Delta-like 1 regulates Bergmann glial monolayer formation during cerebellar development
© Hiraoka et al.; licensee BioMed Central Ltd. 2013
Received: 3 April 2013
Accepted: 19 May 2013
Published: 21 May 2013
Bergmann glia (BG) are unipolar cerebellar astrocytes. The somata of mature BG reside in the Purkinje cell layer and extend radially arranged processes to the pial surface. BG have multiple branched processes, which enwrap the synapses of Purkinje cell dendrites. They migrate from the ventricular zone and align next to the Purkinje cell layer during development. Previously, we reported that Notch1, Notch2, and RBPj genes in the BG play crucial roles in the monolayer formation and morphogenesis of BG. However, it remains to be determined which ligand activates Nocth1 and Notch 2 on BG. Delta-like 1 (Dll1) is a major ligand of Notch receptors that is expressed in the developing cerebellum.
In this study, we used human glial fibrillary acidic protein (hGFAP) promoter-driven Cre-mediated recombination to delete Dll1 in BG. Dll1-conditional mutant mice showed disorganization of Bergmann fibers, ectopic localization of BG in the molecular layer and a reduction in the number of BG.
These results suggest that Dll1 is required for the formation of the BG layer and its morphological maturation, apparently through a Notch1/2-RBPj dependent signaling pathway.
KeywordsBergmann glia Notch signaling Delta like 1 Conditional knockout mouse Monolayer formation
Bergmann glia (BG) are unipolar cerebellar astroglial cells that have their soma in the Purkinje cell layer and extend radially arranged processes to the pial surface . Purkinje cells constitute the sole output of all motor coordination in the cerebellar cortex. Mature BG processes ensheath Purkinje cell somata, dendrites, and both excitatory and inhibitory synapses. BG express GABA and glutamate transporters, which in turn are involved in their clearance from the synapses. A recent study demonstrated that photoactivation of BG can modulate the neuronal activity of Purkinje cells by releasing of glutamate . Thus, BG play important roles in the adult cerebellar cortex. During development, BG are among the earliest cells to develop in the cerebellum, and their radial processes assist in the migration of Purkinje and granule cells during the construction of the cerebellum [3, 4]. BG arise from neuroepithelial cells in the fourth ventricle and migrate from the ventricular zone through the mantle zone in synchrony with the migration of Purkinje cells, where they form a monolayer structure in the Purkinje cell layer from embryonic day (E) 14 to postnatal day (P) 7 in the mouse cerebellum . However, the molecular mechanisms involved in the development and differentiation of BG remain unclear.
The Notch signaling pathway is among the most evolutionally conserved signaling pathways in various animals. The Notch signaling is characterized as mediating cell–cell signaling between adjacent cells. In the central nervous system (CNS), Notch signaling regulates neural progenitor maintenance and differentiation. In the traditional view, Notch receptor activation inhibits neurogenesis in order to maintain neural stem and/or progenitor cell character and that Notch may play an instructive role in promoting glial development [6, 7]. We have previously reported that ablation of Notch1/2 or RBPj in BG results in poor radial fiber extension and defective positioning of BG in the adult cerebellum . This implies that Notch signaling activation is crucial for BG monolayer formation and its morphological development, however, precisely how the Notch pathway is activated remains unclear. During early postnatal development, Dll1 and Jagged-1 are predominant Notch ligands expressed in the cerebellum . It has been reported that conditional knockout of Jagged1 from the neuroepithelial cells of the midbrain–hindbrain boundary causes a reduction in the number and processes of BG, however, the mutant mice did not display abnormal localization of BG in the molecular layer . Therefore, Dll1 is a candidate gene for the ligand that plays an indispensable role in BG monolayer formation. Dll1 mRNAs were present in the molecular and internal granular layer during early postnatal stages, suggesting that Dll1 was expressed in interneurons of the molecular layer, Purkinje cells, BG and granule cells. To address this question, we used a hGFAP-Cre transgenic mouse line and employed the Cre-loxP approach to inactivate Dll1 in BG. Loss of Dll1 resulted in disorganization of Bergmann fibers, irregularities in the BG lining and aberrant localization of BG in the molecular layer, thus displaying phenotypic similarities with Notch1/2 or RBPj conditional knockout mice.
