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Endocrinology Vol. 147, No. 12 5584-5591
Copyright © 2006 by The Endocrine Society

Igf2 Deficiency Results in Delayed Lung Development at the End of Gestation

Delia Silva1, Maria Venihaki1, Wei-Hui Guo and Mary Frances Lopez

Division of Endocrinology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Mary Frances Lopez, Ph.D., Division of Endocrinology, Children’s Hospital, 300 Longwood Avenue, Karp Research Building 04212, Boston, Massachusetts 02115. E-mail: mary.lopez{at}childrens.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-II is a polypeptide hormone with structural homology to insulin and IGF-I. IGF-II plays an important role in fetal growth as mice with targeted disruption of the IGF-II gene (Igf2) exhibit severe growth retardation. The role of IGFs in the fetal lung has been suggested by several studies, including those that have identified IGF mRNA expression, and that of their receptors and binding proteins in the lungs at different stages of development. In this study, we used mice carrying a null mutation of Igf2 (Igf2–/– mice) to determine whether the absence of IGF-II had any effect in fetal lung maturation. Our results showed that the lungs of Igf2–/– fetuses had thicker alveolar septae and poorly organized alveoli when compared with those of Igf2+/+ on d 17.5 and 18.5 of gestation. These morphological alterations may be the result of exposure to lower levels of glucocorticoids because plasma corticosterone levels were significantly lower in Igf2–/– mothers compared with wild-type controls. In support of this, fetuses from homozygous knockout matings, where mothers were treated with 15 µg/ml corticosterone, and Igf2–/– fetuses obtained from heterozygous matings had similar lung histology to those of wild-type fetuses. Finally, we found that IGF-I and SP-B mRNA levels were up-regulated in the lungs of Igf2–/– fetuses at the end of gestation. This study suggests that Igf2 plays an important role in the development of the fetal lung and may affect fetal lung maturation by regulating maternal factors, such as corticosterone levels, during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FETAL LUNG DEVELOPMENT is affected by a variety of factors such as impaired placental function, undernutrition, exposure to tobacco smoke, and maternal and fetal hormones (1, 2, 3). Insulin is a hormone known to affect lung development (4). Infants born from diabetic mothers have high incidence of respiratory distress syndrome (4). IGFs are insulin-related proteins involved in lung development (5). Both IGF-I and -II, their receptors, and binding proteins are expressed in the fetal lung of humans, rodents, and other species (5, 6, 7, 8, 9, 10, 11). IGF-I mRNA expression is found in mesenchymal cells, primarily in those areas surrounding the airway epithelium. IGF-I is detected in the rat lung at embryonic d 15 (e15) and its expression decreases by d 17 and remains at low levels until d 21 of gestation (11). In contrast, IGF-II is expressed predominantly in lung epithelial cells, primarily in the conducting airways. IGF-II expression does not peak until d 19–21 of gestation (11). IGFs are also synthesized in vitro by human and mouse fetal lung explants (12, 13).

The actions of IGF-I and IGF-II are mediated by three receptors, the insulin receptor, the type 1 IGF receptor (IGF1R), and the type 2 IGF receptor (IGF2R) (14). These receptors may compete for the binding of IGFs during fetal development. The insulin receptor and the IGF1R are distinct, but related, receptors that can mediate responses to any of the three ligands. The IGF2R is identical to the cation-independent mannose-6-phosphate receptor, does not bind insulin, and has a low affinity for IGF-I (15). IGF1R expression is widespread throughout the fetal lung and its expression increases during late gestation and early postnatal life. IGF2R expression, on the other hand, is mainly found in mesenchyme and medial layer of intrapulmonary vessels and its expression decreases at the end of gestation (4, 16). The insulin receptor is expressed mainly in the type II cells and its binding affinity for its ligands increases during gestation (4).

