Pulmonary hypoplasia in the connective tissue ... - Wiley Online Library

DEVELOPMENTAL DYNAMICS 237:485– 493, 2008

PATTERNS & PHENOTYPES

Pulmonary Hypoplasia in the Connective Tissue Growth Factor (Ctgf) Null Mouse Mark Baguma-Nibasheka and Boris Kablar*

Connective tissue growth factor (CTGF) is a mediator of growth factor activity, and Ctgf knockouts die at birth from respiratory failure due to skeletal dysplasia. Previous microarray analysis revealed Ctgf downregulation in the hypoplastic lungs of amyogenic mouse embryos. This study, therefore, examined pulmonary development in Ctgfⴚ/ⴚ mouse fetuses to investigate if respiration could also have been impaired by lung abnormalities. The Ctgfⴚ/ⴚ lungs were hypoplastic, with reduced cell proliferation and increased apoptosis. PDGF-B, its receptor and IGF-I, were markedly attenuated and the TTF-1 gradient lost. Type II pneumocyte differentiation was perturbed, the cells depicting excessive glycogen retention and diminished lamellar body and nuclear size, though able to synthesize surfactant-associated protein. However, type I pneumocyte differentiation was not affected by Ctgf deletion. Our findings indicate that the absence of Ctgf and/or its protein product, CTGF, may induce pulmonary hypoplasia by both disrupting basic lung developmental processes and restricting thoracic expansion. Developmental Dynamics 237: 485– 493, 2008. © 2008 Wiley-Liss, Inc. Key words: pulmonary hypoplasia; pneumocyte differentiation; mouse embryo; CTGF Accepted 5 December 2007

INTRODUCTION Embryonic lung development, consisting of growth (increase in size with structural specialization) as well as maturation (cellular and functional differentiation), is subject to various factors, with maturation relying mainly on hormonal factors and growth depending largely upon physical factors and mechanical forces, which, as reviewed by Liu and Post (2000) and Inanlou et al. (2005), affect cell-cycle kinetics and cell differentiation. Mechanical forces caused by intermittent respiratory-like movements in utero, called fetal breathinglike movements (FBMs), appear to

have the principal role here, and their absence impairs lung growth and leads to pulmonary hypoplasia (Tseng at al., 2000; Inanlou and Kablar, 2003, 2005a,b). FBMs are also required for lung maturation, and the final differentiation of the alveolar pneumocyte types I and II, in particular, is not possible in the complete absence of respiratory activity (Nagai et al., 1988; Benachi et al., 1999; Inanlou and Kablar, 2005a,b). Maturation of type II pneumocytes is associated with a decrease in the cytoplasmic glycogen that acts as a substrate for the formation of surfactant-associated proteins and

phospholipids, and an increase in the number of lamellar bodies, the intracellular organelles required for assemblage and storage of surfactant (Ten Have-Opbroek et al., 1990; Batenburg, 1992). In the lungs of fetuses lacking FBMs, type II cells seem unable to utilize glycogen for the synthesis of surfactant, and the number of cytoplasmic lamellar bodies is significantly reduced while the released (intra-alveolar) lamellar bodies are scarce, loose, and disorganized, as are the tubular myelins that act as intermediate structures in the formation of a phospholipid monolayer on the alveolar surface (Nagai et al., 1988;

1 Department of Anatomy and Neurobiology, Dalhousie University Faculty of Medicine, Halifax, Canada Grant sponsor: National Science and Engineering Research Council of Canada; Grant number: 238726-01; Grant sponsor: Canadian Institutes of Health Research; Grant number: MOP-68823; Grant sponsor: Lung Association of Nova Scotia; Grant sponsor: Canada Foundation for Innovation; Grant sponsor: Dalhousie Medical Research Foundation. *Correspondence to: Department of Anatomy and Neurobiology, Dalhousie University Faculty of Medicine, 5850 College Street, Halifax, NS, Canada B3H 1X5. E-mail: [email protected]

DOI 10.1002/dvdy.21433 Published online 16 January 2008 in Wiley InterScience (www.interscience.wiley.com).

© 2008 Wiley-Liss, Inc.