Conditional ablation of Dll1 leads to anatomical abnormalities in the adult cerebellum
Adult Dll1(loxP/loxP)/hGFAP–Cre mice show defects in BG
Lack of Dll1 produces alterations in the arrangement of BG in the purkinje cell layer during early postnatal stages
Ectopic BG were eliminated by apoptosis in Dll1(loxP/loxP)/hGFAP-Cre mice
Ablation of Dll1 from basket and stellate cells had no effect on the maturation of BG
Notch-RBPj dependency of Dll1 signaling in the monolayer formation of BG
In the CNS, Notch signaling is a well-known master regulator of neural stem cells and neural development . In addition, Notch signaling plays instructive roles in promoting the identities of radial glia . In our previous study, we revealed that Notch–RBPj signaling plays an important role in BG development by deleting Notch1 and Notch2 or RBPj. However, the ligand that activates this Notch–RBPj signaling pathway has not been identified. In this study, we found that Dll1 is the functional ligand in Notch–RBPj signaling responsible for BG monolayer formation. Since Cre recombinase is expressed in basket and stellate cells, as well as in the BG of hGFAP-Cre mice , abnormal layering and morphology of the BG observed in Dll1(loxP/loxP)/hGFAP-Cre mice could be the result from ablation of Dll1 form basket and stellate cells. To further investigate, which cell types require Dll1 for appropriate BG development, we used a Ptf1a-Cre mouse line to drive Cre-mediated deletion of Dll1 in stellate and basket cells. However, we did not observe any BG abnormalities in Dll1(loxP/loxP)/Ptf1a-Cre mice. Therefore, the abnormal layering and morphology of BG as observed in the Dll1(loxP/loxP)/hGFAP-Cre mice, was due to the deletion of Dll1 in BG, thus suggesting that Dll1-Notch1/2 interaction between adjacent BG is required for their appropriate development.
Dll1 and Jagged 1 are predominant Notch ligands expressed in the cerebellum during early postnatal development, which is the point at which Dll1(loxP/loxP)/hGFAP-Cre mice display abnormal BG layering . The conditional ablation of Jagged 1 in the cerebellum primordium at mid-embryogenesis did not display any mislocalized BG, although ectopic granular neurons in the molecular layer were observed, as well as a reduction in the number of BG, and truncation of BG fibers . Recently, the Delta/Notch-like EGF-related receptor (DNER) has been identified as a Notch ligand that is expressed on Purkinje cells. DNER-deficient mice display disorganization of BG fibers, as well as the ectopic localization of BG , however, abnormal BG development in DNER-deficient mice became less significant by postnatal week 3 and had improved by the adult stages, which suggests that DNER-Notch signaling is only important for the maturation of BG in the early developmental stages. Taken together, these findings suggest that Dll1-Notch signaling is required for BG monolayer formation, whereas Jagged 1-Notch or DNER-Notch signaling is not crucial for BG arrangement.
It is well known that BG start their preferential radially oriented migration at E14 towards the subpial domain, without axonal scaffold, and form their epithelium-like layer structure in the Purkinje cell layer by the first postnatal week . However, the molecular mechanism underlying this event remains unknown. In this study, we demonstrated that Dll1(loxP/loxP)/hGFAP-Cre mice display ectopic BG after P5, whereas BG migration is normal during the embryonic stages. Furthermore, ectopic BG were negative for BrdU labeling. These results suggest that ectopic BG were not generated in situ but, instead, were disintegrated from the BG layer by overmigration. This implies that Dll1–Notch–RBPj signaling in developing BG may regulate the termination of their migration. Consistent with this finding, it has been reported that the lack of Notch and Numb causes the overmigration of peripheral glia in Drosophila. Further studies are necessary to clarify the molecular mechanisms underlying the termination of BG.
