Fusion Isl1–Lhx3 specifies motor neuron fate by inducing motor neuron and concomitantly suppressing the interneuron programs

Seunghee 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 types spinal cord, which is not exposed to a high concentration of Shh during development and thus likely provide crucial insights into di- (4, 5). Lhx3 alone directs the specification of V2 interneurons rected differentiation of stem cells to a specific cell type. The LIM (V2-INs) by forming the V2-tetramer complex, consisting of two complex composed of Isl1 and Lhx3 directs the specification of spinal Lhx3 and two NLI molecules (Fig. 1A) (4, 6). Thus, the combi- motor neurons (MNs) in embryos. Here, we report that Isl1–Lhx3, natorial action of Isl1 and Lhx3, exerted via the formation of the MN hexamer, is critical to induce MN differentiation without a LIM-complex mimicking fusion, induces a signature of MN tran- fi scriptome and concomitantly suppresses interneuron differentiation triggering V2-IN differentiation. Identi cation of the downstream programs, thereby serving as a potent and specific inducer of MNs in genes that are controlled by the MN hexamer would provide stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the important insights into the developmental processes to generate functionally mature MNs. LIM domain of Lhx3 are crucial for generating MNs without up-reg- – MNs differentiated from stem cells have proven to be useful for ulating interneuron genes. These led us to design Isl1 Lhx3, which developing potential therapies for human MN diseases. Shh, maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domain when combined with retinoic acid (RA), converted ESCs and of Lhx3. Isl1–Lhx3 drives MN differentiation with high specificity and –

induced pluripotent stem cells (iPSCs) to MNs (7 10). However, BIOLOGY efficiency in the spinal cord and embryonic stem cells, bypassing the under this condition, Shh also differentiates ESCs into spinal need for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1– interneurons (7). To develop new methods that resolve this DEVELOPMENTAL Lhx3 induces the expression of a battery of MN genes that control specificity issue and generate spinal MNs from stem cells with various functional aspects of MNs, while suppressing key interneu- higher fidelity and efficiency, we explored the possibility of using ron genes. Our studies uncover a highly efficient method for directed the embryonic transcription program for MN generation, the MN MN generation and MN networks. Our results also demonstrate hexamer, instead of Shh signal. We first investigated the mecha- a general strategy of using embryonic transcription complexes for nisms underlying the MN-hexamer function in MN differentiation producing specific cell types from stem cells. and then applied this information to design a strategy for stem cell differentiation to MNs. Here we report that an equimolar ratio of fi eveloping central nervous system (CNS) produces a vast Isl1 and Lhx3 is critical for speci cally generating MNs without Dnumber of neuronal types, but adult CNS has only limited activating V2-IN pathway and that the LIM domain of Lhx3 is capacity to regenerate neurons. This has prompted great interest in required for the effective recognition of MN-hexamer response elements (HxREs) by the MN hexamer. These findings led us to identifying methods to produce specific neuronal types from stem develop a MN-hexamer mimetic fusion, Isl1–Lhx3, which directs cells. Production of differentiated cell types from pluripotent stem highly specific and efficient differentiation of ESCs into MNs. The cells, such as embryonic stem cells (ESCs), should enable a con- RNA-seq analyses of Isl1–Lhx3-induced MNs revealed that Isl1– tinuous supply of diseased cell types for drug screening and cell Lhx3 up-regulates a battery of genes that control a wide range of replacement therapy and provide valuable insights into the patho- MN functions and concomitantly suppresses the interneuron physiology of human diseases. One important challenge in this ef- fi differentiation programs. To our knowledge, this is a unique fort is to steer stem cells