COUP-TFI coordinates cortical patterning, neurogenesis, and laminar fate and modulates MAPK/ERK, AKT, and beta-catenin signaling

Abstract

A major unsolved question in cortical development is how proliferation, neurogenesis, regional growth, regional identity, and laminar fate specification are coordinated. Here we provide evidence, using loss-of-function and gain-of-function manipulations, that the COUP-TFI orphan nuclear receptor promotes ventral cortical fate, promotes cell cycle exit and neural differentiation, regulates the balance of early- and late-born neurons, and regulates the balanced production of different types of layer V cortical projection neurons. We suggest that COUP-TFI controls these processes by repressing Mapk/Erk, Akt, and beta-catenin signaling.

Figure 1.

Characterization of the D6/COUP-TFI transgenic mouse line. (A) Schematic representation of the construct utilized to generate the transgenic line. (B) RNA in situ hybridization on coronal sections through the telencephalon of WT and the COUP-TFI overexpression line D6/COUP-TFI (D6). D6/COUP-TFI transgene increases COUP-TFI expression in dorsal parts of the cortical VZ (arrowheads) and CP (arrowheads in E15.5 panel). (C) Quantification of the E12.5 in situ hybridization signal intensity shown in (B): normal ventral^high–dorsal^low COUP-TFI gradient (blue line) was modified in the D6/COUP-TFI cortex (red line) due to increased VZ expression in the dorsal pallium (DP). VP, ventral pallium; LP, lateral pallium; MP, medial pallium. Scale bars: 200 μm.

Figure 2.

COUP-TFI dosage regulates molecular patterning of cortical progenitors at E12.5. (A–A′) RNA in situ hybridization for COUP-TFI on coronal sections through the telencephalon of WT (A) and D6/COUP-TFI (D6) (A′), showing the change in expression gradient due to the increase in dorsal regions. (B–C′) Immunohistochemistry and (D–G′) RNA in situ hybridizations on coronal sections through the telencephalon of D6/COUP-TFI, COUP-TFI^–/–, and WT littermate embryos for Pax6 (B–C′), Emx2 (D–E′), and Tbr2 (F–G′), showing that COUP-TFI regulates expression of genes involved in patterning, proliferation, and differentiation (Pax6 and Emx2: B–E′) and in differentiating neurons (Tbr2: F–G′). For each gene marker, arrowheads show the complementary expression pattern between the genotypes. Each in situ hybridization was performed at least 3 times using different litters. Scale bars: 200 μm.

Figure 3.

Increased COUP-TFI dosage negatively regulates proliferation of cortical VZ and SVZ progenitors. (A–A′′) RNA in situ hybridizations on coronal sections through the telencephalon of D6/COUP-TFI, COUP-TFI^–/–, and WT littermate embryos for Cyclin D2, a key regulator of G1-phase progression, showing the complementary effects in the 2 genotypes (arrowheads). Staining intensity in the basal ganglia has been used to normalize Cyclin D2 expression level in the D6/COUP-TFI: Cyclin D2 was decreased of 65% in dorsal pallium. Staining intensity in the medial pallium has been used to normalize Cyclin D2 expression level in the COUP-TFI ^–/–: Cyclin D2 was increased of 40% in lateral/ventral pallium. (B–B′′′) PH3 immunostaining labeling cells in M-phase of the cell cycle in D6/COUP-TFI, COUP-TFI^–/–, and WT at E13.5 littermate embryos. Increasing COUP-TFI dosage decreases the number of PH3⁺ cells in VZ (B′ and quantification in C) and in SVZ (B′, arrowhead, and quantification in D). Loss of COUP-TFI causes an increase of PH3⁺ cells in both VZ and SVZ (B′′ and quantification in C–D). (B′′′ and C–D) The D6/COUP-TFI proliferation defect is partially rescued at E13.5 by introducing 2 copies of the COUP-TFI null allele into the D6/COUP-TFI line. As shown in B–B′′′ and C–D, the D6/COUP-TFI-COUP-TFI KO cortex (D6;KO) shows an intermediate phenotype in the number of PH3⁺ cells compared with the D6 and KO cortex. Statistical analysis performed using Student's t-test. In VZ: WT-KO P < 0.001, WT-D6 P < 1.00E-07, D6-KO P < 5.00E-08, D6-D6;KO P < 0.01. In SVZ: WT-KO P < 0.05, WT-D6 P < 0.02, D6-KO P < 4.00E-05, D6-D6;KO P < 0.05. Two different litters were used.

