Astroglial cells in the external granular layer are precursors of cerebellar granule neurons in neonates

It is well established that cerebellar granule cell precursors (GCPs) initially derive from progenitors in the rhombic lip of the embryonic cerebellar primordium. GCPs proliferate and migrate tangentially across the cerebellum to form the external granule cell layer (EGL) in late embryogenesis and early postnatal development. It is unclear whether GCPs are specified exclusively in the embryonic rhombic lip or whether their precursor persists in the neonate. Using transgenic mice expressing DsRed under the human glial fibrillary acidic protein (hGFAP) promoter, we found 2 populations of DsRed⁺ cells in the EGL in the first postnatal week defined by bright and faint DsRed-fluorescent signal. Bright DsRed⁺ cells have a protein expression profile and electrophysiological characteristics typical of astrocytes, but faint DsRed⁺ cells in the EGL and internal granule cell layer (IGL) express markers and physiological properties of immature neurons. To determine if these astroglial cells gave rise to GCPs, we genetically tagged them with EGFP or βgal reporter genes at postnatal day (P)3–P5 using a hGFAP promoter driven inducible Cre recombinase. We found that GFAP promoter⁺ cells in the EGL are proliferative and express glial and neural stem cell markers. In addition, immature granule cells (GCs) en route to the IGL at P12 as well as GCs in the mature cerebellum, 30 days after recombination, express the reporter protein, suggesting that GFAP promoter⁺ cells in the EGL generate a subset of granule cells. The identification of glial cells which function as neuronal progenitor cells profoundly impacts our understanding of cellular plasticity in the developing cerebellum.     

Radiosensitive populations and recovery in X-ray-induced apoptosis in the developing cerebellum

Sprague-Dawley rats received a single dose of 2 Gy X-rays at the age of 1 or 3 days and were killed at different intervals. Dying cells with the morphological characteristics of apoptosis appeared in the external and internal granular layers (EGL and IGL) and white matter (WM) of the cerebellum, mainly 3–6 h after irradiation, and decreased thereafter to reach normal values between 48 h and 5 days later. This process was curbed by the injection of cycloheximide at a dose of 1 g/g body weight. In addition, the number of mitoses in EGL rapidly decreased after irradiation and did not reach normal values until a few days later. Proliferating cell nuclear antigen (PCNA)-immunoreactive cells, which were chiefly found in EGL but also in IGL and WM, dramatically decreased in number from 3 to 48 h after irradiation. PCNA-immunoreactive cells reappeared and reached age-matched values in the following days. Hu (considered as an early neuronal marker) and vimentin immunocytochemistry disclosed that Hu-nonreactive cells in the upper level of EGL, Hu-immunoreactive cells in the inner level of EGL, Bergmann glia and many astrocytes in WM, as well as many non-typified cells in WM, were radiosensitive populations, whereas Purkinje cells were not. The present results indicate that irradiation at P1 or P3 blocks mitosis in EGL and kills sensitive cells mainly in the late G1 and S phases of the cell cycle, probably by apoptosis through a protein synthesis-mediated process. Radiosensitive cells are germinal cells and neuroblasts in EGL, Bergmann glia, astrocytes in WM, and non-typified cells, probably glial cell precursors, in WM. Surviving cells in EGL and PCNA-immunoreactive cells in other cortical layers and white matter reconstitute the cerebellum following a single dose of X-rays.This work was supported in part by CEC project FI3P-CT92-0015 and FIS grant 93-131. M. Olivé is the recipient of a grant from the Pi i Sunyer Foundation. R. Rivera has a grant from the CIRIT     

Experimental studies on cerebellar foliation. I. A qualitative morphological analysis of cerebellar fissuration defects after neonatal treatment with 6-OHDA in the rat

The present report describes the natural history of defective cerebellar fissuration in the rat after neonatal treatment with 6-hydroxydopamine (6-OHDA). Within 24 hours after an intracisternal (IC) injection of 100 micrograms 6-OHDA cerebellar pial fibroblasts degenerated almost completely and were phagocytosed b macrophages within 2-5 days postinjection (dpi) leaving the cerebellar surface denuded of pia. Bergmann glia end feet at first exhibited morphological signs of gliosis and later formed new sprouts that penetrated the basal lamina and grew into the subarachnoid space covering regenerating pial fibroblasts and also invading ectopic colonies of external granular layer (EGL) cells. Breaches in the basal lamina appeared after the pial fibroblast had been destroyed and were confined to areas where Bergmann glia end feet were absent and where EGL cells were opposed to the basal lamina. EGL cells escaped through these fractures into the subarachnoid space in the fissures, where they proliferated to form large colonies of granule and stellate cells. In those fissures in which EGL ectopia featured, opposing folia fused and fissures were lost. These findings suggest that pial fibroblasts and the basal lamina have an important role in maintaining lobular partition during development of the cerebellum, in establishing cerebellar fissures, and in consolidating Bergmann glia-EGL cell relationships as a prerequisite for orderly migration of EGL cells.     

