Fusion protein Isl1–Lhx3 specifies motor neuron fate byinducing motor neuron genes and concomitantlysuppressing the interneuron programsSeunghee Leea,b, James M. Cuvillierc, Bora Leea, Rongkun Shena,b, Jae W. Leea,1, and Soo-Kyung Leea,b,1

aNeuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Institute, and bVollum Institute, Oregon Health and Science University,Portland, OR 97239; and cDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030

Edited* by Richard H. Goodman, Vollum Institute, Portland, OR, and approved January 18, 2012 (received for review September 2, 2011)

Combinatorial transcription codes generate the myriad of cell typesduring development and thus likely provide crucial insights into di-rected differentiation of stem cells to a specific cell type. The LIMcomplex composed of Isl1 and Lhx3 directs the specification of spinalmotor neurons (MNs) in embryos. Here, we report that Isl1–Lhx3,a LIM-complex mimicking fusion, induces a signature of MN tran-scriptome and concomitantly suppresses interneuron differentiationprograms, thereby serving as a potent and specific inducer of MNs instem cells. We show that an equimolar ratio of Isl1 and Lhx3 and theLIM domain of Lhx3 are crucial for generating MNs without up-reg-ulating interneuron genes. These led us to design Isl1–Lhx3, whichmaintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domainof Lhx3. Isl1–Lhx3 drivesMN differentiationwith high specificity andefficiency in the spinal cord and embryonic stem cells, bypassing theneed for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1–Lhx3 induces the expression of a battery of MN genes that controlvarious functional aspects of MNs, while suppressing key interneu-rongenes. Our studies uncover a highly efficientmethod for directedMNgenerationandMNgenenetworks. Our results alsodemonstratea general strategy of using embryonic transcription complexes forproducing specific cell types from stem cells.

Developing central nervous system (CNS) produces a vastnumber of neuronal types, but adult CNS has only limited

capacity to regenerate neurons. This has prompted great interest inidentifying methods to produce specific neuronal types from stemcells. Production of differentiated cell types from pluripotent stemcells, such as embryonic stem cells (ESCs), should enable a con-tinuous supply of diseased cell types for drug screening and cellreplacement therapy and provide valuable insights into the patho-physiology of human diseases. One important challenge in this ef-fort is to steer stem cells into specific cell types. Recapitulation ofnormal developmental processes using embryonic inductive signalshas been used to drive differentiation of pluripotent stem cells intospecific cell types (1). However, this strategy tends to trigger for-mation of mixed cell types rather than a targeted cell type, becauseeach inductive signal is used in multiple developmental pathways.This shortcoming might be circumvented by using more specific,downstream transcription factors of inductive signals. In thisregard, it should be noted that many transcription factors functionin combination to determine cell fates during development, sug-gesting that coexpression of multiple transcription factors could bea more effective method to generate a particular cell type frompluripotent stem cells.Motor neurons (MNs) in the spinal cord project axons to

muscles and control their contraction. The developmental path-ways to generate MNs have been relatively well studied. In thedeveloping spinal cord, sonic hedgehog (Shh) signal triggers theexpression of two LIM homeodomain (HD) transcription factorsIsl1 and Lhx3 in differentiating MN cells (2, 3). Then, Isl1 andLhx3 form a transcriptional activating MN-hexamer complex, inwhich two Isl1:Lhx3 dimers are assembled into a complex viaa self-dimerizing cofactor nuclear LIM interactor (NLI, alsocalled LDB for LIM domain binding) (Fig. 1A) (4, 5). Thiscomplex is sufficient to induce ectopicMN formation in the dorsal

spinal cord, which is not exposed to a high concentration of Shh(4, 5). Lhx3 alone directs the specification of V2 interneurons(V2-INs) by forming the V2-tetramer complex, consisting of twoLhx3 and two NLI molecules (Fig. 1A) (4, 6). Thus, the combi-natorial action of Isl1 and Lhx3, exerted via the formation of theMN hexamer, is critical to induce MN differentiation withouttriggeringV2-IN differentiation. Identification of the downstreamgenes that are controlled by the MN hexamer would provideimportant insights into the developmental processes to generatefunctionally mature MNs.MNs differentiated from stem cells have proven to be useful for