We show here that Dll1 localized to BG is required for their monolayer formation and morphological maturation in the developing cerebellum, apparently through a Notch1/2 and RBPj dependent signaling pathway.
hGFAP-Cre transgenic mice and Ptf1a-Cre knock-in mice, which have previously been described[16, 17], were cross-bred with floxed Dll1 mice, to obtain conditional knockout mice. These mice were then crossed with ROSA26R mice  or ROSA-tdTomato mice  in order to examine Cre activity. All animal procedures were approved by the Animal Experiment Committee of Tokyo Medical and Dental University (0130166C).
Primary cerebellar astrocytes were isolated from P5 mice cerebella. Dissected cerebella were cut into small pieces and incubated in 2.5% trypsin and 0.01% DNase I, dissolved in phosphate buffered saline (PBS), for 20 min at 37°C. Trypsin activity was blocked by adding fetal bovine serum (FBS), and the tissue was triturated using a micropipette. Cells were washed with Dulbecco’s modified eagle medium (DMEM) containing 10% FBS, plated onto 10-cm tissue culture dishes, and incubated in the same culture medium.
Immunohistochemistry and in situ hybridization
Animals were deeply anesthetized by inhalation of diethyl ether or hypothermia, and transcardially perfused with 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate-buffer (PB), the brains were then dissected out. Brains were postfixed with 4% PFA/PB overnight, and cryoprotected in 30% sucrose. Tissues were embedded in OCT compound (Sakura) and cut into 12-μm-thick sagittal sections using a cryostat. For immunohistochemistry, the sections were permeabilized with 0.5% Triton X100 dissolved in PBS, and then blocked with 5% normal horse serum and 0.3% Triton X100 dissolved in PBS. Sections were stained with either mouse monoclonal anti-β-galactosidase antibody (1:300, Promega), rabbit anti-β-galactosidase antibody (1:1000, Cappel), rabbit anti-GFAP antibody (1:1000, DAKO), mouse monoclonal anti-parvalbumin antibody (1:1000, Sigma), rabbit anti-BLBP (1:300, kindly gifted from Dr.Watanabe) antibody, rabbit anti-GLAST antibody (1:1000, kindly gifted from Dr.Watanabe), mouse monoclonal anti-NeuN antibody (1:400, Chemicon) or rabbit anti-calbindin antibody (1:1000, Sigma). Antibodies were diluted in blocking solution and incubated overnight at 4°C. The sections were then rinsed and incubated in appropriate species-specific secondary antibodies, which were tagged with Alexa-fluor 488, 568, or 633 (Molecular probes). Images were taken using confocal laser microscope (LSM510 META; Carl Zeiss).
In situ hybridization was performed on 12-μm-thick sagittal cryosections using a digoxigenin-labeled Glast- and Hes5-specific cRNA probes, as previously described . Hybridized cRNA probes were detected by using alkaline phosphatase conjugated anti-digoxigenin antibody (1:1000, Roche). The sections were visualized by using NBT/BCIP solution (Roche) for Glast and Fast Red solution (Roche) for Hes5. For the double-staining of Hes5 by in situ hybridization and BLBP by immunohistochemistry, immunohistochemistry was performed after the in situ hybridization process. Images were taken with an Olympus BX60 microscope and an Olympus DP50 digital camera or with confocal laser microscope (LSM510 META; Carl Zeiss).
Dissected brains from perfused animals were submerged in a 3% potassium dichromate solution containing 7.4% formaldehyde for 1 day in the dark, before being transferred to a 3% potassium dichromate solution and incubated for a further 2 days. Tissues were then rinsed with ultra-pure water and submerged in 2% silver nitrate solution for 4 days in the dark. After the tissues were submerged, they were embedded in agarose, and 100-μm-thick sections were cut using a Vibratome. Golgi-stained sections were viewed with an Olympus BX60 microscope, and images were acquired with an Olympus DP50 digital camera.
