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1.
Cell ; 184(21): 5465-5481.e16, 2021 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-34582787

RESUMEN

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.


Asunto(s)
Astrocitos/citología , Diferenciación Celular , Linaje de la Célula , Neuronas/citología , Animales , Astrocitos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/patología , Lesiones Encefálicas/patología , Línea Celular Tumoral , Reprogramación Celular , Dependovirus/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica , Genes Reporteros , Proteína Ácida Fibrilar de la Glía/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Integrasas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo
2.
Cell ; 174(3): 590-606.e21, 2018 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-29961574

RESUMEN

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Animales , Proteínas de Unión al Calcio , Corteza Cerebral/metabolismo , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteína Jagged-2 , Mamíferos/embriología , Ratones , Ratones Endogámicos C57BL , Neocórtex/fisiología , Células-Madre Neurales , Neurogénesis/fisiología , Neuroglía/fisiología , Neuronas , Factor de Transcripción PAX6/metabolismo , Proteínas Represoras , Transducción de Señal , Serpientes/embriología , Proteínas Roundabout
3.
Proc Natl Acad Sci U S A ; 120(39): e2300587120, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37725647

RESUMEN

Oomycetes were recently discovered as natural pathogens of Caenorhabditis elegans, and pathogen recognition alone was shown to be sufficient to activate a protective transcriptional program characterized by the expression of multiple chitinase-like (chil) genes. However, the molecular mechanisms underlying oomycete recognition in animals remain fully unknown. We performed here a forward genetic screen to uncover regulators of chil gene induction and found several independent loss-of-function alleles of old-1 and flor-1, which encode receptor tyrosine kinases belonging to the C. elegans-specific KIN-16 family. We report that OLD-1 and FLOR-1 are both necessary for mounting the immune response and act in the epidermis. FLOR-1 is a pseudokinase that acts downstream of the active kinase OLD-1 and regulates OLD-1 levels at the plasma membrane. Interestingly, the old-1 locus is adjacent to the chil genes in the C. elegans genome, thereby revealing a genetic cluster important for oomycete resistance. Furthermore, we demonstrate that old-1 expression at the anterior side of the epidermis is regulated by the VAB-3/PAX6 transcription factor, well known for its role in visual system development in other animals. Taken together, our study reveals both conserved and species-specific factors shaping the activation and spatial characteristics of the immune response to oomycete recognition.


Asunto(s)
Caenorhabditis elegans , Quitinasas , Animales , Caenorhabditis elegans/genética , Proteínas Tirosina Quinasas Receptoras , Membrana Celular , Alelos
4.
Development ; 149(15)2022 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-35831950

RESUMEN

Vsx2 is a transcription factor essential for retinal proliferation and bipolar cell differentiation, but the molecular mechanisms underlying its developmental roles are unclear. Here, we have profiled VSX2 genomic occupancy during mouse retinogenesis, revealing extensive retinal genetic programs associated with VSX2 during development. VSX2 binds and transactivates its enhancer in association with the transcription factor PAX6. Mice harboring deletions in the Vsx2 regulatory landscape exhibit specific abnormalities in retinal proliferation and in bipolar cell differentiation. In one of those deletions, a complete loss of bipolar cells is associated with a bias towards photoreceptor production. VSX2 occupies cis-regulatory elements nearby genes associated with photoreceptor differentiation and homeostasis in the adult mouse and human retina, including a conserved region nearby Prdm1, a factor implicated in the specification of rod photoreceptors and suppression of bipolar cell fate. VSX2 interacts with the transcription factor OTX2 and can act to suppress OTX2-dependent enhancer transactivation of the Prdm1 enhancer. Taken together, our analyses indicate that Vsx2 expression can be temporally and spatially uncoupled at the enhancer level, and they illuminate important mechanistic insights into how VSX2 is engaged with gene regulatory networks that are essential for retinal proliferation and cell fate acquisition.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Adulto , Animales , Diferenciación Celular/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Factores de Transcripción/metabolismo
5.
Development ; 149(6)2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35224626

