Dissecting lineage priming in mouse embryonic stem cells
Year: 2015
Type: dissertation
Abstract: Embryonic stem (ES) cells derived from the epiblast of mouse blastocysts are characterized by the ability to self-renew and to give rise to all embryonic lineages (a feature known as pluripotency). Therefore, mouse ES cells represent a unique tool to understand how transcription factor networks are modified during embryogenesis. Several knowledge has been accumulated concerning the gene regulatory networks and their transcriptional dynamics during pluripotency and lineage commitment. However, the understanding, at the single cell level, of how pluripotent transcriptional networks are dismantled, while lineage-affiliated genes networks emerge during lineage commitment of ES cells is still scarce. The general aim of this study was to investigate single cell gene expression during neural commitment of pluripotent (Oct4, Sox2, Nanog), stochastic (Car2, Cldn6) and lineage-affiliated genes (Cdh2, Sox3, Crabp2, Fgf5, Dnmt3b, T). For that, I cultured mouse ES cells in conditions that allowed neural differentiation and performed single molecule RNA FISH analysis from cells collected at different time points of the differentiation protocol. Here, I confirm that the highest expression of pluripotency genes occurs when mouse ES cells are cultured in pluripotent conditions. I also show that the pluripotency network is dismantled when cells enter neural lineage differentiation, due to decreased expression of Oct4 and Nanog. In the first days of neural differentiation, neural genes start to be upregulated but their transcription is mainly bursty, similar to genes that are stochastically expressed in the first days of differentiation. Moreover, postimplantation epiblast markers (Fgf5 and Dnmt3b) were found upregulated at day 3, when Nanog is also transiently upregulated, suggesting that a first transition from pluripotent mouse ES cells to a primed state similar to mouse epiblast stem cells occurs during early neural commitment. Later in neural differentiation, neural genes start to be expressed in a constitutive pattern and no expression of stochastic genes is observed, suggesting a second transition to irreversible neural commitment with formation of neural progenitors. Overall, this work reveals molecular transitions and formation of intermediate subpopulations with specific gene signatures when mES cells are driven to neural fate.
Author: Andreia Faria‐Pereira
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2265957867