hGFAP-mediated GLI2 overexpression leads to early death and severe cerebellar malformations with rare tumor formation

Summary The zinc-finger transcription factor GLI2 is frequently amplified in childhood medulloblastoma of the Sonic-hedgehog type (SHH-MB), with or without amplification of NMYC or deletion of TP53. Despite the aggressive tumor behavior, tumorigenesis is not well understood, and adequate mouse models are lacking. Therefore, we generated mice with a GLI2 overexpression under control of the hGFAP-promoter. These mice died within 150 days. The majority only survived until postnatal day 40. They displayed severe cerebellar hypoplasia, cortical malformations, but no brain tumors, except for one out of 23 animals with an undifferentiated hindbrain lesion. Additional loss of p53 did not result in cerebellar tumors, but partially rescued the cerebellar phenotype induced by GLI2 overexpression. Similarly, the combination of GLI2 and NMYC was neither sufficient for the development of SHH-MB. We therefore assume that the development of childhood SHH-MB in mice is either occurring in cellular origins outside the hGFAP-positive lineage or needs additional genetic drivers.


INTRODUCTION
Sonic hedgehog (SHH) signaling is evolutionary conserved and essential for the development and differentiation of different organs, such as the central nervous system. [1][2][3] The Shh signaling pathway comprises two transmembrane proteins Patched (PTCH), a 12-transmembrane protein, and Smoothened (SMO), a 7-transmembrane protein that acts as a signal transducer. PTCH binds Shh to activate the pathway, whereas, in the absence of ligands, PTCH interacts with and inhibits SMO. 4 The hedgehog ligands disassemble PTCH inhibition of the transmembrane protein SMO. Additionally, SUFU (suppressor of fused homolog) acts as a negative intracellular regulator and segregates full-length Gli proteins in the cytoplasm. This all allows the transcriptional activation of GLI target genes. 1,5 The GLI family consists of GLI1, GLI2, and GLI3. 5,6 GLI1 is a downstream target of the SHH pathway. GLI2, also named GLI family zinc finger 2, and GLI3 are transcriptional mediators in SHH activation controlled by posttranscriptional and posttranslational alterations. 1,5-10 GLI2 also plays a key role as transcriptional effector in the SHH pathway, e.g., in the skin, and is known to be frequently amplified and overexpressed in pediatric brain cancer, such as pediatric SHH-medulloblastoma (MB). In SHH-MB, GLI2 occurs in combination with amplifications of NMYC or deletion of TP53. 6,10,11 MB is the most common malignant brain tumor in children, representing 60% of childhood intracranial embryonal tumors and the leading cause of pediatric cancer deaths. 6,12,13 While PTCH1 mutations occur at all ages in SHH-MB, SUFU mutations are enriched in infant SHH-MB, and SHH-MB in adolescence/adulthood often harbors SMO mutations. However, the most aggressive subtype of SHH-MB occurs in childhood. Such tumors often appear histologically as severely anaplastic and are characterized by TP53 mutations as well as NMYC and GLI2 amplifications. 6,9,[14][15][16] GLI2 is therefore believed to function as an important oncogene in SHH-MB. [17][18][19] Regardless of all the research in MB, mechanisms and targetable components in childhood SHH-MB are still not fully understood. Intensive radiotherapy or chemotherapy that may lead to the cure of the disease often shows significant long-term side effects including cognitive deficits and the risk of secondary neoplasms.
