Suppressing Nodal Signaling Activity Predisposes Ectodermal Differentiation of Epiblast Stem Cells

Abstract

SummaryThe molecular mechanism underpinning the specification of the ectoderm, a transient germ-layer tissue, during mouse gastrulation was examined here in a stem cell-based model. We captured a self-renewing cell population with enhanced ectoderm potency from mouse epiblast stem cells (EpiSCs) by suppressing Nodal signaling activity. The transcriptome of the Nodal-inhibited EpiSCs resembles that of the anterior epiblast of embryonic day (E)7.0 and E7.5 mouse embryo, which is accompanied by chromatin modifications that reflect the priming of ectoderm lineage-related genes for expression. Nodal-inhibited EpiSCs show enhanced ectoderm differentiation in vitro and contribute to the neuroectoderm and the surface ectoderm in postimplantation chimeras but lose the propensity for mesendoderm differentiation in vitro and in chimeras. Our findings show that specification of the ectoderm progenitors is enhanced by the repression of Nodal signaling activity, and the ectoderm-like stem cells provide an experimental model to investigate the molecular characters of the epiblast-derived ectoderm.

A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm

“…In analogy with our previous work creating gastrulation-stage patterns in vitro, we reasoned that geometrically confined hESCs treated with appropriately timed exogenous stimuli would create selforganized patterns of fates within the ectodermal germ layer. In both model organisms and embryonic stem cells, Nodal inhibition has been shown to be crucial for preventing mesendoderm differentiation, allowing cells to adopt ectodermal fates, while BMP signaling is responsible for generating the dorsalventral pattern within the ectoderm (Li et al, , 2015Liu et al, 2018). Thus, we hypothesized that following a two-stage protocol where hESCs are initially induced by Nodal inhibition and subsequently stimulated by BMP4 would cause them to exclusively adopt ectodermal fates.…”

“…Although we expect these fates to be spatially disorganized in standard culture, this would provide a starting point for micropatterning experiments that test whether geometric confinement leads to ordered emergence of the same set of fates. Previous work in mouse and hESCs, has shown that prolonged Nodal inhibition leads to commitment to the neural fate (Li et al, 2015;Liu et al, 2018;Smith et al, 2008), so we specifically sought a temporal window in which cells are committed to the ectoderm but not exclusively to neural fates.…”

“…Compared to pluripotent cells, ectodermal progenitors are characterized by lower levels of pluripotency markers OCT4 and NANOG, high levels of SOX2, and the absence of neural specific genes such as PAX6 (Chambers et al, 2009;Li et al, 2015;Liu et al, 2018;Wang et al, 2012). To determine when hESCs enter this state, we examined these markers as a function of the duration of Nodal inhibition, achieved by growing cells in N2B27 media supplemented with 10 μm of SB431542, a small molecule ALK4/5/7 receptor-kinase inhibitor (hereafter called ectodermal induction media).…”

Cdonmutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly

“…while Oct4 and Sox2 mRNA levels were largely unchanged. Expression of the additional 266 SB43152-inhibitable mEpiSC markers Fgf5 and T (Liu et al, 2018) were also reduced by EtOH 267 treatment ( Figure 2C). In contrast, EtOH did not alter levels of Lefty1 or Lefty2 in mEpiSC to differences in SB43152 vs. EtOH treatment, or differences in half-lives of these mRNAs in (e.g., HoxA1, Six1, and Gbx2) was observed in hESCs treated with SB43152 for 2 -6 days 274 (Chng et al, 2010;Vallier et al, 2009).…”

“…Treatment of hESC or mEpiSC cultures with the ALK4 inhibitor, SB43152, led to reduction of 256 Activin/Nodal target gene expression and, eventually, induction of early markers of 257 neuroectoderm differentiation (Chng et al, 2010;Li et al, 2015;Liu et al, 2018;Vallier et al, (Vallier et al, 2009). We assessed expression of these five genes 263 in mEpiSCs treated with EtOH for 6 hours ( Figure 2C).…”