The C terminus of p73 is essential for hippocampal development

Significance Alteration of splicing is emerging as a relevant cause of human disease. The C-terminal region of p73 is subject to complex alternative splicing that can give rise to seven different isoforms. Here, using a newly generated mouse model, we determine the functional consequence of in vivo ectopic switch from the physiologically expressed and most abundant isoform p73α to the shorter p73β isoform. Expression of p73β leads to neurodevelopmental defects with functional and morphological abnormalities in the mouse hippocampus. The ectopic isoform switch results in depletion of Cajal–Retzius (CR) neurons in embryonic stages, leading to aberrant hippocampal architecture. Our study indicates that deregulation in p73 alternative splicing might underlie neurodevelopmental human conditions.


End-point PCR
Mouse genotyping was performed using a RED Extract-N-Amp TM Tissue PCR kit (Sigma) according to the manufacturer's instructions. The primers used are listed in Table 1. P73 C-terminal isoform-specific PCR was performed starting with brain RNA that was extracted using TRIzol ® Reagent (Ambion) according to the manufacturer's instructions. Two micrograms of RNA were reverse transcribed using SuperScript ® III Reverse Transcriptase with oligo-dT (Thermo Fisher) according to the manufacturer's instructions. The primer sequences for all Trp73 C-terminal isoforms are listed in Table 1. Reactions were carried out using the RED Extract-N-Amp TM PCR ReadyMix TM (Sigma). The products were run on a 1-2% agarose gel in TAE buffer and were visualized using a Gene Genius Bio Imaging System (Syngene). The obtained PCR products were sequenced.

Immunofluorescence (IF)
Dissected whole brains were fixed in 4% paraformaldehyde for 48 h and then were embedded in paraffin. Coronal brain sections (5 µm thick) were obtained for analysis of the hippocampus. Sections were deparaffinised by immersion in xylene (Sigma) followed by a gradient of 100%-70% ethanol (Sigma). Antigen retrieval was performed by heating in a microwave at 295 W for 15 minutes in 0.01 M sodium citrate. Tissue was blocked with PBS containing 5% normal goat serum for 1 h at room temperature, and then the tissue was incubated with a primary antibody in PBS containing 5% normal goat serum overnight at 4°C in a humidified atmosphere. See Table 2 for antibody information. Samples were washed 3 times in PBS and incubated for 1 h with Alexa Fluor TM secondary antibodies (Molecular Probes; Invitrogen) at RT. Samples were washed 3× in PBS and mounted using Prolong Gold Antifade mounting media (Thermofisher; P36930). Images were acquired through confocal microscopy (LSM 510, Zeiss). Number of Nestin/GFAP positive cell counting per microscopy field was determined using ImageJ.

Immunohistochemistry (IHC)
Tissue sections were fixed and embedded as described above. IHC was carried out using a Ventana Discovery Ultra platform (Roche). Deparaffinization was performed by immersion in Ventana Discovery Wash buffer (Roche; 950-510) for 3 × 8-minute washes at 69°C. Antigen retrieval was performed by incubation in Ventana CC1 buffer at 95°C for 32 minutes. The slides were blocked by incubation with Discovery goat IgG block (Roche; 760-6008) for 12 min at 37°C. Primary antibody incubations were carried out using EnVision Flex antibody diluent (Dako; DM830). See Table 2 for antibody information. The detection reaction was performed using an UltraMap DAB anti-Rb detection kit (Roche) for single staining of TAp73 or an UltraMap HRP anti-Rb detection kit (Roche) in combination with an UltraMap AP anti-Ms detection kit (Roche) to co-stain TAp73 and Reelin, according to the manufacturer's instructions. H&E staining was performed with the following sequential steps: 2 oven incubations (15 min each), 2 incubations in xylene (2 min each), 3 incubations in 100% IMS (1 min each), wash with water (1 min), incubation in Harri's haematoxylin (15 min), wash with water twice (1 min each), incubation in 1% HCL alcohol (30 secs), wash with water (6 min), wash with water (2 min), incubation in 0.5% eosin (3 min), wash with water (2 min), incubation in 80% IMS (1 min), 3 incubations in 100% IMS (1 min each), and 3 incubations in 3 xylene (1 min each). The images were captured with a NanoZoomer XR digital pathology slide scanner, and images were processed with NDP.view.2.3.1 software (Hamamatsu). Number of Ki67 positive cell counting, normalised on the total number of nuclei, was determined using ImageJ.

