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. 2012;7(11):e48071.
doi: 10.1371/journal.pone.0048071. Epub 2012 Nov 6.

Knockout of insulin-like growth factor-1 receptor impairs distal lung morphogenesis

Ralph Epaud  1 , Flore Aubey, Jie Xu, Zayna Chaker, Maud Clemessy, Alexandre Dautin, Karmène Ahamed, Monique Bonora, Nadia Hoyeau, Jean-François Fléjou, Arnaud Mailleux, Annick Clement, Alexandra Henrion-Caude, Martin Holzenberger
Affiliations

Knockout of insulin-like growth factor-1 receptor impairs distal lung morphogenesis

Ralph Epaud et al. PLoS One. 2012.

Abstract

Background: Insulin-like growth factors (IGF-I and -II) are pleiotropic regulators of somatic growth and development in vertebrate species. Endocrine and paracrine effects of both hormones are mediated by a common IGF type 1 receptor (IGF-1R). Lethal respiratory failure in neonatal IGF-1R knockout mice suggested a particular role for this receptor in pulmonary development, and we therefore investigated the consequences of IGF-1R inactivation in lung tissue.

Methods and findings: We first generated compound heterozygous mutant mice harboring a hypomorphic (Igf1r(neo)) and a null (Igf1r(-)) allele. These IGF-1R(neo/-) mice express only 22% of normal IGF-1R levels and are viable. In adult IGF-1R(neo/-) mice, we assessed lung morphology and respiratory physiology and found normal histomorphometric characteristics and normal breathing response to hypercapnia. We then generated homozygous IGF-1R knockout mutants (IGF-1R(-/-)) and analyzed their lung development during late gestation using histomorphometric and immunohistochemical methods. IGF-1R(-/-) embryos displayed severe lung hypoplasia and markedly underdeveloped diaphragms, leading to lethal neonatal respiratory distress. Importantly, IGF-1R(-/-) lungs from late gestation embryos were four times smaller than control lungs and showed markedly thickened intersaccular mesenchyme, indicating strongly delayed lung maturation. Cell proliferation and apoptosis were significantly increased in IGF-1R(-/-) lung tissue as compared with IGF-1R(+/+) controls. Immunohistochemistry using pro-SP-C, NKX2-1, CD31 and vWF as markers revealed a delay in cell differentiation and arrest in the canalicular stage of prenatal respiratory organ development in IGF-1R(-/-) mutant mice.

