- Research article
- Open Access
Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain
- Andreas Zembrzycki†1, 2,
- Gundula Griesel†1,
- Anastasia Stoykova1, 2 and
- Ahmed Mansouri1, 2, 3Email author
© Zembrzycki et al.; licensee BioMed Central Ltd. 2007
- Received: 22 February 2007
- Accepted: 30 April 2007
- Published: 30 April 2007
The forebrain consists of multiple structures necessary to achieve elaborate functions. Proper patterning is, therefore, a prerequisite for the generation of optimal functional areas. Only a few factors have been shown to control the genetic networks that establish early forebrain patterning.
Results and conclusion
Using conditional inactivation, we show that the transcription factor Sp8 has an essential role in the molecular and functional patterning of the developing telencephalon along the anteroposterior axis by modulating the expression gradients of Emx2 and Pax6. Moreover, Sp8 is essential for the maintenance of ventral cell identity in the septum and medial ganglionic eminence (MGE). This is probably mediated through a positive regulatory interaction with Fgf8 in the medial wall, and Nkx2.1 in the rostral MGE anlage, and independent of SHH and WNT signaling. Furthermore, Sp8 is required during corticogenesis to sustain a normal progenitor pool, and to control preplate splitting, as well as the specification of cellular diversity within distinct cortical layers.
- Additional Data File
- Cortical Arealization
- Medial Ganglionic Eminence
- Ventral Telencephalon
- Cortical Progenitor
The mammalian forebrain, with its components the basal ganglia (subpallium) and cortex (pallium), is a result of advanced evolutionary processes. Although several genetic pathways that establish cell diversity within the developing telencephalon have been identified, only a few factors have been shown to control the earliest steps of anteroposterior (A/P) and dorsoventral (D/V) patterning .
From embryonic stage E7.5 onwards, the telencephalic vesicles are progressively regionalized through complex interactions of secreted ligands from inductive centers, and by the regionalized or graded expression of transcription factors [1–3]. FGF (Fibroblast Growth Factor) signaling acts downstream of SHH (Sonic Hedgehog) and is required to both specify and promote the proliferation and/or survival of ventral cell types in the telencephalon [4–7], while WNT (Wint) signaling apparently elaborates archicortical morphogenesis [8, 9]. Interestingly, modulation of the normal expression gradient of Fgf8 in the early cortical primordium alters the molecular location and the size of cortical domains along the A/P axis [10, 11]. At the beginning of cortical neurogenesis, several transcription factors display graded expression in cortical progenitors along the main axes, which seems to confer cortical regional specificity [1–3]. The transcription factors Emx2 and Pax6 exhibit an opposing expression gradient along the A/P axis of the forebrain. Accordingly, single mutants of either Emx2 or Pax6 show a severe shrinkage of the corresponding cortical area, which normally expresses these genes at high levels . Of note, Emx2 is the only factor that has been shown to additionally affect the innervation of thalamic axons into the cortex [1–3].
The zinc-finger transcription factor Sp8 is expressed in the developing nervous system, limbs and the tail bud. Analysis of Sp8 knockout mice revealed severe truncations of the limbs and tail, while at the midbrain-hindbrain boundary (MHB) a defect of A/P patterning occurred (12, 13, 14). Interestingly, Fgf8 expression is affected in both cases, suggesting that Sp8 may be required for the maintenance of Fgf8 activity in these tissues. Recent evidence indicates that abolishment of Sp8 function (in the ventral telencephalon) provokes enhanced apoptosis in progenitors of the dorsal lateral ganglionic eminence (dLGE), causing the loss of specific olfactory bulb interneurons . Additionally, Sp8 displays a graded expression pattern in cortical progenitors, with highest expression in the medial pallium.
To gain more insights into the role of Sp8 in the developing telencephalon, we created Foxg1-Cre-mediated conditional Sp8 mutants. We present evidence that, in the absence of Sp8, while Shh signaling is normal, the D/V patterning at the medial telencephalic wall is perturbed. Midline derivatives of the subpallium are malformed or completely missing. Additionally, due to the modulation of the graded expression territories of Emx2 and Pax6 in pallial progenitors, a caudalization of cortical areas occurs. Our study indicates that Sp8 function is required to prevent pallial progenitors from apoptosis, and to control the molecular specification of subsets of cortical layer neurons. This is consistent with an essential role for Sp8 in the patterning of the developing forebrain along the A/P and D/V axes. Furthermore, our findings support the idea of a direct interaction between Sp8 and Emx2 proteins.
