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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.


Related in: MedlinePlus

Enrichment of proximal and distal definitive endoderm in EPLEBs. (A) Expression of proximal and distal definitive endoderm gene markers in EPLEBs cultured in KnockOut™ Serum Replacement-containing medium (KOSRM) relative to expression in EPLEBs cultured in serum-containing medium (SCM) by quantitative polymerase chain reaction (qPCR). Gene expression was normalized to β-actin. Error bars represent standard error of the mean; n=7. * Represents a significant change in gene expression when p≤0.05. (B) WISH analysis of the distribution of Spink3, Ttr and Trh transcripts in EPLEBs cultured in SCM or KOSRM, as indicated in the figure. (C) EPLEBs differentiated in SCM or KOSRM for 5 days were transferred to SCM and allowed to differentiate for a further 10 days before analysis for a panel of liver markers by qPCR. Gene expression was normalized to β-actin and expression in EPLEBs initially cultured in SCM has been expressed relative to EPLEBs initially cultured in KOSRM. Error bars represent standard error of the mean; n=3, a significant change in gene expression is denoted as * when the p-value is ≤0.05 and ** when ≤0.01. (D–E) Effects of Activin A, Wnt3a and BMP4 on markers of definitive endoderm in EPLEBs cultured in SCM (C) or KOSRM (D) by qPCR. Gene expression was normalized to β-actin and fold change in gene expression is shown relative to SCM or KOSRM controls. Error bars represent standard error of the mean; n=3 (n=6 KOSRM+Wnt3a). A significant change in gene expression is denoted as * when the p-value is <0.05 and ** when <0.01.
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f6: Enrichment of proximal and distal definitive endoderm in EPLEBs. (A) Expression of proximal and distal definitive endoderm gene markers in EPLEBs cultured in KnockOut™ Serum Replacement-containing medium (KOSRM) relative to expression in EPLEBs cultured in serum-containing medium (SCM) by quantitative polymerase chain reaction (qPCR). Gene expression was normalized to β-actin. Error bars represent standard error of the mean; n=7. * Represents a significant change in gene expression when p≤0.05. (B) WISH analysis of the distribution of Spink3, Ttr and Trh transcripts in EPLEBs cultured in SCM or KOSRM, as indicated in the figure. (C) EPLEBs differentiated in SCM or KOSRM for 5 days were transferred to SCM and allowed to differentiate for a further 10 days before analysis for a panel of liver markers by qPCR. Gene expression was normalized to β-actin and expression in EPLEBs initially cultured in SCM has been expressed relative to EPLEBs initially cultured in KOSRM. Error bars represent standard error of the mean; n=3, a significant change in gene expression is denoted as * when the p-value is ≤0.05 and ** when ≤0.01. (D–E) Effects of Activin A, Wnt3a and BMP4 on markers of definitive endoderm in EPLEBs cultured in SCM (C) or KOSRM (D) by qPCR. Gene expression was normalized to β-actin and fold change in gene expression is shown relative to SCM or KOSRM controls. Error bars represent standard error of the mean; n=3 (n=6 KOSRM+Wnt3a). A significant change in gene expression is denoted as * when the p-value is <0.05 and ** when <0.01.

Mentions: Analysis of the embryo suggests the formation of at least two populations of definitive endoderm, a proximal population that expresses Spink3 and Ttr and a distal population that expresses Trh. Both populations can be detected on EPLEBs. The prevalence of these populations was determined by analyzing the relative transcript levels of Spink3, Ttr, Trh, and Eya2 in EPLEBs cultured in KOSRM or SCM by qPCR (Fig. 6A). Eyes absent 2 homolog (Eya2) is an additional marker of the distal definitive endoderm with an expression pattern similar to that of Trh.14Spink3 and Ttr expression was significantly higher in EPLEBs that were cultured in SCM compared to those that were cultured in KOSRM. In contrast, expression levels of Trh and Eya2 were significantly higher in EPLEBs cultured in KOSRM. Consistent with these data, Spink3+ and Ttr+ cells were more prevalent in EPLEBs differentiated in SCM, whereas more Trh+ cells were detected in EPLEBs differentiated in KOSRM (Fig. 6B). This analysis suggests that SCM and KOSRM support the formation of proximal and distal endoderm, but medium composition impacts the efficiency with which these populations form.


