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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 and Ttr expression in E6.5 to E9.5 mouse embryos. (A, B) Expression of Spink3 in an E6.5 mouse embryo by WISH, shown in wholemount and sagittal section. The position of the embryonic and extra-embryonic boundary in the anterior of the embryo is marked by an asterisk (*). Expression of Spink3(C, D) and Ttr(E, F) in wholemount E7.5 mouse embryos (C, E) and sagittal sections (D, F). The position of the anterior of the embryo is marked by an asterisk (*). Lateral (G), frontal (H), and rear (I) views of an E8.5 embryo stained for Spink3. The yolk sac on one side of the embryo has been removed. Transverse section (J) of the cranial region of the embryo showed expression of Spink3 in the walls of the foregut (fg) and in the yolk sac–embryo boundary (ys-emb; arrow). The position of the anterior of the embryo is marked by an asterisk (*) in H. Expression of Ttr in an E8.5 mouse embryo showing lateral (K), frontal (L), and rear (M) views and in transverse sections of the anterior region of the embryo (N, O). Expression of Spink3 in an E9.5 embryo, as shown by WISH (P), details of expression can be seen in sagittal (Q) and parasagittal (R) sections. fg, foregut; fgo, foregut opening; he, heart; hg, hindgut; hgo, hindgut opening; liv, liver anlage; Post, posterior; ys, yolk sac; ys-emb, yolk sac–embryo boundary.
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f3: Spink3 and Ttr expression in E6.5 to E9.5 mouse embryos. (A, B) Expression of Spink3 in an E6.5 mouse embryo by WISH, shown in wholemount and sagittal section. The position of the embryonic and extra-embryonic boundary in the anterior of the embryo is marked by an asterisk (*). Expression of Spink3(C, D) and Ttr(E, F) in wholemount E7.5 mouse embryos (C, E) and sagittal sections (D, F). The position of the anterior of the embryo is marked by an asterisk (*). Lateral (G), frontal (H), and rear (I) views of an E8.5 embryo stained for Spink3. The yolk sac on one side of the embryo has been removed. Transverse section (J) of the cranial region of the embryo showed expression of Spink3 in the walls of the foregut (fg) and in the yolk sac–embryo boundary (ys-emb; arrow). The position of the anterior of the embryo is marked by an asterisk (*) in H. Expression of Ttr in an E8.5 mouse embryo showing lateral (K), frontal (L), and rear (M) views and in transverse sections of the anterior region of the embryo (N, O). Expression of Spink3 in an E9.5 embryo, as shown by WISH (P), details of expression can be seen in sagittal (Q) and parasagittal (R) sections. fg, foregut; fgo, foregut opening; he, heart; hg, hindgut; hgo, hindgut opening; liv, liver anlage; Post, posterior; ys, yolk sac; ys-emb, yolk sac–embryo boundary.

Mentions: Spink3 was detected in E6.5 embryos in a band of cells that extended posteriorly from the anterior proximal midline (Fig. 3A, B). Expression was restricted to cells located distal to the circumferential constriction that delineates the embryonic and extra-embryonic boundary of the embryo (Fig. 3A). The distal embryonic region of embryo at this developmental stage is encapsulated by squamous embryonic visceral endoderm, while the proximal extra-embryonic region is surrounded by cuboidal extra-embryonic visceral endoderm.31,32Spink3 was expressed specifically by the embryonic visceral endoderm (Fig. 3B). In the E7.5 embryo Spink3 expression was detected in a ring of cells distal to the embryonic–extra-embryonic boundary (Fig. 3C). Sagittal sections of the embryo show Spink3 specifically expressed in the proximal definitive endoderm of the gastrulating embryo, with stronger expression in the anterior region (Fig. 3D). At E8.5 (four somites) Spink3 was predominantly detected along the boundary of the yolk sac and the embryonic embryo and in the lip of the foregut and hindgut opening (Fig. 3G–I). Expression of Spink3 was also detected in the lateral walls of the exposed regions of the foregut and hindgut. Yolk sac on the left side of the embryo was removed to allow visualization of the remaining parts of the embryo (Fig. 3G). Transverse sections of the anterior region of the embryo showed Spink3 expression in the walls of the foregut opening and in the yolk sac–embryo boundary (Fig. 3J). The expression of Spink3 diminishes proximally in the yolk sac. At a more advanced stage of development (eight-somite stage), Spink3 was detected in the ventral wall and rim of the closing gut (Supplementary Fig. S1). By E9.5 Spink3 was detected in the midgut and the rostral part of the hindgut (Fig. 3P-R).


