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

Natalie J. Dorà; Martine Manuel; Dirk-Jan Kleinjan; David J. Price; J. Martin Collinson; Robert E.  Hill; John D. West

doi:10.1186/s13104-018-3812-9

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

Natalie J. Dorà, Martine Manuel, Dirk-Jan Kleinjan, David J. Price, J. Martin Collinson, Robert E. Hill, John D. West (Corresponding Author)

Medical Sciences

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Abstract

Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.
Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.

Original language	English
Article number	705
Journal	BMC Research Notes
Volume	11
DOIs	https://doi.org/10.1186/s13104-018-3812-9
Publication status	Published - 5 Oct 2018

Bibliographical note

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 manuscript

Keywords

cornea
corneal epithelium
Cre-loxP
CAG-CreER
Krt19-CreER
Pax6
mouse
mosaic transgene expression

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10.1186/s13104-018-3812-9Licence: CC BY

A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium
© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Final published version, 1.9 MBLicence: CC BY

Jon Collinson
- School of Medicine, Medical Sciences & Nutrition, Molecular and Cellular Function
- School of Medicine, Medical Sciences & Nutrition, Medical Sciences - Personal Chair
Person: Academic

Cite this

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title = "A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium",

abstract = "Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.",

keywords = "cornea, corneal epithelium, Cre-loxP, CAG-CreER, Krt19-CreER, Pax6, mouse, mosaic transgene expression",

author = "Dor{\`a}, {Natalie J.} and Martine Manuel and Dirk-Jan Kleinjan and Price, {David J.} and Collinson, {J. Martin} and Hill, {Robert E.} and West, {John D.}",

note = "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 manuscript",

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T1 - A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium

AU - Dorà, Natalie J.

AU - Manuel, Martine

AU - Kleinjan, Dirk-Jan

AU - Price, David J.

AU - Collinson, J. Martin

AU - Hill, Robert E.

AU - West, John D.

N1 - 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 manuscript

PY - 2018/10/5

Y1 - 2018/10/5

N2 - Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.

AB - Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.

KW - cornea

KW - corneal epithelium

KW - Cre-loxP

KW - CAG-CreER

KW - Krt19-CreER

KW - Pax6

KW - mouse

KW - mosaic transgene expression

U2 - 10.1186/s13104-018-3812-9

DO - 10.1186/s13104-018-3812-9

M3 - Article

C2 - 30290846

SN - 1756-0500

VL - 11

JO - BMC Research Notes

JF - BMC Research Notes

M1 - 705

ER -

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

Abstract

Bibliographical note

Keywords

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Jon Collinson

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