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Am J Physiol Lung Cell Mol Physiol 294: L665-L675, 2008. First published February 8, 2008; doi:10.1152/ajplung.00027.2008
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Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia

Robin D. Clugston, Wei Zhang, and John J. Greer

Department of Physiology, University of Alberta, Edmonton, Alberta, Canada

Submitted 15 January 2008 ; accepted in final form 3 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Congenital diaphragmatic hernia (CDH) is a frequently occurring birth defect and a source of potentially fatal neonatal respiratory distress. Recently, through the application of detailed karyotyping methods, several CDH-critical regions within the human genome have been identified. These regions typically contain several genes. Here we focused on genes from 15q26, the best-characterized CDH-critical region, as well as FOG2 and GATA4, genes singled out from CDH-critical regions at 8q22–8q23 and 8p23.1, respectively. We tested the hypothesis that these putative CDH-related genes are expressed within the developing diaphragm at the time of the hypothesized initial defect. Our results show that 15q26 contains a cluster of genes that are expressed in the developing rodent diaphragm, consistent with an association between deletions in this region and CDH. We then examined the protein expression pattern of positively identified genes within the developing diaphragm. Two major themes emerged. First, those factors strongly associated with CDH are expressed only in the nonmuscular, mesenchymal component of the diaphragm, supporting the hypothesis that CDH has its origins in a mesenchymal defect. Second, these factors are all coexpressed in the same cells. This suggests that cases of CDH with unique genetic etiology may lead to a common defect in these cells and supports the hypothesis that these factors may be members of a common pathway. This study is the first to provide a detailed examination of how genes associated with CDH are expressed in the developing diaphragm and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.

congenital diaphragmatic hernia-critical genes; pleuroperitoneal fold; mesenchyme

CONGENITAL DIAPHRAGMATIC HERNIA (CDH) is a severe birth defect that causes life-threatening respiratory distress in the neonatal period. It occurs in ~1 of every 2,500 pregnancies, with an overall survival rate of 50–70%, although single specialized centers have reported significantly reduced mortality rates (11, 27, 28, 41, 57, 64). In cases of Bochdalek CDH, which is the most frequent presentation of CDH, incomplete formation of the posterolateral diaphragm allows invasion of the abdominal contents into the thoracic cavity, impeding lung development (32, 65). The pathogenesis of CDH is poorly understood; however, recent research using different animal models of the disorder is beginning to provide insight into its embryonic origins (13, 22, 23). Equally, progress is also being made in understanding the etiology of CDH. In addition to the recently proposed retinoid hypothesis (30), the genetic origins of CDH are also being carefully examined, largely spurred on by the characterization of mutant mice with abnormal diaphragm phenotypes and genetic screening in infants diagnosed with CDH (1, 48, 59).

In the latter case, there has been considerable attention paid toward so-called CDH-critical regions, parts of chromosomes containing several genes where recurring structural abnormalities have been found in multiple cases of CDH (33, 43). These findings suggest that one or more genes in specific regions are essential for normal diaphragm development. The first and best-characterized critical region to be identified is located at 15q26; a deletion of this part of chromosome 15 has been shown to account for ~1.5% of CDH cases and is associated with very high mortality (15, 39, 53). The spectrum of anomalies produced by 15q26 deletion is phenotypically most similar to Fryns syndrome (38, 58). Several other regions of the human genome have also been identified as putative CDH-critical regions, including 1q41–1q42, 4p16.3, 8p23.1, and 8q22–8q23 (33, 60). All of these regions are in the order of several megabases and contain numerous genes; however, how these genes might contribute to diaphragm development and their significance with regard to CDH is unknown. In fact, the need for studies focusing on the molecular origins of how genetic disruptions result in diaphragm defects has recently been highlighted (33). Therefore, the aim of this study was to systematically test the hypothesis that genes identified from CDH-critical regions are expressed in developing rodent diaphragm at the time of the initial defect.

We focused our expression studies on two important stages of rat diaphragm embryogenesis: at embryonic day (E) 13.5, when the pleuroperitoneal folds (PPFs) are developing, and at E16.5, when diaphragm formation is essentially complete (22). Expression patterns within the PPF are of interest, since this is a key structure in the developing diaphragm, particularly in the context of CDH. Muscle precursor cells (MPC) that form the complete musculature of the diaphragm first migrate to the PPF, and it is also the target for pioneer axons of the phrenic nerve that provides the diaphragm with its nervous input (10, 29). With regard to CDH, abnormal PPF development has been shown to underlie diaphragmatic hernia in nitrofen-treated rats, vitamin A-deficient rats, and Wt1-null mutant mice (6, 23). Furthermore, analyses of human CDH postmortem tissue have generated data consistent with primary PPF defects (23). As such, genes found to be expressed in this structure are assumed to be important for normal diaphragm development and, hypothetically, could lead to CDH when abnormally expressed or regulated.