Cell formation in the cortical layers of the developing human cerebellum

Cell proliferation has been studied in the human cerebellar cortex between the 24th gestational week and the 12th postnatal month. Intensive cell formation has been found in the external granular layer (EGL) of the human cerebellum, where the highest cell proliferation rate occurs between the 28th and 34th gestational weeks. This is followed by a gradual decrease that lasts up to the eighth postnatal month. As late in development as the fifth postnatal month, still 30% of cells of the EGL are labeled with the monoclonal antibody Ki-67, which is specific for dividing cells. The width of the EGL remained unchanged from the 28th gestational week to the end of the first postnatal month, when it starts to decrease and completely disappears by the 11th postnatal month. Large number of Ki-67 labeled cells occurs in the internal granular layer (IGL) between the 24th and 28th gestational weeks. From the 36th week onwards, the labeling index is less than 1%, although a few labeled cells have always been found in this layer even in the late postnatal period. Labeled cells are distributed in the entire width of the IGL. However, from the 34th gestational week, almost all labeled cells are found among and directly below the Purkinje cells. Their position, the nuclear features, and their occasionally stained cell processes suggest that those are Bergmann glial cells. There are few Ki-67 labeled cells in the molecular layer (ML) and in the white matter (WM) of the cerebellum throughout the examined period. It is likely that most of these are glial cells. Pyknotic index has been found to be small in all layers of the cerebellum during the examined period.     

Potential of progenitors from postnatal cerebellar neuroepithelium and white matter: lineage specified vs. multipotent fate

Progenitors that migrate through the white matter of the postnatal cerebellum give rise to interneurons, astrocytes, and oligodendrocytes. To investigate the lineage potential of progenitors from the neuroepithelium and the white matter, we performed an in vitro clonal analysis in the presence or absence of various growth factors. Clonal progeny of cells labeled with a green fluorescent protein (GFP)-expressing retrovirus was characterized using morphological features and lineage markers. The large majority of clones were homogeneous, containing astrocytes, oligodendrocytes, neurons, or hybrid progenitors-cells labeled with markers for astrocytes and oligodendrocytes. Heterogeneous clones consisted of astrocytes and oligodendrocytes, with only a few mixed glial-neuronal clones. The neuroepithelium contains a higher number of multipotent progenitors than the white matter, pointing to a lineage specification of most of the cerebellar progenitors before their migration to the white matter.     

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.     

Embryonic cerebellar astroglia in vitro

Three types of astroglia appear during cerebellar development--radial glia and Bergmann glia, which are thought to facilitate neuronal migration, and astrocytes, which are thought to compartmentalize mature granule neurons. Cells resembling Bergmann glia and astrocytes have been described in cultures of cerebellar cells harvested from early postnatal cerebellum. In this study, we have used cell-type specific antisera to visualize embryonic forms of cerebellar astroglia and their interaction with embryonic neurons in vitro. When cells were dissociated from mouse cerebellum on the thirteenth embryonic day (E13), 3 forms of cells were stained with antisera raised against purified glial filament protein ( AbGF ), all of which had more elongated processes and less complex shapes than astroglia from postnatal day 7. The vast majority of embryonic cerebellar neurons did not contact these immature forms of astroglia.     

