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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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Related in: MedlinePlus

RA and FGF4 independently pattern the anterior endoderm.Whole mount in situ hybridization analysis of Hex expression. Ventral view, anterior to the top. (A–D) Analysis of embryos when FGF4 signaling is activated and RA signaling is inhibited. Embryos were treated at stage HH 3+ with DMSO and grafted with PBS beads (A) as control, treated with DMSO and grafted with FGF4 beads (1 mg/ml) (B), treated with10−5 M AGN193109 and grafted with PBS beads (C), or treated with 10−5 M AGN193109 and grafted with FGF4 beads (1 mg/ml) (D). Circles show position of beads in (A–D). (E–H) Analysis of embryos when FGF4 signaling is inhibited and RA signaling is activated. Embryos were treated at stage HH 3+ either with DMSO and ethanol (E) as control, with DMSO and 10−6 M RA (F), with 20 µM SU5402 and ethanol (G), or with 10−6 M RA and 20 µM SU5402 (H).Treatment was done at stage HH3+ and 24 embryos were analyzed 6 hours later at stage HH 4–5. Exact stages of treatment and analysis are indicated in each picture. (I) FGF4 activity is independent of RA. Embryos were treated and analyzed as (E–H). The “length of Hex domain/length of embryo” ratio in % was calculated (DMSO/ethanol n = 10, DMSO/RA n = 8, SU5402/ethanol n = 9, SU5402/RA n = 6). Bars in the diagram represent the mean and error bars display the standard error of the mean. The P-value was less than 0.001 (Student t test) between control and RA treated embryos and between RA treated and RA/SU5402 treated embryos (two asterisks). The P-value was less than 0.05 (Student t test) between control and SU5402 treated embryos (one asterisk). RA/SU5402 showed no significant difference from control embryos.
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pone-0005845-g006: RA and FGF4 independently pattern the anterior endoderm.Whole mount in situ hybridization analysis of Hex expression. Ventral view, anterior to the top. (A–D) Analysis of embryos when FGF4 signaling is activated and RA signaling is inhibited. Embryos were treated at stage HH 3+ with DMSO and grafted with PBS beads (A) as control, treated with DMSO and grafted with FGF4 beads (1 mg/ml) (B), treated with10−5 M AGN193109 and grafted with PBS beads (C), or treated with 10−5 M AGN193109 and grafted with FGF4 beads (1 mg/ml) (D). Circles show position of beads in (A–D). (E–H) Analysis of embryos when FGF4 signaling is inhibited and RA signaling is activated. Embryos were treated at stage HH 3+ either with DMSO and ethanol (E) as control, with DMSO and 10−6 M RA (F), with 20 µM SU5402 and ethanol (G), or with 10−6 M RA and 20 µM SU5402 (H).Treatment was done at stage HH3+ and 24 embryos were analyzed 6 hours later at stage HH 4–5. Exact stages of treatment and analysis are indicated in each picture. (I) FGF4 activity is independent of RA. Embryos were treated and analyzed as (E–H). The “length of Hex domain/length of embryo” ratio in % was calculated (DMSO/ethanol n = 10, DMSO/RA n = 8, SU5402/ethanol n = 9, SU5402/RA n = 6). Bars in the diagram represent the mean and error bars display the standard error of the mean. The P-value was less than 0.001 (Student t test) between control and RA treated embryos and between RA treated and RA/SU5402 treated embryos (two asterisks). The P-value was less than 0.05 (Student t test) between control and SU5402 treated embryos (one asterisk). RA/SU5402 showed no significant difference from control embryos.

Mentions: First, we activated FGF signaling by grafting heparin beads loaded with FGF4 (1 mg/ml) onto the ventral side of the embryos and inhibited RA signaling by including 10−5 M AGN193109 in the culture medium. FGF4 beads alone repressed Hex expression (4/7; Fig. 6B) as previously published [12]. As described above, the RA inhibitor alone caused lateral repression of the Hex domain (3/3, Fig. 3C and Fig. 6C). Activating the FGF signaling while blocking the RA pathway resulted in reduced Hex expression as when FGF4 was activated alone (4/8; Fig. 6D). This result shows that RA signaling is not needed downstream of FGF4 to repress Hex.


