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Emergence of cellular heterogeneity and body plan in early mammalian development

Panavaite, Laura

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In mammalian development, cell-to-cell gene expression heterogeneity first emerges in early embryo when segregation of embryonic and extraembryonic lineages is initiated. Lineage-specific gene expression is especially enhanced within the inner cells of the early blastocyst. I studied this initial phase of cellular heterogeneity to mechanistically understand how the first cell fate decisions are made in the mammalian embryo. To this end, we have performed a gene trap screen and generated mouse lines with Venus reporter expression in one of the two first lineages – inner cell mass and trophectoderm. To quantitatively describe gene expression dynamics at a single-cell resolution in living embryos, we have performed lineage tracking, quantitative gene expression and cell position analyses that allowed us to build a comprehensive lineage map of mouse pre-implantation development. We identified lineage-specific gene regulation that paves the way to embryonic cell fate decisions and determined that first embryonic lineages of the blastocyst – trophectoderm and the inner cell mass – are formed at different timing and by separate mechanisms.

The first phase of lineage-specific gene expression heterogeneity is resolved by segregation of first embryonic lineages and formation of the first embryonic axis within the late blastocyst. Within the inner cell mass of the blastocyst, new cellular heterogeneity arises that will dictate the body axis formation. During subsequent body-patterning phases, an inner cell mass-derived layer of cells called the visceral endoderm provides crucial signals for establishment of anterior-posterior axis. However, due to the difficulty of crossing the implantation barrier in vitro, the mechanism of generating a signaling center within the visceral endoderm remains elusive. In this work, I have presented a 3D peri-implantation culture method that supports continuous mouse embryo development in vitro from pre- to post-implantation stages while preserving its in-vivo-like geometry. Such embryos retain the expression of lineage-specific markers, are minimally delayed in their development, preserve in-vivo-like proportions, and correctly specify anterior-posterior axis in the absence of maternal cues. Observing and manipulating embryos in this culture, we were able to explore the hidden heterogeneity within inner cell mass population that predicts future body patterning. By combining 3D culture, time-lapse light sheet fluorescence microscopy, and single-cell RNA-Seq, we explored the lineage and character of cells that play a crucial role in setting up the anterior-posterior axis, and investigated the role of communication between the epiblast and the visceral endoderm in the development of the peri-implantation embryo and the establishment of the embryonic body plan.

Item Type: Dissertation
Supervisor: Hiiragi, Dr. Takashi
Place of Publication: Heidelberg, Germany
Date of thesis defense: 26 May 2017
Date Deposited: 17 Aug 2017 10:09
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
Uncontrolled Keywords: Mouse embryo, development, body axes, heterogeneity
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