RESUMEN

Pax6 is a well-known regulator of early neuroepithelial progenitor development. Its constitutive loss has a particularly strong effect on the developing prethalamus, causing it to become extremely hypoplastic. To overcome this difficulty in studying the long-term consequences of Pax6 loss for prethalamic development, we used conditional mutagenesis to delete Pax6 at the onset of neurogenesis and studied the developmental potential of the mutant prethalamic neurons in vitro. We found that Pax6 loss affected their rates of neurite elongation, the location and length of their axon initial segments, and their electrophysiological properties. Our results broaden our understanding of the long-term consequences of Pax6 deletion in the developing mouse forebrain, suggesting that it can have cell-autonomous effects on the structural and functional development of some neurons.


Asunto(s)
Proteínas de Homeodominio , Factores de Transcripción Paired Box , Animales , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Neuronas/metabolismo , Factor de Transcripción PAX6/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo
6.
Differentiation ; 137: 100781, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38631141

RESUMEN

Pax6 is a critical transcription factor involved in the development of the central nervous system. However, in humans, mutations in Pax6 predominantly result in iris deficiency rather than neurological phenotypes. This may be attributed to the distinct functions of Pax6 isoforms, Pax6a and Pax6b. In this study, we investigated the spatial and temporal expression patterns of Pax6 isoforms during different stages of mouse eye development. We observed a strong correlation between Pax6a expression and the neuroretina gene Sox2, while Pax6b showed a high correlation with iris-component genes, including the mesenchymal gene Foxc1. During early patterning from E10.5, Pax6b was expressed in the hinge of the optic cup and neighboring mesenchymal cells, whereas Pax6a was absent in these regions. At E14.5, both Pax6a and Pax6b were expressed in the future iris and ciliary body, coinciding with the integration of mesenchymal cells and Mitf-positive cells in the outer region. From E18.5, Pax6 isoforms exhibited distinct expression patterns as lineage genes became more restricted. To further validate these findings, we utilized ESC-derived eye organoids, which recapitulated the temporal and spatial expression patterns of lineage genes and Pax6 isoforms. Additionally, we found that the spatial expression patterns of Foxc1 and Mitf were impaired in Pax6b-mutant ESC-derived eye organoids. This in vitro eye organoids model suggested the involvement of Pax6b-positive local mesodermal cells in iris development. These results provide valuable insights into the regulatory roles of Pax6 isoforms during iris and neuroretina development and highlight the potential of ESC-derived eye organoids as a tool for studying normal and pathological eye development.


Asunto(s)
Ojo , Regulación del Desarrollo de la Expresión Génica , Organoides , Factor de Transcripción PAX6 , Isoformas de Proteínas , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Animales , Ratones , Organoides/metabolismo , Organoides/crecimiento & desarrollo , Organoides/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Organogénesis/genética
7.
J Biol Chem ; 299(3): 103020, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36791914

RESUMEN

Neural stem cells (NSCs) proliferation and differentiation rely on proper expression and posttranslational modification of transcription factors involved in the determination of cell fate. Further characterization is needed to connect modifying enzymes with their transcription factor substrates in the regulation of these processes. Here, we demonstrated that the inhibition of KAT2A, a histone acetyltransferase, leads to a phenotype of small eyes in the developing embryo of zebrafish, which is associated with enhanced proliferation and apoptosis of NSCs in zebrafish eyes. We confirmed that this phenotype is mediated by the elevated level of PAX6 protein. We further verified that KAT2A negatively regulates PAX6 at the protein level in cultured neural stem cells of rat cerebral cortex. We revealed that PAX6 is a novel acetylation substrate of KAT2A and the acetylation of PAX6 promotes its ubiquitination mediated by the E3 ligase RNF8 that facilitated PAX6 degradation. Our study proposes that KAT2A inhibition results in accelerated proliferation, delayed differentiation, or apoptosis, depending on the context of PAX6 dosage. Thus, the KAT2A/PAX6 axis plays an essential role to keep a balance between the self-renewal and differentiation of NSCs.


