Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro.

Abstract

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock-in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet-1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate-based screening and identified BMP type I receptor kinase inhibitors LDN-193189 and DMH1 as activators of luciferase. LDN-193189 induced ES cells to express the genes encoding Pet-1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF-β receptor inhibitor SB-431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN-193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF-β target gene Lefty, whereas the opposite effect was observed with SB-431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF-β receptor signaling are implicated in serotonergic differentiation. Candidate-based screening for serotonergic induction using a rapid assay in mouse embryonic stem cells revealed that the bone morphogenetic protein (BMP) type I receptor kinase inhibitors selectively induce serotonergic differentiation, whereas the TGF-β receptor inhibitor SB-431542 inhibits the differentiation. These results suggest that inhibition of BMP type I receptors and concomitant activation of transforming growth factor-β (TGF-β) receptor signaling are involved in the early trajectory of serotonergic differentiation.