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Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofen-induced congenital diaphragmatic hernia
Journal of Pediatric Surgery xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofen-induced congenital diaphragmatic hernia Toshiaki Takahashi a, Julia Zimmer a, Florian Friedmacher a, Prem Puri a,b,⁎ a b
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 6 August 2016 Accepted 12 September 2016 Available online xxxx Key words: Paired-related homeobox 1 Transcription factor 4 Diaphragm Muscle connective tissue Congenital diaphragmatic hernia Nitrofen
a b s t r a c t Background/purpose: Pleuroperitoneal folds (PPFs) are the source of the primordial diaphragm's muscle connective tissue (MCT), and developmental mutations have been shown to result in congenital diaphragmatic hernia (CDH). The protein paired-related homeobox 1 (Prx1) labels migrating PPF cells and stimulates expression of transcription factor 4 (Tcf4), a novel MCT marker that controls morphogenesis of the fetal diaphragm. We hypothesized that diaphragmatic Prx1 and Tcf4 expression is decreased in the nitrofen-induced CDH model. Methods: Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Prx1 and Tcf4 were analyzed by qRT-PCR. Immunofluorescence double staining for Prx1 and Tcf4 was performed to evaluate protein expression and localization. Results: Relative mRNA expression of Prx1 and Tcf4 was significantly downregulated in PPFs (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy revealed markedly diminished Prx1 and Tcf4 expression in diaphragmatic MCT of nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. Conclusions: Decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, leading to development of CDH in the nitrofen model. Level of evidence: Level 2c (Centre for Evidence-Based Medicine, Oxford). © 2016 Published by Elsevier Inc.
Congenital diaphragmatic hernia (CDH) is a relatively common malformation with international incidence rates currently ranging between 1.93 and 2.3 cases per 10,000 births [1,2]. Severe pulmonary hypoplasia and persistent pulmonary hypertension are considered to be the main reasons for the life-threatening respiratory distress in newborn infants with diaphragmatic defects [3,4]. Despite substantial advances in postnatal resuscitation and modern lung-protective strategies, CDH remains one of the major therapeutic challenges in modern neonatal intensive care, causing high mortality and long-term morbidity for survivors [5–7]. The origin of diaphragmatic defects is considered to lie in the non-muscular parts of the fetal diaphragm. In fact, a proliferative abnormality of the mesenchymal-derived pleuroperitoneal folds (PPFs) has recently been reported in rats with CDH [8,9]. Mesenchymal elements in the developing diaphragm have been shown to mainly comprise of muscle connective tissue (MCT) [9]. Furthermore, there is strong evidence that developmental mutations that inhibit the formation of normal diaphragmatic MCT can result in CDH [10,11]. It has also been suggested that decreased expression of important regulatory proteins for mesenchymal cell proliferation during diaphragmatic morphogenesis leads to ⁎ Corresponding author at: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. Tel.: +353 1 409 6420; fax: +353 1 455 0201. E-mail address: [email protected] (P. Puri).
defective PPFs and eventually diaphragmatic defects [12]. Although the pathogenesis of CDH has been extensively studied, the exact molecular basis of abnormal MCT formation is not clearly understood. The protein paired-related homeobox 1 (Prx1), which labels migrating PPF cells and stimulates the expression of transcription factor 4 (Tcf4), a novel MCT marker that controls growth of the fetal diaphragm, has recently been identified to play a key role during diaphragmatic development [13]. It has further been demonstrated that PPFs are the only source of the primordial diaphragm's MCT and therefore significantly contribute to its myogenesis [13–15]. Mutations in PPF-derived cells have been shown to cause CDH [16,17]. The aim of this study was to investigate the hypothesis that the expression of Prx1 and Tcf4 is decreased in the developing diaphragm in the nitrofen-induced CDH. 1. Material and methods 1.1. Animals, drugs and experimental design Pathogen-free adult Sprague–Dawley rats® (Harlan Laboratories, Shardlow, UK) were mated overnight and the presence of spermatozoids in the vaginal smear of females was considered as embryonic day 0.5 (D0.5). Pregnant animals were then randomly divided into two experimental groups: “nitrofen” and “control”. On D9, dams were
http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007 0022-3468/© 2016 Published by Elsevier Inc.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
briefly anesthetized with 2% volatile isoflurane (Piramal Healthcare Ltd., Morpeth, UK) and either 100 mg of nitrofen (WAKO Chemicals GmbH, Neuss, Germany), dissolved in 1 ml of olive oil, or vehicle alone was administered with a gastric tube. Fetuses were delivered via caesarean section under anesthesia on selected time-points D13 (PPFs), D15 (developing diaphragms) and D18 (fully muscularized diaphragms), and sacrificed by decapitation. After laparotomy, D18 fetuses were inspected under a Leica S8AP0 stereomicroscope (Leica Microsystems AG, Heerbrugg, Switzerland) for diaphragmatic defects. Fetal diaphragms from nitrofen-exposed animals with CDH and controls were dissected under sterile conditions via thoracotomy and stored in a TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) at − 20 °C. Additionally, whole D13 and D15 fetuses as well as D18 trunks were fixed in 10% paraformaldehyde (PFA) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight. In total, 72 fetal diaphragms were used for this study (n = 12 per time-point and experimental group, respectively). All animal procedures were carried out according to the current guidelines for management and welfare of laboratory animals. The experimental protocol was approved by the local research ethics committee (REC668b) and the Department of Health and Children (Ref. B100/ 4378) under the Cruelty to Animals Act, 1876 (as amended by European Communities Regulations 2002 and 2005).
