Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development

NFIA Haploinsufficiency Is Associated with a CNS Malformation Syndrome and Urinary Tract Defects

Complex central nervous system (CNS) malformations frequently coexist with other developmental abnormalities, but whether the associated defects share a common genetic basis is often unclear. We describe five individuals who share phenotypically related CNS malformations and in some cases urinary tract defects, and also haploinsufficiency for the NFIA transcription factor gene due to chromosomal translocation or deletion. Two individuals have balanced translocations that disrupt NFIA. A third individual and two half-siblings in an unrelated family have interstitial microdeletions that include NFIA. All five individuals exhibit similar CNS malformations consisting of a thin, hypoplastic, or absent corpus callosum, and hydrocephalus or ventriculomegaly. The majority of these individuals also exhibit Chiari type I malformation, tethered spinal cord, and urinary tract defects that include vesicoureteral reflux. Other genes are also broken or deleted in all five individuals, and may contribute to the phenotype. However, the only common genetic defect is NFIA haploinsufficiency. In addition, previous analyses of Nfia−/− knockout mice indicate that Nfia deficiency also results in hydrocephalus and agenesis of the corpus callosum. Further investigation of the mouse Nfia+/− and Nfia−/− phenotypes now reveals that, at reduced penetrance, Nfia is also required in a dosage-sensitive manner for ureteral and renal development. Nfia is expressed in the developing ureter and metanephric mesenchyme, and Nfia+/− and Nfia−/− mice exhibit abnormalities of the ureteropelvic and ureterovesical junctions, as well as bifid and megaureter. Collectively, the mouse Nfia mutant phenotype and the common features among these five human cases indicate that NFIA haploinsufficiency contributes to a novel human CNS malformation syndrome that can also include ureteral and renal defects

Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb(−/−) mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb(−/−) mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb(−/−) mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis

Crim1 regulates integrin signaling in murine lens development.

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development. Development 2016 Jan 15; 143(2):346-66

<i>Pax6</i> or <i>Six3</i> expression in G4 mESC cells induces lens marker expression.

<p>(<b>A–F</b>) G4 mES cells transfected with either (<b>A–D</b>) <i>Pax6</i> or (<b>E,F</b>) <i>Six3</i> expression plasmids exhibit γA-crystallin (<b>A,E</b>) and Prox1 (<b>B,F</b>) expression at day 7. <i>Pax6</i>-transfection also results in expression of (<b>C</b>) αB-crystallin, and (<b>D</b>) Tdrd7. (<b>G</b>) Expression of lens markers in <i>Pax6</i>- and <i>Six3</i>-transfected G4 mESC colonies confirmed by RT-PCR. (<b>H–K</b>) In some cases, γA-crystallin positive mES cells accumulate in aggregates at days 7–14, with further expansion into lentoid bodies at 30 days (<b>J,</b> phase; <b>K,</b> γA-crystallin immunofluorscence). Scale bars: <b>A</b> 75 µm; <b>B–F</b> 50 µm; <b>H–I</b> 25 µm; <b>J–K</b> 50 µm.</p

Proximity of lens marker and <i>Pax6-GFP</i> or <i>Six3-GFP</i> expressing mES cells.

<p>(<b>A–F</b>) mES cell cultures transduced with <i>Pax6-GFP</i> under the constitutive E1a promoter show close proximity but generally non-overlapping expression of GFP with γA-crystallin (<b>A–C</b>) or Tdrd7 (<b>D–F</b>) at 21 days. (<b>G–I</b>) Similar results were obtained for E1a driven <i>Six3-GFP</i> transduction and γA-crystallin expression. These results suggest recruitment of undifferentiated mES cells to a lens fate by <i>Pax6</i>- or <i>Six3</i>-expressing cells. Scale bar: <b>A–I</b> 30 µm.</p

The cell adhesion gene PVRL3 is associated with congenital ocular defects.

We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell-cell adhesion mechanism in human ocular development

<i>Pax6-</i> and <i>Six3</i>-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

<div><p>Embryonic stem (ES) cells provide a potentially useful <i>in vitro</i> model for the study of <i>in vivo</i> tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes <i>Pax6</i> and <i>Six3</i> could induce lens cell fate <i>in vitro</i>. To help assess the onset of lens differentiation, we derived a new mES cell line (<i>Pax6</i>-GFP mES) that expresses a GFP reporter under the control of the <i>Pax6</i> P0 promoter and lens ectoderm enhancer. <i>Pax6</i> or <i>Six3</i> expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with <i>Pax6</i> or <i>Six3</i> but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in <i>Pax6</i>-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, <i>Pax6</i> or <i>Six3</i> lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that <i>Pax6</i> or <i>Six3</i> is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.</p></div

<i>Pax6</i> or <i>Six3</i> expression in H1 hES cells induces lens marker expression.

<p>(<b>A–F</b>) H1 hES cells transduced by <i>Pax6</i> lentiviral vector express (<b>A–C</b>) Prox1 in partly overlapping fashion (<b>C</b>) by 14 days post transduction. (<b>D–F</b>) By 24 days post-transduction, (<b>D</b>) γA-crystallin and (<b>E</b>) Tdrd7 are expressed, the latter as cytoplasmic granules. Similar results were obtained following <i>Six3</i> transduction (not shown). (<b>G</b>) RT-PCR confirms induction of lens marker gene expression in <i>Pax6-</i> or <i>Six3-</i>transduced H1 hES cells. Scale bars: <b>A–C</b> 150 µm; <b>D–F</b> 50 µm.</p