Differentiation of hematopoietic lineages in spleen of <i>Zfp148</i>-deficient mice.

<p>Flow cytometric analysis of spleen from P14 pups (<i>n</i> = 3) and adult mice, ages 3-6 months; wild type (<i>n</i> = 6) and <i>Zfp148</i><b>^<i>gt/gt</i></b> (<i>n</i> = 5). (A) Percentage of CD4- or CD8-positive T-cells, CD4-positive T-cells and CD8-positive T-cells among lymphocytes. (B) Representative cytograms from adult spleens. (C) Percentage of CD69-positive cells among lymphocytes. (D) Representative cytograms from adult spleens. (E) CD62L low expressing cells among lymphocytes. (F) Percentage of B220-positive cells among lymphocytes in P14 and adult spleen. (G) Representative cytograms from adult spleens. Lymphocytes were gated based on side scatter (SSC). Values are expressed as mean ± SEM. Significance levels: *P<0.05, **P<0.01.</p

Differentiation of hematopoietic lineages in thymus and bone marrow of <i>Zfp148</i>-deficient mice.

<p>Flow cytometric analysis of bone marrow (A–D) and thymocytes (E–H) from wild type and <i>Zfp148</i>^<i>gt/gt</i> mice. Graphs and representative cytograms show percentage of (A, B) Mac1-, B220- and (C, D) CD4 and/or CD8-positive cells among leukocytes in bone marrow from adult mice, ages 3-6 months; wild type (<i>n</i> = 6) and <i>Zfp148</i><b>^<i>gt/gt</i></b> (<i>n</i> = 5). (E–H) Percentage of (E) double negative (DN) CD4/CD8, (F) double positive (DP) CD4/CD8, (G) CD4 single positive (SP) and CD8 SP cells among thymocytes from P14 pups (left, <i>n</i> = 3) and adult mice (right, <i>n</i> = 5). (H) Representative cytograms from adult thymus. Values are expressed as mean ± SEM. Significance levels: *P<0.05, **P<0.01.</p

Deletion of one or two copies of <i>Trp53</i> rescued <i>Zfp148^gt/gt</i> mice from proliferation arrest, respiratory distress and neonatal lethality.

<p>(A) Photomicrographs showing BrdU labeling of E19.5 lung from wt, <i>Zfp148^gt/gt</i>, <i>Zfp148^gt/gtTrp53^+/−</i> and <i>Trp53^+/−</i> mice. Graphs show quantification of BrdU positive cells per lung area (<i>n</i> = 6 wt, 7 <i>Zfp148^+/gt</i>, 7 <i>Zfp148^gt/gt</i>, 5 <i>Zfp148^gt/gtTrp53^+/−</i>, 4 <i>Trp53^+/−</i>). (B) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from <i>Zfp148^+/+Trp53^+/+</i> (<i>n</i> = 10), <i>Zfp148^gt/gtTrp53^+/+</i> (<i>n</i> = 9), <i>Zfp148^gt/gtTrp53^+/−</i> (<i>n</i> = 9), <i>Zfp148^gt/gtTrp53^−/−</i> (<i>n</i> = 5), and <i>Zfp148^+/+Trp53^−/−</i> (<i>n</i> = 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 <i>Zfp148</i> genotypes of P1 pups of intercrosses between <i>Zfp148^+/gtTrp53^+/+</i> and <i>Zfp148^+/gtTrp53^+/−</i> mice. Scale bars, 100 µm. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Antioxidant rescue of defect lung maturation and neonatal lethality in <i>Zfp148^gt/gt</i> mice.

<p>(<b>A</b>) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from <i>Zfp148^+/+</i>, <i>Zfp148^gt/gt</i>, and NAC treated <i>Zfp148^gt/gt</i> mice, respectively (<i>n</i> = 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>B</b>) Distribution of <i>Zfp148</i> genotypes of P1 pups of intercrosses between <i>Zfp148^+/gt</i> mice with and without NAC treatment. Scale bars, 100 µm. **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>Zfp148</i> deficiency prevented structural maturation of prenatal lungs without effecting epithelial cell differentiation or surfactant production.

