Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

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.

Identification of STAM1 as a novel effector of ventral projection of spinal motor neurons

“…STAM1) and the ISL1-LHX3 complex, our results support the previous prediction made from several genome-wide studies (Lee et al, 2012(Lee et al, , 2013Mazzoni et al, 2013) that the ISL1-LHX3 complex functions as a terminal selector of MN fate by regulating a battery of terminal differentiation genes that establish unique MN properties, such as neurogenesis, axonogenesis, axon guidance, synapse function, and cholinergic neurotransmitter characteristics (Lee et al, 2012(Lee et al, , 2013Cho et al, 2014). Of note, the Stam2 gene was not associated with any ISL1-LHX3 complex-binding peak (data not shown).…”

“…ChIP was performed in mouse embryonic spinal cords as described previously (Cho et al, 2014;Lee et al, 2013). The spinal cords were microdissected from E12.5 mouse embryos and cells were dissociated and subjected to ChIP assays.…”

“…These studies, combined with MN transcriptome analyses (Lee et al, 2012;Mazzoni et al, 2013), revealed that the ISL1-LHX3 complex directly regulates the expression of a wide range of downstream effector genes that function not only in cell fate specification but also in subsequent maturation steps of MN differentiation, such as cell migration, axon pathfinding and synapse formation. As a result, the roles of individual target genes of the ISL1-LHX3 complex in MN specification and differentiation/maturation have finally begun to be dissected (Cho et al, 2014;Lee et al, 2012Lee et al, , 2013Mazzoni et al, 2013). Interestingly, v-MNs in embryos deficient in both LHX3 and LHX4 reorients their axonal projections dorsally, whereas the misexpression of LHX3 in d-MNs reorients their axonal projections ventrally (Sharma et al, 1998).…”

Islet 1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood

“…This and our conditional knockout results point to ISL1 being a terminal differentiation gene that is necessary to establish the specific identity of POMC neurons after these cells are born and accumulate in the mantle zone, giving rise to the arcuate nucleus. Recent work has shown that Isl1 plays pivotal roles in determining the cell fate of GABAergic medium spiny striatofugal neurons (40), cholinergic magnocellular striatal interneurons (41), and cholinergic spinal and cranial motor neurons (42). Interestingly, these different ISL1-dependent neuronal types participate in pyramidal and extrapyramidal circuits that coordinate body movement and locomotion.…”

Transcription factors and effectors that regulate neuronal morphology

“…Similarly, it was recently shown that Fezf2 regulates both glutamatergic identity and axon guidance in corticospinal neurons (Lodato et al, 2014). Isl1, a key regulator of motor axon guidance, promotes cholinergic identity in motor neurons and in a subset of forebrain neurons (Cho et al, 2014). Interestingly, Drosophila Islet also coordinately regulates axon guidance with neural function (Thor and Thomas, 1997;Wolfram et al, 2012).…”