TBR1 directly represses Fezf2 to control the laminar origin and development of the corticospinal tract

Abstract

The corticospinal (CS) tract is involved in controlling discrete voluntary skilled movements in mammals. The CS tract arises exclusively from layer (L) 5 projection neurons of the cerebral cortex, and its formation requires L5 activity of Fezf2 (Fezl, Zfp312). How this L5-specific pattern of Fezf2 expression and CS axonal connectivity is established with such remarkable fidelity had remained elusive. Here we show that the transcription factor TBR1 directly binds the Fezf2 locus and represses its activity in L6 corticothalamic projection neurons to restrict the origin of the CS tract to L5. In Tbr1 null mutants, CS axons ectopically originate from L6 neurons in a Fezf2-dependent manner. Consistently, misexpression of Tbr1 in L5 CS neurons suppresses Fezf2 expression and effectively abolishes the CS tract. Taken together, our findings show that TBR1 is a direct transcriptional repressor of Fezf2 and a negative regulator of CS tract formation that restricts the laminar origin of CS axons specifically to L5.

Spatial specificity of axonal connections is one of the most important prerequisites for normal development (1–3). In mammals, this is especially crucial for axons of the corticospinal (CS) system (4–7). Development of the CS tract is an intricate process that involves the molecular specification of CS neurons and axon pathfinding. All long-range subcortical axons projecting to the brainstem and spinal cord, including those that form the CS tract, originate exclusively from layer (L) 5 projection (pyramidal) neurons of the cerebral cortex (8–11). Projection neurons in other cortical layers give rise to axons that project within the cortex (L2–4) or to the thalamus (L6). How this highly conserved laminar pattern of projections is formed with such perfect accuracy remains elusive.

Previous work revealed that the transcription factor FEZF2 (FEZL, ZFP312) is highly enriched in L5 CS neurons and is critical to the development of the CS tract (12–14). Inactivation of Fezf2 disrupts formation of the CS tract (12–14), whereas misexpression of Fezf2 in upper layer projection neurons induces ectopic subcortical projections (13). These findings indicate that Fezf2 transcription is tightly regulated during development, and that the integrity of normal Fezf2 expression is critical to proper development of the CS tract. Interestingly, Fezf2 is transiently expressed in L6 neurons during early embryonic development, where its transcription is directly repressed by SOX5, thereby establishing a high-in-L5, low-in-L6 postnatal pattern (15, 16). Paradoxically, in Sox5 null mutants, the number of axon projections reaching the brainstem and spinal cord is severely reduced despite increased cortical Fezf2 expression (15). This suggests that Sox5 is required for the formation of these connections independent of its regulation of Fezf2. Furthermore, SOX5 is normally expressed in L5 Fezf2-expressing CS neurons (15). Therefore, the down-regulation of Fezf2 in L6 neurons, and hence the establishment of Fezf2's L5 pattern, likely requires additional molecules. Recently, Bedogni et al. (17) showed that TBR1 exerts positive and negative control over a number of genetic markers of regional (areal) and laminar identity. Notably, Tbr1 null mutants exhibit an increase in Fezf2 expression concomitant with a decrease in Sox5 expression. Those findings, along with the observation that Tbr1 null mutants exhibit axon projection defects with some similarities to those of the Sox5 null phenotype (17, 18), led to the hypothesis that Tbr1 regulates Fezf2 expression indirectly via Sox5.

In the present study, we tested the alternative hypothesis that Tbr1 directly represses Fezf2 transcription and thereby controls the laminar origin and development of the CS tract. We found that TBR1 binds directly to a conserved regulatory element near the Fezf2 gene and selectively represses its activity in L6 corticothalamic projection neurons. Moreover, in Tbr1 null mutants, CS axons originate ectopically from L6 neurons in a Fezf2-dependent manner. Consistent with this, TBR1 was absent from Fezf2-expressing L5 CS tract neurons during development, and the forced expression of Tbr1 in these neurons suppressed Fezf2 and blocked CS tract formation. Taken together, our findings show that TBR1 is a direct transcriptional repressor of Fezf2 that blocks the formation of the CS tract from L6, thereby restricting the origin of CS axons specifically to L5.

Results

TBR1 Binds Directly to a Consensus Site Near Fezf2.

