Osr1 functions downstream of Hedgehog pathway to regulate foregut development

Abstract

During early fetal development, paracrine Hedgehog (HH) ligands secreted from the foregut epithelium activate Gli transcription factors in the surrounding mesenchyme to coordinate formation of the respiratory system, digestive track and the cardiovascular network. Although disruptions to this process can lead to devastating congenital defects, the underlying mechanisms and downstream targets, are poorly understood. We show that the zinc finger transcription factor Osr1 is a novel HH target as Osr1 expression in the foregut mesenchyme depends on HH signaling and the effector of HH pathway Gli3 binds to a conserved genomic loci near Osr1 promoter region. Molecular analysis of mouse germline Osr1 mutants reveals multiple functions of Osr1 during foregut development. Osr1 mutants exhibit fewer lung progenitors in the ventral foregut. Osr is then required for the proper branching of the primary lung buds, with mutants exhibiting miss-located lung lobes. Finally, Osr1 is essential for proper mesenchymal differentiation including pulmonary arteries, esophageal and tracheal smooth muscle as well as tracheal cartilage rings. Tissue specific conditional knockouts in combination with lineage tracing indicate that Osr1 is required cell autonomously in the foregut mesenchyme. We conclude that Osr1 is a novel downstream target of HH pathway, required for lung specification, branching morphogenesis and foregut mesenchymal differentiation.

Keywords: Esophagus; Hedgehog; Lobulation; Lung; Mesenchyme; Odd-skipped; Specification; Trachea.

Fig. 1

Osr1 is a Gli2,3 target and displays dynamic expression pattern during foregut development. (A) Shh in situ staining on E9.0 transverse foregut section. Arrow points to foregut epithelial. (B) LacZ staining in foregut using Gli1^lacZ reporter mouse. (C–D) Osr1 in situ on transverse sections of E9.0 foregut in control and Gli2^−/−;Gli3^−/− double mutant. Yellow dotted lines mark endoderm while the region in between yellow and red dotted lines denotes the splanchnic mesoderm. Arrows point to splanchnic mesenchyme. (E) Whole mount in situ of Osr1 in dissected E10.5 foregut. Scale bar: 500 μm. Dotted lines corresponds to the section levels in Fig. Ea–Ec. (Ea–Ec) Osr1 in situ on transverse sections of foregut region at E10.5 from anterior to posterior. (F) Whole mount Osr1 in situ in E11.5 dissected respiratory tissue. Scale bar: 500 μm. (G–I) Osr1 in situ on transverse sections of foregut at E12.5 from anterior to posterior region. Orange dotted line marks esophagus and red dotted line marks respiratory epithelial cells. (J) Gli1 in situ on transverse sections through trachea and main bronchi. Scale bar: 100 μm. (M) Browser shot of previously published ChIP-seq data in second heart field tissue comparing Gli3-flag ChIP to input around Osr1 region together with mammal pairwise conservation. Blue box indicates the significant Gli3-flag ChIP signal located 40kb downstream of Osr1 transcription start site.

Fig. 2

Osr1 mutants display lung hypoplasia, dysmorphyc lobulation and mesenchymal differentiation defects. (A–B) Bright field view of dissected esophagus, trachea and lung. (C–D) Alcian blue staining of dissected trachea. Arrows show broken and disorganized cartilage rings in Osr1 mutant compared to control. (E–F) Whole mount IF of Nkx2-1 in wildtype and mutant lungs. Arrows point to the anterior extension of the cranial lobe relative to the trachea bifurcation. Scale bar: 200 μm. (G–J) H&E staining of transverse sections lung (G–H, scale bar: 500 μm) and main bronchi (I–J, scale bar: 100 μm) regions. Arrows indicate pulmonary arteries. Med, medial lobe; lef, left lobe; acc, accessory lobe; eso, esophagus; bro, bronchus; cra, cranial lobe.