1.2. Total RNA isolation Following fixation in 10% PFA, paraffin-embedded D13 and D15 fetuses were transversely sectioned at a thickness of 10 μm and mounted on PEN membrane glass slides® (MDS Analytical Technologies, Sunnyvale, CA, USA) in order to obtain total RNA from PPFs and developing diaphragms. All appropriate sections were deparaffinized with xylene, rehydrated through ethanol and distilled water, stained with hematoxylin and dehydrated. D13 PPFs and developing D15 diaphragms were dissected from 9 consecutive sections per fetus by laser capture microdissection (Arcturus XT® Instrument, MDS Analytical Technologies, Sunnyvale, CA, USA) and total RNA was extracted using a High Pure FFPE RNA Micro Kit® (Roche Diagnostics, West Sussex, UK) according to the manufacturer's protocol. After thawing and homogenization of the fully muscularized D18 diaphragms, total RNA was extracted from the TRIzol® suspension using the acid guanidinium thiocyanatephenol-chloroform extraction method. Spectrophotometrical quantification of total RNA was performed with a NanoDrop ND-1000 UV– Vis® Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, NC, USA).
1.3. Complementary DNA synthesis and quantitative real-time polymerase chain reaction Reverse transcription of total RNA was carried out at 85 °C for 3 min (denaturation), at 44 °C for 60 min (annealing), and at 92 °C for 10 min (reverse transcriptase inactivation) using a Transcript High Fidelity cDNA Synthesis Kit® (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer's protocol. The resulting cDNA was used for quantitative real-time polymerase chain reaction using a LightCycler® 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Genespecific primer pairs are listed in Table 1. Following an initialization phase at 95 °C for 5 min, 55 amplification cycles were carried out. Each cycle included an initial denaturation step at 95 °C for 10 s, an annealing step at 60 °C for 15 s and an elongation step at 72 °C for 10 s. The final elongate temperature was 65 °C for 1 min. Relative mRNA expression levels of Prx1 and Tcf4 were measured with a Light Cycler® 480 instrument (Roche Diagnostics, West Sussex, UK) and gene levels were normalized to the housekeeping gene β-actin. All experiments were run duplicated for each sample and primer pair.
Table 1 Gene-specific primer sequences for quantitative real-time polymerase chain reaction. Gene Prx1 Forward Reverse Tcf4 Forward Reverse β-actin Forward Reverse
Sequence (5′-3′)
Product size (bp)
CCA CAT GTG CCA ACA ATA GC GGC ACC TGG TTC CTC TGT AA
106
CGA ATC ACA TGG GTC AGA TG AAA CGG GGT TAA GGA GCA GT
124
TTG CTG ACA GGA TGC AGA AG TAG AGC CAC CAA TCC ACA CA
108
1.4. Immunofluorescence double staining and confocal laser scanning microscopy After fixation in 10% PFA, whole D13 and D15 fetuses as well as D18 trunks were paraffin-embedded, transversely sectioned at a thickness of 5 μm, and mounted on polylysine-coated slides (VWR International, Leuven, Belgium). Resulting tissue sections were deparaffinized with xylene and rehydrated through ethanol and distilled water. Conventional hematoxylin and eosin staining (Sigma Aldrich, Saint Louis, MO, USA) was used to investigate the diaphragmatic histology. All sections for immunofluorescence staining were incubated with phosphatebuffered saline (PBS) containing 1.0% Triton X-100 (Sigma Aldrich Ltd., Arklow, Ireland) for 20 min to improve cell permeabilization. Sections were then washed in PBS + 0.05% Tween (Sigma-Aldrich, St. Louis, MO, USA) and subsequently blocked with 3% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min to avoid non-specific absorption of immunoglobulin. The blocking solution was rinsed off and sections were incubated with affinity-purified primary antibodies either against Prx1 (rabbit polyclonal, sc-21,948-R; 1:100), Tcf4 (goat polyclonal, sc-8631, 1:100) and GATA4 (mouse polyclonal, sc-25,310, 1:100) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight at 4 °C. On the next day, sections washed in PBS + 0.05% Tween and incubated with corresponding secondary antibodies (donkey anti-rabbit Alexa 647-A150067, 1:250, donkey anti-goat Alexa 555-A21432, 1:250 and donkey anti-mouse Alexa 488-A150109, 1:250) (Abcam plc, Cambridge, UK) for 1 h at room temperature. Following another washing step in PBS + 0.05% Tween, sections were counterstained with a DAPI antibody (10,236,276,001, 1:1000) (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min, washed again, and mounted with glass coverslips using Sigma Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA). All sections were scanned with a ZEISS LSM 700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and independently evaluated by two investigators. 1.5. Statistical analysis All numerical data is presented as means ± standard error of the mean. Differences between the two experimental groups were tested using an unpaired Student's t test when the data had normal distribution or a Mann–Whitney U test when the data deviated from normal distribution. A P value b0.05 was considered as statistically significant. 2. Results 2.1. Relative mRNA expression of Prx1 and Tcf4 in rat PPFs and fetal diaphragms The relative mRNA expression levels of Prx1 and Tcf4 were significantly downregulated in PPFs of nitrofen-exposed fetuses on D13, developing diaphragms of nitrofen-exposed fetuses on D15 and fully
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx Table 2 Relative mRNA expression levels of Prx1 and Tcf4 in developing rat diaphragms. Prx1
D13 D15 D18
Tcf4
Control
Nitrofen
Control
Nitrofen
2.32 ± 1.59 2.01 ± 1.44 1.68 ± 0.66
0.87 ± 0.56⁎ 0.87 ± 0.49⁎ 0.97 ± 0.61⁎
3.68 ± 1.61 3.66 ± 1.31 3.21 ± 1.88
2.12 ± 0.78⁎ 2.15 ± 0.69⁎ 1.31 ± 0.65⁎
⁎ P b 0.05 vs. control.
muscularized diaphragms of nitrofen-exposed fetuses with CDH on D18 compared to controls (Table 2).
