Dnmt1 is required for proximal-distal patterning of the lung endoderm and for restraining alveolar type 2 cell fate

Abstract

Lung endoderm development occurs through a series of finely coordinated transcriptional processes that are regulated by epigenetic mechanisms. However, the role of DNA methylation in regulating lung endoderm development remains poorly understood. We demonstrate that DNA methyltransferase 1 (Dnmt1) is required for early branching morphogenesis of the lungs and for restraining epithelial fate specification. Loss of Dnmt1 leads to an early branching defect, a loss of epithelial polarity and proximal endodermal cell differentiation, and an expansion of the distal endoderm compartment. Dnmt1 deficiency also disrupts epithelial-mesenchymal crosstalk and leads to precocious distal endodermal cell differentiation with premature expression of alveolar type 2 cell restricted genes. These data reveal an important requirement for Dnmt1 mediated DNA methylation in early lung development to promote proper branching morphogenesis, maintain proximal endodermal cell fate, and suppress premature activation of the distal epithelial fate.

Figure 1:. Endodermal Dnmt1 is required for lung development.

(A) RNA-seq analysis reveals that Dnmt1 expression is enriched in E12.5 compared to adult lungs (units reflected in fragments per kilobase of transcript per million mapped reads) (B) Immunohistochemical staining for Dnmt1 at E11.5 and E17.5 demonstrates its presence in both the endodermal and mesenchymal compartments prenatally (dashed lines demarcate endoderm)(scale bar: 50μm). (C) Experimental schematic of developmental time points assessed in endodermal Dnmt1 knockout model. (D) Comparison of Dnmt1^EKO and Dnmt1^control lungs at E14.5 (white arrows indicate cystic buds). (E) Hematoxylin and eosin (H&E) staining of histological sections of Dnmt1^EKO lungs demonstrate defective developmental progression by E14.5 and failure by E18.5 (black arrows point to cystic buds)(scale bar: 500μm).

Figure 2:. Endodermal loss of Dnmt1 disrupts proximal-distal patterning.

(A) Immunohistochemical staining for Dnmt1 indicates it is lost in the lung endoderm at E14.5 in Dnmt1^EKO lungs (dashed lines demarcate endoderm)(scale bar: 50μm). (B) Immunohistochemical staining for cleaved caspase-3 indicates that cell death is increased in Dnmt1^EKO lungs at E14.5 (dashed lines demarcate endoderm)(scale bar: 50μm). (C) Quantification of epithelial proliferation by assessment of Cdh1 and Phosphohistone H3 double positive cells shows a significant decrease in endodermal proliferation in Dnmt1^EKO lungs. Cdh1 stain reveals perturbed epithelial cell shape and aberrant twisting of epithelial buds in Dnmt1^EKO lungs (scale bar: 50μm, student’s twotailed t-test, P<0.001). (D) Immunohistochemical staining for Nkx2.1 shows lung epithelial cell specification in both Dnmt1^EKO and Dnmt1^control lungs. Staining for smooth muscle marker Tagln (smooth muscle surrounding vessels is marked by white arrows, smooth muscle surrounding airways is marked by yellow arrows) and endothelial cell marker Pecam1 demonstrates proper differentiation and localization of mesoderm-derived structures in Dnmt1^EKO lungs. Staining for proximal endoderm marker SSEA1 as well as distal endoderm marker Foxp2 reveals perturbed proximal/distal patterning in Dnmt1^EKO lungs (proximal endoderm is marked by a yellow arrow, distal endoderm is marked by a white arrow) (scale bar: 100μm). (E) Immunohistochemical staining for Sox2 and Sox9 in a series of sections from proximal to distal shows expansion of the distal endoderm compartment (proximal endoderm is marked by yellow arrows, distal endoderm is marked by white arrows, cartilage progenitors are marked by black arrows)(scale bar: 100μm).

Figure 3:. Dnmt1 is essential to maintain epithelial-mesenchymal crosstalk and epithelial polarity.

