The Isl1-Lhx3-hexamer is recruited to a battery of cholinergic genes

Given that MNs acquire cholinergic neuronal characteristics as they become specified, we considered the possibility that the Isl1-Lhx3-hexamer, a determinant of the MN fate, regulates expression of a battery of cholinergic genes by directly binding to the enhancer of each cholinergic gene. Intriguingly, our ChIP-seq analysis, which mapped the genomic binding sites of the Isl1-Lhx3-hexamer in mouse embryonic stem cells [20], revealed Isl1-Lhx3-bound peaks in the key cholinergic pathway genes; ChAT, VAChT, high affinity choline transporter (CHT, also known as Slc5a7), a transporter that regulates the uptake of choline from the synaptic cleft into cholinergic neurons, and ATP-citrate lyase (Acly), an enzyme that synthesizes acetyl-CoA (Fig. 1A–C). ChAT has a strong peak within an intronic region that lies downstream of the VAChT gene, which is itself encoded within the intron of the ChAT gene. The Acly gene has two strong peaks in intronic regions and one upstream peak, while the CHT gene has a peak ∼100 kb downstream of its coding region. All the peaks have at least one hexamer response element (HxRE) (Fig. 1C, Fig. S1B) [20].

To test whether the Isl1-Lhx3-hexamer is recruited to Isl1-Lhx3-bound peak regions of the cholinergic genes in vivo, we purified genomic DNA bound by the Isl1-Lhx3-hexamer from E12.5 embryonic spinal cords using ChIP assays with α-NLI, α-Isl1, and α-Lhx3 antibodies. All three components of the Isl-Lhx3-hexamer bound to the peaks in the cholinergic genes, while they did not bind to the genomic regions without the peaks (Fig. 1D), indicating that the endogenous Isl1-Lhx3-hexamer is recruited to the cholinergic pathway genes in the developing spinal cord. Together, our unbiased, genome-wide ChIP-seq data, along with in vivo ChIP results, strongly suggest that the cholinergic pathway genes are directly activated by the Isl1-Lhx3-hexamer during MN fate specification.

The Isl1-Lhx3-hexamer upregulates a battery of cholinergic genes in the spinal cord

To test whether the Isl1-Lhx3-hexamer is capable of inducing the expression of multiple cholinergic genes in embryonic spinal cord, we misexpressed Isl1 and/or Lhx3 in the chick neural tube and monitored the expression of cholinergic genes. Co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of a panel of cholinergic genes, including ChAT, VAChT, Acly and CHT, in the dorsal neural tube, while electroporation of Isl1 or Lhx3 alone did not (Fig. 2A, Fig. S2, data not shown). These data indicate that the Isl1-Lhx3-hexamer is capable of upregulating the cholinergic pathway genes in the developing spinal cord.

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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.

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Isl1 is required for cholinergic gene expression in the developing spinal cord

To test whether Isl1 is needed for the cholinergic neuronal differentiation in the developing CNS, we deleted the Isl1 gene in neural progenitors using nestin-Cre [28], [29]. In E12.5 Isl1^f/f;nestin-Cre mice, Isl1 expression in MNs in the ventral spinal cord was greatly reduced (Fig. 2B). In this condition, expression of cholinergic genes, such as Acly, ChAT, VAChT and CHT, is drastically downregulated (Fig. 2B). The weak signal of VAChT was detected only in the remaining Isl1-expressing cells of Isl1^f/f;nestin-Cre mice (Fig. 2B). These results support a role of Isl1 in controlling cholinergic fate decision in the spinal cord.

Cholinergic enhancers are activated by the Isl1-Lhx3-hexamer

To test whether the Isl1-Lhx3-hexamer binding sites in the cholinergic genes act as enhancers to activate the cholinergic pathway genes in the embryonic spinal cord, we first examined whether the Isl1-Lhx3-hexamer activates the transcription of a reporter gene linked to each cholinergic gene peak, referred to here as ChAT-enh, Acly-enh1 and CHT-enh (Fig. 3A–E), using luciferase reporter assays in mouse embryonic P19 cells. As NLI is expressed endogenously in P19 cells, co-expression of exogenous Isl1 and Lhx3 leads to the formation of the Isl1-Lhx3-hexamer [17], [30]. The co-expression of Isl1 and Lhx3 strongly activated the Acly:LUC, ChAT:LUC and CHT:LUC reporters, but not LUC vector alone, in P19 cells, whereas the expression of Isl1 or Lhx3 alone did not (Fig. 3A–C). The Acly:LUC reporter with point mutations in the HxRE was not activated by the co-expression of Isl1 and Lhx3 (Fig. 3A). The DNA-binding defective forms of Lhx3 or Isl1 failed to synergize to activate Acly:LUC (Fig. 3A), indicating that DNA-binding activity of both Isl1 and Lhx3 is needed to activate the reporter. To further test the role of the HxRE in each enhancer for the potent transcription response to the combination of Isl1 and Lhx3, we generated luciferase reporters that are linked to multiple copies of the HxRE found within Acly-enh1, ChAT-enh, and CHT-enh, respectively. These minimal HxRE reporters were also highly activated by co-expression of Isl1 and Lhx3 (Fig. 3D, E, data not shown), establishing that the HxRE motif mediates activation of the cholinergic enhancers by the Isl1-Lhx3-hexamer. These data establish that the Isl1-Lhx3-hexamer is capable of activating each cholinergic enhancer in the Acly, ChAT/VAChT and CHT genes in heterologous cell types.

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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.

