Figure 1.
Generation of Zfp148gt/gt mice.
(A) Map of the Zfp148 locus indicating the gene-trap insertion site, restriction sites for EcoRI (E), HindIII (H) and KpnI (K), and target sites for Southern blot probes. (B) Southern blot analyses of DNA from XB878 ES-cells and wild-type controls, digested with the restriction enzymes E, H, and K, respectively (asterisk indicates mutant band). Fragment size in kb. (C) PCR amplification across the gene-trap insertion on tail DNA from pups of Zfp148+/gt intercrosses confirmed gene-trap insertion at approximately 8500 base-pairs (bp) downstream of exon 4. The gene-trap specific band of 420 bp was amplified using a forward primer at 8256 bp downstream of exon 4 and a reverse primer in the gene-trap. The wild-type band of 580 bp was amplified using the same forward primer and a reverse primer at 8813 bp downstream of exon 4. (D) Real-time RT-PCR of the exon 4–5 border on cDNA from tissues of adult Zfp148gt/gt and wild-type mice, respectively. (E) Western blots showing expression of Zfp148 in Zfp148gt/gt and wild-type mouse embryonic fibroblasts (MEFs). Actin was used as a loading control. (F) RT-PCR amplification across the indicated exon–exon borders of the Zfp148 cDNA isolated from liver and muscle in Zfp148gt/gt and Zfp148+/gt mice did not indicate cryptic splicing of the mRNA or alternative transcription start sites. Amplification of GAPDH was included as a loading control. (G) Whole mount image of X-Gal stained Zfp148gt/gt mouse at E9.5. (H) Photomicrograph showing X-Gal stained transverse section of E9.5 Zfp148gt/gt mouse. Scale bars, 100 µm. Neural tube (nt), notochord (nc) and paraxial mesoderm (pm).
Figure 2.
Zfp148 deficiency causes lethality in newborn mice and growth retardation and reduced life span in adult mice.
(A) Zfp148-genotype distribution of offspring from heterozygous intercrosses. (B) Photograph of cyanotic Zfp148gt/gt mouse and wt littermate at P1. (C) Body weight of wt, Zfp148+/gt and Zfp148gt/gt mice of mixed genders at E18.5–19.5 (n = 12 wt, 15 Zfp148+/gt, 8 Zfp148gt/gt), P1 (n = 9 wt, 13 Zfp148+/gt, 7 Zfp148gt/gt), P9 (n = 28 wt, 44 Zfp148+/gt, 10 Zfp148gt/gt) and P19–22 (n = 10 wt, 18 Zfp148+/gt, 9 Zfp148gt/gt). (D, E) Body weight curves for adult wt and Zfp148gt/gt male (D) and female (E) mice, respectively (n = 10). (F) Kaplan-Meier plots showing survival of Zfp148gt/gt and wt mice (n = 20). ***P<0.001.
Figure 3.
Zfp148 deficiency prevented structural maturation of prenatal lungs without effecting epithelial cell differentiation or surfactant production.
(A) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from Zfp148gt/gt and wt mice, respectively. (A, B) Graphs show quantification (n = 6) of mean tissue area per total lung area, PAS-positive area with bronchioles excluded, and CC10-positive area with bronchioles excluded in (A) P1 and (B) E19.5 lungs from wt and Zfp148gt/gt mice. a.u., arbitrary units. (C) Real-time RT-PCR showing relative expression levels of markers for type I (T1alpha, Aqp5), type II (Sftpa1, Sftpb, Sftpc, Sftpd), clara (CC-10, Pon1), endothelial (Pecam1, Tie2, Nos3) and smooth muscle (Acta2) cells in Zfp148gt/gt lungs compared to wt at P1 (n = 6). Wt means are represented by the horizontal straight line at 1. (D) Transmission electron microscope (TEM) image of Zfp148gt/gt lung at E18.5–19.5 showing lamellar bodies secreted into the lumen of a terminal sac. (E–G) TEM images showing differentiated cells in Zfp148gt/gt lungs at E18.5–19.5. (E) Apical part of an alveolar type II cell containing typical lamellar bodies (arrowheads), one of which is in the process of exocytosis (arrow), and accumulations of densely contrasted glycogen particles (asterisk). (F) Two ciliated cells (arrowheads) surrounding two Clara cells (arrows) with bulging appearance and mitochondria accumulated in the apical cytoplasm. (G) High power view of the blood-alveolar barrier showing an erythrocyte (asterisk) in close contact with a highly attenuated part of an endothelial cell that shares a basal lamina with the alveolar type I cell. Scale bars, 2 µm. *P<0.05, ***P<0.001.
Figure 4.
Zfp148 deficiency reduced cell proliferation at the saccular stage of lung development.