developing potential therapies for human MN diseases. Shh,when combined with retinoic acid (RA), converted ESCs andinduced pluripotent stem cells (iPSCs) to MNs (7–10). However,under this condition, Shh also differentiates ESCs into spinalinterneurons (7). To develop new methods that resolve thisspecificity issue and generate spinal MNs from stem cells withhigher fidelity and efficiency, we explored the possibility of usingthe embryonic transcription program for MN generation, the MNhexamer, instead of Shh signal. We first investigated the mecha-nisms underlying theMN-hexamer function inMNdifferentiationand then applied this information to design a strategy for stem celldifferentiation to MNs. Here we report that an equimolar ratio ofIsl1 and Lhx3 is critical for specifically generating MNs withoutactivating V2-IN pathway and that the LIM domain of Lhx3 isrequired for the effective recognition of MN-hexamer responseelements (HxREs) by the MN hexamer. These findings led us todevelop a MN-hexamer mimetic fusion, Isl1–Lhx3, which directshighly specific and efficient differentiation of ESCs intoMNs. TheRNA-seq analyses of Isl1–Lhx3-induced MNs revealed that Isl1–Lhx3 up-regulates a battery of genes that control a wide range ofMN functions and concomitantly suppresses the interneurondifferentiation programs. To our knowledge, this is a uniquedemonstration that a fusion mimicking an embryonic transcrip-tion complex can serve as an ideal tool to generate a targeted celltype from stem cells.

ResultsRatio Between Isl1 and Lhx3 Is Critical for the Specific Generation ofMNs. Once Lhx3 is incorporated into the MN hexamer, it cannotform the V2 tetramer (Fig. 1A). Thus, an equimolar expressionof Isl1 and Lhx3 should be critical for MN-specific differentiationof stem cells without inducing V2-IN differentiation. To test this

Author contributions: S.L., J.W.L., and S.-K.L. designed research; S.L., J.M.C., B.L., and S.-K.L.performed research; S.L., R.S., J.W.L., and S.-K.L. analyzed data; and S.L., J.W.L., and S.-K.L.wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: RNA-seq data reported in this paper have been deposited in the GeneExpression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.GSE35510).1To whom correspondence may be addressed. E-mail: leesoo@ohsu.edu or leejae@ohsu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1114515109/-/DCSupplemental.

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idea, we expressed Isl1 with an increasing amount of Lhx3 in thechick neural tube and monitored the ectopic formation of Hb9+

MNs and Chx10+ V2-INs in the dorsal spinal cord (Fig. 1B andFig. S1). When the ratio of Lhx3 to Isl1 was 0.5, only Hb9+ MNs,but no ectopic Chx10+ cells, were formed. However, increasingthe amount of Lhx3 led to the generation of ectopic Chx10+ cellseven in the presence of Isl1 (Fig. 1B and Fig. S1). When the ratioof Lhx3 to Isl1 was 8, several cells acquired MN–V2-IN hybridcharacteristics expressing both Hb9 and Chx10 (Fig. S2). Theectopic generation of Chx10+ cells following coelectroporationof Isl1 and Lhx3 likely results from an excess of Lhx3 molecules,which form the V2-tetramer. Thus, expression levels of Isl1 andLhx3 should be tightly controlled at or close to an equimolarratio to differentiate neural stem cells specifically to MNs.

Isl1–Lhx3 Fusion Is a Specific and Efficient Inducer of the MN Fate. Inkeeping the optimal equimolar ratio of Lhx3 to Isl1, we gener-ated three fusions of Isl1 and Lhx3, which are predicted to mimicthe MN hexamer structurally (Fig. 1C). DD–Isl1HD

–Lhx3HD

consists of the dimerization domain (DD) of NLI fused to theDNA-binding HDs of Isl1 and Lhx3 (4). Isl1–Lhx3HD consists offull-length Isl1 fused to the HD of Lhx3. Isl1–Lhx3 is a fusionprotein of full-length Isl1 and Lhx3. Isl1–Lhx3HD and Isl1–Lhx3

can form MN-hexamer mimetic complexes with widely expressedendogenous NLI (4, 11). To test whether the fusions activate theMN-hexamer target enhancers, we performed luciferase assaysin P19 mouse embryonic cells using the luciferase reporterslinked to HxREs and to the MN-specific enhancer in the Hb9gene, in which the MN-hexamer transcriptionally synergizes withthe proneural basic helix–loop–helix (bHLH) factor NeuroM(NeuroD4) or Ngn2 (Neurog2) (5, 11–13). Isl1–Lhx3 was effi-cient in activating HxRE:LUC, whereas DD–Isl1HD

–Lhx3HD

and Isl1–Lhx3HD were much less effective than Isl1 plus Lhx3(Fig. 1D). Similarly, Isl1–Lhx3 collaborated with NeuroM totrigger a potent activation of the MN enhancer, whereas DD–Isl1HD

–Lhx3HD and Isl1–Lhx3HD showed only a marginal level ofactivation even in the presence of NeuroM (Fig. 1E). Theseresults indicate that Isl1–Lhx3 is a powerful activator of theHxREs and cooperates with NeuroM for the transcriptionalactivation of MN genes.To test whether the fusions induce MN generation in vivo, we

expressed each fusion in chicken embryonic spinal cord using inovo electroporation and monitored the ectopic formation ofHb9+ MNs in the dorsal spinal cord. Consistent with the re-porter assays, Isl1–Lhx3 triggered MN generation significantlymore efficiently than coexpression of Isl1 and Lhx3, and DD–Isl1HD