486 BAGUMA-NIBASHEKA AND KABLAR

Brandsma et al., 1993; Inanlou and Kablar, 2005b). In addition, in the absence of FBMs, no cells with the morphological characteristics of type I pneumocytes are found in the hypoplastic lung, indicating an inability of type II pneumocytes to successfully differentiate into type I pneumocytes (Inanlou and Kablar, 2005b). Restriction of thoracic volume also impairs lung growth and leads to pulmonary hypoplasia (Page and Stocker, 1982; Connor et al., 1985), and previous studies on animal models of human chondrodystrophy with reduced thoracic volume have provided valuable insights into the pathogenesis of pulmonary hypoplasia (Seegmiller et al., 1986; Hepworth and Seegmiller, 1989; Houghton et al., 1989; Hepworth et al., 1990; Foster et al., 1994). However, because neonatal pulmonary hypoplasia can result from many different causes and then develop through many different mechanisms (Page and Stocker, 1982), any additional models for its study would improve our ability to prevent, diagnose, and treat this condition. We, therefore, investigated embryonic lung development in the genetically modified mouse lacking the gene for connective tissue growth factor, which is known to die shortly after birth because of respiratory failure caused by skeletal defects restricting thoracic expansion (Ivkovic et al., 2003). Connective tissue growth factor (CTGF, CCN2) is a prototypic member of the CCN secreted matricellular protein family, which contains cysteinerich protein 61 (Cyr61), CTGF, nephroblastoma overexpressed (Nov), and Wnt-1-induced secreted proteins WISP-1, WISP-2, and WISP-3 (Brigstock, 2003; Perbal, 2004), and is involved in cell proliferation, migration, adhesion, survival, and differentiation as well as apoptosis and the inhibition of cell growth (Babic et al., 1999; Brigstock, 2003; Croci et al., 2004; Gao and Brigstock, 2004; Chien et al., 2006). Although CTGF may have some independent activity, most reviews concur that it principally acts as an autoparacrine downstream mediator for the various modulators (including platelet-derived growth factor, transforming growth factor-␤1, vascular endothelial growth factor, fibroblast growth factor, and bone morphogenic

proteins), which induce its action (Brigstock, 2003; Perbal, 2004; Chaqour and Goppelt-Struebe, 2006; Leask and Abraham, 2006). The ability of CTGF to control mutually exclusive cellular responses (proliferation vs. differentiation; survival vs. death) depends on the inductive upstream signaling pathway and the particular cell type, as well as the existing environmental status like the presence of other growth factors and cytokines (Lau and Lam, 1999; Leask and Abraham, 2003; Desnoyers, 2004; Wu et al., 2006). These conditions determine which of its constituent structural domains will interact with cell surface receptors, and the ensuing intracellular responses may, among several alternative pathways, follow the FAK/PI3K/Akt signal transduction pathway to modulate cytokinesis and cell adhesion, or the Ras/MEK/ERK pathway to regulate cyclin activity and cell proliferation (Abdel-Wahab et al., 2002; Brigstock, 2003; Leask and Abraham, 2003, 2006). Thus, using such post-receptor mechanisms, CTGF plays a role in various biological processes including angiogenesis, extracellular matrix synthesis, osteogenesis, and chondrogenesis (Frazier et al., 1996; Babic et al., 1999; Ivkovic et al., 2003; Arnott et al., 2007). Interestingly, in a recent microarray analysis of gene expression in the lungs of mouse embryos with pulmonary hypoplasia resulting from the absence of skeletal musculature and, consequently, of FBMs, we found that both Ctgf mRNA and CTGF protein were down-regulated in the lungs of the amyogenic embryos (Baguma-Nibasheka et al., 2007). This indicated that CTGF may play some role in pulmonary development and, therefore, warranted further investigation in subsequent attempts to delineate the mechanisms underlying that particular process. Furthermore, it has been shown that Ctgf deletion mutant mice die at birth because of respiratory failure due to skeletal dysplasia (Ivkovic et al., 2003). The Ctgf⫺/⫺ mutants were found to have short, misaligned and inward-bent sterna, and kinked ribs (Ivkovic et al., 2003), possibly due to decreased growth plate angiogenesis, chondrocyte migration, and matrix degradation, resulting in a visibly reduced thoracic volume. At birth,

these mutants attempt to breathe but then exhibit gasping behaviour, become cyanotic, and die within minutes (Dr. K.M. Lyons, unpublished observations). The Ctgf⫺/⫺ mouse is, therefore, a good model for the numerous human chondrodystrophic pulmonary hypoplasias that frequently prove lethal soon after birth. However, besides the skeletal defects, respiration could have been impaired by lung abnormalities present in the Ctgf-deficient newborns. In this study, therefore, we examined the lungs of the Ctgf⫺/⫺ mouse at the stage of fetal life (near term) where our previous research on lung hypoplasia due to the absence of FBMs has revealed differences from normal pulmonary development.

