Analysis of Mll1 deficiency identifies neurogenic transcriptional modules and Brn4 as a factor for direct astrocyte-to-neuron reprogramming.

RESEARCH—LABORATORY RESEARCH—LABORATORY

Matthew B. Potts, MD*‡ Jason J. Siu, BS*‡ James D. Price, BS*‡ Ryan D. Salinas, MD*‡

Analysis of Mll1 Deficiency Identifies Neurogenic Transcriptional Modules and Brn4 as a Factor for Direct Astrocyte-to-Neuron Reprogramming

Mathew J. Cho, BA*‡ Alexander D. Ramos, BS*‡ Junghyun Hahn, PhD§ Marta Margeta, MD, PhD§ Michael C. Oldham, PhD‡¶ Daniel A. Lim, MD, PhD*‡k *Department of Neurological Surgery, ‡The Eli and Edythe Broad Institute of Regeneration Medicine and Stem Cell Research, §Department of Pathology, and ¶Department of Neurology University of California, San Francisco, San Francisco, California; and kSurgical Service, San Francisco Veterans Affairs Medical Center, San Francisco, California Correspondence: Daniel A. Lim, MD, PhD, 35 Medical Center Way, San Francisco, CA 94143-0525. E-mail: [email protected] Received, January 7, 2014. Accepted, May 9, 2014. Published Online, May 30, 2014. Copyright © 2014 by the Congress of Neurological Surgeons.

BACKGROUND: Mixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons. OBJECTIVE: To identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons. METHODS: We performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology. RESULTS: Transcriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons. CONCLUSION: Our results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies. KEY WORDS: Astrocytes, Brn4 (Pou3f4), Cell transdifferentiation, Epigenomics, Mll1, Neural stem cells, Neurogenesis Neurosurgery 75:472–482, 2014

DOI: 10.1227/NEU.0000000000000452

I

n the adult mammalian brain, neural stem cells (NSCs) in the lateral ventricle subventricular zone (SVZ) continue to generate new neurons

WHAT IS THIS BOX? A QR Code is a matrix barcode readable by QR scanners, mobile phones with cameras, and smartphones. The QR Code above links to Supplemental Digital Content from this article.

ABBREVIATIONS: GFP, green fluorescent protein; GO, Gene Ontology; NSC, neural stem cell; Mll1, Mixed lineage leukemia-1; SVZ, subventricular zone; Tuj1, neuronal class III b-tubulin; UCSF, University of California San Francisco Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.neurosurgery-online.com).

472 | VOLUME 75 | NUMBER 4 | OCTOBER 2014

www.neurosurgery-online.com

and glia throughout life.1-6 In mice, SVZ NSCs are a specialized type of astrocyte that can produce both neuronal and glial cell lineages.7 Mixed lineage leukemia-1 (Mll1) encodes an evolutionarily conserved chromatin-modifying factor that is required to maintain lineagespecific gene expression in both embryonic and postnatal stem cell populations.8,9 In SVZ NSCs, Mll1 is required for neurogenesis but not for glial cell differentiation.7,10 Thus, Mll1dependent gene expression may, in part, account for the neurogenic potential of this specialized population of astrocytes in the adult mouse SVZ.


Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited

Mil1-DEPENDENT NEUROGENIC TRANSCRIPTION AND REPROGRAMMING

Interestingly, astrocytes derived from nonneurogenic brain regions such as the cerebral cortex can be directly converted into functional neurons via the overexpression of specific transcription factors.1,3,5,6,11 For instance, enforced expression of Dlx2, Mash1 (also known as Ascl1), or Pax6 in cultured postnatal mouse cortical astroglia results in neuronal transdifferentiation.1,3,5,6 Furthermore, emerging evidence indicates that direct astrocyte-to-neuron reprogramming can also be achieved in vivo,12,13 as is seen in striatal astrocytes where lentiviral transduction of Sox2, along with the expression of specific extrinsic signals, results in the production of mature neurons.12 Given the astrocytic identity of SVZ NSCs, we hypothesized that characterizing their neurogenic gene expression profile may reveal new factors useful for reprogramming astrocytes into neurons. Genome-wide analysis of Mll1-dependent transcription in SVZ NSCs provides an excellent opportunity to distinguish neurogenic gene expression from that required for glial cell differentiation. Whereas wild-type SVZ NSCs produce all 3 major neural cell types (ie, neurons, astrocytes, and oligodendrocytes), Mll1-deficient SVZ cells efficiently generate only the glial lineages.10 However, our understanding of Mll1-dependent gene expression has been limited. Proneural Mash1 and gliogenic Olig2 are expressed in Mll1-deficient SVZ cells during differentiation, but Dlx2 is not expressed, correlating with the impaired neurogenesis.10 In this study, we used microarrays and gene coexpression analysis to identify “modules” of transcribed genes that distinguish neurogenesis from gliogenesis. In contrast to differential gene expression analysis, which compares the mean expression level of individual genes between $2 groups, gene coexpression analysis seeks to identify groups of genes (ie, transcriptional modules) that are expressed in similar patterns across biological samples.14-17 Such approaches enable the discovery of cell type–specific gene expression despite cellular heterogeneity within each sample.14,16 Our coexpression analysis generated a resource that distinguishes gene expression modules related to SVZ neurogenesis from those related to gliogenesis and cell proliferation. To evaluate the neurogenic potential of genes identified by this method, we used lentiviruses to overexpress select module members in Mll1-deficient SVZ cells. Using this approach, we identified genes such as Brn4 (also known as Pou3f4) that belonged to neurogenic modules and partially rescued neuronal differentiation. Finally, to explore whether Mll1-dependent genes in SVZ NSCs can be used to transdifferentiate nonneurogenic astrocytes into neurons, we overexpressed Brn4 in astrocytes cultured from the cerebral cortex. We observed that the overexpression of Brn4 in cortical astrocytes resulted in the production of electrophysiologically active neurons. Taken together, these studies provide insight into the key role that Mll1 plays in postnatal neurogenesis and further indicate that neurogenic factors expressed in SVZ NSCs may be useful for astrocyte-to-neuron reprogramming strategies.