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To identify in vivo cell types in which the cholinergic enhancers activate gene expression in the developing spinal cord, we electroporated the neural tube of chick embryos with the GFP reporters linked to each cholinergic enhancer in ovo at a time when MNs are being specified. Interestingly, the Acly-enh1 drove strong GFP expression in MNs within the developing spinal cord (Fig. 3F, S3A). In contrast, GFP was not expressed in non-MN cell types, despite efficient transfection of those cells following in ovo electroporation (Fig. 3F, Fig. S3A), suggesting that only MNs have the transcriptional machinery that allows activation of Acly-enh1. Additionally, Acly-enh1 with point mutations in the HxRE motif failed to activate target gene expression in MNs (Fig. 3G, S3B), demonstrating that the HxRE motif is responsible for the MN-specific enhancer activity of the Acly-enh1. Furthermore, the multimerized HxRE motifs from the Acly-enh1 were sufficient to drive GFP reporter expression in MNs (Fig. 3H, Fig. S3C). Thus, the HxRE motif is necessary and sufficient for the MN-specific enhancer activity of the Acly-enh1, suggesting that the endogenous Isl1-Lhx3-hexamer is responsible for Acly enhancer activity. Consistent with this idea, co-expression of Isl1 and Lhx3, which assembles the Isl1-Lhx3-hexamer with endogenous NLI and triggers the formation of ectopic MNs in the dorsal spinal cord [14], ectopically activated both Acly-enh1:GFP and Acly-HxRE:GFP reporters, but it failed to activate Acly-enh1 with mutations in HxRE motif (Fig. 3F–H). These data indicate that the Isl1-Lhx3-hexamer is able to activate the Acly enhancer in the dorsal neural tube. The expression of Isl1 or Lhx3 alone failed to increase the transcriptional activity of the Acly-enh1 or Acly-HxRE (Fig. S3D, E). Similarly to the Acly enhancer, the ChAT enhancer also directed gene expression specifically to MNs and became ectopically activated by the co-expression of Isl1 and Lhx3 (data not shown). Together, these data demonstrate that the endogenous Isl1-Lhx3-hexamer binds to the cholinergic enhancers via the HxRE motifs and triggers the transcription of their target cholinergic genes as MNs are specified in embryonic spinal cords, establishing the Isl1-Lhx3-hexamer as a critical determinant of cholinergic neuronal identity in MNs.

Isl1 is co-expressed with Lhx8 and NLI in the ventral telencephalon

The coordinated upregulation of cholinergic pathway genes by the Isl1-Lhx3-hexamer in differentiating MNs, along with the previous loss-of-function studies suggesting that Isl1 and Lhx8 play important roles in the generation of FCNs [13], [24], raises the possibility that Isl1 and Lhx8 might form a complex similar to the Isl1-Lhx3-hexamer that drives cholinergic neuronal fate in the developing forebrain. To understand the role of Isl1 and Lhx8 in cholinergic gene expression in the developing forebrain, we determined the expression pattern of Isl1, Lhx3, and and NLI, which might form an Isl1-Lhx3-hexamer-like complex, using double immunohistochemistry analyses. All FCN precursors arise from the Nkx2.1-expressing MGE and preoptic area (POA) in the ventral telencephalon, and take two distinct migratory pathways; tangential migration to form striatal interneurons in the caudate-putamen (CPu) and radial migration to generate projection neurons in the basal forebrain (Fig. 4A) [21], [31]. In E12.5 forebrain, Lhx8 expression is largely confined within the subventricular zone (SVZ) and mantle zone (MZ) of the MGE, but a few Lhx8⁺ cells were found in the MZ of the lateral ganglionic eminence (LGE), which are likely the cells tangentially migrating from the MGE (Fig. 4B, C). In contrast, Isl1 is more abundantly expressed in the SVZ and MZ of the LGE, but is also expressed in the SVZ and MZ of the MGE (Fig. 4C, D). NLI is highly expressed in both MGE and LGE (Fig. 4D, E). Thus, Isl1, Lhx8 and NLI are co-expressed in a substantial fraction of cells in the MGE and LGE at E12.5. A similar expression pattern for Isl1, Lhx8, and NLI was observed in E13.5 forebrain (data not shown).

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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).

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By E16.5, VAChT⁺ cholinergic neurons were readily detectable in the CPu and basal meganocellular complex (BMC) (Fig. 5A, 6). Although a majority of CPu cells are derived from the LGE, Nkx2.1⁺ progenitors in the MGE produce distinct subtypes of striatal interneurons in the CPu, including cholinergic interneurons [21], [31]. Most cells in BMC, where a subset of cholinergic projection neurons is located, are generated from the Nkx2.1⁺ MGE [21], [31]. In E16.5 brains, Isl1⁺ cells were much more abundant in the CPu than in the BMC, whereas Nkx2.1⁺ and Lhx8⁺ cells were more abundant in the BMC than in the CPu (Fig. 5B, C, F, G), correlated with their expression at earlier developmental time points (Fig. 4). Despite this distinct pattern of gross expression, a number of Isl1⁺ cells co-expressed Nkx2.1 and Lhx8 in both the CPu and BMC, as shown by double immunohistochemistry analyses (Fig. 5B, C, F, G). Given that Nkx2.1⁺ and Lhx8⁺ striatal interneurons are originated in the MGE [22], [31], a subset of Isl1⁺ cells in the CPu, which co-express Nkx2.1 and Lhx8, is likely interneurons that are produced from the MGE.

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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.

https://doi.org/10.1371/journal.pgen.1004280.g005

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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.