2.2. Histology and immunofluorescence evaluation of Prx1, Tcf4 and GATA4 expression in rat PPFs and fetal diaphragms PPFs in the control group were triangular-shaped structures protruding out from the lateral body wall, whereas nitrofen-exposed fetuses had an abnormal PPF structure, characterized by an absence of the dorsally projecting point of the triangular PPF. Immunofluorescence staining for Prx1 and Tcf4 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization of Prx1 and Tcf4 in PPFs and fetal diaphragmatic tissue on D13, D15 and D18. Confocal laser scanning microscopy revealed co-expression of Prx1 and Tcf4 mainly in the diaphragmatic mesenchymal components, and further confirmed the molecular genetic results by showing a markedly diminished Prx1 and Tcf4 immunofluorescence in MCT of nitrofen-exposed PPFs, developing diaphragms and muscularized diaphragms compared to controls (Fig. 1). These findings were associated with a reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal diaphragms with CDH on D13, D15 and D18 compared to controls.
3. Discussion Much of our current understanding of pathophysiology of CDH originates from experimental studies [18]. Nitrofen (2,4-dichloro-phenyl-pnitrophenyl ether) is an herbicide that has been used for many years to create a teratogenic model of CDH. The advantage of this model is that the diaphragmatic defect is induced at the stage when the foregut has just separated into esophagus and trachea and therefore, providing opportunity to carefully study the developmental anatomy of the diaphragm and lungs in CDH [19]. Administration of nitrofen to pregnant rodents between days 8 and 11 after conception results in a high rate of CDH and associated pulmonary hypoplasia and pulmonary vascular abnormalities in the fetuses that is strikingly similar to the human malformation [20]. A fully functional diaphragm requires the coordinated morphogenesis of muscle, MCT and tendon, which have been shown to derive from different embryonic sources [9]. However, because of the large number of complex spatiotemporal processes involving multiple cellular and tissue interactions, normal embryological development of the diaphragm remains incompletely understood. It has been suggested that the origin of the defect in CDH lies in the amuscular mesenchymal components, which mainly comprise of fibrous connective tissue [8,9]. In addition, there is strong evidence that growth of the primordial diaphragm depends on the correct formation of its underlying MCT [10]. Developmental mutations, which inhibit normal expression of extracellular matrix, have recently been demonstrated to cause diaphragmatic defects [11]. Previous findings from our laboratory have indicated that several proteins, which are involved in the development of mesenchymal tissue in the fetal diaphragm, are decreased in the nitrofen-induced CDH model [12,21,22].
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In recent years, the embryogenesis of the PPFs has become a focus for elucidating the pathogenesis of diaphragmatic defects [23]. The PPFs play a pivotal role during diaphragmatic morphogenesis, being the target structures for migrating muscle precursor cells (MPCs) [24]. These MPCs have been reported to form the source for muscular elements in PPFs, which subsequently expand to form a primordial diaphragm [23]. Furthermore, it has been shown that the PPFs are defective in rodents with nitrofen-induced CDH [25]. A recent study has demonstrated by using an experimental CDH model that the primary mechanism of malformed PPFs is caused by a decreased proliferation of mesenchymal cells [26]. However, the exact mechanisms that lead to abnormal PPF development in CDH are largely unclear [9]. Using mouse genetics, Merrell et al. [13] have recently confirmed that the PPFs are the only source of the diaphragm's MCT, thus significantly contributing to diaphragmatic muscle formation. They have also revealed that Prx1 has an essential function during growth of the fetal diaphragm by stimulating the expression of Tcf4, which is a novel marker for MCT cells that critically controls diaphragmatic morphogenesis [13]. Prx1 has been known to maintain the elastic properties of extracellular matrices, which in turn is crucial for differentiation of MPCs [27]. Tcf4 is an important regulator of myogenesis and maturation that is strongly expressed in connective tissue fibroblasts [28,29]. Moreover, it has been shown that MCT expresses the product of the CDHassociated gene Gata4 [16,17,30–32]. Mosaic mutations of Gata4 in PPF-derived MCT fibroblasts have been found to result in the development of localized amuscular regions in the fetal diaphragm, subsequently causing CDH [13]. Taken together, the above findings suggest that PPFs and MCT are fundamental for normal diaphragmatic development. The present study provides strong evidence that the diaphragmatic gene expression of Prx1 and Tcf4 is significantly downregulated in PPFs on D13, developing diaphragms on D15 and fully muscularized diaphragms on D18 in the nitrofen-induced CDH model compared to control littermates. We further demonstrated a co-localized expression of Prx1 and Tcf4 in PPFs on D13, developing diaphragms on D15 and fully muscularized diaphragms on D18, suggesting that these cells have developed from PPF-derived MCT cells. Additionally, immunofluorescence double staining for Prx1 and Tcf4 showed a co-localization with GATA4, which is a central transcription factor during diaphragmatic development and strongly expressed by mesenchymal cells in developing fetal diaphragms [17,30]. Confocal laser scanning microscopy also showed a markedly diminished Prx1 and Tcf4 expression in the diaphragmatic mesenchyme of nitrofen-exposed PPFs and fetuses with CDH on D13, D15 and D18 compared to controls. Thus, these findings confirmed that the quantitative decrease of Prx1 and Tcf4 mRNA transcripts in fetal diaphragms was as well translated to the protein level. Recently, it has been reported that the diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that a decreased GATA4 expression may impair the diaphragmatic development in nitrofen-induced CDH [33]. Besides the markedly diminished Prx1 and Tcf4 expression in nitrofen-exposed PPFs and fetal diaphragms with CDH, we identified a reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18, which indicates a potential disruption in the formation of MCT. In conclusion, our results suggest that decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, and thus contribute to the development of diaphragmatic defects in the nitrofen CDH model. Future studies investigating morphogenetic processes regulating diaphragm's muscle connective tissue morphogenesis may provide new insights into the embryological origins of the diaphragmatic defect in CDH. Conflict of interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Fig. 1. Hematoxylin and eosin (H&E) staining (left image of each time-point and experimental group) as well as specific immunofluorescence staining for Prx1 (yellow), Tcf4 (green) and GATA4 (red) combined with DAPI (blue) in rat PPFs and fetal diaphragms on D13 (a), D15 (b) and D18 (c).
