Functional characterisation of cis-regulatory elements governing dynamic Eomes expression in the early mouse embryo

Abstract

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal Eomes expression in vivo. Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.

CytoCensus: mapping cell identity and division in tissues and organs using machine learning

“…Therefore, we reduced the laser power by approximately a factor of 10 (to ~12µW at the objective for 488 nm, and 7µW for 561 nm), and used post Page Short Title: CytoCensus acquisition patch-based denoising (NDSAFIR, by Kervrann and Boulanger, 2006, implemented as part of PRIISM, with adapt=0, island=4, zt mode and iterations=3 by Carlton et al, 2010) to restore image quality. For details on imaging of mouse embryos ( Figure 6) refer to Simon et al, (2017). Details of organoid imaging can be found in (Eldred et al, 2017).…”

Single cell multi-omics profiling reveals a hierarchical epigenetic landscape during mammalian germ layer specification

“…This finding supports the existence of a 'default' path in the Waddington landscape, providing a potential mechanism for the phenomenon of 'default' differentiation of neurectodermal tissue from ESCs 61-63 . Endoderm and mesoderm are thus actively diverted from the default path by signalling in the primitive streak which presumably induces demethylation and chromatin opening at the corresponding enhancers elements 26,32,64,65 . Hence, at least during gastrulation, lineages are defined by a hierarchical, or asymmetric, epigenetic model ( Figure 4c) .More generally, our discovery has important implications for the role of the epigenome in defining lineage commitment.…”

Genetic dissection of Nodal and Bmp signalling requirements during primordial germ cell development

“…Highest levels retained in the anterior streak function to induce DE and midline fates 10 Eomes in the early PS directly activates Mesp1 expression necessary to allow nascent mesoderm to down-regulate E-Cadherin and undergo EMT 36,50 . Here we demonstrate that Eomes, acting immediately downstream of Nodal within the epiblast 37 , plays an essential role during the early stages of PGC formation. In the absence of Eomes, we observe an expanded population of BV + cells in the proximal epiblast.…”

“…The T-box transcription factor Eomes, acting downstream of Nodal signalling, plays essential roles during gastrulation 36,37 . Conditional deletion in the epiblast (EomesDEpi) results in defective epithelial to mesenchymal transition (EMT) and the failure of nascent mesoderm cells to down-regulate E-cadherin and exit the PS 36 .…”