(A, B) Photomicrographs showing BrdU labeling of E19.5 (A) and E18.5 (B) lungs, respectively, from wt and Zfp148gt/gt mice. (C) DNA content per 10 mg lung tissue (n = 5). (D) Lung wet weight at P1 (n = 12). Scale bars, 100 µm.
Figure 5.
Proliferation arrest in Zfp148gt/gt MEFs.
(A) Quantification of BrdU incorporation in wt, Zfp148+/gt and Zfp148gt/gt MEFs (n = 3). (B) Proliferation of Zfp148gt/gt, Zfp148+/gt, and wt MEFs (n = 4). (C) Apoptosis defined by FACS analyses of Annexin V in wt and Zfp148gt/gt MEFs at passage 2 (n = 3). (D) Photomicrographs of wt and Zfp148gt/gt MEFs at passage 2. (E) Real-time RT-PCR of p16ink4a mRNA in wt and Zfp148gt/gt MEFs (n = 4). (F) Western blot analysis of p16ink4a protein in wt and Zfp148gt/gt MEFs. (G) Cumulative population doublings (CPD) of Zfp148gt/gt and wt MEFs transduced with AdZfp148 or AdNull at passage 0 (n = 3). (H) Western blot analysis of Zfp148 expression levels in wt and Zfp148gt/gt MEF cell lines transduced with AdZfp148. (I) CPD of Zfp148gt/gt and wt MEFs transduced with AdZfp148 or AdNull at passage 2 (n = 3). (J) Cell cycle profile of Zfp148gt/gt MEF (left) and quantification of cell cycle distribution of Zfp148gt/gt and wt MEFs (n = 5) (right) at passage 4 measured by FACS analysis of propidium iodide (PI)–labelled cells. **P<0.01, ***P<0.001.
Figure 6.
Activation of p53 and Trp53-dependent proliferation arrest in Zfp148gt/gt MEFs.
(A) Western blots of MEF lysates showing expression of phospho-p53Ser18 in wt and Zfp148gt/gt MEFs cultured at 21 or 3% oxygen, respectively. Trp53−/− cells were used as a negative control and actin was used as a loading control. Asterisk indicates unspecific band. (B) Real-time RT-PCR of p21 in wt and Zfp148gt/gt MEFs cultured at 21 or 3% oxygen, respectively, and on Trp53+/+, Trp53+/− and Trp53−/− genetic backgrounds (n = 4). (C, D) CPD of Zfp148gt/gt and wt MEFs on Trp53+/+ or Trp53−/− (C) and Trp53+/+ or Trp53+/− (D) genetic backgrounds (n = 3). (E, F) CPD of Zfp148gt/gt and wt MEFs supplemented with n-acetyl-L-cysteine (NAC) (E) in the culture medium (n = 4), or cultured at atmospheric (21%) or low (3%) oxygen concentrations (F) (n = 3). *P<0.05, **P<0.01, ***P<0.001.
Figure 7.
Deletion of one or two copies of Trp53 rescued Zfp148gt/gt mice from proliferation arrest, respiratory distress and neonatal lethality.
(A) Photomicrographs showing BrdU labeling of E19.5 lung from wt, Zfp148gt/gt, Zfp148gt/gtTrp53+/− and Trp53+/− mice. Graphs show quantification of BrdU positive cells per lung area (n = 6 wt, 7 Zfp148+/gt, 7 Zfp148gt/gt, 5 Zfp148gt/gtTrp53+/−, 4 Trp53+/−). (B) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from Zfp148+/+Trp53+/+ (n = 10), Zfp148gt/gtTrp53+/+ (n = 9), Zfp148gt/gtTrp53+/− (n = 9), Zfp148gt/gtTrp53−/− (n = 5), and Zfp148+/+Trp53−/− (n = 10) mice, respectively. Graphs show quantification of mean tissue area per total lung area, PAS-positive area with bronchioles excluded, and CC10-positive area with bronchioles excluded. (C) Distribution of Zfp148 genotypes of P1 pups of intercrosses between Zfp148+/gtTrp53+/+ and Zfp148+/gtTrp53+/− mice. Scale bars, 100 µm. *P<0.05, **P<0.01, ***P<0.001.
Figure 8.
Antioxidant rescue of defect lung maturation and neonatal lethality in Zfp148gt/gt mice.
(A) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from Zfp148+/+, Zfp148gt/gt, and NAC treated Zfp148gt/gt mice, respectively (n = 6). Graphs show quantification of mean tissue area per total lung area, PAS-positive area with bronchioles excluded, and CC10-positive area with bronchioles excluded. (B) Distribution of Zfp148 genotypes of P1 pups of intercrosses between Zfp148+/gt mice with and without NAC treatment. Scale bars, 100 µm. **P<0.01, ***P<0.001.