References [1] McGivern MR, Best KE, Rankin J, et al. Epidemiology of congenital diaphragmatic hernia in Europe: a register-based study. Arch Dis Child Fetal Neonatal Ed 2015; 100:F137–44. [2] Balayla J, Abenhaim HA. Incidence, predictors and outcomes of congenital diaphragmatic hernia: a population-based study of 32 million births in the United States. J Matern Fetal Neonatal Med 2014;27:1438–44. [3] Tovar JA. Congenital diaphragmatic hernia. Orphanet J Rare Dis 2012;7:1. [4] Keijzer R, Puri P. Congenital diaphragmatic hernia. Semin Pediatr Surg 2010;19: 180–5.
[5] McHoney M. Congenital diaphragmatic hernia, management in the newborn. Pediatr Surg Int 2015;31:1005–13. [6] Jeanty C, Kunisaki SM, MacKenzie TC. Novel non-surgical prenatal approaches to treating congenital diaphragmatic hernia. Semin Fetal Neonatal Med 2014;19: 349–56. [7] Losty PD. Congenital diaphragmatic hernia: where and what is the evidence? Semin Pediatr Surg 2014;23:278–82. [8] Greer JJ. Current concepts on the pathogenesis and etiology of congenital diaphragmatic hernia. Respir Physiol Neurobiol 2013;189:232–40. [9] Merrell AJ, Kardon G. Development of the diaphragm – a skeletal muscle essential for mammalian respiration. FEBS J 2013;280:4026–35.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx [10] Maki JM, Sormunen R, Lippo S, et al. Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues. Am J Pathol 2005;167:927–36. [11] Hornstra IK, Birge S, Starcher B, et al. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 2003;278:14387–93. [12] Takahashi T, Friedmacher F, Takahashi H, et al. Kif7 expression is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 2015;50:904–7. [13] Merrell AJ, Ellis BJ, Fox ZD, et al. Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias. Nat Genet 2015;47:496–504. [14] Logan M, Martin JF, Nagy A, et al. Expression of Cre recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 2002;33:77–80. [15] Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999;21:70–1. [16] Longoni M, Lage K, Russell MK, et al. Congenital diaphragmatic hernia interval on chromosome 8p23.1 characterized by genetics and protein interaction networks. Am J Med Genet A 2012;158A:3148–58. [17] Clugston RD, Zhang W, Greer JJ. Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 2008;294:L665–75. [18] Eastwood MP, Russo FM, Toelen J, et al. Medical interventions to reverse pulmonary hypoplasia in the animal model of congenital diaphragmatic hernia: a systematic review. Pediatr Pulmonol 2015;50:820–38. [19] Noble BR, Babiuk RP, Clugston RD, et al. Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 2007;293:L1079–87. [20] Montedonico S, Nakazawa N, Puri P. Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 2008;24:755–61. [21] Takahashi T, Friedmacher F, Zimmer J, et al. Mesenchymal expression of the FRAS1/ FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 2016;32:135–40.
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[22] Takahashi T, Friedmacher F, Takahashi H, et al. Disruption of copper-dependent signaling pathway in the nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 2015;31:31–5. [23] Babiuk RP, Zhang W, Clugston R, et al. Embryological origins and development of the rat diaphragm. J Comp Neurol 2003;455:477–87. [24] Clugston RD, Greer JJ. Diaphragm development and congenital diaphragmatic hernia. Semin Pediatr Surg 2007;16:94–100. [25] Mayer S, Metzger R, Kluth D. The embryology of the diaphragm. Semin Pediatr Surg 2011;20:161–9. [26] Clugston RD, Zhang W, Greer JJ. Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res A Clin Mol Teratol 2010;88:15–24. [27] Ihida-Stansbury K, Ames J, Chokshi M, et al. Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs. Pulm Circ 2015;5:382–97. [28] Mathew SJ, Hansen JM, Merrell AJ, et al. Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 2011;138:371–84. [29] Kardon G, Harfe BD, Tabin CJ. A Tcf4-positive mesodermal population provides a prepattern for vertebrate limb muscle patterning. Dev Cell 2003;5: 937–44. [30] Jay PY, Bielinska M, Erlich JM, et al. Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects. Dev Biol 2007;301:602–14. [31] Arrington CB, Bleyl SB, Matsunami N, et al. A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia. Am J Med Genet A 2012;158A:3137–47. [32] Yu L, Wynn J, Cheung YH, et al. Variants in GATA4 are a rare cause of familial and sporadic congenital diaphragmatic hernia. Hum Genet 2013;132: 285–92. [33] Dingemann J, Doi T, Gosemann JH, et al. Decreased expression of GATA4 in the diaphragm of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res B Dev Reprod Toxicol 2013;98:139–43.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofen-induced congenital diaphragmatic hernia Toshiaki Takahashi a, Julia Zimmer a, Florian Friedmacher a, Prem Puri a,b,⁎ a b
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 6 August 2016 Accepted 12 September 2016 Available online xxxx Key words: Paired-related homeobox 1 Transcription factor 4 Diaphragm Muscle connective tissue Congenital diaphragmatic hernia Nitrofen
a b s t r a c t Background/purpose: Pleuroperitoneal folds (PPFs) are the source of the primordial diaphragm's muscle connective tissue (MCT), and developmental mutations have been shown to result in congenital diaphragmatic hernia (CDH). The protein paired-related homeobox 1 (Prx1) labels migrating PPF cells and stimulates expression of transcription factor 4 (Tcf4), a novel MCT marker that controls morphogenesis of the fetal diaphragm. We hypothesized that diaphragmatic Prx1 and Tcf4 expression is decreased in the nitrofen-induced CDH model. Methods: Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Prx1 and Tcf4 were analyzed by qRT-PCR. Immunofluorescence double staining for Prx1 and Tcf4 was performed to evaluate protein expression and localization. Results: Relative mRNA expression of Prx1 and Tcf4 was significantly downregulated in PPFs (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy revealed markedly diminished Prx1 and Tcf4 expression in diaphragmatic MCT of nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. Conclusions: Decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, leading to development of CDH in the nitrofen model. Level of evidence: Level 2c (Centre for Evidence-Based Medicine, Oxford). © 2016 Published by Elsevier Inc.
