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Spatial Transcriptome of Mouse Embryonic Development Drives Insight into Tissue Formation

NEW YORK – Researchers have generated an atlas of what transcripts are expressed where early in mouse embryonic development.

A University of Chinese Academy of Sciences-led team of researchers used the Geo-seq approach to generate a spatial transcriptome of mouse embryonic germ layer tissues from the pre-gastrulation stage to the late gastrulation stage. After implantation, the different germ layers of the developing embryo emerge and begin to differentiate into various cell lineages that eventually become the mouse's many tissues. As they reported today in Nature, the researchers could trace what tissues cells expressing different genes develop into.

"The spatiotemporal transcriptome opens avenues for exploring the molecular activity of lineage differentiation and tissue patterning in time and space, [and] unravels the principal regulatory mechanisms driving lineage development," the researchers wrote in their paper.

The researchers used the spatial transcriptomics approach Geo-seq to generate a catalog of transcripts from mouse embryonic germ layer tissues from the pre-gastrulation, embryonic day 5.5, to late gastrulation, embryonic day 7.5, stages. They folded in additional RNA-seq data for the morula, inner cell mass of the blastocyst, and the epiblast and primitive endoderm to generate a transcriptome that spans development both before and after implantation.

Using the single-cell regulatory network inference and clustering (SCENIC) pipeline, which measures regulon activities, the researchers grouped their samples into six groups. These broadly represented the major early embryonic tissue types, such as the ectoderm, mesoderm, and endoderm. Additionally, they identified nine transcription-factor-driven regulon groups associated with those six lineage clusters.

Through principal components and t-distributed stochastic neighbor embedding analysis of the tissues, the researchers found the endoderm and principal endoderm clustered together, but not with the epiblast and epiblast derivatives. Meanwhile, the mesoderm and ectoderm clustered together at embryonic day 4.5 but diverged by embryonic day 7.5, and the endoderm of the gastrulating embryo was more similar to the primitive or visceral endoderm than to the primitive streak.

They further noted that the G1 regulon group was enriched in the embryonic day 7.5 mesoderm and posterior epiblast-primitive streak samples, while G9 regulon was enriched in cells from pre-implantation embryos, and the G6 regulon group was enriched in peri-implantation samples.

This suggested to the researchers that the predicted transcription factors associated with the various regulon groups could be involved in the development of the different tissue lineages. When they, for instance, knocked out Sp1, Hmga2, and Hmgb3 from regulon groups G7, G8, and G1 in embryonic stem cells, they found that loss of Sp1 led to the exit of native pluripotency to formative pluripotency during embryonic stem cell differentiation, while the loss of Hmga2 and Hmgb3 led to aberrant expression of primitive hematopoietic and cardiac genes, respectively.

The researchers also examined the role of the Hippo-Yap pathway, which plays a part in trophectoderm segregation. They found that it was only enriched in the endoderm. For instance, they noted that the Hippo-Yap factors like Tead1, Tead4, and Ctgf were specifically expressed in the endoderm, though Yap1 was more widely expressed. Blocking Yap signaling in the endoderm, though, led to the down-regulation of visceral-endoderm markers and the early endoderm-related regulon G3, but had no effect on late endoderm genes. This indicated to them that Hippo signaling is involved in early endoderm development.

The researchers added that the spatiotemporal transcriptome they developed could be applied to additional questions of development. "This dataset will be a valuable resource for guiding future efforts to elucidate the molecular mechanisms of lineage differentiation and morphogenesis of post-implantation embryos, and will enhance the efficiency of directed differentiation of pluripotent stem cells," they wrote in their paper.