Trinucleotide repeat containing 6c (TNRC6c) is essential for microvascular maturation during distal airspace sacculation in the developing lung

Abstract

GW182 (also known asTNRC6) family members are critically involved in the final effector phase of miRNA-mediated mRNA repression. The three mammalian paralogs, TNRC6a, b and c, are thought to be redundant based on Argonaute (Ago) binding, tethering assays, and RNAi silencing of individual members in cell lines. To test this idea, we generated TNRC6a, b and c knockout mice. TNRC6a mutants die at mid-gestation, while b- and c- deleted mice are born at a Mendelian ratio. However, the majority of TNRC6b and all TNRC6c mutants die within 24h after birth, the latter with respiratory failure. Necropsy of TNRC6c mutants revealed normal-appearing airways that give rise to abnormally thick-walled distal gas exchange sacs. Immunohistological analysis of mutant lungs demonstrated a normal distribution of bronchiolar and alveolar cells, indicating that loss of TNRC6c did not abrogate epithelial cell differentiation. The cellular kinetics and relative proportions of endothelial, epithelial, and mesenchymal cells were also not altered. However, the underlying capillary network was simplified and endothelial cells had failed to become tightly apposed to the surface epithelium in TNRC6c mutants, presumably causing the observed respiratory failure. TGFβ family mutant mice exhibit a similar lung phenotype of thick-walled air sacs and neonatal lethality, and qRT-PCR confirmed dynamic downregulation of TGFβ1 and TGFβR2 in TNRC6c mutant lungs during sacculation. VEGFR, but not VEGF-A ligand, was also lower, likely reflecting the overall reduced capillary density in TNRC6c mutants. Together, these results demonstrate that GW182 paralogs are not functionally redundant in vivo. Surprisingly, despite regulating a general cellular process, TNRC6c is selectively required only in the distal lung and not until late in gestation for proper expression of the TGFβ family genes that drive sacculation. These results imply a complex and indirect mode of regulation of sacculation by TNRC6c, mediated in part by dynamic transcriptional repression of an inhibitor of TGFβ family gene expression.

Keywords: Lung alveolar development; MiRNA; Microvascular immaturity; Sacculation; TGFβ; TNRC6.

Fig. 1

TNRC6c mutants develop cyanosis and die of respiratory failure within 24 h after birth. A. qRT-PCR for TNRC6a, b, c, and Gapdh in WT and TNRC6c null mutant mice. Representative gel picture shown above bar graph displaying cumulative results from n = 3–4 mice in each group. *** indicates p < 0.001. B. WT and TNRC6c null pups at PN0. Note cyanosis of null mutant. Scale bar, 4 mm. C. Survival curve showing WT (n = 22) and heterozygotes (n = 46) had no observed lethality, while TNRC6c null mutants (n = 20) all died within 24 h of birth. Dotted line indicates period over which specific time of death was not recorded. D. H & E of WT and TNRC6c null mice at E18.5 showing abnormal, dense appearance of lung tissue in mutant compared with WT (black arrows). Scale bar, 500 µm. E. H& E of WT and TNRC6c null mutant lungs at PN0 shows reduced air sac size and thickened mesenchyme in mutant. Scale bar, 50 µm. E, embryonic day; H & E, hematoxylin and eosin; KO, knockout; PN, postnatal day; WT, wild type.

Fig. 2

Lung epithelial cells in TNRC6c mutants undergo proper histologic and molecular differentiation. A-B. Fluorescent micrographs of lung sections from E18.5 (A) and PN0 (B) WT and TNRC6c null mice immunostained for Club (Secretoglobin 1a1, Scgb1a), ciliated (acetylated tubulin, Acet Tub), AT2 (pro-Surfactant protein C, pSftpc) and AT1 (Podoplanin, Pdpn) cell type markers along with nuclear counterstain (DAPI). Note appropriate localization of airway (Club, ciliated) and gas exchange (AT1, AT2) epithelial cell types in bronchioles (outlined by white dashed line) and distal saccules, respectively. AT2 cells (pSftpc+) are present at E18.5 along with flat AT1 cells (Pdpn+), but exhibit reduced levels of pSftpc staining at PN0 (white arrowheads) in TNRC6c mutants compared with WT and E18.5 mutant lungs. C. Western blots for Nkx2.1 (NK2 Homeobox 1; epithelial cells), pSftpB (pro-Surfactant protein B; mature AT2 cells), and Aqp5 (Aquaporin 5; mature AT1 cells) along with Actb (beta actin) at E18.5 and PN0. Note similar levels of expression in mutant and WT lungs. Scale bar, 100 µm. AT, alveolar epithelial type; E, embryonic day; PN, postnatal day; WT, wild type.