In situ hybridization (ISH)
Mouse brains were embedded in frozen specimen medium Killik (Bio-Optica) after an overnight incubation in 4% paraformaldehyde followed by an overnight incubation in 0.5 M sucrose. Thin sections (14 μm) were cut and mounted on Superfrost glass slides. Slides were then fixed for 10 min in 4% paraformaldehyde and were acetylated for 10 min in triethanolamine/acetic anhydride. Slides were then hybridized overnight at 46°C using 30 nM digoxigenin probes for detection (miRCURY LNA; Exiqon). After hybridization, slides were washed (20 min in 5× SSC, two times for 30 min in Tween 20/SSC at 50°C, two times for 15 min in 0.2× SSC and 15 min in PBS at RT). After 1 h of incubation in a blocking solution at RT, slides were hybridized for 2 h with an alkaline phosphatase-conjugated antidigoxigenin Fab fragment (1:200 dilution; Roche) at RT. After two 20 min washes, detection was performed by incubating 250 μl of nitroblue tetrazolium/BCIP (1-STEP; Thermo Fisher Scientific) together with 2 mM levamisole on the slides for 16 h in the dark at RT. Table 1 lists the primers used to generate the ISH probe.

Neurosphere assay
Whole cortexes from E14.5 mice were carefully dissected and immediately stored in cold Dulbecco's phosphate-buffered saline (StemCell Technologies; without Ca ++ and Mg ++ ) containing 2% glucose. A single cell suspension of cortical neurons was obtained by carefully pipetting the whole cortex (E14.5) in neuronal basal medium supplied with NeuroCult ® Stem Cell Proliferation Supplements (StemCell Technologies). For each passage in culture, 5x10 5 cortical neurons were seeded in 10 mm dishes. Images were acquired with an Axiovert 25 microscope (Zeiss). Neurosphere density and diameter were determined using ImageJ.

Real Time qPCR
RNA was extracted from the hippocampus and cortex using TRIzol ® Reagent (Ambion) according to the manufacturer's instructions. The concentration and purity of RNA were measured using a nanodrop spectrophotometer (Thermo Fisher). One microgram of RNA was used for reverse transcription reaction using a RevertAid minus first strand cDNA synthesis kit (Thermo Fisher) according to the manufacturer's instructions. Primer sequences (Sigma) for TAp73 and ΔNp73 are listed in Table 1. Reactions were carried out in triplicate using Fast SYBR Green PCR Master Mix (Thermofisher #4385612). The relative quantification was obtained using an Applied Biosystems 7500 thermocycler, and quantitation was calculated with the comparative ( ΔΔ Ct) method; expression values were normalised to gapdh, which was used as an endogenous control.

Ultrathin Section Transmission Electron Microscopy (TEM)
Mouse brains were fixed with 2.5% glutaraldehyde (GA) and 2% paraformaldehyde (PFA) in NaHCa buffer (100 mM NaCl, 30 mM HEPES, and 2 mM CaCl2, adjusted to pH 7.4 with NaOH) for more than a few hours at room temperature. The specimens were then subjected to conventional post-fixation treatment with 0.25% osmium tetroxide/0.25% potassium ferrocyanide and 1% tannic acid. After staining en bloc with 5% aqueous uranyl acetate, dehydration with an ethanol series and infiltration were completed for plastic embedding in TER (TAAB epoxy resin). After the polymerization at 65°C for a few days, ultrathin sections (∼60 nm) that were obtained from an Ultramicrotome (Leica Ultracut UCT, Vienna Austria) were mounted in EM grids, stained with lead citrate, and then observed by FEI Talos F200C 200 kV transmission electron microscopy (Thermo Fischer Scientific, Oregon USA) with a Ceta-16M CMOSbased camera (4 k x 4 k pixels under a 16 bit dynamic range).

Western Blot (WB)
Protein extraction was performed on ice. Dissected whole hippocampi were washed in PBS and lysed by homogenization in RIPA buffer (1 M Tris-HCL pH 7.4, 5 M NaCl, 10% sodium deoxycholate, 10% sodium dodecyl sulphate, 1% NP-40, and 0.1 M PMSF) supplemented with a protease inhibitor cocktail (Sigma) and 1 M dithiothreitol (DTT). Debris was pelleted by centrifugation at 10,000 x g for 5 minutes, and the supernatant was collected. Protein concentrations were determined using a BCA assay (Thermo Fisher). Laemmli sample buffer (4x) (BioRad) was added to the protein samples, which was followed by denaturation: heating at 98°C for 10 minutes. Fifteen micrograms of protein was run on an 8% resolving gel (National Diagnostics) at 150 V for 1 h. Protein was transferred to PVDF membranes (Thermo Fisher) in 1x transfer buffer (Thermo Fisher) containing 10% (v/v) methanol by applying 0.8 mA per cm 2 of membrane for 2 h on ice. Membranes were blocked in 5% milk (Marvel) in TBS/0.1% Tween 20 (TBST) for 1 h at RT before incubation with a primary antibody (diluted in 5% milk in TBST) overnight at 4°C. See Table 2 for antibody information. Membranes were washed 3X in TBST and were incubated with a goat anti-mouse/HRP conjugated secondary antibody (Thermofisher #31430) diluted 1:10000 in TBST for 1 h at RT. Membranes were washed 3X in TBST and then were incubated with SuperSignal TM West Dura Extended Duration Substrate (Thermo Fisher) for 2 min before visualisation using radiographic films.