Using microarrays to analyze changes in the Tbr1 null cortical transcriptome, Bedogni et al. (17) found a significant increase in Fezf2 expression, and suggested that this up-regulation is a result of decreased Sox5 expression. Our independent analyses using mRNA-Seq (Fig. 1A), quantitative RT-PCR (qRT-PCR; Fig. 2A), and in situ hybridization (ISH; Fig. 2B) confirmed a significant up-regulation of Fezf2 in Tbr1^−/− neocortex. However, analysis by immunostaining and immunoblotting revealed that SOX5 protein levels were relatively unaltered in late embryonic and neonatal Tbr1^−/− cortex (Fig. S1). This led us to the hypothesis that TBR1 regulates Fezf2 transcription via direct binding to regulatory sequences near Fezf2. To identify genome-wide TBR1 binding sites in an unbiased and hypothesis-independent manner, we analyzed TBR1-immunoprecipitated chromatin using deep sequencing (ChIP-Seq; Fig. 1B). We tested several available anti-TBR1 antibodies and found that none was suitable for immunoprecipitating chromatin of sufficient quality for ChIP-Seq. Thus, we generated a V5-TBR1 fusion construct and expressed it in N2A cells. V5-TBR1 was immunoprecipitated using an anti-V5 antibody. DNA-Seq was performed on the Illumina GAIIx platform, and data were analyzed as described in SI Materials and Methods. A total of 34.5 million reads of 36 bp were generated for V5-precipitated chromatin and 37 million reads were generated for control input DNA, of which 82.9% and 85.1%, respectively, uniquely mapped to the genome. Of immunoenriched regions with a false discovery rate cutoff of 0.02, approximately 62% were mapped within gene loci, and an additional 28% were mapped within 50 kb upstream of transcription start sites (TSSs) or 50 kb downstream of the polyadenylation signal.

Fig. 1.

TBR1 regulates Fezf2 transcription via direct binding and repression of regulatory elements near Fezf2. (A) RNA-Seq of mRNA extracted from neocortices of P0 Tbr1^−/− and Tbr1^+/+ littermates (n = 2 per genotype). Significantly more RNA-Seq reads mapped to Fezf2 exons (gray) in Tbr1^−/− (green; 727 reads) than in Tbr1^+/+ (red, 496 reads; adjusted P value = 1.49E-9). (B) N2a cells were transfected with V5-TBR1, and chromatin was immunoprecipated using an anti-V5 antibody. ChIP-Seq revealed enrichment of mapped reads (blue) compared with input DNA in a region 4.5 kb downstream of the Fezf2 TSS. (C) Schematic of the mouse Fezf2 locus (gray) and mammalian conservation (dark blue) showing the location of the TBR1 consensus binding site (red) defined by ChIP-Seq (detailed in Fig. S2). (D) ChIP–qRT-PCR assays in neurons cultured from E13.5 neocortex transfected with V5-TBR1. DNA precipitated with control rabbit IgG or anti-V5 antibody was analyzed by qRT-PCR using primers flanking the TBR1 binding site and control primers. The TBR1 site was significantly enriched in V5-precipitated DNA. (E) N2A cells were transfected with luciferase plasmids containing the TBR1 binding site. Luciferase assay showed that cotransfection of CAG-Tbr1, but not of empty CAG, repressed the activity of this element. *P < 0.02; **P < 0.005 by unpaired two-tailed t test. ns, not significant (P > 0.05).

Fig. 2.

High Fezf2 expression is aberrantly retained in neonatal L6 neurons in Tbr1^−/− neocortex. (A) qRT-PCR of P0 neocortical mRNA. Normalized to Gapdh, Fezf2 expression levels increased by 1.77 ± 0.08-fold in Tbr1^−/− compared with Tbr1^+/+. (B) Fezf2 ISH of P0 brains showed increased Fezf2 expression in Tbr1^−/− neocortex. (Scale bar: 500 μm.) (C) P0 neocortices of Tbr1^+/+ and Tbr1^−/− littermates transgenic for Fezf2-Gfp. Fezf2-Gfp–expressing neurons were dispersed in the Tbr1^−/−, consistent with previously reported defects in neuronal migration (18). The proportion of neurons expressing Fezf2-Gfp significantly increased from 21.8% in the Tbr1^+/+ to 33.3% in the Tbr1^−/− (1.53 ± 0.27-fold). (Scale bar: 50 μm.) (D) L6 neurons (arrowhead) were stringently defined by expression of the L6 corticothalamic neuron marker ZFPM2 (red) and a lack of incorporation of CldU (blue), multiple doses of which were injected daily from E12.5 to E14.5, when L5 neurons were generated. The number of L6 neurons expressing Fezf2-Gfp increased significantly from 18.5% in the Tbr1^+/+ to 85.9% in the Tbr1^−/− (4.64 ± 0.20-fold). (Scale bar: 10 μm.) *P < 0.01; **P < 0.00005 by unpaired two-tailed t test.

