Fig 2: Tempo ral expression of EFNB2 and EPHB4 in the lungs of the CDH and CON groups. mRNA levels of EFNB2 and EPHB4 in develo ping lungs were determined by reverse transcription-quantitative polymerase chain reaction analysis and results are expressed rela tive to the cont rol at E13.5, E15.5, E17.5, E19.5 and E21.5. Specificity of the products were confirmed by visualization on 2% agarose gels. Results are presented as the mean ± standard error of the mean. *P<0.05, vs. CON at the same time point. #P<0.05, vs. E13.5, inner-group. CDH, congenital diaphragmatic hernia; CON, control; E, embryonic day; EFNB2, ephrin-B2; EPHB4, ephrin type-B receptor 4; M, marker.

Fig 3: Representative immunoblots and densitometric analysis of expression of EFNB2 and EPHB4 proteins in the fetal lungs at E13.5, E15.5, E17.5, E19.5 and E21.5. Results were normalized relative to the expression of β-actin and are presented as the mean ± standard error of the mean. *P<0.05, vs. CON at the same time point. CDH, congenital diaphragmatic hernia; CON, control; E, embryonic day; EFNB2, ephrin-B2; EPHB4, ephrin type-B receptor 4.

Fig 4: Fetal lung explant cultures in vitro. (A) Terminal bud count, (B) explant surface, and (C) explant perimeter were significantly smaller in the E13.5 CDH group than in the CON group (#P<0.05, vs. CON at the same time point). Lung tissue development in the CDH group lagged behind that in the CON group by 96 h of culture. Lung explants of the (D) CON group (E) CDH group, and (F) CDH + EFNB2 group at 0, 24, 48, 72 and 96 h. EFNB2 supplementation promoted branching of rat fetal lung explants. E13.5 lungs were treated with EFNB2 recombinant protein (0.01 μg/ml) daily. Terminal buds counts, explant surface, and perimeter were significantly increased in the EFNB2-treated group, compared with the CDH group. Compared with the untreated lung, EFNB2 treatment markedly increased airway branching morphogenesis, termi nal bud count, explant surface, and explant perimeter at 96 h (*P<0.05, CDH, vs. CDH + EFNB2 at the same time point). After 96 h in vitro, no significant differences in termi nal bud count, explant surface, or explant perimeter were observed between the CDH + EFNB2 group and CON group ($P<0.05, CDH+EFNB2, vs. CON at the same time point). Results are presented as the mean ± standard error of the mean. Original magnification, ×40, scale bar=200 μm (all images at same magnification). CDH, congenital diaphragmatic hernia; CON, control; E13.5, embryonic day 13.5; EFNB2, ephrin-B2.

Fig 5: Expression of EFNB2 and EPHB4. Representative photomicrographs of IHC staining for (A) EFNB2 and (B) EPHB4 in the lung sections from CON and CDH groups at five gestational stages: E13.5, E15.5, E17.5, E19.5 and E21.5 days. EFNB2 and receptor EPHB4 exhibited marked epithelial expression. In the semi-quantitative analysis, at E17.5, E19.5 and E21.5, the expression of EFNB2 and EPHB4 was higher in the CDH group than in the CON group (*P<0.05 vs. CON at the same time point). Original magnification, ×400, scale bar=50 μm. CDH, congenital diaphragmatic hernia; CON, control; E, embryonic day; EFNB2, ephrin-B2; EPHB4, ephrin type-B receptor 4.