Asunto(s)
Células-Madre Neurales , Pez Cebra , Animales , Ratas , Diferenciación Celular/fisiología , Proliferación Celular , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Células-Madre Neurales/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo
8.
EMBO J ; 39(21): e105479, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-32985705

RESUMEN

Structural integrity and cellular homeostasis of the embryonic stem cell niche are critical for normal tissue development. In the telencephalic neuroepithelium, this is controlled in part by cell adhesion molecules and regulators of progenitor cell lineage, but the specific orchestration of these processes remains unknown. Here, we studied the role of microRNAs in the embryonic telencephalon as key regulators of gene expression. By using the early recombiner Rx-Cre mouse, we identify novel and critical roles of miRNAs in early brain development, demonstrating they are essential to preserve the cellular homeostasis and structural integrity of the telencephalic neuroepithelium. We show that Rx-Cre;DicerF/F mouse embryos have a severe disruption of the telencephalic apical junction belt, followed by invagination of the ventricular surface and formation of hyperproliferative rosettes. Transcriptome analyses and functional experiments in vivo show that these defects result from upregulation of Irs2 upon loss of let-7 miRNAs in an apoptosis-independent manner. Our results reveal an unprecedented relevance of miRNAs in early forebrain development, with potential mechanistic implications in pediatric brain cancer.


Asunto(s)
Homeostasis , Proteínas Sustrato del Receptor de Insulina/metabolismo , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Telencéfalo/embriología , Telencéfalo/metabolismo , Uniones Adherentes , Animales , Apoptosis , Proliferación Celular , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Factor de Transcripción PAX6/metabolismo , Proteínas Represoras/genética , Células Madre/metabolismo , Telencéfalo/citología , Factores de Transcripción/metabolismo
9.
Development ; 148(9)2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33982759

RESUMEN

Genetic screens are designed to target individual genes for the practical reason of establishing a clear association between a mutant phenotype and a single genetic locus. This allows for a developmental or physiological role to be assigned to the wild-type gene. We previously observed that the concurrent loss of Pax6 and Polycomb epigenetic repressors in Drosophila leads the eye to transform into a wing. This fate change is not seen when either factor is disrupted separately. An implication of this finding is that standard screens may miss the roles that combinations of genes play in development. Here, we show that this phenomenon is not limited to Pax6 and Polycomb but rather applies more generally. We demonstrate that in the Drosophila eye-antennal disc, the simultaneous downregulation of Pax6 with either the NURF nucleosome remodeling complex or the Pointed transcription factor transforms the head epidermis into an antenna. This is a previously unidentified fate change that is also not observed with the loss of individual genes. We propose that the use of multi-gene knockdowns is an essential tool for unraveling the complexity of development.


Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Animales , Epidermis , Ojo/citología , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Larva , Nucleosomas , Proteínas del Grupo Polycomb/genética , Factores de Transcripción/metabolismo
10.
Stem Cells ; 41(12): 1133-1141, 2023 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-37632794

RESUMEN

Congenital aniridia is caused by heterozygous mutations on the PAX6 gene leading to reduced amount of PAX6 protein (haploinsufficiency), abnormal eye development, and aniridia-associated keratopathy (AAK). This progressive corneal opacification resembles late-onset limbal stem cell (LSC) deficiency, leading to disrupted corneal epithelial renewal. The factors leading to AAK are not known and defects in native LSC differentiation and/or features leading to ocular surface dysfunction like inflammation and loss of innervation could contribute to development of AAK. Here, we produced induced pluripotent stem cells (hiPSC) from 3 AAK patients and examined whether PAX6 haploinsufficiency affects LSC lineage commitment. During LSC differentiation, characterization of the AAK lines showed lowered PAX6 expression as compared to wild type (WT) controls and expression peak of PAX6 during early phase of differentiation was detected only in the WT hiPSC lines. Whether it reflects developmental regulation remains to be studied further. Nevertheless, the AAK-hiPSCs successfully differentiated toward LSC lineage, in line with the presence of LSCs in young patients before cell loss later in life. In addition, patient-specific LSCs showed similar wound healing capacity as WT cells. However, extensive batch-related variation in the LSC marker expression and wound healing efficacy was detected without clear correlation to AAK. As development and maintenance of corneal epithelium involves an interplay between LSCs and their environment, the AAK-hiPSCs generated here can be further used to study the crosstalk between LSCs and limbal niche including, eg, corneal immune cells, stroma cells, and neurons.