There is increasing evidence suggesting that the IGF system plays a pivotal role in the development and differentiation of the fetal lung (7, 17, 18). Mice carrying a null mutation of the IGF-I gene (Igf1–/–) are born 60% the size of their littermates and their lungs have been characterized as atelectatic with increased cellularity (17, 19). Mice that carry a null mutation for the IGF1R (Igf1r–/–) and double Igf1r and Igf2 mutant mice are born 45% and 30% the size of their littermates, respectively, and die at birth of respiratory failure (15, 17, 19). On the other hand, mice lacking the IGF2R gene (Igf2r–/–) are born 140% larger than their wild-type (WT) littermates. This overgrowth is attributed to an increase in IGF-II levels (20). The Igf2r mutant mice have larger lungs than WT and usually die perinatally; however, these mice can be rescued from lethality if crossed with Igf2 or Igf1r-deficient mice (20).

In this study, we analyzed the lungs of Igf2 knockout (Igf2–/–) fetuses at different stages of development and explored possible mechanisms by which Igf2 deficiency may affect lung maturation. We found that the Igf2–/– fetuses had altered lung morphology during late gestation. Lungs from Igf2–/– fetuses on d 17.5 and 18.5 of gestation have thicker alveolar septae and poorly organized alveoli. In addition, surfactant protein B (SP-B) was significantly higher in the fetal lungs of Igf2-deficient mice, suggesting that Igf2 plays a role in the regulation of SP-B during fetal life. We also found significantly higher IGF-I mRNA levels in Igf2–/– fetal lungs, indicative of a compensatory mechanism that may develop in the absence of this gene. Because glucocorticoids play an important role in lung development, we measured corticosterone levels in both mothers and fetuses to determine whether there was any alternation in this hormone in the Igf2–/– mice. We found that corticosterone levels were significantly decreased in Igf2–/– mothers. Treatment of Igf2–/– dams with glucocorticoids resulted in offspring with similar lung morphology to those of WT fetuses. Thus, this mouse model may be proved valuable to understand the role, as well as, the mechanism of IGF-II action in the developing fetal lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mice carrying the inactivated Igf2 were originally provided by Dr. Argiris Efstratiadis through the courtesy of Dr. Lydia Villa-Komaroff (21). Igf2+/+ and Igf2–/– mice were first derived from the same progenitors, using heterozygous x heterozygous matings, and the two genotypes were subsequently maintained and bred separately. In experiments that involved heterozygous matings, the heterozygous mice inherited their Igf2 null mutation from their mothers. Genotyping was performed as described previously (22). Mice were housed on a 12-h light, 12-h dark cycle (lights on at 0700 h) with ad libitum access to rodent chow and water. The mice were housed and cared for according to National Institutes of Health guidelines, and all animal experiments were approved by the Animal Care and Use Committee of Children’s Hospital (Boston, MA).

Timed pregnancies and tissue preparation
Estrous females were mated with stud males. The presence of a vaginal plug on the morning after introduction of the female into the male cage was set as e0.5. Females were subsequently isolated until the time of tissue harvesting to ensure accurate gestational timing. Trunk blood was collected from each gravid female and their fetuses were dissected aseptically from the uterus. Trunk blood was also collected from each embryo on e17.5 and e18.5. In the experiments where pregnant females were treated with glucocorticoids, corticosterone was added in the drinking water beginning on e9.5, at a final concentration of 15 µg/ml. For Northern blot hybridization experiments, fetal lungs were collected and flash frozen in liquid nitrogen. For in situ hybridization histochemistry, whole embryos were rapidly frozen and stored at –80 C. Frozen sagittal sections (14 µm) were cut on a cryostat (Leica, Allendale, NJ), mounted on slides (SuperFrost Plus; Fisher Scientific, Pittsburgh, PA), and stored at –20 C with desiccant until use. To assess the histopathology, lungs were collected on e13.5, e15.5, e17.5, and e18.5. Lungs were fixed in Bouins’ fixative (90 ml aqueous saturated picric acid: 10 ml formaldehyde 40%:10 ml glacial acetic acid) for 24 h, paraffin embedded, sliced (sagittal sections, 5 µm) in a rotary microtome (Reichert-Jung Biocut), and stained with hematoxylin and eosin (H&E).