into speci c cell types. Recapitulation of demonstration that a fusion mimicking an embryonic transcrip- normal developmental processes using embryonic inductive signals tion complex can serve as an ideal tool to generate a targeted cell has been used to drive differentiation of pluripotent stem cells into type from stem cells. specific cell types (1). However, this strategy tends to trigger for- mation of mixed cell types rather than a targeted cell type, because Results each inductive signal is used in multiple developmental pathways. fi fi Ratio Between Isl1 and Lhx3 Is Critical for the Speci c Generation of This shortcoming might be circumvented by using more speci c, MNs. Once Lhx3 is incorporated into the MN hexamer, it cannot downstream transcription factors of inductive signals. In this form the V2 tetramer (Fig. 1A). Thus, an equimolar expression regard, it should be noted that many transcription factors function of Isl1 and Lhx3 should be critical for MN-specific differentiation in combination to determine cell fates during development, sug- of stem cells without inducing V2-IN differentiation. To test this gesting that coexpression of multiple transcription factors could be a more effective method to generate a particular cell type from pluripotent stem cells. Author contributions: S.L., J.W.L., and S.-K.L. designed research; S.L., J.M.C., B.L., and S.-K.L. Motor neurons (MNs) in the spinal cord project axons to 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. muscles and control their contraction. The developmental path- wrote the paper. ways to generate MNs have been relatively well studied. In the The authors declare no conflict of interest. developing spinal cord, sonic hedgehog (Shh) signal triggers the *This Direct Submission article had a prearranged editor. expression of two LIM homeodomain (HD) transcription factors Data deposition: RNA-seq data reported in this paper have been deposited in the Gene Isl1 and Lhx3 in differentiating MN cells (2, 3). Then, Isl1 and Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. Lhx3 form a transcriptional activating MN-hexamer complex, in GSE35510). which two Isl1:Lhx3 dimers are assembled into a complex via 1To whom correspondence may be addressed. E-mail: [email protected] or leejae@ohsu. a self-dimerizing cofactor nuclear LIM interactor (NLI, also edu. A called LDB for LIM domain binding) (Fig. 1 ) (4, 5). This This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex is sufficient to induce ectopic MN formation in the dorsal 1073/pnas.1114515109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1114515109 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 can form MN-hexamer mimetic complexes with widely expressed endogenous NLI (4, 11). To test whether the fusions activate the MN-hexamer target enhancers, we performed luciferase assays in P19 mouse embryonic cells using the luciferase reporters linked to HxREs and to the MN-specific enhancer in the Hb9 gene, in which the MN-hexamer transcriptionally synergizes with the 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 to trigger a potent activation of the MN enhancer, whereas DD– Isl1HD–Lhx3HD and Isl1–Lhx3HD showed only a marginal level of activation even in the presence of NeuroM (Fig. 1E). These results indicate that Isl1–Lhx3 is a powerful activator of the HxREs and cooperates with NeuroM for the transcriptional activation of MN genes. To test whether the fusions induce MN generation in vivo, we expressed each fusion in chicken embryonic spinal cord using in ovo electroporation and monitored the ectopic formation of Hb9+ MNs in the dorsal spinal cord. Consistent with the re- porter assays, Isl1–Lhx3 triggered MN generation significantly more 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 produced several Chx10+ cells in the dorsal spinal cord (Fig. 1G and Fig. S3). These results indicate that the three MN-hexamer mimetic fusions do not form a V2-tetramer–like complex, because the LIM domain of Lhx3 is either deleted or unavailable to bind to NLI (Fig. 1C). Together, these results identify Isl1–Lhx3 as a potent and specific MN inducer that overcomes the specificity Fig. 