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Lung mesenchymal expression of Sox9 plays a critical role in tracheal development.

Turcatel G, Rubin N, Menke DB, Martin G, Shi W, Warburton D - BMC Biol. (2013)

Bottom Line: In situ hybridization showed an altered expression pattern of Tbx4, Tbx5 and Fgf10 genes and marked reduction of Collagen2 expression in the tracheal mesenchyme.Lymphatic vasculature was underdeveloped in the mutant trachea: Prox1, Lyve1, and Vegfr3 were decreased after Sox9 knockout.We also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer P63-positive cells and more CC10-positive cells, fewer goblet cells, and downregulation of surfactant proteins A and C.

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Affiliation: Developmental Biology and Regenerative Medicine Program, Saban Research Institute, Children's Hospital Los Angeles, Keck School of Medicine and Ostrow School of Dentistry, University of Southern California, 4661 Sunset Boulevard, Los Angeles, CA 90027, USA. dwarburton@chla.usc.edu.

ABSTRACT

Background: Embryonic lung development is instructed by crosstalk between mesenchyme and epithelia, which results in activation of transcriptional factors, such as Sox9, in a temporospatial manner. Sox9 is expressed in both distal lung epithelium and proximal lung mesenchyme. Here, we investigated the effect of lung mesenchyme-specific inducible deletion of Sox9 during murine lung development.

Results: Transgenic mice lacking Sox9 expression were unable to breathe and died at birth, with noticeable tracheal defects. Cartilage rings were missing, and the tracheal lumen was collapsed in the mutant trachea. In situ hybridization showed an altered expression pattern of Tbx4, Tbx5 and Fgf10 genes and marked reduction of Collagen2 expression in the tracheal mesenchyme. The tracheal phenotype was increasingly severe, with longer duration of deletion. Lymphatic vasculature was underdeveloped in the mutant trachea: Prox1, Lyve1, and Vegfr3 were decreased after Sox9 knockout. We also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer P63-positive cells and more CC10-positive cells, fewer goblet cells, and downregulation of surfactant proteins A and C.

Conclusion: The appropriate temporospatial expression of Sox9 in lung mesenchyme is necessary for appropriate tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation. We uncovered a novel mechanism of lung epithelium differentiation: tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development. Thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung.

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Lung phenotype after mesenchymal Sox9 knockout. (A) Pups were collected at birth. Mutant pups died less than an hour after birth, and showed gasping, retractions, and cyanosis. (B) Mutant pups did not survive post-natally. (C) Despite the respiratory mortality phenotype, no change in weight at birth was observed between the wild-type pups and the mutant pups. (D-I) Samples of lung at embryonic day (E) 18.5 were collected and analyzed. (E)Sox9Δ/Δ did not show obvious altered lung branching compared with (D) the control. (F, G) Alcian blue staining of (F) E18.5 wild-type and (G) mutant lungs: normal tracheal rings were observed in the control normal lung, Sox9 knockout trachea revealed no tracheal rings. (H, I) Transverse section of E18.5 trachea was stained with Alcian blue. (H) Wild-type and (I) mutant mice.
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Figure 2: Lung phenotype after mesenchymal Sox9 knockout. (A) Pups were collected at birth. Mutant pups died less than an hour after birth, and showed gasping, retractions, and cyanosis. (B) Mutant pups did not survive post-natally. (C) Despite the respiratory mortality phenotype, no change in weight at birth was observed between the wild-type pups and the mutant pups. (D-I) Samples of lung at embryonic day (E) 18.5 were collected and analyzed. (E)Sox9Δ/Δ did not show obvious altered lung branching compared with (D) the control. (F, G) Alcian blue staining of (F) E18.5 wild-type and (G) mutant lungs: normal tracheal rings were observed in the control normal lung, Sox9 knockout trachea revealed no tracheal rings. (H, I) Transverse section of E18.5 trachea was stained with Alcian blue. (H) Wild-type and (I) mutant mice.

Mentions: Perl reported that epithelial Sox9 deletion did not affect lung development [10]. However, more recently, Chang showed that Sox9 deletion in lung epithelium resulted in a smaller lung, with fewer and dilated airway branches [11]. It is possible that the use of different mouse genetic backgrounds is the cause of these contrasting results. The role of mesenchymal Sox9 expression is unknown. In our study, transgenic mice lacking mesenchymal Sox9 expression were born at the expected mendelian frequency and appeared normal at birth, but rapidly became cyanotic and showed marked signs of respiratory obstruction including gasping and retractions (Figure 2A); moreover, not a single transgenic mouse lived more than a few hours after birth (Figure 2B). Despite the lethal respiratory obstruction phenotype, no difference in weight at birth was observed (Figure 2C). At both E15.5 and E18.5, embryonic lung branching did not appear to be affected by lack of mesenchymal Sox9 expression (Figure 2D,E; see Additional file 1: Figure S1A-D). However, transgenic embryos were missing cartilage rings around the trachea and bronchi. (Figure 2F-I; see Additional file 1: Figure S1E,F). Transverse sections of transgenic embryo E18.5 tracheas showed collapsed airway lumen, and a shape and appearance similar to esophagus; however, the esophagus was still present. In 8% of the embryos, rudimentary cartilage spots developed on the ventral side of the trachea (see Additional file 2: Figure S2B black arrowheads). These spots were still expressing Sox9 protein, indicating the existence of another unique population of Sox9+ cells that either did not continue to express the Tbx4 gene or did not express the transgene, or expressed the transgene, but did not recombine (see Additional file 2: Figure S2C). Lung branching was not affected by lack of mesenchyme Sox9 expression (Figure 3A-C), and differentiation of distal lung epithelium and mesenchyme appeared to be normal in the mutants (Figure 3D-G).