<p>(<b>A</b>) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from <i>Zfp148^gt/gt</i> and wt mice, respectively. (<b>A, B</b>) Graphs show quantification (<i>n</i> = 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 <i>Zfp148^gt/gt</i> mice. a.u., arbitrary units. (<b>C</b>) 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 <i>Zfp148^gt/gt</i> lungs compared to wt at P1 (<i>n</i> = 6). Wt means are represented by the horizontal straight line at 1. (<b>D</b>) Transmission electron microscope (TEM) image of <i>Zfp148^gt/gt</i> lung at E18.5–19.5 showing lamellar bodies secreted into the lumen of a terminal sac. (<b>E–G</b>) TEM images showing differentiated cells in <i>Zfp148^gt/gt</i> 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. *<i>P</i><0.05, ***<i>P</i><0.001.</p

<i>Zfp148</i> deficiency causes lethality in newborn mice and growth retardation and reduced life span in adult mice.

<p>(<b>A</b>) <i>Zfp148</i>-genotype distribution of offspring from heterozygous intercrosses. (<b>B</b>) Photograph of cyanotic <i>Zfp148^gt/gt</i> mouse and wt littermate at P1. (<b>C</b>) Body weight of wt, <i>Zfp148^+/gt</i> and <i>Zfp148^gt/gt</i> mice of mixed genders at E18.5–19.5 (<i>n</i> = 12 wt, 15 <i>Zfp148^+/gt</i>, 8 <i>Zfp148^gt/gt</i>), P1 (<i>n</i> = 9 wt, 13 <i>Zfp148^+/gt</i>, 7 <i>Zfp148^gt/gt</i>), P9 (<i>n</i> = 28 wt, 44 <i>Zfp148^+/gt</i>, 10 <i>Zfp148^gt/gt</i>) and P19–22 (<i>n</i> = 10 wt, 18 <i>Zfp148^+/gt</i>, 9 <i>Zfp148^gt/gt</i>). (<b>D, E</b>) Body weight curves for adult wt and <i>Zfp148^gt/gt</i> male (D) and female (E) mice, respectively (<i>n</i> = 10). (<b>F</b>) Kaplan-Meier plots showing survival of <i>Zfp148^gt/gt</i> and wt mice (<i>n</i> = 20). ***<i>P</i><0.001.</p

Activation of p53 and <i>Trp53</i>-dependent proliferation arrest in <i>Zfp148^gt/gt</i> MEFs.

<p>(<b>A</b>) Western blots of MEF lysates showing expression of phospho-p53^Ser18 in wt and <i>Zfp148^gt/gt</i> MEFs cultured at 21 or 3% oxygen, respectively. <i>Trp53^−/−</i> cells were used as a negative control and actin was used as a loading control. Asterisk indicates unspecific band. (<b>B</b>) Real-time RT-PCR of <i>p21</i> in wt and <i>Zfp148^gt/gt</i> MEFs cultured at 21 or 3% oxygen, respectively, and on <i>Trp53^+/+</i>, <i>Trp53^+/−</i> and <i>Trp53^−/−</i> genetic backgrounds (<i>n</i> = 4). (<b>C, D</b>) CPD of <i>Zfp148^gt/gt</i> and wt MEFs on <i>Trp53^+/+</i> or <i>Trp53^−/−</i> (C) and <i>Trp53^+/+</i> or <i>Trp53^+/−</i> (D) genetic backgrounds (<i>n</i> = 3). (<b>E, F</b>) CPD of <i>Zfp148^gt/gt</i> and wt MEFs supplemented with <i>n</i>-acetyl-L-cysteine (NAC) (E) in the culture medium (<i>n</i> = 4), or cultured at atmospheric (21%) or low (3%) oxygen concentrations (F) (<i>n</i> = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p