1. Isl1–Lhx3 is a specific and potent MN inducer. (A) MN-hexamer and issue associated with coexpression of Isl1 and Lhx3. V2-tetramer complexes direct the differentiation of MNs and V2-INs, re- LIM Domain of Lhx3 Is Critical to Induce MN Differentiation. In the spectively, in the developing spinal cord. HxRE, MN-hexamer response ele- – ment; TeRE, V2-tetramer response elements. (B) Hb9+ MN and Chx10+ V2-IN assembly of the MN hexamer, the Isl1 LIM domain functions to specification analyses in chicks electroporated with Lhx3 and Isl1 in indicated bind to NLI, whereas the Lhx3–LIM domain is important to bind ratios. The efficiency of MN and V2-IN induction was quantified by the the C-terminal domain of Isl1 (Fig. 1A) (4). Our results suggest number of ectopic Hb9+ MNs or Chx10+ V2-INs among all Isl1+ electro- an unexpected role of the Lhx3–LIM domain in MN specifica- porated cells. *P < 0.001 in the two-tailed t test. (C) Schematic representa- tion, apart from its known function to bind Isl1. To test whether tion of MN-hexamer mimetic fusions. (D and E) Luciferase reporter assays in the Lhx3–LIM domain within Isl1–Lhx3 is needed to provide P19 cells using HxRE:LUC (D) or MN-enhancer:LUC (E) reporters. (F and G)MN optimal distance between two DNA-binding HDs of Isl1 and and V2-IN specification analyses in chicks electroporated with indicated Lhx3, we made an Isl1–L1–Lhx3 fusion in which the Lhx3–LIM constructs. The efficiency of MN and V2-IN induction was quantified by the domain is replaced by the LIM domain of Lhx1 (Lim1) (Fig. 2A). + number of ectopic MNs or V2-INs among all Lhx3 electroporated cells. Error As the LIM domains of Lhx3 and Lhx1 are highly homologous B D–G bars represent the SD ( and ). with each other (∼70% homology, Fig. S4), the HDs of Isl1 and Lhx3 within Isl1–Lhx3 and Isl1–L1–Lhx3 are similarly spaced in primary sequences. We compared the activation of the MN- idea, we expressed Isl1 with an increasing amount of Lhx3 in the – – – + hexamer target genes by Isl1 Lhx3 and Isl1 L1 Lhx3 using the chick neural tube and monitored the ectopic formation of Hb9 – + HxRE:LUC and MN-enhancer:LUC reporters. Isl1 Lhx3 po- MNs and Chx10 V2-INs in the dorsal spinal cord (Fig. 1B and – – + tently activated both reporters, whereas Isl1 L1 Lhx3 was in- Fig. S1). When the ratio of Lhx3 to Isl1 was 0.5, only Hb9 MNs, effective, despite their comparable expression levels (Fig. 2B). but no ectopic Chx10+ cells, were formed. However, increasing – – – + Similarly, Isl1 Lhx3, but not Isl1 L1 Lhx3, synergized strongly the amount of Lhx3 led to the generation of ectopic Chx10 cells with Ngn2 in stimulating the MN enhancer (Fig. 2C). B even in the presence of Isl1 (Fig. 1 and Fig. S1). When the ratio Next, we tested the ability of Isl1-Lhx3 and Isl1-L1-Lhx3 to – of Lhx3 to Isl1 was 8, several cells acquired MN V2-IN hybrid activate MN genes in the developing spinal cord. The electro- characteristics expressing both Hb9 and Chx10 (Fig. S2). The fi + poration of HxRE:GFP results in MN-speci c GFP expression in ectopic generation of Chx10 cells following coelectroporation chick neural tube as the endogenous MN hexamer in MNs activate of Isl1 and Lhx3 likely results from an excess of Lhx3 molecules, HxREs (5). Isl1–Lhx3, but not Isl1–L1–Lhx3, triggered ectopic which form the V2-tetramer. Thus, expression levels of Isl1 and expression of HxRE:GFP in the dorsal neural tube (Fig. 2D). Lhx3 should be tightly controlled at or close to an equimolar Likewise, Isl1–L1–Lhx3 was inert in ectopic MN generation in ratio to differentiate neural stem cells specifically to MNs. chick neural tube despite its high level of expression, whereas Isl1– Lhx3 induced ectopic MN formation (Fig. 2 E and F). Thus, the Isl1–Lhx3 Fusion Is a Specific and Efficient Inducer of the MN Fate. In Lhx3–LIM domain within Isl1–Lhx3 plays an active role for effi- keeping the optimal equimolar ratio of Lhx3 to Isl1, we gener- cient MN generation, rather than playing a passive role as a spacer. ated three fusions of Isl1 and Lhx3, which are predicted to mimic The LIM domain of Lhx3, but not that of Lhx1, binds to the C- the MN hexamer structurally (Fig. 1C). DD–Isl1HD–Lhx3HD terminal region of Isl1 (14). Thus, it is possible that, within the consists of the dimerization domain (DD) of NLI fused to the Isl1–Lhx3 fusion, the Lhx3–LIM domain interacts with Isl1, DNA-binding HDs of Isl1 and Lhx3 (4). Isl1–Lhx3HD consists of allowing Isl1–Lhx3 to assume the native conformation of the MN- full-length Isl1 fused to the HD of Lhx3. Isl1–Lhx3 is a fusion hexamer. To test this idea, we examined whether the LIM domain protein of full-length Isl1 and Lhx3. Isl1–Lhx3HD and Isl1–Lhx3 of Lhx3, which is fused with the C-terminal region of Isl1, is

