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The Journal of Neuroscience, August 15, 2002, 22(16):7132-7146
Specification of Cerebellar Progenitors After Heterotopic-Heterochronic Transplantation to the Embryonic CNS In Vivo and In Vitro
Barbara Carletti1, Piercesare Grimaldi1, Lorenzo Magrassi2, and Ferdinando Rossi1 1 Department of Neuroscience and "Rita Levi Montalcini Center for Brain Repair", University of Turin, I-10125 Turin, Italy, and 2 Neurosurgery, Department of Surgery, Istituto di Ricerca e Cura a Carattere Scientifico Policlinico S. Matteo, University of Pavia, I-27100 Pavia, Italy
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The different cerebellar phenotypes are generated according to a precise time schedule during embryonic and postnatal development. To assess whether the differentiative potential of cerebellar progenitors is progressively restricted in space and time we examined the fate of embryonic day 12 (E12) or postnatal day 4 (P4) cerebellar cells after heterotopic-heterochronic transplantation into the embryonic rat brain in utero or into organotypic CNS explants in vitro. Donor cells, isolated from transgenic mice overexpressing the enhanced-green fluorescent protein under the control of the
-actin-promoter, engrafted throughout the host brainstem and diencephalon, whereas they rarely incorporated into specific telencephalic structures. In any recipient site, the vast majority of transplanted cells could be recognized as cerebellar phenotypes, and we did not obtain clear evidence that ectopically located cells adopted host-specific identities. Nevertheless, the two donor populations displayed different developmental potentialities. P4 progenitors exclusively generated granule cells and molecular layer interneurons, indicating that they are committed to late-generated cerebellar identities and not responsive to heterotopic-heterochronic environmental cues. In contrast, E12 precursors had the potential to produce all major cerebellar neurons, but the repertoire of adult phenotypes generated by these cells was different in distinct host regions, suggesting that they require instructive environmental information to acquire mature identities. Thus, cerebellar precursors are able to integrate into different foreign brain regions, where they develop mature phenotypes that survive long after transplantation, but they are committed to cerebellar fates at E12. Embryonic progenitors are initially capable, although likely not competent, to generate all cerebellar identities, but their potential is gradually restricted toward late-generated phenotypes.
Key words: differentiation; in utero neural graft; neural precursor; stem cell; cerebellum; mouse; rat
INTRODUCTION
The relative contribution of cell-autonomous mechanisms and environmental signaling to cell specification during CNS development is still poorly understood. Several lines of evidence indicate that the differentiative potential of neural progenitors is progressively restricted both in space, because of regional specification (Barbe and Levitt, 1991
; Alvarado-Mallart 1993
The rodent cerebellum offers a suitable ground to address these issues. All cerebellar phenotypes originate according to a precise time schedule from two germinative neuroepithelia (Ramón y Cajal, 1911
Transplantation studies indicate that mid-hindbrain progenitors become regionally specified between embryonic day 10.5 and 13.5 (Olsson et al., 1997
A preliminary report of this work has been published (Carletti et al., 2001
MATERIALS AND METHODS
Animals and surgical procedures. We transplanted embryonic and postnatal cerebellar progenitors into the telencephalic vesicles of rat embryos in utero or into organotypic explants of mouse brainstem-cerebellum or neocortex. An additional set of in utero experiments was performed using embryonic neocortical precursors. To identify transplanted cells in the recipient tissue, donor cells were isolated from transgenic mice overexpressing the enhanced green fluorescent protein (EGFP) under the control of the
Preparation of cell suspensions. The preparation of donor cerebellar cells was performed as previously described (Jankovski et al., 1996
For both donor cell populations, the collected tissue blocks were incubated for 5 min in trypsin (1% in PBG; Sigma, St. Louis, MO) and DNase (0.1%; Sigma) at 37°C, and then mechanically dissociated to a single cell suspension by means of a fire-polished Pasteur pipette. The obtained suspension was centrifuged and resuspended at a final concentration of 1-2 × 104 cells/µl. An aliquot of the suspension was immediately examined under the microscope to assess cell viability and EGFP expression. In all instances, virtually all the cells displayed an intense fluorescence. The same procedures were applied to dissect and dissociate neocortical donor cells isolated from the dorsal aspect of the telencephalic vesicle of E12 embryos.