Influence of cis-dichlorodiammineplatinum on the structure of the immature rat cerebellum

Groups of 6- to 7-day-old and 10- to 11-day-old rats received cis-dichlorodiammineplatinum (cis-DDP) or inactive trans-DDP subcutaneously and were killed after 1, 6, 10, 15, or 21 days. In both age groups the acute effect (postinjection day 1) was most obvious in the germinal external granular layer (EGL), where many cells underwent shrinkage necrosis (pyknosis, apoptosis); the latter were more frequent within the fissures than on the surface of the cerebellar folia. Cell debris were often seen to be engulfed by macrophages, Bergmann cell fibers, and meningocytes. Bergmann cell bodies were swollen and the nuclei of Purkinje cells as well as of surviving cells of the EGL were enlarged. No comparable effects were observed in animals that had received an injection of trans-DDP in the same dose. Toward postinjection (p.i.) day 6, pyknotic cells disappeared from the EGL or, after the larger doses of cis-DDP, substantially decreased in number. Small nests of pyknotic cells appeared, however, at some places of the internal granular layer (IGL). The EGL was discontinuous or thinner in both age groups. In the IGL and molecular layer (ML) multiple focal fresh hemorrhages appeared together with some macrophages. The packing cell density in the IGL was less than in the controls, especially at the top of the cerebellar folia, i.e., contrary to the distribution of the primary damage in the EGL. Later, at p.i. day 10, ectopic nests of the IGL occurred occasionally in animals injected when 10 to 11 days old and severe atrophy of the ML and Purkinje cell population was observed at some places. At p.i. days 15 to 21, invasion of microglia-like cells appeared in the IGL and in some regions of the ML. Occasionally, subpial hemorrhages occurred at this interval. The acute damage caused by cis-DDP is thus similar to the effect of X rays or some, but not all, drugs with a cytostatic action. In addition, a more profound influence on dividing cells of the EGL within the fissures and distinct capillary lesions, indicated by hemorrhages, were found after cis-DDP. As in other experimental models, the acute cis-DDP damage of the immature cerebellum was partly repaired within a few p.i. days. Spatial difference of the repair process was inferred from the packing cell density in the IGL measured at the top and bottom of the cerebellar folia.     

Effects of prenatal ethanol exposure on the development of Bergmann glia and astrocytes in the rat cerebellum: an immunohistochemical study.

The consequences of prenatal ethanol exposure on the postnatal development of Bergmann glia and astrocytes in the rat cerebellum were investigated by using glial fibrillary acidic protein (GFAP) immunolabeling. Pregnant rats were either fed with an ethanol containing liquid diet (6.7% v/v) or pair-fed with an isocaloric diet throughout gestation. On postnatal day (PD) 15 and 22, parasagittal sections of the cerebellar vermis from female offspring were processed for GFAP immunohistochemistry to assess the development of Bergmann glia and astrocytes in lobules I, VII, and X and astrocytes in the central core of white matter. On PD 15, compared to control animals, ethanol exposed animals had fewer GFAP positive Bergmann glial fibers per unit length of molecular layer; a significantly greater percentage of morphologically immature Bergmann fibers; a significantly greater GFAP positive astrocytic area per unit area of internal granular layer and central white matter; and the astrocytic processes were wider and more closely packed. These glial changes were associated with significantly thicker external granular layer in all 3 lobules. However, no significant differences were seen between the ethanol exposed and control animals on PD 22, indicating "catch-up growth" in the ethanol exposed animals during the third postnatal week. These results suggest that prenatal ethanol exposure causes (1) delayed maturation of Bergmann glia, which in turn contributes to the delayed migration of granule cells; and (2) alterations in the normal postnatal development of astrocytes.     

In vivo characterization of endogenous proliferating cells in adult rat subcortical white matter

Proliferating cells in adult rat subcortical white matter were characterized in vivo using stereotactic injections of a replication-deficient retrovirus containing the construct for βgalactosidase (BAG); BAG was deposited into the cingulum at the level of the septal nuclei. Morphological profiles, generated using Xgal substrate to visualize labeled cells, revealed a population of simple, immature cells. The antigenic profile, generated immunohistochemically with cell-specific markers 2 or 30 days post injection (dpi), showed a population of cells that primarily expressed nestin or an oligodendrocyte-specific glutathione-S-transferase isoform, Yp (GST-Yp) at 2 dpi and nestin, GST-Yp or Rip at 30 dpi. Occasionally, labeled cells differentiated in vivo into myelinating oligodendrocytes 30 dpi. Labeled cells did not express the astrocyte markers GFAP, GST-Yb, or S100β at 2 or 30 dpi. Comparisons of cell distribution 2 and 30 dpi indicated the non-migratory nature of these cells. Cell distribution patterns and nearest neighbor analyses confirmed the emergence of clusters of labeled cells 30 dpi, which bromodeoxyuridine (BrdU) incorporation studies suggested arose from continued proliferation of some labeled cells. In vivo characterization of proliferating cells in the adult revealed a non-migratory, primarily undifferentiated population of cells. © 1996 Wiley-Liss, Inc.     