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114515109 Lee et al. Downloaded by guest on September 27, 2021 Fig. 3. NLI-mediated dimerization of Isl1–Lhx3 is important for MN differ- entiation. (A) Schematic representation of possible complexes. (B) CoIP BIOLOGY assays using HEK293 cells expressing HA–Isl1HD–Lhx3 and Flag–Isl1HD–Lhx3 along with or without HA-tagged NLI-DD. Flag and HA antibodies were used DEVELOPMENTAL for IP and Western blotting, respectively. *, heavy chain bands. (C and E) Fig. 2. The Lhx3–LIM domain is needed for a potent MN-inducing activity of Luciferase reporter assays in P19 cells using HxRE:LUC reporter. Error bars – A B C Isl1 Lhx3. ( ) Schematic representation of various fusions. ( and ) Lucif- represent the SD. (D and F) MN specification analyses in chicks electro- B C erase reporter assays in P19 cells using HxRE:LUC ( ) or MN-enhancer:LUC ( ) porated with indicated constructs. (D) Isl1HD–Lhx3 failed to trigger ectopic D reporters. ( ) GFP expression in chicks electroporated with HxRE:GFP and MN formation in the dorsal neural tube. (F) NLI-DD inhibited Isl1–Lhx3- Isl1–Lhx3 or Isl1–L1–Lhx3. Isl1–Lhx3 triggered the ectopic GFP expression in triggered ectopic MN induction (MNs above the red horizontal line). (G) Gel- the dorsal spinal cord, but Isl1–L1–Lhx3 did not. (E) Hb9+ MN specification shift analyses with indicated above using the MN enhancer (MNe, analyses in chicks electroporated with indicated constructs above. Isl1–Lhx3 32 Upper) and HxRE (Lower)as P-labeled probes. (H) ChIP assays in P19 cells + – – induced ectopic Hb9 MNs above the horizontal lines, whereas Isl1 L1 Lhx3 transfected with fusions indicated above using IgG or α-Lhx3 antibody. Isl1– did not. (F)Efficiency of MN induction was quantified by the number of – – Hb9 Lower + Lhx3, but not Isl1 L1 Lhx3, is recruited to the MNe of the gene. ( ) ectopic MNs among all Lhx3 electroporated cells. Error bars represent the Expression of Isl1–Lhx3 or Isl1–L1–Lhx3 in P19 cells using Western blotting SD (B, C, and F). (G) In vivo GST pull-down assays in HEK293 cells expressing assays with α-Lhx3 antibody. GST–NLI and HA-tagged fusions.

P19 cells nor induced ectopic MNs in chicken embryos (Fig. 3 C and available to interact with NLI. If the LIM domain of Lhx3 is pre- D). These data indicate that the Isl1HD–Lhx3 homodimer is unable occupied due to the intra- or intermolecular interaction with the – – to activate the MN differentiation program, unlike Isl1 Lhx3. Isl1 C-terminal region of the fusion, it would not be available for Isl1–Lhx3 could form both the homodimer via intermolecular NLI interactions. We expressed Isl1HD–Lhx3 or Isl1HD–L1–Lhx3 interactions without NLI and the MN hexamer via NLI self-di- (Fig. 2A) with GST–NLI in HEK293 cells and purified NLI-as- merization (Fig. 3A). To test which of the two complexes func- sociated proteins using glutathione beads. Whereas Isl1HD–L1– fi HD– tions to induce MN differentiation, we used NLI-DD, which Lhx3 ef ciently associated with NLI in cells, Isl1 Lhx3 did not – (Fig. 2G), indicating that the Lhx3–LIM domain within Isl1HD– disrupts assembly of the MN-hexamer, but not formation of Isl1 Lhx3 is not available for NLI interaction. These results support our Lhx3 homodimer (4). NLI-DD strongly inhibited the HxRE ac- – tivation by Isl1–Lhx3 in P19 cells as well as ectopic MN formation idea that, in Isl1 Lhx3, the C-terminal region of Isl1 interacts with – E F the Lhx3–LIM domain intra- or intermolecularly. by Isl1 Lhx3 in the developing spinal cord (Fig. 3 and ). These data establish that the MN-hexamer, not