Ephrin-B1, -B2, and -B4 Expression is Decreased in Developing Diaphragms and Lungs of Fetal Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia

Abstract

Introduction Congenital diaphragmatic hernia (CDH) is assumed to originate from a malformation of the amuscular mesenchymal component of the primordial diaphragm. Mutations in ephrin-B1, a membrane protein that is expressed by mesenchymal cells, have been found in newborn infants with CDH and associated pulmonary hypoplasia (PH), highlighting its important role during diaphragmatic and airway development. Ephrin-B1, -B2, and -B4 are expressed in fetal rat lungs and have been identified as key players during lung branching morphogenesis. We hypothesized that diaphragmatic and pulmonary expression of ephrin-B1, -B2, and -B4 is decreased in the nitrofen-induced CDH model.

Materials and Methods Time-mated rats received nitrofen or vehicle on day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Ephrin-B1, -B2, and -B4 gene expression was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for ephrin-B1, -B2, and -B4 was combined with mesenchymal and epithelial markers (Gata-4/Fgf-10 and calcitonin gene-related peptide) to evaluate protein expression/localization.

Results Ephrin-B1, -B2, and -B4 gene expression was significantly reduced in pleuroperitoneal folds/primordial lungs (D13), developing diaphragms/lungs (D15), and fully muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal laser scanning microscopy demonstrated markedly diminished ephrin-B1 immunofluorescence in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared with controls, whereas ephrin-B2 and -B4 expression was mainly decreased in distal airway epithelium.

Conclusion Decreased ephrin-B1, -B2, and -B4 expression may disrupt diaphragmatic development and lung branching morphogenesis by interfering with epithelial–mesenchymal interactions, thus causing diaphragmatic defects and PH.

