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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.

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Functional analysis of species differences in E4 sequencea, Hierarchical clustering of E4F2 sequences from seven vertebrates. Percentage nucleotide identity relative to mouse E4F2 is indicated in parentheses. b, Analysis of species differences in Sox11 activation of E4F2. Sox11 trans-activated the E4F2 sequence of five vertebrates (≥ 7.3 fold) and Xenopus (2.8 fold). In contrast, the activity of zebrafish E4F2, which is divergent in SB1 and SB2, was not activated by Sox11. c, Murinization (red uppercase nucleotides) of zebrafish SB2 (ZeE4F2-m2) partially rescued the loss of transactivation of zE4F2 by Sox11. d-e, Cell-autonomous rescue of mouse Fezf2 loss-of-function by zebrafish Fezf2 (ZeFezf2). In utero electroporation (IUE) of Fezf2fl/fl neocortical wall at E12.5 with Cre and CRE-responsive Gfp plasmids. Fezf2-deficient L5 neurons do not form CS tract at P0 (d). Co-electroporation of a ZeFezf2 plasmid cell-autonomously rescued the formation of CS tract by Fezf2-deficient neurons (e). Errors bars represent s.e.m. One-tailed Student’s t-test; *P<0.05, **P<0.01, ***P<0.001. n = 4 per condition. Scale bar represents 200 µm.
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Figure 5: Functional analysis of species differences in E4 sequencea, Hierarchical clustering of E4F2 sequences from seven vertebrates. Percentage nucleotide identity relative to mouse E4F2 is indicated in parentheses. b, Analysis of species differences in Sox11 activation of E4F2. Sox11 trans-activated the E4F2 sequence of five vertebrates (≥ 7.3 fold) and Xenopus (2.8 fold). In contrast, the activity of zebrafish E4F2, which is divergent in SB1 and SB2, was not activated by Sox11. c, Murinization (red uppercase nucleotides) of zebrafish SB2 (ZeE4F2-m2) partially rescued the loss of transactivation of zE4F2 by Sox11. d-e, Cell-autonomous rescue of mouse Fezf2 loss-of-function by zebrafish Fezf2 (ZeFezf2). In utero electroporation (IUE) of Fezf2fl/fl neocortical wall at E12.5 with Cre and CRE-responsive Gfp plasmids. Fezf2-deficient L5 neurons do not form CS tract at P0 (d). Co-electroporation of a ZeFezf2 plasmid cell-autonomously rescued the formation of CS tract by Fezf2-deficient neurons (e). Errors bars represent s.e.m. One-tailed Student’s t-test; *P<0.05, **P<0.01, ***P<0.001. n = 4 per condition. Scale bar represents 200 µm.

Mentions: The differences in SOXC-mediated transcription between the mouse and zebrafish E4F2 suggest that species differences in the E4F2 sequence have functional implications. Analysis of SB1-3 sites within the E4F2 sequence in 23 species revealed that SB2 is conserved in all analyzed mammals and the two available non-mammalian amniotes (chicken and lizard) (Supplementary Fig. 13). To investigate the functional consequences, we used luciferase assays in Neuro-2a cells to analyze the activity of E4F2 sequence from different vertebrates (Fig. 5a, b) in response to SOX11, a more powerful trans-activator than SOX4 (Fig. 3). Of the constructs containing an E4F2 sequence of a mammal (human, chimpanzee, macaque, or mouse) or non-mammalian amniote (chicken), which have conserved SB1-3 sequences, the luciferase reporter activity was strongly increased following co-transfection with Sox11. The reporter activity of a non-amniote tetrapod (Xenopous) E4F2 construct was moderately increased compared to the empty or zebrafish luciferase plasmid, but not as high as the increase with mammalian constructs (P = 3.9×10−16, one-tailed Student’s t-test) (Fig. 5b). In contrast, the reporter construct containing the zebrafish E4F2 sequence, which has a highly divergent SB2 sequence from mammals, exhibited only basal level activity similar to the empty control luciferase plasmid. To confirm that this is dependent on sequence variations between species, we mutated zebrafish E4F2 (ZeE4F2-m2) by “murinizing” its SB1, SB2, or SB3 sequence. Reporter activity of the murinized zebrafish SB2 (ZeE4F2-m2), but not SB1 or SB3, was robustly induced by SOX11 (Fig. 5c). ZeE4F2-m2, however, was insufficient to restore the level of expression observed in the wildtype mouse sequence, suggesting the potential contribution of additional sequences. Thus, evolutionary differences in the E4F2 sequence, especially within SB2, are directly related to functional differences in the ability of SOX11 to activate this regulatory element.