Disruption of postnatal progenitor migration and consequent abnormal pattern of glial distribution in the cerebrum following administration of methylmercury

Transplacental administration of methylmercury (MeHg) induces disruption of neuronal migration in the developing cerebral cortex. However, the effects of MeHg on glial progenitor migration remain unclear. To understand this, we performed double administration of MeHg and 5-bromo-2-deoxyuridine (BrdU) to neonatal rat pups on postnatal day 2 (P2), when glial cells are generated from progenitors in the subventricular zone (SVZ). Histopathological examination of a proportion of the MeHg-treated rats on P28 revealed no apparent abnormalities of cytoarchitecture or neuron count in either the primary motor or primary somatosensory cortex of the cerebrum. BrdU immunohistochemistry revealed abnormal accumulation of the labeled cells in the deeper layers of the cortices and underlying white matter of both areas, where an excessive number of astrocytes (glial fibrillary acidic protein- or S-100beta-immunolabeled cells) and oligodendrocytes (2',3'-cyclic-nucleotide 3'-phosphohydrolase-labeled cells) were located. Next, to investigate the migration of individual progenitors from the forebrain SVZ of P2 neonates, we labeled them in vivo with a retrovirus encoding green fluorescent protein (GFP), following administration of MeHg, and then examined the distribution pattern of the GFP-labeled cells in the P28 cerebrum. We found that the labeled cells developed into astrocytes and oligodendrocytes and were accumulated abnormally in the lateral white matter as well as in the adjacent deeper layer of the lateral cortex and lateral side of the striatum. Thus, exposure to MeHg in the gliogenic period induced irregular distribution of glia as a consequence of abnormal migration of the postnatal progenitors.     

Microglial response to the neurotoxicity of 6-hydroxydopamine in neonatal rat cerebellum

Depletion of noradrenaline in newborn rats by 6-hydroxydopamine (6-OHDA) affects the postnatal development and reduces the granular cell area in the neocerebellum (lobules V-VII). During the first postnatal month, Bergmann glial fibers guide the migration of immature granule cells to the internal granule cell layer. Microglia and Bergmann glia may play an important role in this process, but the exact mechanism behind this phenomenon is not known. We studied the effect of systemic administration of 6-OHDA on the expression and localization on microglia and Bergmann glia in the neonatal cerebellum by immunohistochemistry. In the neocerebellum, 6-OHDA treatment caused a significant increase in the number of activated microglia. The increase was observed mainly in the granule cell layer and the cerebellar medulla. Bergmann glial cells in treated brains were abnormally located, did not form intimate associations with Purkinje cells, and the glial fibers were structurally different. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of granule cells, and abnormal migration may be the result of Bergmann glia destruction and the activation of microglia. Activated microglia in the granule cell layer may be used as a marker for an injured cerebellar area.     

Glial responses to neonatal hypoxic-ischemic injury in the rat cerebral cortex.

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic-ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.     

Cellular localization of the full-length isoform of the type 2 corticotropin releasing factor receptor in the postnatal mouse cerebellar cortex

Corticotropin releasing factor (CRF) and its cognate receptors, defined as Type 1 and Type 2 have been localized within the cerebellum. The Type 2 CRF receptor (CRF-R2) is known to have both a full length (CRF-R2alpha) and a truncated (CRF-R2alpha-tr) isoform. A recent study documented CRF-R2alpha primarily in Bergann glia and astrocytes, as well as in populations of Purkinje cells in the adult cerebellum. The goal of the present study is to determine if CRF-R2alpha is present in the postnatal cerebellum, and if so to describe its cellular distribution. RT-PCR data showed that CRF-R2alpha is expressed in the mouse cerebellum from birth through postnatal day 21. Between birth and P14, CRF-R2alpha-immunoreactivity was localized within the somata of Purkinje cells, and migrating GABAergic interneurons. GFAP-immunoreactive astrocytes, including Bergmann glia, also expressed CRF-R2alpha-immunoreactivity from P3-P14. There is a change, however, in CRF-R2alpha immunolabeling within neurons as the cerebellum matures. Compared to its expression in the adult cerebellum, Purkinje cells, and GABAergic interneurons showed more extensive CRF-R2alpha immunolabeling during early postnatal development. We postulate that CRF-R2alpha could be involved in developmental events related to the survival and differentiation of Purkinje cells and GABAergic neurons, whereas in the adult, this isoform of the CRF receptor family is likely involved in modulating Bergmann glia that have been shown to play a role in regulating the synaptic environment around Purkinje neurons.     