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Specific deletion of Isl1 in the MGE leads to a loss of cholinergic interneurons in the CPu

Isl1^f/f;nestin-Cre mice die soon after E12.5, precluding us from observing cholinergic neuronal differentiation in the forebrain. To understand the role of Isl1 in FCN specification in the forebrain, we generated Isl1^f/f;Nkx2.1-Cre mice, in which Isl1 gene is deleted in cells derived from the Nkx2.1-expressing MGE [31]. As expected, in E16.5 Isl1^f/f;Nkx2.1-Cre mice, the number of Isl1⁺ cells was greatly reduced in the BMC, but not in the CPu where most of Isl1⁺ cells were derived from LGE and thus did not express Nkx2.1-Cre (Fig. 5B–I). In the CPu and BMC of the Isl1-conditional mutants, neither Isl1/Nkx2.1-double positive cells nor Isl1/Lhx8-co-expressing cells were found (Fig. 5B–I), indicating that Isl1 is deleted in cells produced from Nkx2.1⁺ MGE and that Isl1/Lhx8-co-expressing cells in the CPu and BMC are derived from the MGE. Interestingly, in the CPu of Isl1^f/f;Nkx2.1-Cre embryos, Lhx8⁺ and Nkx2.1⁺ interneurons were significantly reduced by ∼62% and ∼43%, respectively (Fig. 5J, K), suggesting that Isl1 is required for specification of a subset of Nkx2.1⁺/Lhx8⁺ striatal interneurons.

To monitor cholinergic neuronal differentiation, we performed immunostaining assays with VAChT antibodies. At E16.5, cholinergic neurons were detected in the CPu and BMC, and co-expressed Isl1 and Lhx8 in both areas (Fig. 6A–D). In E16.5 Isl1^f/f;Nkx2.1-Cre mice, however, cholinergic neurons were almost eliminated in the CPu (Fig. 6A, B). While ∼44% Lhx8⁺ cells and ∼35% Nkx2.1⁺ cells were cholinergic in the CPu of control embryos, almost all of Lhx8⁺ and Nkx2.1⁺ neurons did not express VAChT in the CPu of Isl1^f/f;Nkx2.1-Cre mice (Fig. 6E, F). The number of cholinergic neurons in the BMC area of Isl1^f/f;Nkx2.1-Cre embryos appeared to be reduced compared to that in the littermate controls, but the heterogeneity of cholinergic neurons in the BMC made the quantification very challenging (Fig. 6C, D). The remaining cholinergic neurons in the BMC expressed Lhx8 (Fig. 6D). Similar to E16.5, the number of cholinergic neurons remained markedly decreased in the CPu of E17.5 and P2 mice (Fig. S4).

Together, our data indicate that Isl1 and Lhx8 are co-expressed in at least two different populations of FCNs in the CPu and BMC, and that Isl1 function in the MGE-derived cells is required for the specification of cholinergic interneurons in the CPu during forebrain development.

Isl1 forms the Isl1-Lhx8-hexamer complex with Lhx8 and NLI

The co-expression of Isl1 and Lhx8 in FCN precursors and FCNs and requirement of Isl1 and Lhx8 for the specification of a subset of FCNs (Fig. 5, 6, S4) [13], [24] support the possibility that Isl1 and Lhx8 cooperate for the FCN specification by forming a hexamer complex (Fig. 7A), similar to the Isl1-Lhx3-hexamer (Fig. S1). The Isl1-Lhx3-hexamer assembly is dependent on the ability of Lhx3 to interact with Isl1 [14]. Another LIM-HD factor Lhx1 interacts with NLI, a common cofactor of the LIM-HD transcription factors, but does not bind to Isl1, thus forming only a typical LIM tetramer complex consisting of 2NLI:2Lhx1 [14]. Thus, we investigated whether Lhx8 can interact with Isl1, like Lhx3, using in vitro GST-pull down assays (Fig. 7B). As previously shown, Lhx3 interacted with Isl1 as well as NLI, whereas Lhx1 bound only to NLI. Interestingly, Lhx8 strongly associated with both Isl1 and NLI in vitro. Lhx8 also interacted with both Isl1 and NLI in HEK293 cells (Fig. 7C). Combined with the notion that NLI strongly self-dimerizes [32], our data supports a model by which Lhx8, Isl1 and NLI can form a hexameric complex consisting of two NLIs, two Isl1s and two Lhx8 molecules (Figure 7A). We refer to this complex as the Isl1-Lhx8-hexamer.

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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.

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To further test the formation of Isl1-Lhx8-hexamer complex, we examined whether Lhx8 interacts with NLI^DD-Isl1^ΔLIM, in which the dimerization domain (DD) of NLI is fused to LIM domains-deleted Isl1 (Figure 7D). As NLI^DD-Isl1^ΔLIM lacks the LIM-interaction domain (LID) of NLI, it cannot bind to LIM-HD factors via typical interaction interfaces between NLI-LID and LIM-domains of LIM-HD factors, which lead to tetramer formation. Co-immunoprecipitation assays revealed that NLI^DD-Isl1^ΔLIM associated with Lhx8 in cells despite the lack of NLI-LID in this fusion (Fig. 7D), further supporting the formation of the Isl1-Lhx8-hexamer in cells. Together, along with the fact that Lhx8, Isl1 and NLI are co-expressed in FCN precursors in the ventral telencephalon, these results suggest that Lhx8, Isl1, and NLI form the Isl1-Lhx8-hexamer complex in the ventral telencephalon during development.

