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Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons

Abstract

The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype–specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity.

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Figure 1: Fezf2 overexpression in cortical progenitors induces genes that label corticospinal motor neurons.
Figure 2: Fezf2-induced genes identify native CSMN and label subsets of the broad CTIP2-positive population in layer V.
Figure 3: Genome-wide binding analysis for Fezf2 shows preferential association with proximal promoter regions of CSMN genes.
Figure 4: Fezf2 promotes glutamatergic and inhibits GABAergic neurotransmitter pathways.
Figure 5: Fezf2 controls Ephb1 selective expression in CSMN by direct association with the Ephb1 promoter.
Figure 6: Cortical neurons aberrantly project through the anterior commissure in the absence of EphB1.
Figure 7: Ephb1−/− mice recapitulate the axon guidance phenotype observed in Fezf2 null mutants.

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Acknowledgements

We would like to thank A. McMahon and J. Macklis for insightful advice in the early stages of the work; S. McConnell (Stanford University) for her generous sharing of the Fezf2PLAP line; J. Macklis (Harvard University), R. Hevner (Seattle Children's Research Institute), A. Catic (Massachusetts General Hospital) and S. Sykes (Massachusetts General Hospital) for sharing of antibodies and expression vectors; C. O'Donnell and D. Cacchiarelli for discussions on ChIP-seq analysis.; Z. Trayes-Gibson, E. Stronge and A. Iannone for outstanding technical support and for reading the manuscript; and A. Goodwin for careful editing of the manuscript and comments. This work was supported by grants from the US National Institutes of Health (NS062849, MH101268, NS078164 to P.A.), (HD078561 and HD069001 to E.T.), the New York Stem Cell Foundation (Robertson Investigator award to P.A.) and the Harvard Stem Cell Institute to P.A.

Author information

Authors and Affiliations

Authors

Contributions

S.L. and P.A. conceived the work, designed the experiments, analyzed the data and wrote the manuscript. S.L. performed the majority of the experiments. B.J.M. contributed to experimental design and performed microarray analysis. E.Z. performed the ChIP-seq experiments. L.A.G. analyzed the RNA-seq data. H.-H.C. performed the in vitro differentiation experiment and assisted in manuscript preparation. W.Y. performed the electrophoretic mobility shift assay experiment. A.M. assisted with FACS purification and the microarray experiments. E.T. performed the HARDI analysis. S.M. analyzed the ChIP-seq data. D.K.G. and J.L.R. supervised the bioinformatics analyses. P.A. supervised all aspects of the project.

Corresponding author

Correspondence to Paola Arlotta.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 FACS-purification of cortical progenitors electroporated in vivo with Fezf2GFP and CtrlGFP expression constructs at E14.5.

(a) Schematic of the experimental approach. (b) Coronal sections of CtrlGFP and Fezf2GFP-electroporated brains, 48 hours after electroporation, showing GFP-positive progenitors within the proliferative zones. CTIP2 immunohistochemistry (red) marks corticofugal neurons in the developing cortical plate. (c) Dissociated cortical cells before and after FACS purification; only a very small percentage of dissociated cells are electroporated (GFP-positive). FACS purification of GFP-positive cells results in virtually pure populations of labeled cells. CP, cortical plate. Scale bars, 100μm (b).

(Related to Figure 1)

Supplementary Figure 2 Expression profiles of Fezf2-induced genes at multiple embryonic and postnatal stages.

Expression profiles of 12 genes up-regulated by Fezf2 (shown in Figure 1) were examined by in situ hybridization in wild type cortices of E15.5, E18.5, P3, P7 and P14 wild type mice. CP, cortical plate. Scale bars, 50 μm.

(Related to Figure 1).

Supplementary Figure 3 Expression of Fezf2 target genes in Layer Vb subcerebral projection neurons is dependent on Fezf2 expression.

(a) In situ hybridizations for selected Fezf2-induced genes (blue signal) followed by immunocytochemistry for CTIP2 (brown signal) were performed on coronal sections of wild type mouse brains. (b) In situ hybridizations of selected Fezf2-induced genes on coronal sections of Fezf−/− brains (right) and wild type littermate controls (left). CP, cortical plate. Scale bars, 50 μm (a) left panels and (b), and 20 μm (a) right panels.

(Related to Figure 2).

Supplementary Figure 4 Fezf2-induced genes are expressed in nascent CSMN.

