The aim of this study was to report PAX6 disease-causing variants in six Czech families, to describe the associated phenotypes, and to perform functional assessment of the splice site variants. Detailed ophthalmic examination was performed. The PAX6 coding region was directly sequenced in three probands. Two probands were analysed by exome sequencing and one by genome sequencing. The effect of two variants on pre-mRNA splicing was evaluated using an exon trapping assay. Six different heterozygous PAX6 variants were identified, with c.111_120del and c.1183+1G˃T being novel. Both c.1183+1G˃T and c.1032+1G>A were proved to cause aberrant splicing with exon skipping and subsequent frameshift. The phenotypic features were variable between and within families. One individual, aged 31 years, presented with mild unilateral ptosis accompanied by aniridia in the right eye, partial aniridia in the left eye, and bilateral congenital cataracts, without marked foveal hypoplasia. Bilateral microcornea, partial aniridia, congenital cataracts, and a large posterior segment coloboma were found in another proband, aged 32 years. One child, aged 8 years, had bilateral high myopia, optic nerve colobomas, anterior polar cataracts, but no iris defects. Another individual, aged 46 years, had bilateral congenital ptosis, iris hypoplasia, keratopathy with marked fibrovascular pannus, anterior polar cataract, and foveal hypoplasia combined with impaired glucose tolerance. However, his daughter, aged 11 years, showed classical features of aniridia. Our study extends the genetic spectrum of PAX6 disease-causing variants and confirms that the associated phenotypic features may be very broad and different to the 'classical' aniridia.

• MeSH
• aniridie * genetika   MeSH
• dítě MeSH
• dospělí MeSH
• fenotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• oční proteiny genetika   MeSH
• rodokmen MeSH
• sestřih RNA MeSH
• transkripční faktor PAX6 genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• oční proteiny MeSH
• PAX6 protein, human MeSH Prohlížeč
• transkripční faktor PAX6 MeSH

Wnt/β-catenin signaling plays an essential role in the retinal pigment epithelium (RPE) determination. Since activity of Pax6 (together with Pax2) is also required for the RPE determination, we investigated a possible genetic interaction between Pax6 and Wnt/β-catenin signaling pathway by analyzing Pax6, β-catenin, and Pax6/β-catenin conditional knockout mice. Although Pax6 inactivation alone had no impact on initial specification determined by the expression of Mitf and Otx2, melanin pigmentation was reduced in the RPE. This suggests that along with Mitf and Otx2, Pax6 is required for the full differentiation of RPE. Reporter gene assays in vitro suggest that hypopigmentation is at least in part due to the direct regulation of genes encoding enzymes involved in melanin synthesis by Pax6, Mitf, and β-catenin. The RPE of a β-catenin/Pax6 double mutant was differentiated into the neural retina; however, the tissue was thinner than that of the conditional β-catenin mutant due to reduced proliferation. Together, our data demonstrate that Pax6 is required for the RPE differentiation by regulating pigmentation and accountable for hyperproliferation in the transdifferentiated RPE. In this context, Pax6 appears to function as a pleiotropic regulator, directing development of ocular tissues in concert with the signaling pathway and, at the same time, regulating expression of structural component of the eye, such as shielding pigment.

• MeSH
• beta-katenin genetika   metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• oční proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• retina cytologie   metabolismus   MeSH
• retinální pigmentový epitel metabolismus   MeSH
• signální dráha Wnt * MeSH
• transdiferenciace buněk MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• homeodoménové proteiny MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• represorové proteiny MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box MeSH

The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. Here we show that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6(Sey/flox) embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. Our data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner.

• Klíčová slova
• Lens induction, Pax6, Retinal progenitor, mRx-Cre,
• MeSH
• buněčná diferenciace genetika   fyziologie   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• genový knockdown MeSH
• homeodoménové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• kontrolní body buněčného cyklu genetika   fyziologie   MeSH
• myši knockoutované MeSH
• myši transgenní MeSH
• myši MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• oční čočka embryologie   metabolismus   MeSH
• oční proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• represorové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• retina cytologie   embryologie   metabolismus   MeSH
• těhotenství MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box antagonisté a inhibitory   genetika   metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cone rod homeobox protein MeSH Prohlížeč
• homeodoménové proteiny MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• represorové proteiny MeSH
• trans-aktivátory MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box MeSH