the Isl1–Lhx3 homo- fi NLI-Mediated Dimerization of Isl1–Lhx3 Is Important for MN Differ- dimer, is the functional complex that directs MN speci cation. entiation. We considered the possibility that the Lhx3–LIM domain within Isl1–Lhx3 binds to the Isl1–C-terminal domain in another Isl1:Lhx3 Interaction Is Needed for the MN Hexamer to Bind the – Isl1–Lhx3 molecule in trans, leading to the formation of Isl1–Lhx3 HxREs. To test whether the interaction between the Lhx3 LIM – homodimer without NLI, and that this Isl1–Lhx3 homodimer is domain and the Isl1 C-terminal domain aligns the HDs of Isl1 sufficient to activate the MN genes (Fig. 3A). To test this possibility, and Lhx3 in Isl1–Lhx3 for efficient binding to the HxREs, we we examined whether Isl1HD–Lhx3, which contains both the Isl1– monitored the HxRE-binding ability of Isl1–Lhx3 and Isl1–L1– C-terminal domain and the Lhx3–LIM domain, self-dimerizes (Fig. Lhx3. In gel-shift analyses, Isl1–Lhx3 bound to both HxRE and 3A). The CoIP assays revealed that Flag-tagged Isl1HD–Lhx3 asso- the MN enhancer of the Hb9 gene as efficiently as the mixture of ciates with HA-tagged Isl1HD–Lhx3, and that this interaction was Isl1 and Lhx3, but the binding of Isl1–L1–Lhx3 was barely de- not disrupted by the dimerization domain of NLI (NLI-DD), an tectable (Fig. 3G). Similarly, chromatin immunoprecipitation inhibitor of NLI self-dimerization (4) (Fig. 3B). Combined with the (ChIP) assays revealed that Isl1–Lhx3 was recruited to the MN finding that Isl1HD–Lhx3 does not bind NLI (Fig. 2G), these data enhancer of the Hb9 gene in P19 cells, whereas Isl1–L1–Lhx3 suggestthatIsl1HD–Lhx3 fusion forms a homodimer without NLI was not (Fig. 3H). Together, these results uncover that the in- (Fig. 3A). Isl1HD–Lhx3 neither activated the HxRE:LUC reporter in teraction between the Isl1–C-terminal domain and the Lhx3–

Lee et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 LIM domain is important to ensure efficient binding of the MN- conditions: culturing embryoid bodies (EBs) with RA alone, RA hexamer to the HxREs. plus Dox, and RA and a Shh agonist purmorphamine (Fig. 4B). iMN-ESCs cultured with RA alone differentiated to TuJ+/neu- Isl1–Lhx3 Efficiently Directs Differentiation of Mouse ESCs to MNs. rofilament (NF)+ neurons, but failed to form Hb9+ MNs (Fig. 4 C Our results suggest that Isl1–Lhx3 is an ideal tool to specifically and D). Cotreatment of RA and a Shh agonist induced MN dif- induce MN differentiation in stem cells. To test this idea, we ferentiation in ∼40% of TuJ+ neurons in an optimized condition established inducible (i)MN-ESCs, in which Isl1–Lhx3 coding (Fig. 4 C and D). Isl1–Lhx3 expression, without exogenous Shh sequence was inserted downstream of the tetracycline response activation, resulted in differentiation of 77% of TuJ+ neurons to element (TRE) and the reverse tetracycline transactivator Hb9+ MNs, which is substantially more efficient than Shh sig- (rtTA) was integrated into the constitutively active ROSA26 lo- naling activation (Fig. 4 C and D). One of essential characteristics cus (Fig. 4A and Fig. S5) (15). The expression of Isl1–Lhx3 was of MNs is that they use acetylcholine as a neurotransmitter, unlike robustly induced by doxycycline (Dox) treatment (Fig. S6). spinal interneurons that are glutamatergic or GABAergic (16). Given that Isl1–Lhx3 directs ectopic MN formation in the Immunostaining assays revealed that vesicular acetylcholine dorsal spinal cord without additional Shh signaling activation transporter (VAChT), a well-established marker for cholinergic (Figs. 1F and 2E), we hypothesized that expression of Isl1–Lhx3 is neurons, is expressed only in Dox plus RA-treated ESC-derived sufficient to induce MN differentiation in ESCs bypassing the neurons, but not in RA alone-treated ESC-derived neurons (Fig. need of Shh signaling in the conventional method (7). To test this 4E), indicating that Isl1–Lhx3-induced MNs are cholinergic. To- hypothesis, we subjected iMN-ESCs into three differentiation gether, our data demonstrate that Isl1–Lhx3 is capable of pro- moting robust differentiation of ESCs to MNs independently of exogenous activation of Shh signaling.