Overexpression of NMYC or deletion of p53 in combination with GLI2 overexpression is insufficient to drive cerebellar tumor formation Transgenic mice were examined for potential tumor development at different time points as soon as they had to be sacrificed due to symptoms. Representative cerebellar sections (H&E, Ki67, and NeuN staining) of 10-to 31-day-old mice of all generated genotypes are presented in Figure 2. Tumor formation was not observed. Figure 2A demonstrates a 10-day-old hGFAP-cre control mouse with regular structures of the cerebellum. In contrast, all of the 23 hG::Gli2N + mice showed an unstructured cerebellum with ectopic cell clusters (Figure 2B). These mice were sacrificed due to signs, such as lower body weight, smaller than control littermates, strong ataxia, and imbalance, which was most likely caused by the cerebellar dysfunction. Likewise, hG::p53 Fl/+ ;Gli2N + mice ( Figure 2C) showed cerebellar dysfunction. The clinical symptoms were comparable to those of hG::Gli2N + mice. hG::p53 Fl/Fl ;Gli2N + mice displayed a grossly normal structure of the cerebellum except for some multinucleated giant cells that were observed throughout the cerebella and show Ki67 staining, but no NeuN staining ( Figure 2D, arrows). hG::NMYC Fl/+ ;Gli2N + either showed a cerebellar disorder although proliferation was nearly absent at P31 as expected ( Figure 2E) or a phenotype similar to hG::NMYC FlFl+ ;Gli2N + mice. Two out of 13 hG::NMYC Fl/+ ;Gli2N + mice did not show any phenotype. hG::NMYC Fl/Fl ;Gli2N + did neither show any cerebellar abnormalities ( Figure 2F).
To gain insight into the postnatal stages of cerebellar development, we present in Figure 3 Gli2N + , hG::Gli2N + , hG::p53 Fl/l ;Gli2N + , hG::p53 Fl/Fl ;Gli2N + , and hG::NMYC Fl/+ ;Gli2N + mice at postnatal day 7. The cerebellar cortex is arranged in three laminae: molecular layer, Purkinje cell layer (PCL), and granular layer (GL). In Figure 3 the Granule cell layer (GL) and Purkinje cell layer (PCL) are demonstrated. GL and PCL look characteristic for the control mouse ( Figure 3A). For hG::Gli2N + and hG::p53 Fl/+ ;Gli2N + mice in Figures 3B and 3C iScience Article post-mitotic granule neurons. It should be pointed out that giant cells found in hG::p53 Fl/Fl ;Gli2N + mice were already visible at P7 and were positive for Ki67 ( Figure 3D, arrow), while the remaining inner GL was negative. In contrast, NeuN expression was strong in the inner GL, but not in the giant cells itself (Figure 3D, arrows). For hG::NMYC Fl/+ ;Gli2N + mice at p7, the Ki67 staining was strongly visible in the external GL, which was lost at p31 as seen in Figure 2E. Positive calbindin staining of the PCL was demonstrated in all mouse models ( Figures 3A-3E, 6 th column, arrows). Vector plasmid construct, breeding schemes for Gli2 mice, and survival analysis (A) Illustrated is the vector plasmid construct, which was applied for pronuclear injection. Containing a lac-promoter part, the strong CAG-promoter (Cytomegalovirus early enhancer, the first intron of chicken beta-actin gene, the splice acceptor of the rabbit beta-globulin gene), T7 RNA-Polymerase promoter for transcription, the STOP cassette comprising three SV40-derived poly(A) signal repeats flanked by two loxP sites, the truncated GLI2N insert, a ZsGreen sequence (Zoanthus green fluorescent protein), a mammalian terminator bGH (bovine growth hormone), a lacZ gene coding for beta-galactosidase, f1_ori (DNA replication of bacteriophage f1 functions as a signal for initiation of viral strand synthesis and for its termination), and the Amp-promoter for ampicillin resistance. (B) The breeding