–Lhx3HD and Isl1–Lhx3HD were much less effective (Fig.1F and Fig. S3). All three fusions did not induce ectopic Chx10+

cells, unlike coexpression of Isl1 and Lhx3, which producedseveral Chx10+ cells in the dorsal spinal cord (Fig. 1G and Fig.S3). These results indicate that the three MN-hexamer mimeticfusions do not form a V2-tetramer–like complex, because theLIM domain of Lhx3 is either deleted or unavailable to bind toNLI (Fig. 1C). Together, these results identify Isl1–Lhx3 asa potent and specific MN inducer that overcomes the specificityissue associated with coexpression of Isl1 and Lhx3.

LIM Domain of Lhx3 Is Critical to Induce MN Differentiation. In theassembly of the MN hexamer, the Isl1–LIM domain functions tobind to NLI, whereas the Lhx3–LIM domain is important to bindthe C-terminal domain of Isl1 (Fig. 1A) (4). Our results suggestan unexpected role of the Lhx3–LIM domain in MN specifica-tion, apart from its known function to bind Isl1. To test whetherthe Lhx3–LIM domain within Isl1–Lhx3 is needed to provideoptimal distance between two DNA-binding HDs of Isl1 andLhx3, we made an Isl1–L1–Lhx3 fusion in which the Lhx3–LIMdomain is replaced by the LIM domain of Lhx1 (Lim1) (Fig. 2A).As the LIM domains of Lhx3 and Lhx1 are highly homologouswith each other (∼70% homology, Fig. S4), the HDs of Isl1 andLhx3 within Isl1–Lhx3 and Isl1–L1–Lhx3 are similarly spaced inprimary sequences. We compared the activation of the MN-hexamer target genes by Isl1–Lhx3 and Isl1–L1–Lhx3 using theHxRE:LUC and MN-enhancer:LUC reporters. Isl1–Lhx3 po-tently activated both reporters, whereas Isl1–L1–Lhx3 was in-effective, despite their comparable expression levels (Fig. 2B).Similarly, Isl1–Lhx3, but not Isl1–L1–Lhx3, synergized stronglywith Ngn2 in stimulating the MN enhancer (Fig. 2C).Next, we tested the ability of Isl1-Lhx3 and Isl1-L1-Lhx3 to

activate MN genes in the developing spinal cord. The electro-poration of HxRE:GFP results in MN-specific GFP expression inchick neural tube as the endogenousMNhexamer inMNs activateHxREs (5). Isl1–Lhx3, but not Isl1–L1–Lhx3, triggered ectopicexpression of HxRE:GFP in the dorsal neural tube (Fig. 2D).Likewise, Isl1–L1–Lhx3 was inert in ectopic MN generation inchick neural tube despite its high level of expression, whereas Isl1–Lhx3 induced ectopic MN formation (Fig. 2 E and F). Thus, theLhx3–LIM domain within Isl1–Lhx3 plays an active role for effi-cientMNgeneration, rather than playing a passive role as a spacer.The LIM domain of Lhx3, but not that of Lhx1, binds to the C-

terminal region of Isl1 (14). Thus, it is possible that, within theIsl1–Lhx3 fusion, the Lhx3–LIM domain interacts with Isl1,allowing Isl1–Lhx3 to assume the native conformation of the MN-hexamer. To test this idea, we examined whether the LIM domainof Lhx3, which is fused with the C-terminal region of Isl1, is

Fig. 1. Isl1–Lhx3 is a specific and potent MN inducer. (A) MN-hexamer andV2-tetramer complexes direct the differentiation of MNs and V2-INs, re-spectively, in the developing spinal cord. HxRE, MN-hexamer response ele-ment; TeRE, V2-tetramer response elements. (B) Hb9+ MN and Chx10+ V2-INspecification analyses in chicks electroporated with Lhx3 and Isl1 in indicatedratios. The efficiency of MN and V2-IN induction was quantified by thenumber of ectopic Hb9+ MNs or Chx10+ V2-INs among all Isl1+ electro-porated cells. *P < 0.001 in the two-tailed t test. (C) Schematic representa-tion of MN-hexamer mimetic fusions. (D and E) Luciferase reporter assays inP19 cells using HxRE:LUC (D) or MN-enhancer:LUC (E) reporters. (F and G) MNand V2-IN specification analyses in chicks electroporated with indicatedconstructs. The efficiency of MN and V2-IN induction was quantified by thenumber of ectopic MNs or V2-INs among all Lhx3+ electroporated cells. Errorbars represent the SD (B and D–G).