Fetal body and lung weight
Fetuses were weighed on e17.5. Each lung was removed intact from the thoracic cavity, blotted free of excess fluid, and weighed (wet weight). Dry weight was determined after baking at 80 C in a vacuum oven until no further changes in weight were seen on daily weighing.

5-Bromo 3-deoxyuridine (BrdU) and 4',6-diamidino-2-phenylindole (DAPI) staining
Immunohistochemical staining for BrdU was performed using a commercially available BrdU immunohistochemistry kit (Roche, Indianapolis, IN). Igf2+/+ and Igf2–/– females were injected with BrdU labeling reagent (10 µl/g of body weight) on e15.5 and e17.5. Females were killed 3 h after the injection and the fetuses were rapidly frozen in liquid nitrogen. Frozen sagittal 5-µm sections were cut on a cryostat, mounted on slides, and processed for BrdU staining according to the manufacturer’s instructions. A Zeiss Axioskop microscope (Jena, Germany) equipped with a drawing tube was used to analyze the lung sections. Labeled profiles were drawn and counted in randomly selected areas of 1000 x 600 µm. Number of labeled cells per field was compared between genotypes.

For DAPI staining, frozen lung sections were obtained from e15.5 and e17.5 embryos and washed once with cold PBS. Sections were then stained with 1 µg/ml DAPI for 5 min and washed three times with PBS for 5 min. The nuclear morphology of cells was examined using fluorescence microscopy by two observers.

In situ hybridization histochemistry
Mounted tissue sections were fixed with 4% paraformaldehyde in PBS for 15 min, rinsed in 2x SSC (0.3 M sodium chloride/0.03 M sodium citrate), and treated as previously described (23), before performing in situ hybridization histochemistry. Antisense riboprobes were radiolabeled by using [35S] uridine triphosphate (NEN Life Science Products, Life Science Products, Boston, MA). All mouse surfactant apoprotein plasmids used to generate probes were kindly provided by Dr. Whitsett (Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH). After dilution in hybridization buffer [50% deionized formamide, 10% dextran sulfate (Pharmacia LKB, Buckhinghamshire, UK), 0.5 M sodium chloride, 1x Denhardt’s solution (0.1% polyvinylpyrrolidine, 0.1% BSA, 0.1% Ficoll, type 400), 10 mM Tris (pH 8), 1 mM EDTA (pH 8), 500 µg/ml yeast tRNA (Invitrogen, Carlsbad, CA), 10 mM dithiothreitol (Sigma, St. Louis, MO)] to yield 2–3 x 107 cpm/ml, 80–100 µl of a radiolabeled cRNA probe was applied to each slide and hybridized for 16–20 h in a humidified chamber at 60 C. After hybridization, slides were washed twice in 2x SSC at room temperature, subjected to ribonuclease digestion at 37 C for 30 min, and again washed with 0.5x SCC three times for 10 min each time at room temperature and for 30 min at 60 C. Slides were dehydrated in increasing concentrations of ethanol (50%, 70%, 95%, and 100%), air dried, and exposed to X-Omat AR x-ray film (Kodak, Rochester, NY) for 24–48 h. The slides were subsequently dipped in Kodak NTB-2 nuclear emulsion, diluted 1:1, and exposed for 2–3 wk.

