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Multiple conserved regulatory domains promote Fezf2 expression in the developing cerebral cortex.

Eckler MJ, Larkin KA, McKenna WL, Katzman S, Guo C, Roque R, Visel A, Rubenstein JL, Chen B - Neural Dev (2014)

Bottom Line: Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis.We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.These results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA, USA. bchen@ucsc.edu.

ABSTRACT

Background: The genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons.

Results: Using a combination of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) we mapped the binding of four deep-layer-enriched transcription factors previously shown to be important for cortical development. Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis. We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.

Conclusions: These results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.

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Enhancer 434 is active in cortical progenitor cells. (A-C) Immunohistochemistry demonstrated LacZ expression in progenitor cells at E11.5 that co-expressed SOX2 (A), PAX6 (B) and PHH3 (C). LacZ was also expressed in a few postmitotic neurons in the preplate marked by Tuj1 (D). X-Gal staining of whole mount P0 brains (E) and sections (F) demonstrated reporter activity in the VZ. (G, H) Immunohistochemistry using ß-Gal antibody combined with antibodies for RGC markers BLBP (G) and PAX6 (H). (I, K, L) In adult animals, the majority of enhancer 434-LacZ+ cells were in GFAP+ astrocytes throughout the forebrain. A subset of non-astrocyte like cells expressed SATB2 (J, J’). Arrows represent co-expression of LacZ and the indicated markers. Hip, hippocampus; WM, white matter. Scale bars: (D) 50 μm, (E) 250 mm, (F) 500 μm, (G) 25 μm, (H) 10 μm, (I) 500 μm, (J) 100 μm, (J’) 50 μm, (K) 25 μm.
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Figure 4: Enhancer 434 is active in cortical progenitor cells. (A-C) Immunohistochemistry demonstrated LacZ expression in progenitor cells at E11.5 that co-expressed SOX2 (A), PAX6 (B) and PHH3 (C). LacZ was also expressed in a few postmitotic neurons in the preplate marked by Tuj1 (D). X-Gal staining of whole mount P0 brains (E) and sections (F) demonstrated reporter activity in the VZ. (G, H) Immunohistochemistry using ß-Gal antibody combined with antibodies for RGC markers BLBP (G) and PAX6 (H). (I, K, L) In adult animals, the majority of enhancer 434-LacZ+ cells were in GFAP+ astrocytes throughout the forebrain. A subset of non-astrocyte like cells expressed SATB2 (J, J’). Arrows represent co-expression of LacZ and the indicated markers. Hip, hippocampus; WM, white matter. Scale bars: (D) 50 μm, (E) 250 mm, (F) 500 μm, (G) 25 μm, (H) 10 μm, (I) 500 μm, (J) 100 μm, (J’) 50 μm, (K) 25 μm.

Mentions: We generated stable transgenic lines expressing LacZ under the control of enhancer 434 and the hsp68 minimal promoter [37]. At E11.5, ß-gal was expressed in SOX2+ and PAX6+ cortical progenitor cells (Figure 4A-B), including the metaphase cells that were labeled by PHH3 antibody at the ventricular surface (Figure 4C). In addition, Tuj1+ neurons located in the preplate expressed ß-gal (Figure 4D). Whole mount staining of P0 brains revealed strong LacZ activity throughout multiple brain regions (Figure 4E). However, the majority of enhancer 434 activity was in the VZ of the cortex and basal ganglia (Figure 4F). Immunohistochemistry with antibodies for LacZ and the radial glial cell (RGC) marker BLBP demonstrated significant co-localization (Figure 4G). Additionally, some LacZ+ cells in the VZ expressed the RGC marker PAX6 (Figure 4H). Some cells in the cortical plate expressed LacZ, however a minority of these LacZ+ cells expressed the projection neuron markers SOX5 and SATB2 (Additional file 2). Thus, at P0 enhancer 434 activity is strongest in cortical progenitor cells. This strong LacZ signal observed in the VZ is consistent with the activity of enhancer 434 reported by the VISTA Enhancer Browser (http://enhancer.lbl.gov).


