Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium

PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6). Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway. Reporter mice, mosaic for expression of the gene encoding β-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage. Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation. Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh. Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands

A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium

This work was supported by the Wellcome Trust (grant 088876/Z/09/Z to JDW, REH and JMC). The funding body had no role in the design of the study, the collection, analysis and interpretation of data or the preparation of the manuscriptPeer reviewedPublisher PD

Abnormal corneal epithelial maintenance in mice heterozygous for the micropinna microphthalmia mutation Mp

We investigated the corneal morphology of adult Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2(MP) protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2(MP) protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice

Analysis of compound heterozygotes reveals that the mouse floxed Pax6 tm1Ued allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6(tm1Ued) (Pax6(fl)) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6(fl/fl) and heterozygous Pax6(fl/+) mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6(fl/fl) corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6(Sey-Neu) (Pax6(−)) null allele. Pax6(fl/−) compound heterozygotes had more severe eye abnormalities than Pax6(+/−) heterozygotes, implying that Pax6(fl) differs from the wild-type Pax6(+) allele. Immunohistochemistry showed that the Pax6(fl/−) corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6(fl) allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11248-016-9962-4) contains supplementary material, which is available to authorized users

Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence

We thank Drs Kevin Painter (Heriot-Watt University), Steven Morley and Richard Mort for their helpful discussion. We also thank Mr Ronnie Grant for his help with the figures, staff at BRR, University of Edinburgh, for their specialised technical services and Drs Bettina Wilm (Liverpool University), Peter Hohenstein, Richard Mort and Alison Mackinnon for kindly providing founder mice. This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/J015172/1 and BB/J015237/1).Peer reviewedPublisher PD

Hemizygous Le-Cre Transgenic Mice Have Severe Eye Abnormalities on Some Genetic Backgrounds in the Absence of LoxP Sites

Eye phenotypes were investigated in Le-Cre(Tg/-); Pax6(fl/+) mice, which were expected to show tissue-specific reduction of Pax6 in surface ectoderm derivatives. To provide a better comparison with our previous studies of Pax6(+/-) eye phenotypes, hemizygous Le-Cre(Tg/-) and heterozygous Pax6(fl/+)mice were crossed onto the CBA/Ca genetic background. After the Le-Cre transgene had been backcrossed to CBA/Ca for seven generations, significant eye abnormalities occurred in some hemizygous Le-Cre(Tg/-); Pax6(+/+) controls (without a floxed Pax6(fl) allele) as well as experimental Le-Cre(Tg/-); Pax6(fl/+) mice. However, no abnormalities were seen in Le-Cre(-/-); Pax6(fl/+) or Le-Cre(-/-); Pax6(+/+) controls (without the Le-Cre transgene). The severity and frequency of the eye abnormalities in Le-Cre(Tg/-); Pax6(+/+) control mice diminished after backcrossing Le-Cre(Tg/-) mice to the original FVB/N strain for two generations, showing that the effect was reversible. This genetic background effect suggests that the eye abnormalities are a consequence of an interaction between the Le-Cre transgene and alleles of unknown modifier genes present in certain genetic backgrounds. The abnormalities were also ameliorated by introducing additional Pax6 gene copies on a CBA/Ca background, suggesting involvement of Pax6 depletion in Le-Cre(Tg/-); Pax6(+/+) mice rather than direct action of Cre recombinase on cryptic pseudo-loxP sites. One possibility is that expression of Cre recombinase from the Pax6-Le regulatory sequences in the Le-Cre transgene depletes cofactors required for endogenous Pax6 gene expression. Our observation that eye abnormalities can occur in hemizygous Le-Cre(Tg/-); Pax6(+/+) mice, in the absence of a floxed allele, demonstrates the importance of including all the relevant genetic controls in Cre-loxP experiments