In this study we focused on the rodent orthologues of 10 genes located within the region of chromosome 15q26, whose deletion is associated with CDH (Table 1). This list includes chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which is regarded (33) as the most important gene from 15q26 in diaphragm development. We also studied two other specific genes, friend of GATA 2 (FOG2) and GATA-binding protein 4 (GATA4), identified as the most significant genes associated with CDH in the critical regions 8q22–8q23 and 8p23.1, respectively (see Table 1 and Ref. 33). Because of the large number of genes identified from 15q26, in the first part of this study we performed a screen of these candidate genes for their expression in the PPF. Laser capture microdissection was used to precisely isolate PPF tissue, and RT-PCR was performed on extracted mRNA. The protein expression pattern of positively identified genes was then determined using immunohistochemistry. We also determined the protein expression pattern of Fog2 and Gata4 in the PPF. Furthermore, because of the unusual diaphragm phenotype seen in Fog2 and Gata4 mutant mice, we also studied the expression of these factors in the diaphragm at E16.5 to better understand these phenotypes (see DISCUSSION and Refs. 2 and 35). Our protein expression analyses in all cases addressed two questions. First, are these genes expressed in MPC or mesenchymal cells, and second, are they coexpressed in the same cells? In our coexpression studies we also included WT1 into our analysis; this gene is expressed in the PPF and is known to be essential for normal diaphragm development (23, 40).


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  		Table 1. Genes of interest from CDH-critical regions

 
The etiology of CDH is diverse and complex. This study is the first to provide an in-depth examination of how genes associated with CDH are expressed in the developing diaphragm and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Timed pregnant Sprague-Dawley Rats were used for all experiments, in accordance with guidelines established by, and with approval from, the Animal Welfare Committee at the University of Alberta. Noon of the day on which a sperm plug was observed in the breeding cage was determined as E0.5.

Caesarian section and tissue processing. Caesarian section was performed to remove the fetal tissue on the day required. Animals were anesthetized with halothane (~3% delivered in 95% 02 and 5% CO2) prior to surgery. The gestational age of isolated tissue was confirmed by the measurement of fetal crown rump length (7). Fetuses collected for immunohistochemistry were first decapitated and had their hindquarters removed, and the remaining trunk tissue was then fixed by immersion in 4% paraformaldehyde for ≥24 h at 4°C. A dissecting microscope (Leica Wild M3C) was used to isolate whole diaphragms from fixed E16.5 tissue and then rinsed in phosphate-buffered saline (PBS). Isolated E16.5 diaphragms and E13.5 embryos were dehydrated in a graded series of alcohol and embedded in paraffin wax. Transverse sections were cut at 10 µm on a rotary microtome (Leica RM2135) and mounted on presubbed glass slides prior to immunohistochemistry. For RNA isolation, E13.5 embryos were either snap-frozen in liquid nitrogen (for whole embryo RNA) or frozen in OCT (Tissue-Tek) prior to cryostat sectioning (for PPF RNA). E16.5 diaphragms were isolated immediately after cesarean section, rinsed in PBS, and snap-frozen in liquid nitrogen.

PPF laser capture microdissection. Embedded embryos were sectioned at 10 µm using a cryostat (Leica CM1900), mounted on uncoated RNase-free slides, and then stored at –80°C. Prior to laser capture the frozen sections were thawed at room temperature for 30 s and then immersed in 75% ethanol for 30 s. After this fixation the slides were washed for 30 s in diethyl pyrocarbonate-treated water and stained in Mayer's hematoxylin for 1 min. The slides were then dehydrated in graded ethanol concentrations and cleared with xylene. The AutoPix automated laser capture system (Arcturus Bioscience, Mountain View, CA) was used for capturing PPF tissue. Briefly, activation of the laser beam was focused on a thermoplastic film of CapSure HS laser capture microdissection cap, leading to melting of the film bond to selected tissue. As illustrated in Fig. 1, we were able to specifically target the PPF and isolate it without collecting tissue from adjacent structures. After dissection, the captured cells and assembly cap with ExtracSure extraction device were stored at –80°C.


Figure 1
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  		Fig. 1. Isolation of the plueroperitoneal fold (PPF) by laser capture microdissection. A: representative image of a transverse section through an embryonic day (E) 13.5 rat embryo at the lower cervical level, showing the forelimbs (fl), neural tube (nt), and heart (h). B: boxed region from A enlarged to show the distinctive triangular PPFs (*) and the adjacent lung buds. C: high-magnification image showing a single PPF prior to laser capture. D: same region as C seen after removal of the PPF. E: several isolated PPFs following laser capture. Scale bars: A = 500 µm; B = 400 µm; C–E are not to scale.

 
RNA isolation. Total RNA was extracted from whole embryos (n = 6) and diaphragms (n = 10) using TRIzol (Invitrogen Canada, Burlington, ON, Canada) according to the manufacturer's protocol. Total RNA was isolated from collected PPF fragments using the PicoPure RNA isolation kit (Arcturus Bioscience), and due to the small amount of RNA harvested from each PPF, the RNA extracted from ~10 PPFs was pooled for RT-PCR analysis. For RNA extraction, 10 µl of extraction buffer was added to the CapSure-ExtracSure assembly, covered in a 0.5-ml microcentrifuge tube on top of the assembly, and incubated for 30 min at 42°C. After incubation, the tube was centrifuged to collect the cell extract. Before RNA isolation, the purification column was washed with condition buffer at room temperature prior to the mixture of cell extract and the addition of an equal volume of 70% ethanol. After centrifugation and disposal of the flow-through, RNA was bonded onto the surface membrane of the purification column. The column was washed with buffer and centrifuged to remove salt and other contaminants. The column was then transferred to a new 0.5-ml microcentrifuge tube, and pure RNA was eluted from the membrane of the purification column using elution buffer and centrifugation. The concentration of RNA in all cases was measured using a spectrophotometer (NanoDrop ND-1000) and diluted to the same concentration for RT-PCR (50 ng/µl).