The Isl1-Lhx8 complex recognizes a specific DNA motif

To investigate whether the Isl1:Lhx8 dimer, the DNA-binding unit of the Isl1-Lhx8-hexamer, recognizes specific DNA sequences, we performed the unbiased screening method SELEX (for systematic evolution of ligands by exponential enrichment) assay with Isl1, Lhx8, or an Isl1-Lhx8 fusion, in which full-length Isl1 and Lhx8 proteins were linked by a flexible short linker (Fig. 7E). Isl1-Lhx8 highly enriched a 15 nucleotide-long Isl1:Lhx8-binding motif after the third round of SELEX reaction, while Isl1 or Lhx8 failed to enrich any specific DNA sequences. The same motif was also isolated by SELEX with the mixture of Isl1 and Lhx8, which were translated from Isl1-T2A-Lhx8 construct in vitro (Fig. 7F), indicating that the Isl1-Lhx8-binding motif is not an artifact caused by use of the Isl1-Lhx8 fusion protein. These data indicate that Isl1:Lhx8 dimer in the Isl1-Lhx8-hexamer has high affinity to the specific DNA motif. Notably, Isl1-Lhx8-binding motif has a resemblance to the previously identified Isl1:Lhx3-site [17], [20], such as TAAT sequences, but also has unique features (Fig. S5).

The Isl1-Lhx8-hexamer binds to cholinergic enhancers in developing forebrain

Considering the shared function of Isl-Lhx3-hexamer and Isl1-Lhx8-hexamer in inducing cholinergic genes and the similar features of their binding motifs, it is possible that they bind to the same enhancer regions of cholinergic pathway genes. To test whether, in the developing forebrain, the endogenous FCN-hexamer is recruited to the same cholinergic enhancers identified as targets of the Isl1-Lhx3-hexamer in our ChIP-seq analysis, we performed ChIP assays for the Isl1-Lhx8-hexamer using the dissected E15.5 embryonic forebrains and found that Isl1, Lhx8, and NLI, all components of the Isl1-Lhx8-hexamer, bound to the cholinergic enhancers (Fig. 8A). These results suggest that the cholinergic enhancers recruit the Isl1-Lhx8-hexamer in the embryonic forebrain.

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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.

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The Isl1-Lhx8-hexamer activates cholinergic enhancers in the developing forebrain

To test the effect of the Isl1-Lhx8-hexamer on the transcriptional activity of cholinergic enhancers, we performed luciferase reporter assays in P19 cells using the Acly-HxRE:LUC and ChAT-HxRE:LUC reporter constructs. The co-transfection of Isl1 and Lhx8 activated each cholinergic enhancer, while expression of Isl1 or Lhx8 alone had minimal effect (Fig. 8B, C). These results suggest that the Isl1-Lhx8-hexamer complex triggers the transcriptional activity of cholinergic enhancers.

To investigate the activity of the Acly enhancer in the ventral forebrain, we injected the Acly-HxRE:GFP reporter along with the expression vectors encoding LacZ, Isl1 or Lhx8 into the ventral regions of E15.5 brain slices. The brain slices were then electroporated, cultured in vitro for four days, and examined for GFP expression (Fig. 8D). Among many LacZ⁺ electroporated cells, only a small number of basal forebrain cells expressed GFP (data not shown). The co-electroporation of Isl1 and Lhx8 along with the Acly-HxRE:GFP reporter drastically increased the number of GFP⁺ cells and the levels of GFP expression in the ventral forebrain, whereas expression of Lhx8 or Isl1 alone did not exhibit potent effects on Acly-HxRE:GFP. Likewise, the transfection of cortical progenitors using in utero electroporation revealed that the expression of Isl1-Lhx8, but not Isl1 or Lhx8 alone, strongly activates Acly-HxRE in the developing cortex (Fig. S6A). These results indicate that the combinatorial expression of Lhx8 and Isl1 promotes Acly-HxRE enhancer activity in the developing forebrain.

Together, these data suggest that the Isl1-Lhx8-hexamer is sufficient to activate the cholinergic enhancers in heterologous cells and the developing forebrain.

Isl1-Lhx8, but not Isl1-Lhx3, induces cholinergic gene expression in the developing forebrain

The binding and activation of cholinergic enhancers by Isl1-Lhx3 and Isl1-Lhx8 in the spinal cord and forebrain, respectively, prompted us to ask whether both complexes are capable of inducing the cholinergic gene battery irrespective of rostro-caudal positions within the CNS. To address this question, we misexpressed LacZ, Isl1, Lhx8, Lhx3, Isl1-Lhx8 or Isl1-Lhx3, along with EF1 promoter driven-GFP vector to mark the electroporated cells, in the E13.5 mouse cortex using in utero electroporation, and compared the expression levels of cholinergic genes between electroporated and control cerebral hemispheres at E18.5 using quantitative RT-PCR (Fig. 9A). The expression level of transgenes was higher in electroporated sides than in control sides, as expected (Fig. S7A). The expression of Isl1-Lhx8 substantially induced expression of ChAT, VAChT, and CHT in the cortex, compared to expression of Isl1 or Lhx8 alone (Fig. 9B), indicating that Isl1 and Lhx8 function in combination to induce expression of cholinergic genes in the developing forebrain. Interestingly, Isl1-Lhx3 did not trigger cholinergic gene expression in the forebrain (Fig. 9B), despite its potent activity to induce cholinergic pathway genes in the developing spinal cord (Fig. 2A). Moreover, unlike the spinal cord, Isl1-Lhx3 failed to upregulate MN genes, Isl2, Hb9, and chodl [33], in the forebrain (Fig. S7B, data not shown), suggesting that the Isl1-Lhx3-hexamer is unable to turn on the MN gene program in the forebrain. Together, our results strongly support a model whereby the Isl1-Lhx8-hexamer orchestrates upregulation of a battery of cholinergic pathway genes in the developing forebrain.