schemes for the Gli2 mouse models are demonstrated. The transgenic line expressing Cre recombinase under the control of the human glial fibrillary acidic protein (hGFAP) promoter was crossed with CAG-lsl-Gli2N mice. The CAG promoter was applied to drive expression of a construct composed of floxed sites followed by an active form of GLI2 lacking the N-terminal repressor domain (GLI2N), resulting in hGFAP-cre::CAG-lsl-Gli2N transgenic mice. To obtain double-mutant mouse models, hGFAP-cre::p53 mice were intercrossed with CAG-lsl-Gli2N + ;p53 mice to achieve hGFAPcre::p53-CAG-lsl-Gli2N + (heterozygote or homozygote). hGFAP-cre::lsl-NMYC mice were crossed with CAG-lsl-Gli2N; + lsl-NMYC mice, resulting in hGFAP-cre::lsl-NMYC-CAG-lsl-Gli2N + mice, in heterozygous or homozygous results. More simplistic names were given to the mouse models as demonstrated in brackets. In hG::p53 Fl/Fl ;Gli2N + animals ( Figure 4A), giant cells were observed close to the piriform cortex ( Figure 4B), near the hippocampus area and caudate putamen ( Figure 4C), and in the cerebellum ( Figure 4D). These atypical cells expressed Ki67 ( Figures 4B-4D, insets). In Figure 4E, hG::p53 Fl/+ ;Gli2N + mice are  iScience Article demonstrated in a more precise analysis using two further immunohistochemically markers, i.e., Olig2 (Oligodendrocyte transcription factor 2) and Cyclin-D1. [30][31][32][33] Remarkably, in the hG::p53 Fl/+ ;Gli2N + mice, we saw Olig2 expression in white matter as well as in the giant cells described earlier ( Figures 4E and  4G). Besides Olig2, Cyclin-D1 expression was demonstrated in the giant cells ( Figures 4F and 4H). With these observations, we have good evidence that these Ki67-positive cells do not belong to the granule cell lineage.
One out of 23 hG::Gli2N + mice developed a large tumor in the hindbrain ( Figures 4I-4L) containing Ki67positive proliferating cells ( Figure 4K), but hardly any expression of NMYC, which is a typical marker of SHH-MB in humans ( Figure 4L). Also, this tumor appears to arise from the brain stem, but not from the external granular layer of the cerebellum, where SHH-MBs usually appear. One animal out of 13 hG::NMYC Fl/+ ;Gli2N + mice ( Figure 4M) showed proliferative cell clusters in the ventral brain stem (Figure 4N) and in the subventricular zone (SVZ, Figure 4O). These structures show a clear Ki67, but barely  Tumor-like structures demonstrated in the brain stem ( Figure 3N) and in the subventricular zone (SVZ) ( Figure 3O) Figure 4P). These results implicate that regulation of GLI2 is essential for the cerebellar development, but a development of childhood SHH-MB either requires further genetic drivers or arises in cell types outside the hGFAP-positive lineage.
Screening of spinal cords and specific immunohistochemically staining of hG:p53 Fl/+ ; Gli2N + mice We next investigated the spinal cords of the five different mouse models, since 2 out of 14 hGFAP-cre:: CLEG2 Fl/+ mice described by Han et al. showed tumor development in the brain or spinal cord. 28 However, H&E-stained spinal cords of our mice did not show any metastasis or tumor-like structures ( Figure 5).
Western blot analysis verifying murine GLI2, human Gli2N, Gli1, and NMYC expression in mouse cerebellum We further examined GLI2 expression in our mouse models, using western blot analysis ( Figure 6). We detect a predominant band at 190 kDa corresponding to murine GLI2 in mouse brain verifying the expression of murine GLI2 in the transgenic mouse models ( Figure 6A). The GLI2 polyclonal antibody (PA1-28838) corresponds to the amino acids 1193-1209 of mouse Gli2 with homology to human Gli2.