To identify putative TBR1 consensus binding sequences with potential relevance to TBR1 transcriptional activity in the neocortex, we cross-analyzed our mRNA-Seq and ChIP-Seq data. We selected the loci of significantly differentially expressed genes based on mRNA-Seq data to which a significant enrichment of TBR1 ChIP-Seq reads mapped proximally. Analysis of these 181 loci (detailed in SI Material and Methods) revealed a binding sequence motif (Fig. S2) with similarities to the T element in Xenopus (19, 20). Importantly, an enrichment of TBR1 ChIP-Seq reads compared with input DNA was mapped to a highly conserved region ∼4.5 kbp downstream of the Fezf2 TSS. This region contained a core consensus binding site conserved across placental mammalian species (Fig. 1C and Fig. S2C). To confirm this binding in the neocortex, we performed ChIP using cultured embryonic day (E) 13.5 neocortical neurons transfected with V5-TBR1. Analysis of ChIPed DNA by qRT-PCR revealed a significant enrichment of this binding site in DNA precipitated by V5 antibody compared with control IgG (a 6.65 ± 0.37-fold increase; P = 0.00128) (Fig. 1D), confirming that TBR1 binds this region in neocortical neurons. To assess whether this binding affects transcription, we cloned the binding site downstream of the luciferase reporter gene and assayed transfected N2a cells. Forced expression of Tbr1 moderately but significantly repressed the activity of this binding site (a decrease of 31.9% ± 8.6%; P = 0.0105) (Fig. 1E). Collectively, these findings suggest that TBR1 negatively regulates Fezf2 transcription through direct binding to a conserved consensus element.

L6 Neurons Aberrantly Retain High Fezf2 Expression in Tbr1 Null Mutants.

The global increase in Fezf2 expression in the early postnatal Tbr1^−/− neocortex found in the present study and a previous study (17) could result from either up-regulation of Fezf2 in neurons that normally express Fezf2 or ectopic expression of Fezf2 in neurons that do not normally express Fezf2. Consistent with the latter possibility, analysis of TBR1 expression during cortical development revealed that TBR1 and Fezf2 established complementary laminar expression patterns during late embryogenesis (Fig. S3A). To directly assess how TBR1 alters Fezf2 expression at the level of resolution of individual neurons, we bred Tbr1^+/− mice with Fezf2-Gfp transgenic mice. In the postnatal day (P) 0 Tbr1^−/− neocortex, the number of neurons highly expressing Fezf2-Gfp, which did not migrate normally (18), increased significantly from 21.8% in Tbr1^+/+ to 33.3% in Tbr1^−/− (P = 0.0058) (Fig. 2C). This significant increase in the total number of Fezf2-Gfp–expressing neurons in Tbr1^−/− neocortex reflects an aberrant induction of high Fezf2 expression in neurons that do not normally express Fezf2.