Fig. 3

TNRC6c null mutants undergo incomplete mesenchymal thinning but maintain proper epithelial cell proportions and kinetics during sacculation. A. H & E of WT and TNRC6c null lungs at E18.5 and PN0. Note mutant mesenchyme is thicker than WT at E18.5, with evidence of only partial thinning compared with WT at PN0. Scale bar, 50 µm. B. Fluorescent micrographs of distal lung from WT and TNRC6c null mice at E18.5 (left) and PN0 (right) immunostained for epithelial cells (NK2 Homeobox 1, Nkx2.1) and nuclear counterstain (DAPI). Note similar salt and pepper distribution of epithelial cell nuclei in WT and mutant lungs. Scale bar, 50 µm. C. SEM of distal lung at PN0 shows TNRC6c null mutant air sacs are smaller and thicker-walled compared with WT littermate. Thickness of several primary septae between adjacent air sacs indicated by yellow (WT) and red (null mutant) lines. Scale bar, 10 µm. D. Quantification of air sac mesenchyme thickness in TNRC6c mutant (red) and WT (black) littermates, showing incomplete thinning in mutants between E18.5 and PN0. E. Percentage of distal lung epithelial (Nkx2.1+) cells at E18.5 and PN0 shows no difference between WT and mutant. F-G. Similar levels of PCNA (proliferating cell nuclear antigen) at E18.5 (Western blot, F, inset) and low percentage of proliferating (MKi67+) total (F, graph) and non-endothelial (G) cells at E18.5 and PN0 in mutant and WT distal lung. Actb, beta actin. H. Low percentage of cells undergoing apoptosis (cleaved Caspase 3+) at E18.5 and PN0 in WT and mutant distal lung. For D-H, each symbol indicates a distinct data point and each shape a different animal. Horizontal bars indicate the mean value. p-values were calculated with the Wilcoxon Rank Sum test. Ar, pulmonary artery; Br, bronchus; E, embryonic day; H & E, hematoxylin and eosin; NS, non-significant; PN, postnatal day; SEM, scanning electron microscopy; WT, wild type.

Fig. 4

Microvascular immaturity with dynamically reduced expression of TGF beta and VEGF family genes in TNRC6c mutant lungs. A. Representative images of WT and TNRC6c null mutant distal lung regions showing epithelial surface (green; Pdpn, AT1 cells), vasculature (red; endothelial “cocktail”), and nuclei (blue; DAPI) at PN0 (left panel), and without DAPI to highlight AT1 (green) and endothelial (red) cells (middle panel). Pixels representing areas of AT1-endothelial colocalization (yellow, right panel) obtained by element-wise multiplying of thresholded epithelial and vascular pixels in WT and TNRC6c micrographs. Scale bar, 40 µm. B. Plot showing quantification of AT1-endothelial cell colocalization in WT and TNRC6c null mutant animals at PN0. C. Representative maximum intensity projections of 5 optical slices of WT and TNRC6c null distal lung vasculature (endothelial “cocktail”) at PN0 shows simple mesh in mutants with absence of fine projections. Boxed regions in left panels are shown magnified in right panels. Scale bar, 100 µm. D. Data quantifying the microvascular density of WT and TNRC6c lungs, showing significantly reduced density in mutant lungs. E. Representative projection of micrograph of endothelial cells at PN0 in WT and mutant distal lung, immunostained for surface (Endomucin, Emcn) and nuclear (ETS related gene, ERG) markers along with nuclear counterstain (DAPI). Scale, 50 µm. F. Percentage of distal alveolar endothelial cells (ERG+) in WT and mutant lungs at E18.5 and PN0. G. Quantification by qRT-PCR of expression of TGFβ and VEGF family genes in lungs of WT and TNRC6c mutants at E17.5 and E18.5. Note no significant difference between WT and TNRC6c null lungs at E17.5, with dynamic reduction in expression of TGFβ1 and TGFβR2, and of Flt1 and Kdr, at E18.5 in mutants. H. Western Blot for Kdr shows significant reduction in mutant compared with WT lung at E18.5. * indicates p < 0.05, *** indicates p < 0.001, In B, D, F, each symbol indicates a distinct data point and each shape represents an individual animal. Horizontal bars indicate the mean value, and p-value was calculated with the Wilcoxon Rank Sum test. AT, alveolar epithelial type; E, embryonic day; KO, knockout; NS, non-significant; PN, postnatal day; TGFβ, Transforming Growth Factor beta; VEGF, Vascular Endothelial Growth Factor; WT, wild type.