In Figure 6B, the expression of human Gli2N is demonstrated. The GLI2 antibody (Middle Region, ABIN2777474) recognizes the human sequence RNDVHLRTPL LKENGDSEAG TEPGGPESTE ASSTS-QAVED CLHVRAIKTE from human Gli2 in the middle region, which is also found in our Gli2N construct and therefore in the Gli2N expressing mouse models. In western blot analyses, this detection antibody can verify human Gli2N isoform as well as the full-length Gli2. In Figure 6B, all five mouse models show a band at the expected size for Gli2N, albeit the expression in hG::p53 Fl/+ ;Gli2N + and hG::NMYC F/l+ ;Gli2N + mice was weaker compared to the corresponding homozygous mouse model and the hG::Gli2N + mice ( Figure 6B). HEK293T cells were used as negative control and did not show any band as expected. MDA-MB-231 were used as positive control and showed, as expected, a band at the level of the full-length Gli2.
Gli1 is known to be an early target gene induced by SHH signaling, beside PTCH1 and PTCH2. 34 To demonstrate SHH target gene activity in our mouse models, we also did a western blot analysis with a Gli1-specific antibody. All mouse models express Gli1 in the cerebellum ( Figure 6C). However, as also observed in the western blot with the Gli2N antibody, the heterozygous models hG::p53 Fl/+ ;Gli2N + and hG::NMYC Fl/+ ;Gli2N + also show weaker expression here compared to the homozygous mice and hG::Gli2N + mice. To demonstrate the NMYC expression in the different mouse models, a western blot analysis using a specific NMYC antibody was done. All mouse models express NMYC ( Figure 6D; NMYC) showing a clear and strong band. The constitutively expressed housekeeping genes used here were a-tubulin ( Figure 6A), b-actin ( Figures 6B and 6C), and GAPDH ( Figure 6D).

DISCUSSION
Even though it is believed that GLI2 acts as an oncogenic factor in SHH-MB, it is not yet clear how GLI2 mutations act in MB development. 7,10 Therefore, we established the described mouse models using a dominant-active GLI2N lacking the N-terminal repressor domain of GLI2 (missing 328 amino acids). 10 GLI2N is  28,35 both models using the CLEG2 transgene with a constitutively active CAG promoter driving expression of EGFP in absence of Cre expression. A polyA sequence prevents the transcription of the GLI2N transgene, which is found at the 3-terminus of the EGFP cDNA, flanked by loxP sites. 35 In our mouse model, we used the GLI2N DNA from the pCS2-MT-GLI2deltaN plasmid and cloned it into the pCAG-loxPSTOPloxP-ZsGreen vector. 10 By this, our construct was different, and we used ZsGreen for possible tumor detection. Within these unique mouse models, the survival rate of our hG::CAG::Gli2N + mice was comparable to hGFAP-cre::CLEG2 fl/+ mice from Han et al. described previously. 28 In our mouse model, besides Gli2, additional mutations (NMYC and p53) were chosen because of the following reasons: On the one hand, 10% MB cases show a GLI2 activity and 18% also show a genetic event directly targeting the abundance or rather stability of MYCN. In a cohort of SHH-MB patients, GLI2 and MYCN amplifications also occurred simultaneously. 6,9,12 On the other hand, in SHH/TP53 mt MB patients (generally children, 4-17 years), amplification of GLI2, MYCN, or SHH arises, while mutations in SUFU and SMO are rare or absent. 9 Therefore, beside hG::Gli2N + mice, hG::p53;Gli2N + and hG::NMYC;Gli2N + (the last two homozygous or heterozygous) mice were also established. Anyhow, none of our transgenic mice developed MB. In some animals, tumors/ tumor-like structures in the brain occurred, although very infrequently. This is comparable to the observations by Han et al., 28 where only 2 out of 14 hGFAP-cre::CLEG2 Fl/+ mice developed a tumor in the brain or spinal cord, but no MB. In our mouse model, we detected a large tumor near the brain stem, pons, and cerebellum in 1 out of 23 hG::Gli2N + mice, which was comparable to the one described for hGFAP-cre::CLEG2 Fl/+ mice by Han et al. 28 As postulated by Han et al., we agree that it is most likely not an SHH-MB. Apart from the lacking expression of MYCN, the localization of this lesion is not typical for SHH-MB, which usually origin from the external granular layer, i.e., the surface of the cerebellum. Yet, we did not find tumors in the spinal cord. This was not only true for the p53-deficient model but also for the model overexpressing MYCN. This also indicates that overexpression of MYCN in hGFAP-positive cells is not sufficient to drive MYCN-associated ependymoma that were recently described in humans. 36 Additionally, we observed that in hGFAP-expressing cells, MYCN overexpression alone did not result in the development of MB. Also, mice with a single copy of MYCN as well as a combination of a Brg1 knockout and an overexpression of MYCN in multipotent neural stem cells or cerebellar granule neuron precursors were not adequate to drive brain tumor formation such as MB in mice. 33 Ectopic cell clusters in the cerebellum detected in hGFAP-cre::CLEG2 Fl/+ mice were identified in virtually all of our 23 hG::Gli2N + mice and likewise in hG::p53 Fl/Fl ;Gli2N + mice. 28 The latter showed a cerebellar disorder comparable to mice with only GLI2 overexpression.
