Inverse agonism of retinoic acid receptors directs epiblast cells into the paraxial mesoderm lineage

Abstract

We have investigated the differentiation of paraxial mesoderm from mouse embryonic stem cells utilizing a Tbx6-EYFP/Brachyury (T)-Ckerry dual reporter system. Differentiation from the mouse ESC state directly into mesoderm via Wnt pathway activation was low, but augmented by treatment with AGN193109, a pan-retinoic acid receptor inverse agonist. After five days of differentiation, T+ cells increased from 12.2% to 18.8%, Tbx6+ cells increased from 5.8% to 12.7%, and T+/Tbx6+ cells increased from 2.4% to 14.1%. The synergism of AGN193109 with Wnt3a/CHIR99021 was further substantiated by the increased expression of paraxial mesoderm gene markers Tbx6, Msgnl, Meoxl, and Hoxbl. Separate to inverse agonist treatment, when mouse ESCs were indirectly differentiated into mesoderm via a transient epiblast step the efficiency of paraxial mesoderm formation markedly increased. Tbx6+ cells represented 65–75% of the total cell population after just 3 days of differentiation and the expression of paraxial mesoderm marker genes Tbx6 and Msgn increased over 100-fold and 300-fold, respectively. Further evaluation of AGN193109 treatment on the indirect differentiation protocol suggested that RARs have two distinct roles. First, AGN193109 treatment at the epiblast step and mesoderm step promoted paraxial mesoderm formation over other mesoderm and endoderm lineage types. Second, continued treatment during mesoderm formation revealed its ability to repress the maturation of presomitic mesoderm into somitic paraxial mesoderm. Thus, the continuous treatment of AGN193109 during epiblast and mesoderm differentiation steps yielded a culture where —90% of the cells were Tbx6+. The surprisingly early effect of inverse agonist treatment at the epiblast step of differentiation led us to further examine the effect of AGN193109 treatment during an extended epiblast differentiation protocol. Interestingly, while inverse agonist treatment had no impact on the conversion of ESCs into epiblast cells based on the expression of Rexl, Fgf5, and pluripotency marker genes Oct4, Nanog, and Sox2, after three days of differentiation in the presence of AGN193109 caudal epiblast and early paraxial mesoderm marker genes, T, Cyp26al, Fgf8, Tbx6 and Msgn were all highly up-regulated. Collectively, our studies reveal an earlier than appreciated role for RARs in epiblast cells and the modulation of their function via inverse agonist treatment can promote their differentiation into the paraxial mesoderm lineage.

New Insights into the Control of Cell Fate Choices and Differentiation by Retinoic Acid in Cranial, Axial and Caudal Structures

“…A potential target that is repressed by Rarγ2 might be ripply2, a repressor of tbx6; therefore, promoting tbx6 expression [98,99]. Similar results were obtained in a study that differentiated mouse ESCs via Wnt pathway activation [100]. Here the pan-RAR inverse agonist AGN193109, which stabilizes the heterodimeric complex of RA receptors (RAR/RXR) with their transcriptional co-repressors, was applied to inhibit RA signaling during ESC differentiation, beginning at a differentiation stage that corresponds to cells from the CLE.…”

“…A continued treatment with the inverse agonist eventually repressed the maturation of PSM into the somitic mesoderm. This suggests that RARs function in epiblast and early mesoderm progenitor cells-areas where RA is absent-to promote their differentiation into paraxial mesoderm lineage [100].…”

An In Vitro Human Segmentation Clock Model Derived from Embryonic Stem Cells

“…There are several published protocols for differentiation of human pluripotent stem cells (PSCs) into somites and their derivatives (Chal et al, 2015; Loh et al, 2016; Xi et al, 2017; Nakajima et al, 2018; Russell et al, 2018). A recent study using single-cell RNA-sequencing (scRNA-seq) analyses found that the cells pass through a transitory state that displays gene expression signatures similar to somitomeres before specifying into somite cells (Loh et al, 2016); however, no oscillatory gene expression pattern has been reported.…”