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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.

https://doi.org/10.1371/journal.pgen.1004280.g009

The cellular context is critical for the Isl1-Lhx3-hexamer and the Isl1-Lhx8-hexamer to upregulate their target genes

Our results that Isl1-Lhx3 failed to upregulate MN genes and cholinergic genes raise the question of whether Isl1-Lhx8-hexamer is functional in the spinal cord. To address this question, we expressed Lhx8, Isl1, Isl1-Lhx8, or Isl1 plus Lhx8 in chick spinal cord using in ovo electroporation, and monitored cell differentiation and cholinergic gene expression three days post-electroporation. Lhx8 triggered ectopic generation of Chx10⁺ V2a interneurons in the dorsal spinal cord (Fig. 9C, S7C) like Lhx3 [14], underlining the similarity between Lhx8 and Lhx3. Co-expression of Isl1 with Lhx8 blocked Lhx8 from inducing V2a interneurons, suggesting that Isl1 binds to Lhx8 and changes the target gene specificity of Lhx8 as it does with Lhx3 [14] (Fig. 9C, D, S7C). Interestingly, however, co-expression of Isl1 and Lhx8 induced neither ectopic Hb9⁺ MNs nor cholinergic genes in the dorsal spinal cord (Fig. 9C, S7C). Likewise, Isl1-Lhx8 rarely triggered MN formation or cholinergic gene expression in the dorsal spinal cord (Fig. 9C, S7D), indicating that the Isl1-Lhx8-hexamer is ineffective in activating cholinergic gene expression in the spinal cord. Together, our data highlight that the proper cellular context is critical for the Isl1-Lhx3-hexamer and Isl1-Lhx8-hexamer complexes to function in target gene regulation.

Isl1-Lhx8-hexamer induces cholinergic fates in stem cells

Given that the Isl1-Lhx8-hexamer directly regulates the expression of cholinergic gene battery in the developing forebrain, it is possible that the Isl1-Lhx8-hexamer triggers cholinergic neuronal fate in stem cells. To test this possibility, we generated ESCs, in which the expression of Isl1-Lhx8 is induced by doxycycline (Dox), namely Isl1-Lhx8-ESCs (Fig. 10A, B). Isl1-Lhx8 forms the Isl1-Lhx8-hexamer with endogenous NLI in Dox-treated Isl1-Lhx8-ESCs (data not shown). The expression of cholinergic pathway genes, ChAT, VAChT, CHT and Acly, but not a MN gene Hb9, were readily induced by Isl1-Lhx8 under monolayer culture condition (Fig. 10C, D), suggesting that the Isl1-Lhx8-hexamer controls the expression of cholinergic pathway genes in ESCs. We also monitored the cholinergic gene expression in floating culture of embryoid bodies (EBs), which acquire the characteristics of forebrain neural precursors [34]. In the absence of Dox, many TuJ1⁺ neurons were observed in EBs, but VAChT⁺ neurons were hardly detected (Fig. 10E, F). Dox treatment markedly induced VAChT⁺TuJ1⁺ cholinergic neurons in EBs (Fig. 10E, F), suggesting that Isl1-Lhx8 triggers the cholinergic neuronal fate in stem cells. Likewise, RT-PCR also revealed that Isl1-Lhx8 significantly induced the expression of ChAT, VAChT and CHT in EB culture conditions (Fig. 10G). In the same conditions, Isl1-Lhx8 did not induce the expression of MN genes, such as Hb9, Isl2, and Chodl (Fig. 10G, data not shown). In contrast, Isl1-Lhx3 induced Hb9 as well as the cholinergic genes in both monolayer culture and floating embryoid bodies treated with retinoic acid and sonic hedgehog agonist (Fig. 10H, data not shown) [19]. Together, these results indicate that the Isl1-Lhx8-hexamer is capable of triggering the cholinergic neuronal fate, but not MN fate, in stem cells.

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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).

https://doi.org/10.1371/journal.pgen.1004280.g010

Ethics statement

All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at OHSU.

DNA constructs

Rat Isl1, Isl1-N230S, and mouse Lhx3, Lhx3-N211S, Lhx8, Lhx1, Isl1-T2A-Lhx3, Isl1-Lhx3 fusion, Isl1-Lhx8 fusion, NLI, and LacZ genes were cloned into pCS2, pcDNA3 (Invitrogen) containing a HA, Flag or myc-epitope tag, or pCIG for expression in mammalian cells and chick embryos and for in vitro transcription and translation reactions. All of these vectors except Lhx8 were previously described [14], [30], [41]. NLI^DD-Isl1^ΔLIM is a fusion of 1-298aa of NLI containing the self-dimerization domain of NLI and 111-349aa of Isl1, which is a C-terminal region of Isl1 that does not include the LIM domains. Isl1-N230S and Lhx3-N211S are missense mutatnts, which are deficient in their ability to directly bind DNA [14]. Isl1, Lhx3, Lhx8, Isl1-Lhx3 and Isl1-Lhx8 were also cloned into the pCIG-2 vector for electroporation of mouse brains. Isl1 and NLI were cloned into the bacterial expression vector pGEX4T-1 (Amersham) for in vitro GST-pull down experiments. Lhx8 and NLI were cloned into the mammalian GST expression vector pEBG for GST-pull down experiments in cell lines. Isl1-Lhx8 was generated by fusing Isl1 full-length and Lhx8 full-length via flexible linker GGSGGSGGSGG. Isl1-T2A-Lhx8 was generated by inserting T2A sequences between full-length Isl1- and full-length Lhx8-coding sequences.