Pax transcription factors are evolutionarily conserved regulators of eye development and can be distinguished on the basis of three functional domains: two DNA-binding domains (the paired domain and the paired-type homeodomain), and the octapeptide motif. PaxB of the eyed cubozoan jellyfish, Tripedalia cystophora, is characterized by a Pax2-like paired domain and octapeptide, and a Pax6-like homeodomain. In mice, functionally distinct Pax6 and Pax2 proteins have unique as well as redundant roles in eye morphogenesis. Here, we show that expression of the jellyfish PaxB gene in mouse embryonic eye tissues impairs normal development of lens and retina. Our data show that PaxB misexpression leads to a downregulation of endogenous Pax6 protein in the prospective lens and in subsets of cells within the inner nuclear layer of transgenic retina. In addition to Pax6 downregulation, the expression of PaxB leads to an almost complete loss of amacrine cells in the adult transgenic retina, a phenotype that differs from a loss-of-function of the Pax6 gene. The present data suggest that PaxB, due to its Pax2-like paired domain and Pax-6 like homeodomain, disturbs the transcriptional network regulated by Pax6 in the developing lens and retina. Taken together, our data suggest that molecular properties of individual mouse Pax2 and Pax6 proteins are essential determinants of mouse eye development and cannot be substituted for by jellyfish PaxB which possesses elements of vertebrate Pax2 and Pax6.

• MeSH
• down regulace MeSH
• embryo savčí embryologie   metabolismus   MeSH
• fenotyp MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• oči embryologie   metabolismus   MeSH
• oční proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• Scyphozoa genetika   metabolismus   MeSH
• transkripční faktor PAX2 genetika   metabolismus   MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory Otx genetika   metabolismus   MeSH
• transkripční faktory paired box genetika   metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• homeodoménové proteiny MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• represorové proteiny MeSH
• transkripční faktor PAX2 MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory Otx MeSH
• transkripční faktory paired box MeSH

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6 bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

• Klíčová slova
• amphioxus, brain, chordates, evolution, eye, genome editing, pax6,
• Publikační typ
• časopisecké články MeSH

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

• MeSH
• dánio pruhované genetika   MeSH
• ektoderm růst a vývoj   patologie   MeSH
• genové regulační sítě genetika   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• oči růst a vývoj   metabolismus   patologie   MeSH
• oční čočka růst a vývoj   metabolismus   patologie   MeSH
• transkripční faktor Meis1 MeSH
• transkripční faktor PAX6 genetika   metabolismus   MeSH
• vazebná místa MeSH
• vývojová regulace genové exprese MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• homeodoménové proteiny MeSH
• MEIS1 protein, human MeSH Prohlížeč
• Meis1 protein, mouse MeSH Prohlížeč
• Mrg1 protein, mouse MeSH Prohlížeč
• nádorové proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• transkripční faktor Meis1 MeSH
• transkripční faktor PAX6 MeSH

Genetic studies of the last decades strongly indicated that generation of particular retinal cell types is governed by gene regulatory networks of transcription factors and their target genes. The paired and homeodomain transcription factor Pax6 plays a pivotal role in retinal development as its inactivation in the retinal progenitor cell population leads to abolished differentiation of all retinal cell types. However, until now, only a few transcription factors operating downstream of Pax6 responsible for generation of individual retinal cell types have been identified. In this study, we identified two transcription factors of the Onecut family, Onecut1 and Onecut2, as Pax6 downstream-acting factors. Onecut1 and Onecut2 were previously shown to be expressed in developing horizontal cells, retinal ganglion cells and cone photoreceptors; however, their role in differentiation of these cell types is poorly understood. In this study, we show that the horizontal cell genesis is severely disturbed in Onecut-deficient retinae. In single Onecut1 and Onecut2 mutants, the number of horizontal cells is dramatically reduced while horizontal cells are completely missing in the Onecut1/Onecut2 compound mutant. Analysis of genes involved in the horizontal cell genesis such as Foxn4, Ptf1a, Prox1 and Lim1 showed that although horizontal cells are initially formed, they are not maintained in Onecut-deficient retinae. Taken together, this study suggests the model in which Pax6 regulates the maintenance of horizontal cells through the activation of Onecut1 and Onecut2 transcription factors.

• Klíčová slova
• Horizontal cell, Onecut, Pax6, Retina,
• MeSH
• čípky retiny metabolismus   MeSH
• fenotyp MeSH
• hepatocytární jaderný faktor 6 metabolismus   MeSH
• homeodoménové proteiny metabolismus   MeSH
• hybridizace in situ MeSH
• kmenové buňky cytologie   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• myši transgenní MeSH
• myši MeSH
• oční proteiny metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• retina embryologie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• vazebná místa MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hepatocytární jaderný faktor 6 MeSH
• homeodoménové proteiny MeSH
• oční proteiny MeSH
• Onecut1 protein, mouse MeSH Prohlížeč
• ONECUT2 protein, mouse MeSH Prohlížeč
• Pax6 protein, mouse MeSH Prohlížeč
• represorové proteiny MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box MeSH
• transkripční faktory MeSH

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens-specific gene expression in the surface-ectoderm. Supression of canonical Wnt/beta-catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down-regulation of canonical Wnt signaling in the presumptive lens ectoderm.