The role of Pax6 in corneal development and maintenance

PAX77+ mice were shown to express about 150% of wild-type Pax6 levels of corneal epithelial Pax6 and displayed abnormally small corneas due to a failure of postnatal growth and development of the cornea.  The proliferative component of the corneal epithelium was normal, cell cycle progression appears unaffected and there are no abnormal apoptotic events.  Cytokeratin-12 expression was down regulated in the PAX77+ corneal epithelia, implying differentiation is incomplete, and the corneas of PAX77+ were found to display increased fragility in comparison to wild-types. PAX77+ corneas displayed sever wound healing abnormalities which in contrast to Pax6+/- can not be rescued by EGF addition.  Both Pax6+/- and PAX77+ corneal epithelia show no improvement in wound healing with addition of Shh peptide or all-trans retinoic acid (in contrast to wild-type), however in the PAX77+ wound healing abnormalities do not lead to corneal opacity which is seen in Pax6+/-.  Both Pax6+/- and PAX77+corneal epithelia have increased basal levels of retinoic acid signalling in their corneal epithelium when compared to wild-type littermates.  Topical application of Shh increased RA signalling in the corneal epithelium. As described above a severe reduction in corneal size was observed in the PAX77+ mouse: defective lens signalling as a result of Pax6 dosage was postulated to lead to this.  The lens is highly sensitive to Pax6 dosage.  Mouse/chick secondary chimeras were produced, through the grafting of PAX77+ and wild-type mouse lenses into a chicken eye, to investigate the role of altered Pax6 dosage in the lens on anterior eye development.  PAX77+ mouse/chick secondary chimeras, remarkably recapitulated the anterior eye phenotype seen in PAX77+ mice. This supports the role of altered Pax6 dosage in the lens resulting in the corneal abnormalities of the PAX77+ model.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence

The limbal epithelial stem cell (LESC) hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC) hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.Dorà, Natalie J.; West, John D. (2016). Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence, [dataset]. University of Edinburgh Medical School. http://dx.doi.org/10.7488/ds/1341

Partial rescue of adult eye abnormalities by changing the genetic background or increasing Pax6 levels.

<p>(<b>A–F</b>) H & E stained sections of adult eyes from stage 4 crosses (crossed to CBA/Ca for 8 generations and then FVB/N for 2 generations) showing normal morphology of (A) <i>Le-Cre^−/−; Pax6^+/+</i> and (B) <i>Le-Cre^−/−; Pax6^fl/+</i> control eyes and relatively few abnormalities in (C,D) <i>Le-Cre^Tg/−; Pax6^+/+</i> control eyes but more severe abnormalities in (E,F) experimental <i>Le-Cre^Tg/−; Pax6^fl/+</i>eyes. (<b>G–J</b>) H & E stained sections of adult eyes from crosses between stage-3 <i>Le-Cre^Tg/−</i>; <i>Pax6^fl/+</i> mice and CBA-<i>PAX77^Tg/−</i> transgenic mice (with elevated Pax6 levels, congenic on a CBA/Ca genetic background). (<b>G,H</b>) The effect of additional Pax6 from the PAX77 transgene on <i>Le-Cre^Tg/−; Pax6^+/+</i> control genotype is shown by comparing the morphology of (G) the <i>Le-Cre^Tg/−; Pax6^+/+; PAX77^−/−</i> control eye (without the <i>PAX77</i> transgene) and (H) the <i>Le-Cre^Tg/−; Pax6^+/+; PAX77^Tg/−</i> eye (with the <i>PAX77</i> transgene). (<b>I,J</b>) The effect of additional Pax6 from the PAX77 transgene on <i>Le-Cre^Tg/−; Pax6^fl/+</i> experimental genotype is shown by comparing the morphology of (I) the <i>Le-Cre^Tg/−; Pax6^fl/+; PAX77^−/−</i> control eye (without the <i>PAX77</i> transgene) and (J) the <i>Le-Cre^Tg/−; Pax6^fl/+; PAX77^Tg/−</i> eye (with the <i>PAX77</i> transgene). The lenses in A–C, H & J were normal but some were damaged during sectioning. Scale bars A–J = 200 µm. <i>Abbreviations:</i> Cre +/+77 is <i>Le-Cre^Tg/−; Pax6^+/+</i>; <i>PAX77^Tg/</i>; and Cre fl/+77 is <i>Le-Cre^Tg/−; Pax6^fl/+</i>; <i>PAX77^Tg/−</i>. Colour code of panel lettering: black  =  <i>Le-Cre^−/−; Pax6^+/+</i>; blue  =  <i>Le-Cre^−/−; Pax6^fl/+</i>; red  =  <i>Le-Cre^Tg/−; Pax6^+/+</i> or <i>Le-Cre^Tg/−; Pax6^+/+</i>; <i>PAX77^Tg/−</i>; green  =  <i>Le-Cre^Tg/−; Pax6^fl/+</i> or <i>Le-Cre^Tg/−; Pax6^fl/+</i>; <i>PAX77^Tg/−</i>.</p