The location of Isl1-Lhx3-bound ChIP-seq peaks for cholinergic genes in mouse genome (mm9) is the following; ChAT/VAChT, chr14:33256618–33257117; CHT, chr17:54298028–54298480; Acly, chr11:100381966–100382465, chr11:100379377–100379876, and chr11:100395147–100395645. Mouse genomic regions covering the Acly-enhancer, ChAT-enhancer and CHT-enhancer were amplified using PCR, and two or three copies of these enhancers were cloned into TK-LUC or synthetic TATA-GFP reporter vectors. Primers to amplify these genomic enhancers are Acly-enahncer1, forward 5′- GA AGA TCT TGA TAG CAC ACT ACT TTG CTC TGG, reverse 5′- CG GGA TCC CAG TGA CGC ACG GCG AGC GGG AAG; ChAT-enhancer, forward 5′- GA AGA TCT TAC TAA TTG GAT TAA TTG ATT TGC, reverse 5′- CG GGA TCC GGG AAT TAA TAA CTT AGA ATT TGA; CHT-enhancer, forward 5′- GA AGA TCT TGA GCA GCC TAT GCC ACA AGG ACA, reverse 5′-CG GGA TCC AGG AAT CCA TCA CAA AGC TAA GAC. AAGCTGATTA sequences in Acly-enh1 were mutated to CCGCGCGGCC to generate the Acly-enh1-HxRE-mt reporter. Acly-HxRE:LUC, Acly-HxRE:GFP, ChAT-HxRE:LUC and ChAT-HxRE:GFP reporters were created by cloning multiple copies of the following duplex oligonucleotides into synthetic TATA-GFP or TK-LUC vectors. Acly-HxRE, 5′- CAG AGC TAAT CAG CTTG AGTG GGT-3′; ChAT-HxRE 5′- TGG TAC TAAT TGG ATTA ATTG ATT-3′.

Mice

The generation of Isl1^f/f, Nestin-Cre, and Nkx2.1-Cre mice has been described previously [28], [29], [31]. Isl1^f/f mice were crossed with Isl1^f/+;NesticCre mice or Isl1^f/+;Nkx2.1Cre mice to generate Isl1^f/f;NesticCre or Isl1^f/f;Nkx2.1Cre embryos, respectively, for analyses. Mouse embryos were collected at the indicated developmental stages, and fixed in 4% paraformaldehyde, embedded in OCT and cryosectioned in 12 µm thickness for immunohistochemistry assays or 18 µm thickness for in situ hybridization with digoxigenin-labeled probes.

In ovo electroporation

These assays were performed as described [14], [42]. In chick electroporation assays, DNAs were injected into a ∼ Hamburger and Hamilton (HH) stage 13 chick neural tube. The embryos were harvested 3 days post-electroporation and fixed in 4% paraformaldehyde, embedded in OCT and cryosectioned in 12 µm thickness for immunohistochemistry assays or 18 µm thickness for in situ hybridization with digoxigenin-labeled probes. Each set of chick electroporation experiments was repeated independently three to six times with at least three embryos injected with the same combination of plasmids for each experimental set. Representative sets of images from reproducible results were presented.

Immunohistochemistry and in situ hybridization

For immunohistochemistry assays, the following antibodies were used; rabbit anti-Hb9 [43], mouse anti-Mnr2/Hb9 (5C10, DSHB), rabbit anti-Isl1/2 [9], guinea pig anti-Chx10 [43], rabbit anti-Lhx3 [15], guinea pig anti-VAChT (AB1588, Millipore), goat anti-ChAT (AB144P, Millipore), rabbit anti-GFP (A6455, Molecular Probes), rabbit α-Nkx2.1 (Santa Cruz), guinea pig α-Lhx8 (generated using mouse Lhx8 211–367aa region as antigen), rabbit anti-NLI [44], TuJ1 (Covance) and mouse anti-Flag (Sigma).

For in situ hybridization analyses, cDNA for mouse ChAT, Acly and CHT and chick CHT, Acly, VAChT and ChAT were cloned to pBluescript vector and these vectors were used to generate digoxigenin-labeled riboprobes.

Co-immunoprecipitation assays and GST-pull down assays

HEK293T cells were seeded onto 10 cm tissue cultures dishes, cultured in DMEM media supplemented with 10% fetal bovine serum, and transfected using Superfect (Qiagen). 48 hours after transfection, cells were harvested and lysed in IP buffer (20 mM Tris-HCl, pH 8.0, 0.5% NP-40, 1 mM EDTA, 150 mM NaCl, 2 mM PMSF, 10% Glycerol, 4 mM Na₃VO₄, 200 mM NaF, 20 mM Na-pyroPO₄, and protease inhibitor cocktail). In these studies, precipitations were performed with either α-Flag antibody (Sigma) or glutathione sepharose beads (GE-Healthcare). The interactions were monitored by western blotting assays using α-Flag (Sigma) and α-HA (Babco) antibodies. Following western blotting with fluorescence-labeled secondary antibodies, the bound fractions of proteins were scanned by the Odyssey imaging system (Li-Cor) following western blotting with fluorescence-labeled secondary antibodies.

In vitro GST-pull down assays were performed as described [45]. BL21 E. coli were transformed with pGEX vector alone, pGEX-Isl1, or pGEX-NLI to express the GST-fusion proteins and lysed by sonication. The GST-fusion proteins were purified by incubating the lysates with glutathione sepharose beads (GE-Healthcare). The beads were then washed and incubated with the putative interacting partners Lhx8, Lhx3 and Lhx1, which were generated in vitro by using the TnT T7 Quick Coupled transcription/translation system (Promega). Bound proteins were eluted by boiling, and were monitored by western blotting assays using α-HA (Babco) antibodies and Odyssey imaging system (Li-Cor).