(a) Left, ternary plot of gene expression probabilities in VZ, SVZ/IZ and CP for Fezf2-induced targets shows enrichment in the cortical plate. Right, cluster representation of 186 genes induced by Fezf2 and also significantly enriched in the E14.5 CP. Y axis, FPKM (Fragments Per Kilobase RNA per Million mapped reads). Complete gene list is available in Supplementary Table 2. (b) Upper most left panel) In situ hybridization for Fezf2 on an E14.5 embryo shows the position of the cortical plate (CP). In situ hybridizations (www.genepaint.org) for prototypical genes induced by Fezf2 and enriched in the E14.5 CP (insets, enlarged from boxed areas). (c-d) In situ hybridizations for Hivep2 and Lmo3 at E14.5 (left panels) and at P4 (middle panels, http://developingmouse.brain-map.org) show specific expression in the developing CP and in layer V CSMN, confirming that Fezf2 induces expression of CSMN genes from early stages of development. Right panels, expression levels of Hivep2 (c) and Lmo3 (d) in CtrlGFP (blue line) and Fezf2GFP samples (red line) at 24 hours and 48 hours. Error bars indicate standard errors of the mean (SEM). CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone; IZ intermediate zone. Images shown in b; c and d left 2 panels are from the Genepaint database and c and d middle panels are from the Allen Brain Atlas database.

(Related to Figure 2).

Supplementary Figure 5 Fezf2-repressed genes are expressed in E14.5 VZ and SVZ/IZ cortical progenitors and in their progeny.

(a) Left, ternary plot of expression profiles collapsed to probability distributions in VZ, SVZ/IZ and CP for Fezf2-repressed genes shows enrichment in the VZ and the SVZ/IZ. Right, cluster representation of 73 genes repressed by Fezf2 and also significantly enriched in the E14.5 VZ-SVZ/IZ. Y axis, FPKM (Fragments Per Kilobase RNA per Million mapped reads). Complete gene list is available in Supplementary Table 2. (b) In situ hybridizations (www.genepaint.org) for prototypical genes repressed by Fezf2 and preferentially expressed in the E14.5 VZ-SVZ/IZ (insets, enlarged from boxed areas). (c,d) In situ hybridizations for Cux1 and Cux2 at E14.5 (left panels) showing specific expression in cortical progenitors. Middle panel, in situ hybridizations for Cux1 and Cux2 P4 showing specific expression in mature upper layer CPN (http://developingmouse.brain-map.org). Far right, expression levels of Cux1 and Cux2 in CtrlGFP (blue line) and Fezf2GFP samples (red line) are shown at 24 hours and 48 hours. Error bars indicate standard errors of the mean (SEM). CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone; IZ, intermediate zone. Images shown in b; c and d left 2 panels are from the Genepaint database and c and d middle panels are from the Allen Brain Atlas database.

(Related to Figure 2).

Supplementary Figure 6 3xFlag-Fezf2 recapitulates Fezf2 overexpression phenotype in vivo.

N-terminally FLAG-tagged Fezf2, employed for ChIP-seq and RNA-seq experiments, retains the ability to instruct the molecular identity and connectivity of deep layer projection neurons when ectopically expressed in progenitors of the upper layers at E14.5 (a) Experimental design of the ChIP-seq and RNA-seq approach. (b) Schematic of the experimental approach. (c) 3xFLAG-Fezf2 overexpression results in ectopic GFP+ cell aggregates below the corpus callosum, phenocopying untagged Fezf2 overexpression. (d) Immunohistochemistry for GFP, CTIP2 and TBR1 in electroporated brains harvested at P7 shows that 3xFLAG-Fezf2 is sufficient to induce a switch of fate to CTIP2+ and TBR1+ corticofugal projection neurons. (e) Immunohistochemistry for GFP shows axonal projections through the internal capsule (IC) (left panel), toward the thalamus (Th) (middle panel) and through the cerebral peduncle (CP) (right panel) upon 3xFLAG-Fezf2 overexpression. Ctx, cortex; Str, striatum; cc, corpus callosum. Scale bars, 100 μm (b, d), and 20 μm (c).

(Related to Figure 3).

Supplementary Figure 7 Overexpression of Fezf2 in neurosphere-derived neurons in vitro directs transcriptional changes toward the nascent CSMN state.

