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.

Figure 1. ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery.

(A) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. (B) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes ChAT/VAChT, CHT, and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) . (C) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). (D) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the Isl1-Lhx3-hexamer to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT-enhancer (ChAT-enh) and a negative control region lacking the Isl1-Lhx3-binding peak (ChAT-neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.

Figure 2. The Isl1-Lhx3-hexamer plays a crucial role in inducing the expression of cholinergic pathway genes in the developing spinal MNs.

(A) Expression analyses of the cholinergic pathway genes in chick embryos electroporated with Isl1 and Lhx3 using in situ hybridization. The co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of cholinergic genes in the dorsal spinal cord, as marked by brackets. + indicates the electroporated side. (B) Expression analyses of the cholinergic pathway genes using either immunohistochemistry or in situ hybridization on the spinal cord of E12.5 Isl1^f/f;nestinCre and littermate control embryos. The ventral quadrant spinal cord is shown. The cholinergic genes are markedly downregulated in Isl1^f/f;nestinCre mice. The remaining VAChT expression is correlated with the residual Isl1 expression in Isl1^f/f;nestinCre mice, as determined by immunostaining assays.

Figure 3. The cholinergic enhancers are activated by the Isl1-Lhx3-hexamer in the developing spinal cord.

(A–E) Luciferase reporter assays in mouse embryonic P19 cells using Acly-enh1-wt:LUC or Acly-enh1-mt:LUC, in which HxRE motifs are mutated (A), ChAT-enh:LUC (B), CHT-enh:LUC (C), Acly-HxRE:LUC (D), and ChAT-HxRE:LUC (E) reporters with expression vectors as indicated below each graph. The co-expression of Lhx3 wild-type and Isl1 wild-type, indicated as w or +, strongly activated the reporters linked to the cholinergic enhancers, but not the LUC vector alone or Acly-enh1-mt:LUC. Lhx3-N211S and Isl1-N230S, the DNA-binding defective missense mutants of Lhx3 or Isl1 that are indicated as m, failed to activate Acly-enh1-wt:LUC (A). (A–E) Error bars represent the standard deviation in all graphs. Error bars indicate standard deviation. (F–H) GFP reporter activity was monitored in chick embryos electroporated with Acly-enh1:GFP (F), Acly-enh1-HxRE-mt:GFP (G), and Acly-HxRE:GFP (H) reporters with either LacZ or Isl1 plus Lhx3 as indicated above. Acly-enh1 and Acly-HxRE drove MN-specific GFP expression, and were ectopically activated by co-expression of Isl1 and Lhx3 in the dorsal spinal cord (F, H). Acly-enh1-HxRE-mt:GFP did not display GFP expression in MNs and failed to respond to the co-electroporated Isl1 and Lhx3 (G), indicating that the HxRE motif is required for the MN-specific enhancer activity of Acly-enh1. + indicates the electroporated side. The areas of ectopic Hb9⁺ MNs, induced by co-expression of Isl1 and Lhx3, are marked by brackets.

Figure 4. Co-expression of Isl1, Lhx8 and NLI in the developing ventral forebrain.

(A) Schematic representation of the coronal section of E12.5 forebrain. The MGE produces striatal cholinergic interneurons in the CPu and cholinergic projection neurons in the BMC, which take different migratory paths. NCx, neocortex; MGE, medial ganaglionic eminence; LGE, lateral ganglionic eminence; CPu, Caudate-putamen; BMC, basal meganocellular complex. (B–E) Immunohistochemical analyses of expression of Nkx2.1, Isl1, Lhx8, and NLI on coronal sections of E12.5 mouse forebrains. Isl1 is co-expressed with Lhx8 and NLI in the mantle zone of the MGE (yellow asterisk) and LGE (red asterisk).

Figure 5. Isl1 is co-expressed with Nkx2.1 and Lhx8 in the CPu and BMC and is important for the formation of Nkx2.1/Lhx8-expressing striatal interneurons.

