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. 2006 Feb 14;103(7):2208-13.
doi: 10.1073/pnas.0510839103. Epub 2006 Feb 1.

Dicer Function Is Essential for Lung Epithelium Morphogenesis

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Free PMC article

Dicer Function Is Essential for Lung Epithelium Morphogenesis

Kelley S Harris et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

DICER is a key enzyme that processes microRNA and small interfering RNA precursors into their short mature forms, enabling them to regulate gene expression. Only a single Dicer gene exists in the mouse genome, and it is broadly expressed in developing tissues. Dicer-null mutants die before gastrulation. Therefore, to study Dicer function in the later event of lung formation, we inactivated it in the mouse lung epithelium using a Dicer conditional allele and the Sonic Hedgehogcre (Shhcre) allele. Branching arrests in these mutant lungs, although epithelial growth continues in distal domains that are expanded compared with normal samples. These defects result in a few large epithelial pouches in the mutant lung instead of numerous fine branches present in a normal lung. Significantly, the initial phenotypes are apparent before an increase in epithelial cell death is observed, leading us to propose that Dicer plays a specific role in regulating lung epithelial morphogenesis independent of its requirement in cell survival. In addition, we found that the expression of Fgf10, a key gene involved in lung development, is up-regulated and expanded in the mesenchyme of Dicer mutant lungs. Previous studies support the hypothesis that precise localization of FGF10 in discrete sites of the lung mesenchyme serves as a chemoattractant for the outgrowth of epithelial branches. The aberrant Fgf10 expression may contribute to the Dicer morphological defects. However, the mechanism by which DICER functions in the epithelium to influence Fgf10 expression in the mesenchyme remains unknown.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Cre activity and Dicer inactivation. (AC) Cre activity of the Shhcre allele at E9.0 (A) and E9.5 (B) and in an E12.5 lung (C) as assayed by β-gal staining of Shhcre/+;R26R/+ embryos. Arrowheads indicate prospective lung region. (D and E) Dicer RNA expression in E11.75 control (D) and mutant (E) lungs as assayed by RNA in situ hybridization using a probe hybridizing to the exon that is floxed in the Dicer conditional allele. (F) RT-PCR of E11.5 control (c) and Shhcre;Dicer mutant (m) lung epithelium to illustrate that Dicer is inactivated in the mutant at this stage.
Fig. 2.
Fig. 2.
Morphology of Shhcre;Dicer mutant lung. E15.5 lungs shown in whole mount (A and B, left lobes only) or in plastic sections (CF). Each control and Shhcre;Dicer mutant pair is shown at the same magnification. The R26R reporter for Cre activity is included in both the control (Shhcre/+;Dicerflox/+;R26R/+) and mutant (Shhcre/+;Dicerflox/flox;R26R/+) lungs. The epithelium is outlined in blue by β-gal staining. Plastic sections are counterstained with eosin to show mesenchymal cells in pink. To ensure penetration of substrate, a control lung lobe was dissected into small pieces before β-gal staining, followed by plastic sectioning. Hence, only a portion of the control lung is captured in the plastic section shown in C. E and F are magnified views of the boxed areas in C and D, respectively. Insets in E and F are magnified views of the boxed areas within the same panel. The arrowhead in F points to a region where the epithelium is detached from the mesenchyme.
Fig. 3.
Fig. 3.
Epithelial branching patterns in Shhcre;Dicer lungs. Epithelial cells are labeled with anti-E-cadherin antibody. At each stage, control and Shhcre;Dicer mutant lungs are shown at the same magnification. Branching in the mutant was similar to normal control at E12.0 (A and B) but was reduced at E12.5 (C and D) and E13.5 (E and F). The dashed white lines outline the lung lobes. Note that the overall size of the mutant lung lobes was similar to that of controls at all three stages, even though the epithelial surface area was reduced in the mutant at E12.5 and E13.5 because of reduced branching.
Fig. 4.
Fig. 4.
Cell death analysis of Shhcre;Dicer lungs. Cell death was detected by LysoTracker Red staining in whole mount (A–L) or vibratome section (M–P) at the stages indicated. Each control and mutant pair is shown at the same magnification. C, D, G, H, K, and L are magnified views of the boxed areas in A, B, E, F, I, and J, respectively. O and P are bright-field images of sections shown in M and N, respectively. Arrowheads in B and F indicate the distal extent of the cell death that is present in secondary bronchi. Representative proximal (Pr) and distal (Di) axes of the lung are indicated in A and C. In control lungs, cell death was detected in the trachea and primary bronchi at E12.25 but not at E12.5 or later stages. In mutant lungs, cell death was detected in the trachea and primary and secondary bronchi at E12.25 and E12.5. In E13.5 mutant lungs, cell death was detected in the entire lung epithelium but not in the mesenchyme.
Fig. 5.
Fig. 5.
Gene expression in Shhcre;Dicer lungs. Shown is whole-mount RNA in situ analysis of Fgf10, Spry2, and Bmp4 expression in control and mutant lungs at E12.5 (A–F) and E11.75 (G–L). At E12.5, the expression of all three genes was increased in the mutant compared with control. At E11.75, Fgf10 expression was increased in the mutant, whereas Spry2 and Bmp4 expression was not changed compared with control. Note that in B and H, despite Fgf10 up-regulation, there were still regional differences in expression level within each of the mutant lungs, similar to controls in A and G.
Fig. 6.
Fig. 6.
Two models of DICER function in regulating Fgf10 expression during lung epithelium morphogenesis. In both models, DICER protein produced in the lung epithelium (green domain) cleaves precursor miRNAs/siRNAs to their mature form. In one model (A), we hypothesize that mature miRNA/siRNAs travel to the mesenchyme (purple domain) and directly regulate the expression of target gene Fgf10. In an alternative model (B), we hypothesize that the mature forms of one or more of the key miRNAs/siRNAs inhibit the expression of a presently unknown gene X on the RNA or protein level. We propose that gene X encodes a protein that is secreted into the adjacent mesenchyme and functions as a positive regulator of Fgf10 expression. In both models, FGF10 produced in the mesenchyme then acts as a chemoattractant for epithelium outgrowth and branching. When Dicer is inactivated in the lung epithelium, we postulate that the mature form of the key miRNA/siRNA(s) is depleted, leading to an increase of Fgf10 transcripts. This increase may contribute to the morphogenesis phenotype observed in Shhcre;Dicer lungs.

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