Figure 4.

Increasing COUP-TFI expression promotes neurogenesis. (A–F′) D6/COUP-TFI cortex shows increasing PP/CP thickness from E11.5 through E13.5 based on expression of β-III-tubulin (A–C′, red) and Tbr1 (D–F′). At E13.5 (F–F′), the D6/COUP-TFI VZ (F′, asterisk) ectopically expresses Tbr1, a postmitotic marker. Each staining has been performed at least 3 times. (G-H) Quantification of PP and CP width during development (E11.5 to P0 for D6 and E13.5 to E18.5 for COUP-TFI^–/–). In order to quantify CP thickness in the D6 line, we measured dorsal pallium (box in A′, B′, and C′). To quantify CP thickness in the COUP-TFI^–/– line, we measure ventral pallium/lateral pallium (box in Fig. S2B′′). (G) The plot shows the initial (E11.5–E13.5) CP overgrowth in D6 animals, followed by the decrease after E13.5. (H) The plot shows the overall reduced ventral CP thickness in COUP-TFI^–/–.

Figure 5.

BrdU birthdating shows that increased COUP-TFI promotes generation of early-born cells and reduces late-born cells. (A–C′) BrdU injection at indicated stages, analysis at P0. BrdU immunostaining on coronal sections of WT (A–C) and D6/COUP-TFI (D6) (A′–C′) animals. The boxes show a representative region used to count the BrdU⁺ cells. (A–A′) In WT cortex, cells incorporating BrdU at E11.5 populated deep parts (layer VI and subplate) of the CP. In the D6/COUP-TFI cortex, there was ∼3-fold increase in the number of BrdU⁺ cells in deep layers (compare box in A with A′; see chart in D for quantification—cells have been counted in deep layers). (B–B′) Roughly equal numbers of BrdU⁺ cells were generated at E13.5 in WT and D6. Cells have been counted in CP. (C–C′) In WT cortex, cells labeled at E16.5 populated the upper layers II/III of the CP. In D6 animals, these cells were diminished (compare box in C with C′). Cells have been counted in superficial layers. Scale bar: 200 μm. (D) Quantification of BrdU⁺ cells per unit area (200 μm²) in the 3 experiments. Asterisks in (D), t-test statistical analysis: P < 0.0006 for E11.5, P < 0.02 for E16.5, n = 4 (see Materials and Methods).

Figure 6.

COUP-TFI regulates the fraction of cells leaving the cell cycle. (A–A′) RNA in situ hybridization on coronal sections through the telencephalon at E11.5 showing COUP-TFI overexpression in the D6/COUP-TF cortex (box in A′): an equivalent region used for cell counting is shown in B–B′ at higher magnification. (B–B′) Twelve hours before immunofluorescence analysis at E11.5, embryos were exposed to BrdU. The cortex was stained by double labeling (arrowheads in A′) with anti-BrdU (green) and anti-Ki67 (red) antibodies. Arrowheads in B′ show BrdU⁺/Ki67^– cells in the VZ; we suggest that these had exited the cell cycle. (C-D′) Twelve hours before immunofluorescence analysis at E15.5, embryos were exposed to BrdU. Double labeling of the E15.5 pallium with anti-BrdU (green) and anti-Ki67 (red) antibodies. The boxes in C and C′ show the region where the cells have been counted. Arrowheads in D show BrdU⁺/Ki67^– cells that we suggest had exited the cell cycle and had started migration into the CP. (E–F) Ratio of BrdU⁺; Ki67^–/BrdU⁺ cells, showing the fraction of cells that had exited the cell cycle (Q fraction). Asterisk in E: P < 0.00003, asterisk in F: P < 0.0002, Student's t-test). n = 6 sections, on 2 different brains (same result, only 1 shown) for E. n = 5 sections, on 2 different brains (same result, only 1 shown) for F. IZ, intermediate zone;. Scale bars: 200 μm.