Isl1–Lhx3 Is More Specific than Shh Signaling in Driving Motor Neuron Differentiation. Isl1–Lhx3 activates the MN-specific gene program without up-regulating V2-IN genes (Fig. 1 F and G), whereas Shh signaling also triggers specification of ventral interneurons (2). Thus, Isl1–Lhx3 expression likely drives MN differentiation more specifically 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 a Shh agonist. We analyzed the expression profile of cholinergic markers VAChT and choline acetyltransferase (ChAT), a gluta- matergic neuronal marker vesicular glutamate transporter 2 (VGluT2), and a GABAergic neuronal marker GAD1, which encodes the γ-aminobutyric acid (GABA) synthesis enzyme GAD67 (Fig. 4F). RA treatment induced expression of VGluT2 and GAD1, but not VAChT and ChAT genes, consistent with its inability to induce MNs. Interestingly, Isl1–Lhx3 expression not only increased cholinergic but also suppressed VGluT2 and GAD1, compared with RA alone-treated sample. In contrast, RA/Shh-treated cells displayed high levels of GAD1 and VGluT2 as well as cholinergic markers, in agreement with the ability of Shh to specify multiple types of neurons. These results demonstrate that Isl1–Lhx3 promotes cholinergic MN differen- tiation in ESCs at the expense of glutamatergic and GABAergic neuronal cell types. Furthermore, our data indicate that Isl1–Lhx3 drives stem cells to differentiate into cholinergic MNs more spe- cifically than Shh signal.

Isl1–Lhx3-Induced MNs Form Neuromuscular Junctions. To test whether Isl1–Lhx3-induced MNs can form neuromuscular junc- tions with myotubes, we performed MN–myotube coculture assays. Either Shh-induced MNs or Isl1–Lhx3-induced MNs were disso- ciated and plated onto myotubes differentiated from C2C12 cells and cultured for 4 d. TuJ+ motor axons innervated the myotubes and triggered clustering of acetylcholine receptors on myotubes, as detected by patched α-bungarotoxin staining (Fig. 4G), indicating that Isl1–Lhx3-induced MNs establish neuromuscular junctions with muscle cells. Fig. 4. Isl1–Lhx3 expression triggers efficient and specific differentiation of ESCs to MNs. (A) Schematic representation of iMN-ESCs. Dox treatment up- Isl1–Lhx3 Induces the MN Transcriptome and Suppresses the Develop- – regulates Isl1 Lhx3, which induces MN genes in iMN-ESCs. TRE, tetracycline mental Programs for Spinal Interneurons. To investigate how Isl1– response element; rtTA, reverse tetracycline transactivator. (B)Experimen- C D Lhx3 affects the transcriptome during neuronal differentiation of tal design to differentiate ESCs to MNs. ( and ) Cell differentiation ESCs in an unbiased genome-wide manner, we performed RNA- analyses 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+ seq assays with RA alone-treated iMN-ESCs and RA/Dox-treated + P < iMN-ESCs (Fig. 5A). To capture relatively early changes of the MNs among all TuJ neurons. Error bars represent the SD; * 0.001 in two- – tailed t test. (E) Isl1–Lhx3 expression induces VAChT+NF+ cholinergic neu- transcriptome by Isl1 Lhx3 expression, Dox was treated for 2 d. rons. (Scale bar, 25 μm.) (F) RT-PCR analyses to test expression of markers This high-throughput comprehensive analysis revealed that the for neurotransmitter phenotypes. (G)Isl1–Lhx3-induced MNs form neuro- levels of 444 genes were significantly changed by Isl1–Lhx3 ex- muscular junctions (arrows) with myotubes. Clustering of acetylcholine pression, whereas many genes that are commonly expressed in receptors (AChR) was determined by patched α-bungarotoxin staining. neurons, such as β2-tubulin, are highly expressed in both samples (Scale bar, 20 μm.) (Dataset S1 and Fig. S7A). A total of 79% of the significantly

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114515109 Lee et al. Downloaded by guest on September 27, 2021 signaling molecules, which are important for the development of dorsal spinal cord (26), was repressed by Dox treatment (Fig. 5C). These results indicate that Isl1–Lhx3 expression inhibits the gene programs directing spinal interneuron differentiation. Together, our RNA-seq data indicate that Isl1–Lhx3 expression is sufficient to induce a signature of MN transcriptome by triggering MN gene expression while suppressing interneuron differentiation. Discussion During CNS development, many transcription factors function in combination to specify greatly divergent cell types that constitute the functional CNS. Formation of cell type-specific transcription complexes serves as an important mechanism underlying the combinatorial actions of transcription factors. Lhx3 drives either MN fate or V2-IN fate, depending on the transcription complex that it forms in a given cellular context. When coexpressed with Isl1, 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 generation