Morphology of fetal and juvenile eyes and Z/AP reporter expression in adult eyes from mice produced in stage 3.

<p>(<b>A–H</b>) Comparison of morphology in H & E stained histological sections of control <i>Le-Cre^−/−; Pax6^+/+</i> and experimental <i>Le-Cre^Tg/−; Pax6^fl/+</i> fetal and neonatal eyes from (<i>Le-Cre^Tg/−</i>; <i>Pax6^+/+</i> × <i>Le-Cre^−/−</i>; <i>Pax6^fl/+</i>) stage-3 crosses at E12.5 to P2 showing lens abnormalities in <i>Le-Cre^Tg/−; Pax6^fl/+</i> eyes at E16.5 (F) and more extensive ocular abnormalities in <i>Le-Cre^Tg/−; Pax6^fl/+</i> eyes at P2 (H). (<b>I–N</b>) Comparison of morphology in H & E stained histological sections of all three control genotypes from (<i>Le-Cre^Tg/−</i>; <i>Pax6^+/+</i> × <i>Le-Cre^−/−</i>; <i>Pax6^fl/+</i>) stage-3 crosses at P10 showing normal morphology of (I) <i>Le-Cre^−/−; Pax6^+/+</i>, (J) <i>Le-Cre^−/−; Pax6^fl/+</i> and (K) one <i>Le</i>-<i>Cre^Tg/−; Pax6^+/+</i> controls but abnormal morphology in (L) another <i>Le</i>-<i>Cre^Tg/−; Pax6^+/+</i> control and (M,N) eyes from <i>Le-Cre^Tg/−; Pax6^fl/+</i> experimental mice in stage 3 of this study. The lenses in I-K are normal but the lens shown in (J) was damaged during sectioning. (<b>O,P</b>) Histochemical staining for (O) placental alkaline phosphatase (blue endpoint) and (P) β-galactosidase (blue endpoint) in different sections from the same <i>Le-Cre^Tg/−</i>; <i>Z/AP</i> adult reporter eye showing AP staining is restricted to the surface ectoderm derivatives (lens and corneal epithelium). Eye morphology in the <i>Le-Cre^Tg/−</i>; <i>Z/AP</i> eyes was often abnormal in stage 3 (O,P), as it was for some <i>Le</i>-<i>Cre^Tg/−; Pax6^+/+</i> controls in stage 3 (L and Fig. 8). Scale bars: E12.5 and E14.5 (A–D)  = 50 µm; E16.5 and P2 (E–H)  = 200 µm, P10 (I–N)  = 100 µm; 12 weeks (O,P)  = 200 µm. <i>Abbreviations:</i> AP, alkaline phosphatase staining; β-gal  =  β-galactosidase staining. Colour code of panel lettering: black  =  <i>Le-Cre^−/−; Pax6^+/+</i>; blue  =  <i>Le-Cre^−/−; Pax6^fl/+</i>; red  =  <i>Le-Cre^Tg/−; Pax6^+/+</i>; green  =  <i>Le-Cre^Tg/−; Pax6^fl/+</i>; purple  =  <i>Le-Cre^Tg/−</i>; <i>Z/AP.</i></p