Because TBR1 is specifically expressed in L6 neurons at P0, we examined whether L6 neurons aberrantly turned on high Fezf2 expression in the Tbr1^−/− neocortex. We used two stringent criteria to conservatively define L6 neurons in the Tbr1^−/− neocortex, in which the demarcation of distinct layers is complicated by migration defects (ref. 18 and Fig. S4). First, we chose ZFPM2 (FOG2) to molecularly define L6 neurons, because it is a reliable marker of L6 corticothalamic neurons in the early postnatal neocortex (ref. 15 and Fig. S5) and, more importantly, because its expression level is unchanged in Tbr1^−/− neocortex (Fig. S6, mRNA-Seq; P = 0.642 and ref. 17). Second, we used a stringent birth-dating strategy to distinguish L6 neurons from L5 neurons. In the sequential generation of cortical neurons, the same progenitor cells that give rise to L6 neurons subsequently give rise to L5 neurons. Therefore, positive thymidine analog incorporation of L6 neurons will inevitably label a large number of L5 neurons. To circumvent this, we used exclusion of the thymidine analog CldU to distinguish L6 and L5 neurons. CldU was administered to timed-pregnant Tbr1^+/− females daily starting at E12.5, when the first L5 neurons are born, and ending at E14.5, when the last L5 neurons are generated. Using this narrow definition of L6 neurons (ZFPM2⁺, CldU⁻), we observed a significant increase in the number of L6 neurons expressing Fezf2-Gfp (18.5% in Tbr1^+/+ and 85.9% in Tbr1^−/−; P = 0.000031) (Fig. 2D). Thus, in the absence of Tbr1, neurons born at the correct time and with the molecular properties of L6 neurons ectopically expressed high levels of Fezf2.

Ectopic L6 Projections to the Brainstem Are Revealed by Retrograde Tracing in Tbr1 Null Mutants.

Subcortical axon defects have been reported in Tbr1^−/− brains (18). Long-range subcerebral axons and the layer-specific pattern of cortical connectivity have not been specifically examined, however. To test whether aberrantly high expression of Fezf2 in L6 neurons leads to the formation of ectopic projections to the brainstem, we bred Tbr1^+/− mice with Golli-Gfp transgenic mice, which express GFP predominantly in L6 and SP neurons and their axons (21), and with Fezf2-Gfp transgenic mice. In E14.5 normal embryos, Golli-Gfp axons en route to the thalamus entered the upper portion of the striatum, but not the lower portion (Fig. 3A). In E14.5 Tbr1^−/− brains, Golli-Gfp axons exhibited premature overgrowth, ectopically invading the lower portion of the striatum and the primitive internal capsule. At P0, numerous Golli-Gfp axons reached the dorsal thalamus in Tbr1^+/+ (Fig. 3B, solid arrowhead). In the P0 Tbr1^−/− brains, none of the Golli-Gfp axons entered the dorsal thalamus, but many were aberrantly directed toward other subcortical targets, including the hypothalamus (Fig. 3B, arrow) and the midbrain. Similarly, exuberant Fezf-Gfp axons in E16.5 and P0 Tbr1^−/− brains (Fig. 3C, open arrowheads and Fig. S7), ectopically innervated the cerebral peduncles (CPs; Fig. 3D, broken line) and brainstem, indicating that the prematurely outgrown and misguided L6 axons ectopically expressed high levels of Fezf2-Gfp.

Fig. 3.

Premature overgrowth and superabundance of subcortical axons in the brainstem of Tbr1^−/− mice. (A) In E14.5 Tbr1^+/+, Golli-Gfp⁺ axons originating mainly from L6 neurons extended to the upper portion of the striatum (Str; above the dashed line). In Tbr1^−/−, numerous Golli-Gfp⁺ axons extended to the deeper portion of the striatum (below the dashed line) toward the internal capsule. (B) In P0 Tbr1^−/−, Golli-Gfp⁺ axons were not present in the dorsal thalamus (Th), indicating that the normal corticothalamic tract (solid arrowhead) had not formed. Ectopic axons (arrow) were misdirected toward other subcortical targets, including the hypothalamus (Hyp) and the midbrain. (C) In E16.5 Tbr1^+/+ brain, Fezf2-Gfp⁺ axons (solid arrowheads) did not cross the forebrain/midbrain boundary (dashed line). In Tbr1^−/−, numerous Fezf2-Gfp⁺ axons (open arrowheads) extended subcerebrally to invade the midbrain (MB). (D) In E16.5 Tbr1^+/+ brain, only a few Fezf2-Gfp⁺ axons innervated the CPs (dashed lines). Dramatically more Fezf2-Gfp⁺ axons entered the CPs in Tbr1^−/−. (Scale bars: 100 μm.)