RESULTS Ctgfⴚ/ⴚ Embryos Have Pulmonary Hypoplasia The lungs of the Ctgf-deficient embryos were significantly smaller than those of their normal (control) wildtype littermates, with an average weight of 16.8 ⫾ 0.4 mg (mean ⫾ SD) after fixation, versus 19.9 ⫾ 1.3 mg; P ⬍0.05 with Student’s t-test from n ⫽ 3 and 4, respectively. Also, the Ctgf⫺/⫺ embryonic lungs depicted the histopathological features of lung hypoplasia such as dense cellularity, thicker septae, and the absence of expanded saccules, giving the lungs an appearance of being arrested at earlier stages of lung development (Fig. 1A,B). The average area of their potential alveolar airspace was only 9.3 ⫾ 2% (compared to 41.1 ⫾ 12% in the controls) when measured on hematoxylin-eosin stained paraffin sections (Fig. 1C).

Ctgfⴚ/ⴚ Lung Tissue Exhibits Disturbances in Cell Proliferation and Cell Death In order to elucidate the processes underlying the pulmonary hypoplasia, we analyzed cell proliferation and programmed cell death (apoptosis), mechanisms known to be involved in lung growth. The proliferation index (PI), taken as the percentage of cells immunostaining for proliferating cell nu-

PULMONARY HYPOPLASIA IN THE CTGF⫺/⫺ MOUSE 487

clear antigen (PCNA), was found to be significantly lower in both the epithelial and the mesenchymal cell compartments of the mutant lungs than in the lungs of their wild-type littermates (Fig. 1D–F). The average PI in Ctgf⫺/⫺ lungs was 25.6 ⫾ 4% in the epithelium and 33.3 ⫾ 3% in the mesenchyme as compared to 41.6 ⫾ 5 and 47.5 ⫾ 7%, respectively, in the controls. In contrast, the apoptotic index, indicated by caspase-3 labeling, was elevated in both the epithelial and the mesenchymal compartments of the hypoplastic lungs (10.7 ⫾ 1 and 8.1 ⫾ ⫾ 2%, respectively, versus 8.6 ⫾ 1 and 5.5 ⫾ 1% in the controls, Fig. 1G–I). In addition, the distribution pattern of various key regulators of pulmonary growth was examined. Platelet-

Fig. 1.

Fig. 1. Ctgf⫺/⫺ fetuses exhibit pulmonary hypoplasia, decreased lung cell proliferation and increased lung cell apoptosis. Paraffin-embedded sections of the wild-type (A,D,G) and Ctgf⫺/⫺ (B,E,H) lung. Hematoxylin-eosin stained sections show that whereas wild-type lungs have large expanded airspaces or saccules (arrows in A), the airspaces in the Ctgf⫺/⫺ lungs are just narrow and tortuous tubules (arrows in B). The mutant lungs are densely cellular with thicker septae (B), and their potential airspace is greatly diminished (C: *significantly different from wild-type). Lung cell proliferation, indicated by immunostaining for proliferating cell nuclear antigen (PCNA), is reduced in both the epithelial (arrows in D,E) and the mesenchymal (arrowheads) lung compartments of the Ctgf⫺/⫺ embryos (F: *different from corresponding compartment in wild-type). Conversely, staining for caspase-3, indicating apoptosis (bright green fluorescence; arrows for epithelium and arrowheads for mesenchyme in G,H), is increased in Ctgf⫺/⫺ lungs (I: *different from wild-type). Scale bar ⫽ 50 ␮m (A,B) and 20 ␮m (D,E,G,H). For all bar graphs, P ⬍ 0.05 from n ⫽ 4 wild-type and 3 Ctgf⫺/⫺. Fig. 2. Immunostaining for platelet-derived growth factor-B (PDGF-B) and its receptor PDGFR-␤ is reduced in Ctgf⫺/⫺ lungs. The percentage of cells staining positive for PDGF-B (bright red fluorescence; arrows for epithelium and arrowheads for mesenchyme in A,B) is lower in the mesenchyme of Ctgf⫺/⫺ lungs (C: *different from corresponding compartment in wild-type; P ⬍ 0.05 from n ⫽ 4 wild-type and 3 Ctgf⫺/⫺), as is the cytoplasmic staining for PDGFR-␤ (arrows and arrowheads in D,E) in both the epithelium and the mesenchyme (F). Note that because the mutant lung has a much higher total number of cells seen in the section, E appears more stained than D (wild-type). However, of those cells, the actual percent that are red (immunopositive) is higher in D for both the epithelium and the mesenchyme. Scale bar ⫽ 20 ␮m (A,B,D,E).

