Limits...
Cis-regulatory control of corticospinal system development and evolution.

Shim S, Kwan KY, Li M, Lefebvre V, Sestan N - Nature (2012)

Bottom Line: Here we identify a conserved non-exonic element (E4) that acts as a cortex-specific enhancer for the nearby gene Fezf2 (also known as Fezl and Zfp312), which is required for the specification of corticospinal neuron identity and connectivity.We find that SOX4 and SOX11 functionally compete with the repressor SOX5 in the transactivation of E4.These findings reveal that SOX transcription factors converge onto a cis-acting element of Fezf2 and form critical components of a regulatory network controlling the identity and connectivity of corticospinal neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

ABSTRACT
The co-emergence of a six-layered cerebral neocortex and its corticospinal output system is one of the evolutionary hallmarks of mammals. However, the genetic programs that underlie their development and evolution remain poorly understood. Here we identify a conserved non-exonic element (E4) that acts as a cortex-specific enhancer for the nearby gene Fezf2 (also known as Fezl and Zfp312), which is required for the specification of corticospinal neuron identity and connectivity. We find that SOX4 and SOX11 functionally compete with the repressor SOX5 in the transactivation of E4. Cortex-specific double deletion of Sox4 and Sox11 leads to the loss of Fezf2 expression, failed specification of corticospinal neurons and, independent of Fezf2, a reeler-like inversion of layers. We show evidence supporting the emergence of functional SOX-binding sites in E4 during tetrapod evolution, and their subsequent stabilization in mammals and possibly amniotes. These findings reveal that SOX transcription factors converge onto a cis-acting element of Fezf2 and form critical components of a regulatory network controlling the identity and connectivity of corticospinal neurons.

Show MeSH

Related in: MedlinePlus

Sox4 and Sox11 are required for Fezf2 expression and CS tract formationa, The requirement of SOX4 and SOX11 for E4 transactivation was determined in neurons cultured from E14.5 heterozygous littermate control and Sox4;Sox11 cdKO mice and transfected with the E4 luciferase construct. The activity of E4 in double knockout neurons was not significantly above background (1.1 fold. b, Analysis of neocortical Fezf2 expression by qRT-PCR in cortex-specific Sox4 and/or Sox11 mutants. Normalized to Gapdh, Fezf2 mRNA levels were dramatically reduced in cdKO but not Sox4 or Sox11 mutants. c-d, Sagittal (top row) and coronal (bottom row) sections of the control and cdKO pons immunostained for L1CAM (red) and PRKCG (green). The dramatic loss of L1CAM and PRKCG-positive CS tract axons in the cdKO (arrowhead and dashed outline) is similar to that of the cortex-specific Fezf2 mutant. Errors bars represent s.e.m. One-tailed Student’s t-test; NS=Not significant, **P<0.01, ***P<0.001. n = 4 per genotype. Scale bars represent 200 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375921&req=5

Figure 4: Sox4 and Sox11 are required for Fezf2 expression and CS tract formationa, The requirement of SOX4 and SOX11 for E4 transactivation was determined in neurons cultured from E14.5 heterozygous littermate control and Sox4;Sox11 cdKO mice and transfected with the E4 luciferase construct. The activity of E4 in double knockout neurons was not significantly above background (1.1 fold. b, Analysis of neocortical Fezf2 expression by qRT-PCR in cortex-specific Sox4 and/or Sox11 mutants. Normalized to Gapdh, Fezf2 mRNA levels were dramatically reduced in cdKO but not Sox4 or Sox11 mutants. c-d, Sagittal (top row) and coronal (bottom row) sections of the control and cdKO pons immunostained for L1CAM (red) and PRKCG (green). The dramatic loss of L1CAM and PRKCG-positive CS tract axons in the cdKO (arrowhead and dashed outline) is similar to that of the cortex-specific Fezf2 mutant. Errors bars represent s.e.m. One-tailed Student’s t-test; NS=Not significant, **P<0.01, ***P<0.001. n = 4 per genotype. Scale bars represent 200 µm.

