<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

<i>Celf1</i>^{<i>cKO/lacZKI</i>} mouse exhibits mis-expression of key lens genes.

<p>(<b>A</b>) Microarray heat-maps representing genes mis-regulated in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses compared to control (left column, ±2.5 fold-change, <i>p</i><0.05, total 34 genes, indicated by heatmap color gradients (left columns: green, down in <i>Celf1</i>^{<i>cKO/lacZKI</i>}; red, up in <i>Celf1</i>^{<i>cKO/lacZKI</i>}) and their respective enrichment in normal lens compared to whole-embryonic tissue as per <i>iSyTE</i> (right columns, lens-enrichment in fold-change indicated by red color intensity). (<b>B</b>) Differentially expressed genes (DEGs) in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses are plotted on the X-axis as down-regulated (circles) and up-regulated genes (triangles). On the Y-axis, DEGs are separated based on their lens-enrichment. Red and green color gradients represent high and low lens-enrichment, respectively. Genes down-regulated in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses are predominantly highly-lens enriched, while those up-regulated do not exhibit this trend.</p

<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>Celf1</i> deficiency in mouse and fish causes defects in fiber cell morphology.

<p>(<b>A</b>) RT-qPCR analysis confirms significant <i>Actn2</i> down-regulation in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses compared to control. (<b>B</b>) RNA immunoprecipitation (RIP) identifies <i>Actn2</i> as an enriched transcript in Celf1-pulldown in P15 wild-type mouse lens. (<b>C</b>) Cross-linked RNA immunoprecipitation (CLIP) shows <i>Sptb</i> transcripts to be enriched in Celf1-pulldown in wild-type mouse lens. (<b>D</b>) RT-qPCR analysis shows that the high-abundant <i>Sptb</i> isoform (isoform 1 (ENSMUST00000021458)) is reduced, while the low-abundant <i>Sptb</i> isoform (isoform 2 (ENSMUST00000166101)) is abnormally elevated in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses. (<b>E, E’</b>) In mouse, phalloidin staining of lens tissue sections (stage P0) shows uniform F-actin deposition along the fiber cell margins in control, while <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses exhibit abnormal pattern of F-actin (asterisk). (<b>F, F’</b>) In zebrafish, while control lens exhibits normal F-actin deposition, <i>celf1</i>^<i>KD</i> lens (stage 4dpf) exhibits abnormal F-actin pattern (asterisk) in fiber cells. (<b>G</b>-<b>H’</b>) In mouse, scanning electron microscopy analysis of cortical and nuclear fiber cells shows disrupted cell organization (asterisk) in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses (stage P15). Scale bar in D’ is 75 μm and G’ is 2.5μM.</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

<i>Celf1</i> deficiency in mouse and fish causes fiber cell nuclear degradation defects.

<p>(<b>A, B</b>) Histological analysis of control and <i>Celf1</i>^{<i>lacZKI/lacZKI</i>} mouse lenses at post natal day 4 (P4) stage shows abnormal presence of nuclei in centrally located fiber cells only in <i>Celf1</i>^{<i>lacZKI/lacZKI</i>} mice. (<b>A’, B’</b>) High-magnification of the dotted-line area in A, B. Asterisk denote abnormally retained nuclei. (<b>C, D</b>) In zebrafish, compared to control, <i>celf1</i>^<i>KD</i> lens exhibit abnormal presence of nuclei in the central fiber cell region. (<b>C’, D’</b>) High-magnification of the dotted-line area in E, F. Asterisk denote abnormally retained nuclei. (<b>E</b>) RT-qPCR analysis confirms significant <i>Dnase2b</i> down-regulation in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses compared to control. (<b>F</b>) RNA immunoprecipitation (RIP) and (<b>G</b>) cross-linked RNA immunoprecipitation (CLIP) shows <i>Dnase2b</i> to be enriched in Celf1-pulldown in wild-type mouse lens. (<b>H</b>) Celf1 over-expression in NIH3T3 cells, which carry Dnase2b 3’UTR downstream of a luciferase reporter, results in significant increase of luciferase mRNA. Abbr.: f.c., fold-change; NS, not significant. Asterisks in E, G, H indicate a <i>p</i>-value < 0.005.</p

<i>Celf1</i> is required for vertebrate lens development.