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available to interact with NLI. If the LIM domain of Lhx3 is pre-occupied due to the intra- or intermolecular interaction with theIsl1–C-terminal region of the fusion, it would not be available forNLI interactions. We expressed Isl1HD

–Lhx3 or Isl1HD–L1–Lhx3

(Fig. 2A) with GST–NLI in HEK293 cells and purified NLI-as-sociated proteins using glutathione beads. Whereas Isl1HD

–L1–Lhx3 efficiently associated with NLI in cells, Isl1HD

–Lhx3 did not(Fig. 2G), indicating that the Lhx3–LIM domain within Isl1HD

–Lhx3 is not available forNLI interaction. These results support ouridea that, in Isl1–Lhx3, the C-terminal region of Isl1 interacts withthe Lhx3–LIM domain intra- or intermolecularly.

NLI-Mediated Dimerization of Isl1–Lhx3 Is Important for MN Differ-entiation.We considered the possibility that the Lhx3–LIM domainwithin Isl1–Lhx3 binds to the Isl1–C-terminal domain in anotherIsl1–Lhx3 molecule in trans, leading to the formation of Isl1–Lhx3homodimer without NLI, and that this Isl1–Lhx3 homodimer issufficient to activate theMN genes (Fig. 3A). To test this possibility,we examined whether Isl1HD

–Lhx3, which contains both the Isl1–C-terminal domain and the Lhx3–LIM domain, self-dimerizes (Fig.3A). The CoIP assays revealed that Flag-tagged Isl1HD

–Lhx3 asso-ciates with HA-tagged Isl1HD

–Lhx3, and that this interaction wasnot disrupted by the dimerization domain of NLI (NLI-DD), aninhibitor of NLI self-dimerization (4) (Fig. 3B). Combined with thefinding that Isl1HD

–Lhx3 does not bind NLI (Fig. 2G), these datasuggest that Isl1HD

–Lhx3 fusion forms a homodimer without NLI(Fig. 3A). Isl1HD

–Lhx3 neither activated theHxRE:LUC reporter in

P19 cells nor induced ectopicMNs in chicken embryos (Fig. 3C andD). These data indicate that the Isl1HD

–Lhx3 homodimer is unableto activate the MN differentiation program, unlike Isl1–Lhx3.Isl1–Lhx3 could form both the homodimer via intermolecular

interactions without NLI and the MN hexamer via NLI self-di-merization (Fig. 3A). To test which of the two complexes func-tions to induce MN differentiation, we used NLI-DD, whichdisrupts assembly of the MN-hexamer, but not formation of Isl1–Lhx3 homodimer (4). NLI-DD strongly inhibited the HxRE ac-tivation by Isl1–Lhx3 in P19 cells as well as ectopic MN formationby Isl1–Lhx3 in the developing spinal cord (Fig. 3 E and F). Thesedata establish that the MN-hexamer, not the Isl1–Lhx3 homo-dimer, is the functional complex that directs MN specification.

Isl1:Lhx3 Interaction Is Needed for the MN Hexamer to Bind theHxREs. To test whether the interaction between the Lhx3–LIMdomain and the Isl1–C-terminal domain aligns the HDs of Isl1and Lhx3 in Isl1–Lhx3 for efficient binding to the HxREs, wemonitored the HxRE-binding ability of Isl1–Lhx3 and Isl1–L1–Lhx3. In gel-shift analyses, Isl1–Lhx3 bound to both HxRE andthe MN enhancer of the Hb9 gene as efficiently as the mixture ofIsl1 and Lhx3, but the binding of Isl1–L1–Lhx3 was barely de-tectable (Fig. 3G). Similarly, chromatin immunoprecipitation(ChIP) assays revealed that Isl1–Lhx3 was recruited to the MNenhancer of the Hb9 gene in P19 cells, whereas Isl1–L1–Lhx3was not (Fig. 3H). Together, these results uncover that the in-teraction between the Isl1–C-terminal domain and the Lhx3–

Fig. 2. The Lhx3–LIM domain is needed for a potent MN-inducing activity ofIsl1–Lhx3. (A) Schematic representation of various fusions. (B and C) Lucif-erase reporter assays in P19 cells using HxRE:LUC (B) or MN-enhancer:LUC (C)reporters. (D) GFP expression in chicks electroporated with HxRE:GFP andIsl1–Lhx3 or Isl1–L1–Lhx3. Isl1–Lhx3 triggered the ectopic GFP expression inthe dorsal spinal cord, but Isl1–L1–Lhx3 did not. (E) Hb9+ MN specificationanalyses in chicks electroporated with indicated constructs above. Isl1–Lhx3induced ectopic Hb9+ MNs above the horizontal lines, whereas Isl1–L1–Lhx3did not. (F) Efficiency of MN induction was quantified by the number ofectopic MNs among all Lhx3+ electroporated cells. Error bars represent theSD (B, C, and F). (G) In vivo GST pull-down assays in HEK293 cells expressingGST–NLI and HA-tagged fusions.

