Identifying gene regulatory networks during neuronal differentiation of iPSCs

Abstract

Neural induction is the earliest step in the formation of the human nervous system. However, the regulatory signals underlying neural induction are largely unknown due to ethical, technical and legal restrictions that limit access to live human cells during this stage. Dual SMAD inhibition induces ESC/iPSCs cells to acquire neural fate, providing an exceptional in vitro system to analyze neural induction. Transcriptome analyses enabled identification of sets of genes down-regulated during the initial 2 days of neural differentiation including pluripotency markers OCT4, NANOG and MYC. Subsequently, numerous genes are activated, including the neuroectodermal markers PAX6, ZEB2 and SOX11 and genes that have not being previously related with neural differentiation. Genes with similar expression profiles regulate biological processes such us cell-cell adhesion, required for the transition from pluripotency to neural competence. Statistical and mathematical approaches enable to infer time delay regulatory interactions of a set of transcription factors with high connectedness and model a gene regulatory network with 9 principal hubs (JUN, MYC, FOS, PAX6, SP3, CDC6, SMAD2, HDAC6, and LEF1). The network modules regulate activation or inhibition of biological processes associated with neural induction, such us cell proliferation (MYC), cell cycle progression (CDC6) and regulation of CNS development (PAX6). Single cell RNASeq demonstrated that neural induction gave rise to a largely homogeneous neuro-ectodermal cells population. Inhibition of WNT signaling during neural induction leads to re-specification of neuroectodermal cells to a placodal fate, which subsequently differentiate into GnRH neurons. Transcriptome analysis revealed a unique set of genes activated and inhibited during neural induction in the presence of WNT inhibition, some of which may be essential for re-specification to a placodal fate. These studies go some way to identifying genes and gene modules that pay a role during human neural induction thereby offering insight into a basic human developmental process and providing a foundation for understanding how specific genetic variations may give rise to neurodevelopmental disorders.

Date of Award	2018
Original language	English
Awarding Institution	King's College London
Supervisor	Jack Price (Supervisor) & Noel Buckley (Supervisor)