RT-PCR. RT-PCR was performed using a One-Step RT-PCR kit (Qiagen Canada, Mississauga, ON, Canada). Primer sequences, annealing temperatures, and amplicon sizes are presented in Table 2. Reactions were performed in 25-µl volumes, with the final reaction mixture containing 5 µl of 5x RT-PCR buffer, 5 µl of "Q" solution, 1 µl of dNTP, 1.5 µl of primer solution (containing forward and reverse primers, final concentration 0.6 µM), 10.5 µl of water, 1 µl of enzyme mix, and 1 µl of template RNA. The RT-PCR protocol used was reverse transcription at 50°C for 30 min and PCR activation at 95°C for 15 min followed by 30–35 cycles of 94°C for 30-s denaturation, 30-s annealing at the temperature indicated in Table 2, and 30 s at 72°C extension. A final extension step of 72°C for 10 min was also carried out. Reaction products were run on a 1.2% agarose gel containing ethidium bromide and bands visualized using a 312-nm transilluminator (FBTI 88; Fisher Scientific, Pittsburgh, PA). At the beginning of the experimental series, bands for each gene of interest were extracted using a gel extraction kit (Qiagen Canada) and their identities confirmed by DNA sequencing. For each reaction series, β-actin was included as a positive control, and H2O was used instead of RNA as a negative control to assay for contaminating RNA. For each gene of interest, reactions were carried out in triplicate and repeated at least two times.


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  		Table 2. Primer sequences, annealing temperature, and amplicon size for CDH-associated genes found in region 15q26

 
Immunohistochemistry. Protein expression patterns were determined using antibodies raised against Coup-tfII (monoclonal mouse anti-human, 1:250 dilution; PPMX Perseus Proteomics, Tokyo, Japan), IGF-I receptor (Igf1r; polyclonal rabbit anti-human, sc-713, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), repulsive guidance molecule A (Rgma; polyclonal goat anti-human, sc-46481, 1:50 dilution; Santa Cruz Biotechnology), Fog2 (polyclonal rabbit anti-mouse, sc-10755, 1:50–100 dilution; Santa Cruz Biotechnology), Gata4 (polyclonal goat anti-mouse, sc-1237, 1:250 dilution; Santa Cruz Biotechnology), and Wt1 (monoclonal mouse anti-human, M3561, 1:50 dilution; Dako Diagnostics Canada, Mississauga, ON, Canada). Anti-Pax3 (goat polyclonal anti-human, sc-7748, 1:100 dilution; Santa Cruz Biotechnology) and anti-MyoD (monoclonal mouse anti-human, M3512, 1:50 dilution; Dako Diagnostics Canada) antibodies were also used to visualize muscle precursor cells. Experiments were repeated in triplicate, including negative controls, to confirm the pattern of staining. The pattern of expression observed was consistent between embryos, and representative examples were chosen for publication. A series of immunohistochemistry experiments were carried out, the basic protocol for which is described below.

Paraffin-embedded embryos were cut at 10 µm with a rotary microtome (Leica RM2135), and serial sections were mounted on presubbed glass slides. Sections were dewaxed in xylene then rehydrated using a graded series of alcohol. Sections were rinsed in PBS and then microwaved in 0.01 M sodium citrate buffer, pH 6, at 600 W for 5 min and then pretreated with 1% hydrogen peroxide in 100% methanol for 30 min. Sections were treated with 1% bovine serum albumin (BSA) in 0.4% Triton X-100-PBS for 30 min prior to incubation with the appropriate primary antibody. All primary antibodies were diluted in PBS with 0.1% BSA and 0.4% Triton X-100; the antibodies were left to incubate overnight at room temperature. After incubation with primary antibodies the sections were washed with PBS and then incubated with a mixture of fluorophore-conjugated secondary antibodies diluted in PBS and 0.1% BSA for 2 h. Three different fluorophore-conjugated secondary antibodies were used depending upon the primary antibodies used: Cy3-conjugated donkey anti-mouse (1:200 dilution; Jackson ImmunoResearch, West Groove, PA), Cy5-conjugated donkey anti-goat (1:200 dilution; Jackson ImmunoResearch), and a combination of a biotinylated donkey anti-mouse antibody (Jackson ImmunoResearch) followed by a separate incubation with streptavidin 488 (1:200 dilution; Molecular Probes, Eugene, OR). After incubation with the secondary antibody, sections were washed in PBS and coverslipped with Fluorsave mounting medium (Calbiochem, San Diego, CA). In cases where double labeling was being carried out and the two primary antibodies were from the same species of origin, we used a tyramide signal amplification kit (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's protocol.