Normal L6 projection neurons project axons only as far as the thalamus, never reaching the brainstem (8). Our data are consistent with the intriguing possibility that L6 neurons in the Tbr1^−/− aberrantly project axons to the brainstem due to increased Fezf2 expression. Although the Golli-Gfp transgene is a useful marker of L6 neurons, a small number of neurons near the L5/L6 border of the cingulate cortex express Golli-Gfp and project axons to the brainstem (15). Therefore, the presence of exuberant Golli-Gfp axons in the Tbr1^−/− brainstem does not unequivocally indicate that the axons originated from L6 neurons. Thus, we performed retrograde axonal tracing by injecting rhodamine-conjugated latex microspheres (LMS) into the pons of Tbr1^+/+ and Tbr1^−/− newborn mice. Analysis was carried out after 18–24 h of survival to allow for transport of the tracer, a time frame that is limited by the perinatal lethality of the Tbr1^−/− (22). In addition to the highly stringent criteria described above for identifying L6 neurons using ZFPM2 immunostaining and exclusion of E12.5–E14.5 CldU incorporation, we further narrowed our definition using Golli-Gfp, which is mostly restricted to L6 neurons in the somatosensory cortical areas that we analyzed.

In the Tbr1^+/+, projection neurons labeled retrogradely from the pons were restricted to L5, and most incorporated CldU and expressed neither Golli-Gfp nor ZFPM2 (Fig. 4B, open arrowheads). A limited number of labeled neurons were positive for Golli-Gfp or lacked CldU, but none expressed ZFPM2. Most importantly, as defined by the highly conservative triple criteria (Golli-Gfp⁺, ZFPM2⁺, and CldU⁻), no L6 neurons were labeled by the retrograde tracer in any of the traced Tbr1^+/+ brains (n = 8). In contrast, defined by the same stringent criteria, a considerable number of L6 neurons in the Tbr1^−/− somatosensory cortex were clearly labeled by the retrograde tracer, which encircled neuronal nuclei and entered apical dendrites (Fig. 4C, solid arrowheads; n = 4 brains). Similar to reported retrograde tracing from the CPs of the Tbr1^−/− (18), retrograde tracing from the pons was absent from the most medial cortical areas. Thus, the somatosensory cortex was consistently used for quantification, which revealed a significant increase in the number of labeled L6 neurons in the Tbr1^−/− for each of the three criteria used (Golli-Gfp⁺, P = 0.000009; ZFPM2⁺, P = 0.000007; CldU⁻, P = 0.000049) (Fig. 4D). Notably, the numbers of retrogradely labeled Golli-Gfp⁻ and LSM⁺ neurons were unaltered in the Tbr1^−/− somatosensory cortex (Fig. 4E), suggesting that L5 projections to the pons were mostly normal. We also used nuclear size to confirm the identity of the CS neurons projecting to the brainstem and spinal cord. Normal L5 CS neurons (LMS⁺, ZFPM2⁻, and Golli-Gfp⁻) have larger nuclei compared with L6 corticothalamic neurons (LMS⁻, ZFPM2⁺, and Golli-Gfp⁺; P = 2.25E-17) (Fig. 4F). Analysis of retrogradely traced L6 neurons (LMS⁺, ZFPM2⁺, and Golli-Gfp⁺) in the Tbr1^−/− revealed a nuclear diameter indistinguishable from that of normal L6 corticothalamic neurons (P = 0.168), but significantly smaller than that of L5 CS neurons (P = 2.11E-13). These data indicate that in the Tbr1^−/− neocortex, L6 neurons, as stringently defined by marker expression, birth-dating, and nuclear size, project axons ectopically to the brainstem instead of to the thalamus and express high levels of Fezf2, key features of normal L5 neurons.

Fig. 4.

