Cell- and developmental stage-specific Dicer1 ablation in the lung epithelium models cystic pleuropulmonary blastoma

Abstract

Inherited syndromes provide unique opportunities to identify key regulatory mechanisms governing human disease. We previously identified germline loss-of-function DICER1 mutations in a human syndrome defined by the childhood lung neoplasm pleuropulmonary blastoma (PPB), which arises during lung development. DICER1 regulates many biological processes critical in development and disease pathogenesis. Significant challenges in defining the role of DICER1 in human disease are identifying cause-effect relationships and generating manipulatable systems that model the complexity of organ development and disease pathogenesis. Here we report the generation of a murine model for PPB and demonstrate that precise temporal and cell type-specific Dicer1 ablation is necessary and sufficient for the development of cystic lungs that histologically and phenotypically model PPB. Dicer1 ablation in the distal airway epithelium during early stages of lung development resulted in a cystic lung phenotype indistinguishable from PPB, whereas DICER1 function was not required for development of the proximal airway epithelium or during later stages of organogenesis. Mechanistic studies demonstrate that Dicer1 loss results in epithelial cell death, followed by cystic airway dilatation accompanied by epithelial and mesenchymal proliferation. These studies define precise temporal and epithelial cell type-specific DICER1 functions in the developing lung and demonstrate that loss of these DICER1 functions is sufficient for the development of cystic PPB. These results also provide evidence that PPB arise through a novel mechanism of non-cell-autonomous tumour initiation, in which the genetic abnormality initiating the neoplasm does not occur in the cells that ultimately transform, but rather occurs in a benign-appearing epithelial cell component that predisposes underlying mesenchymal cells to malignant transformation.

Keywords: Dicer1; lung development; pleuropulmonary blastoma.

Figure 1. Dicer1 ablation targeted to the developing lung epithelium results in neonatal death and a PPB-like phenotype

(a) PPB resected from a 10 day old infant (left panel) showing cystic spaces separated by septae with expanded mesenchyme (arrows) adjacent to normal bronchiole (arrowheads) and alveoli. In the normal neonatal lung (center panel), DICER1 expression is detected in the bronchiolar (arrowheads) and alveolar (arrows) epithelium as well as in the mesenchymal cells by immunohistochemistry. In PPB (right panel), DICER1 expression is lost in the epithelial (arrows) but not mesenchymal component of the neoplasm (200×, inset 400×). (b) Dicer1 ablation targeted to the lung epithelium in SPC-rtTA/Cre double transgenic Dicer1^f/f mice (Dicer1 Ablated, n=8) resulted in death by postnatal day 5, in contrast to surviving double transgenic Dicer1^w/f mice (Dicer1 haploinsufficient, n=16) and control littermates including double transgenic Dicer1^w/w (n=16) and Dicer1^f/f, Dicer1^w/f or Dicer1^w/w mice lacking one or both transgenes required for Dicer1 recombination (n=51). (c) Recombination of Dicer1^f/f alleles (Rec) by PCR analysis of lung DNA from E18.5 Dicer1-ablated but not double transgenic Dicer1^w/w littermates. The wild type Dicer1 allele (WT) was detected in lung DNA from Dicer1^w/w mice. (d) Dicer1 mRNA levels were decreased in lungs from E18.5 Dicer1-ablated as compared to double transgenic Dicer1^w/w controls by quantitative RT-PCR. Data represented as mean ± SEM (n=5–6 per group; ****p<0.0001). (e) Normal human neonatal lung and adjacent Type I PPB (top row), and E18.5 lung from Dicer1-proficient double transgenic Dicer1^w/w control and littermate with Dicer1-ablated lung epithelium (bottom row) showing cysts and expanded mesenchyme morphologically similar to Type I PPB. Cyst lining epithelial cells stain for pancytokeratin by immunohistochemistry (human lung 40×, PPB 40×, 1000×, 100×, inset 400×; mouse lung 100×, Dicer1-ablated lung 100×, 1000×, 200×, inset 1000×).