Fig. 2.


derived growth factor-B (PGDF-B) is known to stimulate lung cell proliferation (Liu et al., 1995, 1999) and to prevent apoptosis (Desai and Gruber, 1999). Its receptor, PDGF receptor beta (PDGFR-␤), is also involved in lung cell growth (Liu et al., 1995). Our present data show that PDGF-B immunostaining was strikingly reduced in the mesenchyme of Ctgf⫺/⫺ lungs (37.9 ⫾ 8 versus 62.5 ⫾ 5% in the wild-type), though fairly equivalent in the epithelia (Fig. 2 A–C). PDGFR-␤ staining, on the other hand, was markedly attenuated in both the epithelium and the mesenchyme of the mutant lungs (17.9 ⫾ 6 and 22.5 ⫾ 4%, respectively, vs. 36.4 ⫾ 4 and 42.2 ⫾ 6% in controls, Fig. 2D–F). Insulin-like growth factor-I (IGF-I), another proliferative and anti-apoptotic promoter of lung growth (Baxter, 1988; Barres et al., 1992), was examined next. We noted a reduction in the number of cells immunopositive for IGF-I in the epithelia of the Ctgf-deficient lungs compared to the controls (23.5 ⫾ 4 vs. 32.1 ⫾ 4%, respectively, Fig. 3A–C). IGF-I labeling in the mesenchyme, however, was similar in the two lung types. The final modulator of lung organogenesis and differentiation examined was thyroid transcription factor-1 (TTF-1) (Minoo et al., 1995; Kelly et al., 1996). Although the precise mechanism by which TTF-1 influences lung development is still unclear, it has been well established that in normal lung development, TTF-1 expression is suppressed in the epithelium of the proximal conductive ducts but persists in the epithelia of the lung periphery, as shown in Figure 3D, giving a proximal-to-distal increasing expression gradient (Zhou et al., 2001). In pulmonary hypoplasia, however, this gradient is lost, with the epithelial cells of the proximal conductive ducts continuing to express TTF-1 (Zhou et al., 2001, Inanlou and Kablar, 2003, 2005a,b). The TTF-1 gradient was found to be disturbed in Ctgf⫺/⫺ embryonic lungs, with almost all the columnar epithelial cells of the proximal conductive ducts still staining strongly for TTF-1 near term (Fig. 3E). Collectively, our results indicate that deletion of the Ctgf gene modifies both proliferation and apoptosis of

pulmonary cells and also changes the distribution and abundance of factors that regulate the two processes.