Sp8 mutant brains exhibit multiple malformations
Homozygous Sp8 mutants died at birth. Mutant E10.5 forebrains lacked detectable levels of Sp8 mRNA (Figure 1d). At midgestation cKO embryos showed strong craniofacial abnormalities (data not shown). Nissl-stained histological sections revealed that cKO embryos displayed a dysgenesis of the olfactory bulbs (data not shown) and SE, including an almost complete absence of the midline (15% penetrance, n = 25) that resulted in a mild rostral holoprosencephaly (Figure 1e, e', e''; Additional data file 2). In addition, the thickness of the cortical plate of the cKO cortex was reduced (Figure 1g, g'; 63.5% ± 5.1% of control, n = 10). The basal ganglia consisted of a single eminence (Additional data file 2) with a barely discernable constriction between the LGE and MGE. Corticofugal fiber tracts did not cross the midline and instead formed probst bundles (Figure 1e, e', e''). Caudally, neuronal fibers formed bundles between the internal and external capsules (arrows in Figure 1f, f', f'').
Sp8 modulates the D/V patterning of the medial telencephalon
To study whether the Sp8 mutation might affect pattern formation through the Wnt signaling pathway , we examined the expression of Wnt3a, Wnt5a and Wnt7b in the cortical hem and the Wnt antagonist Sfrp2 in the antihem. No change in the expression of these markers was apparent (data not shown). We concluded that Sp8 might act downstream or independently of Wnt signaling [5, 7, 10].
Abnormal cortical arealization and thalamic innervation in Sp8 mutants
To study whether the observed molecular caudalization of the Sp8cKO cortex reflects alterations of the cortical area identity, we performed retrograde labeling of thalamocortical (TCA) projections by placing crystals of the lypophilic dies DiI (red) and DiO (green) in the presumptive visual cortex or somatosensory area, respectively (insets in Figure 4g, g'). In controls, the red dye was exclusively present in cells within the dorsal lateral geniculate nucleus (dLGE; dorsal from the dashed line in Figure 4g), while the DiO labels (in green) cells in the ventroposterior complex (VP; ventral from the dashed line in Figure 4g). In the Sp8KO brain, the dLGE was labeled by TCA projections coming from both visual and somatosenory cortex (Figure 4g'). These results suggest that the molecular caudalization of the Sp8KO cortex causes a partial change in the cortical area identity (somatosensory to visual fate).
To elucidate whether the regulation of Emx2 by Sp8 might be direct or indirect, we performed biochemical in vitro experiments. Glutathione S-transferase (GST)-pull down assays revealed that truncated Emx2 protein, lacking the homeobox, does not interact with GST-Sp8 or GST-Sp8 without zinc fingers (Figure 4h, lanes 3 and 4). However, we found that GST-Sp8 as well as GST-Sp8 lacking zinc fingers are able to bind the full-length Emx2 protein in vitro (Figure 4h, lanes 7 and 8). Taken together, this indicates that the Sp8 expression gradient in cortical progenitors plays an important role in the correct positioning of distinct cortical domains along the A/P axis of the developing cortex by modulating the expression level of Emx2. Furthermore, our findings support the idea of a direct interaction between Sp8 and Emx2 proteins.
Sp8 controls cell survival in the developing forebrain
We next wanted to know whether the observed loss of progenitors might affect the generation of specifically early- or late-born cortical neurons . By injecting BrdU at E12.5 or E15.5 and sampling at E18.5, we created specimens that had BrdU exclusively incorporated in early- (E12.5) or late-born (E15.5) neurons. Analysis of the samples injected at E12.5 identified cells populating deep and intermediate positions of the CP (Figure 5f). In accordance with the detected apoptosis, the number of BrdU+ nuclei in mutant brains was reduced (Figure 5f' ; 64.7 ± 7.8% of wild type, n = 3), pointing to a diminished progenitor pool. Cells labeled with BrdU at E15.5 populated mainly deep compartments (VZ, subventricular zone (SVZ), intermediate zone (IZ)) of the cortex of both genotypes (Figure 5g, g'). However, the amount of BrdU+ cells within the putative SVZ, which were recently shown to generate exclusively upper cortical layers [33–35], appeared reduced in the mutants (yellow arrow in Figure 5g'). Conversely, more BrdU-labeled cells were detected in superficial positions in the CP (white arrow in Figure 5g'), most probably reflecting the reduction in the distance from the ventricular to the marginal zone in the mutant cortex.
The loss of Sp8 results in defective preplate splitting
Furthermore, we used a cell labeling approach consisting of injecting BrdU at E11 to label SP cells [36, 40] and then harvesting tissue after a visible separation of SP and CP at E15 (Figure 6a). In addition, the co-detection of BrdU and Tbr1 enabled us to follow the laminar position of the double positive cells, which in controls were found in the SP (arrow in Figure 6b). In contrast, in cKO these cells were located in virtually the most superficial part of the cortical plate (compare Figure 6b, b'). We conclude that proper PPL splitting does not occur in Sp8cKO.