Confocal microscopy. Immunostained sections were scanned with a Zeiss LSM510 laser-scanning microscope (Oberkochen, Germany) configured to a computer running LSM510 software. For Cy3 fluorescence, excitation (HeNe, 1 mV) was set to 543 nm, and emissions were collected using a 560-nm long-pass filter. For Cy5 fluorescence, excitation (HeNe, 1 mV) was set to 633 nm, and emissions were collected using a 630-nm long-pass filter. For Alexa fluor 488 fluorescence, excitation (Argon, 40 mV) was set to 488 nm, and emissions were collected with a 505-nm long-pass filter. Acquired images were exported in bitmap format and prepared for publication in Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
RT-PCR analysis of 15q26 CDH-critical genes in the developing diaphragm. A systematic RT-PCR analysis of the genes from the CDH-critical region found at 15q26 was performed. Total RNA extracted from the whole embryo, PPF, and whole diaphragm were assayed for each gene of interest (Table 3). Consistent amplification between replicates and experiments was considered to reflect positive expression in the target tissue. The inability to produce a detectable PCR product was considered to be an absence of expression. We were able to amplify mRNA for all of the genes studied from whole embryo extracts at E13.5. The following genes were found to be expressed in the PPF at E13.5: ST8 {alpha}-N-acetyl-neuraminide-{alpha}-2,8-sialyltransferase 2 (St8sia2), Rgma, Coup-tfII, arrestin domain containing 4 (Arrdc4), Igf1r, and leucine rich repeat containing 28 (Lrrc28). We were unable to amplify transcripts for chromodomain helicase DNA-binding protein 2 (Chd2), multiple C2 domains, transmembrane 2 (Mctp2), desmuslin (Dmn), and tetratricopeptide repeat domain 23 (Ttc23), which suggests that they are not expressed in this tissue. Similarly, St8sia2, Rgma, Coup-tfII, Arrdc4, Igf1r, and Lrrc28 were found to be expressed in the whole diaphragm at E16.5; additionally Chd2 was also found to be expressed at this age. Similar to our results for PPF RNA, we could not amplify Mctp2, Dmn, and Ttc23 transcripts from the whole diaphragm.


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  		Table 3. Results of RT-PCR analysis of CDH-associated genes

 
Immunohistochemical expression of Coup-tfII, Igf1r, and Rgma within the PPF. Immunohistochemical staining for Coup-tfII, Igf1r, and Rgma was performed to visualize the expression pattern of these proteins within the developing PPF of the rat at E13.5 (Fig. 2). There were no commercial antibodies available for St8sia2, Arrdc4, and Lrrc28; therefore, we could not examine the expression of these proteins. The expression pattern of Coup-tfII within the PPF has been partially described previously (23) and is extended upon here. In agreement with previous data, Coup-tfII expression is found in the nuclei of cells throughout the PPF and in the mesenchyme of the adjacent lung (Fig. 2A). Igf1r is expressed in the cytoplasm of every cell within the PPF as well as in the adjacent lung mesenchyme and bronchi (Fig. 2B). The expression of Rgma was more restricted in the PPF (Fig. 2C); punctate staining for this protein was seen throughout the PPF, although a smaller proportion of cells was labeled compared with Coup-tfII and Igf1r. There was also obvious Rgma staining in the lung bronchi. The transcription factor Pax3 is a marker of muscle precursor cells within the PPF (10). Double-labeling experiments with Pax3 were performed to determine whether Coup-tfII, Igf1r, and Rgma were expressed in the mesenchyme of the PPF or in its muscular component. We found no colocalization between Coup-tfII positive nuclei and Pax3-positive nuclei, indicating that Coup-tfII is expressed only in the mesenchyme of the PPF (Fig. 2D). In contrast, Igf1r expression was found in the cytoplasm of Pax3-positive muscle precursors and mesenchyme cells throughout the PPF (Fig. 2E). Rgma was not expressed in association with Pax3-positive muscle precursors, suggesting that this protein is associated only with the mesenchymal cells of the PPF (Fig. 2F). A second series of double-labeling experiments was performed to determine whether Coup-tfII, Igf1r, and Rgma were coexpressed within the same cells. Consistent with the widespread expression of Igf1r in the PPF, we found that it was coexpressed in Coup-tfII (Fig. 2G) and Rgma (Fig. 2H) immunopositive cells. With regard to the coexpression of Rgma and Coup-tfII, cells that expressed both proteins were observed; however, singly labeled cells for both proteins were also observed (Fig. 2I). Alhough one of the functions ascribed to Rgma is in axon guidance, we found no association between Rgma staining and the phrenic nerve within the PPF (visualized by immunostaining for neurofilament; data not shown).


Figure 2
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  		Fig. 2. Chicken ovalbumin upstream promoter-transcription factor II (Coup-tfII), IGF-I receptor (Igf1r), and repulsive guidance molecule A (Rgma) expression in the PPF. A: single channel confocal image showing Coup-tfII-immunopositive cells in the PPF. B: Igf1r immunostaining in the PPF. C: Rgma immunostaining in the PPF. D: high-magnification images of double labeling for Pax3 (red) and Coup-tfII (green). E: Pax3 (red) and Igf1r (green) expression. F: Pax3 (red) and Rgma (green) expression. G: double labeling for Igf1r (red) and Coup-tfII (green). H: double labeling for Igf1r (red) and Rgma (green). I: double labeling for Rgma (red) and Coup-tfII (green). Scale bars: A, B, and C = 75 µm; D–I = 25 µm.

