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.

Figure 1

Fezf2 overexpression in cortical progenitors induces genes that label corticospinal motor neurons. Left, in situ hybridizations on coronal sections of the cerebral cortex at different embryonic (E15.5, E17.5 and E18.5) and postnatal stages (P3, P7 and P14) (insets enlarged from boxed areas). Right, expression levels (normalized microarray intensity, see Methods) for each selected gene in Ctrl^GFP (blue) and Fezf2^GFP (red) in utero electroporated cortical progenitors that were collected at 24 h (n = 3 litters per condition) and 48 h (n = 4 litters per condition). Vertical axes are normalized intensities (arbitrary units). Error bars indicate s.e.m. LV, lateral ventricle; Str, striatum. Scale bars, 100 μm; 50 μm in insets. Source data are shown in Supplementary Table 1.

Figure 2

Fezf2-induced genes identify native CSMN and label subsets of the broad CTIP2-positive population in layer V. (a) In situ hybridization showing expression of Adcyap1 in E13.5 cortical progenitors (left) and young postmitotic subcerebral neurons in developing cortical plate (middle and right). (b–d) Immunocytochemistry for CTIP2 combined with in situ hybridization for Adcyap1, Kif26a and Tmem163 (boxed area enlarged in panel to the right). Examples of double-positive cells (arrowheads) are indicated in the right column. CP, cortical plate; LGE, lateral ganglionic eminence; LV, lateral ventricle; Str, striatum. Scale bars, 100 μm (a; b–d left panels), 20 μm (b–d, middle panels), 10 μm (b–d, right panels). The complete gene list is given in Supplementary Table 3.

Figure 3

Genome-wide binding analysis for Fezf2 shows preferential association with proximal promoter regions of CSMN genes. (a) Fezf2 binding events preferentially occur in proximity to promoter regions, within 5 kb of the TSS of annotated genes. (b–i) Examples of 3xFlag-Fezf2 peaks at proximal promoters for early (Sox5 and Ctip2; b,c), middle (Crim1) (d) and late (Pcp4, Opn3, Diap3, S100a10 and Cdh22; e–i) CSMN genes. In situ hybridizations are shown for Crim1 (P21), Pcp4 (P21), Opn3 (P21), S100a10 (P21), Diap3 (P14) and Cdh22 (P21). Immunohistochemistry results are shown for SOX5 (E18.5) and CTIP2 (P1). Scale bars, 100 μm (b–i). (j) The “zero cross” area represents genes where the Cuffdiff2 test statistic is equal to 0 and no appropriate rank information is available. The color scales used for the rank density represent kernel density estimate of gene rank positions (white = 0, red/blue=max density). GSEA for CSMN and CPN signature gene sets. Signature gene sets (in red) and the corresponding subsets bound by Fezf2 (in blue) were assessed for enrichment in Fezf2-overexpressing neurospheres. Both CSMN signature genes and the subset bound by Fezf2 were significantly enriched in Fezf2-overexpressing neurospheres. Neither set of CPN signature genes showed significant enrichment. Source data are shown in Supplementary Table 4.

Figure 4

Fezf2 promotes glutamatergic and inhibits GABAergic neurotransmitter pathways. (a) ChIP-seq trace shows that 3xFlag-Fezf2 binds specifically to the promoters of Vglut1 (Slc17a7) and Gad1 but not Vglut2 (Slc17a6). (b) RNA-seq analysis shows the effect of 3xFlag-Fezf2 overexpression on these three genes in neural stem cells in vitro. (c) Inducible expression of single-copy Fezf2-IRES-GFP is show by immunoblotting and fluorescence microscopy. Uncropped original immunoblots are shown in Supplementary Figure 10. (d) Schematic representation of directed differentiation protocol used to instruct embryonic stem cell (ES) differentiation into mixed populations of cortical neurons. (e) RNA-seq analysis showing the effect of Fezf2 expression on Vglut1, Vglut2 and Gad1 in ES-derived neurons. Clones used for RNA-seq were from the differentiation of n = 2 independently generated iGFP and n = 2 iFIG lines. the error bars represent 95% confidence intervals for the Cuffdiff2 model expression estimate as defined in Methods and described in Trapnell et al., 2013.