Maturation of Type II Pneumocytes Is Altered in Ctgf Deletion Mutants For the lung to perform its normal function, it requires a mature alveolar epithelium composed of two cell types: type I pneumocytes, responsible for gas exchange, and type II pneumocytes, responsible for surfactant production and also thought to be progenitors for the type I cells (Adamson and Bowden, 1975). Therefore, to investigate if functional maturity is also compromised in Ctgf-deficient lungs, we examined the development of both types of pneumocytes. Type II differentiation was studied by evaluating the immunoreactivity for surfactant protein-C (SP-C), a specific biochemical indicator of type II cell differentiation and with the ability to synthesize surfactant (Phelps and Floros, 1991). Our analysis found that SP-C levels were about the same in both wild-type and Ctgf⫺/⫺ lungs (Fig. 4A,B). An assessment of intracellular glycogen, the substrate for surfactant synthesis (Batenburg, 1992), was also conducted, using the periodic acidSchiff (PAS) reaction. This, however, revealed that the proportion of PASpositive epithelial cells in Ctgf-deficient lungs was quadruple that in control lungs (85.0 ⫾ 14 vs. 21.2 ⫾ 5%, respectively, Fig. 4C–E). Since the above biochemical findings indicated that, despite their evident ability to synthesize SP-C, the Ctgf⫺/⫺ pneumocytes may have some maturational defects curtailing their ability to utilize glycogen for this surfactant synthesis, we used transmission electron microscopy (TEM) to look at the morphology of these cells. The type II cells in the Ctgf-deficient lungs were found to have smaller nuclei and large cytoplasmic accumulations of glycogen (Table 1 and Fig. 5B). Their cytoplasmic lamellar bodies (surfactant storage bodies), although smaller (Table 1; Fig. 5B), appeared structurally similar to those in the type II cells of the wild-type lungs (Fig. 5A,B). The number of these lamellar bodies was, in addition, the same as that in the type II cells of the

wild-type controls (Table 1). Similarly, the surfactant secretory bodies released into the alveolar fluid (intraalveolar lamellar bodies, later transforming to myelin tubules before forming the surfactant monolayer on the alveolar surface), were not morphologically different from those in the control lungs (Fig. 5C,D), and neither were the myelin tubules (Fig. 5E,F). Put together, these observations (excessive retention of glycogen indicating limited surfactant synthesis; diminished lamellar body size indicating reduced surfactant storage) point to disturbances in the final stages of type II pneumocyte differentiation.

Type I Pneumocyte Differentiation Appears Similar in Wild-type and Ctgfⴚ/ⴚ Lungs Next, the development of type I alveolar cells was examined. Podoplanin, also known as aggrus, Gp38, and T1␣, is a specific type I pneumocyte marker (Ramirez et al., 2003; Williams, 2003; Mishima et al., 2006). We found that podoplanin immunohistochemistry in the Ctgf-deficient lungs was indistinguishable from that in the wild-type lungs (Fig. 6A,B). Subsequent TEM examination confirmed the presence of squamous epithelial cells with the typical extended cytoplasm and flattened nucleus of type I cells in both the wild-type and the mutant lungs (Fig. 6C,D). These findings indicate that the differentiation of type I pneumocytes is not affected by Ctgf deletion.

DISCUSSION The mechanochemical signal transduction pathways that translate mechanical stimuli to meaningful gene instructions for final pulmonary cell differentiation are still unclear. In a recent experiment, therefore, we used oligonucleotide microarrays to identify genes possibly involved in pneumocyte differentiation in amyogenic mouse embryos (Baguma-Nibasheka et al., 2007). Of the down-regulated genes identified in that experiment, we chose further investigation of Ctgf for several reasons. First, being a growth factor involved in regulating


Fig. 3. Alterations in the distribution patterns of insulin-like growth factor-I (IGF-I) and thyroid transcription factor-1 (TTF-1) in Ctgf⫺/⫺ fetal lungs. The number of cells immunopositive for IGF-I (bright green fluorescence; arrows for epithelium and arrowheads for mesenchyme in A,B) is attenuated in the epithelium of Ctgf⫺/⫺ lungs (C: *different from corresponding compartment in wild-type; P ⬍ 0.05 from n ⫽ 4 wild-type and 3 Ctgf⫺/⫺). Also, whereas TTF-1 staining is observed in the cuboidal epithelial cells of the distal conductive ducts (arrows in D,E) but not in columnar epithelial cells of the larger proximal ducts (asterisk) in wild-type lungs (D), TTF-1 immunoreactive cells are clearly evident in both the distal (arrows) and the proximal ducts (asterisk) in Ctgf⫺/⫺ lungs (E). Scale bar ⫽ 20 ␮m (A,B,D,E).