In accordance with defective SP formation [37–40], Gap43 antibody staining revealed that the subcortical connectivity and the axonal wiring appeared abnormal in mutants. In contrast to controls, Gap43+ fibers in Sp8 mutants formed aberrant bundles within the internal capsule, with some axons projecting ectopically towards the MZ (arrows and arrowheads in Figure 6d, d') and basal telencephalon (Additional data file 2 (d, d'')).
Perturbed specification of distinct cortical layer neurons in Sp8 mutants
The orphan nuclear receptor RzR-β was utilized to follow layer IV genesis . At E18.5, RzR-β transcripts were completely missing in the CP of mutants (compare Figure 7f, f'). Moreover, the expression of Cux proteins in SVZ progenitors was recently shown to promote the fate specification of late-born neurons [34, 35]. Accordingly, we found that in the mutant, although Cux2 expression was reduced (Figure 7d, d'), Cux1 mRNA could not be detected at E18.5 (Figure 7c, c'). Along the same line of evidence, the expression of an additional upper layer neuron marker, Lhx2, is also highly down-regulated in the cortical plate of Sp8 mutants (Figure 7e, e'). This suggests that a reduction in the generation of late-born/upper cortical layer neurons occurs. We assayed Tbr2 immmunoreactivity. Tbr2 is a specific marker for basal/SVZ progenitors , which predominantly generate the upper cortical layers [34, 35]. In Sp8 mutants, the population of Tbr2+ (basal) progenitors was significantly reduced at E18.5 (49.4 ± 4.3% of controls, n = 3; Figure 7g, g'). This is consistent with a diminished pool of late progenitors, resulting in a diminished generation of upper cortical layer neurons in conditional Sp8 mutants.
The MZ mostly consists of Reelin+ Cajal-Retzius cells [1, 9, 37]. Using in situ hybridization (ISH) for Reelin mRNA (and a Reelin antibody for quantification) we found more Reelin+ neurons in cKO than in control littermates (142.2 ± 6.4% of controls; Figures 6c, c' and 7b, b'). In summary, these findings support the idea that the lack of Sp8 function during early neurogenesis is responsible for a severe depletion of the early and the late cortical progenitor pool, resulting in a misspecification of distinct cortical neuron subtypes, such as Cux1+, Lhx2+, RzR-β+, and ER81+ lineages.
We used conditional inactivation to study the role of Sp8, the ortholog of the Drosophila transcription factor buttonhead , during murine forebrain development. We report that the absence of Sp8 provokes a morphological dysplasia of the rostromedial forebrain, perturbs A/P patterning and enhances apoptosis of neuronal progenitors. A marker analysis further revealed that although the layering of the mutant cerebral cortex seems normal, Sp8 function is required for the specification of neuronal subpopulations.
Sp8 has an essential role in the formation of the telencephalic midline
One morphologically apparent defect was the dysgenesis of the septum. On the molecular level, this might result from the ventral expansion of Emx2, Pax6 and Ngn2 expression territories. As a consequence, the expression of several ventral markers, such as Fgf8, Mash1, Dlx1, and most importantly, Nkx2.1 is regionally down-regulated or completely abolished. Interestingly, Shh and Wnt expression seems to be preserved in the mutant, suggesting that the observed perturbation is independent of these signaling pathways. Our findings suggest that Sp8 might have a critical role in the maintenance of gene activity at the mPSB, since early marker expression is not affected. Interestingly, both Sp8 and Fgf8 are expressed in the septum anlage at early developmental stages. Recent findings demonstrate that FGF signaling is acting downstream of Shh to propagate ventral telencephalic cell types and to promote their survival . Our study reveals that, while preserved in the midline and septum anlage until E10.5, the expression of Fgf8 (in the septum) and Nkx2.1 (in the septum and rostral MGE) is completely abolished at E12.5. Moreover, a recent report provides evidence that Fgf8 may regulate Nkx2.1 expression, interfering with the axial patterning of the telencephalon , therefore suggesting that the midline defect in cKO could be mediated through Fgf8.
Although Sp8 is expressed in the dLGE, the patterning at the PSB is not disturbed. Two possibilities might be envisioned: first, the D/V patterning at the PSB is established before E9, 5; and second, functional redundancy may exist between Sp8 and the closely related transcription factor Sp9  in the ventral telencephalon (Additional data file 1).