<p>(<b>A</b>) In zebrafish, <i>celf1</i> transcripts are detected in the lens at 1 day post fertilization (1dpf) by <i>in situ</i> hybridization (ISH). (<b>B</b>) In <i>X</i>. <i>laevis</i>, ISH indicates strong <i>celf1</i> expression (arrow) in the embryonic St. 30 eye (arrow) and lens (indicated by broken line area). (<b>C</b>) In mouse, ISH shows strong <i>Celf1</i> transcript expression in the lens at embryonic day 12.5. (<b>D</b>) In mouse lens, Celf1 protein is expressed at (<b>D</b>) E11.5 (<b>E</b>) E14.5 and (<b>F</b>) E16.5 predominantly in fiber cells (f) and to a lower extent in epithelial cells (e). (<b>G</b>, <b>G’</b>) In zebrafish, while control eyes are normal, <i>celf1</i> knockdown (KD) results in microphthalmia and clouding of lens (asterisk) by 4dpf. (<b>H, H’</b>) In <i>X</i>. <i>laevis</i>, compared to control, <i>celf1</i> KD results in microphthalmia. (<b>I, I’</b>) In mouse, compared to control, <i>Celf1</i>^{<i>cKO/cKO</i>} lens exhibits severe cataract (asterisk). (<b>J</b>-<b>K’</b>) Compared to control, refraction errors (asterisk) are observed in <i>Celf1</i>^{<i>cKO/cKO</i>} lens under dark-field and light-field microscopy. (<b>L</b>, <b>L’</b>) At E16.5 stage, the mouse <i>Celf1</i>^{<i>cKO/cKO</i>} lens exhibits abnormal spaces (asterisk) in the fiber cell region. Scale bar in F is 75 μm.</p

Celf1-mediated post-transcriptional control of mitotic machinery components facilitates lens fiber cell nuclear degradation.

<p>(<b>A, A’</b>) Compared to control lens, phosphorylation of Lamin A/C (pLamin A/C) is reduced in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens. Dotted-line areas of the fiber cell regions in control (<b>B</b>-<b>D</b>) and <i>Celf1</i>^{<i>cKO/lacZKI</i>} mice (<b>B’</b>-<b>D’</b>) lenses are shown at high-magnification. Arrows in B, C indicate examples of high pLamin A/C expressing nuclei, while no nuclei are observed in D as this area represents a normal nuclear-free zone in the control lens. Asterisks in B’-D’ indicate reduced signals of pLamin A/C in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens fiber cell nuclei. Note the presence of nuclei in D’ in the centrally located fiber cells due to the nuclear degradation defects in the <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens. (<b>E</b>, <b>E’</b>) Unlike in the control lens where p27^Kip1 protein is restricted to cells of the transition zone and cortical fiber cells (arrow), high levels of p27^Kip1 protein are detected in the entire fiber cell compartment (asterisks) including the central region in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens. (<b>F, F’</b>) High-magnification of dotted-line areas in E and F. Asterisks indicate elevated signals of p27^Kip1 protein. (<b>G</b>) Quantification of the p27^Kip1 immunofluorescence signals from control and <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses shows significantly increased p27^Kip1 protein levels in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lenses. (<b>H</b>) Western blot analysis shows increased levels of p27^Kip1 protein in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens compared to control. (<b>I</b>) Compared to control, p27^Kip1 mRNA levels are not significantly altered in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens. (<b>J, K</b>) RIP and CLIP assays, respectively, identify p27^Kip1 mRNA to be highly enriched in Celf1-pulldown in wild-type mouse lens. (<b>L</b>) A potential Celf1 binding GU-rich region in present in the mouse p27^Kip1 5’ UTR. (<b>M</b>) Activity of firefly luciferase fused downstream of p27^Kip1 5’ UTR is significantly elevated in Celf1-knockdown (Celf1-KD) mouse lens cell line compared to control (firefly luciferase normalized to <i>Renilla</i> luciferase (<i>Rluc</i>)). (<b>N</b>-<b>O’</b>) Compared to control, p21^Cip1 mRNA and protein is abnormally elevated in <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens. Asterisk in O’ indicate high expression of p21^Cip1 protein in the <i>Celf1</i>^{<i>cKO/lacZKI</i>} lens. Abbr.: e, epithelial cells; f, fiber cells; tz, transition zone; NS, not significant. Scale bar in A’, E’, O’ is 75 μm and in D’ and F’ is 12 μm. Asterisks in G, K, M and N indicate a <i>p</i>-value < 0.0005, 0.005, 0.05 and 0.005, respectively.</p

Model for Celf1-mediated post-transcriptional gene expression control in the lens.

<p>In normal lens development, Celf1 is required for nuclear degradation and proper cell morphology in fiber cell differentiation. Celf1 positively regulates the nuclease Dnase2b (being necessary for its high mRNA levels) and negatively regulates the cyclin-dependent kinase inhibitors p21^Cip1 (being necessary for its low mRNA levels) and p27^Kip1 (by inhibiting its translation into protein). Inhibition of p21^Cip1 and p27^Kip1 allows the activation of Cdk1, which phosphorylates Lamin A/C to initiate nuclear envelope breakdown in fiber cells. Thus, Celf1 controls the nuclease (Dnase2b) as well as its access to nuclear DNA, to regulate nuclear degradation in lens fiber cells. These findings show how mitotic machinery components–normally involved in nuclear envelope disassembly during cell division–are post-transcriptionally rewired by RNA-binding proteins to regulate cell differentiation in lens development. Additionally, Celf1 controls the splice isoform abundance of the membrane-organization protein Sptb (β-spectrin) and high transcript levels of the F-actin-binding protein Actn2 (α-actinin 2), to regulate fiber cell morphology. Abbr.: Epi, epithelium; TZ, transition zone; FC, fiber cells.</p

<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