Stringently defined L6 neurons aberrantly formed subcerebral/CS projections in Tbr1^−/−. (A) Schematic of the experimental design. CldU was administered daily between E12.5 and E14.5 to label L5 neurons in timed-pregnant Tbr1^+/− females transgenic for Golli-Gfp. Retrograde tracer (LMS) was injected into the pons of live neonates. L6 neurons were narrowly defined by lack of CldU incorporation and coexpression of Golli-Gfp and ZFPM2. (B and C) Confocal images of the somatosensory cortex were obtained at an optical thickness of 3 μm. (B) In Tbr1^+/+, all retrogradely labeled neurons were positioned in L5, and most were Golli-Gfp⁻ and ZFPM2⁻ and incorporated CldU (open arrowhead). No neurons meeting our stringent definition of L6 neurons were retrogradely traced in any of the Tbr1^+/+ brains analyzed (n = 8). (Scale bar: 50 μm.) (C) In Tbr1^−/−, a significant number of L6 neurons meeting our criteria were labeled retrogradely (solid arrowhead; n = 3 brains). Ectopic CS projections of these L6 neurons indicate a projectional fate switch. (Scale bar: 50 μm). (D and E). Quantitative analysis revealed that the number of traced L6 neurons increased significantly in Tbr1^−/− compared with Tbr1^+/+ for each of the criteria used. However, no difference in the number of traced Golli-GFP⁻ L5 neurons was detected. (F) Analysis of nucleus diameter. In Tbr1^+/+ neocortex, L5 CS neurons (LMS⁺) were significantly larger in nuclear diameter compared with L6 corticothalamic neurons (CTh; ZFPM2⁺ and Golli-Gfp⁺). In Tbr1^−/−, nuclei of L6 CS neurons (LMS⁺, ZFPM2⁺, and Golli-Gfp⁺) were indistinguishable in size from Tbr1^+/+ L6 CTh neurons but significantly smaller than Tbr1^+/+ L5 CS neurons. *P < 0.00005 by unpaired two-tailed t test. ns, not significant (P > 0.05).

Ectopic Tbr1 Expression in L5 Neurons Effectively Abolishes Fezf2 Expression and CS Tract Formation.

TBR1 is normally excluded from L5 Fezf2-expressing CS neurons during embryonic and postnatal development (Fig. S3 A and B). To investigate whether forced misexpression of Tbr1 in L5 neurons is able to down-regulate Fezf2 and suppress CS projections, we performed in utero electroporation at E13.5 to transfect L5 neurons with CAG-Tbr1 or control empty CAG plasmid DNA, together with CAG-Rfp to label neurons generated by electroporated progenitor cells. To analyze Fezf2 expression in transfected cells, we carried out these experiments in Fezf2-Gfp transgenic mice. The vast majority of L5 RFP⁺ neurons electroporated with empty CAG plasmid expressed high levels of Fezf2-Gfp (85.3% ± 4.3%) (Fig. 5 A and B). Remarkably, when Tbr1 was misexpressed, the number of L5 RFP⁺ neurons expressing high levels of Fezf2-Gfp decreased dramatically, to 10.8% ± 4.2% (P = 0.000003) (Fig. 5 A and B). This indicates that Tbr1 misexpression in L5 neurons is sufficient to suppress Fezf2 expression.

Fig. 5.

TBR1 misexpression in L5 neurons abolished Fezf2 expression and ablated CS axons. (A and B) CAG-Tbr1 or empty CAG plasmid DNA was delivered together with CAG-Rfp into E13.5 embryos expressing Fezf2-Gfp using in utero electroporation. RFP-expressing L5 neurons were examined at P0. Of the L5 neurons electroporated with empty CAG plasmid, 85.3% ± 4.3% expressed Fezf2-Gfp. In contrast, only 10.8% ± 4.2% of the Tbr1-misexpressing L5 neurons expressed Fezf2-Gfp, a significant reduction compared with empty CAG. P = 0.000003. (C–F) CAG-Tbr1 or CAG-empty plasmid DNA was coelectroporated with CAG-Gfp into WT E13.5 embryos. Brains were collected for analysis at P0. (C) The majority of neurons expressing CAG-Tbr1 migrated normally to settle in all cortical layers, including L5. In all examined brains that overexpressed Tbr1, ectopic neuropils and cells were clustered in the white matter (*); however, most axons entered the white matter and innervated the corpus callosum (D). Neurons electroporated with empty CAG plasmid projected numerous subcerebral/CS axons into the ventral pons (empty arrowhead) and medullary pyramids. Misexpression of Tbr1 ablated all CS axons. A few GFP⁺ axons reached the ventral pons (E), but none reached the medullary pyramids (F). This indicates that misexpression of TBR1 in L5 neurons substantially down-regulated Fezf2-Gfp and blocked formation of the CS tract. (Scale bars: 20 μm in A; 200 μm in C–E; 50 μm in F.)

