ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

Abstract

Background Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. Methods The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot. Results Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4. Conclusions These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Amniotic fluid stem cell extracellular vesicles promote fetal lung branching and cell differentiation in experimental congenital diaphragmatic hernia

“…As we had previously demonstrated in fetal lung explants harvested at the pseudoglandular stage [7], AFSC-EVs administered to explants harvested at both canalicular and saccular stages rescued the expression of both FGF10 and FGFR2 back to control levels. The ROBO/SLIT pathway is also essential for normal lung development, as shown in knock-out studies [34,35], and is downregulated in nitrofen-exposed lungs of fetuses with CDH [36]. In the present study, we confirmed the upregulation of ROBO1 and the corresponding downregulation of ROBO2 and SLIT2 expression in nitrofen-exposed lungs at both timepoints, which were rescued by AFSC-EV treatment to control levels.…”

Treatment with Amniotic Fluid Stem Cell Extracellular Vesicles Promotes Fetal Lung Branching and Cell Differentiation at Canalicular and Saccular Stages in Experimental Pulmonary Hypoplasia Secondary to Congenital Diaphragmatic Hernia

“…The ROBO/SLIT pathway is also essential for normal lung development, as shown in knockout studies, 36 , 37 and is downregulated in nitrofen-exposed lungs of fetuses with CDH. 38 In the present study, we confirmed the upregulation of ROBO1 and the corresponding downregulation of ROBO2 and SLIT2 expression in nitrofen-exposed lungs at both time points, which were rescued by AFSC-EV treatment to control levels. When we assessed factors involved in sending guidance cues for lung branching, we confirmed that the Ephrin pathway was downregulated at the saccular stage, as previously reported, 39 and rescued by AFSC-EV treatment to control levels.…”

Robo2 Predicts a Better Prognosis and Inhibits Malignant Behavior in Vivo in Pancreatic Ductal Adenocarcinoma

“…ROBO2 has been found to regulate different physiological functions and tumor development. Gonçalves et al [18] revealed an important role of ROBO2 in lung development. Escot et al [19] found that ROBO2 plays an important role in pancreas cell identity and plasticity.…”