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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.


Colocalization of 3,3′-diaminobenzidine (DAB)-stained visceral endoderm and Spink3+/Ttr+/Trh+ endoderm in E7.5 embryos. E7.5 mouse embryo showing the distribution of DAB+ endoderm alone (A) and colocalization of DAB+ and Spink3+ endoderm (B). Sagittal (C, D) and coronal (E, F) sections of a double stained E7.5 mouse embryo. The position of Spink3+ DAB− cells is bracketed by arrowheads in D and F. An E7.5 mouse embryo showing the colocalization of DAB+ and Trh+ endoderm in wholemount (G), sagittal (H, I), and coronal (J, K) sections. The border between the Trh+ cells and DAB+ cells is indicated by the arrowheads. An E7.5 mouse embryo showing the colocalization of DAB+ and Ttr+ endoderm in wholemount (L), sagittal (M, N), and coronal (O, P) sections. The position of Ttr+ DAB− cells is bracketed by arrowheads in N and P. The dashed line indicates the position of the embryonic and extra-embryonic boundary in all images. *Anterior.
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f4: Colocalization of 3,3′-diaminobenzidine (DAB)-stained visceral endoderm and Spink3+/Ttr+/Trh+ endoderm in E7.5 embryos. E7.5 mouse embryo showing the distribution of DAB+ endoderm alone (A) and colocalization of DAB+ and Spink3+ endoderm (B). Sagittal (C, D) and coronal (E, F) sections of a double stained E7.5 mouse embryo. The position of Spink3+ DAB− cells is bracketed by arrowheads in D and F. An E7.5 mouse embryo showing the colocalization of DAB+ and Trh+ endoderm in wholemount (G), sagittal (H, I), and coronal (J, K) sections. The border between the Trh+ cells and DAB+ cells is indicated by the arrowheads. An E7.5 mouse embryo showing the colocalization of DAB+ and Ttr+ endoderm in wholemount (L), sagittal (M, N), and coronal (O, P) sections. The position of Ttr+ DAB− cells is bracketed by arrowheads in N and P. The dashed line indicates the position of the embryonic and extra-embryonic boundary in all images. *Anterior.

Mentions: Disparity in endocytotic capabilities of definitive endoderm and visceral endoderm allows these two cell types to be distinguished in mouse embryos using a colormetric endocytosis assay.13,24,34–36 An HRP endocytosis assay was performed on freshly dissected E7.5 mouse embryos. DAB+ visceral endoderm was localized in the extra-embryonic region and in the most proximal domain of the embryonic region (Fig. 4A). Subsequent staining by WISH demonstrated that Spink3 is expressed in cells in the proximal domain of the embryonic region, below the amnion and overlapping with the region of HRP uptake (Fig. 4B). Sagittal (Fig. 4C, D) and coronal (Fig. 4E, F) sections showed two populations of Spink3+ cells. Spink3+ DAB+ visceral endoderm was detected in the anterior, posterior, and lateral parts of the embryonic region beneath the amnion. Spink3+ DAB− definitive endoderm was detected subjacent to the Spink3+ DAB+ visceral endoderm in the anterior, lateral, and posterior embryonic region. Spink3− DAB+ visceral endoderm was confined to the extra-embryonic region above the amnion.


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)

Colocalization of 3,3′-diaminobenzidine (DAB)-stained visceral endoderm and Spink3+/Ttr+/Trh+ endoderm in E7.5 embryos. E7.5 mouse embryo showing the distribution of DAB+ endoderm alone (A) and colocalization of DAB+ and Spink3+ endoderm (B). Sagittal (C, D) and coronal (E, F) sections of a double stained E7.5 mouse embryo. The position of Spink3+ DAB− cells is bracketed by arrowheads in D and F. An E7.5 mouse embryo showing the colocalization of DAB+ and Trh+ endoderm in wholemount (G), sagittal (H, I), and coronal (J, K) sections. The border between the Trh+ cells and DAB+ cells is indicated by the arrowheads. An E7.5 mouse embryo showing the colocalization of DAB+ and Ttr+ endoderm in wholemount (L), sagittal (M, N), and coronal (O, P) sections. The position of Ttr+ DAB− cells is bracketed by arrowheads in N and P. The dashed line indicates the position of the embryonic and extra-embryonic boundary in all images. *Anterior.
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Related In: Results  -  Collection

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f4: Colocalization of 3,3′-diaminobenzidine (DAB)-stained visceral endoderm and Spink3+/Ttr+/Trh+ endoderm in E7.5 embryos. E7.5 mouse embryo showing the distribution of DAB+ endoderm alone (A) and colocalization of DAB+ and Spink3+ endoderm (B). Sagittal (C, D) and coronal (E, F) sections of a double stained E7.5 mouse embryo. The position of Spink3+ DAB− cells is bracketed by arrowheads in D and F. An E7.5 mouse embryo showing the colocalization of DAB+ and Trh+ endoderm in wholemount (G), sagittal (H, I), and coronal (J, K) sections. The border between the Trh+ cells and DAB+ cells is indicated by the arrowheads. An E7.5 mouse embryo showing the colocalization of DAB+ and Ttr+ endoderm in wholemount (L), sagittal (M, N), and coronal (O, P) sections. The position of Ttr+ DAB− cells is bracketed by arrowheads in N and P. The dashed line indicates the position of the embryonic and extra-embryonic boundary in all images. *Anterior.
Mentions: Disparity in endocytotic capabilities of definitive endoderm and visceral endoderm allows these two cell types to be distinguished in mouse embryos using a colormetric endocytosis assay.13,24,34–36 An HRP endocytosis assay was performed on freshly dissected E7.5 mouse embryos. DAB+ visceral endoderm was localized in the extra-embryonic region and in the most proximal domain of the embryonic region (Fig. 4A). Subsequent staining by WISH demonstrated that Spink3 is expressed in cells in the proximal domain of the embryonic region, below the amnion and overlapping with the region of HRP uptake (Fig. 4B). Sagittal (Fig. 4C, D) and coronal (Fig. 4E, F) sections showed two populations of Spink3+ cells. Spink3+ DAB+ visceral endoderm was detected in the anterior, posterior, and lateral parts of the embryonic region beneath the amnion. Spink3+ DAB− definitive endoderm was detected subjacent to the Spink3+ DAB+ visceral endoderm in the anterior, lateral, and posterior embryonic region. Spink3− DAB+ visceral endoderm was confined to the extra-embryonic region above the amnion.

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.