SELEX

SELEX was performed as described [46] with proteins in vitro transcribed and translated from the following vectors; Flag- tagged Isl1-Lhx8 fusion, Flag-Isl1, Flag-Lhx8, and Isl1-T2A-Lhx8 which produce both Flag-Isl1 and HA-Lhx8 proteins. The proteins, which were generated by using the TnT T7 Quick Coupled transcription/translation system (Promega), were incubated with a pool of double-stranded oligonucleotides containing a central core region of 22 random nucleotides with identical 5′- and 3′-flanking regions. For each SELEX reaction, ∼30 clones were randomly selected and sequenced. The motif analysis was conducted using Multiple Em for Motif Elicitation (MEME) [47].

Cell culture and luciferase assays

P19 embryonic carcinoma cells were cultured in α-minimal essential media supplemented with 2.5% fetal bovine serum (FBS) and 7.5% bovine calf serum. For luciferase assays, P19 cells were seeded and incubated for 24 hours, and transient transfections were performed using Lipofectamine 2000 (Invitrogen). An actin promoter-β-galactosidase plasmid was cotransfected for normalization of transfection efficiency, and empty vectors were used to equalize the total amount of transfected DNA. Cells were harvested 36–40 hours after transfection. Cell extracts were assayed for luciferase activity and the values were normalized with β-galactosidase activity. Data are presented as means of triplicate values obtained from representative experiments. All transfections were repeated independently at least four times. Luciferase reporter data are shown in relative activation fold (mean +/− standard deviation).

Ex vivo embryonic mouse brain electroporation followed by organotypic slice culture and in utero electroporation

The overall procedures for ventral forebrain electroporation and organotypic slice culture were previously described [48]. E15.5 mouse embryos were harvested and brains were dissected and embedded in 3% low melting point agarose dissolved in complete Hanks Balanced Salt solution (cHBSS). 250 µm thick slices of the brains were generated using a Leica VT1200 vibratome. Slices containing the appropriate regions of the ventral forebrain were focally injected with combinations of plasmids. The slices were then mounted on the anode above a 1 mm agarose slice and cHBSS was used to gap the cathode, and electroporated using ECM 830 electroporator (BTX) under the following condition; 60 mV, 5 ms interval pulse, 500 ms delay, and 5 pulses. Immediately after the electroporation, the slices were transferred to transwell inserts (0.4 µm pore size) and cultured for three to five days in vitro with slice media containing 5% heat inactivated horse serum added below the insert at 37°C with 5% CO₂. Slices were fixed in 4% paraformaldehyde, washed in PBS and analyzed post-fix using immunofluorescence histochemistry.

The overall procedures for ex vivo brain electroporation and organotypic slice culture were previously described [49]. E15.5 mouse embryos were harvested and then the heads were removed and placed in cHBSS. Each combination of DNA constructs mixed with 0.5% Fast Green (Sigma) were injected into the lateral ventricles of isolated E15.5 mouse heads using a Picospritzer III microinjector. The electroporation was carried out on whole heads using ECM 830 electroporator (BTX) under the following condition; 30 mV, 100 ms intervals, 4 pulses, and 100 ms delay. For organotypic slice culture, brains were dissected immediately following electroporation, and embedded in 3% low melting point agarose dissolved in cHBSS. 250 µm thick slices of the brains were generated using a Leica VT1200 vibratome and transferred to transwell inserts (0.4 µm pore size). The slices were then cultured for three to five days in vitro with slice media containing 5% heat inactivated horse serum added below the insert at 37°C with 5% CO₂. Slices were fixed in 4% paraformaldehyde and analyzed for GFP expression.

Each set of mouse brain electroporation experiments was repeated independently three to six times. For each set of mouse brain electroporation, three to four brain slices were electroporated per condition. Reproducible results were presented in the figures. Confocal images were acquired using a Nikon Eclipse Ti inverted microscope with perfect focus and a motorized stage coupled to a 4 laser line A1 scanning confocal system. Representative sets of images were presented.

For in utero electroporation, timed-pregnant C57BL/6N females were anesthetized at stage E13.5 with isoflurane (4% during induction, 2.5% during surgery), and the uterine horns were exposed by way of laparotomy. 1 µℓ of the expression vector in PBS containing 0.05% fast green (Sigma-Aldrich, St Louis, MO, USA) was injected into the lateral ventricle of the embryo using a 100 mm glass capillary (1B100-4, World Precision Instruments, Inc., USA). Electroporation was performed using Tweezertrodes (diameter, 5 mm; BTX, Holliston, MA, USA) with 5 pulses of 45 V for 50 millisecond duration and 950 millisecond intervals using a square-wave pulse generator (ECM 830; BTX). The uterine horns were then returned to the abdominal cavity, the wall and skin were sutured, and embryos were allowed to continue their normal development and collected for the further analyses at indicated stages.