NEUROSURGERY

METHODS Tissue Preparation and Culture of Primary SVZ NSCs and Cortical Astrocytes All use of animals conformed to guidelines established by the Institutional Animal Care and Use Committee at the University of California San Francisco (UCSF). Mll1-deleted SVZ NSCs were obtained from hGFAP-Cre;Mll1f/f mice, which exhibit Cre-mediated recombination of the Mll1f/f alleles in cells expressing the hGFAP-Cre transgene. hGFAP-Cre;Mll11/1 littermates were used as wild-type controls. The brains from mice at postnatal days 5 to 7 were removed from the skull and placed in ice-cold Leibovitz L15 media (Gibco, catalog No. 11415064). Coronal slabs containing the SVZ were obtained, and the meninges were removed. The lateral SVZ and cortical gray matter were then microdissected, with care taken to prevent cross-contamination. Dissected tissue was then dissociated with 0.25% trypsin as previously described.10 The suspension of cortical astrocytes was also treated with DNase I (Roche Diagnostics, catalog No. ROCZR139). SVZ single-cell suspensions were plated in N5 medium18 containing Dulbecco modified Eagle medium/F12 with Glutamax (Gibco, catalog No. 10565-018), 5% fetal bovine serum (Hyclone, catalog No. SH30070.03), N2 supplement (Gibco, catalog No. 17502-048), 35 mg/mL bovine pituitary extract (Gibco, catalog No. 13028.014), 20 ng/mL recombinant human epidermal growth factor (Peprotech, catalog No. AF-100-15), 20 ng/mL recombinant human fibroblast growth factor (Peprotech, catalog No. 100-18B-1mg), and antibiotic-antimycotic (Gibco, catalog No. 15240062). SVZ cultures were maintained with serial passages as previously described.18 At the first or second passage, SVZ cells were seeded onto 16-well chamber slides and immunostained for mouse MLL1 (1:500; Millipore, catalog No. 05-764) to confirm $90% deletion before proceeding with RNA isolation for subsequent experiments. Cortical astrocyte single-cell suspensions were resuspended in medium containing Dulbecco modified Eagle medium/F12, 10% fetal bovine serum, 5% horse serum (UCSF Cell Culture Facility, catalog No. CCFAW001), B27 supplement (Gibco, catalog No. 17504-044), 3.5 mmol/L glucose, 10 ng/mL epidermal growth factor, 10 ng/mL fibroblast growth factor, and antibiotic-antimycotic. Astrocytes were then plated on culture dishes precoated with 0.1 mg/mL poly-D-lysine (Sigma-Aldrich, catalog No. P6407) and 2 mg/mL laminin (Life Technologies, catalog No. 23017015) and allowed to reach confluence. SVZ cultures were differentiated after 5 to 7 passages. For differentiation, the medium was changed to a serum-free differentiation medium consisting of Dulbecco modified Eagle medium/F12 with Glutamax, N2 supplement, bovine pituitary extract, and antibiotic-antimycotic.18 Astrocyte differentiation was performed after the first passage by changing to a medium consisting of Dulbecco modified Eagle medium/F12, 3.5 mmol/L glucose, B27 supplement, antibiotic-antimycotic, and 20 ng/mL brain derived neurotrophic factor (Peprotech, catalog No. 450-02).

RNA Isolation and Microarray Analysis Microarray analysis of wild-type and Mll1-deleted SVZ NSC cultures was performed before differentiation (0 days) and at 1, 2, and 4 days after differentiation (biologic triplicates at each time point). SVZ cultures were homogenized with the use of QIAshredder columns (QIAgen, catalog No. 79654) and RNA was extracted with the QIAgen RNeasy Plus Mini Kit (catalog No. 74134). RNA quality and integrity were assessed with the Agilent BioAnalyzer 2100 before samples were converted to cDNA. Affymetrix Mouse Gene 1.0 ST microarrays were used to measure gene

VOLUME 75 | NUMBER 4 | OCTOBER 2014 | 473


POTTS ET AL

expression levels and scanned by the UCSF Genomics Core. Raw data (.CEL files) were preprocessed with the Affymetrix Power Tools (APT) software package (http://www.affymetrix.com/partners_programs/ programs/developer/tools/powertools.affx). Expression data were background corrected, summarized, and quantile normalized by use of the robust multiarray average algorithm19 in Affymetrix Power Tools. Sample heterogeneity was assessed with the SampleNetwork R function20; the mean intersample adjacency was 0.968, and no outliers (Z.K ,23) were present.

Gene Coexpression Analysis Gene coexpression modules were identified with a 4-step approach. First, pairwise Pearson correlation coefficients were calculated across all samples for all transcripts (n = 25,232) that were perfectly and uniquely targeted by their microarray probe sequence (Affymetrix CrossHyb Type = 1). Second, transcripts were clustered by use of the flashClust17 implementation of a hierarchical clustering procedure with complete linkage and 1 2 correlation coefficient as a distance measure. The resulting dendrogram was cut at a static height of 0.258, corresponding to the top 1% of pairwise correlations for the entire dataset. Third, all clusters consisting of at least 15 members were identified and summarized by their module eigengene (ie, the first principal component obtained via singular value decomposition).14,15 Fourth, highly similar modules were merged if the Pearson correlation coefficients of their module eigengenes exceeded an arbitrary threshold (0.85). This procedure was performed iteratively so that the pair of modules with the highest correlation .0.85 were merged, followed by recalculation of all module eigengenes, then followed by recalculation of all correlations, until no pairs of modules exceeded the threshold. After these steps, 14 coexpression modules were identified. The strength of the module membership for each probe on the microarray was calculated by correlating its expression pattern across all samples with each module eigengene.15,16

Module Enrichment Analysis Module enrichment analysis with curated gene sets was performed by use of a 1-sided Fisher exact test for overrepresentation with gene symbols as a common identifier. Curated gene sets included experimentally validated markers of astrocytes (Table S7 from Bachoo et al21), oligodendrocytes (from Nielsen et al,22 reanalyzed as previously described16; from Cahoy et al,23 restricted to genes expressed .10 times higher in oligodendrocytes vs other cell types in the adult mouse forebrain; and from Doyle et al,24 consisting of markers of mouse cerebellar Olig21 oligodendrocytes), and neurons (from Cahoy et al,23 restricted to genes expressed .10 times higher in neurons vs other cell types in the adult mouse forebrain). Modules were defined as consisting of all transcripts that were positively correlated with the module eigengene at a significance threshold of P , 1.26e207. This threshold corresponds to a Bonferronicorrected P value of .05/(the total number of probes times the total number of modules). Gene Ontology (GO, http://www.geneontology. org) analysis was performed with DAVID (Database for Annotation, Visualization, and Integrated Discovery; http://david.abcc.ncifcrf.gov/). Probe identifications of module genes were uploaded and converted to gene lists using as a background all transcripts (n = 25,232) that were perfectly and uniquely targeted by their microarray probe sequence (Affymetrix CrossHyb Type = 1). To determine whether modules were Mll1 dependent or independent, analysis of variance was performed in R (http://cran.us.r-project.org/) on multivariate linear regression models that related each module eigengene to treatment condition (wild-type vs


Mll1-deleted), the number of days in culture, and the interaction between these 2 terms. Mll1 dependence was defined as P , .05 for the treatment effect.