RNA extraction and Northern blot hybridization
RNA from each lung was isolated using the TRI reagent (Sigma). RNA (20 µg) was separated on a 1.4% formaldehyde/agarose gel and transferred to GeneScreen (NEN Life Science Products, Life Science Products) following standard protocols (24). cRNA SP-A, SP-B, and SP-D riboprobes were labeled with [32P] uridine triphosphate (NEN Life Science Products, Life Science Products) and T7 (for SP-D and SP-B), and SP6 (for SP-A) polymerases as previously described (24). A complementary to mouse IGF-I DNA (cDNA) fragment (purchased by American Type Culture Collection, Manassas, VA) was randomly labeled using [32P]-deoxy-CTP. Hybridizations were carried out at 65 C for at least 16 h with 106 cpm riboprobe/lane. After washing, the membranes were exposed to Kodak X-Omat AR x-ray film at room temperature for 30 min for surfactants or at –80 C for 8 d for IGF-I. Quantitation of the signal was performed using the public-domain analysis software NIH Image (http://rsb.info.nih.gov/nih-image/) (National Institutes of Health, Bethesda, MD) by analysis of scanned images of the autoradiography film.

Corticosterone RIA
Blood obtained from individual pregnant females and fetuses was centrifuged at 1900 x g at 4 C for 10 min, and plasma was collected and stored at –20 C until use. Plasma corticosterone levels were measured by using a commercial RIA kit (ICN, Costa Mesa, CA).

Statistics
Data were analyzed using the unpaired, two-tailed Student’s t test for comparison of two groups and ANOVA for comparison of more than two groups followed by post hoc multiple comparison tests. Significance was accepted at P < 0.05. Data were expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histological evaluation of the fetal Igf2–/– lung
To determine differences in lung morphology between Igf2+/+ and Igf2–/– fetuses, fetal lungs were collected from homozygous matings on e13.5, e15.5, e17.5, and e18.5. A delay in the fetal lung development was observed in the Igf2–/– mice starting on e15.5 (Fig. 1Go). It is known that during early stages of lung development, the undifferentiated columnar cells that line the branching tubules of the primordial lungs differentiate and proliferate (25). By e15.5, the columnar cells in the Igf2+/+ lungs have begun to differentiate and proliferate giving the lung its dense appearance (Fig. 1Go, E and G). The Igf2–/– lungs of e15.5 fetuses, on the other hand, remained similar in appearance to those of Igf2+/+ lungs on e13.5, marking the beginning of delay in lung development observed in the Igf2–/– mouse (Fig. 1Go, F and H). At e17.5, lungs from Igf2+/+ fetuses displayed normal development with formation of sac-like structures. The Igf2–/– fetuses, on the other hand, maintained a more dense pseudoglandular appearance; they appeared to have less saccular space and more mesenchyme than those of Igf2+/+ lungs (Fig. 1Go, I–L). Similar differences were also observed on e18.5, when the Igf2+/+ fetal lungs showed additional airspace formation and thinning of the alveolar septae, whereas the Igf2–/– lungs showed less additional airspace formation (Fig. 1Go, M–P). These observations suggest that Igf2 deficiency can lead to a delay in lung maturation during late gestation.


Figure 1
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  		FIG. 1. Representative histological sections of Igf2+/+ (A, C, E, G, I, K, M, and O) and Igf2–/– (B, D, F, H, J, L, N, and P) fetal lungs obtained from Igf2+/+ x Igf2+/+ and Igf2–/– x Igf2–/– matings, respectively, on d 13.5 (A–D), 15.5 (E–H), 17.5 (I–L), and 18.5 (M–P) of gestation stained with H&E. Notice the two first columns were taken at lower magnification (x4), whereas the last two columns were taken at higher magnification (x40). Scale bar, 400 µm (A, B, E, F, I, J, M, and N), 40 µm (C, D, G, H, K, L, O, and P).