To investigate whether this repressed Fezf2 expression functionally affects the axonal projections of L5 CS neurons, we performed in utero electroporation to transfect L5 neurons with CAG-Tbr1 or empty CAG plasmid DNA, together with CAG-Gfp, because the higher solubility of GFP better facilitates the analysis of long-distance CS axons. At E13.5, neurons electroporated with empty CAG plasmid projected corticocortical axons into the corpus callosum (Fig. 5D) and projected numerous CS axons into the ventral pons and medullary pyramids (Fig. 5 E and F). In contrast, GFP⁺ axons originating from L5 neurons misexpressing Tbr1 were almost completely absent from the pons and pyramids. This disruption of axons was specific to the CS tract, however. Tbr1-misexpressing neurons projected abundant GFP⁺ axons into the corpus callosum (Fig. 5D). Together, these findings demonstrate that the misexpression of Tbr1 is sufficient to suppress Fezf2 expression in L5 neurons and effectively abolish formation of the CS tract.

Induction of Ectopic L6 CS Axons in the Tbr1 Null Mutants Is Dependent on Fezf2.

To examine whether the induction of ectopic axons originating from L6 neurons in the Tbr1^−/− cortex depends on up-regulation of Fezf2, we engineered a conditional Fezf2 allele (Fig. S8 A and B) for the generation of a cortex-specific Tbr1;Fezf2 double-null mutant. Fezf2^flox/flox mice were crossed with Emx1-Cre PAC transgenic mice (23) to specifically inactivate Fezf2 in the cortex (Fig. S8 A and B). To confirm that our cortex-specific conditional Fezf2 null mutant was phenotypically similar to germ-line Fezf2^−/− mice (12, 14), we crossed Emx1-Cre PAC; Fezf2^flox/flox mice with CAG-Cat-Gfp transgenic mice (24) to reveal the pancortical pattern of axonal connectivity. Similar to germ-line Fezf2^−/−, the CS tract was nearly completely absent from the brainstem (Fig. S8 C–E). For analysis of L6 neurons, we bred in the Golli-Gfp transgene to generate cortex-specific Tbr1;Fezf2 double-null mutants (Tbr1^−/−, Fezf2^fl/fl, Emx1-Cre, and Golli-Gfp). LMS was injected in the pons of Golli-Gfp double-null mice and littermate controls at P0. Remarkably, neither L5 nor L6 projection neurons were retrogradely labeled from the pons of the double-null mutants (Fig. 6). Collectively, these data indicate that cortical Fezf2 activity is required for the formation of ectopic L6 projections to the pons, including the CS system axons in Tbr1 null mutants, and that Tbr1 restricts the laminar origin of the CS tract by repressing Fezf2 in L6 neurons in normal mice.

Fig. 6.

Aberrant L6 projections to the brainstem in the Tbr1^−/− require Fezf2. Cortex-specific double deletion of Fezf2 and Tbr1 was achieved using the Emx1-Cre transgene. LMS was injected into the pons of neonatal mice of the indicated genotype. (A) In Tbr1^+/+ mice, retrogradely labeled neurons were positioned in L5 and did not express Golli-Gfp or ZFPM2 (open arrowheads). (B) In Tbr1^−/− mice, many L6 neurons expressing Golli-Gfp and ZFPM2 were retrogradely labeled by LMS (solid arrowheads). (C) In Tbr1^−/−;Fezf2^fl/fl;Emx1-Cre cortex-specific double mutant, no Golli-Gfp⁺ or ZFPM2⁺ L6 neurons were retrogradely labeled. These data indicate a partial rescue of the Tbr1^−/− phenotype in the absence of cortical Fezf2 and suggest that the L6 CS projections of Tbr1^−/− depend on cortical Fezf2. (Scale bars: 200 μm.)

Discussion

We have shown that TBR1 directly binds a conserved consensus site near the Fezf2 locus and represses Fezf2 expression in L6 neocortical projection neurons. In the absence of Tbr1, postnatal L6 neurons aberrantly express high levels of Fezf2 and ectopically project to the brainstem. These ectopic L6 projections to the brainstem, including the CS axons, are Fezf2-dependent. Moreover, TBR1 misexpression is sufficient to repress Fezf2 in L5 neurons and block the formation of the CS tract. These findings (summarized in Fig. S9) demonstrate that TBR1 is a direct transcriptional repressor of Fezf2 and a negative regulator of the CS tract that restricts the tract's laminar origin specifically to L5.