Enforced Expression of Mll1-Dependent Factors cDNA of genes of interest (Dlx2, Brn4, Tlx, Zdhhc23, and Shisa3) was cloned into the lentiviral pUltra vector (Addgene), which contains a green fluorescent protein (GFP) reporter. Lentivirus was then generated from HEK293T cells with 36 mg plasmid containing a given cDNA of interest, 9 mg VSVg coat plasmid, 7.5 mg pRSV, and 7.5 mg pMDL helper plasmids using the FuGENE 6 transfection system (Promega, catalog No. E2693). One day after transfection, cell culture media was changed to viral production media containing Lonza Ultraculture media (12-1725F), 110 ng/mL sodium pyruvate, and 5 mmol/L sodium butyrate. Cells were grown in viral production media for 48 hours before pelleting of virus at 22,000 rpm by ultracentrifuge for 2 hours. Viral pellet was resuspended in phosphate-buffered saline and stored at 280C.

Immunocytochemistry At the time points of interest, cells were fixed in 4% paraformaldehyde for 30 minutes and then washed 3 times in phosphate-buffered saline. Blocking was performed for 2 hours at room temperature in blocking buffer containing phosphate-buffered saline with 1% bovine serum albumin, 5% normal goat serum, 0.3% Triton-X-100 (Sigma), 0.3 mol/L glycine, and 0.03% sodium azide. All antibody incubations were also performed in blocking buffer at room temperature. Primary antibodies included mouse anti–neuronal class III b-tubulin (Tuj1, 1:500; Covance, catalog No. MMS-435P), mouse anti–microtubule-associated protein 2 (1:200; Millipore, catalog No. MAB364), guinea pig anti-doublecortin (1:400; Millipore, catalog No. AB2253), rabbit anti-Brn4 (1:500; Millipore, catalog No. AB15456), rabbit anti-glial fibrillary acidic protein (1:500; Dako, catalog No. Z0334), chicken anti-GFP (1:500; Aves, catalog No. GFP-1020), mouse anti-O4 (1:100; Millipore, catalog No. MAB345), rabbit anti-NG2 (1:400; Millipore, catalog No. AB5320), mouse antiGAD67 (1:1000; Millipore, catalog No. MAB5406), and rabbit anticalbindin (1:200; Swant, catalog No. CB 38). Appropriate secondary antibody incubations were performed for 30 minutes in blocking buffer at room temperature and included a nuclear counterstain (DAPI). Imaging was performed with a Leica DMA4000 B inverted fluorescent microscope. Cell counts for a given condition were performed on 7 nonoverlapping regions imaged at ·20. The totals of each region were then summed and averaged over biological triplicates.

Electrophysiology Whole-cell current-clamp recordings25 were made with a Multiclamp 700B amplifier (Molecular Devices). Signals were filtered at 1 kHz and sampled at 10 kHz with a Digidata 1440A analog-to-digital converter (Molecular Devices). All data were recorded and analyzed with pClamp 10 software (Molecular Devices). The liquid junction potential was measured with 3 mol/L KCl and adjusted. Cells on coverslips were placed in a bath solution (containing the following (in mmol/L): 135 NaCl, 5 KCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, 10 glucose at 300 mOsm, pH 7.4) and visualized with an Olympus IX71 microscope with differential interface contrast optics at ·40. Patch electrodes (3-4 MV) contained (in mmol/L) 125 KCl, 10 Na2PO4, 10 HEPES, 2 Mg3ATP, 0.3 Na2GTP at 290 mOsm (pH 7.4). Currents were manually injected to hold the membrane




potential around 270 mV to record voltage responses. Tetrodotoxin (1 mmol/L; Tocris, catalog No. 1078) was applied through a gravityfed perfusion system.

RESULTS Mll1 Is Required for the Expression of Transcriptional Modules Related to SVZ Neurogenesis In the adult and postnatal mouse brain, SVZ NSCs produce neuroblasts that migrate to the olfactory bulb where they differentiate into interneurons and integrate into local circuits.26,27 SVZ NSCs can also generate astrocytes in response to local injury,28 as well as oligodendrocytes, in both normal and pathological conditions.29-31 When cultured as monolayers, SVZ NSC cultures express nestin and Sox210,18 and can recapitulate the development of these distinct neural lineages,10,18,32 with wild-type SVZ NSCs efficiently generating neurons, astrocytes, and oligodendrocytes (Figure 1A). In contrast, Mll1-deleted SVZ NSCs (which are derived from hGFAP-Cre;Mll1f/f mice and are 95% deleted for Mll110) survive, proliferate, and produce glia, but they are defective for neurogenesis (Figure 1B).10 To identify Mll1-dependent gene expression, we performed microarray analysis of wild-type and Mll1-deleted SVZ cultures in selfrenewal conditions and after 1, 2, and 4 days of differentiation (each performed in biological triplicate). By analyzing gene coexpression relationships,14-16 we identified 14 transcriptional “modules” (labeled by color), each representing groups of genes with expression levels that were highly correlated across the 24 samples (see Figure, Supplemental Digital Content 1, http:// links.lww.com/NEU/A658 and selected modules in Figure 2A). Each module was related to specific biological processes and neurobiological cell types via enrichment analysis for GO terms (Table 1) and curated biological gene sets.