 
Evaluation of cell proliferation
To further investigate the dense appearance of the Igf2–/– lungs in late gestation, we weighed Igf2+/+ and Igf2–/– fetuses and their respective lungs on e17.5 (Table 1Go). Because Igf2–/– fetuses are smaller than their WT littermates (19), we normalized the lung weight to their corresponding body weight. We performed both dry and wet weight measurements. We found that the average ratio of wet lung to body weight was significantly smaller in Igf2–/– than in Igf2+/+ fetuses (Table 1Go). However, no significant differences were found when this ratio was obtained using dry weights. To gain a more detailed insight into the proliferative activity of the Igf2–/– lungs, we performed BrdU labeling experiments. No significant differences were found in the number of proliferating cells between genotypes on e15.5 (Igf2+/+ 61.2 ± 4.07 cells and Igf2–/– 61.5 ± 3.77 cells; P > 0.05), suggesting that the increased cell density seen in the Igf2–/– lungs is perhaps due to a delay in cell differentiation and not to an increase in the proliferation index. To determine whether differences in cell death were seen between genotypes, lung sections were stained with DAPI to detect nuclear integrity. However, no differences in cell death were observed between Igf2+/+ and Igf2–/– fetal lungs on d 15.5 and 17.5 of gestation (data not shown), suggesting that the differences in lung morphology between genotypes are not due to differences in cell death.


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  		TABLE 1. Lung weights on d 17.5 of gestation in Igf2+/+ and Igf2–/– fetuses

 
Evaluation of IGF-I mRNA expression
To determine whether Igf2 deficiency resulted in any changes in lung IGF-I expression, we measured IGF-I mRNA levels in the lungs of Igf2+/+ and Igf2–/– fetuses on e17.5 and e18.5. We found that the levels of IGF-I mRNA were very low in the fetal lung, as previously reported in the fetal rat (8). However, after an 8-d exposure of the blot, we found that the levels of IGF-I mRNA were significantly higher in Igf2–/– than in Igf2+/+ control lungs on e18.5 (Fig. 2Go). Although the differences between genotypes did not reach significance on e17.5, there was a trend showing lower levels of IGF-I in Igf2+/+ than in Igf2–/– fetal lungs (0.336 ± 0.13 and 1.442 ± 0.58 arbitrary units, respectively; Fig. 2BGo). The levels of IGF-I mRNA were significantly lower in Igf2+/+ than in Igf2–/– fetal lungs on e18.5 (0.410 ± 0.08 and 2.11 ± 0.57 arbitrary units, respectively; P < 0.05).


Figure 2
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  		FIG. 2. Lung IGF-I mRNA levels in Igf2+/+ and Igf2–/– fetuses. A, IGF-I Northern blot of lungs obtained from Igf2+/+ and Igf2–/– fetuses on d 17.5 and 18.5 of gestation. B, Northern blot analysis quantification; Igf2+/+ (hatched bars) and Igf2–/– (black bars). IGF-I densitometric values were normalized to the corresponding 28 S RNA densitometric values. Bars indicate means ± SEM of four different lungs/genotype. Quantitation was performed using NIH Image. *, P < 0.05

 
Evaluation of surfactant protein mRNA levels
The expression of the surfactant proteins in the fetal lung is characterized by a gradual increase in mRNA and protein abundance as gestation nears term. This is thought to reflect type II pneumocyte maturation (26, 27). To investigate whether differences in lung morphology in Igf2–/– fetuses were associated with changes in the profile of surfactant proteins mRNA expression, Northern blots and in situ hybridization experiments were performed in lungs collected on d 17.5 and 18.5 of gestation. Expression of SP-A and SP-D were similar between the two genotypes on e17.5, but the levels of SP-A mRNA levels were significantly lower in Igf2–/– than in Igf2+/+ fetal lungs on e18.5 (Igf2+/+, 5.06 ± 0.81 and Igf2–/– 3.76 ± 0.27 arbitrary units, P < 0.05; Fig. 3AGo). Surprisingly, the levels of SP-B mRNA were significantly higher in the lungs of Igf2–/– mice on both embryonic days tested (Fig. 3BGo). On e17.5 and e18.5, SP-B mRNA levels in Igf2–/– lungs were almost double the levels of WT fetuses (e17.5: Igf2+/+, 1.14 ± 0.12 and Igf2–/– 1.96 ± 0.24 arbitrary units, e18.5: Igf2+/+, 1.96 ± 0.45 and Igf2–/– 3.60 ± 0.27 arbitrary units; P < 0.05).