hG::p53 Fl/Fl ;Gli2N + mice showed Olig2 expression in the so-called giant cells, which were also Cyclin-D1 positive. Olig2 was identified as transcription factor for expression of myelin-associated genes in cells of the oligodendrocyte lineage. 30 Cyclin-D1, a transcriptional target in the SHH pathway, directly regulates proliferative and immature states of cerebellar granule cell progenitors. 31 Furthermore, Gli1 also showed strong expression in cell lysates of hG::Gli2N + and hG::p53 Fl/Fl ;Gli2N + mice as well as all other described mouse models. Gli1 is a target and mediator of Shh signaling in e.g., ventral neuronal differentiation. It was initially acknowledged as an amplified nuclear oncogene in human sarcomas or gliomas. 34 Although the SHH signaling pathway is most likely activated, a heterozygous loss of P53 additionally to the GLI2 overexpression did not lead to development of MB, but to neurological symptoms such as ataxia, paralysis, and bodyweight loss, also observed for hG::Gli2N + mice.
60% of hG::p53 Fl/Fl ;Gli2N + mice showed proliferating giant cells in the brain. Indeed, loss of TP53 is frequently observed in large cell MB as it is in so-called giant cell glioblastoma. 32 iScience Article the giant cells in the hG::p53 Fl/Fl ;Gli2N + mice also showed positive Olig2, Cyclin-D1, and Ki67 staining, but no NeuN staining. With these observations, we have good evidence that these giant cells do not belong to the granule cell lineage. One could also postulate that these giant cells are early progenitor oligodendrocytes.
As mentioned previously, apart from models with loss of P53, we developed hG::NMYC Fl/Fl ;Gli2N + and hG::NMYC Fl/+ ;Gli2N + mice. In the latter, we observed in one animal tumor-like structures that proliferate in the SVZ and brain stem as well as cerebellar disorder in 4 out of 13 mice. We observed a pathological unstructured cerebellum only in hG::NMYC Fl/+ ;Gli2N + mice or in mice with a heterozygous deletion of p53. Homozygous expression of NMYC or homozygous deletion of p53 did not lead to this cerebellar structure. One could postulate here that the overexpression (Gli2N) and knockdown/loss (p53) were potentially not exactly parallel events in our models and influence each other. Nevertheless, we demonstrate Gli2N and NMYC expression in all mouse models described here, although a markedly weaker expression of Gli1 and Gli2N was detectable in hG::p53 Fl/+ ;Gli2N + and hG::NMYC Fl/+ ;Gli2N + animals.
Since more than a quarter of MB patients show activation of the SHH signaling pathway shown by GLI2/1 immunopositivity, 11,39 activated forms of GLI2 preserve pathway activity and support survival of the tumor cells, indicating GLI2 activity as a key driver for MB. 40 High level of Gli2 expression has been shown in  iScience Article several mouse models, but as mentioned, the overexpression alone is not sufficient to drive MB development, [20][21][22] but basal cell carcinoma (BCC) in the skin. 25 In contrast, an orthotropic granule neuron progenitors (GNP) transplantation mouse model was shown to develop MB development after Gli1 expression.
