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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

Expression pattern of potential definitive endoderm markers in in vitro models of differentiation. Gene expression of potential definitive endoderm markers was analyzed in EBs (A), EPLEBs (B), and EBMs (C) by RT-PCR. Gapdh was used as a loading control. n=3, a representative result is shown. EB, embryoid body; EPLEBs, EPL cell-derived EBs; EBM, embryoid body cultured and maintained in MEDII; EPL, early primitive ectoderm-like cells cultured for 2 days; ES, embryonic stem cells cultured for 2 days; −RT, negative control in the absence of reverse transcriptase; NTC, no template control.
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f1: Expression pattern of potential definitive endoderm markers in in vitro models of differentiation. Gene expression of potential definitive endoderm markers was analyzed in EBs (A), EPLEBs (B), and EBMs (C) by RT-PCR. Gapdh was used as a loading control. n=3, a representative result is shown. EB, embryoid body; EPLEBs, EPL cell-derived EBs; EBM, embryoid body cultured and maintained in MEDII; EPL, early primitive ectoderm-like cells cultured for 2 days; ES, embryonic stem cells cultured for 2 days; −RT, negative control in the absence of reverse transcriptase; NTC, no template control.

Mentions: A set of potential definitive endoderm markers was identified from a microarray analysis comparing EPLEBs on day 2 and 4 of differentiation. A group of genes up regulated on day 4, and likely to be expressed by nascent mesoderm and nascent definitive endoderm16,17 (Supplementary Table S3), was further refined by eliminating genes that were previously reported to be expressed in the mesoderm and ectoderm to identify those genes most likely to be expressed in the definitive endoderm. A total of 10 genes were shortlisted for further analysis: Tgfb1i1, Tdo2, Amot, Spink3, Ttr, P2ry5, Sox7, Map3k8, Sparc, and Lman2 (Supplementary Table S3). Three in vitro models of differentiation, EBs, EPLEBs, and EBMs, differing in the repertoire of cells formed, were analyzed by RT-PCR to characterize further the expression patterns of potential definitive endoderm markers (Fig. 1). EBs are widely documented to form cells representative of the three germ lineages and the extra-embryonic endoderm18,25–28; EPLEBs form derivatives of the primitive streak, mesoderm, and definitive endoderm and are deficient in ectodermal lineages and extra-embryonic endoderm16,17; while differentiation in EBMs is restricted to the formation of the ectodermal lineages.29 Definitive endoderm markers would be expected to be expressed in EBs and EPLEBs, and not EBMs or pluripotent cells. Differentiation in EPLEBs is advanced in comparison to EBs, with the onset of molecular gastrulation occurring on day 2 compared to day 4,16,17 such that the expression of prospective definitive endoderm markers expression would be expected to occur earlier, relative to day 0, in EPLEBs. Molecular gastrulation is used here to describe the events in EBs that recapitulate gastrulation in the embryo and can be recognized by the up-regulation of primitive streak markers such as T and Mixl1.


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)

Expression pattern of potential definitive endoderm markers in in vitro models of differentiation. Gene expression of potential definitive endoderm markers was analyzed in EBs (A), EPLEBs (B), and EBMs (C) by RT-PCR. Gapdh was used as a loading control. n=3, a representative result is shown. EB, embryoid body; EPLEBs, EPL cell-derived EBs; EBM, embryoid body cultured and maintained in MEDII; EPL, early primitive ectoderm-like cells cultured for 2 days; ES, embryonic stem cells cultured for 2 days; −RT, negative control in the absence of reverse transcriptase; NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048981&req=5

f1: Expression pattern of potential definitive endoderm markers in in vitro models of differentiation. Gene expression of potential definitive endoderm markers was analyzed in EBs (A), EPLEBs (B), and EBMs (C) by RT-PCR. Gapdh was used as a loading control. n=3, a representative result is shown. EB, embryoid body; EPLEBs, EPL cell-derived EBs; EBM, embryoid body cultured and maintained in MEDII; EPL, early primitive ectoderm-like cells cultured for 2 days; ES, embryonic stem cells cultured for 2 days; −RT, negative control in the absence of reverse transcriptase; NTC, no template control.
Mentions: A set of potential definitive endoderm markers was identified from a microarray analysis comparing EPLEBs on day 2 and 4 of differentiation. A group of genes up regulated on day 4, and likely to be expressed by nascent mesoderm and nascent definitive endoderm16,17 (Supplementary Table S3), was further refined by eliminating genes that were previously reported to be expressed in the mesoderm and ectoderm to identify those genes most likely to be expressed in the definitive endoderm. A total of 10 genes were shortlisted for further analysis: Tgfb1i1, Tdo2, Amot, Spink3, Ttr, P2ry5, Sox7, Map3k8, Sparc, and Lman2 (Supplementary Table S3). Three in vitro models of differentiation, EBs, EPLEBs, and EBMs, differing in the repertoire of cells formed, were analyzed by RT-PCR to characterize further the expression patterns of potential definitive endoderm markers (Fig. 1). EBs are widely documented to form cells representative of the three germ lineages and the extra-embryonic endoderm18,25–28; EPLEBs form derivatives of the primitive streak, mesoderm, and definitive endoderm and are deficient in ectodermal lineages and extra-embryonic endoderm16,17; while differentiation in EBMs is restricted to the formation of the ectodermal lineages.29 Definitive endoderm markers would be expected to be expressed in EBs and EPLEBs, and not EBMs or pluripotent cells. Differentiation in EPLEBs is advanced in comparison to EBs, with the onset of molecular gastrulation occurring on day 2 compared to day 4,16,17 such that the expression of prospective definitive endoderm markers expression would be expected to occur earlier, relative to day 0, in EPLEBs. Molecular gastrulation is used here to describe the events in EBs that recapitulate gastrulation in the embryo and can be recognized by the up-regulation of primitive streak markers such as T and Mixl1.

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