Mentions: To assess the requirement of Sox4 and Sox11 in E4-mediated activity, we transfected primary cortical neurons cultured from heterozygous littermate control or Sox4; Sox11 cdKO embryos with control or E4-containing luciferase constructs (Fig. 4a). In control neurons, the presence of E4 increased luciferase activity by 2.5±0.3 fold (P=1.9×10−4, one-tailed Student’s t-test). This increase was abolished in neurons culture from Sox4; Sox11 cdKO (1.1±0.1 fold; P=0.866), indicating that the two SOXC TFs are major activators of the enhancer in cortical neurons. Next, using qRT-PCR, we found a significant reduction in neocortical Fezf2 expression in cdKO compared to control or single cKO littermates at postnatal day 0 (P0) (Fig. 4b). In addition, immunostaining for L1CAM and PRKCG revealed a dramatic loss of CS axons in Sox4; Sox11 cdKO mice but not single cKO littermates at P0 and P6 (Fig. 4c, d, Supplementary Fig. 8), whereas the organization of other brainstem tracts was not affected (Fig. 3c, d). Because the absence of L1CAM and PRKCG staining could reflect their down-regulated expression in the cdKO, as opposed to loss of CS axons, we confirmed the absence of the CS tract using the CRE-responsive CAG-Cat-Gfp transgenic line to express GFP in all cortical projection neurons and their axons39,40 (Supplementary Fig. 9). Two additional observations indicate that the loss of Fezf2 and CS axons in these mice was not due to an absence of L5 neurons. First, only a moderate increase in cell death was detected in the motor-sensory areas, from which CS axons originate (Supplementary Fig. 10). Second, many BCL11B-immunopositive L5 neurons were present in the cdKO motor-sensory areas, albeit lightly immunostained (Supplementary Fig. 11), indicating that neurons that would normally give rise to the CS tract were present. Analysis of additional laminar markers further revealed an inversion of cortical layers similar to the reeler mutant mouse10–12 (Supplementary Fig. 11), indicating that Sox4 and Sox11 are also required for proper laminar positioning of neurons. Previous studies have shown that the CS tract is present in reeler mice12, indicating that this laminar phenotype occurs independently of Fezf2 and probably in response to defects in the RELN signaling pathway13,14. In support of this possibility, RELN was absent from cdKO Ncx (Supplementary Fig. 12). Thus, the combined deletion of Sox4 and Sox11 leads to defects in laminar position of neurons and molecular specification and connectivity of CS neurons.


Cis-regulatory control of corticospinal system development and evolution.

Shim S, Kwan KY, Li M, Lefebvre V, Sestan N - Nature (2012)

Sox4 and Sox11 are required for Fezf2 expression and CS tract formationa, The requirement of SOX4 and SOX11 for E4 transactivation was determined in neurons cultured from E14.5 heterozygous littermate control and Sox4;Sox11 cdKO mice and transfected with the E4 luciferase construct. The activity of E4 in double knockout neurons was not significantly above background (1.1 fold. b, Analysis of neocortical Fezf2 expression by qRT-PCR in cortex-specific Sox4 and/or Sox11 mutants. Normalized to Gapdh, Fezf2 mRNA levels were dramatically reduced in cdKO but not Sox4 or Sox11 mutants. c-d, Sagittal (top row) and coronal (bottom row) sections of the control and cdKO pons immunostained for L1CAM (red) and PRKCG (green). The dramatic loss of L1CAM and PRKCG-positive CS tract axons in the cdKO (arrowhead and dashed outline) is similar to that of the cortex-specific Fezf2 mutant. Errors bars represent s.e.m. One-tailed Student’s t-test; NS=Not significant, **P<0.01, ***P<0.001. n = 4 per genotype. Scale bars represent 200 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375921&req=5

