ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen

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RESEARCH

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen‑induced congenital diaphragmatic hernia Ana N. Gonçalves1,2  , Jorge Correia‑Pinto1,2,3 and Cristina Nogueira‑Silva1,2,4*

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 struc‑ ture 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 branch‑ ing morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanismdependent 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. Keywords:  Branching, CDH, Epithelial progenitors, ROBO, SOX

*Correspondence: [email protected] 1 Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710‑057 Gualtar, Braga, Portugal Full list of author information is available at the end of the article

Background Respiratory function depends on proximal or conducting airways that allow the continuous passageway of air and the distal or respiratory airways, where the gas exchange takes place [1, 2]. Interestingly, a family of transcription

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 Internationa