Asunto(s)
Aniridia , Enfermedades de la Córnea , Epitelio Corneal , Células Madre Pluripotentes Inducidas , Limbo de la Córnea , Humanos , Córnea , Epitelio Corneal/metabolismo , Enfermedades de la Córnea/genética , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Aniridia/genética
11.
Exp Eye Res ; 243: 109916, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38679224

RESUMEN

The conjunctiva is a non-keratinized, stratified columnar epithelium with characteristics different from the cornea and eyelid epidermis. From development to adulthood, a distinguishing feature of ocular versus epidermal epithelia is the expression of the master regulator PAX6. A conditionally immortalized conjunctival epithelial cell line (iHCjEC) devoid of stromal or immune cells established in our laboratory spontaneously manifested epidermal metaplasia and upregulated expression of the keratinization-related genes SPRR1A/B and the epidermal cytokeratins KRT1 and KRT10 at the expense of the conjunctival trait. In addition, iHCjEC indicated a significant decrease in PAX6 expression. Dry eye syndrome (DES) and severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome, cause the keratinization of the entire ocular surface epithelia. We used iHCjECs as a conjunctiva epidermal metaplasia model to test PAX6, serum, and glucocorticoid interventions. Reintroducing PAX6 to iHCjECs resulted in upregulating genes related to cell adhesion and tight junctions, including MIR200CHG and CLDN1. The administration of glucocorticoids or serum resulted in the downregulation of epidermal genes (DSG1, SPRR1A/B, and KRT1) and partially corrected epidermal metaplasia. Our results using an isolated conjunctival epidermal metaplasia model point toward the possibility of rationally "repurposing" clinical interventions, such as glucocorticoid, serum, or PAX6 administration, for treating epidermal metaplasia of the conjunctiva.


Asunto(s)
Conjuntiva , Metaplasia , Conjuntiva/patología , Conjuntiva/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Glucocorticoides/uso terapéutico , Regulación de la Expresión Génica , Epidermis/patología , Epidermis/metabolismo , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Línea Celular
12.
Exp Eye Res ; 248: 110105, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-39303843

RESUMEN

Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6+/-). In the corneal epithelium of PAX6+/- mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p≤0.008), while the decrease in PHD1 was limited to AN-LFCs (p = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p = 0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p = 0.005), SOD1 (p = 0.005), GPX1 (p = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p = 0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p≥0.151) and protein expression (p ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl2 treatment.


Asunto(s)
Aniridia , Western Blotting , Fibroblastos , Limbo de la Córnea , Estrés Oxidativo , Fibroblastos/metabolismo , Humanos , Células Cultivadas , Aniridia/genética , Aniridia/metabolismo , Aniridia/patología , Limbo de la Córnea/metabolismo , Limbo de la Córnea/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Regulación de la Expresión Génica , Femenino , Masculino , Adulto , Reacción en Cadena en Tiempo Real de la Polimerasa , Catalasa/metabolismo , Catalasa/genética , Cobalto/farmacología , Cobalto/toxicidad
13.
Hum Genomics ; 17(1): 45, 2023 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-37269011

RESUMEN

BACKGROUND: Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches. RESULTS: Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1. CONCLUSIONS: In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.