Sp8 affects cortical arealization along the A/P axis
We recently have shown that Sp8 knockout mice display a patterning defect at the MHB . In the present study we demonstrate that, in addition to its role in the formation of a normal mPSB, Sp8 is necessary for the molecular arealization of the cerebral cortex along the A/P axis. The arealization of the early cortical primordium is dependent on the regionalized expression of ligands belonging to the FGF, WNT/BMP and epidermal growth factor signaling pathways, produced by the anterior neural ridge (ANR), cortical hem, roof plate and antihem, respectively [1, 3]. Such ligands are assumed to control the graded expression of transcription factors, encoding positional pattern, and specific for distinct cortical fields. So far, only a few regionally enriched transcription factors have been shown to be critical for this process. For instance, in mice where either Pax6 or Emx2 is not functional, the corresponding rostral and caudal cortical regions, where these genes display highest expression, appear malformed and cortical areas are displaced in opposite directions [2, 46].
Our findings suggest that the inactivation of Sp8 in the forebrain causes a prominent caudalization of the molecular properties of the cortical neuroepithelium, as highlighted by the ectopic rostral expansion of the expression domains of the regionally enriched marker genes Coup-TF1, EphrinA5 and EphA7. Furthermore, Sp8cKO cortices show an enhanced Emx2 and a reciprocally down-regulated Pax6 expression gradient. This supports the notion that the genetic interplay between Pax6 and Emx2 is controlling the establishment of their normal expression gradients . However, the loss of Emx2 or Pax6 function does not affect the expression of Sp8, suggesting that Sp8 acts upstream of these genes (Additional data file 1).
FGF signaling from the ANR plays a crucial role in the patterning along the cortical A/P axis . Evidence has been presented that Emx2 might indirectly control cortical arealization, through the regulation of Fgf8 . Recent data, however, challenged such a view by demonstrating that Emx2 may operate directly, and independent of, Fgf8 to specify cortical areas . In addition, it was shown that thalamocortical connectivity is affected only in the absence of proper Emx2 function , and conversely does not change in Fgf8 hypomorphic cortices in vivo . In good agreement with these findings we observe defects in thalamocortical projections in Sp8cKO cortices. Notably, the early FGF signaling from the ANR does not seem to be affected in the Sp8cKO forebrain until at least E10.5. Taken together, our findings strongly suggest leading roles for Emx2 and Sp8 in cortical arealization.
In agreement with findings in Drosophila, we show a direct interaction between Sp8 and Emx2 proteins in vitro, indicating a conservation of this regulatory pathway .
Sp8 plays a critical role in cortical neurogenesis
Enhanced apoptosis was detected in the limbs  and basal telencephalon in Sp8 loss-of-function mice . We show here that the forebrain hypoplasia in Sp8cKO mice is primarily due to cell death, affecting both early and late progenitor pools of dorsal and ventral telencephalon.
We further found that when the function of Sp8 is abolished, the preplate splitting is defective, and the MZ contains more Reelin+ cells , possibly due to the enhanced Emx2 expression in the mutant cortex. The basic lamination of the cortex is not compromised in cKO. However, the specification of particular neuronal subtypes (such as ER81-, RzR-β-, and Cux1-positive) is affected. Similarly, conditional ablation of Sp8 in the basal telencephalon results in misspecification of a subset of interneurons .
Our findings provide evidence that the transcription factor Sp8 is required for both the early patterning of the forebrain and the specification and survival of ventral cell types of the telencephalon. Our findings support the idea that a direct interaction between Sp8 and Emx2 controls D/V patterning of the medial telencephalon, functional arealization along the A/P axis, and the specification of subpopulations of cortical layers.
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  (cKO). Cre activity was monitored using R26R reporter mice . 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.
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' Bam HI and 3' Eco RI 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 Bam HI and Eco RI 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' Eco RI and 3' Not I 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 Eco RI and Not I 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.
We would like to thank A Driehorst, H Fett, and the BTL animal facility crew for the excellent animal caretaking and S. Blanke for helping with the Pull-down assay experiments. We are indebted to R Hevner for kindly providing Tbr1/2 antibodies and to A Goffinet for providing Reelin antibody and fruitful discussion and J Ericson for the Nkx6.2 probe. We further would like to thank SK McConnell for providing the Foxg1-Cre mice and A Mallamaci for providing the Emx2 cDNA. We additionally want to thank J Butler and H Fukumitsu for critically reading the manuscript. Finally, we would like to thank P. Gruss for constant support and encouragement. This work was supported by the Max Planck Society, the Dr Helmut Storz and Alte Leipziger Stiftung, the German Ministry for Education and Research BMBF (01GN0510), and the DFG Center for Molecular Physiology of the Brain (CMPB).
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