(a) Left, ternary plot of expression profiles collapsed to probability distributions in VZ, SVZ/IZ and CP for Fezf2-induced genes (identified from RNA-seq analysis) shows enrichment in the CP. Genes are classified as CP-specific (red), VZ-specific (blue), SVZ-specific (green), or non-specific (grey) using a specificity score threshold of 0.65 (detailed in Methods). Right, top 10 Gene Ontology categories from GO analysis of 224 genes up-regulated by Fezf2 in vitro. (b) Left, ternary plot of expression profiles collapsed to probability distributions in VZ, SVZ/IZ and CP for Fezf2-repressed genes (identified from RNA-seq analysis) shows enrichment in the VZ- SVZ/IZ. Right, top 10 Gene Ontology categories from GO analysis of 155 genes down-regulated by Fezf2 in vitro and specifically expressed in E14.5 VZ or SVZ/IZ. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone.

(Related to Figure 3).

Supplementary Figure 8 Fezf2 directly controls expression of its transcriptional targets via association with their promoter regions.

(a) Bar plots showing the percentage of FEZF2-bound genes among all genes (grey), Fezf2-regulated genes (green), Fezf2-induced genes (red), and Fezf2-repressed genes (blue) in in vitro cortical progenitors. (b) Table of gene counts, percentages of total, and p-values (hypergeometric test and bootstrap method) showing significant enrichment for FEZF2-bound genes in both the Fezf2-induced and Fezf2-repressed significant gene sets from in vitro cortical progenitors. (c) Bar plots showing the percentage of FEZF2-bound genes among all genes (grey), Fezf2-regulated genes (green), Fezf2-induced genes (red), and Fezf2-repressed genes (blue) in in vivo purified cortical progenitors. (d) Table of gene counts, percentages of total, and p-values (χ2-test) demonstrating significant enrichment for FEZF2-bound genes in both the Fezf2-induced and Fezf2-repressed significant gene sets from in vivo purified E14.5 cortical progenitors collected at 24h and 48h after Fezf2 over-expression. (e) Examples of 3xFLAG-Fezf2 peaks at the proximal promoters of CSMN-specific, Fezf2-induced genes (red peaks) and CPN-specific, Fezf2-repressed genes (blue peaks). Source data is shown in Supplementary Table 6.

(Related to Figure 3).

Supplementary Figure 9 Fezf2 directly associates with a CG-rich consensus sequence.

(a) The CG-rich FEZF2 consensus motif identified by GEM differs from two previously predicted FEZF2 motifs (SELEX and hPDI). (b) The motif-independent approach GPS found the GEM-defined motif more represented at the FEZF2-bound sites compared to the two previously defined motifs. FEZF2-bound Transcriptional Start Sites (TSSs) were also found preferentially associated with the GEM-defined motif compared to unbound TSSs. (c) Coomassie blue staining of purified GST-tFEZF2 protein (see Methods). The expected molecular weight of the GST-fusion truncated protein is 43.5 kDa, (arrow). (d) Electrophoretic mobility shift assay of probes for regions within the promoters of Ascl1 and EphB1 in the presence of GST-tFEZF2. Probes containing FEZF2 consensus motifs (EphB1-positive and Ascl1-positive) showed specific interaction with GST-tFEZF2 compared to probes without the consensus motifs in neighboring region of the same promoters (Ephb1-negative and Ascl1-negative). Arrow, probes bound to GST-tFEZF2; Asterisk, unbound probes.

(Related to Figure 3).

Supplementary Figure 10 Generation of inducible Fezf2 mouse ES cell lines.

(a) Strategy used for derivation of TET-ON Fezf2 mouse ES cell lines using the inducible cassette exchange method (see Methods). (b) Uncropped original immunoblots of Figure 4c.

Supplementary Figure 11 Axon guidance signaling molecules are highly represented in the GO analysis of Fezf2-induced genes and include the tyrosine kinase receptor Ephb1.

(a) GO analysis using Fezf2-induced genes at 24 hours and 48 hours identifies “axon guidance signaling” among the most significant categories (ranking as the most significant category at 48 hours). (b, far left panel) Schematic representation of the EphB1 gene targeting strategy (Deltagen Inc.). Arrows show positions and orientations of the primers employed for genotyping PCR. (b, middle panel) Genotyping PCR using primers flanking the targeted region identifies a 350 bp product corresponding to the wild type allele and a 300 bp product corresponding to the EphB1−/− allele. (b, most right panel) Western blot of brain lysate from P1 wild type, EphB1+/– pups, and EphB1−/− P1 pups using anti-EPHB1 antibody shows absence of EPHB1 protein (expected MW=100 kDa) in the EphB1−/− sample and a reduction in the EphB1+/– sample compared to wild type. Anti-β-tubulin is used as loading control. (c) Uncropped original immunoblots shown in panel (b, most right panel).

(Related to Figure 5).

Supplementary Figure 12 Expression patterns of EphB ligands in the mouse brain.