Asunto(s)
Aniridia , Factores de Transcripción Paired Box , Humanos , Factores de Transcripción Paired Box/genética , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Aniridia/genética , Inversión Cromosómica , Mutación
14.
FASEB J ; 37(2): e22776, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36688817

RESUMEN

AQP5 plays a crucial role in maintaining corneal transparency and the barrier function of the cornea. Here, we found that in the corneas of Aqp5-/- mice at older than 6 months, loss of AQP5 significantly increased corneal neovascularization, inflammatory cell infiltration, and corneal haze. The results of immunofluorescence staining showed that upregulation of K1, K10, and K14, and downregulation of K12 and Pax6 were detected in Aqp5-/- cornea and primary corneal epithelial cells. Loss of AQP5 aggravated wound-induced corneal neovascularization, inflammation, and haze. mRNA sequencing, western blotting, and qRT-PCR showed that Wnt2 and Wnt6 were significantly decreased in Aqp5-/- corneas and primary corneal epithelial cells, accompanied by decreased aggregation in the cytoplasm and nucleus of ß-catenin. IIIC3 significantly suppressed corneal neovascularization, inflammation, haze, and maintained corneal transparent epithelial in Aqp5-/- corneas. We also found that pre-stimulated Aqp5-/- primary corneal epithelial cells with IIIC3 caused the decreased expression of K1, K10, and K14, the increased expression of K12, Pax6, and increased aggregation in the cytoplasm and nucleus of ß-catenin. These findings revealed that AQP5 may regulate corneal epithelial homeostasis and function through the Wnt/ß-catenin signaling pathway. Together, we uncovered a possible role of AQP5 in determining corneal epithelial cell fate and providing a potential therapeutic target for corneal epithelial dysfunction.


Asunto(s)
Neovascularización de la Córnea , Vía de Señalización Wnt , Ratones , Animales , Acuaporina 5/genética , Neovascularización de la Córnea/metabolismo , beta Catenina/metabolismo , Córnea/metabolismo , Células Epiteliales/metabolismo , Inflamación/metabolismo
15.
Cell Biol Int ; 2024 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-39308152

RESUMEN

Pax-6 emerges as a critical transcription factor that guides the fate of stem cells towards neural lineages. Its expression influences the differentiation of neural progenitors into diverse neuronal subtypes, glial cells, and other neural cell types. Pax-6 operates with other regulatory factors to ensure the precise patterning and organization of the developing nervous system. The intricate interplay between Pax-6 and other signaling pathways, transcription factors, and epigenetic modifiers underpins the complicated balance between stem cell maintenance, proliferation, and differentiation in neuroectodermal and ocular contexts. Dysfunction of Pax-6 can lead to a spectrum of developmental anomalies, underscoring its importance in these processes. This review highlights the essential role of Pax-6 expression in neuroectodermal and ocular stem cells, shedding light on its significance in orchestrating the intricate journey from stem cell fate determination to the emergence of diverse neural and ocular cell types. The comprehensive understanding of Pax-6 function gained from a developmental biology perspective offers valuable insights into normal development and potential therapeutic avenues for neuroectodermal and ocular disorders.