Here, primary GNPs expressing GLI1/GFP were injected and resulted in cerebellar tumor formation. 41 Since this was not a transgenic mouse model, it is difficult to compare to our mouse models, because it is another model system. But, transgenic mice with a loss of Sufu alone do not show MB formation, because GLI2 activation is inadequate. 7 However, Sufu loss (knockout-background) and Spop (E3 ubiquitin ligase) re-establishes GLI2 activation and MB is induced. 7 Therefore, we postulated that our mouse models could be an option for a MB-mouse model, which mimics GLI2 overexpression and additional mutations in the SHH pathway, since Gli2N expression could be measured in all five mouse models, but in both heterozygous models it was weaker compared to the others.
Nevertheless, this work adds valuable information to the research for transgenic MB mouse models and origination of MB development and paves the way for further investigation.

Limitations of the study
Limitations of the study include the following: (1) the low number of hG::NMYC Fl/Fl ;Gli2N + mice. In the matings where one expected hG::NMYC Fl/Fl ;Gli2N + mice, less pups were born compared to the births after the mating for the other described mouse models. Additionally, pre-weaning loss was a problem. Since we stick to the three Rs principle, we disclaimed unessential breeding's to minimize an excess of mice with a wrong genotype. (2) The combination of GLI2 and NMYC or GLI2 and p53 was neither sufficient for the development of SHH-MB. The regulation of GLI2 is therefore crucial for proper cerebellar development. Nevertheless, these data suggest that the development of childhood SHH-MB is either occurring in cellular origins outside the hGFAP-positive lineage or needs an additional genetic driver modification. Regarding the last point, we are currently working on new models.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: iScience Article the correct construct, sequencing analyses were done using specific oligonucleotides. The cre-responsive fluorescent reporter plasmid contains a STOP-cassette flanked by loxP sites followed by ZsGreen. The activity of Cre-driver candidates in mice, which is e.g. Gli2N in our model, can be detected using a ZsGreen reporter plasmid, based on the STOP cassette of the CAG-floxed ZsGreen plasmid. In principle, one could see a specific expression of ZsGreen in the appropriate tissues as prescribed by the expression pattern of Cre recombinase. So in the presence of Cre recombinase, loxP site-specific excision of the STOP cassette occurs which results in expression of the ZsGreen gene driven by the ubiquitously active chicken b-actin promoter, which is linked with a CMV early enhancer (CAG). Pronuclear injection of the SapI/NotI-fragment into C57BL/6J mice was performed according to standard procedures (Transgenic Mouse Facility, ZMNH/UKE, Hamburg, Germany). Positive founders or heterozygous animals were selected by genotyping and identified by PCR using the following primer pairs: GCCTCTGCTAACCATGTTCATGCCTTC and GATCTAGCTTGGGCTGCAGGTCGAG, CCCGC CTGGAGAACCTGAAGACA and CTCTCGGTCTTGATGGCTCTGACGT, CATCATGGATGATGGCGATCAC TCGAG and CCTTGGTCAGGCCGTGCTTGGACT. PCR genotyping was performed with DreamTaq Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) using standard protocols. The founder animal was chosen for further breeding upon the following criteria: No phenotype, good breeder, no leaky expression before removal of the Stop-cassette, sufficient expression of the transgene after removal of the Stop-cassette.

