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
1. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan;2. Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, Suita, Osaka, Japan;3. Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Osaka University, Suita, Osaka, Japan;4. iPS Cell‐based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan;5. Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Osaka, Japan;6. Department of Molecular Neuropsychiatry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan;7. Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA;8. Intellectual Development and Disabilities Research Center, The David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA;9. Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan;10. School of Pharmacy, Hyogo University of Health Sciences, Chuo‐ku, Kobe, Hyogo, Japan
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.