Congenital diaphragmatic hernia (CDH) is a relatively common malformation with international incidence rates currently ranging between 1.93 and 2.3 cases per 10,000 births [1,2]. Severe pulmonary hypoplasia and persistent pulmonary hypertension are considered to be the main reasons for the life-threatening respiratory distress in newborn infants with diaphragmatic defects [3,4]. Despite substantial advances in postnatal resuscitation and modern lung-protective strategies, CDH remains one of the major therapeutic challenges in modern neonatal intensive care, causing high mortality and long-term morbidity for survivors [5–7]. The origin of diaphragmatic defects is considered to lie in the non-muscular parts of the fetal diaphragm. In fact, a proliferative abnormality of the mesenchymal-derived pleuroperitoneal folds (PPFs) has recently been reported in rats with CDH [8,9]. Mesenchymal elements in the developing diaphragm have been shown to mainly comprise of muscle connective tissue (MCT) [9]. Furthermore, there is strong evidence that developmental mutations that inhibit the formation of normal diaphragmatic MCT can result in CDH [10,11]. It has also been suggested that decreased expression of important regulatory proteins for mesenchymal cell proliferation during diaphragmatic morphogenesis leads to ⁎ Corresponding author at: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. Tel.: +353 1 409 6420; fax: +353 1 455 0201. E-mail address: [email protected] (P. Puri).
defective PPFs and eventually diaphragmatic defects [12]. Although the pathogenesis of CDH has been extensively studied, the exact molecular basis of abnormal MCT formation is not clearly understood. The protein paired-related homeobox 1 (Prx1), which labels migrating PPF cells and stimulates the expression of transcription factor 4 (Tcf4), a novel MCT marker that controls growth of the fetal diaphragm, has recently been identified to play a key role during diaphragmatic development [13]. It has further been demonstrated that PPFs are the only source of the primordial diaphragm's MCT and therefore significantly contribute to its myogenesis [13–15]. Mutations in PPF-derived cells have been shown to cause CDH [16,17]. The aim of this study was to investigate the hypothesis that the expression of Prx1 and Tcf4 is decreased in the developing diaphragm in the nitrofen-induced CDH. 1. Material and methods 1.1. Animals, drugs and experimental design Pathogen-free adult Sprague–Dawley rats® (Harlan Laboratories, Shardlow, UK) were mated overnight and the presence of spermatozoids in the vaginal smear of females was considered as embryonic day 0.5 (D0.5). Pregnant animals were then randomly divided into two experimental groups: “nitrofen” and “control”. On D9, dams were
http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007 0022-3468/© 2016 Published by Elsevier Inc.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
briefly anesthetized with 2% volatile isoflurane (Piramal Healthcare Ltd., Morpeth, UK) and either 100 mg of nitrofen (WAKO Chemicals GmbH, Neuss, Germany), dissolved in 1 ml of olive oil, or vehicle alone was administered with a gastric tube. Fetuses were delivered via caesarean section under anesthesia on selected time-points D13 (PPFs), D15 (developing diaphragms) and D18 (fully muscularized diaphragms), and sacrificed by decapitation. After laparotomy, D18 fetuses were inspected under a Leica S8AP0 stereomicroscope (Leica Microsystems AG, Heerbrugg, Switzerland) for diaphragmatic defects. Fetal diaphragms from nitrofen-exposed animals with CDH and controls were dissected under sterile conditions via thoracotomy and stored in a TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) at − 20 °C. Additionally, whole D13 and D15 fetuses as well as D18 trunks were fixed in 10% paraformaldehyde (PFA) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight. In total, 72 fetal diaphragms were used for this study (n = 12 per time-point and experimental group, respectively). All animal procedures were carried out according to the current guidelines for management and welfare of laboratory animals. The experimental protocol was approved by the local research ethics committee (REC668b) and the Department of Health and Children (Ref. B100/ 4378) under the Cruelty to Animals Act, 1876 (as amended by European Communities Regulations 2002 and 2005).