Seven of the 14 coexpression modules exhibited similar temporal patterns in wild-type and Mll1-deleted cells, indicating that some SVZ gene expression programs do not require Mll1 (see Table 1, Supplemental Digital Content 2, http://links. lww.com/NEU/A659). For example, the turquoise module (Figure 2B) consisted of genes that were progressively downregulated under differentiation conditions in both wild-type and Mll1-deleted cells. This module was highly enriched for GO terms related to cell-cycle regulation (Table 1), and these findings are consistent with the observation that Mll1-deleted cultures proliferate at rates similar to wild-type SVZ NSCs.10 The tan module (Figure 2B) consisted of genes that were quickly upregulated under differentiation conditions in both wild-type and Mll1-deleted SVZ cells. This module contained Gfap and was enriched for experimentally validated markers of astrocytes21 (P = 2.76 · 1028); indeed, both Mll1-deleted and wild-type NSCs efficiently generate GFAP1 astrocytes.10 The green-yellow module consisted of genes with higher expression levels in Mll1-deleted cells vs wild-type cells at the 4day time point (Figure 2B), and its relationship to oligodendrocytes was revealed by GO analysis (Table 1) and significant enrichment with experimentally validated markers of oligodendrocytes from Cahoy et al23 (P = 1.11 · 10229), Nielsen et al22 (P = 3.64 · 10213), and Doyle et al24 (P = 3.02 · 10214). Interestingly, a trend toward increased oligodendrogliogenesis is observed with Mll1 deletion both in vivo and in vitro.10 Other coexpression modules exhibited temporal patterns of transcriptional upregulation that were strongly Mll1 dependent. For example, the black and magenta modules (Figure 2C) consisted of genes that were strongly upregulated in wild-type but not Mll1-deleted SVZ cells after 2 days (black) or 1 day (magenta) of differentiation (see Table 2, Supplemental Digital Content 3, http://links.lww.com/NEU/A660). Both modules

FIGURE 1. Mll1 is required for neurogenesis but not for gliogenesis. A, differentiation of wild-type subventricular zone (SVZ) neural stem cells (NSCs) over a 7-day time course. B, differentiation of Mll1-deleted SVZ NSCs. Neuronal class III b-tubulin (Tuj1)–positive neurons are shown in i through iv; O41 and NG21 oligodendrocytes are shown in v and vi. i through iv, glial fibrillary acidic protein (GFAP; green), Tuj1 (red), and DAPI (blue). v and vi, O4 (green) and NG2 (red). Scale bar = 50 mm. Ai through Aiv were originally published in Gonzalez-Roybal and Lim.50

NEUROSURGERY



POTTS ET AL

FIGURE 2. Gene coexpression analysis identifies distinct transcriptional modules. A, hierarchical clustering of gene coexpression modules. This dendrogram was produced using average linkage hierarchical clustering with 1 2 the Pearson correlation of the module eigengenes15 (first principal components) as a distance measure. B and C, select gene coexpression modules showing representative heat maps and first principal components at 0 (self-renewal conditions), 1, 2, and 4 days of differentiation in wildtype (WT) and Mll1-deleted (Mll1D/D) NSCs. B, nonneurogenic modules identified through gene coexpression analysis. C, Mll1dependent modules that are enriched for neurogenic gene expression. Lowercase letters in B and C indicate independent biological replicates.

were enriched for GO terms related to neurogenesis (Table 1), and the black module was strongly significant for experimentally validated markers of mature neurons from Cahoy et al23 (P = 2.77 · 10215). Black module gene members include Dlx5, Dlx6, Sp8, and Sp9, all of which encode transcription factors expressed in SVZ neuroblasts and olfactory bulb interneurons.33,34 Furthermore,


the presence of glutamic acid decarboxylase-1 (Gad1) and GABA(A) receptor-associated protein like 1 (Gabarapl1) in the black module suggest a GABAergic identity of neurons within the population of differentiating cells. The magenta module contained genes that are expressed at earlier stages of neurogenesis (see Table 2, Supplemental Digital




TABLE 1. Gene Ontology Terms Associated With Select Transcriptional Modules Module Turquoise

Tan

Green-yellow

Black

Magenta

Top 5 Gene Ontology Terms

P Value

GO:0007049: cell cycle GO:0022402: cell-cycle process GO:0022403: cell-cycle phase GO:0000279: M phase GO:0000278: mitotic cell cycle GO:0019725: cellular homeostasis GO:0019228: regulation of action potential in neuron GO:0010975: regulation of neuron projection development GO:0019226: transmission of nerve impulse GO:0001508: regulation of action potential GO:0007272: ensheathment of neurons GO:0008366: axon ensheathment GO:0019228: regulation of action potential in neuron GO:0001508: regulation of action potential GO:0048709: oligodendrocyte differentiation GO:0031175: neuron projection development GO:0048666: neuron development GO:0030182: neuron differentiation GO:0030030: cell projection organization GO:0007409: axonogenesis GO:0030900: forebrain development GO:0021537: telencephalon development GO:0021879: forebrain neuron differentiation GO:0006928: cell motion GO:0021872: generation of neurons in the forebrain

6.44E-42 9.04E-39 1.95E-37 1.51E-34 2.93E-33 4.51E-03 4.76E-03 5.39E-03 7.43E-03 7.93E-03 1.09E-06 1.09E-06 1.96E-06 4.11E-06 7.26E-06 1.97E-06 4.01E-06 1.36E-05 5.68E-05 1.13E-04 1.35E-03 1.93E-03 4.60E-03 5.25E-03 6.01E-03

Content 3, http://links.lww.com/NEU/A660). For instance, the expression of Dlx1 generally precedes that of Dlx5 and Dlx6.35 Consistent with this temporal relationship, Dlx1 was a member of the magenta module, whereas Dlx5 and Dlx6 were members of the later-upregulated black module. Dlx2, which is also temporally expressed with Dlx1, was most strongly associated with the magenta module. Other magenta module genes included Arx, a homeobox transcription factor that regulates the proliferative expansion of immature neuronal progenitor cells in the developing cortex (P = 3.45E-08),36 and Tlx (also known as Nr2e1), which is required for SVZ NSC self-renewal.37 Finally, Brn4, which has also been associated with SVZ neurogenesis,38 was also identified in the magenta module.