 
Immunohistochemical expression of Fog2 and Gata4 in the PPF. We performed a systematic immunohistochemical evaluation of Fog2 and Gata4 expression in the PPF at E13.5, finding that both proteins are expressed in the nuclei of cells throughout this structure (Fig. 3, A and B, respectively). The basic pattern of Fog2 expression observed closely resembled that described previously in mice (3). To determine whether either Fog2 or Gata4 was expressed in the pool of diaphragmatic MPCs that are localized within the PPF, double-labeling experiments with Pax3 were carried out. Neither Fog2 (Fig. 3C) nor Gata4 (Fig. 3D) was found to colocalize with Pax3-expressing cells within the PPF, and thus the expression of these genes is restricted to the mesenchymal component of this structure. However, as could be expected from their partnership in gene regulation, double labeling for Fog2 and Gata4 revealed almost complete colocalization within the PPF (see Fig. 3E and Ref. 18). Expression of both of these proteins within the lung was observed at E13.5; however, a detailed description of their expression in this tissue has been described elsewhere (3, 35).


Figure 3
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  		Fig. 3. Friend of GATA 2 (Fog2) and GATA-binding protein 4 (Gata4) expression in the PPF. A: Fog2-immunopositive cells in the PPF. B: Gata4 immunopositive cells in the PPF. C: Fog2 (red) and Pax3 (green) expression does not colocalize in the PPF. D: Gata4 (red) and Pax3 (green) expression does not colocalize in the PPF. E: Fog2 (red) and Gata4 (green) immunostaining colocalizes in the PPF (yellow staining). Scale bars: A and B = 100 µm; C–E = 25 µm.

 
Expression of Fog2 and Gata4 within the whole diaphragm. In addition to their expression pattern in the PPF, Fog2 and Gata4 expression was also studied in the whole diaphragm at E16.5 (Fig. 4). Whole diaphragm immunohistochemistry performed at this stage was used to determine the expression of these factors in the muscularized part of the diaphragm and the central tendon. Both Fog2 and Gata4 were found to be expressed in the muscularized regions of the diaphragm (Fig. 4, C and B, respectively); however, they had a different cellular localization. Gata4 staining was localized to nuclei, but Fog2 expression appeared to be cytoplasmic, representing an unexpected shift from the nuclear expression observed in the PPF. The cytoplasmic expression of Fog2 is paradoxical given its widely recognized function as a cofactor for Gata4 controlling gene transcription in the nucleus. We found only one reference to cytoplasmic Fog2 expression in the literature, which Bielinska et al. (14) described as having "unclear significance." By focusing on the boundary between the central tendon and the muscularized region of the diaphragm, we observed that, although Gata4 is expressed throughout the entire diaphragm (Fig. 4D), Fog2 expression was restricted to the muscularized part of the diaphragm and not the central tendon (Fig. 4E). This result is in contrast to a previous report (35), which noted that Fog2 expression overlapped with Gata4 in the central tendon of fetal mice. Double-labeling studies in diaphragm cross-sections demonstrated that Gata4 and MyoD are not coexpressed in the same cells, and thus Gata4 expression is restricted to the mesenchymal substrate of the diaphragm (Fig. 4F). No overlapping expression between Fog2 and MyoD was observed (Fig. 4G); however, when viewed at higher power (Fig. 4, I–K) it became obvious that Fog2 was expressed in the cytoplasm of MyoD-positive muscle precursors and MyoD-negative mesenchymal cells of the diaphragm. Double labeling for Fog2 and Gata4 revealed no overlapping expression of these transcription factors; careful study of high magnification images failed to find any obvious Fog2 staining in the cytoplasm of Gata4-positive cells (not shown).


Figure 4
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  		Fig. 4. Fog2 and Gata4 expression in the diaphragm. A: schematic diagram of the diaphragm (plan view). Box 1 indicates the region represented in B and C, detailing the muscularized part of the diaphragm; box 2 indicates the region represented in D and E, detailing the boundary between the diaphragm muscle and the central tendon; line 3 indicates the plane of section through the diaphragm used in F–K. B: Gata4-immunopositive cells within the muscularized part of the diaphragm. C: Fog2 immunopositive cells within the muscularized part of the diaphragm. D: Gata4-immunopositive cells are found in the central tendon and the muscularized part of the diaphragm. E: Fog2 immunostaining is restricted to the muscularized part of the diaphragm and is not seen in the central tendon. F: Gata4 (red) and MyoD (green) immunostaining does not colocalize. G: double labeling for Fog2 (red) and MyoD (green) shows no colocalized staining. H: double labeling for Gata4 (green) and Fog2 (red) does not colocalize. I: high-magnification image of Fog2 expression showing cytoplasmic labeling. J: high-magnification image of nuclear MyoD expression. K: merged image of Fog2 (red) and MyoD (green) expression; Fog2 expression is found in MyoD-negative (arrowhead) and MyoD-positive cells (arrow). Scale bars: B and C = 50 µm; D and E = 200 µm; F–H = 20 µm; I–K = 10 µm.