Figure 5

Fezf2 controls Ephb1 selective expression in CSMN by direct association with the Ephb1 promoter. (a) In situ hybridization for Ephb1 in the forebrain at different stages of embryonic and postnatal development shows highest expression in developing cortical plate at E15.5, the time of initial CSMN axonal extension, and its restricted expression in developing layer V, until approximately E18.5. (b) In situ hybridization for Ephb1 and immunocytochemistry for TBR1 and APC show maintained expression of Ephb1 at postnatal stages in corticothalamic neurons (TBR1-positive in layer VI) and oligodendrocytes (APC-positive). (c) β-galactosidase immunocytochemistry in Ephb1 heterozygous mice at P1 shows that, within layer V, expression colocalizes with CTIP2, and not with SATB2 (arrows upper panel). The dotted rectangle area in the left panel is shown in high magnification in the four panels on the right. Retrograde labeling of ScPN from the pons of P1 Ephb1 heterozygous mice shows colocalization of FluoroGold with β-galactosidase in layer Vb (arrows). Retrograde labeling of CPN in P2 Ephb1 heterozygous mice shows no colocalization of FluoroGold with β-galactosidase in callosal neurons of layer Va (arrowheads). Confocal images of layer V were combined to produce three-dimensional reconstructions (Lower right panels). Sidebars represent projections along the x–z axes (right) and the y–z axes (below). (d) Left, in situ hybridization for Ephb1 on E18.5 wild-type (left inset) and Fezf2^−/−(right inset) littermates shows that Ephb1 levels specifically decrease in layer V of the mutant cortices. Right, DNA regions spanning 5′ UTR and first exon of the Ephb1 gene show enrichment of 3xFlag-Fezf2 binding (q = 10⁻¹⁵) compared to control. Str, striatum. Scale bars, 100 μm (a; c, far left panels), 20 μm (b), 50 μm (c, top right panels; d), 20 μm (c, bottom right panels).

Figure 6

Cortical neurons aberrantly project through the anterior commissure in absence of EphB1. (a–d) Immunocytochemistry for myelin basic protein (MBP) on coronal sections of P28 wild-type and Ephb1^−/− littermates shows internal capsule reduction and defasciculation, accompanied by an expansion of both the external capsule (arrows) and the anterior commissure (arrowheads). (e) DiI anterograde injections in deep layers of the somatosensory cortex of P2 wild-type (n = 3) and Ephb1^−/− (n = 3) pups show axons ectopically crossing at the anterior commissure and extending ventrally and rostrally (red arrows) in the Ephb1 mutants but not in wild-type animals. (f) Color-coded three-dimensional reconstructions based on HARDI of P28 wild-type and Ephb1^−/− littermates show distinct axon fibers originating in dorsal areas of the neocortex merging abnormally with the anterior commissure in the Ephb1^−/− (top right) compared to wild-type (top left) brains. Red represents the anterior–posterior direction; green represents the medial–lateral direction; blue represents the dorsal–ventral direction. Green arrows indicate axon tracts penetrating into the cortex. AC, anterior commissure; ACa, anterior part of the anterior commissure; ACp, posterior part of the anterior commissure; cc, corpus callosum; dCtx, dorsal cortex; lCtx, lateral cortex; EC, external capsule; IC, internal capsule. Scale bars: 50 μm (a), 20 μm (b–d) 100 μm (e).

Figure 7

Ephb1^−/− mice recapitulate the axon guidance phenotype observed in Fezf2 null mutants. (a–c) PLAP-positive axons inappropriately project through the anterior commissure in Ephb1^−/− mutants (b, arrows), mimicking the Fezf2^PLAP/PLAP axonal phenotype (c, arrows). Enlargement of the anterior commissure in Ephb1^−/− mice is accompanied by a reduction of PLAP-positive axons found in the cerebral peduncle (b, middle panel) and the cervical spinal cord (b, bottom), canonical targets of the corticospinal tract in wild-type mice (a, middle and bottom). No axons are found in the cerebral peduncle or spinal cord of the Fezf2^PLAP/PLAP mice (c, middle and bottom). AC, anterior commissure; Cp, cerebral peduncle; Hp, hippocampus; CST, corticospinal tract. C6, cervical vertebra. Scale bars: 100 μm (a–c, top panels), 50 μm (a–c, middle and bottom).