Figure 3
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  		FIG. 3. Northern blot analysis quantification of RNA collected from Igf2+/+ (hatched bars) and Igf2–/– (black bars) lungs on d 17.5 and 18.5 of gestation. A, SP-A (top) and SP-D mRNA (bottom); B, SP-B mRNA Northern blot analysis quantification (top) and representative whole body sagittal sections from SP-B in situ hybridization experiments (bottom). Arrows indicate location of lung. Quantitation was performed using NIH Image. *, P < 0.05 (n = 3–4 lungs/group).

 
Maternal and fetal corticosterone levels
We measured glucocorticoids levels, a known regulator of lung maturation, to determine whether the delayed maturation observed in the Igf2–/– lungs was due to corticosterone deficiency. Both maternal and fetal corticosterone levels were measured on d 17.5 and 18.5 of gestation. Plasma corticosterone levels of both pregnant Igf2+/+ and Igf2–/– mice were significantly higher than those of non pregnant females of the same genotype (nonpregnant Igf2+/+, 3.3 ± 1.3 µg/dl and nonpregnant Igf2–/– 1.4 ± 0.09 µg/dl; P > 0.05). However, corticosterone levels of Igf2–/– pregnant mice were significantly lower than those of Igf2+/+ mothers (pregnant Igf2+/+, 124.7 ± 11.58 µg/dl and pregnant Igf2–/– 63.6 ± 12.8 µg/dl at e17.5, P < 0.05; Fig. 4AGo). The lower levels of corticosterone in Igf2–/– pregnant mice did not result in lower fetal corticosterone levels because no significant differences were found between fetuses of both genotypes (Fig. 4BGo). To further determine the importance of maternal corticosterone levels in the IGF-II-deficient lungs, we analyzed lungs from Igf2+/+ and Igf2–/– fetuses obtained from the same heterozygous mother. Histological examination showed that the lungs of Igf2+/+ and Igf2–/– fetuses, which developed within the same heterozygous uterus and were exposed to the same maternal corticosterone, have similar architecture (Fig. 5Go). To confirm the relationship between maternal glucocorticoids levels and lung development, we determined the effect of exogenous administration of corticosterone to both pregnant homozygous Igf2+/+ and Igf2–/– dams. We found that administration of 15 µg/ml corticosterone, from e9.5 to e17.5, resulted in Igf2–/– fetuses with lung architecture similar to those of WT controls, which were derived from WT dams not treated with corticosterone (Fig. 6Go). We also found that when homozygous Igf2+/+ mothers are treated with corticosterone, the lungs of their offspring are further developed than those that received no treatment (Fig. 6Go). These results indicate the importance of maternal corticosterone in lung development and suggest that the lower levels of maternal corticosterone in the Igf2–/– pregnancies may affect the level of lung maturation of their offspring.


Figure 4
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  		FIG. 4. Maternal and fetal glucocorticoids levels collected on d 17.5 and 18.5 of gestation. A, Plasma corticosterone levels in Igf2+/+ (hatched bars) and Igf2–/– (black bars) pregnant females mated with males of the same genotype (n = at least 4 animals/group); B, Plasma corticosterone levels in Igf2+/+ (hatched bars) and Igf2–/– (black bars) fetuses (n = at least 8 fetuses from three different litters). *, P < 0.05

 

Figure 5
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  		FIG. 5. Representative H&E stained lung sections from e17.5 Igf2+/+ (A and C) and Igf2–/– (B and D) fetuses collected from the uterus of heterozygous females mated to heterozygous males. Notice that the lung morphology is similar in both genotypes and unlike those collected from homozygous matings. Scale bar, 100 µm.