NEUROSURGERY

Interestingly, at the time that the magenta module was first upregulated (1 day of differentiation), both wild-type and Mll1deleted cultures lacked Tuj1-positive neuroblasts and contained many GFAP-positive astroglia (Figure 1). This early-differentiation expression of the magenta module suggests its relationship to early stages of neuronal lineage commitment in wild-type SVZ NSCs, which at that time still have astroglial characteristics. Enforced Expression of Select Magenta Module Genes Partially Rescues Neurogenesis in Mll1-Deleted SVZ Cells To evaluate the neurogenic potential of genes associated with the magenta module, we tested their ability to rescue neuronal differentiation of Mll1-deleted SVZ cells. For each of these genes (Dlx2, Tlx, Zdhhc23, and Brn4), we generated an overexpression lentivirus that coexpresses the GFP marker (Table 2). To provide a negative control, we produced a lentiviral vector that expresses only the marker proteins GFP and alkaline phosphatase (LVhPLAP-GFP). Mll1-deleted SVZ cultures were infected with each lentivirus, and after 4 days of differentiation, neuronal differentiation was assessed by immunocytochemistry (Figure 3A-3F) and quantified (Figure 3G). In control virus-infected cultures (Figure 3A), approximately 10% of the GFP-positive cells were immunopositive for the Tuj1 neuronal marker (Figure 3G), representing the lower levels of neurogenesis of Mll1-deleted SVZ cells. Compared with this control, cultures infected with the Dlx2 lentivirus (Figure 3B) resulted in a 5-fold increase in GFPpositive, Tuj1-positive cells (Figure 3G), which is similar to the rescue previously observed with transient Dlx2 overexpression.10 Individual overexpression of Brn4, Tlx, and Zdhhc23 also increased the production of neurons from Mll1-deleted SVZ cells (Figure 3C-3E), albeit with approximately 2-fold lower efficiency than that of Dlx2 (Figure 3G). We also generated a lentivirus that expresses Shisa3, a gene that was not found in the neurogenic modules, and infection with this vector did not significantly increase neuronal differentiation (Figure 3, F and G). Thus, when overexpressed, select members of the magenta module can induce neurogenesis in Mll1-deleted SVZ cells.

TABLE 2. Lentiviral Expression Vectorsa Magenta module genes (neurogenic) LV-Dlx2-GFP LV-Brn4-GFP LV-Tlx-GFP LV-Zdhhc23-GFP Mll1-independent gene (nonneurogenic) LV-Shisa3-GFP Control vector LV-hPLAP-GFP a

GFP, green fluorescent protein.



POTTS ET AL

FIGURE 3. Rescue of neurogenesis by overexpression of select transcription factors. A through F, immunocytochemistry of hGFAP-Cre;Mll1f/f subventricular zone (SVZ) neural stem cell (NSC) cultures after 4 days of differentiation and enforced expression with lentiviral constructs containing select Mll1-dependent factors: control (A), Dlx2 (B), Brn4 (C), Tlx (D), Zdhhc23 (E), and Shisa3 (F). Green fluorescent protein (GFP; green), neuronal class III b-tubulin (Tuj1; red), and DAPI (blue). G, quantification of infected (GFP1) cells that are also Tuj11 (n = 3 wells). Error bars = standard deviation. One-way analysis of variance with the post hoc Dunnett test was used to compare each experimental factor with hPLAP control. Scale bar = 50 mm.

Enforced Expression of Brn4 in Cortically Derived Primary Astroglial Cultures Induces Neurogenesis Astroglia cultured from the postnatal cortex can be transdifferentiated into neurons via the overexpression of certain single transcription factors, including Dlx2 and Pax6,1,3 both of which are members of the magenta module. To test if another magenta module member could likewise induce neurogenesis from cortical astroglia, we enforced Brn4 expression using LV-Brn4-GFP and analyzed the cell fate of GFP-positive cells after 20 days of differentiation. Although Brn4 has been associated with SVZ neurogenesis,38 its ability to transdifferentiate astrocytes into neurons was unknown. As a control, we also followed the fate of astroglial cells infected with LV-hPLAP-GFP. Before Brn4 overexpression, cortical astroglial cultures consisted of GFAPpositive cells (Figure 4A), and no Tuj1-positive neuronal cells were observed (data not shown). After 10 days of differentiation, LV-Brn4-GFP–infected cultures contained GFP-positive, Tuj1positive cells (data not shown). After 20 days of differentiation, these cultures contained GFP-positive, Tuj1-positive cells with neuronal morphology (Figure 4B). As expected, we did not observe any GFP-positive neuronal cells in the control LV-hPLAP-GFP–infected cultures (quantified in Figure 4G). LV-Brn4-GFP infection also generated GFP-positive cells that coexpressed microtubule-associated protein 2 (Figure 4C) or doublecortin (Figure 4D), further supporting the neuronal identity of these cells. In contrast, LV-hPLAP-GFP control infection did not produce any GFP-positive cells expressing the doublecortin and microtubule-associated protein 2 neuronal antigens (quantified in Figure 4G). Furthermore, some neuronal cells induced by


LV-Brn4-GFP expressed GAD67 (Figure 4E), an enzyme present in GABAergic neurons, and calbindin (Figure 4F), a marker of specific neuron subtypes. This immunocytochemistry analysis indicates that LV-Brn4-GFP infection induces the neuronal transdifferentiation of cortical astrocytes in vitro. To further characterize the identity of these LV-Brn4-GFP– induced neurons, we performed whole-cell patch-clamp recordings (Figure 5). Resting membrane potential was recorded in current clamp mode without injecting current and found to be negative in each cell tested (241 6 2 mV; n = 3), indicating the expression of ion channels and the regulation of ionic gradients across the cellular membrane. We then depolarized cells by injecting a series of depolarizing current steps, and all cells tested fired action potentials that appeared after a threshold membrane potential in an all-or-none fashion (Cm = 28 6 0.1 pA/pF; n = 3; Figure 5B, left). Additionally, these action potentials were reversibly eliminated by the addition of tetrodotoxin, a specific blocker of voltage-gated Na1 channels (Figure 5B, middle and right). Taken together, these data indicate that neurons transdifferentiated from cortical astroglia via LV-Brn4-GFP infection are electrophysiologically functional.

