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Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation.

Woo J, Miletich I, Kim BM, Sharpe PT, Shivdasani RA - PLoS ONE (2011)

Bottom Line: This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi.The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1(-/-) foregut.Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT
Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1(-/-) mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1(-/-) esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1(-/-) foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.

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Anatomy and marker expression in E10.5 thoracic foregut of E10.5 Barx1+/+ (A–J) and Barx1−/− (K–T) embryos.Anatomic structures and the domains of Nkx2.1 (red) and Sox2 (purple) expression are depicted in diagrams in A and K, where axial levels of each row of micrographs are marked with dotted lines. The left column shows hematoxylin and eosin-stained tissue sections, with dashed lines demarcating the undivided foregut (Fo), esophagus (Es), trachea (Tr) and mainstem bronchi (Br); these dashed lines carry over into the immunohistochemical micrographs for NKX2.1 (middle) and SOX2 (right column). In each image, dorsal is on top and ventral on the bottom. The results reveal ectopic NKX2.1 (red arrowhead in S) and loss of SOX2 (purple arrowhead in T) expression in the ventral endoderm of the undivided Barx1−/− foregut, corresponding to tissue that shows SOX2 (purple arrow in J) but no Nkx2.1 (red arrow in I) expression in Barx1+/+ littermates. Results are representative of experiments with 3 embryos of each genotype.
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pone-0022493-g002: Anatomy and marker expression in E10.5 thoracic foregut of E10.5 Barx1+/+ (A–J) and Barx1−/− (K–T) embryos.Anatomic structures and the domains of Nkx2.1 (red) and Sox2 (purple) expression are depicted in diagrams in A and K, where axial levels of each row of micrographs are marked with dotted lines. The left column shows hematoxylin and eosin-stained tissue sections, with dashed lines demarcating the undivided foregut (Fo), esophagus (Es), trachea (Tr) and mainstem bronchi (Br); these dashed lines carry over into the immunohistochemical micrographs for NKX2.1 (middle) and SOX2 (right column). In each image, dorsal is on top and ventral on the bottom. The results reveal ectopic NKX2.1 (red arrowhead in S) and loss of SOX2 (purple arrowhead in T) expression in the ventral endoderm of the undivided Barx1−/− foregut, corresponding to tissue that shows SOX2 (purple arrow in J) but no Nkx2.1 (red arrow in I) expression in Barx1+/+ littermates. Results are representative of experiments with 3 embryos of each genotype.

Mentions: To investigate the role of Barx1 in anterior foregut development, we assessed tissue morphology and expression of the transcription factors Nkx2.1, Sox2 and p63 in mutant and control embryos. The homeodomain protein Nkx2.1 is one of the earliest markers of developing respiratory endoderm, including trachea and lungs [11]; Sox2 is a useful marker of the dorsal foregut, esophagus, and anterior stomach [12], whereas p63 is required for development of many stratified epithelia, including that of the esophagus [12], [13]. Compared to wild-type controls, in which the esophagus and trachea were well separated by E10.5 (Fig. 2E–G), Barx1−/− embryos showed contiguity of the Nkx2.1-expressing tracheal epithelium and Sox2-expressing esophageal epithelium in a single luminal structure, indicating failure of foregut septation (Fig. 2 M–N, P–Q, S–T). This pattern of marker expression was well reflected in the tissue morphology, as Barx1−/− embryos showed a single elongated foregut tube instead of distinct tracheal and esophageal structures; absence of separation was evident over the full length of the thoracic foregut, extending caudal to the coronal level of the mainstem bronchi (Fig. 2 L, O, R). The presence of a strong Nkx2.1 signal in the bronchial epithelium points to intact lung bud formation and lung endoderm specification in Barx1−/− embryos. Furthermore, Nkx2.1-expressing lung progenitors, present in the ventral thoracic foregut, continued into the esophagus-stomach junction (Fig. 2S, red arrowhead). By contrast, Sox2-expressing esophageal endoderm was confined to the dorsal surface and excluded from the ventral esophagus and stomach (Fig. 2T, purple arrowhead). The stratified epithelial marker p63, present in wild-type E10.5 esophagus, was undetectable in Nkx2.1-expressing dorsal foregut cells in Barx1−/− mutants (blue arrowhead in Fig. S1E), suggesting inadequate squamous cell differentiation.


Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation.

