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Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

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.

SUBMITTER: Goh HN

PROVIDER: S-EPMC4048981 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMany species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).ResultsWe hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent.ConclusionWe have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.

Project description:BACKGROUND: The Spemann/Mangold organizer is a transient tissue critical for patterning the gastrula stage vertebrate embryo and formation of the three germ layers. Despite its important role during development, there are still relatively few genes with specific expression in the organizer and its derivatives. Foxa2 is a forkhead transcription factor that is absolutely required for formation of the mammalian equivalent of the organizer, the node, the axial mesoderm and the definitive endoderm (DE). However, the targets of Foxa2 during embryogenesis, and the molecular impact of organizer loss on the gastrula embryo, have not been well defined. RESULTS: To identify genes specific to the Spemann/Mangold organizer, we performed a microarray-based screen that compared wild-type and Foxa2 mutant embryos at late gastrulation stage (E7.5). We could detect genes that were consistently down-regulated in replicate pools of mutant embryos versus wild-type, and these included a number of known node and DE markers. We selected 314 genes without previously published data at E7.5 and screened for expression by whole mount in situ hybridization. We identified 10 novel expression patterns in the node and 5 in the definitive endoderm. We also found significant reduction of markers expressed in secondary tissues that require interaction with the organizer and its derivatives, such as cardiac mesoderm, vasculature, primitive streak, and anterior neuroectoderm. CONCLUSION: The genes identified in this screen represent novel Spemann/Mangold organizer genes as well as potential Foxa2 targets. Further investigation will be needed to define these genes as novel developmental regulatory factors involved in organizer formation and function. We have placed these genes in a Foxa2-dependent genetic regulatory network and we hypothesize how Foxa2 may regulate a molecular program of Spemann/Mangold organizer development. We have also shown how early loss of the organizer and its inductive properties in an otherwise normal embryo, impacts on the molecular profile of surrounding tissues.

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Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

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