Figure 2. Cyst lining cells in PPB and Dicer1-ablated lungs have a distal respiratory epithelial cell phenotype, increased proliferation and ectopic apoptosis

(a) Epithelial cells lining cysts in PPB (top row) and E18.5 Dicer1-ablated lungs have positive immunohistochemical staining for Nkx2-1 and type I (Type I (T1)-alpha) and type II (proSPC and LPCAT1) cell markers. Only rare epithelial cells stain for the conducting airway epithelial cell marker, Sox2. Arrows and insets highlight positive staining (PPB 100×, insets 400×; Dicer1-ablated 200×, insets 1000×). (b) Epithelial cell proliferation and (c) apoptosis were increased in conducting and distal parenchymal epithelia in Dicer1-ablated lungs as compared to Dicer1-haploinsufficient (Dicer1^f/w) and Dicer1-proficient (Dicer1^w/w) double transgenic controls by immunohistochemical staining for phosphorylated histone H3 (pHH3) and TUNEL staining, respectively (arrows and inset). (n=5–7 per group; *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant; 200×, insets 1000×).

Figure 3. Lungs with Dicer1 ablation targeted to the developing proximal conducting airway are morphologically similar to Dicer1-proficient control lungs

(a) Dicer1 ablation targeted to the developing conducting airway epithelium using the rat CCSP promoter driving the rt-TA (Dicer1^CCSPcKO) resulted in lungs with morphology similar to Dicer1-proficient littermates lacking one or both transgenes (Control) (top row, 100×, bottom row, 400×). (b) Conducting airway epithelial cells were targeted in double transgenic Dicer1^w/w (Dicer1 WT) and Dicer1^CCSPcKO lungs treated with doxycycline throughout gestation as indicated by whole mount and immunohistochemical β-galactosidase staining of lungs from mice containing the ROSA26 reporter locus (+) but not in control lungs from mice lacking the Cre transgene (C). Whole mount staining of lung lobes from two Dicer1^CCSPcKO and two control mice are shown (whole mounts 8×, microscopic images 200×, inset 1000×). Recombination of Dicer1^f/f alleles (Rec) was detected by PCR analysis of lung DNA from E18.5 Dicer1^CCSPcKO (Ablated) but not Dicer1-proficient littermate controls lacking one or both transgenes (Control). PCR controls include wild type (w, WT) and recombined (r, Rec) Dicer1 alleles. NS=nonspecific band.

Figure 4. Dicer1 loss targeted to the lung epithelium results in ectopic cell death and cystic airway dilation followed by increased cellular proliferation

(a) E13.5 double transgenic SPC-rtTA/Cre Dicer1^f/f lungs (Dicer1-ablated) but not Dicer1-proficient controls lacking one or both transgenes (Control) showed morphologic evidence of apoptosis in distal airway epithelial cells (arrows) (top row 100×, bottom row 1000×). (b) Dicer1 mRNA levels were decreased in distal airway epithelial cells but not in the surrounding mesenchyme obtained from E13.5 Dicer1-ablated lungs as compared to Dicer1-proficient controls by quantitative RT-PCR (n=3–6 per group; **p<0.01). (c) E15.5 Dicer1-ablated lungs had dilated distal airways (arrowheads) and morphologic evidence of apoptosis in epithelial cells lining distal airways (arrows) (top row 40×, bottom row 1000×). (d) Mature, but not primary, miR-17 was decreased in Dicer1-ablated E15.5 lungs as compared to Dicer1-proficient controls by quantitative RT-PCR (n=4 per group; *p<0.05). (e) Northern blot analysis showed decreased mature miR-17 in Dicer1-ablated as compared to Dicer1-proficient E15.5 lungs. Relative Dicer1 levels as determined by quantitative RT-PCR and miR-17 densitometry values relative to matched littermates are shown. Densitometry values were normalized to the U6 loading control. Levels of pre-miR-17 and a 23 bp RNA oligonucleotide marker indicating the migration position of mature miR-17 are also shown. Low level miR-17 expression is detected in Dicer1-ablated lungs since miR-17 is expressed in both mesenchymal and epithelial cells with relatively higher expression in the epithelium. (f) Apoptosis was increased in distal airway epithelial cells (arrows) in Dicer1-ablated E13.5 and E15.5 lungs as compared to Dicer1-proficient controls by TUNEL analysis (n=3–10 per group; *p<0.05, ****p<0.0001; E15.5 lungs shown, 200×, insets 1000×). (g) Distal epithelial cell proliferation was similar in Dicer1-ablated and control E13.5 and E15.5 lungs but increased in Dicer1-ablated E16.5 lungs as compared to Dicer1-proficient controls by immunohistochemical staining for pHH3 (arrows) (n=4–10 per group; ***p<0.001; E16.5 lungs shown, 200×, insets 1000×).