1.2. Total RNA isolation Following fixation in 10% PFA, paraffin-embedded D13 and D15 fetuses were transversely sectioned at a thickness of 10 μm and mounted on PEN membrane glass slides® (MDS Analytical Technologies, Sunnyvale, CA, USA) in order to obtain total RNA from PPFs and developing diaphragms. All appropriate sections were deparaffinized with xylene, rehydrated through ethanol and distilled water, stained with hematoxylin and dehydrated. D13 PPFs and developing D15 diaphragms were dissected from 9 consecutive sections per fetus by laser capture microdissection (Arcturus XT® Instrument, MDS Analytical Technologies, Sunnyvale, CA, USA) and total RNA was extracted using a High Pure FFPE RNA Micro Kit® (Roche Diagnostics, West Sussex, UK) according to the manufacturer's protocol. After thawing and homogenization of the fully muscularized D18 diaphragms, total RNA was extracted from the TRIzol® suspension using the acid guanidinium thiocyanatephenol-chloroform extraction method. Spectrophotometrical quantification of total RNA was performed with a NanoDrop ND-1000 UV– Vis® Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, NC, USA).
1.3. Complementary DNA synthesis and quantitative real-time polymerase chain reaction Reverse transcription of total RNA was carried out at 85 °C for 3 min (denaturation), at 44 °C for 60 min (annealing), and at 92 °C for 10 min (reverse transcriptase inactivation) using a Transcript High Fidelity cDNA Synthesis Kit® (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer's protocol. The resulting cDNA was used for quantitative real-time polymerase chain reaction using a LightCycler® 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Genespecific primer pairs are listed in Table 1. Following an initialization phase at 95 °C for 5 min, 55 amplification cycles were carried out. Each cycle included an initial denaturation step at 95 °C for 10 s, an annealing step at 60 °C for 15 s and an elongation step at 72 °C for 10 s. The final elongate temperature was 65 °C for 1 min. Relative mRNA expression levels of Prx1 and Tcf4 were measured with a Light Cycler® 480 instrument (Roche Diagnostics, West Sussex, UK) and gene levels were normalized to the housekeeping gene β-actin. All experiments were run duplicated for each sample and primer pair.
Table 1 Gene-specific primer sequences for quantitative real-time polymerase chain reaction. Gene Prx1 Forward Reverse Tcf4 Forward Reverse β-actin Forward Reverse
Sequence (5′-3′)
Product size (bp)
CCA CAT GTG CCA ACA ATA GC GGC ACC TGG TTC CTC TGT AA
106
CGA ATC ACA TGG GTC AGA TG AAA CGG GGT TAA GGA GCA GT
124
TTG CTG ACA GGA TGC AGA AG TAG AGC CAC CAA TCC ACA CA
108
1.4. Immunofluorescence double staining and confocal laser scanning microscopy After fixation in 10% PFA, whole D13 and D15 fetuses as well as D18 trunks were paraffin-embedded, transversely sectioned at a thickness of 5 μm, and mounted on polylysine-coated slides (VWR International, Leuven, Belgium). Resulting tissue sections were deparaffinized with xylene and rehydrated through ethanol and distilled water. Conventional hematoxylin and eosin staining (Sigma Aldrich, Saint Louis, MO, USA) was used to investigate the diaphragmatic histology. All sections for immunofluorescence staining were incubated with phosphatebuffered saline (PBS) containing 1.0% Triton X-100 (Sigma Aldrich Ltd., Arklow, Ireland) for 20 min to improve cell permeabilization. Sections were then washed in PBS + 0.05% Tween (Sigma-Aldrich, St. Louis, MO, USA) and subsequently blocked with 3% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min to avoid non-specific absorption of immunoglobulin. The blocking solution was rinsed off and sections were incubated with affinity-purified primary antibodies either against Prx1 (rabbit polyclonal, sc-21,948-R; 1:100), Tcf4 (goat polyclonal, sc-8631, 1:100) and GATA4 (mouse polyclonal, sc-25,310, 1:100) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight at 4 °C. On the next day, sections washed in PBS + 0.05% Tween and incubated with corresponding secondary antibodies (donkey anti-rabbit Alexa 647-A150067, 1:250, donkey anti-goat Alexa 555-A21432, 1:250 and donkey anti-mouse Alexa 488-A150109, 1:250) (Abcam plc, Cambridge, UK) for 1 h at room temperature. Following another washing step in PBS + 0.05% Tween, sections were counterstained with a DAPI antibody (10,236,276,001, 1:1000) (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min, washed again, and mounted with glass coverslips using Sigma Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA). All sections were scanned with a ZEISS LSM 700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and independently evaluated by two investigators. 1.5. Statistical analysis All numerical data is presented as means ± standard error of the mean. Differences between the two experimental groups were tested using an unpaired Student's t test when the data had normal distribution or a Mann–Whitney U test when the data deviated from normal distribution. A P value b0.05 was considered as statistically significant. 2. Results 2.1. Relative mRNA expression of Prx1 and Tcf4 in rat PPFs and fetal diaphragms The relative mRNA expression levels of Prx1 and Tcf4 were significantly downregulated in PPFs of nitrofen-exposed fetuses on D13, developing diaphragms of nitrofen-exposed fetuses on D15 and fully
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx Table 2 Relative mRNA expression levels of Prx1 and Tcf4 in developing rat diaphragms. Prx1
D13 D15 D18
Tcf4
Control
Nitrofen
Control
Nitrofen
2.32 ± 1.59 2.01 ± 1.44 1.68 ± 0.66
0.87 ± 0.56⁎ 0.87 ± 0.49⁎ 0.97 ± 0.61⁎
3.68 ± 1.61 3.66 ± 1.31 3.21 ± 1.88
2.12 ± 0.78⁎ 2.15 ± 0.69⁎ 1.31 ± 0.65⁎
⁎ P b 0.05 vs. control.
muscularized diaphragms of nitrofen-exposed fetuses with CDH on D18 compared to controls (Table 2).