 
Relative expression of proteins implicated in CDH in the PPF. We have described in this article the expression pattern of several genes that have been implicated in the development of CDH and how they relate to each other and the myogenic cells of the PPF. Here we present further data describing the relative expression of Coup-tfII, Fog2, Gata4, and Wt1 to each other. Through our double-labeling experiments we found that Coup-tfII strongly colocalizes with Gata4 and Fog2 within the PPF (Fig. 5, A and B, respectively). Furthermore, we found that Wt1 also strongly colocalizes with Gata4 and Fog2 within this structure (Fig. 5, C and D, respectively). In addition to the data presented here, it has previously been reported that Coup-tfII and Wt1 also colocalize within the PPF (23). The extent of colocalization throughout the PPF in all cases is such that there is an almost complete overlap in expression of all these factors.


Figure 5
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  		Fig. 5. Relative expression of Coup-tfII, Fog2, Gata4, and Wt1 in the PPF. Each section shows a column of 3 confocal microscope images obtained from the same section. Top: green channel; middle: red channel; bottom: merged image (yellow staining represents colocalization). A: Coup-tfII (green) and Gata4 (red). B: Coup-tfII (green) and Fog2 (red). C: Wt1 (green) and Gata4 (red). D: Wt1 (green) and Fog2 (red). Scale bar: A–D = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
15q26 Contains a cluster of genes expressed in the developing diaphragm. In the first part of this study we tested the hypothesis that genes from region 15q26 are expressed in the developing diaphragm. Several different groups have reported subtly different boundaries of the minimally deleted region at 15q26 associated with CDH, and there is currently no consensus on its exact boundary (19, 39, 55). Therefore, for the purpose of this study we considered the largest deleted area so as not to exclude any genes unnecessarily. Our RT-PCR analysis identified six genes from 15q26 that were expressed in the PPF and seven genes in the more developed diaphragm. Thus we can conclude that region 15q26 contains a cluster of genes that are expressed in the developing diaphragm. This finding may help to explain why chromosomal deletions encompassing this region frequently result in CDH.

In the next step we determined the expression pattern of positively identified genes from 15q26 in the PPF. This was critical for determining the type of PPF cell in which the proteins were expressed and whether there was a common pattern among the gene products. Commercial antibodies were unavailable for three of the genes (ST8SIA2, ARRDC4, and LRRC28), and they were excluded from further analysis in this study; however, future studies examining the expression of these genes by in situ hybridization are planned. The function of these excluded genes and the proteins they encode are not fully understood. Therefore, it is difficult to speculate on their role in diaphragm development, although they certainly require further investigation. ARRDC4 may be of particular interest; by including data from one infant with a small 15q26 deletion, a minimally deleted region that contained only this gene was defined. Furthermore, Slavotinek et al. (60) also identified an infant with CDH who had a unique nucleotide substitution in ARRDC4, although they were unable to conclude that this was causal. ST8SIA2 is also an interesting candidate; this gene encodes a polysialyltransferase enzyme that is thought to play a role in the polysialylation of the neural cell adhesion molecule (21). This is of interest because polysialylated neural cell adhesion molecule is thought to play a role in the guidance of the phrenic nerve to the PPF and has been shown to be differentially expressed during myotube separation in the diaphragm, although the significance of this with regard to the development of CDH is unclear, because the pathogenesis of Bochdalek CDH does not appear to be related to diaphragm myogenesis and innervation (4, 5, 9). Below, we discuss the expression pattern of Igf1r, Rgma, and Coup-tfII in the PPF and the significance of this with regard to CDH.

The full name for Igf1r is insulin-like growth factor I receptor (Igf1r); this gene encodes a membrane-bound receptor, the primary ligand for which is Igf1. Signaling by Igf1 through Igf1r mediates many of the effects of growth hormone during development and postnatal life (36). Igf1r is widely expressed in the embryo throughout gestation, including, as our results show, the PPF (17). With respect to CDH, mice with a targeted deletion of Igf1r do not have diaphragmatic hernia; however, the majority of these animals do die at birth from respiratory failure. The explanation for this respiratory failure was not determined, and no lung abnormality could be found. The diaphragm in these mice was hypoplastic, although this is set in the context of widespread muscular dystrophy and is not specific to the diaphragm (42, 49). Igf1r is clearly important in the differentiation of skeletal muscle, including the diaphragm; however, the phenotype of null mutant mice suggests that its deletion is not directly related to the diaphragmatic hernia observed in infants with 15q26 deletion. Rather, deletion of IGF1R is thought to contribute to the intrauterine growth retardation that is a common clinical feature associated with this deletion (38, 53).

Rgma is a membrane-bound signaling molecule that binds to the neogenin receptor, mediating a chemorepulsive axon guidance response; it is also thought to have further roles in neuronal differentiation and survival (24, 44, 45). Recently, the family of repulsive guidance molecule proteins has also been identified as coreceptors in the bone morphogenetic signaling pathway (31). Sequencing of critical region genes in infants with CDH revealed two instances of unique sequence variations in RGMA, although these were not conclusively proven to be causal in the formation of diaphragmatic hernia (60). However, Rgma knockout mice die at birth due to early failure of cephalic neural tube closure, leading to exencephaly. There were no reports of diaphragm abnormalities, and 50% of mutants are viable (46). Rgma is not essential for diaphragm formation in mice; however, it is expressed in the rodent PPF, and its association with CDH in humans justifies further clarification of its role in diaphragm development.

