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. 2017 Jul 1;427(1):12-20.
doi: 10.1016/j.ydbio.2017.05.012. Epub 2017 May 13.

miR-205 is a critical regulator of lacrimal gland development

D'Juan T Farmer  1 , Jennifer K Finley  2 , Feeling Y Chen  3 , Estefania Tarifeño-Saldivia  1 , Nancy A McNamara  3 , Sarah M Knox  2 , Michael T McManus  4
Affiliations

miR-205 is a critical regulator of lacrimal gland development

D'Juan T Farmer et al. Dev Biol. .

Abstract

The tear film protects the terrestrial animal's ocular surface and the lacrimal gland provides important aqueous secretions necessary for its maintenance. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an important gene for lacrimal gland development. Mice lacking miR-205 fail to properly develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal gland development. Specifically, more than half of knockout lacrimal glands never initiated, suggesting a critical role of miR-205 at the earliest stages of lacrimal gland development. RNA-seq analysis uncovered several up-regulated miR-205 targets that may interfere with signaling to impair lacrimal gland initiation. Supporting this data, combinatorial epistatic deletion of Fgf10, the driver of lacrimal gland initiation, and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways related to Fgf10 in order to regulate glandular development. These data show that a single microRNA is a key regulator for early lacrimal gland development in mice and highlights the important role of microRNAs during organogenesis.

Keywords: Fgf10; Lacrimal gland; MiR-205; MicroRNAs.

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Figures

Fig. 1.
Fig. 1.. Deletion of miR-205 results in ocular defects.
(A) Construct strategy for mouse generation. (B) Images of representative control and miR-205−/− mice. Mice were imaged at four months of age.
Fig. 2.
Fig. 2.. Loss of miR-205 impairs tear secretion.
(A) Whisker plot of tear collection quantification after stimulation (*** p-value <0.0001, t-test, SEM error bars). Black points indicate outliers. (B) Quantification of gland phenotypes in adult mice (p-value, <0.001, Fisher’s exact test). Green: normal, yellow: runted, and blue: absent glands.
Fig. 3.
Fig. 3.. miR-205 controls lacrimal gland initiation.
(A) Expression analysis of miR-205 in the lacrimal gland. Lacrimal gland (lg), meibomian gland (mg), eye (e). Surface ectoderm is indicated by arrowhead. Scattered lacZ staining around ducts (d) and acini cells (a) is indicated by arrowhead. Scale bars are 100 μM. (B) Representative images of E15.5 embryos with lacZ reporter. Dashed lines indicate the location of the lacrimal gland. Scale bars are 500 μM. (C) Quantification of phenotypes in control and miR-205−/− mice (p-value<0.001, Fisher’s exact test). Green: normal glands, yellow: runted glands, and blue: absent glands.
Fig. 4.
Fig. 4.. miR-205 is required within the epithelium of the lacrimal gland.
(A) Representative images of phenotypes observed in miR-205flox:flox;cre embryos and timed controls (p-value<0.001, Fisher’s exact test). Dashed lines highlight the lacrimal gland position. Scale bars are 500 μM (B) Quantification of phenotypes in conditional miR-205 knockout mice. Green: normal glands, yellow: runted glands, and blue: absent glands. (C) Confocal imaging of E15.5 lacrimal glands and quantification of Ki67+ Ecad+ positive cells. Scale bars are 100 μM (ns=not significant, t-test, SEM error bars). (D) Confocal imaging of P0 lacrimal glands and quantification of Ki67+ Ecad+ positive cells. Scale bars are 100 μM (ns=not significant, t-test, SEM error bars).
Fig. 5.
Fig. 5.. RNA-seq of miR-205−/− tissues uncovers relevant miR-205 targets and pathways.
(A) Schematic overview of experimental method for tissue isolation and RNA sequencing. (B) Overlap between up-regulated genes and predicted miR-205 targets expressed in the surface ectoderm. (C) Luciferase UTR assays for subset of genes up-regulated in miR-205 knockout samples (SEM error bars). No binding site (NS), a perfect binding site (PS) control. (D) Luciferase UTR assays for miR-205 targets with wildtype UTRs (normalized to one) and mutated UTRs.
Fig. 6.
Fig. 6.. miR-205 controls the fidelity of Fgf signaling during lacrimal gland development.
(A) Representative images of combinatorial deletions in P0 mice imaged with Krt5GFP reporter. Dashed lines indicate the location of the lacrimal gland. (B) Quantification of lacrimal gland defects in the Fgf10 heterozygous background (p=0.0048, Fisher’s exact test). Green: normal, yellow: runted, and blue: absent glands. (C) Working model for the role of miR-205 in lacrimal gland development. In wildtype mice, FGF10 is secreted by the mesenchyme to stimulate epithelial budding and miR-205 targets several genes to ensure efficient lacrimal gland initiation. In the absence of the microRNA, miR-205 targets are up-regulated, interfere with Fgf10 signaling and impair lacrimal gland development.
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