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The authors show that the transcription factor Sp8 has an essential role …


Biology Articles » Developmental Biology » Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain » Materials and methods

Materials and methods
- Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain

Generation of Sp8 conditional mutant mice

Animal treatment and housing was in agreement with the regulations of LAVES (Landesamt für Verbraucherschutz und Lebensmittelsicherheit) in Oldenburg. The different alleles of Sp8 are represented in Additional data file 1. Exons are indicated by black boxes, LoxP sites by black triangles and FRT (Flip recombination target) sites by white triangles. The LoxP-FRT-PGKneo-FRT cassette was inserted 5' to exon 1 and the second LoxP site 3' to exon 3. Southern blot analysis, using probes A, B and C (Figure 1b) identified recombinant clones. Hybridization with probe D (Additional data file 1) confirmed the deletion of the wild-type Sp8 allele. Homozygous Sp8 floxed mice were maintained on a C57/BL6 background. Conditional Sp8 knockout animals were generated by mating Sp8 floxed mice with Foxg1-Cre mice [16] (cKO). Cre activity was monitored using R26R reporter mice [51]. Cre+ cells were traced in triple transgenic mice, obtained by crossing Sp8 floxed heterozygous mice (positive for the Cre recombinase) with R26R mice (cKO-R26R).

Genotyping was done by PCR using the following primers: Sp8 (1: CCA-ATG-GGA-GGA-AAA-CAC-ACC-CCC-TCT-TAC-TCC-TC, 2: CCA-GCT-TCC-TGG-ACT-CTT-TCA-GTA-TAG-TTT-TGA-AG, 3: GCG-TGC-AAT-CCA-TCT-TGT-TCA-ATG-GCC-GAT-C); Cre (creF: ATG-CTT-CTG-TCC-GTT-TGC-CG, creR: CCT-GTT-TTG-CAC-GTT-CAC-CG); β-galactosidase (lacZF: TTG-GCG-TAA-GTG-AAG-CGA-C, lacZR: AGC-GGC-TGA-TGT-TGA-ACT-G).

Embryo recovery and tissue sampling

Pregnancy of mated mice was determined by the appearance of the vaginal plug and defined as day E0.5. Staging of embryos was done according to the plug date. Mice were killed by cervical dislocation. Embryos or tissues were dissected, washed in cold phosphate-buffered saline (PBS) and fixed in 4% PFA/PBS (paraformaldehyde) for several hours overnight. After rinsing in PBS, tissues were processed for standard paraffin- or cryo-embedding. Tissues were cryo-protected by overnight incubation in 30% sucrose/PBS at 4°C. Embedding was done in tissue tec (Jung, Nussloch, Germany) and freezing on dry ice. For whole mount ISH, dissected embryos were processed through a methanol series and kept at -20°C.

Immunohistochemistry, X-Gal staining and in situ hybridization

Immunohistochemistry was performed on 18 μm cryosections, or paraffin embedded sections of 5 μm to 10 μm thickness. Antigens were generally unmasked by boiling in citrate buffer (Vector, Burlingame, CA, USA), as described elsewhere. Primary antibodies were μ-Pax6 (Babco, Richmond, CA, USA), μ-Gap43, μ-Tuj (Covance, Berkeley, CA, USA), μ-BrdU, μ-phospho-HistoneH3 (Abcam, Cambridge, UK), μ-BrdU/IdU (Caltag, Burlingame, CA, USA), μ-Tbr1+2 (gift from R Hevner), and μ-Reelin (gift from A Goffinet). Paraffin sections were dewaxed, rehydrated and rinsed in PBS. Cryosections were washed in PBS and postfixed in 4% PFA/PBS. After unmasking, sections were blocked in a solution containing PBT (PBS + 0.1% TritonX) and 10% FCS (fetal calf serum) for 30 minutes. Primary antibodies were incubated overnight at 4°C in blocking solution. Secondary antibodies were diluted 1:500 in blocking solution and incubated for 1–2 hours at room temperature. Secondary antibodies were Alexa594- or Alexa488-conjugated and raised against mouse-, rabbit- or rat antigens (Molecular Probes, Karlsruhe, Germany). Before mounting, sections were rinsed three times in PBT and sealed with Vectashield mounting medium, containing DAPI as nuclear counterstain (Vector).

X-Gal staining was performed on whole mount tissue or 18 μm cryosections. β-Galactosidase activity was developed in staining solution (PBS, 1 mg/ml X-Gal, 2 mM MgCl2, 0.01% SDS, 0.02% NP40, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6) for several hours overnight at 37°C. Specimens were then washed in PBS and postfixed in PFA. Sections were counterstained in a solution containing 0.1% neutral red.

ISH on 12 μm to 18 μm cryosections and whole mount ISH were performed using digoxygenin labeled riboprobes, as described previously [13,14]. Histological analysis was done on nissl stained or neutral red counterstained sections, following standard procedures.