DISCUSSION The differentiation of neurons from multipotent NSCs requires the concerted expression of distinct sets of genes that promote neuronal identity. Such lineage-specific gene expression is, in part, coordinated by epigenetic regulators such as MLL1, a histone methyltransferase and member of the trithorax group of chromatin




FIGURE 4. Brn4 induces neurogenesis in cortically derived astroglia. Immunocytochemistry of cortically derived astroglial cultures before (A) and 20 days after (B-F) neuronal differentiation and enforced lentiviral expression of Brn4. B through F, examples of cells with neuronal morphology expressing various neuronal markers: neuronal class III b-tubulin (Tuj1; B), microtubule-associated protein 2 (MAP2; C), doublecortin (DCX; D), GAD67 (E), and calbindin (CalB; F). Glial fibrillary acidic protein (GFAP; red, A), DAPI (blue, A-F), and green fluorescent protein (GFP; green, B-F). No cells with neuronal morphology expressing these neuronal markers were identified in control-infected cultures. G, quantification of infected (GFP1) cells that were also Tuj11, MAP21, or DCX1 (from B, C, and D, respectively; compared with hPLAP controls; n = 3 wells for each). Error bars = standard deviation. The Student t test was used to compare Brn4 and hPLAP for each stain. Scale bar = 50 mm (A) and 25 mm (B-F).

regulators.8,9 In human patients, MLL1 mutations have been linked to Wiedemann-Steiner syndrome, a rare developmental disorder characterized by intellectual disability, including autism.39 We demonstrate that Mll1 is required for the expression of neurogenic transcriptional modules in SVZ NSCs, indicating its central role in the gene expression required for normal neural development. Furthermore, genes selected from the neurogenic modules can promote neuronal differentiation from gliogenic cell populations. Our study thus provides new insights into the role that Mll1 plays in neural development and further suggests that specific neurogenic module genes may be useful for direct astrocyte-to-neuron reprogramming strategies. Mll1 deletion targeted to SVZ NSCs results in defective neurogenesis, and in a previous study, we found that this lineagespecific deficiency relates to the lack of proper Dlx2 expression.10 In the present study, we used microarray and gene coexpression

NEUROSURGERY

analyses to further characterize the Mll1-dependent transcription that corresponds to neurogenesis. We identified 2 neurogenic gene coexpression modules, black and magenta, and our analysis suggests that the magenta module is involved in earlier stages of neuronal lineage commitment. The magenta module was upregulated in wild-type cultures after just 1 day of differentiation and before the appearance of Tuj1-positive neuroblasts, suggesting that gene expression in this module does not simply reflect transcription related to the presence of neurons. Indeed, Dlx2, which is most closely associated with the magenta module, is first expressed in SVZ transit amplifying cells, before their differentiation into Tuj1-positive migratory neuroblasts.33 In addition, the magenta module defined 127 other genes with a similar pattern of expression (see Table 2, Supplemental Digital Content 3), providing many novel candidates for the study of genes involved in early stages of neurogenesis.



POTTS ET AL

FIGURE 5. Electrophysiological analysis of neurons induced by Brn4 overexpression in cortically derived astroglia. A, phasecontrast image of a sample recorded neuron. B, action potentials were elicited by a series of current injections ranging from 240 to 80 pA in 20-pA increments. Action potentials were reversibly blocked with 1 mmol/L tetrodotoxin (TTX).

Previously, we showed that transient overexpression of Dlx2 partially rescues Mll1-dependent neurogenesis. For this study, we used lentiviral expression vectors to enforce the expression of Dlx2, Tlx, Zdhhc23, and Brn4 in Mll1-deleted SVZ NSCs, and the resultant increase in neuronal differentiation induced by each vector demonstrated the neurogenic potential of these magenta module genes. Furthermore, the ability to induce neuronal differentiation of Mll1-deleted cells suggests that this trithorax group chromatin regulator is not required for the transcription downstream of key neurogenic genes, at least when they are overexpressed. Tlx encodes a nuclear receptor that is expressed in SVZ NSCs, and Tlx deficiency reduces NSC self-renewal and SVZ neurogenesis.37,40 Interestingly, transgenic overexpression of Tlx in vivo results in the expansion of the NSC population and glioma-like lesions40; it is therefore possible that the increased number of neurons observed with Tlx overexpression in Mll1deleted cultures relates to increased proliferation of neurogenic progenitor cells. Zdhhc23 encodes a zinc-finger–containing palmitoyl-transferase41 that is associated with the synaptic membrane.42 The function of this molecule is poorly understood, and to the best of our knowledge, it has not been previously associated with neurogenesis or Mll1. Brn4 is a member of the POU domain transcription factor family. Brn4 knockout mice exhibit deafness, vestibular abnormalities, and gait impairments.43 Brn4 is expressed in the SVZ of the adult rodent,44 and results of a recent study suggest that Brn4 is a component of a transcriptional network downstream of Pax6 that drives SVZ neurogenesis.38 The Brn4 locus is a direct target for the PAX6 transcription factor, and Pax6 overexpression upregulates Brn4.38 Given this transcriptional interrelationship and the Mll1 dependence of Pax6 expression, it is possible that the activation of the Brn4 locus during neurogenesis is not directly dependent on MLL1 and instead relates to activation by PAX6. Future studies of MLL1 protein enrichment and related chromatin state changes, as has been performed to demonstrate the role of MLL1 at the Dlx2 locus,10 will help clarify whether Brn4 and other neurogenic genes are directly regulated by this chromatin-modifying factor. Our study identified Brn4 as a transcription factor that can induce neuronal transdifferentiation from cultured astrocytes.


This finding contributes to the relatively small collection of factors that have been shown to promote the conversion of astrocytes into neurons.1,3,5,6 Pax6 can induce neurons from astrocytes, and interestingly, this transcription factor was also a member of the magenta module. Similarly, Dlx2, which also induces neurons from astrocytes, was most strongly associated with the magenta module. We did not detect Ngn2 in an SVZ neurogenic module; however, the overexpression of Ngn2 induces the production of glutamatergic neurons,3 a cell type that is not commonly produced from SVZ NSCs. We also did not find Mash1 as a member of a neurogenic module. Consistent with previous results, Mash1 expression was not Mll1 dependent.10 Although Mash1 overexpression induces transdifferentiation into GABAergic neurons, this transcription factor also plays a role in normal SVZ gliogenesis.45 Recent studies of direct fibroblast-toneuron reprogramming indicate that Mash1 acts as a “pioneer” factor that promotes the recruitment of other transcription factors.46 Indeed, astrocytes with both Mash1 and Dlx2 overexpression generate greater numbers of neurons than either transcription factor alone.3 Future combinatorial strategies (eg, Mash1, Brn4, and Dlx2 together) may further improve the efficiency of neuronal reprogramming observed both in vitro and in vivo. Importantly, these findings may serve as a basis for future studies of neuronal reprogramming in human cells. Several studies have already demonstrated that human-derived cells can be successfully reprogrammed.47-49 Corti et al6 have even converted human astrocytes into neurons using OCT4, SOX2, or NANOG. Interestingly, they also show that this process is epigenetically regulated through DNA methylation. Given the important role of Mll1 in murine SVZ neurogenesis, it will be worthwhile to explore both its role in human neurogenesis and cell reprogramming. Neurosurgeons are in the unique position to do this through access to intraoperatively derived human tissue.