Conclusions/significance: We found that low levels of IGF-1R were sufficient to ensure normal lung development in mice. In contrast, complete absence of IGF-1R significantly delayed end-gestational lung maturation. Results indicate that IGF-1R plays essential roles in cell proliferation and timing of cell differentiation during fetal lung development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. IGF-1R protein levels, lung histology and respiratory function in adult IGF-1Rneo/− mice. A
, Western immunoblot of IGF-1R in lung from mice with distinct combinations of mutant IGF-1R alleles. Total proteins were extracted from lung tissue from IGF-1Rneo/−, IGF-1R+/−, IGF-1R+/neo and IGF-1R+/+ mice (n = 3 for each genotype), and IGF-1R−/− embryo (negative control), and were probed with anti-IGF-1Rβ (upper panel) or anti-β-actin antibodies (lower panel). IGF-1Rneo/− mice have 22% of receptor levels present in IGF-1R+/+ mice (quantified in B), IGF-1R+/− have 50%, IGF-1R+/neo mice are between 70 and 80%, and IGF-1R−/− mice lack IGF-1R completely. B, IGF-1R abundance determined in lung tissue. Bar graph shows IGF-1R levels relative to β-actin from 5 IGF-1R+/+ and 6 IGF-1Rneo/− individuals (Error bars SEM; Student’s t-test). Image shows 4 representative lanes from western immunoblot. C, Hematoxylin-eosin stained lung sections from IGF-1R+/+ and IGF-1Rneo/− males. D, Alveolar airspace, E, alveolar boundary length density, F, alveolar wall thickness, in IGF-1R+/+ (n = 4) and IGF-1Rneo/− mice (n = 4). Error bars indicate SEM; Wilcoxon Mann-Whitney U test. G-I, Respiratory function in adult IGF-1Rneo/− mice. Mice were challenged with 6% and 8% CO2. G, Minute ventilation (VE), H, tidal volume (VT), and I, respiratory frequency (BR) were measured in 6 individuals per group. Differences between room air and hypercapnia were significant, but no significant differences were found between genotypes. Values labeled b were different from a (P<0.005); Error bars indicate SEM; Wilcoxon Mann-Whitney U test.
Figure 2
Figure 2. Growth retardation in IGF-1R−/− embryos affects lung more than other tissue.
Values represent organ weight relative to body weight (mean ± SEM), normalized to the stage-specific mean of the control (IGF-1R+/+) group. Organ/body weight ratio was calculated from data in Table 1. * P<0.05; ** P<0.01; *** P<0.001, compared with normalized IGF-1R+/+ data (Norm) of the same developmental stage; Student’s t-test; ND, not determined.
Figure 3
Figure 3. Lung development in late gestation IGF-1R−/− mice.
A–L, Lungs prepared from IGF-1R+/+ and IGF-1R−/− embryos at developmental stages E14.5, E17.5 and E19.5. A–F, Ventral view of whole lungs. G–L, Rim of lung lobe. Abbreviations: AL, apical lobe; AzL, azygous lobe; CL, cardiac lobe; DL, diaphragmatic lobes; LL, left lobe. M–X, Lung histology of IGF-1R+/+ versus IGF-1R−/− embryos. H&E stained lung sections at developmental stages E14.5 (M–P), E17.5 (Q–T) and E19.5 (U–X), showing that saccular walls are thicker and acinar buds smaller in IGF-1R−/− embryos as compared with controls of the same stage. Note that histomorphological appearance is similar when comparing E19.5 IGF-1R−/− (V, X) with two days younger E17.5 IGF-1R+/+ lungs (Q, S).
Figure 4
Figure 4. Lung histomorphology and cell turnover in the absence of IGF-1R.
A, Saccular airspace and B, saccular wall thickness (mean ± SEM) at developmental stages E17.5 in IGF-1R+/+ (n = 4) and IGF-1R−/− embryos (n = 4). Wilcoxon Mann-Whitney U test. C–F, Extended gestation period and lung histology. H&E stain of lung tissue from embryos at 19.5 (C, D) and 21.5 days (E, F). To extend gestation period up to 21.5 days, pregnant mothers were treated with progesterone from E17.5 onwards. Note the presence of red blood cell extravasation in E21.5 lung samples from IGF-1R+/+ and IGF-1R−/− mice. G–O, Cell turnover in IGF-1R−/− embryonic lung at E17.5. Lung histology from IGF-1R+/+ embryos (G, J and M) and IGF-1R−/− embryos (H, K and N) at E17.5. Bar graphs (I, L and O) show quantification (mean ± SEM; n = 3–7 individuals per group; Student’s t-test). G–I, Cells were counted using DAPI staining (blue signal). J–L, Cell proliferation was measured using phospho–histone H3 immunohistochemistry (brown staining). M–O, Apoptosis was detected using cleaved caspase-3 immunohistochemistry (brown staining). Cleaved caspase-3 (P-U) and phospho-histone H3 labeling (V-AA) at high magnification showing examples for IHC-positive epithelial (red arrows), vascular endothelial (blue) and mesenchymal cells (green), as identified by their anatomical location. Note that many of the proliferating cells are located in areas that are composed of mostly mesenchymal cells.
Figure 5
Figure 5. Immunohistochemistry of lung differentiation markers.
A and B, Representative tissue sections from IGF-1R+/+ and IGF-1R−/− embryos at stage E17.5 showing CD31-immunoreactivity specific for capillary endothelia. C, Morphometric comparison of CD31 signal between genotypes (n = 5 per group; two-tailed t-test). D–F, Capillary complexity was estimated calculating the density of capillary junctions from CD31 IHC. G–J, Sections from IGF-1R+/+ and IGF-1R−/− embryos at E17.5 and E19.5 show IHC of blood vessel-specific von Willebrand protein. Arrows (I, J) point to small blood vessels developing in saccular walls. Large blood vessels were similarly marked in all specimen. K–M, Representative lung histology from IGF-1R+/+ and IGF-1R−/− embryos at E17.5. NKX2-1 distal-to-proximal IHC signal ratio was measured in 6 IGF-1R+/+ and 5 IGF-1R−/− embryos. NS, not significant; Wilcoxon Mann-Whitney U test. N-Q, Epithelial cell-specific NKX2-1 transcription factor was detected in IGF-1R+/+ and IGF-1R−/− embryos at E17.5 and E19.5. R–Y, IHC of type 2-specific pro-SP-C at low (R-U) and high magnification (V-Y). Interestingly, for NKX2-1 and pro-SP-C, the IHC pattern of IGF-1R−/− lungs at E19.5 resembles controls at E17.5 (panel N versus Q, R versus U, and V versus Y), suggesting an approximately 2-day developmental delay in IGF-1R−/− end-gestational lungs.
Figure 6
Figure 6. Development of diaphragm and chest in the absence of IGF-1R.
A, Hematoxylin-eosin stained transversal section of thoracic wall and diaphragm in control (left) and IGF-1R−/− embryos (right) at E17.5. Bar graphs compare B, diaphragm thickness, C, rib diameter, and D, diaphragm-to-rib ratio (mean ± SEM) in IGF-1R+/+ embryos (n = 4) and IGF-1R−/− embryos (n = 4). R, Rib; D, diaphragm. Wilcoxon Mann-Whitney U test.
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This work was supported by EU Network of Excellence LifeSpan (036894) and ANR (NT05-3 42491). Both funders supported salary and reagents. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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