Svep1 expression pattern and Svep1-/- hypoplastic lung lobes anomalies.

(a) In situ hybridization of lungs from E14.5 wild type embryos showed strong Svep1 mRNA expression in the mesenchyme at the lung periphery and around bifurcating distal airway tips (right inset outlined in red, arrowhead). (b) Arrowhead shows Mesenchymal expression of SVEP1 protein in normal E12.5 lung. White arrows indicate the location of the SVEP1 protein on the epithelial membrane stained with ECAD (purple) in a distal airway bud. (c) SVEP1 expression is strong in the mesenchyme towards the lobe tip of the E14.5 lungs and branching cleft sites (arrowheads). (d) SVEP1 protein localizes in the lung parenchyma including the primary septa (upper box) and adjacent to the proximal airway epithelium (lower box). (e) microCT scan of E18.5 embryos showing multiple defects including thinning of the diaphragm and lung hypoplasia (red arrows). (f) Ventral view of E18.5 of Svep1+/+ and Svep1-/- lungs. The arrowheads indicate the tips of the lung lobes, which are short and rounded (highlight in black) in Svep1-/- mice and have like a cauliflower morphology. (g) H&E-stained lung sections embryos demonstrate that E16.5 lungs from Svep1-/- embryos are smaller and have irregular lobe edges. Black arrowheads indicate left lobes with highlighted surface. (h) The red arrowheads show the dorsal side of the E18 lungs, whereas the black arrows highlight the ventral side. Svep1-/- embryos show defective saccular development mainly at the edge on the ventral side of the lung (inserts arrowheads). Co-localization of ECAD and SOX9 confirms high expression of SOX9 in distal airways (white arrowheads) in the lung of Svep1-/- embryos at E18.5. (i) Plots showing Sox9 relative mRNA expression at E14.5, E16.5, and E18.5. mRNA expression is significantly high at E18.5 in Svep1-/- embryos (average number of tips per area ± SEM; p < 0.05; n ≥ 5). Right cranial (RCr), Right Caudal (RCa), Right Medial (RMe), and Right Accessory (RAc) lobes. Scale bars: 50μm (a, b, c, d), 100 μm.

Airway organization defects and abnormal branching patterns in Svep1-/- embryonic lungs resulting in lung lobe shape anomalies.

(a) Co-localization of epithelial (ECAD) and epithelial progenitor (SOX9) markers showing distal airways in lungs of E16.5 mice. (a’) The blue highlights in inserts represent the distal tip of the normal and mutant RAc lung lobes, with the mutant left lobe showing multiple branching. (a’’) Inserts illustrate airway density at the lung periphery in WT and mutants, with mutants having higher density. (a’’’) In Svep1-/- lungs, distal airway tips are organized in packed airway rosettes on the ventral side of the lung lobe. (b) Illustration of the three branching modes that pattern the airways of the lung (domain branching, planar bifurcation, orthogonal bifurcation) and distal trifurcation. (c-f) Representative Z-stack images show SOX9 immunofluorescence (c) E13.5 Whole lungs of normal and Svep1-/- embryos. The images show Svep1-/- lungs with abnormal RCa lobe morphology (arrowheads), RAc lobe distal tip branching (arrows), and secondary branching pattering (orange arrowheads). (d) Pictures of E13.5 Svep1+/+ and Svep1-/- embryos showing ectopic branching/budding anomalies in mutant embryos (areas outlined in pink in lower inserts). (e) E14.5 left lung lobes of Svep1+/+ and Svep1-/- embryos. SOX9+ buds highlighted in pink and cartoon show airway tip arrangement in normal and mutants. Arrow shows defective tip arrangement resulting in abnormal lung tip morphology in Svep1-/- embryos. (f) Cleared tissue immunofluorescence of SOX9 in the left lung lobe at E14.5 reveals airway disorganization in Svep1-/- embryos vs Svep1+/+ embryos. Distal lung tips highlighted show branching trifurcations in the Svep1-/- embryo (white arrow) and aberrant distal tip airway. (g) Representation of the left lung lobe Z-stack for distal airway quantification. Whole lungs of Svep1-/- embryos at E14.5 have a statistically significant increase in the total number of SOX9 positive airway tips and ratio of tip and area compared to wild type embryos (average number of tips for area ± SEM; p < 0.05; n ≥ 5). Right cranial (RCr), Right Caudal (RCa), Right Medial (RMe), and Right Accessory (RAc) lobes. Scale bars: 100μm (a-e).