Woo J, Miletich I, Kim BM, Sharpe PT, Shivdasani RA - PLoS ONE (2011)

Anatomy and marker expression in E10.5 thoracic foregut of E10.5 Barx1+/+ (A–J) and Barx1−/− (K–T) embryos.Anatomic structures and the domains of Nkx2.1 (red) and Sox2 (purple) expression are depicted in diagrams in A and K, where axial levels of each row of micrographs are marked with dotted lines. The left column shows hematoxylin and eosin-stained tissue sections, with dashed lines demarcating the undivided foregut (Fo), esophagus (Es), trachea (Tr) and mainstem bronchi (Br); these dashed lines carry over into the immunohistochemical micrographs for NKX2.1 (middle) and SOX2 (right column). In each image, dorsal is on top and ventral on the bottom. The results reveal ectopic NKX2.1 (red arrowhead in S) and loss of SOX2 (purple arrowhead in T) expression in the ventral endoderm of the undivided Barx1−/− foregut, corresponding to tissue that shows SOX2 (purple arrow in J) but no Nkx2.1 (red arrow in I) expression in Barx1+/+ littermates. Results are representative of experiments with 3 embryos of each genotype.
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pone-0022493-g002: Anatomy and marker expression in E10.5 thoracic foregut of E10.5 Barx1+/+ (A–J) and Barx1−/− (K–T) embryos.Anatomic structures and the domains of Nkx2.1 (red) and Sox2 (purple) expression are depicted in diagrams in A and K, where axial levels of each row of micrographs are marked with dotted lines. The left column shows hematoxylin and eosin-stained tissue sections, with dashed lines demarcating the undivided foregut (Fo), esophagus (Es), trachea (Tr) and mainstem bronchi (Br); these dashed lines carry over into the immunohistochemical micrographs for NKX2.1 (middle) and SOX2 (right column). In each image, dorsal is on top and ventral on the bottom. The results reveal ectopic NKX2.1 (red arrowhead in S) and loss of SOX2 (purple arrowhead in T) expression in the ventral endoderm of the undivided Barx1−/− foregut, corresponding to tissue that shows SOX2 (purple arrow in J) but no Nkx2.1 (red arrow in I) expression in Barx1+/+ littermates. Results are representative of experiments with 3 embryos of each genotype.
Mentions: To investigate the role of Barx1 in anterior foregut development, we assessed tissue morphology and expression of the transcription factors Nkx2.1, Sox2 and p63 in mutant and control embryos. The homeodomain protein Nkx2.1 is one of the earliest markers of developing respiratory endoderm, including trachea and lungs [11]; Sox2 is a useful marker of the dorsal foregut, esophagus, and anterior stomach [12], whereas p63 is required for development of many stratified epithelia, including that of the esophagus [12], [13]. Compared to wild-type controls, in which the esophagus and trachea were well separated by E10.5 (Fig. 2E–G), Barx1−/− embryos showed contiguity of the Nkx2.1-expressing tracheal epithelium and Sox2-expressing esophageal epithelium in a single luminal structure, indicating failure of foregut septation (Fig. 2 M–N, P–Q, S–T). This pattern of marker expression was well reflected in the tissue morphology, as Barx1−/− embryos showed a single elongated foregut tube instead of distinct tracheal and esophageal structures; absence of separation was evident over the full length of the thoracic foregut, extending caudal to the coronal level of the mainstem bronchi (Fig. 2 L, O, R). The presence of a strong Nkx2.1 signal in the bronchial epithelium points to intact lung bud formation and lung endoderm specification in Barx1−/− embryos. Furthermore, Nkx2.1-expressing lung progenitors, present in the ventral thoracic foregut, continued into the esophagus-stomach junction (Fig. 2S, red arrowhead). By contrast, Sox2-expressing esophageal endoderm was confined to the dorsal surface and excluded from the ventral esophagus and stomach (Fig. 2T, purple arrowhead). The stratified epithelial marker p63, present in wild-type E10.5 esophagus, was undetectable in Nkx2.1-expressing dorsal foregut cells in Barx1−/− mutants (blue arrowhead in Fig. S1E), suggesting inadequate squamous cell differentiation.

Bottom Line: This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi.The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1(-/-) foregut.Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT
Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1(-/-) mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1(-/-) esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1(-/-) foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.

Show MeSH
Related in: MedlinePlus