16.
Mol Biol Rep ; 51(1): 1096, 2024 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-39460795

RESUMEN

BACKGROUND: Medulloblastoma is a pediatric malignant brain tumor associated with an aberrantly activated Shh pathway. The Shh pathway acts via downstream effector molecules, including Pax6 and Nkx2.2. Transcription factor Nkx2.2 plays crucial roles during early embryonic patterning and development. In this study, we aimed to determine the role of transcription factor Nkx2.2 in medulloblastoma development. METHODS AND RESULTS: Here, whole transcriptome levels and suppressive effect of transcription factor Nkx2.2 on Pax6 were assessed using one normal human brain and three surgically removed medulloblastoma samples. Additionally, protein levels of Shh, Gli1, Pax6, and Nkx2.2 and co-expression patterns of Pax6 and Nkx2.2 were assessed in 14 medulloblastoma samples. Quantitative reverse transcription-polymerase chain reaction revealed the suppressive effect of Nkx2.2 on Pax6. D283 cells were treated with the Shh pathway activator, SAG, and Gli1 inhibitor, GANT61, which revealed Pax6-Nkx2.2 regulation. Increased cell proliferation was observed in D283 cells transfected with Nkx2.2 small interfering RNA. Moreover, mRNA expression levels of Shh, Pax6, Nkx2.2, and Gli1 were assessed in Daoy cells transfected with Gli1 and Nkx2.2 small interfering RNAs using quantitative reverse transcription-polymerase chain reaction. Pax6 levels were increased in Nkx2.2 siRNA-transfected cells. CONCLUSIONS: Aberrantly activated Shh pathway leads to the ectopic expression of Pax6 in granular cells, inducing medulloblastoma development. Moreover, Nkx2.2 transcription factor acts as a suppressor of Pax6 during medulloblastoma development and maintenance. Overall, this study provides novel insights for the development of effective therapeutic strategies and suggests potential targets for medulloblastoma.


Asunto(s)
Proliferación Celular , Proteínas Hedgehog , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Meduloblastoma , Factor de Transcripción PAX6 , Transducción de Señal , Factores de Transcripción , Proteínas de Pez Cebra , Proteína con Dedos de Zinc GLI1 , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Humanos , Factor de Transcripción PAX6/metabolismo , Factor de Transcripción PAX6/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Regulación Neoplásica de la Expresión Génica/genética , Animales , Pirimidinas/farmacología , Piridinas/farmacología , Proteínas Nucleares
17.
Bioessays ; 44(12): e2200163, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36317531

RESUMEN

Pioneering molecular work on chelicerate visual system development in the horseshoe crab Limulus polyphemus surprised with the possibility that this process may not depend on the deeply conserved retinal determination function of Pax6 transcription factors. Genomic, transcriptomic, and developmental studies in spiders now reveal that the arthropod Pax6 homologs eyeless and twin of eyeless act as ancestral determinants of the ocular head segment in chelicerates, which clarifies deep gene regulatory and structural homologies and recommends more unified terminologies in the comparison of arthropod visual systems. Following this phylotypic stage, chelicerate visual system development differs fundamentally from other arthropods during the compartmentalization of the ocular segment in that eye and optic neuropil primordia originate independently from each other. Comparative analyses of this phase identified further gene regulatory homologies but also major differences, most notably the possibly compensatory replacement of Pax6 by Pax2 in lateral eye specification. Also see the video abstract here: https://youtu.be/Hdfr3z5kEXU.


Asunto(s)
Ojo , Cangrejos Herradura , Animales , Genoma , Cangrejos Herradura/genética , Factor de Transcripción PAX6/genética
18.
BMC Ophthalmol ; 24(1): 157, 2024 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-38594720

RESUMEN

BACKGROUND: Aniridia is a rare eye disorder with a high incidence of glaucoma, and surgical intervention is often needed to control the intraocular pressure (IOP). Here, we reported a case of illuminated microcatheter-assisted circumferential trabeculotomy (MAT) performed on an aniridic glaucoma patient following a previous failed angle surgery. The surgical procedures for aniridic glaucoma were also reviewed. CASE PRESENTATION: A 21-year-old man, diagnosed with aniridic glaucoma, came to our hospital consulting for the poor control of left eye's IOP despite receiving goniotomy surgery 3 years ago. The IOP was 26 mmHg with maximum topical antiglaucoma eyedrops. The central cornea was opaque and the majority of iris was absent. The gonioscopy and ultrasound biomicroscopy (UBM) demonstrated that 360° anterior chamber angle was closed. The whole exome sequencing of peripheral blood confirmed a 13.39 Mb copy number loss at chromosome 11p15.1p13, containing PAX6 and WT1 gene. The 360° MAT surgery was performed on his left eye. At 1-year follow-up, the IOP was 19mmHg with 2 kinds of topical antiglaucoma medications, and the postoperative UBM demonstrated the successful incision of the anterior chamber angle. CONCLUSIONS: The case presented here exhibited a case of aniridic glaucoma treated by MAT surgery. The MAT surgery may be an effective option for IOP control in aniridic glaucoma patients following a previous failed angle surgery.