Multiple conserved regulatory domains promote Fezf2 expression in the developing cerebral cortex.

Eckler MJ, Larkin KA, McKenna WL, Katzman S, Guo C, Roque R, Visel A, Rubenstein JL, Chen B - Neural Dev (2014)

Enhancer 434 is active in cortical progenitor cells. (A-C) Immunohistochemistry demonstrated LacZ expression in progenitor cells at E11.5 that co-expressed SOX2 (A), PAX6 (B) and PHH3 (C). LacZ was also expressed in a few postmitotic neurons in the preplate marked by Tuj1 (D). X-Gal staining of whole mount P0 brains (E) and sections (F) demonstrated reporter activity in the VZ. (G, H) Immunohistochemistry using ß-Gal antibody combined with antibodies for RGC markers BLBP (G) and PAX6 (H). (I, K, L) In adult animals, the majority of enhancer 434-LacZ+ cells were in GFAP+ astrocytes throughout the forebrain. A subset of non-astrocyte like cells expressed SATB2 (J, J’). Arrows represent co-expression of LacZ and the indicated markers. Hip, hippocampus; WM, white matter. Scale bars: (D) 50 μm, (E) 250 mm, (F) 500 μm, (G) 25 μm, (H) 10 μm, (I) 500 μm, (J) 100 μm, (J’) 50 μm, (K) 25 μm.
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Figure 4: Enhancer 434 is active in cortical progenitor cells. (A-C) Immunohistochemistry demonstrated LacZ expression in progenitor cells at E11.5 that co-expressed SOX2 (A), PAX6 (B) and PHH3 (C). LacZ was also expressed in a few postmitotic neurons in the preplate marked by Tuj1 (D). X-Gal staining of whole mount P0 brains (E) and sections (F) demonstrated reporter activity in the VZ. (G, H) Immunohistochemistry using ß-Gal antibody combined with antibodies for RGC markers BLBP (G) and PAX6 (H). (I, K, L) In adult animals, the majority of enhancer 434-LacZ+ cells were in GFAP+ astrocytes throughout the forebrain. A subset of non-astrocyte like cells expressed SATB2 (J, J’). Arrows represent co-expression of LacZ and the indicated markers. Hip, hippocampus; WM, white matter. Scale bars: (D) 50 μm, (E) 250 mm, (F) 500 μm, (G) 25 μm, (H) 10 μm, (I) 500 μm, (J) 100 μm, (J’) 50 μm, (K) 25 μm.
Mentions: We generated stable transgenic lines expressing LacZ under the control of enhancer 434 and the hsp68 minimal promoter [37]. At E11.5, ß-gal was expressed in SOX2+ and PAX6+ cortical progenitor cells (Figure 4A-B), including the metaphase cells that were labeled by PHH3 antibody at the ventricular surface (Figure 4C). In addition, Tuj1+ neurons located in the preplate expressed ß-gal (Figure 4D). Whole mount staining of P0 brains revealed strong LacZ activity throughout multiple brain regions (Figure 4E). However, the majority of enhancer 434 activity was in the VZ of the cortex and basal ganglia (Figure 4F). Immunohistochemistry with antibodies for LacZ and the radial glial cell (RGC) marker BLBP demonstrated significant co-localization (Figure 4G). Additionally, some LacZ+ cells in the VZ expressed the RGC marker PAX6 (Figure 4H). Some cells in the cortical plate expressed LacZ, however a minority of these LacZ+ cells expressed the projection neuron markers SOX5 and SATB2 (Additional file 2). Thus, at P0 enhancer 434 activity is strongest in cortical progenitor cells. This strong LacZ signal observed in the VZ is consistent with the activity of enhancer 434 reported by the VISTA Enhancer Browser (http://enhancer.lbl.gov).

Bottom Line: Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis.We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.These results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA, USA. bchen@ucsc.edu.

ABSTRACT

Background: The genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons.

Results: Using a combination of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) we mapped the binding of four deep-layer-enriched transcription factors previously shown to be important for cortical development. Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis. We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.

Conclusions: These results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.

Show MeSH