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doi: 10.1242/10.1242/dev.00345

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Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development

Jun-ichi Sakiyama1, Atsushi Yamagishi1,2 and Atsushi Kuroiwa1,*

1 Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
2 CREST, Japan Science and Technology Corporation (JST), Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan



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  		Fig. 1. Schematic representation of the chick respiratory tract development and expression of Tbx4, Fgf10 and Nkx2.1 in the respiratory tract of the chick embryo. (A) The chick respiratory tract development. (B-H) Expression of Tbx4 (B,D-H) and Nkx2.1 (C-E) in the chick embryonic foregut at stage 15 (B-D), 18 (E), 19 (F) and 24 (G,H) visualized by whole-mount in situ hybridization. (B,C,F), Views from the left side. (D,E,G), Frontal views. (H) Cross-section as indicated in G. (D) Two-color whole-mount in situ hybridization with probes for Tbx4 (blue) and Nkx2.1 (purple). (E) Two-color whole-mount in situ hybridization with probes for Tbx4 (red), Foxa2 (red) and Nkx2.1 (purple). Endodermal red staining in E indicates the expression of Foxa2, a pan-endoderm marker gene. Note that the respiratory endoderm (Nkx2.1-expressing endoderm, purple stained endoderm) also expresses Foxa2. Arrowheads and arrows in B,C indicate the position of the anterior and posterior boundaries of Tbx4 expression, respectively. Double arrow in C indicates the position of the anterior boundaries of Nkx2.1 expression. Blue arrowhead in D and red arrowhead in E indicate the position of the anterior boundary of Tbx4 expression. Blue arrow fringed with purple in D indicates the position of the posterior boundaries of Tbx4 and Nkx2.1 expression. Red arrow in E indicates the position of the posterior boundary of Tbx4 expression. Purple double arrow in D and black double arrow in E indicate the position of the anterior boundaries of Nkx2.1 expression. Broken lines in C indicate the outline of the endodermal tube. The broken yellow line in G represents the bifurcation pattern of the endodermal layer of the respiratory tract. (I-K) Expression of Fgf10 (purple) and Nkx2.1 (blue) in the chick embryonic foregut at stage 15. (I) View from the left side. (J) Frontal view. (K) Cross-section as indicated in I. Top of the K is the dorsal side. Purple arrowheads in I,J indicate the position of the anterior boundary of Fgf10 expression. Blue arrows fringed with purple in I,J indicate the position of the posterior boundaries of Fgf10 and Nkx2.1 expression. Blue double arrows in I,J indicate the position of the anterior boundaries of Nkx2.1 expression. en, endoderm; es, esophagus; eso-res, esophagus-respiratory region; res en, respiratory endoderm; st, stomach; lt, laryngotracheal groove; ph, pharynx; tr, trachea; vm, visceral mesoderm; LL, left lung bud; RL, right lung bud.

 


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  		Fig. 2. Misexpression of Tbx4 causes ectopic endodermal budding and tracheo-esophageal fistula. Tbx4 misexpression induced ectopic endodermal budding in the underlying endoderm. (A-F) The foreguts from a Tbx4 misexpressed embryo 2 days after electroporation. (A,E,F) Frontal views. (B,C) Dorsal views. (D) View from the right. (A) Ectopic endodermal bud (yellow arrowhead) formed in the ventral distal esophagus. The endoderm was visualized by whole-mount in situ hybridization with a Nkx2.1 probe. (B,C) Ectopic endodermal bud (yellow arrowhead) formed in the distal esophagus as in A. (C) Tbx4-transfected mesoderm monitored by GFP fluorescence. (D) Ectopic endodermal bud (yellow arrowhead) formed in the dorsal distal esophagus. The endoderm was visualized by whole mount in situ hybridization with probes for Nkx2.1 (purple) and Foxa2 (red), a pan-endoderm marker gene. (E,F) Ectopic endodermal bud formed between the primary bud, which results in trifurcation of the bronchi. (F) Tbx4-transfected mesoderm monitored by GFP fluorescence. (G) Cross-section of the foregut of the Tbx4 misexpressed embryo showing tracheo-esophageal fistula. The endoderm was visualized by whole-mount in situ hybridization with a Shh probe, followed by sectioning. The arrow indicates fusion of the esophagus and tracheal endoderm. cont, control; exp, experimental.

 


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  		Fig. 3. Fgf10 and Bmp4 expression in the Tbx4 misexpressing foregut. (A-C) Induction of ectopic Fgf10 expression in the Tbx4-misexpressing mesoderm. (A) The Tbx4-transfected mesoderm was monitored by GFP fluorescence from the dorsal view 2 days after electroporation. (B) Fgf10 expression in the same specimen from the same viewpoint as in A. Blue arrowheads indicate the esophagus mesoderm misexpressing exogenous Tbx4. (C) Cross-section as indicated in B. (D,E) Tbx4 misexpression does not affect Bmp4 expression. (D) The Tbx4-transfected region was monitored by GFP fluorescence 2 days after electroporation. (E) Bmp4 expression in the same specimen from the same view point as in D. Red arrowheads in D,E indicate the esophagus mesoderm misexpressing exogenous Tbx4. es, esophagus; ph, pharynx; st, stomach; tr, trachea; LL, left lung; RL, right lung; Lbr, left main bronchus; Rbr, right main bronchus.