Fig. 3. NLI-mediated dimerization of Isl1–Lhx3 is important for MN differ-entiation. (A) Schematic representation of possible complexes. (B) CoIPassays using HEK293 cells expressing HA–Isl1HD–Lhx3 and Flag–Isl1HD–Lhx3along with or without HA-tagged NLI-DD. Flag and HA antibodies were usedfor IP and Western blotting, respectively. *, heavy chain bands. (C and E)Luciferase reporter assays in P19 cells using HxRE:LUC reporter. Error barsrepresent the SD. (D and F) MN specification analyses in chicks electro-porated with indicated constructs. (D) Isl1HD–Lhx3 failed to trigger ectopicMN formation in the dorsal neural tube. (F) NLI-DD inhibited Isl1–Lhx3-triggered ectopic MN induction (MNs above the red horizontal line). (G) Gel-shift analyses with proteins indicated above using the MN enhancer (MNe,Upper) and HxRE (Lower) as 32P-labeled probes. (H) ChIP assays in P19 cellstransfected with fusions indicated above using IgG or α-Lhx3 antibody. Isl1–Lhx3, but not Isl1–L1–Lhx3, is recruited to the MNe of the Hb9 gene. (Lower)Expression of Isl1–Lhx3 or Isl1–L1–Lhx3 in P19 cells using Western blottingassays with α-Lhx3 antibody.

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LIM domain is important to ensure efficient binding of the MN-hexamer to the HxREs.

Isl1–Lhx3 Efficiently Directs Differentiation of Mouse ESCs to MNs.Our results suggest that Isl1–Lhx3 is an ideal tool to specificallyinduce MN differentiation in stem cells. To test this idea, weestablished inducible (i)MN-ESCs, in which Isl1–Lhx3 codingsequence was inserted downstream of the tetracycline responseelement (TRE) and the reverse tetracycline transactivator(rtTA) was integrated into the constitutively active ROSA26 lo-cus (Fig. 4A and Fig. S5) (15). The expression of Isl1–Lhx3 wasrobustly induced by doxycycline (Dox) treatment (Fig. S6).Given that Isl1–Lhx3 directs ectopic MN formation in the

dorsal spinal cord without additional Shh signaling activation(Figs. 1F and 2E), we hypothesized that expression of Isl1–Lhx3 issufficient to induce MN differentiation in ESCs bypassing theneed of Shh signaling in the conventional method (7). To test thishypothesis, we subjected iMN-ESCs into three differentiation

conditions: culturing embryoid bodies (EBs) with RA alone, RAplus Dox, and RA and a Shh agonist purmorphamine (Fig. 4B).iMN-ESCs cultured with RA alone differentiated to TuJ+/neu-rofilament (NF)+ neurons, but failed to formHb9+MNs (Fig. 4Cand D). Cotreatment of RA and a Shh agonist induced MN dif-ferentiation in ∼40% of TuJ+ neurons in an optimized condition(Fig. 4 C and D). Isl1–Lhx3 expression, without exogenous Shhactivation, resulted in differentiation of 77% of TuJ+ neurons toHb9+ MNs, which is substantially more efficient than Shh sig-naling activation (Fig. 4 C andD). One of essential characteristicsofMNs is that they use acetylcholine as a neurotransmitter, unlikespinal interneurons that are glutamatergic or GABAergic (16).Immunostaining assays revealed that vesicular acetylcholinetransporter (VAChT), a well-established marker for cholinergicneurons, is expressed only in Dox plus RA-treated ESC-derivedneurons, but not in RA alone-treated ESC-derived neurons (Fig.4E), indicating that Isl1–Lhx3-induced MNs are cholinergic. To-gether, our data demonstrate that Isl1–Lhx3 is capable of pro-moting robust differentiation of ESCs to MNs independently ofexogenous activation of Shh signaling.