provides a useful model to test the strategy to differentiate stem cells to a specific neuronal type by expressing a defined set of transcription factors. Indeed, the coexpression of Isl1 and Lhx3, Fig. 5. Isl1–Lhx3 induces a gene expression profile of MNs, while sup- along with other transcription factors that induce neurogenesis, pressing the developmental programs for spinal interneurons. (A) Experi- is capable of directing differentiation of ESCs, iPSCs, and mental design to prepare samples for RNA-seq analyses. (B) Scatter plot to fibroblasts to MNs (27, 28). However, a major challenge remains show RNA-seq results. x axis indicates the mean value of the normalized that each individual has its own activity in number of reads for each gene transcript in logarithm scale. y axis shows the cell lineage determination, distinct from a combinatorial func- log fold change between Dox-treated and control samples. Red spots rep-

tion, and thus the expression ratio of the transcription factors BIOLOGY fi – resent the signi cantly induced genes by Isl1 Lhx3 expression, and blue should be optimized. Here we demonstrated that Isl1–Lhx3, fi – DEVELOPMENTAL spots indicate the signi cantly down-regulated genes by Isl1 Lhx3 expres- a fusion molecule mimicking the MN hexamer, directs highly sion. Cutoff is false discovery rate <10%. Arrows mark corresponding spots efficient and reproducible generation of MNs from ESCs. Our for several MN genes (red spots) and interneuron genes (blue spots). (C) List of significantly altered genes by Isl1–Lhx3 expression. y axis shows the log approach offers two critical advantages over the conventional method that uses combined exposure of ESCs to RA and Shh fold change between Dox-treated and control samples. Induced genes and fi repressed genes are marked in red and blue, respectively. signals (7, 8). First, by using the MN-hexamer, a MN-speci c transcription complex downstream of the multifunctional Shh signal, we were able to minimize differentiation of ESCs to changed genes exhibited induction and 21% displayed reduction, mixed repertoire of neuronal cell types, which arise due to the consistent with the notion that the MN hexamer functions as broad spectrum of biological activities of Shh. Second, our a transcription activator (5, 11). Isl1–Lhx3 highly induced the ex- strategy maximizes the specificity and efficiency of MN genera- pression of prototypical markers of MNs (Fig. 5 B and C). Hb9, tion from stem cells by intrinsically maintaining the required 1:1 a direct target of the MN hexamer (11), was induced by 28-fold. ratio of Lhx3 to Isl1. This suppresses erroneous formation of the ChAT, VAChT, and a high-affinity choline transporter CHT, were V2-tetramer by excess Lhx3 protein, which can drive stem cells to induced by 218-, 46-, and 203-fold, respectively, indicating that an unwanted V2-IN pathway. Isl1–Lhx3 directs the cholinergic differentiation. Lhx4, a LIM-HD How are the proper stoichiometry of Isl1 and Lhx3 and selec- factor functioning redundantly with Lhx3 for MN differentiation tive formation of the MN-hexamer over the V2-tetramer ensured (17), two LIM-only proteins (LMOs) that are expressed in MNs, during MN differentiation in vivo? Selective degradation of Isl1, LMO1 and LMO4 (5, 18), Nkx6.2, a marker of the lateral motor Lhx3, or NLI, depending on their status in complex formation, column (LMC) type of MNs (16), and Isl2 are also significantly might contribute to achieving the proper stoichiometry. For in- up-regulated. The induction of Hb9 and Isl1/2 proteins was con- stance, single-stranded DNA-binding proteins (SSBPs) and an E3 firmed using immunoblotting analyses (Fig. S7B). Isl1–Lhx3 ex- ubiquitin ligase RLIM/Rnf12 regulate the abundance of LIM-HD pression also triggered the expression of a battery of genes factors (29, 30). In addition, LMOs play important roles in reg- involved in axon guidance and cell adhesion, many of which have ulating the assembly of LIM-HD complexes (5, 6, 31, 32). Spe- been shown to control motor axon guidance, cell body positions, cifically, LMO4 inhibits the assembly of V2-tetramer in MNs, and MN survival (Fig. 5C). These genes include Met, Neuropilin1 thereby promoting MN-hexamer formation (5). These multiple (Nrp1), FGF9, Neurotrophin-3 (NTF3), PlexinA4 (PlxnA4), and layers of in vivo stoichiometric regulation might not fully operate Semaphorins (19–24). This induced gene profile suggests that in stem cells, and thus it is important to keep the correct ratio of Isl1–Lhx3 directs the expression of MN genes that are important transcription factors in generating a specific type of neurons from for functional maturation and survival of MNs. stem cells by expressing combinations of transcription factors. The expression of and Pou3f3 (Brn1), transcription