Fezf2 promotes neuronal differentiation through localised activation of Wnt/β-catenin signalling during forebrain development

Brain regionalisation, neuronal subtype diversification and circuit connectivity are crucial events in the establishment of higher cognitive functions. Here we report the requirement for the transcriptional repressor Fezf2 for proper differentiation of neural progenitor cells during the development of the Xenopus forebrain. Depletion of Fezf2 induces apoptosis in postmitotic neural progenitors, with concomitant reduction in forebrain size and neuronal differentiation. Mechanistically, we found that Fezf2 stimulates neuronal differentiation by promoting Wnt/β-catenin signalling in the developing forebrain. In addition, we show that Fezf2 promotes activation of Wnt/β-catenin signalling by repressing the expression of two negative regulators of Wnt signalling, namely lhx2 and lhx9. Our findings suggest that Fezf2 plays an essential role in controlling when and where neuronal differentiation occurs within the developing forebrain and that it does so by promoting local Wnt/β-catenin signalling via a double-repressor model.

plasmids were linearised with Not I (NEB). pSPORT6-Msx1 was linearised with Not I. Ambion® mMessenger SP6 kit and MegaClear Spin columns were used for in vitro transcription of all mRNA transcripts.
Animal cap explants were isolated from mid-blastula (stage 8-8.5) embryos and cultured in ¾ NAM until specified stages. Collected embryos or explants were stored in Ambion® RNAlater solution and extracted using Qiagen RNEasy mini kit. For injection embryos or explants subjected to qPCR analysis, three replicates were used generate statistical results. qPCR analysis was performed using ABI high-capacity RNA-to-cDNA reverse transcription kit, ABI SYBR Fast or ABI TaqMan Fast reagent depending on experiment requirements. Data collection was performed on an ABI StepOnePlus machine.

Morpholino (MO) design and injection
All MO experiments were performed in X. tropicalis with MOs specifically designed against the X. tropicalis genes and supplied by Gene Tools Inc. Some MOs were FITC-labelled to provide information on injection quality and to provide a measure of lineage labelling. The control MO was the standard Gene Tools control 25-mer (5'-CCTCTTACCTCAGTTACAATTTATA-3'). The fezf2 e3i3 splicing MO sequence was 5'-AGGATTTCCCCCGTTCCGTACCTGT-3'.

Restriction enzyme mediated integration (REMI) and fluorescence stereoscope imaging
Construct assembly for REMI experiments was done using the pTransgenesis recombination system (Love et al., 2011). Briefly, either an NβT-tauGFP or an 7LEF-dEGFP (Denayer et al., 2006) cassette was placed in the p1 position such that both promoters transcribed in opposite directions. The 3kb fezf2 core promoter was isolated and placed in p3 site. Several p3 constructs were made from a starting vector containing a 2A construct flanked by two pairs of restriction enzyme sites. The VP16-Fezf2 construct has been described. The stabilised β-catenin construct was made by removing the ~270 bp region encoding the N-terminal GSK3β binding domain (Aberle et al., 1997). The dominant-negative Tcf3 construct was made by removing the ~150 bp N-terminal β-catenin binding domain (Molenaar et al., 1996). The constitutively-active GSK3β was made by mutating the serine 9 into alanine thus preventing its inhibition by PI3K/AKT signalling (Stambolic and Woodgett, 1994;Sutherland et al., 1993). Only VP16-Fezf2 was inserted upstream of the 2A short peptide sequence, while the other inserts were all placed downstream of the 2A sequence. The p1, p2, and p3 constructs were integrated into the pDEST R4-R3 destination vector and subsequently linearised using Aat II (NEB) for integration.
Standard REMI was performed as described (Ishibashi et al., 2008). After REMI, embryos were incubated in 16°C for 4 days (for in situ hybridisation) or 7 days (for immunofluorescence microscopy). Images were taken using a Leica M165FC stereoscope with colour CCD module attached.

Western blot and in vivo co-immunoprecipitation
For Western Blot analyses on the phosphorylation status of signalling molecules, each 7 injected X. laevis embryos were collected as a group at gastrula stage (10.5), extracted, and homogenized with SDS loading buffer for Western blot analysis. The Western blot protocol for analysing endogenous 1/5/8 and Smad2/3 has been described (Dorey and Hill, 2006). For RNA injection amounts, wild-type fezf2: 1ng; For in vivo co-immunoprecipitation, 50 X. laevis embryos were injected with tagged mRNA at 1-2 cell stage as amounts followed: fezf2-HA, 500 pg; ∆Eh1-fezf2-HA, 500 pg; tle1-FLAG, 500 pg; tle2-FLAG, 500 pg; tle4-FLAG, 500 pg; tes-FLAG, 500pg; wt-Fezf2-FLAG, 500 pg; Each injection was supplemented with GFP mRNA to achieve a total of 2 ng mRNA. Embryos were collected and homogenised at stage 10.5 using a modified IP-lysis buffer (50mM Tris-acetate pH 7.5, 300 mM NaCl, 1 mg/ml BSA, 2% NP40, protease inhibitor, and phosphatase inhibitor) followed by Freon extraction to remove excessive lipid in the extract. 10 µl of supernatant was kept as total input. 2 µg of anti-HA (Invitrogen) or anti-FLAG M2 (Sigma-Aldrich) antibody was added into 500µl extract and rotated for 2 hours at 4°C. Following the addition of 30 µl pre-incubated Protein A/G Sepharose (Santa Cruz), samples were rotated overnight at 4°C. Four rounds of washings were performed with washing buffer (50mM Tris-acetate pH 7.5, 300 mM NaCl, 1 mg/ml BSA, 0.5% NP40, protease inhibitor, and phosphatase inhibitor) and Sepharose beads were boiled with 20 µl of SDS-loading buffer for 10 minutes. The bead/buffer mix was loaded on a 10% SDS-PAGE gel. Western blot was performed on a semi-dry system according to the manufacturer's instructions (Hoefer). PVDF membrane (Millipore) was blocked with 5% dry milk powder in TBS containing 0.1% Tween 20 for 1 hour followed by incubation with anti-Flag-HRP (1:10000, Sigma) or anti-HA-HRP (1:50000, Roche) antibodies in blocking buffer overnight. Following three washing steps in TBS containing 0.1% Tween 20. Blots were developed using Immobilon ECL reagents (Millipore).

Cell culture transfections
Mouse c17.2 neuronal progenitor cells (ATCC) were a kind gift from Nancy Papalopulu. Briefly, cells were maintained in DMEM supplemented with 10% FBS with penicillin/streptomycin. Mouse Fezf2 and ∆NTcf3 plasmids were cloned using primers described into pCS107 vector. Plasmid DNA constructs were transfected into cells plated in LAB-TEK II chambered slides (NUNC) and allowed to proliferate until 10-15% confluence before transfection using Lipofectamin 2000 following manufacturer's instruction (Invitrogen). After transfection, the cells were incubated in DMEM with 10% FBS for 1 day and medium was subsequently changed to DMEM with 1% FBS to allow differentiation for 4 days. The following amounts of plasmid were used: mouse fezf2, 1 µg; mouse ∆NTcf3, 1 µg. Each transfection was supplemented with empty pCS107 vector to a total of 2 µg DNA.