Figure 7.

Regulation of RTKs and β-catenin signaling. (A–B′) Immunohistochemistry on coronal sections at E11.5 for pErk (A–A′) and Akt-pS (B–B′), showing the early response of RTK signaling to COUP-TFI overexpression. The boxes show the area used for the high magnification picture on the right. This is the area where COUP-TFI is highly overexpressed (See Fig. 1A–A′). (A′′–B′′) Quantification of pErk⁺ (A′′) and Akt-pS⁺ (B′′) cells in A–B′. Asterisk in A′′: P < 0.00005; asterisk in B′′: P < 0.005. (C–C′) RNA in situ hybridization on coronal sections at E11.5 for Fgfr3, showing its upregulation. (D–D′) β-Galactosidase staining on coronal sections at E11.5 for β-catenin activation of the BAT-gal transgene. In D6/COUP-TFI (D6) brains β-catenin activation is reduced. (E–F′′) Immunofluorescence on coronal sections at E13.5 for pErk (E–E′′) and Akt-pS (F–F′′) in WT (E, F), D6/COUP-TFI (E′, F′), and COUP-TFI^–/– (E′′, F′′), showing the complementary effects on these 2 signaling pathways by changing COUP-TFI dose. (E′′′–F′′′) Quantification of pErk⁺ (E′′′) and Akt-pS⁺ (F′′′) cells in E–F′′. Asterisks in E′′′: P < 0.0005 for D6 and KO; asterisk in F′′′: P < 0.007. (G–G′′) RNA in situ hybridization on coronal sections through the telencephalon at E13.5 for Fgfr3, showing its complementary regulation in D6/COUP-TFI (G′) and COUP-TFI^–/– (G′′). Scale bars: 200 μm.

Figure 8.

D/V patterning and layer V neurons are modified by COUP-TFI dosage. (A–A′) β-Galactosidase staining on coronal sections through the telencephalon of WT;BAT-gal (A), D6/COUP-TFI;BAT-gal (A′), and COUP-TFI^–/–;BAT-gal (A′′) at E15.5. β-Catenin activation follows a dorsal^high–ventral^low gradient that is disrupted in opposing ways in the 2 genotypes (compare arrowheads in A–A′′). (B–D′′) RNA in situ hybridizations on coronal sections through the telencephalon of WT (B–D), D6/COUP-TFI (B′–D′), and COUP-TFI^–/– (B′′–D′′) for ER81 (B–B′′), Fezl (C–C′′), and p75 (D–D′′). The D/V expression pattern of these markers is affected by altering COUP-TFI gene dosage: arrowheads show the complementary D/V changes in D6 and COUP-TFI^–/– animals (E-F″) RNA in situ hybridizations on coronal sections through the telencephalon of WT (E, F), D6/COUP-TFI (E′-F′), and COUP-TFI^−/− (E″-F″) for ER81 (E′E″) and Fez-l (F-F″) at E18.5. COUP-TFI dosage affects milecular markers of different layer V neurons.

Figure 9.

Model for COUP-TFI function in regulating proliferation, differentiation, and patterning. (A) COUP-TFI dosage regulates RTK signaling pathways through downregulation of Mapk/Erk and PI3K/Akt activity. These changes promote progenitor cells to leave the cell cycle (reducing the numbers of both VZ and SVZ cells) and to differentiate into Tbr2+ and Tbr1+ neurons. D6/COUP-TFI promotes expression of Fgfr3; we hypothesize that this Fgf receptor may participate in the proliferation-to-differentiation switch. (B) COUP-TFI promotes ventral fate in the cortical primordium. Disruption of the COUP-TFI gradient changes D/V molecular properties in the CP, as revealed by the expression of ventral (ER81 and p75, green) and dorsal (β-catenin and Fezl, violet) markers.