High resolution temporal transcriptomics of mouse embryoid body development reveals complex expression dynamics of coding and noncoding loci

Brian S Gloss,Marcel E Dinger,Seth W Cheetham,Dominik C Kaczorowski,Bethany Signal,Andrew C Perkins,Franziska Gruhl

doi:10.1038/s41598-017-06110-5

Abstract

Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course.

Highlights

Over the past decade, transcriptomic investigations into the of nature embryonic stem cell (ESC) differentiation have elucidated key biochemical features of stemness and differentiation
Microarray and RNA sequencing have provided a means to characterize the molecular transitions in gene expression underlying ESC biology and more recently single cell transcriptomic studies have provided the first glimpses into the molecular history of these cells[2]
The gene expression patterns were broadly consistent with published studies (Supplementary Fig. S1B), there were changes in expression on less than 24 hourly timeframes that could not be attributed to stochastic expression changes

Summary

Introduction

Transcriptomic investigations into the of nature embryonic stem cell (ESC) differentiation have elucidated key biochemical features of stemness and differentiation. Various expression profiling studies, using both microarrays and RNA-seq[15,16,17,18], have been used to explore the molecular changes occurring during ES cell development, typically at 24-hourly or more This potentially has lead to incomplete gene expression relationships through the phenomenon of temporal aggregation bias whereby each time point is assumed to represent all the signaling changes occurring in that time window[19]. In contrast to single cell based approaches- which provide insight into the state of individual cells - examinations of whole cell populations provides system-wide behavior and a practical means to explore gene expression dynamics across time The combination of these techniques has recently shed light on the molecular framework of cellular differentiation[20]. High temporal resolution has provided valuable insights into transcriptional annotation and regulation in drosophila[22, 23], Xenopus[24] and C.elegans[25]

Methods

Results

Conclusion