Integrative and perturbation based analysis of the transcriptional dynamics of TGFβ/BMP system components in transition from embryonic stem cells to neural progenitors.

Ruben Dries, Agata Stryjewska, Kathleen Coddens, Satoshi Okawa, Tineke Notelaers, Judith Birkhoff, Mike Dekker, Catherine M. Verfaillie, Antonio del Sol, Eskeatnaf Mulugeta, Andrea Conidi, Frank G. Grosveld, Danny Huylebroeck

Abstract

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by Transforming Growth Factor type β (TGFβ) family ligands (eg, Bone Morphogenetic Proteins [BMPs] and Activins/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and tran- scriptional interplay of the TGFβ system. To understand intra-family transcriptional regulation as part of this system’s actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interac- tions and single-cell expression heterogeneity in mouse embryonic stem cells (mESCs) transiting to neural progenitors. Interrogation at 24 hour intervals identified 4 types of temporal gene transcription profile that capture all stages, that is, pluripotency, epi- blast formation and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intri- cate system of multi-level regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demon- strates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.