 


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  		Fig. 4. Misexpression of Fgf10 causes ectopic endodermal budding, tracheo-esophageal fistula and ectopic Tbx4 expression. (A-C) Fgf10 misexpression induced ectopic endodermal budding in the underlying endoderm. Foregut of the Fgf10 misexpressing embryo 2 days after electroporation. The endoderm was visualized by whole-mount in situ hybridization with Nkx2.1 (purple) and Foxa2 (red) probes. (A,B) Views from the right. (C) Frontal view. (A,B), Ectopic endodermal buds (yellow arrowheads) formed in the dorsal distal esophagus viewed from the right. The Fgf10-transfected mesoderm is visualized by merging with the picture of GFP fluorescence (green) in B. (C) Ectopic endodermal bud (yellow arrowhead) formed in the ventral distal esophagus. (D-F) Fgf10 misexpression induced ectopic Tbx4 expression. Foregut of the Fgf10 misexpressing embryo 2 days after electroporation. (D) The Fgf10-transfected mesoderm monitored by GFP fluorescence. (E) Tbx4 expression in the same specimen as D. Red arrowheads indicate ectopic Tbx4 expression. Yellow arrowhead indicates the Fgf10-transfected mesoderm. (F) Cross-section as indicated in E. (G) Cross-section of the foregut of the Fgf10 misexpressed embryo showing tracheo-esophageal fistula. The endoderm was visualized by whole-mount in situ hybridization with Nkx2.1 (purple) and Foxa2 (red) probes, followed by sectioning. Arrow indicates fusion of the esophagus and tracheal endoderm. en, endoderm; es, esophagus; tr, trachea; vm, visceral mesoderm; LL, left lung bud; RR, right lung bud; cont, control; exp, experimental.

 


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  		Fig. 5. Misexpression of variant forms of Tbx4 in ovo. (A-F,I,J), Frontal views of the foregut 1 day after electroporation. (H) Dorsal view of the foregut 2 days after electroporation. (A,B), Tbx4 misexpression. (A) The Tbx4-transfected mesoderm monitored by GFP fluorescence. (B) Fgf10 expression in the same specimen as in A. (C,D), Tbx4-VP16 misexpression. (C) The Tbx4-VP16-transfected mesoderm monitored by GFP fluorescence. (D) Fgf10 expression of the same specimen as in C. (E-H) Tbx4-EnR misexpression. (E) The Tbx4-EnR-transfected mesoderm monitored by GFP fluorescence. Red arrows in E-G indicate the distal esophagus mesoderm, which expresses ectopic Fgf10. Blue arrows in E-G indicate the distal esophagus mesoderm on the contralateral side. (F) Fgf10 expression in the same specimen as in E. (G) Schematic representation of F. Darkblue area indicates ectopic Fgf10 expression in the distal esophagus mesoderm. Light blue areas indicate endogenous Fgf10 expression. Green stippled area indicates Tbx4-EnR/GFP-expressing mesoderm. (H) Nkx2.1 expression. The Tbx4-EnR-transfected mesoderm is visualized by merging with the picture of GFP fluorescence (green). Arrowhead indicates ectopic endodermal bud in the distal esophagus. (I,J) GFP misexpression. (I) The GFP-transfected mesoderm monitored by GFP fluorescence. (J) Fgf10 expression in the same specimen as in I. des, distal esophagus; tl, tip of the lung; LL, left lung bud; RL, right lung bud.

 


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  		Fig. 6. Nkx2.1 expression in the Tbx4 or Fgf10 misexpressing foregut. (A-F) Nkx2.1 expression in the foregut 2 days after Tbx4 or Fgf10 electroporation. (A-C) Tbx4 misexpression induces ectopic Nkx2.1 expression in the distal esophagus endoderm. (A,B) The same specimen viewed from the dorsal (A) and ventral (B) sides. The Tbx4-transfected mesoderm is visualized by merging with the picture of GFP fluorescence (green) in A. Yellow arrowheads in A,B indicate ectopic endodermal buds. (C) Cross-section of the distal esophagus showing that ectopic Nkx2.1 expression is restricted to the ventral endoderm on the Tbx4-transfected side. (D-F) Fgf10 misexpression induces ectopic Nkx2.1 expression in the distal esophagus endoderm. (D) The Fgf10-transfected mesoderm monitored by GFP fluorescence from the right side. (E) Nkx2.1 expression in the same sample and from the same viewpoint as in D. Blue arrowhead indicates ectopic Nkx2.1 expression in the distal esophagus endoderm. Orange arrowheads indicate the overproliferation of the mesoderm. Broken lines indicate the outline of the endodermal tube. (F) Cross-section of the distal esophagus showing that ectopic Nkx2.1 expression is restricted to the ventral endoderm. (G,H) Normal Nkx2.1 (purple) and Foxa2 (red) expression at stage 20. (G) Frontal view. (H) View from the right side. des, distal esophagus; es, esophagus; st, stomach; tr, trachea; LL, left lung bud; RL, right lung bud; cont, control; exp, experimental.

 

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