) and hsa-miR-328-3p with optical density of macular pigment. Materials and Methods: We evaluated 112 individuals (34 with moderate myopia, eight with high-degree myopia, and 70 healthy individuals). The optical density of macular pigment was measured using single-wavelength reflectometry. DNA and RNA were extracted from whole blood samples. Expression of hsa-miR-328-3p and genotyping of single-nucleotide polymorphism of PAX6 (rs662702) were performed using Applied Biosystems 7900HT real-time polymerase chain reaction system. Optical density of retinal pigment epithelial cells was evaluated using Fundus plus camera. Results: In the group with myopia, with increasing ∆Ct hsa-miR-328-3p, the median optical density of the retinal pigment epithelium decreased statistically significantly (p

Rationale: Transforming Growth Factor-beta (TGF-β) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown. Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function. Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro. Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Background Exposure to traffic-related air pollution (TRAP) during development and/or in adulthood has been associated in many human studies with both neurodevelopmental and neurodegenerative diseases, such as autism spectrum disorder (ASD) and Alzheimer’s disease (AD) or Parkinson’s disease (PD). Methods In the present study, C57BL/6 J mice were exposed to environmentally relevant levels (250+/−50 μg/m3) of diesel exhaust (DE) or filtered air (FA) during development (E0 to PND21). The expression of several transcription factors relevant for CNS development was assessed on PND3. To address possible mechanistic underpinnings of previously observed behavioral effects of DE exposure, adult neurogenesis in the hippocampus and laminar organization of neurons in the somatosensory cortex were analyzed on PND60. Results were analyzed separately for male and female mice. Results Developmental DE exposure caused a male-specific upregulation of Pax6, Tbr1, Tbr2, Sp1, and Creb1 on PND3. In contrast, in both males and females, Tbr2+ intermediate progenitor cells in the PND60 hippocampal dentate gyrus were decreased, as an indication of reduced adult neurogenesis. In the somatosensory region of the cerebral cortex, laminar distribution of Trb1, calbindin, and parvalbumin (but not of Ctip2 or Cux1) was altered by developmental DE exposure. Conclusions These results provide additional evidence to previous findings indicating the ability of developmental DE exposure to cause biochemical/molecular and behavioral alterations that may be involved in neurodevelopmental disorders such as ASD.

PAX6 is considered the master regulator of eye development, the majority of variants affecting this gene cause the pan-ocular developmental eye disorder aniridia. Although no genotype-phenotype correlations are clearly established, missense variants affecting the DNA-binding paired domain of PAX6 are usually associated with non-aniridia phenotypes like microphthalmia, coloboma or isolated foveal hypoplasia. In this study, we report two missense heterozygous variants in the paired domain of PAX6 resulting in isolated foveal hypoplasia with nystagmus in two independent families: c.112 C > G; p.(Arg38Gly) and c.214 G > C; p.(Gly72Arg) in exons 5 and 6, respectively. Furthermore, we provide evidence that paternal postzygotic mosaicism is the cause of inheritance, with clinically unaffected fathers and reduced affected allele fraction. This work contributes to increase the phenotypic spectrum caused by PAX6 variants, and to our knowledge, is the first report to describe the presence of postzygotic parental mosaicism causing isolated foveal hypoplasia with nystagmus. These results support the growing evidence that suggest an overestimation of sporadic cases with PAX6 variants, which has strong implications for both genetic counselling and family planning.

