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Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

Goh HN, Rathjen PD, Familari M, Rathjen J - Biores Open Access (2014)

Bottom Line: This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh).Moreover, further differentiation suggested that the potential of these populations differed.These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Melbourne , Victoria, Australia .

ABSTRACT
Endoderm formation in the mammalian embryo occurs first in the blastocyst, when the primitive endoderm and pluripotent cells resolve into separate lineages, and again during gastrulation, when the definitive endoderm progenitor population emerges from the primitive streak. The formation of the definitive endoderm can be modeled using pluripotent cell differentiation in culture. The differentiation of early primitive ectoderm-like (EPL) cells, a pluripotent cell population formed from embryonic stem (ES) cells, was used to identify and characterize definitive endoderm formation. Expression of serine peptidase inhibitor, Kazal type 3 (Spink3) was detected in EPL cell-derived endoderm, and in a band of endoderm immediately distal to the embryonic-extra-embryonic boundary in pregastrula and gastrulating embryos. Later expression marked a region of endoderm separating the yolk sac from the developing gut. In the embryo, Spink3 expression marked a region of endoderm comprising the distal visceral endoderm, as determined by an endocytosis assay, and the proximal region of the definitive endoderm. This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh). Endoderm expressing either Spink3 or Trh could be formed during EPL cell differentiation, and the prevalence of these populations could be influenced by culture medium and growth factor addition. Moreover, further differentiation suggested that the potential of these populations differed. These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.

No MeSH data available.


Localization of mRNA transcripts of potential definitive endoderm markers in EPLEBs by wholemount in situ hybridization (WISH). The expression pattern of potential definitive endoderm markers was analyzed in day 5 EPLEBs. Foxa2 was included in the analysis as a positive control. Representative 8 μm sections are shown.
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f2: Localization of mRNA transcripts of potential definitive endoderm markers in EPLEBs by wholemount in situ hybridization (WISH). The expression pattern of potential definitive endoderm markers was analyzed in day 5 EPLEBs. Foxa2 was included in the analysis as a positive control. Representative 8 μm sections are shown.

Mentions: Previous characterization has shown that cells on the surface of EPLEBs on day 5 resemble definitive endoderm by gene expression (specifically by Sox17 expression), morphology, and endocytosis ability, while cells located internally express markers of mesoderm.16 The location of cells expressing Amot, Spink3, Tgfb1i1, and Ttr in EPLEBs was determined by WISH (Fig. 2). Foxa2, a commonly used definitive endoderm marker,26,30 was included. Although often used as a marker of the definitive endoderm, Foxa2 is expressed in the mesoderm as it forms in the embryo.30Spink3 (Fig. 2A) and Ttr (Fig. 2B) were expressed specifically in a squamous layer of cells encapsulating the EPLEB, the cell layer that has been previously identified as definitive endoderm.16Amot (Fig. 2C), Tgfb1i1 (Fig. 2D), and Foxa2 (Fig. 2E) transcripts were detected in the squamous cells on the surface of EPLEBs and in the mesenchyme in the core of EPLEBs, demonstrating expression in the definitive endoderm and newly formed mesoderm. A satisfactory signal could not be obtained from EPLEBs analyzed for the expression of Tdo2 (data not shown).


Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

Goh HN, Rathjen PD, Familari M, Rathjen J - Biores Open Access (2014)

Localization of mRNA transcripts of potential definitive endoderm markers in EPLEBs by wholemount in situ hybridization (WISH). The expression pattern of potential definitive endoderm markers was analyzed in day 5 EPLEBs. Foxa2 was included in the analysis as a positive control. Representative 8 μm sections are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4048981&req=5

f2: Localization of mRNA transcripts of potential definitive endoderm markers in EPLEBs by wholemount in situ hybridization (WISH). The expression pattern of potential definitive endoderm markers was analyzed in day 5 EPLEBs. Foxa2 was included in the analysis as a positive control. Representative 8 μm sections are shown.
Mentions: Previous characterization has shown that cells on the surface of EPLEBs on day 5 resemble definitive endoderm by gene expression (specifically by Sox17 expression), morphology, and endocytosis ability, while cells located internally express markers of mesoderm.16 The location of cells expressing Amot, Spink3, Tgfb1i1, and Ttr in EPLEBs was determined by WISH (Fig. 2). Foxa2, a commonly used definitive endoderm marker,26,30 was included. Although often used as a marker of the definitive endoderm, Foxa2 is expressed in the mesoderm as it forms in the embryo.30Spink3 (Fig. 2A) and Ttr (Fig. 2B) were expressed specifically in a squamous layer of cells encapsulating the EPLEB, the cell layer that has been previously identified as definitive endoderm.16Amot (Fig. 2C), Tgfb1i1 (Fig. 2D), and Foxa2 (Fig. 2E) transcripts were detected in the squamous cells on the surface of EPLEBs and in the mesenchyme in the core of EPLEBs, demonstrating expression in the definitive endoderm and newly formed mesoderm. A satisfactory signal could not be obtained from EPLEBs analyzed for the expression of Tdo2 (data not shown).

Bottom Line: This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh).Moreover, further differentiation suggested that the potential of these populations differed.These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Melbourne , Victoria, Australia .

ABSTRACT
Endoderm formation in the mammalian embryo occurs first in the blastocyst, when the primitive endoderm and pluripotent cells resolve into separate lineages, and again during gastrulation, when the definitive endoderm progenitor population emerges from the primitive streak. The formation of the definitive endoderm can be modeled using pluripotent cell differentiation in culture. The differentiation of early primitive ectoderm-like (EPL) cells, a pluripotent cell population formed from embryonic stem (ES) cells, was used to identify and characterize definitive endoderm formation. Expression of serine peptidase inhibitor, Kazal type 3 (Spink3) was detected in EPL cell-derived endoderm, and in a band of endoderm immediately distal to the embryonic-extra-embryonic boundary in pregastrula and gastrulating embryos. Later expression marked a region of endoderm separating the yolk sac from the developing gut. In the embryo, Spink3 expression marked a region of endoderm comprising the distal visceral endoderm, as determined by an endocytosis assay, and the proximal region of the definitive endoderm. This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh). Endoderm expressing either Spink3 or Trh could be formed during EPL cell differentiation, and the prevalence of these populations could be influenced by culture medium and growth factor addition. Moreover, further differentiation suggested that the potential of these populations differed. These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.

No MeSH data available.