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.

The motor neuron (MN)-hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN-hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN-hexamer-bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN-hexamer, enhancing the transcriptional activity of the MN-hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN-hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord.he combinatorial action of transcription factors is a prevalent strategy for achieving cellular complexity in the CNS. However, how the combinatorial action of transcription factors leads to the expression of distinct batteries of terminal differentiation genes, which together establish a specific cellular identity; how the cell fate-specifying transcription factors interact with extracellular cues remain unclear. To address these questions, it is essential to identify both the cis-regulatory elements in the genome, which recruit a specific combination of transcription factors, and the target genes associated with those cis-regulatory elements.One of the best examples of combinatorial transcription codes has emerged from studies of spinal motor neuron (MN) development (1). Two LIM-homeodomain (LIM-HD) factors, LIM homeobox 3 (Lhx3) and Islet-1 (Isl1), are vital for directing MN fate specification in the developing spinal cord (2-5). During this process, two Isl1:Lhx3 dimers bind to nuclear LIM interactor (NLI, also known as LDB for LIM domain binding) that has a self-dimerization domain, thereby forming the MN-hexamer complex ( Fig. 1A and Fig. S1A) (2, 6). The combinatorial expression of Lhx3 and Isl1 is capable of triggering MN specification in chick spinal cord, ES cells (ESCs), and induced pluripotent stem cells (2,(6)(7)(8). In contrast to MNs, during the specification of V2 interneurons, two Lhx3s and two NLIs form a tetrameric complex, which directs the V2-interneuron fate (Fig. S1A) (2, 9). Thus, the combinatorial action of Lhx3 and Isl1, via the formation of the MN-hexamer, is critical to determine MN identity over V2-interneuron fate. However, key questions remain unanswered. First, does the MN-hexamer directly control terminal differentiation genes that are required for consolidating the functional identity of MNs? Second, does the MN-hexamer collaborate with other transcription factors and/or extracellu...

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