Lung mesenchymal expression of Sox9 plays a critical role in tracheal development.

Turcatel G, Rubin N, Menke DB, Martin G, Shi W, Warburton D - BMC Biol. (2013)

Lung phenotype after mesenchymal Sox9 knockout. (A) Pups were collected at birth. Mutant pups died less than an hour after birth, and showed gasping, retractions, and cyanosis. (B) Mutant pups did not survive post-natally. (C) Despite the respiratory mortality phenotype, no change in weight at birth was observed between the wild-type pups and the mutant pups. (D-I) Samples of lung at embryonic day (E) 18.5 were collected and analyzed. (E)Sox9Δ/Δ did not show obvious altered lung branching compared with (D) the control. (F, G) Alcian blue staining of (F) E18.5 wild-type and (G) mutant lungs: normal tracheal rings were observed in the control normal lung, Sox9 knockout trachea revealed no tracheal rings. (H, I) Transverse section of E18.5 trachea was stained with Alcian blue. (H) Wild-type and (I) mutant mice.
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Figure 2: Lung phenotype after mesenchymal Sox9 knockout. (A) Pups were collected at birth. Mutant pups died less than an hour after birth, and showed gasping, retractions, and cyanosis. (B) Mutant pups did not survive post-natally. (C) Despite the respiratory mortality phenotype, no change in weight at birth was observed between the wild-type pups and the mutant pups. (D-I) Samples of lung at embryonic day (E) 18.5 were collected and analyzed. (E)Sox9Δ/Δ did not show obvious altered lung branching compared with (D) the control. (F, G) Alcian blue staining of (F) E18.5 wild-type and (G) mutant lungs: normal tracheal rings were observed in the control normal lung, Sox9 knockout trachea revealed no tracheal rings. (H, I) Transverse section of E18.5 trachea was stained with Alcian blue. (H) Wild-type and (I) mutant mice.
Mentions: Perl reported that epithelial Sox9 deletion did not affect lung development [10]. However, more recently, Chang showed that Sox9 deletion in lung epithelium resulted in a smaller lung, with fewer and dilated airway branches [11]. It is possible that the use of different mouse genetic backgrounds is the cause of these contrasting results. The role of mesenchymal Sox9 expression is unknown. In our study, transgenic mice lacking mesenchymal Sox9 expression were born at the expected mendelian frequency and appeared normal at birth, but rapidly became cyanotic and showed marked signs of respiratory obstruction including gasping and retractions (Figure 2A); moreover, not a single transgenic mouse lived more than a few hours after birth (Figure 2B). Despite the lethal respiratory obstruction phenotype, no difference in weight at birth was observed (Figure 2C). At both E15.5 and E18.5, embryonic lung branching did not appear to be affected by lack of mesenchymal Sox9 expression (Figure 2D,E; see Additional file 1: Figure S1A-D). However, transgenic embryos were missing cartilage rings around the trachea and bronchi. (Figure 2F-I; see Additional file 1: Figure S1E,F). Transverse sections of transgenic embryo E18.5 tracheas showed collapsed airway lumen, and a shape and appearance similar to esophagus; however, the esophagus was still present. In 8% of the embryos, rudimentary cartilage spots developed on the ventral side of the trachea (see Additional file 2: Figure S2B black arrowheads). These spots were still expressing Sox9 protein, indicating the existence of another unique population of Sox9+ cells that either did not continue to express the Tbx4 gene or did not express the transgene, or expressed the transgene, but did not recombine (see Additional file 2: Figure S2C). Lung branching was not affected by lack of mesenchyme Sox9 expression (Figure 3A-C), and differentiation of distal lung epithelium and mesenchyme appeared to be normal in the mutants (Figure 3D-G).

Bottom Line: In situ hybridization showed an altered expression pattern of Tbx4, Tbx5 and Fgf10 genes and marked reduction of Collagen2 expression in the tracheal mesenchyme.Lymphatic vasculature was underdeveloped in the mutant trachea: Prox1, Lyve1, and Vegfr3 were decreased after Sox9 knockout.We also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer P63-positive cells and more CC10-positive cells, fewer goblet cells, and downregulation of surfactant proteins A and C.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology and Regenerative Medicine Program, Saban Research Institute, Children's Hospital Los Angeles, Keck School of Medicine and Ostrow School of Dentistry, University of Southern California, 4661 Sunset Boulevard, Los Angeles, CA 90027, USA. dwarburton@chla.usc.edu.

ABSTRACT

Background: Embryonic lung development is instructed by crosstalk between mesenchyme and epithelia, which results in activation of transcriptional factors, such as Sox9, in a temporospatial manner. Sox9 is expressed in both distal lung epithelium and proximal lung mesenchyme. Here, we investigated the effect of lung mesenchyme-specific inducible deletion of Sox9 during murine lung development.

Results: Transgenic mice lacking Sox9 expression were unable to breathe and died at birth, with noticeable tracheal defects. Cartilage rings were missing, and the tracheal lumen was collapsed in the mutant trachea. In situ hybridization showed an altered expression pattern of Tbx4, Tbx5 and Fgf10 genes and marked reduction of Collagen2 expression in the tracheal mesenchyme. The tracheal phenotype was increasingly severe, with longer duration of deletion. Lymphatic vasculature was underdeveloped in the mutant trachea: Prox1, Lyve1, and Vegfr3 were decreased after Sox9 knockout. We also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer P63-positive cells and more CC10-positive cells, fewer goblet cells, and downregulation of surfactant proteins A and C.

Conclusion: The appropriate temporospatial expression of Sox9 in lung mesenchyme is necessary for appropriate tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation. We uncovered a novel mechanism of lung epithelium differentiation: tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development. Thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung.

Show MeSH
Related in: MedlinePlus