PAF-AH Catalytic Subunits Modulate the Wnt Pathway in Developing GABAergic Neurons

Abstract

Platelet-activating factor acetylhydrolase 1B (PAF-AH) inactivates the potent phospholipid platelet-activating factor (PAF) and is composed of two catalytic subunits (alpha1 and alpha2) and a dimeric regulatory subunit, LIS1. The function of the catalytic subunits in brain development remains unknown. Here we examined their effects on proliferation in the ganglionic eminences and tangential migration. In alpha1 and alpha2 catalytic subunits knockout mice we noticed an increase in the size of the ganglionic eminences resulting from increased proliferation of GABAergic neurons. Our results indicate that the catalytic subunits act as negative regulators of the Wnt signaling pathway. Overexpression of each of the PAF-AH catalytic subunits reduced the amount of nuclear beta-catenin and provoked a shift of this protein from the nucleus to the cytoplasm. In the double mutant mice, Wnt signaling increased in the ganglionic eminences and in the dorsal part of the cerebral cortex. In situ hybridization revealed increased and expanded expression of a downstream target of the Wnt pathway (Cyclin D1), and of upstream Wnt components (Tcf4, Tcf3 and Wnt7B). Furthermore, the interneurons in the cerebral cortex were more numerous and in a more advanced position. Transplantation assays revealed a non-cell autonomous component to this phenotype, which may be explained in part by increased and expanded expression of Sdf1 and Netrin-1. Our findings strongly suggest that PAF-AH catalytic subunits modulate the Wnt pathway in restricted areas of the developing cerebral cortex. We hypothesize that modulation of the Wnt pathway is the evolutionary conserved activity of the PAF-AH catalytic subunits.

Keywords: Wnt; beta-catenin; ganglionic eminences; platelet-activating factor acetylhydrolase IB.

Figure 1

Double KO mice exhibited increased GE area where the PAF-AH catalytic subunit genes are expressed. (A,B) Sections from E13.5 double KO (A) or control mice (B) were immunostained with anti-pH3. The area of the proliferating zone was marked (black line), and the cells were counted. There were more pH3 positive cells in the double KO mice. (C,D) In situ hybridization was conducted at E13.5 using PAF-AH α1 (C) or α2 (D) probes. Scale bar is 1 mm. (E,F) High magnification of (C) and (D), respectively. Scale bar is 0.5 mm.

Figure 2

Positioning of PAH-AH in the Wnt pathway. PAF-AH (highlighted in yellow) interacting proteins (highlighted in light blue) connected with green lines were detected in a large yeast two hybrid screen, additional interactions are connected by orange lines (Stelzl et al., 2005) and associated site: http://141.80.164.19/y2h_network/ppi_search.php (enter official gene symbol: pafah1b3). The BRD7 interactions were added from (Kim et al., 2003), and the interactions between CTNNB, TLE1 or TCF proteins were taken from (Daniels and Weis, 2005), these are marked with black lines.

Figure 3

PAFAH α1 and α2 catalytic subunits repress TCF/LEF-dependent transcription induced by CA β-catenin (A) or disheveled (Dvl) (B). HEK293 cells were transfected with either TOPFLASH, a reporter vector for the activation of the Wnt signaling, or FOPFLASH, a negative control reporter. In addition, different combinations of α1 and α2 on top of either TLE1 (A), or CRMP1 (B). Results are normalized as fold of induction relative to basal luciferase activity. Histograms represent the mean of three experiments ± S.E.M. Stars indicate the p values: **P < 0.01, ***P < 0.001.

Figure 4

PAFAH subunits affect subcellular localization of GFP-CA β-catenin. COS-7 cells were transfected with GFP-CA β-catenin (A) alone or together with DsRedα1 (B,C) or DsRedα2 (D,E), or both PAF-AH catalytic subunits (F,G). Cells were fixed 48 h after transfection and nuclear DNA was stained with DAPI (shown in blue). Overexpression of GFP-CA β-catenin demonstrated nuclear localization (A), whereas the addition of either DsRedα1 (C) or DsRedα2 (E) or both (G) shifted β-catenin to the cytoplasm (B,D,F). Scale bars are 20 μm.

Figure 5

Double KO brain sections exhibit an increased area of nuclear β-catenin. Double KO (A–C) exhibit more cells with nuclear β-catenin in comparison with control (B–F). The white boxes define the higher magnifications areas. Scale bars: (A,D) 200, (B,E) 100, (C,F) 40 μm.

Figure 6

Double KO brains exhibit increased and expanded expression of downstream and upstream components of the Wnt pathway using in situ hybridization. (A,B) Expression of Cyclin D1 in double KO (A) or control (B). Note the expansion in the area expressing Cyclin D1 in the GE. (C,D) Expression of Tcf4 in double KO (C) or control (D). Arrowheads mark the comparable GE area, as well as cerebral cortex area (E,F) Expression of Tcf3 in double KO (E) or control (F). Note the mutually exclusive pattern of expression between the Tcf3 and Tcf4. (G,H) Expression of Wnt7B in double KO (G) or control (H). The area that expressed high levels of nuclear β-catenin is marked with arrowheads. Scale bars are 0.5 mm.

Figure 7

Double KO mice exhibit earlier thalamocortical projections. (A–D) TAG-1 immunostaining of double KO (A,C) and control (B,D) mice. Scale bars: (A,B) – 100; (C,D) – 40 μm. The white boxes in (A) and (B) define the area shown in (C) and (D). (E,F) DiI stained thalamocortical fibers exhibit earlier projections in double KO brains (E) in comparison to the control brains (F). Scale bars are 500 μm.

Figure 8

Double KO mice exhibit advanced tangential migration. (A–D) GAD67-GFP-labeled interneurons are found in more advanced positions in double KO brain sections (A,B) in comparison with control sections (C,D). Scale bars in (A) and (C) is 100 μm, and in (B) and (D) is 40 μm. (E,F) Calbindin immunostaining, which identifies a subgroup of interneurons, demonstrates advanced positioning of these interneurons in the double KO (E,F) versus control (G,H). Scale bars in (E) and (G) is 100 μm, and in (F) and (H) is 40 μm. (I,J) Sdf-1 expression by in situ hybridization is expanded in the cortex of double KO (I) versus the control cortex (J). (K,L) In situ hybridization of Netrin-1 reveals an expanded expression in the striatum of double KO (K) versus the control striatum (L). Scale bars in (I–L) are 0.5 mm.