Isl1–Lhx3 Is More Specific than Shh Signaling in Driving Motor NeuronDifferentiation. Isl1–Lhx3 activates the MN-specific gene programwithout up-regulating V2-IN genes (Fig. 1 F andG), whereas Shhsignaling also triggers specification of ventral interneurons (2).Thus, Isl1–Lhx3 expression likely drives MN differentiation morespecifically than Shh signal in ESCs. To test this idea, we moni-tored induction of various neurotransmitter phenotypes in iMN-ESC–derived neurons using RA alone, RA plus Dox, or RA plus aShh agonist. We analyzed the expression profile of cholinergicmarkers VAChT and choline acetyltransferase (ChAT), a gluta-matergic neuronal marker vesicular glutamate transporter 2(VGluT2), and a GABAergic neuronal marker GAD1, whichencodes the γ-aminobutyric acid (GABA) synthesis enzymeGAD67 (Fig. 4F). RA treatment induced expression of VGluT2and GAD1, but not VAChT and ChAT genes, consistent with itsinability to induce MNs. Interestingly, Isl1–Lhx3 expression notonly increased cholinergic gene expression but also suppressedVGluT2 and GAD1, compared with RA alone-treated sample. Incontrast, RA/Shh-treated cells displayed high levels of GAD1 andVGluT2 as well as cholinergic markers, in agreement with theability of Shh to specify multiple types of neurons. These resultsdemonstrate that Isl1–Lhx3 promotes cholinergic MN differen-tiation in ESCs at the expense of glutamatergic and GABAergicneuronal cell types. Furthermore, our data indicate that Isl1–Lhx3drives stem cells to differentiate into cholinergic MNs more spe-cifically than Shh signal.

Isl1–Lhx3-Induced MNs Form Neuromuscular Junctions. To testwhether Isl1–Lhx3-induced MNs can form neuromuscular junc-tions with myotubes, we performedMN–myotube coculture assays.Either Shh-induced MNs or Isl1–Lhx3-induced MNs were disso-ciated and plated onto myotubes differentiated from C2C12 cellsand cultured for 4 d. TuJ+ motor axons innervated the myotubesand triggered clustering of acetylcholine receptors on myotubes, asdetected by patched α-bungarotoxin staining (Fig. 4G), indicatingthat Isl1–Lhx3-induced MNs establish neuromuscular junctionswith muscle cells.

Isl1–Lhx3 Induces the MN Transcriptome and Suppresses the Develop-mental Programs for Spinal Interneurons. To investigate how Isl1–Lhx3 affects the transcriptome during neuronal differentiation ofESCs in an unbiased genome-wide manner, we performed RNA-seq assays with RA alone-treated iMN-ESCs and RA/Dox-treatediMN-ESCs (Fig. 5A). To capture relatively early changes of thetranscriptome by Isl1–Lhx3 expression, Dox was treated for 2 d.This high-throughput comprehensive analysis revealed that thelevels of 444 genes were significantly changed by Isl1–Lhx3 ex-pression, whereas many genes that are commonly expressed inneurons, such as β2-tubulin, are highly expressed in both samples(Dataset S1 and Fig. S7A). A total of 79% of the significantly

Fig. 4. Isl1–Lhx3 expression triggers efficient and specific differentiation ofESCs to MNs. (A) Schematic representation of iMN-ESCs. Dox treatment up-regulates Isl1–Lhx3, which induces MN genes in iMN-ESCs. TRE, tetracyclineresponse element; rtTA, reverse tetracycline transactivator. (B) Experimen-tal design to differentiate ESCs to MNs. (C and D) Cell differentiationanalyses in iMN-ESCs treated with RA alone, RA plus Dox, or RA plus Shh.(D) Efficiency of MN induction was quantified by the number of Hb9+TuJ+

MNs among all TuJ+ neurons. Error bars represent the SD; *P < 0.001 in two-tailed t test. (E) Isl1–Lhx3 expression induces VAChT+NF+ cholinergic neu-rons. (Scale bar, 25 μm.) (F) RT-PCR analyses to test expression of markersfor neurotransmitter phenotypes. (G) Isl1–Lhx3-induced MNs form neuro-muscular junctions (arrows) with myotubes. Clustering of acetylcholinereceptors (AChR) was determined by patched α-bungarotoxin staining.(Scale bar, 20 μm.)

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changed genes exhibited induction and 21% displayed reduction,consistent with the notion that the MN hexamer functions asa transcription activator (5, 11). Isl1–Lhx3 highly induced the ex-pression of prototypical markers of MNs (Fig. 5 B and C). Hb9,a direct target of the MN hexamer (11), was induced by 28-fold.ChAT, VAChT, and a high-affinity choline transporter CHT, wereinduced by 218-, 46-, and 203-fold, respectively, indicating thatIsl1–Lhx3 directs the cholinergic differentiation. Lhx4, a LIM-HDfactor functioning redundantly with Lhx3 for MN differentiation(17), two LIM-only proteins (LMOs) that are expressed in MNs,LMO1 and LMO4 (5, 18), Nkx6.2, a marker of the lateral motorcolumn (LMC) type of MNs (16), and Isl2 are also significantlyup-regulated. The induction of Hb9 and Isl1/2 proteins was con-firmed using immunoblotting analyses (Fig. S7B). Isl1–Lhx3 ex-pression also triggered the expression of a battery of genesinvolved in axon guidance and cell adhesion, many of which havebeen shown to control motor axon guidance, cell body positions,and MN survival (Fig. 5C). These genes include Met, Neuropilin1(Nrp1), FGF9, Neurotrophin-3 (NTF3), PlexinA4 (PlxnA4), andSemaphorins (19–24). This induced gene profile suggests thatIsl1–Lhx3 directs the expression of MN genes that are importantfor functional maturation and survival of MNs.The expression of Sox2 and Pou3f3 (Brn1), transcription fac-

