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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.


Spink3+, Ttr+, and Trh+ endoderm cells that are not DAB-stained in EPLEBs resemble definitive endoderm. Typical EPLEBs showing the lack of DAB+ cells (brown) and presence of Spink3+(A), Ttr+(B), and Trh+(C) cells. DAB+ cells (brown) are detected on the surface of rare EPLEBs within a population: Spink3+DAB−(D), Ttr+DAB−(E), and Trh+DAB−(F) endoderm cells resemble definitive endoderm while Spink3−DAB+, Ttr+DAB+(E), and Trh−DAB+(F) show a cuboidal morphology and represent visceral endoderm.
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f5: Spink3+, Ttr+, and Trh+ endoderm cells that are not DAB-stained in EPLEBs resemble definitive endoderm. Typical EPLEBs showing the lack of DAB+ cells (brown) and presence of Spink3+(A), Ttr+(B), and Trh+(C) cells. DAB+ cells (brown) are detected on the surface of rare EPLEBs within a population: Spink3+DAB−(D), Ttr+DAB−(E), and Trh+DAB−(F) endoderm cells resemble definitive endoderm while Spink3−DAB+, Ttr+DAB+(E), and Trh−DAB+(F) show a cuboidal morphology and represent visceral endoderm.

Mentions: Analysis of endoderm marker expression pattern has revealed the presence of multiple endoderm populations within the embryonic region of the gastrulating embryo. To understand the populations that are formed during pluripotent cell differentiation, day 5 EPLEBs were double-stained by HRP endocytosis uptake assay and Spink3, Trh, or Ttr WISH (Fig. 5). As expected, the number of EPLEBs containing cells capable of endocytosis, indicating the presence of visceral endoderm, was low.16,17Spink3 and Trh expression was detected in a layer of squamous cells on the surface of EPLEBs and the expression of these genes and HRP endocytosis was mutually exclusive (Fig. 5A, C, D, F). The majority of cells that expressed Ttr did not endocytose HRP, but cells capable of endocytosis did express Ttr as expected (Fig. 5E).


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)

Spink3+, Ttr+, and Trh+ endoderm cells that are not DAB-stained in EPLEBs resemble definitive endoderm. Typical EPLEBs showing the lack of DAB+ cells (brown) and presence of Spink3+(A), Ttr+(B), and Trh+(C) cells. DAB+ cells (brown) are detected on the surface of rare EPLEBs within a population: Spink3+DAB−(D), Ttr+DAB−(E), and Trh+DAB−(F) endoderm cells resemble definitive endoderm while Spink3−DAB+, Ttr+DAB+(E), and Trh−DAB+(F) show a cuboidal morphology and represent visceral endoderm.
© Copyright Policy
Related In: Results  -  Collection

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

f5: Spink3+, Ttr+, and Trh+ endoderm cells that are not DAB-stained in EPLEBs resemble definitive endoderm. Typical EPLEBs showing the lack of DAB+ cells (brown) and presence of Spink3+(A), Ttr+(B), and Trh+(C) cells. DAB+ cells (brown) are detected on the surface of rare EPLEBs within a population: Spink3+DAB−(D), Ttr+DAB−(E), and Trh+DAB−(F) endoderm cells resemble definitive endoderm while Spink3−DAB+, Ttr+DAB+(E), and Trh−DAB+(F) show a cuboidal morphology and represent visceral endoderm.
Mentions: Analysis of endoderm marker expression pattern has revealed the presence of multiple endoderm populations within the embryonic region of the gastrulating embryo. To understand the populations that are formed during pluripotent cell differentiation, day 5 EPLEBs were double-stained by HRP endocytosis uptake assay and Spink3, Trh, or Ttr WISH (Fig. 5). As expected, the number of EPLEBs containing cells capable of endocytosis, indicating the presence of visceral endoderm, was low.16,17Spink3 and Trh expression was detected in a layer of squamous cells on the surface of EPLEBs and the expression of these genes and HRP endocytosis was mutually exclusive (Fig. 5A, C, D, F). The majority of cells that expressed Ttr did not endocytose HRP, but cells capable of endocytosis did express Ttr as expected (Fig. 5E).

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.