Cis-regulatory control of corticospinal system development and evolution.

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

Functional analysis of species differences in E4 sequencea, Hierarchical clustering of E4F2 sequences from seven vertebrates. Percentage nucleotide identity relative to mouse E4F2 is indicated in parentheses. b, Analysis of species differences in Sox11 activation of E4F2. Sox11 trans-activated the E4F2 sequence of five vertebrates (≥ 7.3 fold) and Xenopus (2.8 fold). In contrast, the activity of zebrafish E4F2, which is divergent in SB1 and SB2, was not activated by Sox11. c, Murinization (red uppercase nucleotides) of zebrafish SB2 (ZeE4F2-m2) partially rescued the loss of transactivation of zE4F2 by Sox11. d-e, Cell-autonomous rescue of mouse Fezf2 loss-of-function by zebrafish Fezf2 (ZeFezf2). In utero electroporation (IUE) of Fezf2fl/fl neocortical wall at E12.5 with Cre and CRE-responsive Gfp plasmids. Fezf2-deficient L5 neurons do not form CS tract at P0 (d). Co-electroporation of a ZeFezf2 plasmid cell-autonomously rescued the formation of CS tract by Fezf2-deficient neurons (e). Errors bars represent s.e.m. One-tailed Student’s t-test; *P<0.05, **P<0.01, ***P<0.001. n = 4 per condition. Scale bar represents 200 µm.
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Figure 5: Functional analysis of species differences in E4 sequencea, Hierarchical clustering of E4F2 sequences from seven vertebrates. Percentage nucleotide identity relative to mouse E4F2 is indicated in parentheses. b, Analysis of species differences in Sox11 activation of E4F2. Sox11 trans-activated the E4F2 sequence of five vertebrates (≥ 7.3 fold) and Xenopus (2.8 fold). In contrast, the activity of zebrafish E4F2, which is divergent in SB1 and SB2, was not activated by Sox11. c, Murinization (red uppercase nucleotides) of zebrafish SB2 (ZeE4F2-m2) partially rescued the loss of transactivation of zE4F2 by Sox11. d-e, Cell-autonomous rescue of mouse Fezf2 loss-of-function by zebrafish Fezf2 (ZeFezf2). In utero electroporation (IUE) of Fezf2fl/fl neocortical wall at E12.5 with Cre and CRE-responsive Gfp plasmids. Fezf2-deficient L5 neurons do not form CS tract at P0 (d). Co-electroporation of a ZeFezf2 plasmid cell-autonomously rescued the formation of CS tract by Fezf2-deficient neurons (e). Errors bars represent s.e.m. One-tailed Student’s t-test; *P<0.05, **P<0.01, ***P<0.001. n = 4 per condition. Scale bar represents 200 µm.
Mentions: The differences in SOXC-mediated transcription between the mouse and zebrafish E4F2 suggest that species differences in the E4F2 sequence have functional implications. Analysis of SB1-3 sites within the E4F2 sequence in 23 species revealed that SB2 is conserved in all analyzed mammals and the two available non-mammalian amniotes (chicken and lizard) (Supplementary Fig. 13). To investigate the functional consequences, we used luciferase assays in Neuro-2a cells to analyze the activity of E4F2 sequence from different vertebrates (Fig. 5a, b) in response to SOX11, a more powerful trans-activator than SOX4 (Fig. 3). Of the constructs containing an E4F2 sequence of a mammal (human, chimpanzee, macaque, or mouse) or non-mammalian amniote (chicken), which have conserved SB1-3 sequences, the luciferase reporter activity was strongly increased following co-transfection with Sox11. The reporter activity of a non-amniote tetrapod (Xenopous) E4F2 construct was moderately increased compared to the empty or zebrafish luciferase plasmid, but not as high as the increase with mammalian constructs (P = 3.9×10−16, one-tailed Student’s t-test) (Fig. 5b). In contrast, the reporter construct containing the zebrafish E4F2 sequence, which has a highly divergent SB2 sequence from mammals, exhibited only basal level activity similar to the empty control luciferase plasmid. To confirm that this is dependent on sequence variations between species, we mutated zebrafish E4F2 (ZeE4F2-m2) by “murinizing” its SB1, SB2, or SB3 sequence. Reporter activity of the murinized zebrafish SB2 (ZeE4F2-m2), but not SB1 or SB3, was robustly induced by SOX11 (Fig. 5c). ZeE4F2-m2, however, was insufficient to restore the level of expression observed in the wildtype mouse sequence, suggesting the potential contribution of additional sequences. Thus, evolutionary differences in the E4F2 sequence, especially within SB2, are directly related to functional differences in the ability of SOX11 to activate this regulatory element.

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