Fig. 3

Osr1 is required for sufficient respiratory specification and accurate lobe formation. (A) Whole mount immunostaining of foregut with respiratory marker (Nkx2-1), esophageal marker (Sox2), proliferation (p-HH3) and apoptosis (C-caspase 3) markers in dissected foreguts. (B) Quantification of respiratory specification marked by Nkx2-1+ cells normalized by the entire foregut epithelial cells in between pharynx and liver at E9.5 and the total number of Nkx2-1+ cells at E10.5. (C) In situ staining of Axin2, Wnt2, 2b and Bmp4 and IF stainging of p-Smad 1,5,9 in wildtypes and Osr1 mutants. (D) Whole mount IF with dissected lungs. Yellow lines indicate the distance between cranial lobe and trachea bifurcation. Cra, cranial lobe; med, medial lobe; cau, caudal lobe; lef, left lobe; acc, accessory lobe. Asterisks indicate the distances from cranial lobe to tracheal bifurcation and from accessory lobe to medial lobe are both significantly changed. Arrows indicate cranial lobe locations. Scale bars: 100 μm.

Fig. 4

Mesenchymal development including pulmonary arteries and smooth muscle differentiation in foregut is dependent on Osr1. (A) Whole mount IF staining in E10.5 embryos. Yellow arrows mark the vascular plexus connecting the out flow tract to the lungs. Scale bar: 50 μm. (B–C) αSMA whole mount staining of dissected foregut. Epithelial staining (not shown) is used to guide the identification of different smooth muscle cell structures. Different regions of the staining are isolated and pseudo-colored using Imaris. (D–E) Whole mount staining of the dissected foregut with the heart attached. Arrows point to the pulmonary arteries. Scale bars: 100 μm. (F–I) IF on transverse sections at the trachea level (F–G) and the main bronchi level (H–I). Purple arrows indicate esophagus SMC, green arrows indicate tracheal SMC, while red arrows indicate pulmonary arteries SMC. OFT, out flow tract; p. art, pulmonary artery; tra, trachea; eso, esophagus; bro, bronchi. Scale bars: 50 μm.

Fig. 5

Osr1 is required in the mesenchyme to support foregut development. (AD) Confirmation of loss of Osr1 in the mesenchyme. Exon specific in situ detecting either exon1 which is not floxed, and exon2 which is within the floxed region. (E–F) In the mesenchymal conditional knockout of Osr1, cranial lobe anterior shifting is evident at E12.5 (the relative distance ratio of yellow to green line), though to a lesser extent compared to the germline Osr1 mutant. Scale bars: 100 μm. (G–H) At E15.5, the mesenchymal conditional knockout lung demonstrates very similar hypoplasia and misshaping as the germline mutant. Scale bar: 2 mm. (I–J) Whole mount IF shows compromised pulmonary artery (dotted line) development in mesenchymal Osr1 knockout. Scale bar: 100 μm. (K–N) Longitudinal (K,L) and transverse (M–N) sections demonstrate failure of two layers of smooth muscle (red arrows) formation surrounding esophagus without Osr1 in mesenchyme, sparse trachea smooth muscle (yellow arrows) formation as well as disorganized tracheal cartilage (yellow arrowheads). Scale bars: 50 μm.

Fig. 6

Osr1 expressing progenitors contribute to multiple mesenchymal lineages. (A) GFP lineage labeling is co-stained with multiple mesenchymal lineage markers at E12.5. The upper panel shows co-staining while the lower panel shows the lineage markers alone. Arrowheads indicate co-stainings. Art, artery; eso, esophagus; tra, trachea. Scale bars: 15 μm. (B) β-gal co-staining with Sox9 on transverse sections. Scale bar: 100 μm. (C–D) β-gal co-staining with αSMA on transverse sections. Red arrows indicate esophageal smooth muscle, while yellow arrows indicate pulmonary artery. Scale bar: 50 μm. Scale bars in inserts: 10 μm.

Fig. 7

In conclusion, Osr1 functions downstream of HH pathway and regulate multiple aspects of foregut development. (A) during lobulation, Osr1, which expression is restricted in the medial mesenchyme, prevents medial/anterior shifting of cranial lobe; (B) during trachea and esophagus mesenchyme differentiation, Osr1 is expressed in the peripheral mesenchyme surrounding trachea and esophagus, supporting esophagus and trachea smooth muscle differentiation, trachea cartilage ring formation and pulmonary arteries development. Blue spaces indicate Osr1 expression domains; red dotted line outlines the splanchnic mesenchyme. Yellow marks the endoderm; red spaces mark esophageal smooth muscle in the dorsal and tracheal smooth muscle in the ventral; green space marks the cartilage; red circles show pulmonary artery smooth muscle. D, dorsal; V, ventral; A, anterior; P, posterior.