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 and Ttr expression in E6.5 to E9.5 mouse embryos. (A, B) Expression of Spink3 in an E6.5 mouse embryo by WISH, shown in wholemount and sagittal section. The position of the embryonic and extra-embryonic boundary in the anterior of the embryo is marked by an asterisk (*). Expression of Spink3(C, D) and Ttr(E, F) in wholemount E7.5 mouse embryos (C, E) and sagittal sections (D, F). The position of the anterior of the embryo is marked by an asterisk (*). Lateral (G), frontal (H), and rear (I) views of an E8.5 embryo stained for Spink3. The yolk sac on one side of the embryo has been removed. Transverse section (J) of the cranial region of the embryo showed expression of Spink3 in the walls of the foregut (fg) and in the yolk sac–embryo boundary (ys-emb; arrow). The position of the anterior of the embryo is marked by an asterisk (*) in H. Expression of Ttr in an E8.5 mouse embryo showing lateral (K), frontal (L), and rear (M) views and in transverse sections of the anterior region of the embryo (N, O). Expression of Spink3 in an E9.5 embryo, as shown by WISH (P), details of expression can be seen in sagittal (Q) and parasagittal (R) sections. fg, foregut; fgo, foregut opening; he, heart; hg, hindgut; hgo, hindgut opening; liv, liver anlage; Post, posterior; ys, yolk sac; ys-emb, yolk sac–embryo boundary.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048981&req=5

f3: Spink3 and Ttr expression in E6.5 to E9.5 mouse embryos. (A, B) Expression of Spink3 in an E6.5 mouse embryo by WISH, shown in wholemount and sagittal section. The position of the embryonic and extra-embryonic boundary in the anterior of the embryo is marked by an asterisk (*). Expression of Spink3(C, D) and Ttr(E, F) in wholemount E7.5 mouse embryos (C, E) and sagittal sections (D, F). The position of the anterior of the embryo is marked by an asterisk (*). Lateral (G), frontal (H), and rear (I) views of an E8.5 embryo stained for Spink3. The yolk sac on one side of the embryo has been removed. Transverse section (J) of the cranial region of the embryo showed expression of Spink3 in the walls of the foregut (fg) and in the yolk sac–embryo boundary (ys-emb; arrow). The position of the anterior of the embryo is marked by an asterisk (*) in H. Expression of Ttr in an E8.5 mouse embryo showing lateral (K), frontal (L), and rear (M) views and in transverse sections of the anterior region of the embryo (N, O). Expression of Spink3 in an E9.5 embryo, as shown by WISH (P), details of expression can be seen in sagittal (Q) and parasagittal (R) sections. fg, foregut; fgo, foregut opening; he, heart; hg, hindgut; hgo, hindgut opening; liv, liver anlage; Post, posterior; ys, yolk sac; ys-emb, yolk sac–embryo boundary.
Mentions: Spink3 was detected in E6.5 embryos in a band of cells that extended posteriorly from the anterior proximal midline (Fig. 3A, B). Expression was restricted to cells located distal to the circumferential constriction that delineates the embryonic and extra-embryonic boundary of the embryo (Fig. 3A). The distal embryonic region of embryo at this developmental stage is encapsulated by squamous embryonic visceral endoderm, while the proximal extra-embryonic region is surrounded by cuboidal extra-embryonic visceral endoderm.31,32Spink3 was expressed specifically by the embryonic visceral endoderm (Fig. 3B). In the E7.5 embryo Spink3 expression was detected in a ring of cells distal to the embryonic–extra-embryonic boundary (Fig. 3C). Sagittal sections of the embryo show Spink3 specifically expressed in the proximal definitive endoderm of the gastrulating embryo, with stronger expression in the anterior region (Fig. 3D). At E8.5 (four somites) Spink3 was predominantly detected along the boundary of the yolk sac and the embryonic embryo and in the lip of the foregut and hindgut opening (Fig. 3G–I). Expression of Spink3 was also detected in the lateral walls of the exposed regions of the foregut and hindgut. Yolk sac on the left side of the embryo was removed to allow visualization of the remaining parts of the embryo (Fig. 3G). Transverse sections of the anterior region of the embryo showed Spink3 expression in the walls of the foregut opening and in the yolk sac–embryo boundary (Fig. 3J). The expression of Spink3 diminishes proximally in the yolk sac. At a more advanced stage of development (eight-somite stage), Spink3 was detected in the ventral wall and rim of the closing gut (Supplementary Fig. S1). By E9.5 Spink3 was detected in the midgut and the rostral part of the hindgut (Fig. 3P-R).

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.