Axon tracing

E18.5 brains were fixed in PFA overnight and dissected from the skull. After hemi-sectioning the brains with a blade, crystals of DiI and DiO (Molecular Probes) were placed into multiple, but comparable, locations of the cortex [11,48] across genotypes (n = 6 hemi-sections per genotype and experiment). The diffusion of the tracers was allowed to proceed for 4 weeks in PFA at 37°C. After embedding the brains in 5% LMP-Agarose, 100 μm coronal sections were cut on a vibratome and counterstained with DAPI containing mounting medium (Vector).

BrdU labeling and TUNEL assay

BrdU and IdU (Sigma, Seelze, Germany) uptake experiments were done by intraperitoneal injection (50 μg/g body weight) of nucleotides into pregnant mice. For pulse labeling, injected mice were sacrificed 30 minutes after injection. Tissues of BrdU-injected embryos were processed for paraffin embedding. Subsequently, 5–10 μm sections were used for immunohistochemistry.

The S-Phase labeling index was estimated by dividing DAPI+ from BrdU+ cells on complete forebrain sections. For fate mapping purposes, BrdU was injected at varying time points from E10.5 to E15.5. Tissues were dissected on E15.5 to E18.5, respectively. The cell cycle length was estimated by sequential BrdU/IdU injection, according to [30,31]. The (cell cycle) leaving fraction was counted by dividing IdU+/BrdU+ cells from IdU+ only cells on forebrain sections.

TUNEL assay was performed on 5 μm paraffin sections of E10, E12, E15 and E18 brains using a ApopTag Red In Situ Apoptosis Detection kit (Chemicon, Hampshire, UK) and following the manufacturer's advice. The amount of TUNEL+ cells in control specimens of every stage was defined as 100%. Apoptotic cell clusters were identified in cKO from E12.5 to E18.5 and counted as one apoptotic cell.

In vitro pull-down assay

The cDNA encoding Sp8 (Sp8FL, AA 1–486) and Sp8 lacking zinc fingers (Sp8ΔZn, AA 1–355) were amplified by PCR (MGI-Clone 2443471) using the following PCR primers, adding 5' BamHI and 3' EcoRI restriction sites: Sp8FL forward and Sp8ΔZn forward, G CGC GGA TCC ATG CTT GCT GCT ACC TGT AAT AAG ATC; Sp8FL reverse, G CGC GAA TTC CTC CAG GCC GTT GCG GTG; Sp8ΔZn reverse, G CGC GAA TTC CAG CCC TTT GCG ACG CAG GC. PCR products were then subcloned in frame into the BamHI and EcoRI restriction sites of the pGEX-4T-3 expression vector (Promega, Madison, WI, USA). GST and GST-Sp8 fusion proteins were expressed in Escherichia coli and purified following standard protocols. Equal amounts of GST-Sp8 proteins or GST were incubated with gluthathione-sepharose beads (Amersham, Piscataway, NJ, USA) and washed in PBS.

The cDNA encoding Emx2 (Emx2FL, AA 1–253) and Emx2 lacking the homeobox (Emx2ΔHox, AA 1–144) were amplified by PCR using the following PCR primers, adding 5' EcoRI and 3' NotI restriction sites: Emx2FL forward and Emx2ΔHox forward, G CGC GAA TTC ATG TTT CAG CCG GCG CC; Emx2FL reverse, GCG CGC GGC CGC ATC GTC TGA GGT CAC ATC; Emx2ΔHox reverse, GCG CGC GGC CGC GCC AGG GGT AGA AGG TGG ACG. Template DNA containing the Emx2 open reading frame was kindly provided by A Mallamaci. Amplified PCR products were then subcloned into the EcoRI and NotI restriction sites of the pCMV-TNT vector (Promega). [35S]-methionine labeling of Emx2 proteins was performed using the TNT Quick Coupled Transcription/Translation system (Promega).

The GST and GST-Sp8 bound beads were then incubated with [35S]-methionine-labeled Emx2 protein isoforms in 0.4 ml binding buffer (0.1 M NaCl, 0.01% NP-40). After incubation for 2 hours at 4°C, beads were washed five times in 400 μl binding buffer and then boiled in 40 μl of 10 × SDS-PAGE loading buffer (10% SDS, 10 mM β-mercaptoethanol, 20% glycerol, 0.2 M Tris-HCl, pH 6.8, 0.05% bromophenolblue). Solubilized proteins were separated by 15% SDS-PAGE and the radiolabeled proteins visualized by autoradiography.

Statistics and data processing

Statistics were calculated with Microsoft Excel. Quantifications always represent the mean values of tested specimens. Analysis included error bars and the mean deviation. The total sample number is indicated in the corresponding figure legends or text sections. The different parameters were counted on captured images. Images were processed with Adobe Photoshop 7.0.


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