Interlaminar astrocytes (ILAs) are a subset of cortical astrocytes that reside in layer I, express GFAP, have a soma contacting the pia, and contain long interlaminar processes that extend through several cortical layers. We studied the prenatal and postnatal development of ILAs in three species of primates (rhesus macaque, chimpanzee, and human). We found that ILAs are generated prenatally likely from radial glial (RG) cells, that ILAs proliferate locally during gestation, and that ILAs extend interlaminar processes during postnatal stages of development. We showed that the density and morphological complexity of ILAs increase with age, and that ILAs express multiple markers that are expressed by RG cells (Pax6, Sox2, and Nestin), specific to inner and outer RG cells (Cryab and Hopx), and astrocyte markers (S100β, Aqp4, and GLAST) in prenatal stages and in adult. Finally, we demonstrated that rudimentary ILAs in mouse also express the RG markers Pax6, Sox2, and Nestin, but do not express S100β, Cryab, or Hopx, and that the density and morphological complexity of ILAs differ between primate species and mouse. Together these findings contribute new information on astrogenesis of this unique class of cells and suggest a lineal relationship between RG cells and ILAs.

Intra-retinal axon guidance involves a coordinated expression of transcription factors, axon guidance genes, and secretory molecules within the retina. Pax6, the master regulator gene, has a spatio-temporal expression typically restricted till neurogenesis and fate-specification. However, our observation of persistent expression of Pax6 in mature RGCs led us to hypothesize that Pax6 could play a major role in axon guidance after fate specification. Here, we found significant alteration in intra-retinal axon guidance and fasciculation upon knocking out of Pax6 in E15.5 retina. Through unbiased transcriptome profiling between Pax6fl/fl and Pax6−/− retinas, we revealed the mechanistic insight of its role in axon guidance. Our results showed a significant increase in the expression of extracellular matrix molecules and decreased expression of retinal fate specification and neuron projection guidance molecules. Additionally, we found that EphB1 and Sema5B are directly regulated by Pax6 owing to the guidance defects and improper fasciculation of axons. We conclude that Pax6 expression post fate specification of RGCs is necessary for regulating the expression of axon guidance genes and most importantly for maintaining a conducive ECM through which the nascent axons get guided and fasciculate to reach the optic disc.

BackgroundThe HEART Pathway combines a History ECG Age Risk factor (HEAR) score and serial troponins to risk stratify patients with acute chest pain. However, it is unclear whether patients with HEAR scores of <1 require troponin testing. The objective of this study is to measure the major adverse cardiac event (MACE) rate among patients with <1 HEAR scores and determine whether serial troponin testing is needed to achieve a miss rate MethodsA secondary analysis of the HEART Pathway Implementation Study was conducted. HEART Pathway risk assessments (HEAR scores and serial troponin testing at 0 and 3 hours) were completed by the providers on adult patients with chest pain from three US sites between November 2014 and January 2016. MACE (composite of death, myocardial infarction (MI) and coronary revascularisation) at 30 days was determined. The proportion of patients with HEAR scores of <1 diagnosed with MACE within 30 days was calculated. The impact of troponin testing on patients with HEAR scores of <1 was determined using Net Reclassification Improvement Index (NRI).ResultsProviders completed HEAR assessments on 4979 patients and HEAR scores<1 was 97.8% (95%CI 94.5% to 99.4%) and 99.1% (95% CI 97.7% to 99.8%), respectively, and were not improved by troponin testing. Troponin testing in patients with HEAR <1 correctly reclassified two patients diagnosed with MACE, and was elevated among seven patients without MACE yielding an NRI of 0.9% (95%CI −0.7 to 2.4%).ConclusionThese data suggest that patients with HEAR scores of 0 and 1 represent a very low-risk group that may not require troponin testing to achieve a missed MACE rate Trial registration numberNCT02056964

Crinoids are considered as the most basal extant echinoderms. They retain aboral nervous system with a nerve center, which has been degraded in the eleutherozoan echinoderms. To investigate the evolution of patterning of the nervous systems in crinoids, we examined temporal and spatial expression patterns of three neural patterning-related homeobox genes, six3, pax6, and otx, throughout the development of a feather star Anneissia japonica. These genes were involved in the patterning of endomesodermal tissues instead of the ectodermal neural tissues in the early planktonic stages. In the stages after larval attachment, the expression of these genes was mainly observed in the podia and the oral nervous systems instead of the aboral nerve center. Our results indicate the involvement of these three genes in the formation of oral nervous system in the common ancestor of the echinoderms and suggest that the aboral nerve center is not evolutionally related to the brain of other bilaterians.