Fig. 4.

the cell cycle (Abdel-Wahab et al., 2002), it would have an important role in the development of all organs, including the lung. The occasional presence of CTGF in the nucleus (Wahab et al., 2001; Baguma-Nibasheka et al., 2007) suggests transcription factor activity and, therefore, possible effects on many developmental pathways. Secondly, CTGF is immunodetectable as early as day 13.5 in the murine embryonic lung (Surveyor and Brigstock, 1999), and the gene remains well expressed in the adult mouse lung (Su et al., 2002), indicating continuing developmental and physiologic necessity. Also, since it is involved in cytoskeletal organization (Crean et al., 2004), it could affect the ability of type II pneumocytes to give rise to the flattened type I cells. In addition, null mutants for the Ctgf gene have been shown to die within minutes of birth due to respiratory failure related to skeletal defects (Ivkovic et al., 2003). We, therefore, examined lung development in Ctgf⫺/⫺ mouse embryos to determine if, besides the skeletal defects, respiration could have been impaired by lung abnormalities present in the Ctgf-deficient newborns. The study revealed, in summary, that the Ctgf⫺/⫺ lungs were hypoplastic, with reduced cell proliferation and increased apoptosis. Immunostaining for PDGF-B, its receptor (PDGFR-␤) and IGF-I, were markedly attenuated, while the TTF-1 gradient was lost. Type II pneumocyte differentiation was perturbed, with the cells depicting excessive glycogen retention and diminished lamellar body and nuclear size, though able to synthesize surfactant-associated protein. However, type I pneumocyte differentiation was not affected by Ctgf deletion. The finding, in our present study, of

Fig. 4. Ctgf⫺/⫺ lung cells exhibit normal levels of surfactant protein but contain more glycogen. Epithelial cell immunostaining for surfactant-associated protein-C (SP-C; arrows in A,B) is similar in wild-type and mutant lungs. However, the proportion of epithelial cells positive for the periodic acid-Schiff (PAS) stain (arrows in C,D), indicating cytoplasmic glycogen, is greatly enhanced in the Ctgf-deficient lungs (E: *different from wild-type; P ⬍⬍ 0.05 from n ⫽ 4 wild-type and 3 Ctgf⫺/⫺). Scale bar ⫽ 20 ␮m (A–D).


TABLE 1. Characteristicsa of Type II Pneumocytes in Wild-type and Ctgfⴚ/ⴚ Embryos at E18.5

2

Nuclear size (␮ ) Lamellar body size (␮2) Lamellar bodies per cell

Wild-type

Ctgfⴚ/ⴚ

19.9 ⫾ 0.4 1.0 ⫾ 0.2 6.8 ⫾ 0.6

14.7 ⫾ 1.6* 0.5 ⫾ 0.1* 5.0 ⫾ 0.7

Mean ⫾ SD. * ⫽ significantly different from wild-type; p ⬍ 0.05 from n ⫽ 2 wild-type and 3 Ctgf⫺/⫺. a

Fig. 5. Type II pneumocytes in Ctgf⫺/⫺ embryos have large accumulations of glycogen but normal surfactant protein bodies. Electron micrographs of type II cells showing large glycogen vesicles (gly) flanking the nucleus (N) in the Ctgf⫺/⫺ lung cell (B) but not in the wild-type (A). The structure of intracellular lamellar bodies (arrows in A,B) is similar in the two types of embryos, though their size is smaller in the mutants (B). The released (intra-alveolar) lamellar bodies (arrows in C,D) also appear similar in wild-type (C) and Ctgf⫺/⫺ (D) lungs, as do the tubular myelins (arrows in E,F). Scale bar ⫽ 500 nm (A–D [original magnification, ⫻25,000] and E,F [⫻40,000]).

hypoplastic lungs seemingly arrested at the canalicular stage of development supports our hypothesis that, besides the skeletal defects, respiration in the Ctgf-deficient mouse could be impaired by lung abnormalities caused by the restriction of thoracic expansion and compounded by the limitation of FBMs. The hypoplasia was most probably due to the observed decrease in cell proliferation and increase in apoptosis in both the epithe-

lial and the mesenchymal pulmonary cells, since similar upsets in cell-cycle kinetics have been seen as a mechanism leading to pulmonary hypoplasia (Tseng et al., 2000; Inanlou and Kablar, 2003, 2005a,b). The cell cycle dyskinesia, in turn, could be largely explained by the observed alteration of cell cycle modulators known to be involved in lung development, particularly PDGF-B, PDGFR-␤, IGF-I, and TTF-1 (Baxter,