Our data confirm and extend those of previous studies showing the role of Tbr1 in the formation of subcortical projections (17, 18). We show here that TBR1 directly represses Fezf2 and TBR1 overexpression in L5 neurons abolishes CS projections. However, given that L5 CS neurons normally extend collateral axon branches to the dorsal thalamus (25), exogenous expression of TBR1 in these neurons cannot be used to address whether TBR1 is sufficient to confer a corticothalamic projection fate. Notably, the upper-layer neurons, which do not project to the thalamus, normally express TBR1 starting at an early postnatal age. Therefore, it is likely that TBR1, although necessary for normal corticothalamic projections (17, 18), is not sufficient to induce this projection fate.

Using the Fezf2-Gfp transgene to reveal the global pattern of corticofugal connections, we found that at P0, the Tbr1^−/− CS tract is largely of normal size compared with controls (Fig. S7). The additional axons projecting to the brainstem from L6 neurons might be expected to increase the size of this tract in the Tbr1^−/−; however, this increase is likely offset by the role of Tbr1 in areal patterning (17, 18), in which it is required by neurons in the medial cortex to project subcortical axons. Taken together, these findings indicate that TBR1 is a critical determinant of multiple aspects of cortical development, including neuronal migration, laminar and areal identity, and axonal projection.

The dynamic pattern of Fezf2 exemplifies how precise temporal and spatial regulation of gene expression has functional consequences in neocortical development and axonal connections. Furthermore, the exclusively postmitotic expression pattern of Tbr1 (17, 18) indicates that layer-specific molecular and projectional identities are not firmly established during neurogenesis, but undergo postmitotic refinement to establish their mature patterns. This fine-tuning of neuronal identity and connectivity provides a mechanism by which the precise configuration of neural circuits can be modified after neurons integrate into the cortex.

TBR1 is not the only molecule that postmitotically controls the dynamic pattern of Fezf2 expression. Another transcription factor, SOX5, contributes to this process as well (15, 16). In contrast to the Tbr1 null mutant, in the Sox5 null mutants projections to the brainstem are nearly completely abolished (15), indicating that Sox5 is independently required for the formation of CS tract. Moreover, SOX5 is normally expressed in Fezf2-expressing L5 CS neurons (15). These results and our analysis of TBR1 binding sites by ChIP-Seq and ChIP–qRT-PCR are consistent with the hypothesis that TBR1 regulation of Fezf2 transcription is direct rather than secondary to changes in SOX5 levels, as was previously suggested. Collectively, these data indicate that both SOX5 and TBR1 are required in the regulation of Fezf2 transcription. Whether the two transcription factors physically interact or cooperate in alternative ways in this role remain unknown, however.

All projections to the brainstem, including the CS system, originate from L5 neurons in all mammalian species examined (4–6). The high degree of conservation indicates that this specific pattern of connectivity is likely critical to the survival and evolutionary success of mammals. It also suggests that the regulatory pathways that establish this pattern are well conserved. Consistent with this, the prenatal neocortical expression patterns of Fezf2, Tbr1, and Sox5 are similar in different mammals, including humans (15, 26, 27). Therefore, TBR1-mediated down-regulation of Fezf2 in L6 is most likely a conserved mechanism for restricting the laminar origin of the CS tract to L5.

Materials and Methods

Animals.

All experiments were carried out in accordance with a protocol approved by Yale University's Committee on Animal Research. Tbr1 mice were a generous gift from John Rubenstein (22). Fezf2-Gfp mice were obtained from GENSAT (28). Golli-Gfp, Emx1-Cre PAC, and CAG-Cat-Gfp mice were generous gifts from Anthony Campagnoni (21), Takuji Iwasato (23) and Melissa Colbert (24), respectively.

Further experimental details can be found in SI Materials and Methods.

Note Added in Proof.

After the approval of this paper for publication, similar findings by McKenna et al. (29) showed that Tbr1 and Fezf2 regulate the alternate corticofugal identities of projection neurons.