Figure 5. Dicer1 loss targeted to the lung epithelium results in a transient increase in mesenchymal proliferation

(a) Proliferation of mesenchymal cells surrounding the distal airways was transiently increased in Dicer1-ablated E16.5 lungs as compared to Dicer1-proficient controls by immunohistochemical staining for pHH3 and BrdU (arrows and insets). (b) Distal mesenchymal cell apoptosis (arrows and insets) was similar in E13.5, E15.5 and E18.5 Dicer1-ablated and proficient control lungs by TUNEL analysis. (n=4–10 per group; *p<0.05; E15.5 lungs shown for TUNEL analysis, 200×, insets 1000×)).

Figure 6. Dicer1 ablation at different stages of lung development results in distinct phenotypes

(a) Dicer1-ablated and proficient control E18.5 lungs treated with doxycycline throughout development (E0.5–E18.5), during early development (E0.5–E14.5) or during late development (E14.5–E18.5). Dicer1 ablation throughout lung development and in early lung development resulted in cystic lungs with expanded mesenchyme resembling Type I PPB. Dicer1 ablation during late development resulted in lungs that were morphologically similar to Dicer1-proficient controls expect for rare epithelial cells with morphologic features of apoptosis (inset, arrow) (top row 100×, bottom row 400×, inset 1000×). (b) Conducting airway and parenchymal epithelial cells were targeted in control double transgenic Dicer1^w/w (Dicer1 WT) and Dicer1^f/f (Dicer1 Ablated) lungs treated with doxycycline (dox) throughout development as well as during early and late development by whole mount and immunohistochemical β-galactosidase staining of lungs from mice containing the ROSA26 reporter locus. Whole mount staining of lung lobes from two double transgenic (+) and two Cre transgene negative control (C) mice are shown (whole mounts 8×, microscopic images 200×, inset 1000×). (c) Recombination of Dicer1^f/f alleles (Rec) was detected by PCR analysis on lung DNA from E18.5 Dicer1-ablated (Ablated) but not Dicer1-proficient littermate controls lacking one or both transgenes (Control) treated with dox during early and late development. PCR controls include the wild type (w, WT) and recombined (r, Rec) Dicer1 alleles. NS=nonspecific band. Dicer1 mRNA levels were decreased in lungs from Dicer1-ablated as compared to control lungs from mice treated with dox during early and late development by quantitative RT-PCR. (n=4 per group; *p<0.05, **p<0.01). (d) Whole mount beta-galactosidase staining of lung lobes from three littermates containing the ROSA26 reporter locus and treated with dox during early development showing typical diffuse conducting airway and parenchymal epithelial staining of lungs from double transgenic Dicer1^f/f mice (+, left) and a rare lung with less uniform staining (+, right). No specific staining was detected in lungs from littermate controls lacking the Cre transgene (C) (8×). Recombination of Dicer1^f/f alleles (Rec) was detected by PCR analysis on lung DNA from the single surviving Dicer1-ablated adult mouse treated with doxycycline during early development (Ablated) but not in lung DNA from a littermate control lacking the Cre transgene (Control). Dicer1^f/f alleles were detected in both Dicer1-ablated and control lungs. PCR controls include wild type (w, WT), recombined (r, Rec) and floxed (f, Floxed) Dicer1 alleles. NS=nonspecific band. (e) Lungs from surviving Dicer1-ablated adult mouse and Dicer1-proficient littermate control showing that Dicer1 ablation resulted in cystic lungs lacking the mesenchymal expansion seen in E18.5 lungs morphologically identical to regressed or non-progressing cystic Type I PPB (20×).