Expression of Kv3.1 and Kv4.2 genes in developing cerebellar granule cells.

The expression of voltage-gated potassium channels plays an important role in the acquisition of membrane excitability in neurons. We examined the expression pattern of genes in developing cerebellar granule neurons in vivo and in vitro. In situ hybridization of Kv3.1 mRNA demonstrated that the gene was expressed at high levels in the external granule layer (EGL) as well as in the internal granule layer (IGL) at all postnatal stages (P) examined (from P3 to P10). In contrast, Kv4.2 mRNA was detected in the premigratory zone (PMZ) of the EGL, but not in the proliferative zone (PLZ), in addition to the IGL. This indicates that Kv4.2 gene expression initiates in the postmitotic migrating neurons. We also examined the expression of the channel genes in microexplant culture systems. Kv3.1 polypeptide was detected in parallel fibers of granule cells at 2 days in vitro, and the expression continued in later stages. The signal of Kv4.2 protein was very low at 2 days in vitro; however, the number of positive cells and the intensity of the signals were increased at 6 days in vitro. These in vitro observations matched those in vivo and our previous electrophysiological studies in which we demonstrated that delayed- rectifier-type current was predominant in the immature granule cells followed by the later appearance of A-type current. The patterns of K(+) channel expression suggest that sequential expression of these channel genes primarily determines the membrane excitability.     

Immunocytological localization of the highly polysialylated form of the neural cell adhesion molecule during development of the murine cerebellar cortex

The expression of the highly polysialylated form of the neural cell adhesion molecule (N-CAM)--the so-called embryonic N-CAM (E-N-CAM)--was investigated in the developing and adult mouse cerebellar cortex by immunohistology and immunocytology at the light and electron microscopic levels. E-N-CAM was never (from embryonic day 14 to postnatal day 15) detectable in the germinal zone of neuroblasts destined to form or forming the external granular layer and was only observed once small cerebellar interneurons had become postmitotic before the beginning of granule cell migration. Granule cells expressed E-N-CAM on cell bodies, axons, and leading and trailing processes also during migration but ceased to reveal detectable levels of E-N-CAM at the end of migration after having reached their final position in the internal granular layer. Other cerebellar cell types, such as Purkinje cells, Bergmann glia, astrocytes, oligodendrocytes, and most prominently, stellate and basket cells, also expressed E-N-CAM, but became E-N-CAM-negative during the third and fourth postnatal weeks, coinciding with overt cessation of cerebellar histogenesis. Thus, except for neuroblasts, E-N-CAM appeared characteristic of growing and moving cellular structures, in agreement with the notion that the highly polysialylated form of N-CAM is less adhesive than the adult form.     

Glial responses to neonatal hypoxic–ischemic injury in the rat cerebral cortex

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic–ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.     

Neurotrophin‐3 promotes cerebellar granule cell exit from the EGL

In the cerebellum, the mRNAs for neurotrophin-3 (NT-3) and its high-affinity tyrosine kinase receptor trkC are expressed by both the differentiated granule cells of the internal granule cell layer (IGL) and their precursors in the external germinal layer (EGL). We have investigated the effects of chronic application of exogenous NT-3 in vivo on cerebellar granule cell genesis and differentiation. NT-3 was applied to the posterior surface of the rat cerebellum from P6 onwards using Elvax implants. At P10 the EGL of cerebellar lobules VII and VIII was significantly reduced in thickness in NT-3 implanted rats when compared with controls. Immunocytochemical analysis of the EGL using antibodies to proliferating cell nuclear antigen (PCNA) revealed that the number of postmitotic, premigratory (PCNA-immunonegative) granule cell precursors was preferentially reduced in the NT-3 implanted rats. In situ DNA fragmentation labelling confirmed that this was not accompanied by increased cell death in the EGL. These results suggest that NT-3 promotes the differentiation of postmitotic, premigratory granule cell precursors, accelerating cell exit from the EGL.