Transplantation in utero. The surgical manipulation of rat embryos in utero was performed according to a previously described approach (Cattaneo et al., 1994
Transplantation in vitro. Organotypic explants of brainstem-cerebellum were prepared as described by Chédotal et al. (1997)
The explants were cultured on the membrane of a 30 mm Millipore culture insert (Millicell; Millipore, Bedford, MA; pore size, 0.4 µm) in 10 cm culture dishes containing 3 ml of a medium composed of 50% basal medium with Earle's salts (Life Technologies, Gaithersburg, MD), 2.5% HBSS (Invitrogen, Gaithersburg, MD), horse serum (25% first week, 15% following weeks; Invitrogen), 22.5% water, L-glutamine (1 mM; Invitrogen), penicillin-streptomycin (200 U/ml; Invitrogen), and glucose (5 mg/ml). The cultures were kept at 37°C in a humidified atmosphere with 5% CO2, and they were fixed after 15-25 d in vitro. On the whole we analyzed 21 cerebellar and 10 neocortical explants grafted with postnatal cerebellar cells and 19 cerebellar and 14 neocortical explants receiving embryonic donor cells.
Histological procedures. Under deep general anesthesia (as above), the rats were transcardially perfused with 1 l of 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2-7.4. The brains were immediately dissected, stored for 2 hr in the same fixative at 4°C, and finally transferred in 0.12 M phosphate buffer. The brain was cut in 100-µm-thick vibratome parasagittal slices, whereas the cerebellum was sectioned on the frontal plane. The sections were collected in PBS and immediately examined under the microscope to localize the transplanted cells. Sections containing EGFP-positive cells where incubated with different primary antibodies (in some instances the vibratome slices were further cut in 10-µm-thick sections directly collected on gelatinized slides). The explants were fixed 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2-7.4, for 3 hr at room temperature, washed in PBS, and processed for immunocytochemistry.
To detect the expression of cell-specific markers we applied a set of primary antibodies: anti-calbindin (Purkinje cells; Celio, 1990
6 subunit of the GABAA receptor (granule cells; Kato, 1990
BrdU labeling. To evaluate the proliferative activity of donor cells in the host brain, BrdU (50 µg/g of body weight, dissolved at 5 mg/ml in 0,007N NaOH in normal saline) was injected intraperitoneally to the pregnant mother after transplantation (three injections during 48 hr) or to the live-born recipient rats 24-48 hr (three injections) before the end of the survival time. The brains were fixed and vibratome-sectioned as above. Sections containing transplanted cells were incubated in 2N HCl for 1 hr at room temperature, exposed to anti-BrdU antibodies (1:500; monoclonal; Sigma) overnight at 4°C, and then reacted with biotinylated second antibody and streptavidin Texas Red conjugate as above.
In situ hybridization of RU49 expression. As an additional marker to identify transplanted granule cells, we examined the expression of RU49 mRNA, which is selectively expressed in cerebellar and hippocampal granule cells and olfactory bulb interneurons (Yang et al., 1996
Data analysis. The histological preparations were examined by means of a Zeiss Axiophot light microscope. Digital micrographs were taken by means of a Nikon Coolpix 950 digital camera attached to the same microscope. The material was also examined with an Olympus (Hamburg, Germany) Fluoview 300 confocal microscope. Digital images were processed with Adobe Photoshop 6.0 to adjust contrast and to assemble the final plates. Quantitative and morphometric evaluations were made using the Neurolucida software (MicroBrightField Inc., Colchester, VT) connected to an E-800 Nikon microscope via a color CCD camera.
To evaluate the distribution of heterotopically transplanted cells in the host CNS in vivo, we selected three representative cases transplanted with postnatal or embryonic cerebellar cells, killed 3-5 weeks after transplantation, when grafted cells had completed their development. For each case, the outline of several serial sections, encompassing mesencephalon and forebrain, was reproduced using the Neurolucida system, and the position of every EGFP-positive cell were carefully mapped. To allow a better comparison between different cases, the obtained maps were reported on corresponding sections from a stereotaxic atlas of the rat brain (Paxinos and Watson, 1982
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Figure 1. Distribution of postnatal (P4-A-C) and embryonic (E12-A-C) cerebellar cells transplanted to the embryonic rat brain in utero. For each case, four serial sections, modified from Paxinos and Watson (1982)
The mature phenotypes adopted by transplanted cells were investigated by labeling with different cell specific markers (see above). However, although all the selected markers are distinctive for different cerebellar cell populations, some of them are also expressed in extracerebellar regions. Hence, phenotype identification of transplanted cells was based on several concurrent criteria: (1) marker expression; (2) typical morphological features; (3) morphometric parameters. This approach allowed us to unequivocally identify glial cells, Purkinje cells, granule cells, and molecular layer interneurons. The other transplanted neurons, a class of medium-sized or large multipolar neurons, in part labeled by different markers (see Results), included presumptive deep nuclei neurons plus a minority of other types that could not be precisely classified (see Results). All these cells were grouped into a single category of "multipolar neurons."