Postnatal mice were injected intraperitoneally with BrdU (50 μg/g body weight; Sigma) dissolved in saline. The mice were sacrificed 3 h after injection. Sagittal cryosections (12-μm-thick) were then treated with 2N hydrochloric acid at 37°C for 30 min and neutralized with 0.1 M sodium borate for 10 min. Afterwards, the sections were immunostained with an anti-BrdU antibody (Oxford Biomedical Research) and with anti-BLBP and anti-β-galactosidase antibodies.
The Terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) staining was done using the DeadEnd colorimetric TUNEL system (Promega) according to the manufacturer's instructions. Sagittal cryosections (12-μm-thick) were used for this staining technique. Horseradish peroxidase-conjugated streptavidin and diaminobenzidine were used for bright field detection, and an Alexa-fluor 568-conjugated streptavidin (Molecular Probes) and an Alexa-fluor 488-conjugated secondary antibody were used for fluorescence detection of TUNEL-positive cells, alongside immunohistochemical staining for GLAST.
Fluorescence Activated Cell Sorting (FACS)
Cerebellar interneurons were isolated from P2 mice cerebella. Cerebella were dissected from hypothermia-induced anesthetized mice. Papain dissociation system (Worthington Biochemical Corporation) was used for the dissociation, according to manufacturer’s instructions. The cell suspension was passed through a 40 μm filter, and resuspended in Hank’s balanced salt solution. Dispersed cells were separated into tdTomato fluorescence positive and negative cells using a cell sorter (MoFlo XDP; Beckman Coulter), and each cell population was saved for RNA extraction.
Total RNA was isolated from cell culture samples or FACS sorted cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Total RNA was reverse-transcribed into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen), according to the manufacturer’s instructions. For cDNA detection, the following primers were used:
Glast FWD: AGA ATT CTG ACC TGA ACT TTG GCA GAT TA
Glast REV: TGG ATC CTC TTG AAA GTT GAT TTT AAA ACT
Dll1 FWD: GAG AGA ATT CGG TCA GTG CAG TAC TGG CCT
Dll1 REV: GAG ACT CGA GAC AGT AGC GGC CGC ACA GAC
β-actin FWD: ATA TCG CTG CGC TGG TCG TC
β-actin REV: TCA CTT ACC TGG TGC CTA GGG
Map2 FWD: TTC CTC AGC TTG TCT CTA AC
Map2 REV: GCT TCA GCT GTG ACT ACT TG
βIII tubulin FWD: ATG TCT ATG AAG GAG GTG GAC G
βIII tubulin REV: TCT CGG CCT CGG TGA ACT C
Gfap FWD: TTC TCC TTG TCT CGA ATG AC
Gfap REV: GGT TTC ATC TTG GAG CTT CT
To examine the number of cells, at least 3 mice from each genotypic group were used and 3 sections from all individual mice were analyzed. Brain lipid binding protein (BLBP) positive cells were counted in order to assess the number of BG in the adult mice cerebella. To examine the number of BG in developing cerebellum, GLAST positive cells were counted. To examine the proliferative rate of BG, the ratio of BLBP, β-galactosidase, and BrdU triple positive cells to the total number of BLBP and β-galactosidase double positive cells in the Purkinje cell layer was calculated. In order to quantify the number of apoptotic cells in the EGL, TUNEL and GLAST double positive cells and TUNEL single positive cells were counted. The data are presented as means ± standard deviation of the mean. The P-values were calculated using Student’s t-test.
Brain lipid binding protein
Central nervous system
Delta like 1
External granular layer
Glial fibrillary acidic protein
glutamate aspartate transporter
Internal granular layer
Microtubule associated protein 2
Neuronal nucleic protein
Pancreas transcription factor 1 subunit alpha
Recombining binding protein suppressor of hairless
Purkinje cell layer
Terminal deoxynucleotidyl transferase–mediated nick-end labeling.
We thank Y. Kawaguchi and R. Kageyama for the Ptf1a-Cre mice and Hes5-specific cRNA probe, respectively. This study results in part from “Understanding of molecular and environmental bases for brain health” executed under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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