Asunto(s)
Aniridia , Glaucoma , Trabeculectomía , Humanos , Masculino , Adulto Joven , Estudios de Seguimiento , Glaucoma/diagnóstico , Glaucoma/cirugía , Gonioscopía , Presión Intraocular , Factor de Transcripción PAX6 , Estudios Retrospectivos , Trabeculectomía/métodos , Resultado del Tratamiento
19.
Indian J Clin Biochem ; 39(1): 47-59, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38223000

RESUMEN

Meningioma is a common brain tumour which has neither a specific detection nor treatment method. The Sonic hedgehog (Shh) cell signaling pathway is a crucial regulatory pathway of mammalian organogenesis and tumorigenesis including meningioma. Shh cell signalling pathway cascade function by main transcription factor Gli1 and which further regulates in its downstream to Pax6 and Nkx2.2. This current study is aimed to explore the regulation of the Sonic hedgehog-Gli1 cell signaling pathway and its potential downstream targets in meningioma samples. A total of 24 surgically resected meningioma samples were used in this current study.Cytological changes were assessed using electron microscopic techniques as well as hematoxylin & eosin and DAPI staining. The expression pattern of Gli1, Nkx2.2 and Pax6 transcription factors were determined by using immunohistochemistry. The mRNA expression was assessed using RT-qPCR assays. Later, the whole transcriptome analysis of samples was performed with the amploseq technique. Results were compared with those obtained in normal human brain tissue (or normal meninges). Compared to the normal human brain tissue, meningioma samples showed crowded nuclei with morphological changes. Transcription factor Nkx2.2 expressed highly in all samples (24/24, 100%). Twenty-one of the 24 meningiomas (88%) showed high Gli1 and Pax6 expression. Whole transcriptome analysis of two meningioma samples also exhibited a very high increase in Gli1 expression signal in meningioma samples as compare to normal control. Hence, we may conclude that the Shh-Gli1 pathway is aberrantly activated in meningioma cells and is canonically upregulating the expression of transcription factors Pax6 and Nkx2.2. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01085-1.

20.
Pak J Med Sci ; 40(3Part-II): 509-513, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38356808

RESUMEN

Objective: To investigate the correlation of maternal PDX1 methylation, NGN3 and Pax6 expression levels with neonatal blood sugars and birth weight in pregnant women with GDM and non GDM. Methods: This was a prospective cohort study. Total 80 pregnant women who were examined and delivered in the Department of Obstetrics of Affiliated Hospital of Hebei University from January 2019 to June 2022 were recruited and divided into two groups according to the results of oral glucose tolerance test (OGTT): the control group and the observation group, with 40 cases in each group. PDXl methylation rate was measured by the methylation-specific PCR method, whereas gene expression levels of PDX1, NGN3 and Pax6 were measured by RT-PCR meanwhile, neonatal blood glucose and hemoglobin A1c (HbA1c) levels were also measured. Results: In comparison with the control group, the observation group had higher levels of FBG, 2-hour postprandial blood glucose (2hPBG) and HbA1c (P<0.05). Neonatal birth weight and insulin levels in the observation group were significantly higher than those in the control group, while Apgar scores and blood glucose were lower than those in the control group(P<0.05). Moreover, the observation group had significantly lower gene expression levels of PDX1, NGN3 and Pax6, and a higher PDX1 methylation rate than the control group (P<0.05). Correlation analysis revealed a negative correlation between neonatal blood glucose levels and PDX1, NGN3 and Pax6 levels in the observation group, with statistical significance (P<0.05). Conclusion: Changes in maternal PDX1 methylation, NGN3 and Pax6 expression levels may lead to abnormal glucose metabolism in neonates, which has a close bearing on neonatal hypoglycemia and blood glucose levels caused by GDM.

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