To quantify the phenotypic repertoire generated by ectopically located donor cells we examined parvalbumin immunostained material, where Purkinje cells and molecular layer interneurons could be identified for both morphology and labeling (Celio et al., 1988
To further define the phenotype of the EGFP-positive multipolar neurons, we determined the number of these cells stained by anti-neurofilament SMI32 or anti-parvalbumin antibodies on three vibratome sections from four different cases. The fraction of SMI32-positive neurons was also estimated on a sample of 452 nerve cells from the deep cerebellar nuclei of two adult EGFP mice. In addition, we compared the cell body size of EGFP-positive multipolar neurons in cerebellar explants with that of SMI32-positive host neurons located in the deep nuclei of the same organotypic cultures. To this aim, by means of the Neurolucida system at 40× magnification, we measured the size of the perikaryon of 88 transplanted and 82 host neurons from three different explants grafted with embryonic donor cells. The same approach was used to compare the size of parvalbumin-immunopositive molecular layer interneurons. In this case we sampled 59 transplanted and 47 host cells from three different explants, receiving postnatal donor cells. Statistical comparisons between the obtained values were made by the Student's t test.
Finally, to estimate the relative numbers of the different types of host cerebellar neurons in the cerebellar explants, we sampled parvalbumin-immunopositive Purkinje cells and molecular layer interneurons and SMI32-positive deep nuclei neurons. The analysis was performed on three hemicerebella from different explants stained by each antibody. The cells were counted on the computer screen in every third microscopic field by moving across the cerebellar region of the explant in a raster manner with the motorized stage of the Neurolucida system (magnification was 20×).
RESULTS
To assess the differentiative potential of cerebellar progenitors at different developmental stages, we examined the fate of heterotopically-heterochronically transplanted cells taken from E12 cerebellar anlage or P4 cerebellar cortex. E12 cerebellar cells include proliferating progenitors destined to generate all cerebellar phenotypes, whereas those from the postnatal cortex produce granule cells, molecular layer interneurons, and glia (Miale and Sidman, 1961
Fate of postnatal cerebellar progenitors transplanted to the embryonic CNS in utero
Successful transplantation was observed in ~65% of the treated embryos, in which numerous EGFP-positive cells were dispersed through the host brain or gathered in clusters of variable size and density. The vast majority of transplanted cells were well integrated within extracerebellar regions of the recipient CNS, with very rare elements remaining on the ventricular surface. The distribution of heterotopically located cells was determined by mapping their position on several sections of the recipient brain (Fig. 1). In all the examined animals, transplanted cells were consistently found throughout the host brainstem, mesencephalon, and diencephalon. In addition, EGFP-positive cells were also seen in basal ganglia and corpus callosum, but they were never found in the neocortex, hippocampal formation, or olfactory bulb.
Postnatal cerebellar cells that engrafted in extracerebellar regions of the host CNS generated both neurons and glia. The majority of glial cells were different types of GFAP-positive astrocytes (Fig. 2A), well integrated into the recipient glial network. In addition, transplanted cells also produced oligodendrocytes. Immature cells of this category could be identified by anti-NG2 labeling (Fig. 2B), whereas their mature counterparts displayed the typical morphology with several processes aligned to host white matter tracts (Fig. 2C).