In situ hybridization for ephrin B1, ephrin B2 and ephrin B3 on horizontal wild type brain sections at E18.5 shows expression of ephrin B3 at the ventral forebrain midline. AC, anterior commissure. Scale bars, 100 μm.

(Related to Figure 7).

Supplementary Figure 13 Class-specific identity and distribution of cortical projection neurons is unaltered in Ephb1−/− mice.

(a) Schematic representation of the axon pathfinding phenotypes observed in both EphB1−/− and Fezf2−/− mice. (b) Schematic drawing of a P7 brain showing the position of area imaged. (c-d) Immunocytochemistry for CTIP2, TBR1, CUX1 and SATB2 show no difference in the layer distribution of these markers in EphB1−/− (d) compared to wild type mice (c). Scale bars, 100 μm.

(Related to Figure 7).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 2814 kb)

Supplementary Methods Checklist (PDF 395 kb)

Supplementary Table 1

Genes induced and repressed upon Fezf2 overexpression in E14.5 cortical progenitors in vivo as shown in Figure 1. Gene lists include Fezf2- induced genes (fold change >1.5; p value <0.001) at 24 hours (263 genes), at 48 hours (441 genes), and all unique genes induced at both time points (589 genes); Fezf2-repressed genes (fold change >1.5; p value <0.001) at 24 hours (90 genes), at 48 hours (89 genes), and all unique genes repressed at both time points (153 genes). Relevant and complete Gene Ontology terms for each gene are tabulated. Results from in situ hybridization across multiple developmental stages are summarized from data shown in Fig. 1 and Supplementary Fig. 2. (XLSX 134 kb)

Supplementary Table 2

Fezf2-induced and Fezf2-repressed genes that are expressed in the E14.5 developing cortex in Supplementary Figures 4 and 5. Lists include genes induced by Fezf2 and enriched in E14.5 CP, and genes repressed by Fezf2 and enriched in E14.5 VZ and SVZ progenitors. Each transcript is assigned to a cluster, which includes genes with analogous expression profiles in VZ, SVZ, and CP at E14.5. (XLSX 16 kb)

Supplementary Table 3

Early postnatal (P3/P6) CSMN-specific genes induced by Fezf2 overexpression in vivo including those shown in Figure 2. 30 genes preferentially expressed in purified CSMN3 P3 & P6, p<0.0001, CSMN/CPN >1.5) that are also induced by Fezf2 overexpression in vivo (p<0.001, Fezf2GFP/CtrlGFP >1.5). (XLSX 9 kb)

Supplementary Table 4

Genes bound by Fezf2 at their TSS and signature genes used for GSEA analysis as shown in Figure 3. High-confidence genes bound by 3xFLAG-Fezf2 in two independent ChIP-seq replicates (3xFLAG-Fezf2 bound genes) are listed with Ensembl IDs. A separate list of CSMN genes3 and one of axon guidance signaling molecules induced by Fezf2 in vivo are shown with related FEZF2 promoter-binding information. CSMN signature genes are defined as genes with 3 fold or higher expression levels in CSMN than CPN at both P3 and P6 (103 genes, CPN/CSMN ≤ 1/3). CPN signature genes are defined as genes with 3 fold or higher expression levels in CPN than CSMN at both P3 and P6 (144 genes, CPN/CSMN ≥ 3). Signature genes that are also bound by 3xFLAG-Fezf2 are shown in red and in a separate column (47 CSMN signature genes and 77 CPN signature genes). (XLSX 381 kb)

Supplementary Table 5

Fezf2-regulated genes identified from RNA-seq analysis of cortical progenitors (cultured as neurospheres) in vitro that are also differentially expressed between E14.5 CP and SVZ/VZ as shown in Supplementary Figure 7. Lists include genes that are up-regulated and down-regulated by Fezf2 in vitro, genes that are both up-regulated by Fezf2 in vitro and enriched in E14.5 CP, and genes that are both down-regulated by Fezf2 and enriched in VZ- SVZ/IZ. (XLSX 95 kb)

Supplementary Table 8

Combined analysis of in vivo Fezf2-overexpression microarray and in vitro Fezf2 ChIP-seq as shown in Supplementary Figure 8. χ2 test results of the enrichment for FEZF2-bound genes in both the Fezf2-induced and Fezf2-repressed significant gene lists. (XLSX 14 kb)

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Lodato, S., Molyneaux, B., Zuccaro, E. et al. Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons. Nat Neurosci 17, 1046–1054 (2014). https://doi.org/10.1038/nn.3757

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