Disrupted branching programs and altered airway architecture in Svep1-/- RAc lung lobes at E14.5.

(a) Compilation of a Z-stack image series for tissue-cleared whole RAc lung lobes labeled with ECAD (green) and SOX9 (purple) highlight disorganized branching in Svep1-/- embryos at E14.5. Arrows (lower right) point to branching airways with rosette-like arrangements and ectopic airway tips growing in random directions. Arrowheads (left panels) at the RCa lobe tips indicate a single tip in the lobe of Svep1+/+ embryos but multi branched tips in Svep1 knockout embryos. (b) Schematic illustrating representative domain (blue), planar (green), and orthogonal (red) branching program types of Svep1+/+ and Svep1-/- RAc lobes. Lungs of Svep1-/- embryos lack planar bifurcations and have many trifurcating branch tips (red highlighted in black). (c-d) Graphs displaying the percentages of branching modes, orthogonal bifurcations and trifurcations (c) and ectopic budding (d) in E14.5 Rac lobes of wild type and Svep1-/- embryos. (e) Representative single Z-stack images of RAc lobes at E14.5, stained with ECAD (green) and SOX9 (purple), show bifurcations (white dotted lines and white arrowhead) in Svep1+/+ embryos, but trifurcations (white dotted lines and white arrows) in Svep1-/- mice. (f) Single Z-stack image of RAc lobes stained with ECAD (green) and SOX9 (red) display ectopic domain branching in Svep1-/- mice (arrow). (g) Lung explants from E12.5 embryos after 48 hours in organ culture. Explants from Svep1+/+ embryos show normal bifurcation (black arrowheads) whereas explants from Svep1-/- embryos show trifurcations (black arrows) and ectopic budding (red arrowheads). (Average number of tips per area ± SEM; p < 0.05; n ≥ 5) (c,d). Scale bars 100μm (a, g); 50μm (e).

SVEP1 treatment modulates airway branching in E12.5 lung explants

(a) Images of lung explants from E12.5 Svep1+/+ embryos cultured for 48 hrs. Images of lung explants from E12.5 Svep1+/+ embryos cultured for 48 hrs. show a significant reduction of branching (control, top row) when treated with SVEP1 peptide (bottom row). (b) Plot showing peripheral airway counts of a left lung lobe after 48 hrs cultured with and without SVEP1 treatment. (c) Svep1-/- lung explants treated with SVEP1 for 48 hours show a reduction in trifurcations compared to untreated lung explants. Black arrowheads indicate bifurcation; arrows indicate trifurcations. (Average counts ± SEM; t test p < 0.05 n≤ 6). Scale bars 100 µm (a,c).

Impaired smooth muscle and airway epithelium differentiation in Svep1-/- embryonic lungs.

(a) Interactome of downregulated genes in lungs of E18.5 Svep1-/- embryos with clusters labeled by enriched annotation terms. (b) Gene expression heatmap from RNA sequencing shows significantly downregulated genes in lungs from Svep1-/- embryos are associated with collagen and smooth muscle genes. (c) ACTA2 and Calponin staining at E16.5. (d) ACTA2 and calponin localization in lung at E18.5 (white arrowheads indicate distal respiratory bronchioles; orange arrows indicate proximal bronchioles; white arrows indicate blood vessels). (e) ACTA2 staining (red) in wild type and Svep1-/- E12.5 lung explants each cultured for six days, showed a reduction of smooth muscle cell formation in secondary and tertiary bronchi of the mutants (white arrows). (f) SOX2 Immunofluorescence staining marking the progenitor cells of the epithelium in the proximal airways. Arrow marks the proximal airway at the core of the Svep1-/- lung. (g) Immunofluorescence of the club cell marker SCGB1A1 (green) highlights the small proximal airways in mutant lungs. SCGB1A1 (green) colocalization with the ciliated cell marker FOXJ1 (pink) reveals a multicellular airway epithelium composed primarily of club cells. In Svep1-/- embryos, the bronchiolar lumina are narrowed than Svep1+/+ (highlighted in white). Quantification shows an increased average of number of club cells (SCGB1A1) but not of ciliated cells (FOXJ1). (Average counts ± SEM; t test p < 0.05 n≤ 3). Scale bars 200 µm or 100 µm