Coup-tfII was the third gene whose expression pattern we studied in the PPF. Coup-tfII encodes a nuclear transcription factor with a critical role in mouse development. Coup-tfII-null mutant mice die at midgestation, exhibiting defects in angiogenesis and heart development (47). Specific ablation of Coup-tfII in tissues expressing Nkx3–2 induces diaphragmatic hernia phenotypically identical to Bochdalek CDH (68). However, unlike some of the other genes from this region, no specific CDH-causing mutation in the coding region of this gene has been identified (60). Its chromosomal location in the region of 15q26 associated with CDH and the phenotype of mice lacking Coup-tfII provide strong evidence that this gene is essential for diaphragm development.

Fog2 and Gata4 expression in the developing diaphragm. In the second part of our study, we shifted our focus from the genes of 15q26 to the expression of two other genes, FOG2 and GATA4, both of which have been linked with CDH in humans and animal models. It is important to point out here that FOG2 is a transcriptional coregulator of GATA4 transcriptional activity such that FOG2 can enhance and/or inhibit GATA4 activity, depending on the cellular context (18). FOG2 is considered a strong candidate responsible for the development of Bochdalek hernia in infants harboring microdeletions encompassing 8q22–8q23, and this hypothesis is supported by the recent identification of two infants with unique sequence variations in FOG2 who had isolated Bochdalek CDH (16, 33, 43). GATA4 has also been singled out as an important gene in the context of infants harboring microdeletions in the region of 8p23.1 (33, 43). If we interpret our PPF expression studies in the context of Bochdalek CDH in humans, then our data support a role for these genes in the basic development of the diaphragm and the etiology of CDH.

In addition to their expression pattern in the PPF and their association with Bochdalek CDH, we were also interested in looking at rodent FOG2 and GATA4 protein expression later in diaphragm development because of an association between mutations in these genes and other types of diaphragm defects. With regard to FOG2, there has been a case report of an infant with a specific mutation in the FOG2 gene who had diaphragm eventration. This infant harbored a de novo mutation in exon 4 of FOG2, leading to the production of a truncated protein that contains none of the zinc finger DNA-binding domains essential for normal protein function (2, 61). Mutant mice carrying a point mutation in Fog2 produce a similar truncated protein, and, significantly, they also have abnormal muscularization of the diaphragm. Thus, not only is deletion and mutation of FOG2 in humans associated with Bochdalek CDH, but mutation of the gene leading to the expression of a truncated peptide is associated with diaphragm eventration. This finding can be rationalized when the expression pattern of Fog2 described in this article is considered. In rodents, Fog2 expression in the PPF is exclusive to its nonmuscular component, which is consistent with a role in early diaphragm development and the hypothesis that a malformation of the PPF precedes Bochdalek CDH. However, the expression of Fog2 in the fully formed diaphragm is intimately linked to the developing musculature, the disruption of which leads to diaphragm eventration. This hypothesis suggests that Fog2 may have two roles in diaphragm development, an early role in helping to establish the basic structure of the diaphragm and a later role linked to its muscularization. Therefore, genetic redundancy or residual function of the truncated Fog2 protein could be enough to support early diaphragm development, with the mutation only manifesting itself during diaphragm muscularization. The fact that these mice do not have as severe a phenotype as Fog2-null mutant mice suggests that some function of this protein is preserved, or compensated for, which may be able to support early diaphragm development (2, 62, 63). Although Fog2 certainly has an important role in diaphragm development, the generation of conditional Fog2 mutant mice will be helpful in establishing the exact role of this gene in diaphragm formation.

Similarly, in addition to GATA4's association with Bochdalek CDH in humans, a low percentage (~30%) of mice heterozygous for a deletion in this gene (Gata4+/–) has recently been reported to have diaphragm abnormalities that are phenotypically consistent with the rare central tendon defect seen in humans; however, the literature contains no link between babies with central tendon defects and GATA4 mutations (35). The appearance of an abnormal phenotype in these mice with only one copy of Gata4 is consistent with the observation (67) that there is a threshold of Gata4 activity required for normal control of gene expression, below which abnormal development can occur. Other than establishing that Gata4 is expressed in the central tendon, our results cannot explain the occurrence of central tendon defects in these mice. However, review of the literature raises one possibility. Although they do not survive long enough to study the diaphragm, it is known that Gata4–/– mice lack an epicardium. Interestingly, it is this structure that forms part of the pericardium that adheres to the central tendon of the diaphragm. Thus the central tendon defects seen in Gata4+/– mice could result from a congenital weakness of the central tendon associated with abnormal epicardial development rather than a primary defect in diaphragm formation (66). This possibility suggests that development of central tendon defects in Gata4+/– mice have distinct embryonic origins from the posterior defects seen in humans with 8p23.1 microdeletions. A thorough examination of the central tendon defects of Gata4+/– mice beyond that of the original description is required to address this issue (35). What is clear is that the development of central tendon defects in Gata4+/– mice is independent from Fog2; not only have we shown that Fog2 is not expressed in the central tendon, but mice heterozygous for a mutant copy of Gata4 that cannot physically interact with Fog2 and can therefore only function independently from it were born at the expected ratio and were morphologically normal (25). Despite Fog2 and Gata4's known partnership in gene regulation, the differing phenotype of mice mutant for these genes, coupled with our expression data, suggests that they may function independently in the later stages of diaphragm development.