Largazole is a potent class I selective histone deacetylase inhibitor prodrug with anticancer activity against solid tumors in preclinical models. Largazole possesses in vitro activity against glioblastoma multiforme (GBM) cells and sufficiently crosses the blood-brain barrier based on measurement of the active species, largazole thiol, to achieve therapeutically relevant concentrations in the mouse brain. The effective dose resulted in pronounced functional responses on the transcript level based on RNA sequencing and quantitative polymerase chain reaction after reverse transcription (RT-qPCR), revealing desirable expression changes of genes related to neuroprotection, including Bdnf and Pax6 upregulation, extending the applicability of largazole to the treatment of brain cancer and neurodegenerative disorders. The largazole-induced modulation of Pax6 unifies both activities, since Pax6 expression suppresses GBM proliferation and invasion and inversely correlates with GBM tumor grade, while it is also implicated in neurogenesis, neuronal plasticity, and cognitive ability. Our results suggest that largazole could be repurposed for diseases of the brain.

WAGRO syndrome is a rare genetic syndrome that includes Wilms tumor, aniridia, genitourinary system abnormalities, mental retardation, and obesity. The syndrome is associated with deletions of the short arm of chromosome 11 (11p), where the PAX6 and WT1 genes are located. We present the clinical case of a 7-year-old boy with aniridia, polar cataract, and concomitant neuromotor, psychomotor, and speech delays. Evaluation of the childs karyotype followed by confirmation using multiplex ligation-dependent probe amplification showed the presence of a deletion including in the 11p13-p14 region.

BackgroundAniridia is a severe autosomal dominant panocular disorder associated with pathogenic sequence variants of the PAX6 gene or 11p13 chromosomal aberrations encompassing the coding and/or regulatory regions of the PAX6 gene in a heterozygous state. Patients with aniridia display several ocular anomalies including foveal hypoplasia, cataract, keratopathy, and glaucoma, which can vary in severity and combination.MethodsA cohort of 155 patients from 125 unrelated families with identified point PAX6 pathogenic variants (118 patients) or large chromosomal 11p13 deletions (37 patients) was analyzed. Genetic causes were divided into 6 types. The occurrence of 6 aniridic eye anomalies was analyzed. Fisher’s exact test was applied for 2×2 contingency tables assigning numbers of patients with/without each sign and each type of the PAX6 variants or 11p13 deletions with Benjamini–Hochberg correction. The age of patients with different types of mutation did not differ.ResultsPatients with 3′-cis-regulatory region deletions had a milder aniridia phenotype without keratopathy, nystagmus, or foveal hypoplasia. The phenotypes of the patients with other rearrangements involving 11p13 do not significantly differ from those associated with point pathogenic variants in the PAX6 gene. Missense mutations and genetic variants disrupting splicing are associated with a severe aniridia phenotype and resemble loss-of-function mutations. It is particularly important that in all examined patients, PAX6 mutations were found to be associated with multiple eye malformations. The age of patients with keratopathy, cataract, and glaucoma was significantly higher than the age of patients without these signs.ConclusionWe got clear statistically significant genotype-phenotype correlations in congenital aniridia and evident that aniridia severity indeed had worsened with age.

