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Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis.

Bayha E, Jørgensen MC, Serup P, Grapin-Botton A - PLoS ONE (2009)

Bottom Line: Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm.These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm.Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT

Background: Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.

Methodology/principal findings: RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA(+) cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity.

Conclusions/significance: Our work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.

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Electroporation of dominant negative RARs abolishes CdxA expression and pancreas formation.Electroporation of pCIG (A,B,G,I,K) or pCIG-DNRAR (C–F,H,J,L). Whole mount in situ hybridization on stage HH 19 embryos shows CdxA expression (blue) in the closed duodenum and the open midgut. CdxA expression is repressed either partially (n = 7/11, C) or completely (n = 3/11, F) by DN-RAR. Cells expressing the expression construct are labeled by subsequent immunocytochemistry for GFP expressed from the bicistronic construct (Green in D, F and H, masked by blue CdxA staining in B, DAB-brown in G), demonstrating that repression extends to the neighbors of targeted cells. Whole mount immunocytochemistry on stage HH 20 embryos shows that dominant negative RAR (traced with GFP, green) represses pancreas progenitor emergence (traced with Nkx6.1, red) in a non-cell autonomous manner (J) as compared to control embryos electroporated with empty vector (I). Glucagon+ cells could still differentiate (blue). (K and L) Selected optical sections of embryos displayed in (I) and (J), respectively. Scale bar 200 µm.
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pone-0005845-g005: Electroporation of dominant negative RARs abolishes CdxA expression and pancreas formation.Electroporation of pCIG (A,B,G,I,K) or pCIG-DNRAR (C–F,H,J,L). Whole mount in situ hybridization on stage HH 19 embryos shows CdxA expression (blue) in the closed duodenum and the open midgut. CdxA expression is repressed either partially (n = 7/11, C) or completely (n = 3/11, F) by DN-RAR. Cells expressing the expression construct are labeled by subsequent immunocytochemistry for GFP expressed from the bicistronic construct (Green in D, F and H, masked by blue CdxA staining in B, DAB-brown in G), demonstrating that repression extends to the neighbors of targeted cells. Whole mount immunocytochemistry on stage HH 20 embryos shows that dominant negative RAR (traced with GFP, green) represses pancreas progenitor emergence (traced with Nkx6.1, red) in a non-cell autonomous manner (J) as compared to control embryos electroporated with empty vector (I). Glucagon+ cells could still differentiate (blue). (K and L) Selected optical sections of embryos displayed in (I) and (J), respectively. Scale bar 200 µm.

Mentions: The expression of retinoic acid receptors and reporter genes for pathway activity such as Cyp26A1 suggest direct activity in endoderm. We directly addressed the question by electroporating dominant negative retinoic acid receptors in endoderm. We observed that CdxA expression was either abolished (n = 3/11, highly electroporated embryos, not shown) or down-regulated (n = 7/11, lowly electroporated embryos) in endodermal cells expressing dominant negative receptors as compared to embryos electroporated with control plasmids (n = 16) (Fig. 5A–H). These results show that RA signaling is required directly in endoderm for CdxA expression. Moreover, CdxA expression was also repressed in neighboring endodermal cells several cell diameters away from the cells expressing dominant negative retinoic acid receptors (Fig. 5H). This shows that signaling between CdxA expressing cells is normally needed to maintain its expression.


Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis.

Bayha E, Jørgensen MC, Serup P, Grapin-Botton A - PLoS ONE (2009)

Electroporation of dominant negative RARs abolishes CdxA expression and pancreas formation.Electroporation of pCIG (A,B,G,I,K) or pCIG-DNRAR (C–F,H,J,L). Whole mount in situ hybridization on stage HH 19 embryos shows CdxA expression (blue) in the closed duodenum and the open midgut. CdxA expression is repressed either partially (n = 7/11, C) or completely (n = 3/11, F) by DN-RAR. Cells expressing the expression construct are labeled by subsequent immunocytochemistry for GFP expressed from the bicistronic construct (Green in D, F and H, masked by blue CdxA staining in B, DAB-brown in G), demonstrating that repression extends to the neighbors of targeted cells. Whole mount immunocytochemistry on stage HH 20 embryos shows that dominant negative RAR (traced with GFP, green) represses pancreas progenitor emergence (traced with Nkx6.1, red) in a non-cell autonomous manner (J) as compared to control embryos electroporated with empty vector (I). Glucagon+ cells could still differentiate (blue). (K and L) Selected optical sections of embryos displayed in (I) and (J), respectively. Scale bar 200 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690404&req=5

pone-0005845-g005: Electroporation of dominant negative RARs abolishes CdxA expression and pancreas formation.Electroporation of pCIG (A,B,G,I,K) or pCIG-DNRAR (C–F,H,J,L). Whole mount in situ hybridization on stage HH 19 embryos shows CdxA expression (blue) in the closed duodenum and the open midgut. CdxA expression is repressed either partially (n = 7/11, C) or completely (n = 3/11, F) by DN-RAR. Cells expressing the expression construct are labeled by subsequent immunocytochemistry for GFP expressed from the bicistronic construct (Green in D, F and H, masked by blue CdxA staining in B, DAB-brown in G), demonstrating that repression extends to the neighbors of targeted cells. Whole mount immunocytochemistry on stage HH 20 embryos shows that dominant negative RAR (traced with GFP, green) represses pancreas progenitor emergence (traced with Nkx6.1, red) in a non-cell autonomous manner (J) as compared to control embryos electroporated with empty vector (I). Glucagon+ cells could still differentiate (blue). (K and L) Selected optical sections of embryos displayed in (I) and (J), respectively. Scale bar 200 µm.
Mentions: The expression of retinoic acid receptors and reporter genes for pathway activity such as Cyp26A1 suggest direct activity in endoderm. We directly addressed the question by electroporating dominant negative retinoic acid receptors in endoderm. We observed that CdxA expression was either abolished (n = 3/11, highly electroporated embryos, not shown) or down-regulated (n = 7/11, lowly electroporated embryos) in endodermal cells expressing dominant negative receptors as compared to embryos electroporated with control plasmids (n = 16) (Fig. 5A–H). These results show that RA signaling is required directly in endoderm for CdxA expression. Moreover, CdxA expression was also repressed in neighboring endodermal cells several cell diameters away from the cells expressing dominant negative retinoic acid receptors (Fig. 5H). This shows that signaling between CdxA expressing cells is normally needed to maintain its expression.

Bottom Line: Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm.These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm.Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT

Background: Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.

Methodology/principal findings: RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA(+) cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity.

Conclusions/significance: Our work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.

Show MeSH
Related in: MedlinePlus