 

Figure 6
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  		FIG. 6. Lung morphology of e17.5 Igf2+/+ (A and B) and Igf2–/– (C and D) fetuses collected from homozygous matings where dams did not receive (A and C) or received (B and D) 15 µg/ml corticosterone in their drinking water starting on d 9.5 of gestation. Notice that corticosterone not only improved lung maturation in Igf2–/– fetuses (D), but also accelerated lung maturation in Igf2+/+ fetuses (B). n = 3 lungs/genotype/treatment. Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that Igf2 deficiency leads to a delay in fetal lung maturation, an increase in SP-B and IGF-I mRNA expression levels, and a decrease in maternal plasma corticosterone levels. Histological analysis of fetuses obtained from Igf2–/– pregnancies showed a delay in lung maturation during d 17.5 of gestation, a time when there is a progression from the early to the late canalicular phase. During this phase, development of the distal airways into definitive primary acinar structures (such as respiratory bronchioles, alveolar ducts, and rudimentary alveoli) occurs. The Igf2-deficient lungs were characterized by a dense pseudoglandular appearance, with a marked decrease in airspace formation when compared with WT controls. This lung phenotype is similar to that of Igf1-deficient mice, which also appears hypercellular and with less alveolar separation (28). Mice with Igf2 x Igf1 or Igf1r x Igf2 double mutation die of respiratory failure at birth (29), suggesting that IGF-II mediates its biological functions in the fetal lung via the IGFIR and supporting the importance of the IGF system in proper lung maturation and survival after birth.

IGFs play a role in cell cycle progression, and it is thought that one of the reasons why IGF mutants are born smaller than normal is because they undergo less proliferation cycles (17). We have previously demonstrated that placentas from Igf2-deficient mice have fewer numbers of cells than WT placentas (22). Control of cell number in the null mice could be mediated by mechanisms affecting the length of the cell cycle or the incidence of programmed cell death. We believe that different mechanisms might operate at different developmental stages or in different tissues. In the present study, we found that Igf2-deficient lungs were smaller than those of WT fetuses, but this reduction was proportional to that of its body weight. In addition, we were not able to find any differences in the proliferation rate of pulmonary cells, thus suggesting that IGF-II is not an essential factor for pulmonary cell proliferation to occur on d 15.5 and 17.5 of gestation.

It is well accepted that surfactants are important for respiration by lowering the surface tension of the air-liquid interface and by stabilizing of alveoli during low lung volumes (30). Several growth factors and hormones such as glucocorticoids regulate surfactant protein expression (26, 31). However, we are not aware of any studies that have focused on determining the role of the IGF system on surfactant expression in the fetus. In addition, immunohistochemical studies in Igf1R-deficient lungs have reported similar levels of surfactant apoproteins with those of their WT littermates (19). In our studies, we found that SP-A is significantly decreased in Igf2–/– mice on d 18.5 of gestation. Because SP-A is mainly known for its function in host defense (26) and mice with a targeted mutation in the gene for SP-A survive without obvious lung abnormalities (27), we do not believe that the lower levels of SP-A found in the Igf2–/– fetuses would result in delayed lung maturation. It is more likely that the lower levels of SP-A may lead to other consequences such as a decreased ability to clear bacteria from their lung spaces. However, this remains to be determined. To our surprise, SP-B mRNA levels were significantly increased in Igf2–/– fetal lungs compared with those of WT controls as shown by both Northern blot analysis and in situ hybridization. Insulin is known to inhibit SP-B gene expression in the fetal lung (32). It is possible, that, like insulin, IGF-II may also inhibit SP-B in the fetus, and in its absence the expression of SP-B is up-regulated. Because SP-B is required by the lung to achieve optimal surface tension along the alveolar surface (33, 34, 35), it is possible that SP-B overexpression allows the Igf2-deficient fetus to achieve complete stabilization of its alveoli before birth.

In this study, we also report higher IGF-I mRNA levels in the lungs of Igf2–/– fetuses compared with those of WT controls, suggesting that the elevated levels of IGF-I may be sufficient to ensure adequate lung development and survival at birth. It is also likely that each ligand can compensate, to a certain degree, for the absence of the other. In fact, Igf1 x Igf2 double mutants die of respiratory failure at birth (19). It is thought that these double mutant mice fail to breathe due to muscle hypoplasia (19). We cannot rule out, however, the possibility that failure to breathe may be due to alveolar collapse at birth. Because IGF-I mRNA levels are extremely low in the fetal lung (36), and we only see a slight but significant increase in the Igf2–/– fetuses on e17.5, further studies need to be performed to detect whether this increase in mRNA translates into an increase in protein levels and to determine the physiological importance of this observation.

