Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes Hyong-Ho Cho 1,2 , Francesca Cargnin 1 , Yujin Kim 1 , Bora Lee 1 , Ryuk-Jun Kwon 1 , Heejin Nam 3 , Rongkun Shen 1,4 , Anthony P. Barnes 1,5 , Jae W. Lee 1,5 , Seunghee Lee 3 *, Soo-Kyung Lee 1,4,5 * 1 Pediatric Neuroscience Research Program, Pape ´ Family Pediatric Research Institute, Department of Pediatrics, Portland, Oregon, United States of America, 2 Department of Otolaryngology–Head and Neck Surgery, Chonnam National University Medical School, Gwangju, Korea, 3 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea, 4 Vollum Institute, Oregon Health & Science University, Portland, Oregon, United States of America, 5 Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, United States of America Abstract The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development. Citation: Cho H-H, Cargnin F, Kim Y, Lee B, Kwon R-J, et al. (2014) Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes. PLoS Genet 10(4): e1004280. doi:10.1371/journal.pgen.1004280 Editor: Bennett G. Novitch, University of California Los Angeles, United States of America Received July 25, 2013; Accepted February 18, 2014; Published April 24, 2014 Copyright: ß 2014 Cho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from NIH/NINDS (R01 NS054941), March of Dimes Foundation, and Christopher and Dana Reeve Foundation (to SKL), NIH/NIDDK (R01 DK064678) (JWL), and Basic Science Research Program (2012R1A1A1001749) and Bio & Medical Technology Development Program (2012M3A9C6050508) of the National Research Foundation (NRF) funded by the Korean government (MEST) and National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (1220120) (to SL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: leeseung@snu.ac.kr (SL); leesoo@ohsu.edu (SKL) Introduction The choice of neurotransmitter is one of the most fundamental aspects of neuronal fate decision. Cholinergic neurons are located in diverse regions of the CNS, which do not share the developmental origin, and regulate complex behaviors. In the spinal cord, cholinergic motor neurons (MNs) control locomo- tion, whereas in the forebrain, cholinergic neurons regulate cognitive processes [1,2]. Defects in function or survival of cholinergic neurons result in severe human pathologies, including spinal cord injuries, diseases associated with impaired motor function and cognitive disorders resulting from the loss of forebrain cholinergic neurons (FCNs) [3]. Despite the crucial roles of cholinergic neurons in human physiology and pathology, the mechanisms that specify cholinergic neuronal cell fate throughout the CNS during vertebrate development remain largely unknown. The cholinergic neurotransmission system requires the function of several key factors that are highly expressed in all cholinergic neurons, termed cholinergic pathway genes (Fig. 1A) [4,5]. Understanding the gene regulatory mechanisms that control the expression of cholinergic pathway genes in different groups of cholinergic neurons will provide crucial insights into the process of cholinergic fate specification in CNS development. Given that each of the cholinergic pathway genes is essential for efficient cholinergic neurotransmission, it is probable that they are up- regulated in a coordinated fashion as neurons acquire cholinergic neuronal identity during vertebrate development. Supporting this possibility, the vesicular acetylcholine transporter (VAChT, also known as Slc18a3) gene is encoded within an intron of the choline PLOS Genetics | www.plosgenetics.org 1 April 2014 | Volume 10 | Issue 4 | e1004280