CONCLUSION This study provides new insights into the Mll1-dependent expression that underlies postnatal SVZ neurogenesis. Gene coexpression analysis identifies transcriptional modules that distinguish neurogenesis from gliogenesis, and enforced expression




of select neurogenic factors can promote neurogenesis from Mll1deleted SVZ NSCs. Brn4 is identified as a factor for direct astrocyte-to-neuron transdifferentiation. These data thus serve as a resource for future studies of Mll1 in neural development and direct astrocyte-to-neuron reprogramming strategies. Disclosures This work was supported by a VA BSLRD Merit Award (I01 BX000252), a National Institutes of Health New Innovator Award (DP2 OD006505), and a UCSF REAC/CTSI pilot award to Dr Lim; National Institutes of Health NSRA (F32 NS073173) to Dr Potts; a National Institutes of Health grant (R01NS073765) to Dr Margeta; the UCSF Program for Breakthrough Biomedical Research, which is funded in part by the Sandler Foundation, to Dr Oldham; and facilities and resources provided by the San Francisco Veterans Affairs Medical Center. The other authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.

REFERENCES 1. Heins N, Malatesta P, Cecconi F, et al. Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci. 2002;5(4):308-315. 2. Hwang WW, Alvarez-Buylla A, Lim DA. Glial nature of adult neural stem cells: neurogenic competence in adult astrocytes. In: Rao MS, Carpenter M, Vemuri MC, eds. Neural Development and Stem Cells. 3rd ed. New York: Springer; 2012:149-172. 3. Heinrich C, Blum R, Gascón S, et al. Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol. 2010;8(5):e1000373. 4. Fuentealba LC, Obernier K, Alvarez-Buylla A. Adult neural stem cells bridge their niche. Cell Stem Cell. 2012;10(6):698-708. 5. Heinrich C, Gascón S, Masserdotti G, et al. Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nat Protoc. 2011;6 (2):214-228. 6. Corti S, Nizzardo M, Simone C, et al. Direct reprogramming of human astrocytes into neural stem cells and neurons. Exp Cell Res. 2012;318(13):1528-1541. 7. Kriegstein A, Alvarez-Buylla A. The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci. 2009;32:149-184. 8. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G. Genome regulation by Polycomb and trithorax proteins. Cell 2007;128(4):735-745. 9. Zhang P, Bergamin E, Couture J-F. The many facets of MLL1 regulation. Biopolymers. 2013;99(2):136-145. 10. Lim DA, Huang YC, Swigut T, et al. Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells. Nature. 2009;458 (7237):529-533. 11. Berninger B, Costa MR, Koch U, et al. Functional Properties of neurons derived from in vitro reprogrammed postnatal astroglia. J Neurosci. 2007;27(32):8654-8664. 12. Niu W, Zang T, Zou Y, et al. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nat Cell Biol. 2013;15(10):1164-1175. 13. Torper O, Pfisterer U, Wolf DA, et al. Generation of induced neurons via direct conversion in vivo. Proc Natl Acad Sci U S A. 2013;110(17):7038-7043. 14. Oldham MC, Horvath S, Geschwind DH. Conservation and evolution of gene coexpression networks in human and chimpanzee brains. Proc Natl Acad Sci U S A. 2006;103(47):17973-17978. 15. Horvath S, Dong J. Geometric interpretation of gene coexpression network analysis. PLoS Comput Biol. 2008;4(8):e1000117. 16. Oldham MC, Konopka G, Iwamoto K, et al. Functional organization of the transcriptome in human brain. Nat Neurosci. 2008;11(11):1271-1282. 17. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559. 18. Scheffler B. Phenotypic and functional characterization of adult brain neuropoiesis. Proc Natl Acad Sci U S A. 2005;102(26):9353-9358. 19. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31(4):e15. 20. Oldham MC, Langfelder P, Horvath S. Network methods for describing sample relationships in genomic datasets: application to Huntington’s disease. BMC Syst Biol. 2012;6:63.

