Fig 1: Effect of ROBO1 or ROBO2 functional impairment in branching morphogenesis. a The upper panel is representative of untreated lung explants (0 ng/mL) at D0; the bottom panel represents lung explants treated with recombinant IgG protein (0 ng/mL) and several doses of recombinant ROBO1 or ROBO2 proteins at day 4 (D4). b Morphometric analysis of the number of peripheral airway buds of fetal rat lung explants treated with increasing concentrations of recombinant ROBO1 (black) and ROBO2 (gray) proteins. Results are expressed as the D4/D0 ratio. c Examples of representative blots are showed for each analysed protein. Protein expression levels of d ROBO; e SOX2; f SOX9; g total β-Catenin; h non-phospho (active) β-Catenin; and i BMP4 in normal explant cultures treated with recombinant rat ROBO1 Fc Chimera for ROBO1 inhibition (black line) or recombinant human ROBO2 Fc Chimera for ROBO2 functional impairment (gray line). Recombinant human IgG Fc was used as control (IgG). n ≥ 9 per protein/condition. Each lane represents a pooled-tissue sample, and relative expression was determined against β-Tubulin and IgG. The data are presented as means ± SEM. p < 0.05: αvs. IgG
Fig 2: a Overview of the main changes in spatiotemporal distribution of ROBO1, ROBO2, SOX2 and SOX9 at pseudoglandular (E17.5) and saccular stages (E21.5) in hypoplastic (hyp) fetal lungs (lower panel). w/o without b A proposed model of ROBO regulation of ex vivo branching morphogenesis through BMP4, β-Catenin, SOX2 and SOX9
Fig 3: ROBO1 expression profile in normal and hypoplastic lungs. a representative immunohistochemical evidence for the presence of ROBO1 during normal (aA–aH) and hypoplastic (hyp, aa–ah) fetal lung development at the distinct gestational ages. Symbols in each figure identify the distinct pulmonary structures: *bronchi; ¤primordia of bronchiole; ¥terminal bronchiole; {bronchioalveolar duct junction; &alveolar duct. Data is representative of n ≥ 3 independent experiments per stage/protein. b ROBO1 stained cells quantified by pulmonary structure and developmental stage. Results are presented as mean ± SEM. Symbols indicate the main effects and non-redundant interactions of one-way ANOVA. αp < 0.05. Scale bar 50 µm
Fig 4: Distinct expression profiles for receptors and epithelial progenitors in normal and hypoplastic lungs. a Examples of representative blots are shown for each protein analyzed. Western blot analysis of b ROBO1; c ROBO2; d SOX2; and e SOX9 relative expression levels in normal (ctrl) and hypoplastic (hyp) lungs at selected gestational ages from E13.5-to-E21.5. Each lane represents a pooled-tissue sample, and relative expression was determined against β-Tubulin. Semi-quantitative analysis of three independent experiments for each protein is plotted (n ≥ 9 per timepoint and experimental groups, respectively). Results are presented as mean ± SEM. Symbols indicate the main effects and non-redundant interactions of the two-way ANOVA. p < 0.05: αvs. ctrl; βvs. E13.5- and 15.5-ctrl; Ψvs E13.5- and E15.5-hyp; γvs E15.5-ctrl; µvs E15.5-hyp; λvs E17.5-ctrl; εvs E17.5-hyp; δvs E19.5-hyp
Fig 5: Dopaminergic misprojection in mouse embryos lacking either Robo1/Robo2 or Slit1/Slit2 is accompanied by dramatic alterations in the GAD65 axon bundles. Brains from control (A,D,G), Robo1/Robo2 double knock out (dko) (B,E,H), or Slit1/Slit2 dko (C,F,I) E12 mouse embryos were double immunostained for TH (green) and GAD65 (red) and hemibrains were mounted flat for observation. Normal projection at this early stage of TH+ axon pathfinding can be observed in (A) (control). Dramatic reduction and misprojection of TH axons is observed in (B) (Robo1/Robo2 dko), and (C) (Slit1/Slit2 dko). (D–F) Show GAD65 immunostaining of the same areas; in (D), normal GAD65 axon scaffolds are observed while notable alterations in the scaffold are observed in double knockouts (dko) (E,F) being more dramatic in Robo1/Robo2 dko (E). (G–I) Show single focal planes of the same regions with ortohogonal projections at the red and green lines shown. Apposition of TH axons on the orthogonal projections shown in (G–I) was quantified and is shown in (J). Significance (*) in (J) is p < 0.05. Scale bar: 100 μm.
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