(A) A model of Dnmt1^control (top) vs. Dnmt1^EKO (bottom) lungs demonstrates the expansion of the distal compartment, perturbed epithelial cell shape, and abnormal twisting of epithelial buds in Dnmt1^EKO lungs. (B) RNA fluorescence in situ hybridization reveals a decrease in Fgfr2 expression in Dnmt1 mutant endoderm and an expansion of Fgf10 expression in the mesoderm. Bmp4 and Shh expression are expanded in Dnmt1^EKO lungs consistent with expansion of the distal endodermal compartment (white dashed line demarcates endoderm, white arrows indicate distal buds of the endoderm) (scale bar: 50μm). (C) Immunohistochemistry for β-catenin reveals loss of characteristic columnar shape of Dnmt1 mutant epithelial cells (scale bars: 25μm). (D) Immunohistochemistry for ZO-1 (white arrows) and laminin (yellow arrows) shows the proper localization of ZO-1 to the apical membrane of Dnmt1^control endodermal cells and deposition of laminin in the basement membrane compared to ectopic localization of ZO-1 to the lateral and basal membranes of Dnmt1^EKO endodermal cells and abnormal localization of laminin despite also being present in the basement membrane (scale bars from left to right: 50μm, 50 μm, 5 μm). (E) Immunohistochemical staining for Cdh1 demonstrates perturbed epithelial cell shape and aberrant localization of Cdh1 protein to the apical membrane of Dnmt1^EKO endodermal cells (scale bars: 25μm). (F) Immunohistochemistry for Dlg1 (white arrows) and laminin (yellow arrows) shows the proper localization of Dlg1 to the basal membrane of Dnmt1^control endodermal cells and deposition of laminin in the basement membrane compared to strong cytosolic and abnormal lateral membrane staining of Dlg1 in Dnmt1^EKO endodermal cells. Laminin is again present in the basement membrane and is abnormally localized in Dnmt1^EKO endodermal cells (scale bars from left to right: 50μm, 50 μm, 5 μm).

Figure 4:. Loss of Dnmt1 impacts endodermal differentiation.

(A) H&E and immunohistochemical staining for Nkx2.1 and DAPI reveal cystic sacs in place of a normal branched network with a paucity of Nkx2.1 cells in Dnmt1^EKO lungs (scale bars: 500μm). (B) Immunohistochemical staining for Tagln and Pecam1 demonstrate E18.5 lungs are vascularized and that large vessels are surrounded with smooth muscle (vessels are marked by white arrows). Smooth muscle also lines the large cysts in Dnmt1^EKO lungs (yellow arrow (top image) marks smooth muscle surrounding an airway yellow arrow (bottom image) marks smooth muscle lining cystic lung buds)(scale bars: 25μm). (C) A magnified image of immunohistochemistry for Nkx2.1 shows epithelial cells are present at E18.5 (first image marked by white arrows). Airway epithelial cell types marked by Scgb1a1 and Foxj1 (second image, white and yellow arrows respectively) are rare in Dnmt1^EKO lungs, while alveolar type 1 cells marked by Hopx (third image, white arrows) and alveolar type 2 cells marked by Sftpc (fourth image, white arrows) are more common (scale bar: 25μm).

Figure 5:. Dnmt1 is required for proper branching morphogenesis.

(A) Dnmt1 protein is lost in the lung endoderm in Dnmt1^EKO lungs at E12.5 (dashed lines demarcate endoderm)(scale bar: 50μm). (B) Dnmt1^EKO lungs display a subtle branching defect by H&E staining (black lines demarcate endoderm) (scale bar: 100μm). (C) Immunohistochemistry for Sox2 and Sox9 reveal no major disturbance of the proximal-distal axis at E12.5 (scale bar: 100μm). (D) Quantification of epithelial proliferation by assessment of Cdh1 and Phosphohistone H3 double positive cells shows no significant difference between Dnmt1^EKO and Dnmt1^control lungs (scale bar: 50μm, student’s two-tailed t-test, P>0.05). (E) Branch point analysis of E12.5 lungs demonstrates a statistically significant decrease in branching in Dnmt1^EKO lungs (scale bars: 150μm, student’s one-tailed t-test, P<0.05). (F) Still images from ex vivo live imaging of E11.5 lungs at 0, 24, and 72 hours of culture reveal that the disturbed branching patterns and formation of cystic sacs in Dnmt1^EKO lungs are lung intrinsic defects. White arrow indicates loss of a domain branch in Dnmt1^EKO lungs (scale bar: 500μm).

Figure 6:. Dnmt1 knockout lungs exhibit precocious AT2 differentiation signature.

(A) PCA demonstrates E12.5 Dnmt1^EKO and control lungs cluster separately. (B/C) Visualization of differentially expressed transcripts in Dnmt1^EKO versus control lungs by volcano plot and heat map show distal endoderm markers and surfactant genes enriched in Dnmt1^EKO lungs. (D/E) GO category analysis reveals alveolar lamellar body related genes to be among the most up-regulated in Dnmt1^EKO lungs. (F) qPCR verification of Nkx2.1, Sox2, and Sox9 expression confirms no significant difference in expression between Dnmt1^EKO and control lungs, while expression of Dnmt1 is significantly decreased and Sftpc is significantly enriched in Dnmt1^EKO lungs. (G) Endodermal cells stain positive for Sftpb and Sftpc protein in Dnmt1^EKO lungs at E12.5. At E14.5 Sftpb staining is expanded throughout the endoderm of Dnmt1^EKO lungs and Sftpc staining is more abundant in Dnmt1^EKO compared to Dnmt1^control lungs (scale bar: 50μm).