tors that establish neural progenitor identity (25), was suppressedby Dox treatment (Fig. 5C and Fig. S7B), suggesting that Isl1–Lhx3 facilitates differentiation of neural progenitors to neurons.Interestingly, Isl1–Lhx3 expression repressed class II progenitorfactors, whose expression is suppressed by Shh signal, such as Irx3/5, Dbx1/2, and Pax3/6 (2, 3) (Fig. 5C). These progenitor factorsmark the progenitor domains giving rise to spinal interneurons.Likewise, Isl1–Lhx3 expression also inhibited many transcriptionfactors that determine the identity of spinal interneurons, such asPtf1a, Olig3, Gsx1/2 (Gsh1/2), Msx3, FoxD3, Pou4f1 (Brn3A),Pax2/8, and bHLHe22 (bHLH5) (Fig. 5C) (16, 26). In addition,the expression of bone morphogenetic protein (BMP) and Wnt

signaling molecules, which are important for the development ofdorsal spinal cord (26), was repressed by Dox treatment (Fig. 5C).These results indicate that Isl1–Lhx3 expression inhibits the geneprograms directing spinal interneuron differentiation.Together, our RNA-seq data indicate that Isl1–Lhx3 expression

is sufficient to induce a signature ofMN transcriptome by triggeringMN gene expression while suppressing interneuron differentiation.

DiscussionDuring CNS development, many transcription factors function incombination to specify greatly divergent cell types that constitutethe functional CNS. Formation of cell type-specific transcriptioncomplexes serves as an important mechanism underlying thecombinatorial actions of transcription factors. Lhx3 drives eitherMN fate or V2-IN fate, depending on the transcription complexthat it forms in a given cellular context. When coexpressed withIsl1, Lhx3 forms the MN-hexamer that activates MN genes,whereas Lhx3 without Isl1 turns on V2-IN genes (Fig. 1A) (4, 5).This combinatorial action of Lhx3 and Isl1 in MN generationprovides a useful model to test the strategy to differentiate stemcells to a specific neuronal type by expressing a defined set oftranscription factors. Indeed, the coexpression of Isl1 and Lhx3,along with other transcription factors that induce neurogenesis,is capable of directing differentiation of ESCs, iPSCs, andfibroblasts to MNs (27, 28). However, a major challenge remainsthat each individual transcription factor has its own activity incell lineage determination, distinct from a combinatorial func-tion, and thus the expression ratio of the transcription factorsshould be optimized. Here we demonstrated that Isl1–Lhx3,a fusion molecule mimicking the MN hexamer, directs highlyefficient and reproducible generation of MNs from ESCs. Ourapproach offers two critical advantages over the conventionalmethod that uses combined exposure of ESCs to RA and Shhsignals (7, 8). First, by using the MN-hexamer, a MN-specifictranscription complex downstream of the multifunctional Shhsignal, we were able to minimize differentiation of ESCs tomixed repertoire of neuronal cell types, which arise due to thebroad spectrum of biological activities of Shh. Second, ourstrategy maximizes the specificity and efficiency of MN genera-tion from stem cells by intrinsically maintaining the required 1:1ratio of Lhx3 to Isl1. This suppresses erroneous formation of theV2-tetramer by excess Lhx3 protein, which can drive stem cells toan unwanted V2-IN pathway.How are the proper stoichiometry of Isl1 and Lhx3 and selec-

tive formation of the MN-hexamer over the V2-tetramer ensuredduring MN differentiation in vivo? Selective degradation of Isl1,Lhx3, or NLI, depending on their status in complex formation,might contribute to achieving the proper stoichiometry. For in-stance, single-stranded DNA-binding proteins (SSBPs) and an E3ubiquitin ligase RLIM/Rnf12 regulate the abundance of LIM-HDfactors (29, 30). In addition, LMOs play important roles in reg-ulating the assembly of LIM-HD complexes (5, 6, 31, 32). Spe-cifically, LMO4 inhibits the assembly of V2-tetramer in MNs,thereby promoting MN-hexamer formation (5). These multiplelayers of in vivo stoichiometric regulation might not fully operatein stem cells, and thus it is important to keep the correct ratio oftranscription factors in generating a specific type of neurons fromstem cells by expressing combinations of transcription factors.Our RNA-seq data from Isl1–Lhx3-induced MNs provide