1988; Liu et al., 1995; Minoo et al., 1995). Disturbances in these same mediators have also been noted in our previous studies of pulmonary hypoplasia associated with perturbed lung distension (Inanlou and Kablar, 2003, 2005a,b), and mechanical stretch is known to increase PDGF-B, PDGFR-␤, and IGF-I expression (Liu et al., 1995, 1999; Joe et al., 1997; Liu and Post, 2000). One notable difference in the current study was that PDGF-B was down-regulated in the mesenchyme but not in the epithelia of the Ctgf⫺/⫺ lungs. However, PDGF-B is known to be a mesenchymal growth factor (Buch et al., 1991), and other researchers have shown that the mechanical stretch-induced fetal lung cell proliferation is determined mainly by the responsiveness of the mesenchymal cells, specifically fibroblasts (Xu et al., 1998). Thus, a lower mesenchymal PDGF-B level could be a sufficient contribution to the progression of lung hypoplasia. Moreover, the PDGF-B receptor, PDGFR-␤, was attenuated in both epithelium and mesenchyme, and therefore PDGF-B effects could, in turn, be attenuated in both compartments. The mechanism through which TTF-1 induces and maintains lung morphogenesis and differentiation of epithelial cells is not fully established, though TTF-1 might act by regulating the expression of the growth factor midkine (Reynolds et al., 2003). However, our current data on Ctgf⫺/⫺ embryonic lungs do reinforce the previously established fact that in pulmonary hypoplasia, the normal proximal-to-distal increasing TTF-1 expression gradient is disturbed, with the columnar epithelial cells of the proximal conductive ducts continuing to stain strongly for TTF-1 near term (Zhou et al., 2001; Inanlou and Kablar, 2003, 2005a,b). Regarding alveolar cytodifferentiation, morphometric analysis using TEM showed that the Ctgf-deficient mouse type II pneumocytes had smaller nuclei and large cytoplasmic glycogen vesicles. Their cytoplasmic lamellar bodies, the organelles in which pulmonary surfactant is stored prior to secretion, were half the size of those in the control lungs’ pneumocytes. This reduction in lamellar body size, coupled with the great reduction


Fig. 6. Normal differentiation of type I pneumocytes in Ctgf⫺/⫺ embryos. Immunolabeling for podoplanin, a specific type I pneumocyte marker, on the apical and lateral surfaces of the epithelial cells (arrows in A,B) is indistinguishable between wild-type and Ctgf⫺/⫺ lungs; and squamous cells with the typical extended cytoplasm and flattened nucleus (N) of type I cells are clearly discernible under the electron microscope in both lung types (C,D). Scale bar ⫽ 20 ␮m (A,B) and 2 ␮m (C,D [original magnification, ⫻12,000]).

of available alveolar space in the mutant mouse lungs, makes it very likely that the actual total amount of surfactant produced by the type II cells is also reduced. The high level of PAS staining, reflecting high levels of retained cytoplasmic glycogen, also promotes the probability of reduced surfactant production. The mutant type II pneumocytes may, however, be capable of implementing their functional maturity to some level, as shown by their ability to synthesize SP-C and to release structurally wellorganized and normal-appearing individual surfactant secretory bodies. Inspection of type II pneumocytes indicates that the degree of their differentiation in pulmonary hypoplasia depends to a large extent upon the causation of that hypoplasia. For instance, although earlier investigations had intimated that thoracic restriction does not inhibit the differentiation of type II cells or their production of surfactant (Hepworth and Seegmiller, 1989; Foster et al., 1994), mechanical lung distension has subsequently been shown to decrease the number of type II cells and the

amount of surfactant they synthesize (Joe et al., 1997), as well as the expression of SP-C, a specific marker for them (Gutierrez et al., 1999), possibly by promoting their differentiation into type I. Studies with the limitation or absence of FBMs, on the other hand, consistently show that final type II pneumocyte differentiation requires these movements (Nagai et al., 1988; Joe et al., 1997; Benachi et al., 1999; Tseng et al., 2000; Inanlou and Kablar, 2005a,b). Also, the simulation of FBMs by intermittent mechanical strain increases SP-C expression and, indeed, continuous mechanical contraction seems to favor the expression of the type II rather than the type I cell phenotype (Gutierrez et al., 2003). It is, therefore, possible that in Ctgf⫺/⫺ embryos, the restriction of thoracic expansion and the limitation of FBMs cancelled each other out as far as their effect on SP-C expression was concerned. As for type I pneumocytes, previous research has shown that FBMs are essential for these cells to complete their morphological and functional differentiation (Inanlou and Kablar,

