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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

Exogenous RA activates ectopic gene transcription in the endoderm.(A) is a schematic illustration showing the initial grafting position of the RA bead in stage HH 4− or HH 10 chick embryos. (B–I) show whole mount in situ hybridization analysis of Cyp26A1 in control (B,F), RA bead grafted (C,G) or inhibitor treated embryos (D,H), ventral view, anterior to the top. Beads were soaked in ethanol (control) or ethanol containing 10−3 M RA and were grafted onto the endoderm and analyzed 6 hours later (approximately HH 5 or HH 11, respectively). For inhibition, embryos were treated with 10−5 M AGN193109 added to the culture medium at HH 3+ or HH 10, incubated 6 hours and then analyzed (approximately HH 4–5 or HH 11, respectively). Exact stage of grafting and analysis are given in each picture. Position of beads is marked by a circle and lines give the plane of sections shown in (E and I), ventral side down. Scale bars are 100 µM. Note broad induction in (E) and unilateral induction ventrally in (I). A, anterior; h, hours; P, posterior.
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pone-0005845-g002: Exogenous RA activates ectopic gene transcription in the endoderm.(A) is a schematic illustration showing the initial grafting position of the RA bead in stage HH 4− or HH 10 chick embryos. (B–I) show whole mount in situ hybridization analysis of Cyp26A1 in control (B,F), RA bead grafted (C,G) or inhibitor treated embryos (D,H), ventral view, anterior to the top. Beads were soaked in ethanol (control) or ethanol containing 10−3 M RA and were grafted onto the endoderm and analyzed 6 hours later (approximately HH 5 or HH 11, respectively). For inhibition, embryos were treated with 10−5 M AGN193109 added to the culture medium at HH 3+ or HH 10, incubated 6 hours and then analyzed (approximately HH 4–5 or HH 11, respectively). Exact stage of grafting and analysis are given in each picture. Position of beads is marked by a circle and lines give the plane of sections shown in (E and I), ventral side down. Scale bars are 100 µM. Note broad induction in (E) and unilateral induction ventrally in (I). A, anterior; h, hours; P, posterior.

Mentions: To investigate whether endoderm responds to exogenous RA, we made use of the direct RA target gene Cyp26A1. Either at stage HH 4 (late primitive streak stage) or at stage HH 10 (10 somite stage), we grafted beads soaked in RA (10−3 M or 10−4 M) onto the endoderm in modified New cultures (Fig. 2A) [28]. After 6 hours of incubation, these embryos were assayed for the expression of Cyp26A1. We chose this relatively short incubation period to reduce the possibility of indirect target gene induction via RA activation in neighboring cells, notably in mesoderm. At HH 5, embryos exposed to RA-loaded beads showed broad Cyp26A1 induction around the bead in endoderm and in the epiblast (Table 1; Fig. 2C, E) as well as in endoderm and surface ectoderm at HH 11 (Table 1; Fig. 2G, I). The range of Cyp26A1 induction around a bead soaked with 10−3 M RA can be estimated to be half of the embryo during gastrulation and to diffuse to the length of 3 somites during somitogenesis. Lower concentrations (10−4 M) of RA induced Cyp26A1 mRNA in a shorter range (not shown). Likewise, either 10−6 M and 10−7 M RA included into the medium either at gastrula or somitic stage resulted in elevated Cyp26A1 expression in its endogenous domains. However, induction in ectopic areas was not observed.


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)

Exogenous RA activates ectopic gene transcription in the endoderm.(A) is a schematic illustration showing the initial grafting position of the RA bead in stage HH 4− or HH 10 chick embryos. (B–I) show whole mount in situ hybridization analysis of Cyp26A1 in control (B,F), RA bead grafted (C,G) or inhibitor treated embryos (D,H), ventral view, anterior to the top. Beads were soaked in ethanol (control) or ethanol containing 10−3 M RA and were grafted onto the endoderm and analyzed 6 hours later (approximately HH 5 or HH 11, respectively). For inhibition, embryos were treated with 10−5 M AGN193109 added to the culture medium at HH 3+ or HH 10, incubated 6 hours and then analyzed (approximately HH 4–5 or HH 11, respectively). Exact stage of grafting and analysis are given in each picture. Position of beads is marked by a circle and lines give the plane of sections shown in (E and I), ventral side down. Scale bars are 100 µM. Note broad induction in (E) and unilateral induction ventrally in (I). A, anterior; h, hours; P, posterior.
© Copyright Policy
Related In: Results  -  Collection

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pone-0005845-g002: Exogenous RA activates ectopic gene transcription in the endoderm.(A) is a schematic illustration showing the initial grafting position of the RA bead in stage HH 4− or HH 10 chick embryos. (B–I) show whole mount in situ hybridization analysis of Cyp26A1 in control (B,F), RA bead grafted (C,G) or inhibitor treated embryos (D,H), ventral view, anterior to the top. Beads were soaked in ethanol (control) or ethanol containing 10−3 M RA and were grafted onto the endoderm and analyzed 6 hours later (approximately HH 5 or HH 11, respectively). For inhibition, embryos were treated with 10−5 M AGN193109 added to the culture medium at HH 3+ or HH 10, incubated 6 hours and then analyzed (approximately HH 4–5 or HH 11, respectively). Exact stage of grafting and analysis are given in each picture. Position of beads is marked by a circle and lines give the plane of sections shown in (E and I), ventral side down. Scale bars are 100 µM. Note broad induction in (E) and unilateral induction ventrally in (I). A, anterior; h, hours; P, posterior.
Mentions: To investigate whether endoderm responds to exogenous RA, we made use of the direct RA target gene Cyp26A1. Either at stage HH 4 (late primitive streak stage) or at stage HH 10 (10 somite stage), we grafted beads soaked in RA (10−3 M or 10−4 M) onto the endoderm in modified New cultures (Fig. 2A) [28]. After 6 hours of incubation, these embryos were assayed for the expression of Cyp26A1. We chose this relatively short incubation period to reduce the possibility of indirect target gene induction via RA activation in neighboring cells, notably in mesoderm. At HH 5, embryos exposed to RA-loaded beads showed broad Cyp26A1 induction around the bead in endoderm and in the epiblast (Table 1; Fig. 2C, E) as well as in endoderm and surface ectoderm at HH 11 (Table 1; Fig. 2G, I). The range of Cyp26A1 induction around a bead soaked with 10−3 M RA can be estimated to be half of the embryo during gastrulation and to diffuse to the length of 3 somites during somitogenesis. Lower concentrations (10−4 M) of RA induced Cyp26A1 mRNA in a shorter range (not shown). Likewise, either 10−6 M and 10−7 M RA included into the medium either at gastrula or somitic stage resulted in elevated Cyp26A1 expression in its endogenous domains. However, induction in ectopic areas was not observed.

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