Figure 4: Sox4 and Sox11 are required for Fezf2 expression and CS tract formationa, The requirement of SOX4 and SOX11 for E4 transactivation was determined in neurons cultured from E14.5 heterozygous littermate control and Sox4;Sox11 cdKO mice and transfected with the E4 luciferase construct. The activity of E4 in double knockout neurons was not significantly above background (1.1 fold. b, Analysis of neocortical Fezf2 expression by qRT-PCR in cortex-specific Sox4 and/or Sox11 mutants. Normalized to Gapdh, Fezf2 mRNA levels were dramatically reduced in cdKO but not Sox4 or Sox11 mutants. c-d, Sagittal (top row) and coronal (bottom row) sections of the control and cdKO pons immunostained for L1CAM (red) and PRKCG (green). The dramatic loss of L1CAM and PRKCG-positive CS tract axons in the cdKO (arrowhead and dashed outline) is similar to that of the cortex-specific Fezf2 mutant. Errors bars represent s.e.m. One-tailed Student’s t-test; NS=Not significant, **P<0.01, ***P<0.001. n = 4 per genotype. Scale bars represent 200 µm.
Mentions: To assess the requirement of Sox4 and Sox11 in E4-mediated activity, we transfected primary cortical neurons cultured from heterozygous littermate control or Sox4; Sox11 cdKO embryos with control or E4-containing luciferase constructs (Fig. 4a). In control neurons, the presence of E4 increased luciferase activity by 2.5±0.3 fold (P=1.9×10−4, one-tailed Student’s t-test). This increase was abolished in neurons culture from Sox4; Sox11 cdKO (1.1±0.1 fold; P=0.866), indicating that the two SOXC TFs are major activators of the enhancer in cortical neurons. Next, using qRT-PCR, we found a significant reduction in neocortical Fezf2 expression in cdKO compared to control or single cKO littermates at postnatal day 0 (P0) (Fig. 4b). In addition, immunostaining for L1CAM and PRKCG revealed a dramatic loss of CS axons in Sox4; Sox11 cdKO mice but not single cKO littermates at P0 and P6 (Fig. 4c, d, Supplementary Fig. 8), whereas the organization of other brainstem tracts was not affected (Fig. 3c, d). Because the absence of L1CAM and PRKCG staining could reflect their down-regulated expression in the cdKO, as opposed to loss of CS axons, we confirmed the absence of the CS tract using the CRE-responsive CAG-Cat-Gfp transgenic line to express GFP in all cortical projection neurons and their axons39,40 (Supplementary Fig. 9). Two additional observations indicate that the loss of Fezf2 and CS axons in these mice was not due to an absence of L5 neurons. First, only a moderate increase in cell death was detected in the motor-sensory areas, from which CS axons originate (Supplementary Fig. 10). Second, many BCL11B-immunopositive L5 neurons were present in the cdKO motor-sensory areas, albeit lightly immunostained (Supplementary Fig. 11), indicating that neurons that would normally give rise to the CS tract were present. Analysis of additional laminar markers further revealed an inversion of cortical layers similar to the reeler mutant mouse10–12 (Supplementary Fig. 11), indicating that Sox4 and Sox11 are also required for proper laminar positioning of neurons. Previous studies have shown that the CS tract is present in reeler mice12, indicating that this laminar phenotype occurs independently of Fezf2 and probably in response to defects in the RELN signaling pathway13,14. In support of this possibility, RELN was absent from cdKO Ncx (Supplementary Fig. 12). Thus, the combined deletion of Sox4 and Sox11 leads to defects in laminar position of neurons and molecular specification and connectivity of CS neurons.

Bottom Line: Here we identify a conserved non-exonic element (E4) that acts as a cortex-specific enhancer for the nearby gene Fezf2 (also known as Fezl and Zfp312), which is required for the specification of corticospinal neuron identity and connectivity.We find that SOX4 and SOX11 functionally compete with the repressor SOX5 in the transactivation of E4.These findings reveal that SOX transcription factors converge onto a cis-acting element of Fezf2 and form critical components of a regulatory network controlling the identity and connectivity of corticospinal neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

ABSTRACT
The co-emergence of a six-layered cerebral neocortex and its corticospinal output system is one of the evolutionary hallmarks of mammals. However, the genetic programs that underlie their development and evolution remain poorly understood. Here we identify a conserved non-exonic element (E4) that acts as a cortex-specific enhancer for the nearby gene Fezf2 (also known as Fezl and Zfp312), which is required for the specification of corticospinal neuron identity and connectivity. We find that SOX4 and SOX11 functionally compete with the repressor SOX5 in the transactivation of E4. Cortex-specific double deletion of Sox4 and Sox11 leads to the loss of Fezf2 expression, failed specification of corticospinal neurons and, independent of Fezf2, a reeler-like inversion of layers. We show evidence supporting the emergence of functional SOX-binding sites in E4 during tetrapod evolution, and their subsequent stabilization in mammals and possibly amniotes. These findings reveal that SOX transcription factors converge onto a cis-acting element of Fezf2 and form critical components of a regulatory network controlling the identity and connectivity of corticospinal neurons.

Show MeSH
Related in: MedlinePlus