2.2. Histology and immunofluorescence evaluation of Prx1, Tcf4 and GATA4 expression in rat PPFs and fetal diaphragms PPFs in the control group were triangular-shaped structures protruding out from the lateral body wall, whereas nitrofen-exposed fetuses had an abnormal PPF structure, characterized by an absence of the dorsally projecting point of the triangular PPF. Immunofluorescence staining for Prx1 and Tcf4 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization of Prx1 and Tcf4 in PPFs and fetal diaphragmatic tissue on D13, D15 and D18. Confocal laser scanning microscopy revealed co-expression of Prx1 and Tcf4 mainly in the diaphragmatic mesenchymal components, and further confirmed the molecular genetic results by showing a markedly diminished Prx1 and Tcf4 immunofluorescence in MCT of nitrofen-exposed PPFs, developing diaphragms and muscularized diaphragms compared to controls (Fig. 1). These findings were associated with a reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal diaphragms with CDH on D13, D15 and D18 compared to controls.
3. Discussion Much of our current understanding of pathophysiology of CDH originates from experimental studies [18]. Nitrofen (2,4-dichloro-phenyl-pnitrophenyl ether) is an herbicide that has been used for many years to create a teratogenic model of CDH. The advantage of this model is that the diaphragmatic defect is induced at the stage when the foregut has just separated into esophagus and trachea and therefore, providing opportunity to carefully study the developmental anatomy of the diaphragm and lungs in CDH [19]. Administration of nitrofen to pregnant rodents between days 8 and 11 after conception results in a high rate of CDH and associated pulmonary hypoplasia and pulmonary vascular abnormalities in the fetuses that is strikingly similar to the human malformation [20]. A fully functional diaphragm requires the coordinated morphogenesis of muscle, MCT and tendon, which have been shown to derive from different embryonic sources [9]. However, because of the large number of complex spatiotemporal processes involving multiple cellular and tissue interactions, normal embryological development of the diaphragm remains incompletely understood. It has been suggested that the origin of the defect in CDH lies in the amuscular mesenchymal components, which mainly comprise of fibrous connective tissue [8,9]. In addition, there is strong evidence that growth of the primordial diaphragm depends on the correct formation of its underlying MCT [10]. Developmental mutations, which inhibit normal expression of extracellular matrix, have recently been demonstrated to cause diaphragmatic defects [11]. Previous findings from our laboratory have indicated that several proteins, which are involved in the development of mesenchymal tissue in the fetal diaphragm, are decreased in the nitrofen-induced CDH model [12,21,22].
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In recent years, the embryogenesis of the PPFs has become a focus for elucidating the pathogenesis of diaphragmatic defects [23]. The PPFs play a pivotal role during diaphragmatic morphogenesis, being the target structures for migrating muscle precursor cells (MPCs) [24]. These MPCs have been reported to form the source for muscular elements in PPFs, which subsequently expand to form a primordial diaphragm [23]. Furthermore, it has been shown that the PPFs are defective in rodents with nitrofen-induced CDH [25]. A recent study has demonstrated by using an experimental CDH model that the primary mechanism of malformed PPFs is caused by a decreased proliferation of mesenchymal cells [26]. However, the exact mechanisms that lead to abnormal PPF development in CDH are largely unclear [9]. Using mouse genetics, Merrell et al. [13] have recently confirmed that the PPFs are the only source of the diaphragm's MCT, thus significantly contributing to diaphragmatic muscle formation. They have also revealed that Prx1 has an essential function during growth of the fetal diaphragm by stimulating the expression of Tcf4, which is a novel marker for MCT cells that critically controls diaphragmatic morphogenesis [13]. Prx1 has been known to maintain the elastic properties of extracellular matrices, which in turn is crucial for differentiation of MPCs [27]. Tcf4 is an important regulator of myogenesis and maturation that is strongly expressed in connective tissue fibroblasts [28,29]. Moreover, it has been shown that MCT expresses the product of the CDHassociated gene Gata4 [16,17,30–32]. Mosaic mutations of Gata4 in PPF-derived MCT fibroblasts have been found to result in the development of localized amuscular regions in the fetal diaphragm, subsequently causing CDH [13]. Taken together, the above findings suggest that PPFs and MCT are fundamental for normal diaphragmatic development. The present study provides strong evidence that the diaphragmatic gene expression of Prx1 and Tcf4 is significantly downregulated in PPFs on D13, developing diaphragms on D15 and fully muscularized diaphragms on D18 in the nitrofen-induced CDH model compared to control littermates. We further demonstrated a co-localized expression of Prx1 and Tcf4 in PPFs on D13, developing diaphragms on D15 and fully muscularized diaphragms on D18, suggesting that these cells have developed from PPF-derived MCT cells. Additionally, immunofluorescence double staining for Prx1 and Tcf4 showed a co-localization with GATA4, which is a central transcription factor during diaphragmatic development and strongly expressed by mesenchymal cells in developing fetal diaphragms [17,30]. Confocal laser scanning microscopy also showed a markedly diminished Prx1 and Tcf4 expression in the diaphragmatic mesenchyme of nitrofen-exposed PPFs and fetuses with CDH on D13, D15 and D18 compared to controls. Thus, these findings confirmed that the quantitative decrease of Prx1 and Tcf4 mRNA transcripts in fetal diaphragms was as well translated to the protein level. Recently, it has been reported that the diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that a decreased GATA4 expression may impair the diaphragmatic development in nitrofen-induced CDH [33]. Besides the markedly diminished Prx1 and Tcf4 expression in nitrofen-exposed PPFs and fetal diaphragms with CDH, we identified a reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18, which indicates a potential disruption in the formation of MCT. In conclusion, our results suggest that decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, and thus contribute to the development of diaphragmatic defects in the nitrofen CDH model. Future studies investigating morphogenetic processes regulating diaphragm's muscle connective tissue morphogenesis may provide new insights into the embryological origins of the diaphragmatic defect in CDH. Conflict of interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Fig. 1. Hematoxylin and eosin (H&E) staining (left image of each time-point and experimental group) as well as specific immunofluorescence staining for Prx1 (yellow), Tcf4 (green) and GATA4 (red) combined with DAPI (blue) in rat PPFs and fetal diaphragms on D13 (a), D15 (b) and D18 (c).