2005b). Other investigators have also reported an inhibition of the expression of RTI40 (the rat analog of Gp38, [T1␣, podoplanin] and a marker for the type I cell phenotype), as well as a decrease in the air space surface area covered by type I cells relative to type II, in fetal oligohydramnios, which reduces the normal distension of the lungs (Kitterman et al., 2002). Our present results, however, indicate that, despite the limitation of saccular expansion in the Ctgf knockout lungs, Ctgf is not essential for type I pneumocyte differentiation, as shown by the normal podoplanin staining and by the TEM observation of cells with characteristic type I features. In conclusion, our findings indicate that the absence of Ctgf and/or its protein product, CTGF, may induce pulmonary hypoplasia both directly (by disrupting some basic molecular lung cell developmental process) and indirectly (by restricting thoracic expansion), and that CTGF could be directly involved in the transduction of mechanical stimuli into specific biomolecular instructions for growth and differentiation of the lungs.

EXPERIMENTAL PROCEDURES Animal Breeding and Fetal Collection Homozygous Ctgf-deficient (Ctgf⫺/⫺) fetuses were obtained by the interbreeding of heterozygous (Ctgf⫹/⫺) parents, as previously described (Ivkovic et al., 2003). All fetuses were collected by Cesarean section at embryonic day 18.5 and genotyped by RT-PCR as described in Ivkovic et al. (2003). Animal use and care was in accordance with all institutional guidelines.

Immunohistochemistry and Morphometry Immunohistochemistry was performed as previously described (Inanlou and Kablar, 2005a,b) on paraffin-embedded 4-␮m sections. Hematoxylin-eosin and PAS staining followed the protocols recommended in their Sigma kits. Immunostaining used the following primary antibodies: Dako mouse monoclonal against PCNA, 1 ␮g/ml; Chemicon rab-


bit polyclonal against caspase-3, 2 ␮g/ ml; Santa Cruz rabbit polyclonals against PDGF-B, PDGFR-␤ and TTF-1, 4 ␮g/ml and goat polyclonals against SP-C and podoplanin, 2 ␮g/ml; and R&D Systems goat polyclonal against IGF-I, 5 ␮g/ml. Secondary antibodies were from Zymed (amino ethyl carbazol [AEC] staining) and Molecular Probes (fluorescent staining). Hematoxylin nuclear counterstaining followed the AEC immunostains. Control staining to eliminate antibody non-specificity was performed by application of secondary antibodies without prior exposure of the cells to the primaries. In morphometry, the cells for each animal were counted in seven randomly selected fields lacking large airways and blood vessels and with at least 150 cells each.

Transmission Electron Microscopy (TEM) Samples of lung tissue for electron microscopic analysis were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3), post-fixed in 1% osmium tetroxide, and dehydrated in an ascending series of ethanol before embedding in LR White acrylic resin. The LKB ultramicrotome was used to make 100-nm-thick sections, which were then placed on 300-mesh copper grids and stained with 2% aqueous uranyl acetate and lead citrate. The sections were examined in a JEOL JEM 1230 electron microscope equipped with a Hamamatsu ORCA-HR digital camera. Morphometry was performed on cells in four randomly selected grid squares covering an area of 0.03 mm2 for each embryo.

Statistical Analysis The samples used for morphometric analyses were from four wild-type and three Ctgf⫺/⫺ embryos for paraffin sections, and two wild-type and three Ctgf⫺/⫺ for TEM. The resulting counts were compared using the Student’s t-test, with differences of P ⬍ 0.05 considered significant. Data are presented as mean ⫾ standard deviation (SD).

ACKNOWLEDGMENTS Our grateful appreciation goes to Drs. Karen M. Lyons and Rommelda A. DeYoung at the Department of Molec-

ular, Cell and Developmental Biology, University of California, Los Angeles, for kindly providing the lung tissues, and for critical reading of the manuscript (K.M.L.). Dr. Mohammad R. Inanlou provided expert technical assistance. This work was funded by operating grants from the National Science and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Lung Association of Nova Scotia, the Canada Foundation for Innovation, and the Dalhousie Medical Research Foundation to B.K.

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