• MeSH
• beta-katenin genetika   metabolismus   MeSH
• ektoderm metabolismus   MeSH
• embryo savčí metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• morfogeneze genetika   MeSH
• myši transgenní MeSH
• myši MeSH
• oční čočka embryologie   metabolismus   MeSH
• oční proteiny genetika   metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• retina metabolismus   MeSH
• signální transdukce genetika   MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• homeodoménové proteiny MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• proteiny Wnt MeSH
• represorové proteiny MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory paired box MeSH

Mammalian corneal development is a multistep process, including formation of the corneal epithelium (CE), endothelium and stroma during embryogenesis, followed by postnatal stratification of the epithelial layers and continuous renewal of the epithelium to replace the outermost corneal cells. Here, we employed the Cre-loxP system to conditionally deplete Pax6 proteins in two domains of ocular cells, i.e., the ocular surface epithelium (cornea, limbus and conjunctiva) (OSE) or postnatal CE via K14-cre or Aldh3-cre, respectively. Earlier and broader inactivation of Pax6 in the OSE resulted in thickened OSE with CE and limbal cells adopting the conjunctival keratin expression pattern. More restricted depletion of Pax6 in postnatal CE resulted in an abnormal cornea marked by reduced epithelial thickness despite increased epithelial cell proliferation. Immunofluorescence studies revealed loss of intermediate filament Cytokeratin 12 and diffused expression of adherens junction components, together with reduced tight junction protein, Zonula occludens-1. Furthermore, the expression of Cytokeratin 14, a basal cell marker in apical layers, indicates impaired differentiation of CE cells. Collectively, our data demonstrate that Pax6 is essential for maintaining proper differentiation and strong intercellular adhesion in postnatal CE cells, whereas limbal Pax6 is required to prevent the outgrowth of conjunctival cells to the cornea.

• Klíčová slova
• Conditional knockout, Cornea, Development, E-cadherin, Eye, Keratins, Pax6,
• MeSH
• keratin-12 metabolismus   MeSH
• keratin-14 metabolismus   MeSH
• keratiny metabolismus   MeSH
• proteiny těsného spoje metabolismus   MeSH
• rohovka * metabolismus   MeSH
• rohovkový epitel * metabolismus   MeSH
• savci metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• keratin-12 MeSH
• keratin-14 MeSH
• keratiny MeSH
• proteiny těsného spoje MeSH

The paired domain, DNA-binding domain of Pax6 and other Pax transcription factors, is composed of two subdomains (PAI and RED), each recognizing distinct half-sites of the bipartite binding site in adjacent major grooves of the DNA helix. The alternatively spliced Pax6(5a) isoform containing 14 extra amino acids within the PAI domain recognizes the 5aCON sequence consisting of four interdigitated 5' half-sites of the bipartite consensus sequence. A genome database search for similar tetrameric Pax6(A) recognition sequences led to the identification of a Pax6-binding site in the lens-specific enhancer of the mouse E- and F-crystallin genes. This binding site combines the properties of bipartite and tetrameric recognition sequences and, by mutational analysis, is shown to mediate Pax6-dependent regulation of the E- and F-crystallin promoter constructs both in primary chicken lens cells and in chicken embryo fibroblasts. The Pax6-binding site is adjacent to a previously identified retinoic acid response element and is itself required for retinoic acid induction of the F- and E-crystallin genes, suggesting that Pax proteins and retinoic acid receptors cooperate in transcriptional regulation. In summary, our protein-DNA binding and transactivation studies suggest that -crystallin genes are under the control of a multifunctional enhancer element that mediates Pax6 regulation as well as retinoic acid-mediated induction.

• MeSH
• DNA vazebné proteiny genetika   fyziologie   MeSH
• down regulace MeSH
• epitelové buňky metabolismus   MeSH
• homeodoménové proteiny genetika   fyziologie   MeSH
• krystaliny genetika   MeSH
• kultivované buňky MeSH
• kuřecí embryo MeSH
• luciferasy genetika   metabolismus   MeSH
• oční čočka cytologie   metabolismus   MeSH
• oční proteiny MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• receptory kyseliny retinové genetika   fyziologie   MeSH
• regulace genové exprese účinky léků   MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   fyziologie   MeSH
• retinoidní X receptory MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• transfekce MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory bHLH MeSH
• transkripční faktory paired box MeSH
• transkripční faktory genetika   fyziologie   MeSH
• tretinoin farmakologie   MeSH
• vazebná místa genetika   MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• gro protein, Drosophila MeSH Prohlížeč
• homeodoménové proteiny MeSH
• krystaliny MeSH
• luciferasy MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• receptory kyseliny retinové MeSH
• rekombinantní fúzní proteiny MeSH
• represorové proteiny MeSH
• retinoidní X receptory MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory bHLH MeSH
• transkripční faktory paired box MeSH
• transkripční faktory MeSH
• tretinoin MeSH