fac- Our RNA-seq data from Isl1–Lhx3-induced MNs provide tors that establish neural progenitor identity (25), was suppressed important insights into the MN gene networks regulated by the by Dox treatment (Fig. 5C and Fig. S7B), suggesting that Isl1– MN-hexamer. Isl1–Lhx3 up-regulates the expression of Isl2 and Lhx3 facilitates differentiation of neural progenitors to neurons. Lhx4, which function redundantly with Isl1 and Lhx3, re- Interestingly, Isl1–Lhx3 expression repressed class II progenitor spectively, in forming the hexamer complex and inducing MN factors, whose expression is suppressed by Shh signal, such as Irx3/ differentiation (32–34). The level of NLI was also increased by 5, Dbx1/2, and Pax3/6 (2, 3) (Fig. 5C). These progenitor factors Isl1–Lhx3 expression. Thus, Isl1–Lhx3 expression leads to the mark the progenitor domains giving rise to spinal interneurons. higher levels of the MN-hexamer complexes, indicating a positive Likewise, Isl1–Lhx3 expression also inhibited many transcription regulatory feedback. Isl1–Lhx3 also induces the expression of factors that determine the identity of spinal interneurons, such as Hb9 and LMO4, which inhibit the transcription of V2-IN genes Ptf1a, Olig3, Gsx1/2 (Gsh1/2), Msx3, FoxD3, Pou4f1 (Brn3A), and the V2-tetramer assembly, respectively (5, 35, 36), rein- Pax2/8, and bHLHe22 (bHLH5) (Fig. 5C) (16, 26). In addition, forcing the previous model that the MN-hexamer actively blocks the expression of bone morphogenetic protein (BMP) and Wnt the V2-IN differentiation pathway (5). Once MNs are specified,

Lee et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 Lhx3 expression is down-regulated in all MNs except MMCm- scription complex mimetic fusions can be explored as a strategy type MNs, which innervate axial musculature (17). Although it to direct stem cell differentiation to a specific cell type. This remains to be determined whether Isl1–Lhx3 is capable of method is relatively free from the specificity issue associated with directing generation of all subtypes of MNs, it is interesting to the application of widely acting inductive signals or with coex- note that Isl1–Lhx3 induces not only the MMCm genes, such as pression of individual transcription factors in cells. In addition, Lhx3, Lhx4, and LMO4, but also the genes enriched in LMC- our approach of using inducible ESCs provides powerful model type MNs innervating limb muscles, such as Nkx6.2 (16). It is system to define target genes and downstream events of de- noteworthy that Isl1–Lhx3 induces a panel of genes that control velopmental transcription factors. MN cell body position, motor axonal trajectory, cholinergic neurotransmission, and MN survival, suggesting that the MN- Materials and Methods hexamer controls a wide range of MN functions beyond initial SI Materials and Methods fi Details are provided in . For differentiation assays, cell-type speci cation. This result suggests a role of the MN- the EBs of iMN-ESCs were treated with RA (0.5 μM) alone for 2 d and then hexamer as a master regulator of the MN fate. The tran- cultured without or with Dox (2 μg/mL) in the presence of RA for 2–3d.In scriptome analyses also uncovered that the MN-hexamer blocks ovo electroporation, immunohistochemistry, gel-shift and ChIP assays were the spinal interneuron differentiation programs by suppressing performed as described (4, 5, 11). RNA-seq libraries were prepared according the expression of many key determinants of multiple interneuron to the Illumina TruSeq protocol and sequenced on an Illumina HiSeq 2000. fates. Whether the MN-hexamer actively suppresses the inter- neuron genes, for instance via induction of microRNAs that ACKNOWLEDGMENTS. We are very grateful to Michael Kyba for sharing the block the interneuron gene expression, or passively represses the tetracycline-inducible ESC system, Thomas Zwaka for help with ESC experi- interneuron programs by driving MN generation at the expense ments, and Seongkyung Seo for excellent technical support. This research of interneurons, remains to be studied. In the future, it will also was supported by Grants from National Institutes of Health (NIH)/National be interesting to investigate which of the induced and repressed Institute of Diabetes and Digestive and Kidney Diseases (R01 DK064678) (to J.W.L.), NIH/National Institute of Neurological Disorders and Stroke (R01 genes are direct targets of the MN-hexamer complex. NS054941), NIH (P01 GM81672), Pew Scholars Program, Mrs. Clifford Elder In summary, our study demonstrates that the activation of an White Graham Endowed Research Fund, March of Dimes Foundation, and embryonic differentiation program using developmental tran- Christopher and Dana Reeve Foundation (to S.-K.L.).

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