Keywords

ephrin - diaphragm - lung - congenital diaphragmatic hernia - nitrofen

• 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 (02) F137-F144
• 2 Rocha G, Azevedo I, Pinto JC, Guimarães H. Follow-up of the survivors of congenital diaphragmatic hernia. Early Hum Dev 2012; 88 (04) 255-258
• 3 van den Hout L, Reiss I, Felix JF. , et al; Congenital Diaphragmatic Hernia Study Group. Risk factors for chronic lung disease and mortality in newborns with congenital diaphragmatic hernia. Neonatology 2010; 98 (04) 370-380
• 4 Ameis D, Khoshgoo N, Keijzer R. Abnormal lung development in congenital diaphragmatic hernia. Semin Pediatr Surg 2017; 26 (03) 123-128
• 5 Harting MT. Congenital diaphragmatic hernia-associated pulmonary hypertension. Semin Pediatr Surg 2017; 26 (03) 147-153
• 6 Greer JJ. Current concepts on the pathogenesis and etiology of congenital diaphragmatic hernia. Respir Physiol Neurobiol 2013; 189 (02) 232-240
• 7 Merrell AJ, Kardon G. Development of the diaphragm -- a skeletal muscle essential for mammalian respiration. FEBS J 2013; 280 (17) 4026-4035
• 8 Mäki JM, Sormunen R, Lippo S, Kaarteenaho-Wiik R, Soininen R, Myllyharju J. 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 (04) 927-936
• 9 Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 2003; 278 (16) 14387-14393
• 10 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 (01) 15-24
• 11 Metzger RJ, Klein OD, Martin GR, Krasnow MA. The branching programme of mouse lung development. Nature 2008; 453 (7196): 745-750
• 12 Roth-Kleiner M, Post M. Similarities and dissimilarities of branching and septation during lung development. Pediatr Pulmonol 2005; 40 (02) 113-134
• 13 Warburton D, Bellusci S, De Langhe S. , et al. Molecular mechanisms of early lung specification and branching morphogenesis. Pediatr Res 2005; 57 (5 Pt 2): 26R-37R
• 14 Friedmacher F, Gosemann JH, Fujiwara N, Takahashi H, Hofmann A, Puri P. Expression of Sproutys and SPREDs is decreased during lung branching morphogenesis in nitrofen-induced pulmonary hypoplasia. Pediatr Surg Int 2013; 29 (11) 1193-1198
• 15 Friedmacher F, Fujiwara N, Hofmann AD, Takahashi H, Gosemann JH, Puri P. Expression of Eya1 and Six1 is decreased in distal airways of rats with experimental pulmonary hypoplasia. J Pediatr Surg 2014; 49 (02) 301-304
• 16 Peixoto FO, Pereira-Terra P, Moura RS, Carvalho-Dias E, Correia-Pinto J, Nogueira-Silva C. The role of ephrins-B1 and -B2 during fetal rat lung development. Cell Physiol Biochem 2015; 35 (01) 104-115
• 17 Wilkinson GA, Schittny JC, Reinhardt DP, Klein R. Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity. Dev Dyn 2008; 237 (08) 2220-2234
• 18 Srisupundit K, Brady PD, Devriendt K. , et al. Targeted array comparative genomic hybridisation (array CGH) identifies genomic imbalances associated with isolated congenital diaphragmatic hernia (CDH). Prenat Diagn 2010; 30 (12-13): 1198-1206
• 19 Hogue J, Shankar S, Perry H, Patel R, Vargervik K, Slavotinek A. A novel EFNB1 mutation (c.712delG) in a family with craniofrontonasal syndrome and diaphragmatic hernia. Am J Med Genet A 2010; 152A (10) 2574-2577
• 20 Wallis D, Lacbawan F, Jain M. , et al. Additional EFNB1 mutations in craniofrontonasal syndrome. Am J Med Genet A 2008; 146A (15) 2008-2012
• 21 Varisco BM, Sbragia L, Chen J. , et al. Excessive reversal of epidermal growth factor receptor and ephrin signaling following tracheal occlusion in rabbit model of congenital diaphragmatic hernia. Mol Med 2016; 22: 398-411
• 22 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 (04) L1079-L1087
• 23 Eastwood MP, Russo FM, Toelen J, Deprest J. Medical interventions to reverse pulmonary hypoplasia in the animal model of congenital diaphragmatic hernia: a systematic review. Pediatr Pulmonol 2015; 50 (08) 820-838
• 24 Chiu PP. New insights into congenital diaphragmatic hernia - a surgeon's introduction to CDH animal models. Front Pediatr 2014; 2: 36
• 25 Babiuk RP, Zhang W, Clugston R, Allan DW, Greer JJ. Embryological origins and development of the rat diaphragm. J Comp Neurol 2003; 455 (04) 477-487
• 26 Merrell AJ, Ellis BJ, Fox ZD, Lawson JA, Weiss JA, Kardon G. Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias. Nat Genet 2015; 47 (05) 496-504
• 27 Takahashi T, Friedmacher F, Takahashi H, Hofmann AD, Puri P. Kif7 expression is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 2015; 50 (06) 904-907
• 28 Takahashi T, Friedmacher F, Takahashi H, Daniel Hofmann A, Puri P. Lysyl oxidase expression is decreased in the developing diaphragm and lungs of nitrofen-induced congenital diaphragmatic hernia. Eur J Pediatr Surg 2015; 25 (01) 15-19
• 29 Weaver M, Dunn NR, Hogan BL. Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis. Development 2000; 127 (12) 2695-2704
• 30 De Langhe SP, Sala FG, Del Moral PM. , et al. Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev Biol 2005; 277 (02) 316-331
• 31 Vadivel A, van Haaften T, Alphonse RS. , et al. Critical role of the axonal guidance cue EphrinB2 in lung growth, angiogenesis, and repair. Am J Respir Crit Care Med 2012; 185 (05) 564-574
• 32 Bennett KM, Afanador MD, Lal CV. , et al. Ephrin-B2 reverse signaling increases α5β1 integrin-mediated fibronectin deposition and reduces distal lung compliance. Am J Respir Cell Mol Biol 2013; 49 (04) 680-687
• 33 Klein R. Eph/ephrin signalling during development. Development 2012; 139 (22) 4105-4109
• 34 Kullander K, Klein R. Mechanisms and functions of Eph and ephrin signalling. Nat Rev Mol Cell Biol 2002; 3 (07) 475-486
• 35 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 (02) 602-614
• 36 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 (04) L665-L675
• 37 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 (03) 285-292
• 38 Dingemann J, Doi T, Gosemann JH, Ruttenstock EM, Nakazawa N, Puri P. Decreased expression of GATA4 in the diaphragm of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res B Dev Reprod Toxicol 2013; 98 (02) 139-143
• 39 Sekine K, Ohuchi H, Fujiwara M. , et al. Fgf10 is essential for limb and lung formation. Nat Genet 1999; 21 (01) 138-141
• 40 Lebeche D, Malpel S, Cardoso WV. Fibroblast growth factor interactions in the developing lung. Mech Dev 1999; 86 (1-2): 125-136
• 41 Teramoto H, Yoneda A, Puri P. Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats. J Pediatr Surg 2003; 38 (07) 1021-1024
• 42 Keijzer R, Liu J, Deimling J, Tibboel D, Post M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 2000; 156 (04) 1299-1306
• 43 Gattone II VH, Morse DE. Pleuroperitoneal canal closure in the rat. Anat Rec 1984; 208 (03) 445-460