(A) Schematic representation of the coronal section of E16.5 forebrain. CPu, Caudate-putamen; BMC, basal meganocellular complex. (B–I) Immunohistochemical analyses on the CPu and BMC of E16.5 Isl1^f/f;Nkx2.1Cre and littermate control embryos. Isl1 is co-expressed with Nkx2.1 and Lhx8 in subsets of neurons in the CPu and the BMC (B, C, F, G). The dotted circles depict Isl1/Nkx2.1-double positive cells (D, F). In Isl1^f/f;Nkx2.1Cre embryos, the number of Nkx2.1 or Lhx8-expressing interneurons in the CPu is reduced (B–E), and Isl1⁺ cells in the BMC drastically decreased (F–I). (J, K) Quantification of the number of Lhx8- and Nkx2.1-expressing cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p<0.0005 in Student's t-test.

Figure 6. Isl1 is required for the formation of striatal cholinergic interneurons in the developing forebrain.

Immunohistochemical analyses on the CPu (A, B) and BMC (C, D) of E16.5 Isl1^f/f;Nkx2.1Cre and littermate control embryos. VAChT⁺ cholinergic interneurons in the CPu failed to form in the MGE-specific Isl1-null embryos. (E, F) Quantification of the number of Lhx8⁺VAChT⁺ (E) or Nkx2.1⁺VAChT⁺ cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p<0.0005 in Student's t-test.

Figure 7. The formation of the Isl1-Lhx8-hexamer complex.

(A) Schematic representation of the Isl1-Lhx8-hexamer consisting of Isl1, Lhx8 and NLI. The model depicts that the Isl1-Lhx8-complex regulates the cholinergic genes via binding to HxREs. (B) In vitro GST-pull down assays. Lhx8 and Lhx3 bind to both Isl1 and NLI with high affinity, whereas Lhx1 binds to only NLI, but not to Isl1. (C) GST-pull down assays in HEK293 cells transfected with Flag- and GST-tagged constructs as indicated above. Lhx8 interacts with both Isl1 and NLI in cells. (D) CoIP assays in HEK293 cells transfected with Flag-Lhx8 and HA-tagged NLI^DD-Isl1^ΔLIM. Lhx8 interact with NLI^DD-Isl1^ΔLIM, forming the FCN-hexamer-mimicking complex. (E, F) The SELEX methods revealed the high affinity binding sites for Isl1-Lhx8 fusion (E-value, 2.5e-79) and the mixture of Isl1 and Lhx8 (E-value, 2.8e-65). The bottom sequence logo shows reverse complementary sequences of the upper logo.

Figure 8. The Isl1-Lhx8-hexamer activates the cholinergic genes.

(A) ChIP assays with IgG, α-Isl1, α-Lhx8, and α-NLI antibodies in dissected E15.5 embryonic forebrains. The location of two sets of primers in the ChAT gene is indicated (arrows). The FCN-hexamer is recruited to the cholinergic enhancers. (B, C) Luciferase reporter assays in P19 cells using Acly-HxRE:LUC (B), and ChAT-HxRE:LUC (C) reporters with vectors indicated below each graph. The co-expression of Lhx8 and Isl1 strongly activated these reporters. Error bars represent the standard deviation in all graphs (A–C). (D) Schematic representation of ex vivo electroporation of the ventral forebrain. Sections containing the appropriate regions of the ventral forebrain were focally injected with combinations of plasmids and subjected to slice electroporation, followed by slice culture. The area of transfection was indicated in green. Due to the electroporation process with slices, part of cortex was also transfected with plasmids. (E–H) Activation of Acly-HxRE:GFP reporter in the ventral forebrains electroporated with constructs, LacZ (E), Lhx8 (F), Isl1 (G), and Isl1 and Lhx8 (H). The co-expression of Isl1 and Lhx8 strongly activated Acly-HxRE in the forebrain.

Figure 9. Isl1-Lhx8 induces the expression of cholinergic gene battery in the forebrain, but not in the spinal cord.