Enhanced FGF signaling in Svep1-/- embryos contributes to smooth muscle differentiation and branching defects and FGF9 rescues SVEP1 inhibition of branching.

(a) In situ hybridization demonstrates increased expression of Fgfr2 in Svep1-/- mutants. (b) Relative transcript abundance of Fgfr2 (and its isoforms Fgf2b and Fgfr2c), Fgf10, Fgf9, and Kras showing increased expression in Svep1-/- mutant embryos at E18.5. (c) Svep1+/+ and Svep1-/- lung explants from E12.5 embryos treated ex vivo with the FGF signaling inhibitor SU5402 (2.5 µg/ml). Black arrows point to normal tip morphology in Svep1+/+ lungs. The black arrowhead points to distal tip trifurcations that persist in lung explants from Svep1-/- embryos. SOX9 (green) and ACTA2 (purple) staining confirm the branching anomalies (highlighted in the white dotted areas) in Svep1-/- lung explants. White arrows mark normal bifurcations, white arrowheads indicate trifurcations and distal budding; the red arrowhead highlights ectopic budding not affected by the induction of FGF. (d) E12.5 lung explants treated with SU5402 (2.5 µg/ml) for 4 days demonstrate reduced ACTA2 localization in distal airways of Svep1-/- lungs compared to wild type lungs. (e) Fgf9 mRNA expression in wild type (E14.5) and mutant lung (E16.5). (f) Wild type E12.5 lung explants after 48 hours: untreated control (top left); treated with SVEP1 alone (top right); FGF9 alone (bottom left); combination of SVEP1 and FGF9 (bottom right). The combination restores a normal bud tip phenotype. (Average counts ± SEM; t-test p < 0.01 and <0.05, n >3). Scale bars 100 µm (a, c, d, e).

Saccular cellular and structural defects in Svep1-/- embryos at E18.5.

(a) Expression heatmaps of selected genes associated with alveolar AT1 and AT2 cells show that Svep1-/- lungs have lower expression of AT1 genes compared to Svep1+/+ lungs and higher expression of AT2 associated genes. (b) Immunofluorescence of PDPN, HOPX, Pro-SFPC and ABCA3 in the lungs of E18.5 Svep 1+/+ and Svep1-/- embryos. Arrows indicate airway bud stalks. (c) PDGRFA+ (green) staining at E18.5. Plots of the average number of PDGFRA+ cells (average counts ± SEM; t test p < 0.05 n=3). (d) Immunofluorescence of the mesothelial progenitor marker WT1 (red) with KI67 (green) staining indicating actively dividing WT1+ progenitors (white arrows). Plot of the number of WT1+ and WT+/KI67+ cells at E16.5 and E18.5. Svep1-/- lungs have more WT1+ cells than Svep1+/+ lungs at E18.5; however, the number of WT1+ KI67 lungs is unchanged between Svep1+/+ and Svep1-/- lungs. (e) labeling of endothelial cells with CD31 illustrating the double layer of the saccular microvasculature in normal lungs (arrowhead; and disorganized microvasculature in Svep1-/- lungs at E18.5. Scale bars 250 µm (B); 100 µm (c,d) 5 µm (e,h). Scale bars 25 µm (c,e); 50 µm (b) 100 µm (c).

Graphical summary o normal and Svep1-/- embryonic lung phenotypes during branching morphogenesis and saccular formation.

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