Generation of mouse models and genotyping
hGFAP-cre animals (JAX #004600) were bred with CAG-lsl-Gli2N animals. CAG-lsl-Gli2N mice were bred with lsl-p53 e2-e10 mice (JAX #008462) or with lsl-NMYC mice. 43 For double mutant mice, the CAG-lsl-Gli2N mice interbred with p53 e2-e10 mice were crossed with hGFAP-cre::p53 animals, resulting in hGFAP-cre::p53-CAG-lsl-Gli2N animals. Similarly, lsl-NMYC::lsl-CAG-Gli2Nmice were crossed with hGFAP-cre::lsl-NMYC mice, resulting in hGFAP-cre::lsl-NMYC-CAG-lsl-Gli2N mice. Mice of both sexes were used for the experiments and were kept in individually ventilated cages (IVC) on a constant light-dark rhythm of 12/12 hours, water and food were given ad libitum. The experimental procedures were performed in accordance with the German Animal Welfare Act and approved by the Government of Hamburg, Germany. The termination criteria which were use in the here described mouse model were weight loss >15% over 24 h, altered grooming condition, shaggy coat, changed posture, segregation from the group lasting for more than 2 days, paralysis which means partial or complete loss of function of one or more extremities, akinesia which means lack of movement to immobility, motor disorders such as gait (ataxia), and balance disturbances. Additionally decreased motor activity (decreased reaction to touching the mouse) and indications of pain (e.g. increased respiratory rate, biting, aggressiveness, lethargy, photophobia, prolonged sleep, increased defensive reaction) are part of the stop criteria. To determine the genotype of the mice, DNA was extracted from tail tips at postnatal day 3 (P3). Genotyping PCR analysis was done with the following oligo nucleotides: Gli2 CATCATGGATG ATGGCGATCACTCGAG, CATCATGGATGATGGCGATCACTCGAG and CCTTGGTCAGGCCGTGCTTG GACT, p53 GCACCTTTGATCCCAGCACATA and CACAAAAAACAGGTTAAACCCAGC, NMYC ACCACA AGGCCCTCAGTACC, TGGGACGCACAGTGATGG, CTGAGTGACAGCACCCCTTT, GTTTCCTCCGTGGT GAGGTT, CTCTTCCCTCGTGATCTGCAACTCC and CATGTCTTTAATCTACCTCGATGG.

Immunohistochemistry of brain and spine sections
Mouse brains and spines were fixed in 4% paraformaldehyde for at least 24 h. Spines were decalcified in Osteosoftâ (MerckChemicals, Darmstadt, Germany) for at least 48 h additionally. The tissue for paraffinembedded sections was dehydrated, embedded, and cut in 2-5 mm-thick sections. Sections were then H&E-stained or stained on a Ventana Benchmark XT System using standardized protocols titrated and optimized for each antibody ((ultraView Universal DAB Detection Kit and OptiView DAB Detection Kit (Roche)). The used antibodies were rabbit anti-Ki67 (ab15580, Abcam, Cambridge, UK, dilution 1:100), rabbit anti-Nmyc (tech517055, Cell signaling, Frankfurt, Germany, dilution 1:50), rabbit anti-NeuN (MAB 377, Sigma Aldrich, Taufkirchen, Germany, dilution 1:50), goat anti-Olig2 (AF2418, R&D Systems, Minneapolis, MN, USA, dilution 1:50) and rabbit anti-Calbindin (NCL-CALBINDIN, Novacastra/Leica Biosystems, Wetzlar, Germany, dilution 1:400). Histological photomicrographs were recorded using an Olympus B43 or a Leica M165 FC microscope. Staining with Cyclin D1 antibody (ab134175, Abcam, Cambridge, UK, dilution 1:100) was done as followed: Heat-mediated antigen retrieval at 99 C in 10 mM sodium citrate buffer (pH 6.0) for 25 min was made. Afterwards, the sections were incubated in 10% hydrogen peroxide to inactivate endogenous peroxidase activity and washed in PBS two times. Prior to the incubation with the primary antibody (Cyclin-D1) the sections were blocked for 30 min (I-Block, Life Technologies, Waltham, MA. United States). The incubation with primary antibodies took place over night followed by a washing step in PBS subjected