important insights into the MN gene networks regulated by theMN-hexamer. Isl1–Lhx3 up-regulates the expression of Isl2 andLhx4, which function redundantly with Isl1 and Lhx3, re-spectively, in forming the hexamer complex and inducing MNdifferentiation (32–34). The level of NLI was also increased byIsl1–Lhx3 expression. Thus, Isl1–Lhx3 expression leads to thehigher levels of the MN-hexamer complexes, indicating a positiveregulatory feedback. Isl1–Lhx3 also induces the expression ofHb9 and LMO4, which inhibit the transcription of V2-IN genesand the V2-tetramer assembly, respectively (5, 35, 36), rein-forcing the previous model that the MN-hexamer actively blocksthe V2-IN differentiation pathway (5). Once MNs are specified,

Fig. 5. Isl1–Lhx3 induces a gene expression profile of MNs, while sup-pressing the developmental programs for spinal interneurons. (A) Experi-mental design to prepare samples for RNA-seq analyses. (B) Scatter plot toshow RNA-seq results. x axis indicates the mean value of the normalizednumber of reads for each gene transcript in logarithm scale. y axis shows thelog fold change between Dox-treated and control samples. Red spots rep-resent the significantly induced genes by Isl1–Lhx3 expression, and bluespots indicate the significantly down-regulated genes by Isl1–Lhx3 expres-sion. Cutoff is false discovery rate <10%. Arrows mark corresponding spotsfor several MN genes (red spots) and interneuron genes (blue spots). (C) Listof significantly altered genes by Isl1–Lhx3 expression. y axis shows the logfold change between Dox-treated and control samples. Induced genes andrepressed genes are marked in red and blue, respectively.

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Lhx3 expression is down-regulated in all MNs except MMCm-type MNs, which innervate axial musculature (17). Although itremains to be determined whether Isl1–Lhx3 is capable ofdirecting generation of all subtypes of MNs, it is interesting tonote that Isl1–Lhx3 induces not only the MMCm genes, such asLhx3, Lhx4, and LMO4, but also the genes enriched in LMC-type MNs innervating limb muscles, such as Nkx6.2 (16). It isnoteworthy that Isl1–Lhx3 induces a panel of genes that controlMN cell body position, motor axonal trajectory, cholinergicneurotransmission, and MN survival, suggesting that the MN-hexamer controls a wide range of MN functions beyond initialcell-type specification. This result suggests a role of the MN-hexamer as a master regulator of the MN fate. The tran-scriptome analyses also uncovered that the MN-hexamer blocksthe spinal interneuron differentiation programs by suppressingthe expression of many key determinants of multiple interneuronfates. Whether the MN-hexamer actively suppresses the inter-neuron genes, for instance via induction of microRNAs thatblock the interneuron gene expression, or passively represses theinterneuron programs by driving MN generation at the expenseof interneurons, remains to be studied. In the future, it will alsobe interesting to investigate which of the induced and repressedgenes are direct targets of the MN-hexamer complex.In summary, our study demonstrates that the activation of an

embryonic differentiation program using developmental tran-

scription complex mimetic fusions can be explored as a strategyto direct stem cell differentiation to a specific cell type. Thismethod is relatively free from the specificity issue associated withthe application of widely acting inductive signals or with coex-pression of individual transcription factors in cells. In addition,our approach of using inducible ESCs provides powerful modelsystem to define target genes and downstream events of de-velopmental transcription factors.

Materials and MethodsDetails are provided in SI Materials and Methods. For differentiation assays,the EBs of iMN-ESCs were treated with RA (0.5 μM) alone for 2 d and thencultured without or with Dox (2 μg/mL) in the presence of RA for 2–3 d. Inovo electroporation, immunohistochemistry, gel-shift and ChIP assays wereperformed as described (4, 5, 11). RNA-seq libraries were prepared accordingto the Illumina TruSeq protocol and sequenced on an Illumina HiSeq 2000.

ACKNOWLEDGMENTS. We are very grateful to Michael Kyba for sharing thetetracycline-inducible ESC system, Thomas Zwaka for help with ESC experi-ments, and Seongkyung Seo for excellent technical support. This researchwas supported by Grants from National Institutes of Health (NIH)/NationalInstitute of Diabetes and Digestive and Kidney Diseases (R01 DK064678) (toJ.W.L.), NIH/National Institute of Neurological Disorders and Stroke (R01NS054941), NIH (P01 GM81672), Pew Scholars Program, Mrs. Clifford ElderWhite Graham Endowed Research Fund, March of Dimes Foundation, andChristopher and Dana Reeve Foundation (to S.-K.L.).

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