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Figure 2. A-P, Fate of postnatal cerebellar cells engrafted in ectopic regions of the embryonic brain in utero. The confocal pictures A-C show glial phenotypes generated by postnatal cerebellar precursors, including GFAP-positive astrocytes (A), NG2-positive immature oligodendrocytes (B, taken from an animal killed at P1), and mature oligodendrocytes properly integrated in host white matter tracts (C). A large aggregate of small, densely packed transplanted cells is shown in D (taken from an animal killed at P1); BrdU labeling of the same microscopic field (E) reveals the intense proliferative activity of these cells (numerous host cells outside the cluster are also labeled). Migrating cells, with typical leading and trailing processes, radiate from such clusters (F) and likely continue to divide, as shown by BrdU incorporation (G, arrowheads point to some double-labeled cells). At the end of their migratory phase (H), the cells show several short processes radiating from the perikaryon (e.g., arrowhead) and eventually acquire the typical morphology of granule cells (e.g., arrow). Mature granule cells can be found scattered or clustered in aggregates (arrowheads in I) with bundles of parallel fibers (arrows). The identification of these cells is further supported by the expression of RU49 mRNA (J) as well as
All EGFP-positive neurons generated by grafting postnatal cerebellar progenitors could be classified in two distinct phenotypes, identified as granule cells and molecular layer interneurons. Both cell types were distributed throughout the different recipient regions, but granule cells always prevailed.
Granule cells were generated through a peculiar developmental sequence. They originated from large clusters made of several hundreds tightly packed small cells (Fig. 2D,E), which were a typical feature of the postnatal cerebellar transplants. Large streams of migratory elements, with characteristic leading and trailing processes, radiated from these clusters into the host parenchyma (Fig. 2F). BrdU injections, either before birth or immediately before killing up to several weeks after transplantation, labeled many cells inside the clusters (Fig. 2D,E) or migrating into the host parenchyma (Fig. 2G). In contrast, EGFP-positive cells in such migratory streams were never labeled by any of the applied glial markers, confirming that they belonged to a neuronal lineage.
At the end of the migratory stream the cells displayed transitional morphologies, with ramified processes radiating from the cell body (Fig. 2H), and eventually acquired the structural features of mature granule cells (Fig. 2I,M) with small round perikarya (8-10 µm in diameter), short-clawed dendrites, and thin varicose axons, which sometimes bore the typical T-shaped bifurcation of parallel fibers (Fig. 2N). The identification of these cells was further confirmed by labeling for the
On the basis of this sequence of proliferation, migration, and terminal differentiation, we concluded that the clusters of actively dividing cells represent EGL progenitors that generate mature granule neurons after a migratory phase. Granule neurons were either scattered through the recipient parenchyma or clustered as in Figure 2I. However, confocal scanning throughout several of such clusters confirmed that, with very rare exceptions, they exclusively contained cells of this type.
EGFP-positive cells belonging to the second phenotype were small multipolar neurons with slender dendrites and a short axon ending in the vicinity of the parent cell body. These structural characteristics, together with the strong anti-parvalbumin immunolabeling shown by these neurons (Fig. 2O,P), were distinctive features of molecular layer interneurons, i.e., basket and stellate cells. Indeed, even when such cells were close to parvalbumin-positive host neurons they were always clearly recognizable for their size, morphology, and intensity of immunostaining (see Celio, 1990
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Figure 3. A-K, Fate of embryonic cerebellar cells engrafted in ectopic regions of the embryonic brain in utero. Parvalbumin-immunolabeled Purkinje cells with large dendritic trees (arrowheads) are illustrated in A; arrows point to adjacent parvalbumin-positive host neurons. Another calbindin-immunolabeled transplanted Purkinje cell, bearing less extended dendrites, is shown in the confocal image B. In C a presumptive EGFP-positive Purkinje axon (arrowheads), recognized by the particularly intense fluorescence, terminates on a neurofilament immunostained host neuron. Frequently, Purkinje axons (arrowheads in D point to the parvalbumin-immunolabeled terminal branches) enwrap the somatodendritic surface of EGFP-positive multipolar neurons. E and F show a small cluster of these neurons (asterisks), which are labeled by anti-neurofilament antibodies (F) and covered by strongly fluorescent Purkinje axons (arrowheads in E). The morphological features of EGFP-multipolar neurons, bearing slender dendrites with long ramifications, are illustrated in the confocal picture G; note that this neuron is also double labeled for parvalbumin. H and I show small parvalbumin-immunopositive neurons, classified as molecular layer interneurons. Arrowheads in J point to another two of such neurons settled in the host striatum; note the different size and morphology shown by transplanted and recipient neurons (arrows). A cluster of transplanted granule cells in the host dorsal cochlear nucleus is displayed by the confocal picture K. NF, Neurofilament SMI32; PV, parvalbumin; CaBP, calbindin; EGFP, enhanced green fluorescent protein. Scale bars: K, 10 µm; C, E, F, 15 µm; B, D, 20 µm; A, 25 µm; G-J, 30 µm.