Objective We aimed to find crucial microRNAs (miRNAs) associated with the development of atrial fibrillation (AF), and then try to elucidate the possible molecular mechanisms of miRNAs in AF. Methods The miRNA microarray, GSE68475, which included 10 right atrial appendage samples from patients with persistent AF and 11 samples from patients with normal sinus rhythm, was used for the analysis. After data preprocessing, differentially expressed miRNAs were screened using limma. Target genes of miRNAs were predicted using miRWalk2.0. We then conducted functional enrichment analyses for miRNA and target genes. Protein-protein interaction (PPI) network and module analyses for target genes were performed. Finally, transcription factors (TFs)-target genes regulatory network was predicted and constructed. Results Seven genes, including CAMK2D, IGF2R, PPP2R2A, PAX6, POU3F2, YWHAE, and AP2A2, were targeted by TFs. Among these seven genes, CAMK2D (targeted by miR-31-5p), IGF2R (targeted by miR-204-5p), PAX6 (targeted by miR-223-3p), POU3F2 (targeted by miR-204-5p), YWHAE (targeted by miR-31-5p), and AP2A2 (targeted by miR-204-5p) belonged to the top 10 degree genes in the PPI network. Notably, MiR-204-5p, miR-31-5p, and miR-223-3p had more target genes. Besides, CAMK2D was enriched in some pathways, such as adrenergic signaling in cardiomyocytes pathway and cAMP signaling pathway. YWHAE was enriched in the Hippo signaling pathway. Conclusion miR-31-5p played a crucial role in cardiomyocytes by targeting CAMK2D and YWHAE via cAMP and Hippo signaling pathways. miR-204 was involved in the progression of AF by regulating its target genes IGF2R, POU3F2, and AP2A2. On the other hand, miR-223-3p functioned in AF by targeting PAX6, which was associated with the regulation of apoptosis in AF. This study would provide a theoretical basis and potential therapeutic targets for the treatment of AF. (Anatol J Cardiol 2020; 23: 324-33).

Background Age-related macular degeneration (AMD) represents the leading cause of visual impairment in the aging population. The goal of this study was to identify aberrantly-methylated, differentially-expressed genes (MDEGs) in AMD and explore the involved pathways via integrated bioinformatics analysis. Methods Data from expression profile GSE29801 and methylation profile GSE102952 were obtained from the Gene Expression Omnibus database. We analyzed differentially-methylated genes and differentially-expressed genes using R software. Functional enrichment and protein–protein interaction (PPI) network analysis were performed using the R package and Search Tool for the Retrieval of Interacting Genes online database. Hub genes were identified using Cytoscape. Results In total, 827 and 592 genes showed high and low expression, respectively, in GSE29801; 4117 hyper-methylated genes and 511 hypo-methylated genes were detected in GSE102952. Based on overlap, we categorized 153 genes as hyper-methylated, low-expression genes (Hyper-LGs) and 24 genes as hypo-methylated, high-expression genes (Hypo-HGs). Four Hyper-LGs (CKB, PPP3CA, TGFB2, SOCS2) overlapped with AMD risk genes in the Public Health Genomics and Precision Health Knowledge Base. KEGG pathway enrichment analysis indicated that Hypo-HGs were enriched in the calcium signaling pathway, whereas Hyper-LGs were enriched in sphingolipid metabolism. In GO analysis, Hypo-HGs were enriched in fibroblast migration, membrane raft, and coenzyme binding, among others. Hyper-LGs were enriched in mRNA transport, nuclear speck, and DNA binding, among others. In PPI network analysis, 23 nodes and two edges were established from Hypo-HGs, and 151 nodes and 73 edges were established from Hyper-LGs. Hub genes (DHX9, MAPT, PAX6) showed the greatest overlap. Conclusion This study revealed potentially aberrantly MDEGs and pathways in AMD, which might improve the understanding of this disease.

Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia., Graphical Abstract, Aniridia is a rare genetic disease that reduces vision and progresses to blindness due to unmet therapeutic needs. The authors first created a new aniridic mouse model that allows quantification of genome-based therapies. They then successfully developed a CRISPR strategy to correct this mouse’s germline mutation and completely rescued the blindness.