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)

Enrichment of proximal and distal definitive endoderm in EPLEBs. (A) Expression of proximal and distal definitive endoderm gene markers in EPLEBs cultured in KnockOut™ Serum Replacement-containing medium (KOSRM) relative to expression in EPLEBs cultured in serum-containing medium (SCM) by quantitative polymerase chain reaction (qPCR). Gene expression was normalized to β-actin. Error bars represent standard error of the mean; n=7. * Represents a significant change in gene expression when p≤0.05. (B) WISH analysis of the distribution of Spink3, Ttr and Trh transcripts in EPLEBs cultured in SCM or KOSRM, as indicated in the figure. (C) EPLEBs differentiated in SCM or KOSRM for 5 days were transferred to SCM and allowed to differentiate for a further 10 days before analysis for a panel of liver markers by qPCR. Gene expression was normalized to β-actin and expression in EPLEBs initially cultured in SCM has been expressed relative to EPLEBs initially cultured in KOSRM. Error bars represent standard error of the mean; n=3, a significant change in gene expression is denoted as * when the p-value is ≤0.05 and ** when ≤0.01. (D–E) Effects of Activin A, Wnt3a and BMP4 on markers of definitive endoderm in EPLEBs cultured in SCM (C) or KOSRM (D) by qPCR. Gene expression was normalized to β-actin and fold change in gene expression is shown relative to SCM or KOSRM controls. Error bars represent standard error of the mean; n=3 (n=6 KOSRM+Wnt3a). A significant change in gene expression is denoted as * when the p-value is <0.05 and ** when <0.01.
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Related In: Results  -  Collection

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f6: Enrichment of proximal and distal definitive endoderm in EPLEBs. (A) Expression of proximal and distal definitive endoderm gene markers in EPLEBs cultured in KnockOut™ Serum Replacement-containing medium (KOSRM) relative to expression in EPLEBs cultured in serum-containing medium (SCM) by quantitative polymerase chain reaction (qPCR). Gene expression was normalized to β-actin. Error bars represent standard error of the mean; n=7. * Represents a significant change in gene expression when p≤0.05. (B) WISH analysis of the distribution of Spink3, Ttr and Trh transcripts in EPLEBs cultured in SCM or KOSRM, as indicated in the figure. (C) EPLEBs differentiated in SCM or KOSRM for 5 days were transferred to SCM and allowed to differentiate for a further 10 days before analysis for a panel of liver markers by qPCR. Gene expression was normalized to β-actin and expression in EPLEBs initially cultured in SCM has been expressed relative to EPLEBs initially cultured in KOSRM. Error bars represent standard error of the mean; n=3, a significant change in gene expression is denoted as * when the p-value is ≤0.05 and ** when ≤0.01. (D–E) Effects of Activin A, Wnt3a and BMP4 on markers of definitive endoderm in EPLEBs cultured in SCM (C) or KOSRM (D) by qPCR. Gene expression was normalized to β-actin and fold change in gene expression is shown relative to SCM or KOSRM controls. Error bars represent standard error of the mean; n=3 (n=6 KOSRM+Wnt3a). A significant change in gene expression is denoted as * when the p-value is <0.05 and ** when <0.01.
Mentions: Analysis of the embryo suggests the formation of at least two populations of definitive endoderm, a proximal population that expresses Spink3 and Ttr and a distal population that expresses Trh. Both populations can be detected on EPLEBs. The prevalence of these populations was determined by analyzing the relative transcript levels of Spink3, Ttr, Trh, and Eya2 in EPLEBs cultured in KOSRM or SCM by qPCR (Fig. 6A). Eyes absent 2 homolog (Eya2) is an additional marker of the distal definitive endoderm with an expression pattern similar to that of Trh.14Spink3 and Ttr expression was significantly higher in EPLEBs that were cultured in SCM compared to those that were cultured in KOSRM. In contrast, expression levels of Trh and Eya2 were significantly higher in EPLEBs cultured in KOSRM. Consistent with these data, Spink3+ and Ttr+ cells were more prevalent in EPLEBs differentiated in SCM, whereas more Trh+ cells were detected in EPLEBs differentiated in KOSRM (Fig. 6B). This analysis suggests that SCM and KOSRM support the formation of proximal and distal endoderm, but medium composition impacts the efficiency with which these populations form.

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.


Related in: MedlinePlus