References [1] McGivern MR, Best KE, Rankin J, et al. Epidemiology of congenital diaphragmatic hernia in Europe: a register-based study. Arch Dis Child Fetal Neonatal Ed 2015; 100:F137–44. [2] Balayla J, Abenhaim HA. Incidence, predictors and outcomes of congenital diaphragmatic hernia: a population-based study of 32 million births in the United States. J Matern Fetal Neonatal Med 2014;27:1438–44. [3] Tovar JA. Congenital diaphragmatic hernia. Orphanet J Rare Dis 2012;7:1. [4] Keijzer R, Puri P. Congenital diaphragmatic hernia. Semin Pediatr Surg 2010;19: 180–5.
[5] McHoney M. Congenital diaphragmatic hernia, management in the newborn. Pediatr Surg Int 2015;31:1005–13. [6] Jeanty C, Kunisaki SM, MacKenzie TC. Novel non-surgical prenatal approaches to treating congenital diaphragmatic hernia. Semin Fetal Neonatal Med 2014;19: 349–56. [7] Losty PD. Congenital diaphragmatic hernia: where and what is the evidence? Semin Pediatr Surg 2014;23:278–82. [8] Greer JJ. Current concepts on the pathogenesis and etiology of congenital diaphragmatic hernia. Respir Physiol Neurobiol 2013;189:232–40. [9] Merrell AJ, Kardon G. Development of the diaphragm – a skeletal muscle essential for mammalian respiration. FEBS J 2013;280:4026–35.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx [10] Maki JM, Sormunen R, Lippo S, et al. Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues. Am J Pathol 2005;167:927–36. [11] Hornstra IK, Birge S, Starcher B, et al. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 2003;278:14387–93. [12] Takahashi T, Friedmacher F, Takahashi H, et al. Kif7 expression is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 2015;50:904–7. [13] Merrell AJ, Ellis BJ, Fox ZD, et al. Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias. Nat Genet 2015;47:496–504. [14] Logan M, Martin JF, Nagy A, et al. Expression of Cre recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 2002;33:77–80. [15] Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999;21:70–1. [16] Longoni M, Lage K, Russell MK, et al. Congenital diaphragmatic hernia interval on chromosome 8p23.1 characterized by genetics and protein interaction networks. Am J Med Genet A 2012;158A:3148–58. [17] Clugston RD, Zhang W, Greer JJ. Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 2008;294:L665–75. [18] Eastwood MP, Russo FM, Toelen J, et al. Medical interventions to reverse pulmonary hypoplasia in the animal model of congenital diaphragmatic hernia: a systematic review. Pediatr Pulmonol 2015;50:820–38. [19] Noble BR, Babiuk RP, Clugston RD, et al. Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 2007;293:L1079–87. [20] Montedonico S, Nakazawa N, Puri P. Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 2008;24:755–61. [21] Takahashi T, Friedmacher F, Zimmer J, et al. Mesenchymal expression of the FRAS1/ FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 2016;32:135–40.
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[22] Takahashi T, Friedmacher F, Takahashi H, et al. Disruption of copper-dependent signaling pathway in the nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 2015;31:31–5. [23] Babiuk RP, Zhang W, Clugston R, et al. Embryological origins and development of the rat diaphragm. J Comp Neurol 2003;455:477–87. [24] Clugston RD, Greer JJ. Diaphragm development and congenital diaphragmatic hernia. Semin Pediatr Surg 2007;16:94–100. [25] Mayer S, Metzger R, Kluth D. The embryology of the diaphragm. Semin Pediatr Surg 2011;20:161–9. [26] Clugston RD, Zhang W, Greer JJ. Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res A Clin Mol Teratol 2010;88:15–24. [27] Ihida-Stansbury K, Ames J, Chokshi M, et al. Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs. Pulm Circ 2015;5:382–97. [28] Mathew SJ, Hansen JM, Merrell AJ, et al. Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 2011;138:371–84. [29] Kardon G, Harfe BD, Tabin CJ. A Tcf4-positive mesodermal population provides a prepattern for vertebrate limb muscle patterning. Dev Cell 2003;5: 937–44. [30] Jay PY, Bielinska M, Erlich JM, et al. Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects. Dev Biol 2007;301:602–14. [31] Arrington CB, Bleyl SB, Matsunami N, et al. A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia. Am J Med Genet A 2012;158A:3137–47. [32] Yu L, Wynn J, Cheung YH, et al. Variants in GATA4 are a rare cause of familial and sporadic congenital diaphragmatic hernia. Hum Genet 2013;132: 285–92. [33] Dingemann J, Doi T, Gosemann JH, et al. Decreased expression of GATA4 in the diaphragm of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res B Dev Reprod Toxicol 2013;98:139–43.
Please cite this article as: Takahashi T, et al, Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofeninduced congenital diaphragmatic hernia, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.09.007