Congenital PAX6-aniridia is a rare panocular disease resulting from limbal stem cell deficiency. In PAX6-aniridia, the downregulation of the retinol-metabolizing enzymes ADH7 (All-trans-retinol dehydrogenase 7) and ALDH1A1/A3 (Retinal dehydrogenase 1, Aldehyde dehydrogenase family 1 member A3) have been described in limbal epithelial cells (LECs) and conjunctival epithelial cells. The aim of this study was to identify the role of retinol derivates in the differentiation of human LEC and its potential impact on aniridia-associated keratopathy development. Human LEC were isolated from healthy donor corneas and were cultured with retinol, retinoic acid, or pan-retinoic acid receptor antagonist (AGN 193109) acting on RARα, β, γ (NR1B1, NR1B2 NR1B3) or were cultured with pan-retinoid X receptor antagonist (UVI 3003) acting on RXR α, β, γ (retinoid X receptor, NR2B1, NR2B2, BR2B3). Using qPCR, differentiation marker and retinoid-/fatty acid metabolism-related mRNA expression was analysed. DSG1 (Desmoglein 1), KRT3 (Keratin 3), and SPINK7 (Serine Peptidase Inhibitor Kazal Type 7) mRNA expression was downregulated when retinoid derivates were used. AGN 193109 treatment led to the upregulation of ADH7, KRT3, and DSG1 mRNA expression and to the downregulation of KRT12 (Keratin 12) and KRT19 (Keratin 19) mRNA expression. Retinol and all-trans retinoic acid affect some transcripts of corneal LEC in a similar way to what has been observed in the LEC of PAX6-aniridia patients with the altered expression of differentiation markers. An elevated concentration of retinol derivatives in LEC or an altered response to retinoids may contribute to this pattern. These initial findings help to explain ocular surface epithelia differentiation disorders in PAX6-aniridia and should be investigated in patient cells or in cell models in the future in more detail.

Purpose Foveal hypoplasia (FVH) is defined as the lack of fovea with a relatively preserved neuroretina, occurring either as an isolated FVH (IFVH) condition or associated with other diseases. This study aimed to systemically molecularly characterize IFVH. Methods Genetic defects in 33 families with IFVH were analyzed by exome sequencing. Variants in three genes (PAX6, SLC38A8, and AHR) were selected and evaluated with multistep bioinformatic tools. Results Mutations in the three genes were identified in 69.7% (23/33) of families with IFVH and infantile nystagmus, including 18 families with PAX6 mutations, 5 with SLC38A8 mutations, but none with AHR mutations. Clinical data from 32 patients in the 23 families showed FVH, infantile nystagmus, and full iris. Careful follow-up visits revealed subtle changes in iris in 9 of 14 patients with PAX6 variants. The PAX6 variants of the 18 families (15 missense and one stop-loss) were mostly located in the C-terminal region of the paired box domain. Variants in AHR, SLC38A8, and PAX6 contributed to IFVH in one (2%), 25 (45%), and 30 (53%) families with identified genetic defects (23 families in this study and 33 reported previously), respectively. Conclusions PAX6 and SLC38A8 mutations are the main cause of IFVH based on our data and a systematic review. IFVH-associated PAX6 variants are mostly missense with a specific location, indicating a specific correlation of these variants with IFVH but not with typical aniridia. Full iris with subtle structural abnormalities is more common in patients with PAX6-associated IFVH, suggesting a potential diagnostic indicator.

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2β (AP-2β) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2β is conditionally deleted in the NCCs (AP-2β NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2β deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2β NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/β-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.