Glucocorticoids enhance lung development and type II epithelial cell differentiation, promote expression of surfactants, influence clearance of lung liquid, and promote tissue remodeling before birth (25, 31, 37, 38). In this study, we found that maternal plasma corticosterone levels were significantly reduced in Igf2–/– pregnant mice during late gestation. These findings suggest that low corticosterone levels seen in the Igf2–/– mother may affect the lung maturity of its offspring. Further evidence that supports the importance of circulating maternal corticosterone on fetal lung maturation, comes from experiments where we used heterozygous matings. In these experiments, fetuses inherited the null mutation from their mother; thus, the phenotype of the heterozygous mice used was identical with those of WT mice. Thus, the lungs obtained from Igf2+/+ and Igf2–/– fetuses derived from the same heterozygous uterus and exposed to the same maternal corticosterone levels had similar morphology. In addition, exposure of pregnant homozygous Igf2+/+ and Igf2–/– females to exogenous levels of corticosterone, led to an increase in lung maturation in both Igf2+/+ and Igf2–/– offspring. Other studies that provide support for the importance of proper maternal corticosterone comes from the CRH (Crh)-deficient fetuses. These mice display a reduced pulmonary septal thinning and airway formation, and their lungs appear condensed and hypercellular during late gestation and die at birth (39). If, however, the Crh-deficient mice originate from heterozygous mothers their lungs have normal morphology and are able to survive (39). In addition, just like the Igf2–/– fetuses, treatment of Crh–/– pregnant females with corticosterone, resulted in offsprings with normal lung development (23). Mice that overexpressd Igf2 have increased levels of corticosterone (40), suggesting that Igf2 may play a role in the regulation of corticosterone. Whereas some studies in the rat show that dexamethasone inhibits IGF-II mRNA levels in the placenta (41), others have shown that it increases IGF-II mRNA expression in the fetal lung (18, 42). It has been demonstrated that one way by which glucocorticoids may regulate IGF-II expression is by regulating one of the promoters of the mouse Igf2 gene (43). In our study, we see differences in corticosterone levels only during pregnancy and not under basal conditions. It is possible that the adrenal gland of Igf2–/– pregnant mice does not have the capacity to work on high demands such as those observed during pregnancy because IGF-I and -II are known to play a key role in steroidogenesis by inducing steroidogenesis and mitogenesis in adrenocortical cells in vitro (44, 45, 46). It is also possible that IGF-II may be involved in the regulation of corticosterone secretion during gestation.

In summary, our study indicates that lack of Igf2 during fetal life results in a delay in lung development and differentiation. We showed that the absence of Igf2 might affect normal lung development by altering maternal factors such as corticosterone and/or fetal SP-B and IGF-I mRNA expression. These results demonstrate the complexity of the interactions within the IGF and other systems and also the importance of normal IGF-II expression during the development of the fetal lung.


   Acknowledgments
 
We thank Pieter Dikkes and Beah Hatcher for their technical support.


   Footnotes
 
This research was supported by National Institutes of Health Grants R01 GM071046 and K01 DK02653 (to M.F.L.).

Current address: Department of Clinical Chemistry-Biochemistry, School of Medicine, University of Crete, Crete, Greece.

First Published Online September 7, 2006

1 D.S. and M.V. made equal contributions. Back

Abbreviations: BrdU, 5-Bromo 3-deoxyuridine; DAPI, 4',6-diamidino-2-phenylindole; e, embryonic day; H&E, hematoxylin and eosin; IGF1R, type 1 IGF receptor; IGF2R, type 2 IGF receptor; WT, wild type.

Received April 18, 2006.

Accepted for publication August 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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