(A) Schematic representation of in utero electroporation of the cortex, followed by quantitative RT-PCR (qRT-PCR) analyses. E13.5 brains were subjected to electroporation after each combination of constructs was injected into the lateral ventricle. The GFP⁺ region of electroporated (+) cortex and the comparable region of unelectroporated (−) cortex were micro-dissected and analyzed for gene expression. (B) Expression analyses of the cholinergic pathway genes, ChAT, VAChT and CHT, in mouse cortices electroporated with constructs, as indicated by color bars. Y-axis indicates the relative expression levels of each cholinergic gene on the electropoated side over the control side. The expression of Isl1-Lhx8 led to upregulation of the cholinergic genes in the cortex, whereas that of Isl1-Lhx3 failed to induce cholinergic genes in this context. Error bars indicate standard deviation. ** p<0.0005 in Student's t-test. (C) Cell differentiation assays in chick embryos electroporated with constructs as indicated on top. Expression of either Lhx8 or Lhx3 led to the ectopic formation of Chx10⁺ V2 interneurons in the dorsal spinal cord. The electroporation of Isl1 plus Lhx3, but neither Isl1-Lhx8 fusion nor Isl1 plus Lhx8, generated ectopic Hb9⁺VAChT⁺ MNs. Only the electroporated side of the chick spinal cord is shown. Brackets indicate ectopic Chx10⁺ V2 interneurons or Hb9⁺VAChT⁺ MNs, which were formed above the dotted line of endogenous V2 interneurons (Chx10) or MNs (Hb9, VAChT). (D) Quantification of ectopic Chx10⁺ V2 interneurons in chick spinal cord upon electroporation of constructs indicated below the graph. Error bars indicate standard deviation. ** p<0.0005 in Student's t-test.

Figure 10. Isl1-Lhx8 induces a cholinergic fate in ESC-derived neurons.

(A, B) In Isl1-Lhx8-ESCs, the expression of Flag-tagged Isl1-Lhx8 was induced by Dox, as detected by western blotting assays with α-Flag antibodies (A) and immunohistochemistry assays with α-Isl1 and α-Lhx8 antibodies (B). (C, D) Quantitative RT-PCR analyses in Isl1-Lhx8-ESCs when cultured as a monolayer. Cholinergic genes, but not the MN gene Hb9, were induced by Isl1-Lhx8. (E–G) Cell differentiation analyses in floating EBs derived from Isl1-Lhx8-ESCs, cultured with or without Dox, which triggers expression of Isl1-Lhx8. Immunohistochemical analyses show that Isl-Lhx8 expression induces differentiation of VAChT⁺TuJ⁺ cholinergic neurons (E, F). Quantitative RT-PCR analyses show that cholinergic pathway genes, but not MN genes Hb9 and Isl2, were induced by Isl1-Lhx8 (G). (H) Quantitative RT-PCR analyses of Hb9 expression in Isl1-Lhx3-ESCs. Hb9 was induced by Dox treatment, which induces the expression of Isl1-Lhx3 in Isl1-Lhx3-ESCs, when cultured in either monolayer (M) or spinal neuronal differentiation (SN) conditions. Error bars represent the standard deviation in all graphs (C, D, G, H).

Figure 11. Isl1-Lhx8-hexamer and Isl1-Lhx3-hexamer complexes establish a cholinergic neuronal identity in FCNs and spinal MNs, respectively, by directly upregulating cholinergic gene battery.

During CNS development, the cholinergic genes recruit the Isl1-Lhx8-hexamer in the forebrain and the Isl1-Lhx3-hexamer in spinal cord via hexamer-response elements. This recruitment leads to concerted induction of the cholinergic genes, therefore enabling MNs and FCNs to acquire the cholinergic neuronal identity. The Isl1-Lhx8-hexamer and Isl1-Lhx3-hexamer likely induce unique sets of target genes in FCNs and MNs. These hexamers may cooperate with other transcription factors (TFs) in establishing cell type-specific gene expression patterns.