Neural stem cells (NSCs) have great potential in the application of neurodegenerative disease therapy, drug screening and disease modeling. NSC can be generated by reprogramming from terminally differentiated cells with transcription factors or small molecules. However, current methods for producing NSCs involve the danger of integrating foreign genes into the genome and the problem of low efficiency. Here, we report an efficient method to generate NSCs from human skin-derived fibroblasts with microRNA (mir-302a) in 2-3 days. The induced NSCs (iNSCs) have more than 90% of purity. Their morphology is similar to regular NSCs, expressing key markers including Nestin, Pax6 and Sox2, and can be expanded for more than 20 passages in vitro. They can also differentiate into functional neuron progeny, astrocytes and oligodendrocytes as well. Those cells can elicit action potential, can be xeno-transplanted into the brain of immune-deficient mice, and can survive and differentiate in vivo without tumor formation. This study shows that a single part of pluripotency-inducing mir-302 cluster can drive fibroblasts reprogramming, providing a general platform for high-efficiency generation of individual-specific human NSCs for studies of neuron system development and regenerative cell therapy.

The prethalamic eminence (PThE) is the most dorsal subdomain of the prethalamus, which corresponds to prosomere 3 (p3) in the prosomeric model for vertebrate forebrain development. In mammalian and avian embryos, the PThE can be delimited from other prethalamic areas by its lack of Dlx gene expression, as well as by its expression of glutamatergic-related genes such as Pax6, Tbr2 and Tbr1. Several studies in mouse embryos postulate the PThE as a source of migratory neurons that populate given telencephalic centers. Concerning the avian PThE, it is visible at early embryonic stages as a compact primordium, but its morphology becomes cryptic at perinatal stages, so that its developmental course and fate are largely unknown. In this report, we characterize in detail the ontogeny of the chicken PThE from 5 to 15 days of development, according to morphological criteria, and using Tbr1 as a molecular marker for this structure and its migratory cells. We show that initially the PThE contacts rostrally the medial pallium, the pallial amygdala and the paraventricular hypothalamic alar domain. Approximately from embryonic day 6 onwards, the PThE becomes progressively reduced in size and cell content due to massive tangential migration of many of its neuronal derivatives towards nearby subpallial and hypothalamic regions. Our analysis supports that these migratory neurons from the avian PThE target telencephalic centers such as the commissural septal nuclei, as previously described in mammals, but also the diagonal band and preoptic areas, and hypothalamic structures in the paraventricular hypothalamic area.

Background: CCL2 (also known as monocyte chemoattractant protein 1) and CX3CR1 (also known as Fractalkine receptor)-deficient mice have damaged photoreceptors. Objectives: We examined the interaction of SDF1 and CXCR4 on the differentiation of retinal progenitors into rhodopsin-positive photoreceptors. Methods: Cloned retinal progenitors were obtained by Pax6 gene transfection of mouse iPS cells followed by serial dilution. Clones were selected by expression of nestin, Musashi1, Six3, and Chx10 mRNA. Cell surface protein expression was analyzed by flow cytometry. The levels of mRNA and intracellular protein were examined by real-time PCR and immunochemistry, respectively. Transient transfection experiments of retinal progenitors were conducted using a human rhodopsin promoter luciferase plasmid. Results: We selected 10 clones that expressed Six3, Chx10, Crx, Rx1, Nrl, CD73, and rhodopsin mRNA, which, except for rhodopsin, are photoreceptor precursor markers. Clones expressed both CD73 and CXCR4 on the cell surface and differentiated into rhodopsin-positive photoreceptors, which was reinforced by the addition of exogenous SDF1. A CXCR4 inhibitor AMD3100 blocked SDF1-mediated differentiation of progenitors into photoreceptors. SDF1 enhanced human rhodopsin promoter transcription activity, possibly via the NFκB pathway. Addition of SDF1 to the cell culture induced nuclear translocation of NFκB on retinal progenitor cell clones. Neonatal and newborn mouse retinas expressed SDF1 and CXCR4. Cells in the outer nuclear layer where photoreceptors are located expressed CXCR4 at P14 and P56. Cells in the inner nuclear layer expressed SDF1. Conclusions: These findings suggest that retinal progenitor cell differentiation was at least partly regulated by SDF1 and CXCR4 via upregulation of NFκB activity.