CoupTFI interacts with retinoic acid signaling during cortical development

Abstract

We examined the role of the orphan nuclear hormone receptor CoupTFI in mediating cortical development downstream of meningeal retinoic acid signaling. CoupTFI is a regulator of cortical development known to collaborate with retinoic acid (RA) signaling in other systems. To examine the interaction of CoupTFI and cortical RA signaling we utilized Foxc1-mutant mice in which defects in meningeal development lead to alterations in cortical development due to a reduction of RA signaling. By analyzing CoupTFI(-/-);Foxc1(H/L) double mutant mice we provide evidence that CoupTFI is required for RA rescue of the ventricular zone and the neurogenic phenotypes in Foxc1-mutants. We also found that overexpression of CoupTFI in Foxc1-mutants is sufficient to rescue the Foxc1-mutant cortical phenotype in part. These results suggest that CoupTFI collaborates with RA signaling to regulate both cortical ventricular zone progenitor cell behavior and cortical neurogenesis.

Figure 1. Sampling Window Used For Cell Counts.

The location of the 150 µm sampling window in the medial-lateral dimension of the cortex spanning from the ventricular to pial surface of the dorsal cortex is identified by the black box in this coronal forebrain section of an E14.5. Scale bar = 500 µm.

Figure 2. CoupTFI And CoupTFII Expression Is Not Downregulated Or Misexpressed In The Cortex Of Foxc1-Mutants At E14.5.

In situ hybridization of coronal sections of control (A, C, E, G, I, K, M, O) and Foxc1^H/L (B, D, F, H, J, L, N, P) at E14.5 with CoupTFI probe (A–H) and CoupTFII probe (I–P). Higher magnification panels in E, F, K, L correspond to boxed regions in G, H, O, P. Scale bar: 500 µm in AF, I–N, 100 µm in G, H, O, P. Arrow in L, N points to the cortical hem. * in D, F, L, N indicates the ventral progenitor population. MZ: marginal zone.

Figure 3. CoupTFI Is Required For The RA Mediated Rescue Of Foxc1-Mutants Animals.

Cresyl Violet staining of E14.5 coronal forebrain sections of untreated and RA treated (E10–E14.5) embryos (A–H). Asterisk in D, H signifies the medial wall. Pax6 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5 embryos) (I–P). Tbr2 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5) embryos (Q–X). Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5) embryos (Y–FF). Sections are counterstained with DAPI (blue) (I–FF). Scale bar: 500 µm in A–H, 50 µm in I–FF. VZ: ventricular zone; SVZ: subventricular zone; PC: progenitor cell; CP: cortical plate.

Figure 4. CoupTFI Is Required For The RA Mediated Rescue Of Foxc1-Mutants Animals.

Quantification of dorsal forebrain length at E14.5 (A), Pax6 cell number at E14.5 (B), Tbr2 cell number at E14.5 (C), Ctip2 cell number at E14.5 (D), Tbr1 cell number at E14.5 (E), area of the counting window (F), density of DAPI⁺ cells in the counting window (G). Error bars represent SEM. A–G were analyzed by two way ANOVA: A: genotype (F_(3,25) = 55.86, p<0.001), treatment (F_(1,25) = 27.84, p<0.001), interaction (F_(3,25) = 49.98, p<0.001); B: genotype (F_(3,26) = 9.5, p<0.001), treatment (F_(1,26) = 48.66, p<0.01), interaction (F_(3,26) = 11.67, p<0.001); C: genotype (F_(3,27) = 19.23, p<0.001), treatment (F_(1,27) = 8.71, p<0.01), interaction (F_(3,27) = 14.55, p<0.001); D: genotype (F_(3,26) = 40.83, p<0.001), treatment (F_(1,26) = 18.45, p<0.001), interaction (F_(3,26) = 14.75, p<0.001); E: genotype (F_(3,26) = 47.99, p<0.001), treatment (F_(1,26) = 48.66, p<0.001), interaction (F_(3,26) = 14.91, p<0.001); F: genotype (F_(3,24) = 24.77, p<0.001), treatment (F_(1,24) = 8.158, p<0.01), interaction (F_(3,24) = 13.38, p<0.001); G: genotype (F_(3,25) =10.33, p<0.001), treatment (F_(1,25) =0.53, p = 0.47), interaction (F_(3,25) = 1.62, p = 0.2). *p<0.05, ***p<0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.

Figure 5. Dose response of CoupTFI in the RA-mediated rescue of Foxc1-mutants.

Quantification of dorsal forebrain length at E14.5 (A), Pax6 cell number at E14.5 (B), Tbr2 cell number at E14.5 (C), Ctip2 cell number at E14.5 (D), and Tbr1 cell number at E14.5 (E) in RA treated animals. Note: the CoupTFI^+/+;Foxc1^H/L and CoupTFI^−/−;Foxc1^H/L data are the same as in Figure 4, but are being compared to CoupTFI^+/−;Foxc1^H/L for this analysis. A–E were analyzed by one-way ANOVA: A: F_(2,13) = 3.7, p = 0.06; B: F_(2,12) = 1.3, p = 0.3; C: F_(2,10) = 8.5, p<0.01; D: F_(2,10) = 7.1, p<0.05; E: F_(2,10) = 6.9, p<0.05. *p<0.05 and indicates significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from CoupTFI^+/+Foxc1^H/L group.

Figure 6. Overexpression Of CoupTFI In Cortical Progenitor Cells Partially Rescues The Cortical Phenotype Of Foxc1-Mutants.

Cresyl Violet staining of E14.5 coronal forebrain sections (A–D). Quantification of dorsal forebrain length at E14.5 (E). Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E14.5 (F–I). Quantification of Ctip2 and Tbr1 cell number at E14.5 (J). Pax6 (red) immunohistochemistry of the dorsal cortex at E14.5 (K–N) Quantification of Pax6 cell number at E14.5 (O). Tbr2 (red) immunohistochemistry of the dorsal cortex at E14.5 (P–S). Quantification of Tbr2 cell number at E14.5 (T). Sections are counterstained with DAPI (blue) (F–I, K–N, P–S). Scale bar: 500 µm in A–D; 50 µm in F–I, K–N, P–S. E, J, O, P were analyzed by one way ANOVA: E: F_(3,12) = 40.87, p<0.001; J: F_(3,12) = 62.28, p<0.001; F_(3,12) = 48.8, p<0.001; O: F_(3,12) = 74.38, p<0.001; P: F_(3,11) = 76.71, p<0.001. *p<0.05, **p<0.01; ***p<0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.

Figure 7. Overexpression Of CoupTFI In Cortical Progenitor Cells Increases Early Neuorgenesis.

Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E12.5 (A–D). Sections are counterstained with DAPI (blue). Quantificaiton of Ctip2 and Tbr1 cell number at E12.5 (E). Quantification of the Q-fraction at E12.5 (F) and E14.5 (G). E–G were analyzed by one way ANOVA: E: Ctip2 F_(3,11) = 35.5, p<0.001; Tbr1: F_(3,12) = 66.1, p<0.001; F: F_(3,12) = 5.2, p<0.001; G: F_(3,12) = 54.6, p<0.001. *p<0.05, **p<0.01; ***p<0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.