NEUROSURGERY

21. Bachoo RM, Kim RS, Ligon KL, et al. Molecular diversity of astrocytes with implications for neurological disorders. Proc Natl Acad Sci U S A. 2004;101(22): 8384-8389. 22. Nielsen JA, Maric D, Lau P, Barker JL, Hudson LD. Identification of a novel oligodendrocyte cell adhesion protein using gene expression profiling. J Neurosci. 2006;26(39):9881-9891. 23. Cahoy JD, Emery B, Kaushal A, et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci. 2008;28(1):264-278. 24. Doyle JP, Dougherty JD, Heiman M, et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell. 2008;135(4):749-762. 25. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391(2):85-100. 26. Lim DA, Huang Y-C, Alvarez-Buylla A. Adult subventricular zone and olfactory bulb neurogenesis. In: Gage FH, Kempermann G, Song H, eds. Adult Neurogenesis. New York: Cold Spring Harbor Laboratory Press; 2008:175-206. 27. Ming GL, Song H. Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron. 2011;70(4):687-702. 28. Benner EJ, Luciano D, Jo R, et al. Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4. Nature. 2013;497(7449):369-373. 29. Menn B. Origin of oligodendrocytes in the subventricular zone of the adult brain. J Neurosci. 2006;26(30):7907-7918. 30. Picard-Riera N, Decker L, Delarasse C, et al. Experimental autoimmune encephalomyelitis mobilizes neural progenitors from the subventricular zone to undergo oligodendrogenesis in adult mice. Proc Natl Acad Sci U S A. 2002;99(20): 13211-13216. 31. Nait-Oumesmar B, Decker L, Lachapelle F, Avellana-Adalid V, Bachelin C, Baron-Van Evercooren A. Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci. 1999;11(12):4357-4366. 32. Merkle FT, Mirzadeh Z, Alvarez-Buylla A. Mosaic organization of neural stem cells in the adult brain. Science. 2007;317(5836):381-384. 33. Long JE, Garel S, Álvarez-Dolado M, et al. Dlx-dependent and -independent regulation of olfactory bulb interneuron differentiation. J Neurosci. 2007;27(12): 3230-3243. 34. Allen ZJ II, Waclaw RR, Colbert MC, Campbell K. Molecular identity of olfactory bulb interneurons: transcriptional codes of periglomerular neuron subtypes. J Mol Histol. 2007;38(6):517-525. 35. Panganiban G, Rubenstein JL. Developmental functions of the Distal-less/Dlx homeobox genes. Development. 2002;129(19):4371-4386. 36. Colasante G, Collombat P, Raimondi V, et al. Arx is a direct target of Dlx2 and thereby contributes to the tangential migration of GABAergic interneurons. J Neurosci. 2008;28(42):10674-10686. 37. Shi Y, Chichung Lie D, Taupin P, et al. Expression and function of orphan nuclear receptor TLX in adult neural stem cells. Nature. 2004;427(6969):78-83. 38. Ninkovic J, Steiner-Mezzadri A, Jawerka M, et al. The BAF complex interacts with Pax6 in adult neural progenitors to establish a neurogenic cross-regulatory transcriptional network. Cell Stem Cell. 2013;13(4):403-418. 39. Jones WD, Dafou D, McEntagart M, et al. De novo mutations in MLL cause Wiedemann-Steiner syndrome. Am J Hum Genet. 2012;91(2):358-364. 40. Liu H-K, Wang Y, Belz T, et al. The nuclear receptor tailless induces long-term neural stem cell expansion and brain tumor initiation. Genes Dev. 2010;24(7):683-695. 41. Tian L, McClafferty H, Knaus HG, Ruth P, Shipston MJ. Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels. J Biol Chem. 2012;287(18):14718-14725. 42. Saitoh F, Tian QB, Okano A, Sakagami H, Kondo H, Suzuki T. NIDD, a novel DHHC-containing protein, targets neuronal nitric-oxide synthase (nNOS) to the synaptic membrane through a PDZ-dependent interaction and regulates nNOS activity. J Biol Chem. 2004;279(28):29461-29468. 43. Phippard D, Lu L, Lee D, Saunders JC, Crenshaw EB III. Targeted mutagenesis of the POU-domain gene Brn4/Pou3f4 causes developmental defects in the inner ear. J Neurosci. 1999;19(14):5980-5989. 44. Mathis JM, Simmons DM, He X, Swanson LW, Rosenfeld MG. Brain 4: a novel mammalian POU domain transcription factor exhibiting restricted brain-specific expression. EMBO J. 1992;11(7):2551-2561. 45. Parras CM, Galli R, Britz O, et al. Mash1 specifies neurons and oligodendrocytes in the postnatal brain. EMBO J. 2004;23(22):4495-4505.



POTTS ET AL

46. Wapinski OL, Vierbuchen T, Qu K, et al. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell. 2013;155(3):621-635. 47. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872. 48. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917-1920. 49. Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008;451(7175):141-146. 50. Gonzales-Roybal G, Lim DA. Chromatin-based epigenetics of adult subventricular zone neural stem cells. Front Genet. 2013;4:194.

transdifferentiation from cultured murine astrocytes. This work adds to the body of literature supporting the importance of Mll-dependent expression to postnatal neurogenesis. The addition of an electrophysiological analysis also enforces the reliability of their claims and confirms its potential clinical implications. Future efforts by neurosurgeonneuroscientists such as the senior author may pave the road for therapies driven by epigenetic reprogramming of human-derived cells.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.neurosurgery-online.com).

O

Pengbo Zhang Shaanxi, China

verall, this is an excellent, well-written, and well-referenced article evaluating the transcriptional and epigenetic regulation of glial and neuronal differentiation in the subventricular zone. The authors appear to have used solid methods that are both innovative and complementary. The confirmation of neuronal phenotype with electrophysiology data is an added bonus, and the authors attempt to relate Mll to a human condition in their discussion. The article is a testament to fantastic science that can be performed by individuals in our field of neurosurgery while harnessing the insight of molecular and cellular events surrounding glial/neuronal differentiation. The statistical data suggest that a good amount of care was spent selecting data that are conservative and not an overrepresentation of the group’s data. The authors should be commended on their sound experimental and statistical methods.

he authors present a comprehensive in vitro study demonstrating a role for Brn4 as a transcription factor relevant to neural

Tim Vogel Cincinnati, Ohio

COMMENTS

T

he authors used microarray, gene coexpression, and module enrichment analysis to find out that Mll1 is required for the expression of neurogenic transcriptional modules in neural stem cells and to indicate that Brn4 may be useful for astrocyte-to-neuron reprogramming strategies. This is an interesting study of neurogenic transcription and reprogramming that will provide some important clues for using adult cells as injury repair strategies.

T

Nader Sanai Phoenix, Arizona




Co-expression network analysis identifies transcriptional modules in the mouse liver.

An integrated systems biology approach identifies positive cofactor 4 as a factor that increases reprogramming efficiency.

Chromatin and Transcriptional Analysis of Mesoderm Progenitor Cells Identifies HOPX as a Regulator of Primitive Hematopoiesis.

Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence.

FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence.

Deficiency of transcription factor Brn4 disrupts cochlear gap junction plaques in a model of DFN3 non-syndromic deafness.

TRIM28 as a novel transcriptional elongation factor.

The reprogramming of transcriptional competence.

Direct Reprogramming: Bypassing Stem Cells for Therapeutics.

Direct Cardiac Cellular Reprogramming for Cardiac Regeneration.

Bmi1-mediated epigenetic signature acts as a critical barrier for direct reprogramming to mature cardiomyocytes.

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy.

Masticatory deficiency as a risk factor for cognitive dysfunction.

Direct Reprogramming of RESTing Astrocytes.

Transcriptional reprogramming in cellular quiescence.

Systematic microRNA analysis identifies ATP6V0C as an essential host factor for human cytomegalovirus replication.

Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions.

Nuclear Actin in Development and Transcriptional Reprogramming.

Transcriptional Profiling Identifies the Signaling Axes of IGF and Transforming Growth Factor-b as Involved in the Pathogenesis of Osteosarcoma.

Smart Capture Modules for Direct Sensor-to-FPGA Interfaces.

rTRM-web: a web tool for predicting transcriptional regulatory modules for ChIP-seq-ed transcription factors.

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

Systematic tracking of dysregulated modules identifies disrupted pathways in narcolepsy.