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)

RA and FGF4 independently pattern the anterior endoderm.Whole mount in situ hybridization analysis of Hex expression. Ventral view, anterior to the top. (A–D) Analysis of embryos when FGF4 signaling is activated and RA signaling is inhibited. Embryos were treated at stage HH 3+ with DMSO and grafted with PBS beads (A) as control, treated with DMSO and grafted with FGF4 beads (1 mg/ml) (B), treated with10−5 M AGN193109 and grafted with PBS beads (C), or treated with 10−5 M AGN193109 and grafted with FGF4 beads (1 mg/ml) (D). Circles show position of beads in (A–D). (E–H) Analysis of embryos when FGF4 signaling is inhibited and RA signaling is activated. Embryos were treated at stage HH 3+ either with DMSO and ethanol (E) as control, with DMSO and 10−6 M RA (F), with 20 µM SU5402 and ethanol (G), or with 10−6 M RA and 20 µM SU5402 (H).Treatment was done at stage HH3+ and 24 embryos were analyzed 6 hours later at stage HH 4–5. Exact stages of treatment and analysis are indicated in each picture. (I) FGF4 activity is independent of RA. Embryos were treated and analyzed as (E–H). The “length of Hex domain/length of embryo” ratio in % was calculated (DMSO/ethanol n = 10, DMSO/RA n = 8, SU5402/ethanol n = 9, SU5402/RA n = 6). Bars in the diagram represent the mean and error bars display the standard error of the mean. The P-value was less than 0.001 (Student t test) between control and RA treated embryos and between RA treated and RA/SU5402 treated embryos (two asterisks). The P-value was less than 0.05 (Student t test) between control and SU5402 treated embryos (one asterisk). RA/SU5402 showed no significant difference from control embryos.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690404&req=5

pone-0005845-g006: RA and FGF4 independently pattern the anterior endoderm.Whole mount in situ hybridization analysis of Hex expression. Ventral view, anterior to the top. (A–D) Analysis of embryos when FGF4 signaling is activated and RA signaling is inhibited. Embryos were treated at stage HH 3+ with DMSO and grafted with PBS beads (A) as control, treated with DMSO and grafted with FGF4 beads (1 mg/ml) (B), treated with10−5 M AGN193109 and grafted with PBS beads (C), or treated with 10−5 M AGN193109 and grafted with FGF4 beads (1 mg/ml) (D). Circles show position of beads in (A–D). (E–H) Analysis of embryos when FGF4 signaling is inhibited and RA signaling is activated. Embryos were treated at stage HH 3+ either with DMSO and ethanol (E) as control, with DMSO and 10−6 M RA (F), with 20 µM SU5402 and ethanol (G), or with 10−6 M RA and 20 µM SU5402 (H).Treatment was done at stage HH3+ and 24 embryos were analyzed 6 hours later at stage HH 4–5. Exact stages of treatment and analysis are indicated in each picture. (I) FGF4 activity is independent of RA. Embryos were treated and analyzed as (E–H). The “length of Hex domain/length of embryo” ratio in % was calculated (DMSO/ethanol n = 10, DMSO/RA n = 8, SU5402/ethanol n = 9, SU5402/RA n = 6). Bars in the diagram represent the mean and error bars display the standard error of the mean. The P-value was less than 0.001 (Student t test) between control and RA treated embryos and between RA treated and RA/SU5402 treated embryos (two asterisks). The P-value was less than 0.05 (Student t test) between control and SU5402 treated embryos (one asterisk). RA/SU5402 showed no significant difference from control embryos.
Mentions: First, we activated FGF signaling by grafting heparin beads loaded with FGF4 (1 mg/ml) onto the ventral side of the embryos and inhibited RA signaling by including 10−5 M AGN193109 in the culture medium. FGF4 beads alone repressed Hex expression (4/7; Fig. 6B) as previously published [12]. As described above, the RA inhibitor alone caused lateral repression of the Hex domain (3/3, Fig. 3C and Fig. 6C). Activating the FGF signaling while blocking the RA pathway resulted in reduced Hex expression as when FGF4 was activated alone (4/8; Fig. 6D). This result shows that RA signaling is not needed downstream of FGF4 to repress Hex.

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