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. 2013 Oct 7;8(10):e76634.
doi: 10.1371/journal.pone.0076634. eCollection 2013.

Partially penetrant postnatal lethality of an epithelial specific MicroRNA in a mouse knockout

D'Juan T Farmer  1 , Nikki Shariat, Chong Yon Park, Huey Jiin Liu, Anastasia Mavropoulos, Michael T McManus
Affiliations

Partially penetrant postnatal lethality of an epithelial specific MicroRNA in a mouse knockout

D'Juan T Farmer et al. PLoS One. .

Abstract

MicroRNAs are small noncoding RNAs thought to have pivotal roles in numerous diseases and developmental processes. However, a growing body of literature indicates that in vivo elimination of these tiny RNAs usually has little to no observable consequence, suggesting functional redundancy with other microRNAs or cellular pathways. We provide an in-depth analysis of miR-205 expression and define miR-205 as an epithelial-specific microRNA, and for the first time show that ablation of this microRNA knockout exhibits partially penetrant lethality in a constitutive mouse knockout model. Given the role of this microRNA in cancer and development, this mouse model will be an incredible reagent to study the function and mechanisms of miR-205 in epithelial tissue development and disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A targeted miR-205-lacZ reporter is functional in mouse embryos.
A. Two targeting strategies were used for miR-205 analyses. All lacZ stained embryos were generated in a mixed background crossed directly to Actin-Cre. All mice phenotypically analyzed were crossed to Actin-FLP, followed by Actin Cre, and backcrossed seven generations to C57/Bl mice. B. miR-205 is developmentally regulated. Expression is evident in the branchial arches of E11.5 embryos and by E15.5, embryos demonstrate high expression in skin.
Figure 2
Figure 2. miR-205 is expressed in squamous stratified epithelium derived organs.
X-gal staining of E14.5 revealed expression in the thymus [t] (A), stomach [s] and pancreas [p] (B), ureters [u] and bladder [b] (C). In the E18.5 embryo head, expression was salient in the several cranial organs, including the skin [sk], lacrimal glands [la], parotid glands [pa], and salivary glands [sm]. Staining was also detected in the oesophagus [o] and trachea [tr] (D). In addition, staining was found in the meibomian glands [m] of the eyelid (E). Staining was maintained in the oesophagus of the E18.5 embryo (F). LacZ activity is found in the ureters [u] and faint staining was also detected in the kidneys [k] (G).
Figure 3
Figure 3. LacZ Reporter provides high resolution and accurate readout of mature miR-205.
Section regions and developmental stage are indicated in the schematics on the left. (A-C) Transverse sections of E14.5 reveal defined expression in several cranial tissues. (D-E) E18.5 sectioning confirms maintained expression in tissues and refined and restricted expression in the stomach. Images in C and E are higher magnifications of the dashed box shown in B and D. (F-G) miR-205 LNA in situs on stomach sections from wild type E14.5 embryos. (H) Expression of miR-205 assayed by qRT-PCR of total RNA from different organs of wild type E18.5 embryos. Sno202 was used as a loading control. The error bars shown are S.E.M. Tongue [T], nasal cavity [NC], whisker follicles [W], and salivary glands [Sm], oesophagus [O], trachea [Tr], stomach [S], glandular region of the stomach [G], non-glandular region of the stomach [NG]
Figure 4
Figure 4. miR-205 knockout mice demonstrate postnatal lethality.
(A) Image of WT (flox/flox, left) and KO (d/d, right) demonstrating striking differences at P10. Clear skin defects are observed with a gross decrease in the overall size of the KO. (B) KO mice are born normally but about 50% die before two weeks after birth (blue, flox/flox n = 20, red, d/d, n = 22). (C) Non-surviving KOs weigh significantly less than littermate controls at P7, averaging about half the weight of littermates. Surviving KOs show minor decrease in weight at P7 compared to littermate controls (flox/flox, n = 13, flox/d, n = 26, surviving d/d, n = 13, non-surviving d/d, n = 12), standard error reported.
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