Mesenchymal expression of genes associated with CDH in the PPF. It has been hypothesized that the diaphragm abnormality in Bochdalek CDH arises from a defect in the nonmuscular, mesenchymal cells of the PPF and is independent of myogenesis (10, 22, 23). Therefore, we were interested in determining whether the genes associated with CDH described above were mesenchymally expressed. Insightfully, our data from this and a previous study (23) indicate that neither Coup-tfII, Fog2, Gata4, nor Wt1 expression colocalize with pax3-positive muscle precursor cells within the PPF and are therefore expressed only in the nonmuscular, mesenchymal component of the PPF. Given the association between these factors and CDH, coupled with their exclusive expression in the mesenchyme of the PPF, we interpret the current data as supporting the hypothesis that Bochdalek CDH arises from a defect in the mesenchyme of the PPF and is independent of myogenesis. In this regard, future studies into the embryological origins of Bochdalek diaphragmatic hernia in our laboratory are focused on the nonmuscular, mesenchymal cells of this structure.

Genes associated with CDH are coexpressed in the PPF. With regard to understanding how aberrant expression of certain key genes contributes to the development of an abnormal diaphragm, we were also interested in examining how the expression of Coup-tfII, Fog2, Gata4, and Wt1 related to each other. Wt1 was included in this analysis because it is already known to be expressed in the PPF. Wt1-null mutant mice have Bochdalek CDH, and there are several syndromes that include CDH within their spectrum of abnormalities that are caused by a WT1 mutation (23, 26, 40, 50, 51, 54). Significantly, we found that all of these factors are coexpressed within the same mesenchymal cells of the PPF. This is interesting with regard to the etiology of CDH because it suggests that different genetic mutations affect the same cells within the PPF, representing a point of convergence for distinct genetic insults; i.e., the same cells within the developing diaphragm are being affected in individuals with distinct genetic etiologies. With regard to the clinical management of CDH, it is also important to observe that several of these genes are also expressed in the developing lung; thus their mutation may affect not only diaphragm development but also that of the lung.

Interestingly, Coup-tfII, Fog2, Gata4, and Wt1 all regulate gene transcription. In this regard, it is possible to hypothesize that they are members of the same gene regulatory network. In support of this hypothesis, it has previously been shown that Fog2 can act as a corepressor for Coup-tfII and that Fog2, Gata4, and Wt1 act together to regulate the expression of some genes (34, 52). If these factors really are members of a common or overlapping gene regulatory network in the developing diaphragm, it will be important to identify what the downstream genes are and how their dysregulation might contribute to the development of diaphragm abnormalities. Furthermore, it will be intriguing to explore any potential links with the retinoid signaling pathway, perturbation of which has been hypothesized to cause CDH (30). In this regard, it is known that retinoids are involved in controlling the expression of Gata4, Wt1, and Coup-tfII (8, 12). There is also a precedent for a model that combines these two hypotheses. The expression of phosphoenolpyruvate carboxykinase is sensitive to retinoids, and its promoter contains composite binding sites for various transcription factors, most notably Coup-tfII and the retinoid receptors (56). Furthermore, retinoids can regulate the expression of cardiogenic genes by directly interacting with Fog2 and Gata4 via the retinoid x receptor-{alpha} (20). The hypothesis that distinct genetic insults might contribute to the development of CDH by disrupting different components of a common pathway is attractive because it provides a unified model to understand the complex etiology of CDH and may allow therapeutic targets to be identified (37).

Summary. It is implied from genetic studies in humans and mice that the loss of function of critical genes can lead to CDH. Our analysis of the candidate genes from the critical region at 15q26 revealed that this region contains a cluster of genes that are expressed in the developing rodent diaphragm. Although there is compelling evidence to suggest that Coup-tfII is essential for normal diaphragm development, our data also indicate that there are other genes in this region that may contribute to the formation of this structure. The significance of our study into the protein expression pattern of specific candidate genes is twofold. First, we have shown that Coup-tfII, Fog2, Gata4, and Wt1 are all expressed in the nonmuscular mesenchyme of the PPF, therefore supporting the hypothesis that it is abnormalities in the mesenchymal cells of the developing diaphragm that lead to the formation of Bochdalek CDH. Second, we have demonstrated that these genes are all coexpressed within the same population of cells, supporting the hypothesis that occurrences of CDH with distinct genetic etiologies arise from abnormalities in the same cell population and possibly the same pathway within these cells. To conclude, the genetic origins of CDH are diverse and complex. However, our analysis has revealed unifying patterns of expression that point to a common pathogenic mechanism for the development of diaphragmatic hernia.


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This study was supported by the Canadian Institutes of Health Research and the March of Dimes. J. J. Greer and R. D. Clugston received Scientist and Studentship Awards, respectively, from the Alberta Foundation for Medical Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Greer, Univ. of Alberta, Dept. of Physiology, 513 HMRC, Edmonton, AB, Canada T6G 2S2 (e-mail: john.greer{at}ualberta.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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