RNA extraction and RT-PCR analysis

Total RNAs were extracted using the Trizol (Invitrogen) and reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control (FAM Dye/MGB Probe, Non-Primer Limited). In addition, the following primers were used with the SYBR green kit (11762-500, Invitrogen) and Mx3000P (Stratagene). Hb9, 5′-GTT GGA GCT GGA ACA CCA GT, 5′-CTT TTT GCT GCG TTT CCA TT; ACLY, 5′-GAA GCT GAC CTT GCT GAA CC, 5′-CTG CCT CCA ATG ATG AGG AT; ChAT, 5′-CCT GCC AGT CAA CTC TAG CC, 5′-GGA AGC CTT TAT GAT GAG AA; CHT, 5′-GTG GTC TAG CTT GGG CTC AG, 5′-GGC AAT GAG TGC AGA GAC AA; VAChT, 5′-TTG ATC GCA TGA GCT ACG AC, 5′-CCA CTA GGC TTC CAA AGC TG; Hb9, 5′-GTT GGA GCT GGA ACA CCA GT, 5′-CTT TTT GCT GCG TTT CCA TT; Isl2, 5′-GCA AAC TCG CTG AGT GCT TTC, 5′-ACC ATA CTG TTG GGG GTG TC; Chodl, 5′-CAG TGG AAT GAC GAC AGG TG, 5′-GGT TCC CAA AGC AAC CAG TA; Isl1, 5′-GAC ATG ATG GTG GTT TAC AGG C, 5′- GCT GTT GGG TGT ATC TGG GAG; Lhx3, 5′-AGA GCG CCT ACA ACA CTT CG, 5′-GGC CAG CGT CTT TCT TCA GT; Lhx8, 5′-CAG TTC GCT CAG GAC AAC AA, 5′-AGC CAT TTC TTC CAA CAT GG; GAPDH, 5′-ACC ACA GTC CAT GCC ATC AC, 5′-TCC ACC ACC CTG TTG CTG TA; and Cyclophilin A, 5′-GTC TCC TTC GAG CTG TTT GC, 5′-GAT GCC AGG ACC TGT ATG CT. RT-PCR experiments were performed with three or four independent sets of samples. Data are represented as the mean of duplicate or triplicate values obtained from representative experiments. Error bars represent standard deviation.

Chromatin immunoprecipitation (ChIP) assays

The ChIP-seq data used for the analysis in this paper has been deposited in the Gene Expression Omnibus (GEO) database (assession no. GSE50993) [20].

To perform the ChIP assays with mouse embryonic tissues, we dissected E12.5 spinal cords or E15.5 forebrains. The microdissected spinal cords from five E12.5 embryos or the forebrains of three E15.5 embryos were combined together for each ChIP reaction with a specific antibody. The tissues were dissociated completely, fixed by 1% formaldehyde for 10 min at room temperature, and quenched by 125 mM glycine. Next, cells were washed with Buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Hepes, pH 6.5) and Buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Hepes, pH 6.5) sequentially. Then, cells were lysed with lysis buffer (0.5% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 8.0, Protease inhibitor cocktail) and were subjected to sonication for DNA shearing. Next, cell lysates were diluted 1∶10 in ChIP buffer (0.5% Triton X-100, 2 mM EDTA, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0, Protease inhibitor cocktail) and, for immunoclearing, were incubated with IgG and protein A agarose beads for one hour at 4°C. Supernatant was collected after quick spin and incubated with appropriate antibodies and protein A agarose beads to precipitate the hexamer/chromatin complex for overnight at 4°C. After pull-down of the hexamer/chromatin complex/antibody complex with protein A agarose beads, the beads were washed with TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), TSE II (same components as in TSE I except 500 mM NaCl) and Buffer III (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) sequentially for 10 minutes at each step. Then the beads were washed with TE buffer three times. The hexamer/chromatin complexes were eluted in elution buffer (1% SDS, 1 mM EDTA, 0.1M NaHCO₃, 50 mM Tris-HCl, pH 8.0) and decross-linked by incubating at 65°C overnight. Eluate was incubated at 50°C for more than two hours with Proteinase K. Next, DNA was purified with Phenol/chloroform and DNA pellet was precipitated by ethanol and resolved in water. The purified final DNA samples were subjected to quantitative PCR reactions using the SYBR green kit (11762-500, Invitrogen) and Mx3000P (Stratagene). The total input was used for normalization. All ChIP experiments were repeated independently at least three times. Data are represented as the mean of duplicate or triplicate values obtained from representative experiments, and error bars represent standard deviation.

The following primers were used for ChIP-PCR.

ChAT-enhancer

forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC

reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA

ChAT-negative

forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG

reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA

Acly-enh1

forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG

reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG

CHT-enhancer

forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA

reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA

The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 [9], rabbit anti-Lhx3 [15], rabbit anti-NLI [44], and goat anti-Lhx8 (sc-22216, Santa Cruz).

Generation of Dox-inducible embryonic stem cells (ESCs) and differentiation of ESCs

The generation of Isl1-Lhx3-ESCs was described previously [19]. To generate Isl1-Lhx8-ESCs, the A172LoxP ES cell line [50] was maintained in an undifferentiated state on 0.1% gelatin-coated dishes in the ESC growth medium that consisted of Knockout DMEM, 10% FBS, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol and recombinant leukemia inhibitory factor (LIF, 1000 units/ml, Chemicon). Flag-tagged Isl1-Lhx8 fusion was inserted into Tet-inducible plasmid p2Lox. The Isl1-Lhx8 vector was co-transfected with pSALK-Cre into the A172LoxP ES cell line using Lipofectamine 2000 (Invitrogen). Stable transfectants were isolated by selection with neomycin (G418, 400 µg/ml) for seven days. Dox-dependent induction of Flag-Isl1-Lhx8 expression was monitored by western blotting and immunohistochemical analyses using α-Isl1, α-Lhx8 and α-Flag antibodies.

To induce cell differentiation, Embryoid bodies (EBs) were formed and cultured for 2 days using the hanging drop method (1×10³ ESCs per 20 µℓ drop). Hanging drops were transferred to suspension culture in 6 well low attachment dishes and cultured. EBs were cultured without or with doxycycline (2 µg/ml) for 2–5 days in the ESC medium without LIF or in the differentiation medium that contains KnockOut serum replacement (Life technologies). Then, EBs were collected for either RT-PCR or immunohistochemical analyses.