To quantitatively estimate the phenotypic repertoire generated by grafted cells, we counted all the cells encountered in three to five representative vibratome sections from three different brains (see Fig. 4, light gray bars). On the whole, we examined 7057 EGFP-positive cells, including 6858 neurons and 199 glial cells. Among neuronal phenotypes there were 6624 granule cells (96.6%) and 234 molecular layer interneurons (3.4%), uniformly distributed among the different animals. Thus, postnatal cerebellar progenitors are able to engraft in several heterotopic regions of the host CNS, but they exclusively adopt late-generated cerebellar identities.
Fate of embryonic cerebellar progenitors transplanted to the embryonic CNS in utero
The results obtained with P4 cerebellar progenitors indicate that they are committed to cerebellar fates. Hence, we asked whether E12 cerebellar precursors have a wider differentiative potential. Previous observations on the expression of the En-1 gene in heterotopically transplanted mid-hindbrain progenitors indicate that these cells become regionally specified between E10.5 and E13.5 (Olsson et al., 1997
As shown in Figure 1, the distribution of embryonic cerebellar cells in extracerebellar regions of the recipient brain was essentially equivalent to that observed with postnatal donor cells, although the large cell aggregates present in the latter animals were not observed. In addition, in four animals of this set scattered EGFP-positive neurons were also occasionally encountered in the cerebral cortex. In all recipient regions the transplanted cells generated neurons and glia. Among neuronal phenotypes, numerous Purkinje cells could be identified by their morphology and expression of specific markers, such as parvalbumin or calbindin (Fig. 3A,B). They displayed highly ramified dendritic trees (Fig. 3A) or, more frequently, less developed arbors surrounding the cell body (Fig. 3B), similar to those of the rare ectopic Purkinje cells present in the intact brain (De Camilli et al., 1984
In addition to Purkinje cells, the most frequent EGFP-positive nerve cells were a population of medium-sized or large multipolar neurons, characterized by a fainter fluorescence (Fig. 3D-G). These neurons displayed triangular or spindle-shaped cell bodies and long ramified dendrites (Fig. 3D,G), which were frequently covered by the terminal branches of Purkinje axons (Fig. 3D-F). Many of these neurons could be stained by anti-neurofilament antibodies (55%, of 72 sampled neurons) (Fig. 3E,F). The SMI32 antibodies stain deep nuclei neurons (Jankovski et al., 1996
Thus, although all these features indicate that many EGFP-positive multipolar nerve cells were deep nuclei neurons, part of these transplanted cells could not be definitely classified. Nevertheless, EGFP-positive neurons with these characteristics were present in many different regions of the host CNS, and they did not display clear site-specific features. For instance, most of the parvalbumin-immunopositive neurons were not in the vicinity of recipient neurons stained by the same antibody. Thus, on the basis of these considerations, as well as of the results of the in vitro experiments (see below), we concluded that these transplanted cells comprise different populations of deep nuclei neurons plus a minority of other cell types, likely including other cerebellar phenotypes such as Golgi and Lugaro cells, and, possibly, cells that had acquired extracerebellar features.
The embryonic cerebellar cells also produced parvalbumin-immunopositive molecular layer interneurons (Fig. 3H-J), although this phenotype was not present in all the examined cases (Fig. 4). As for postnatal transplants, these cells could be readily identified for their intense immunolabeling and morphology even when they were intermingled with parvalbumin-immunolabeled host neurons (Fig. 3J). In contrast, EGL progenitors or mature granule cells were never observed in extracerebellar regions of the host CNS, except for the dorsal region of the lower brainstem and, most notably, the dorsal cochlear nucleus (Fig. 3K), a structure bearing strict neuroanatomical (Mugnaini et al., 1980
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Figure 4. Quantitative analysis of the phenotypic repertoire generated by embryonic (dark gray) and postnatal (light gray) cerebellar cells transplanted to the embryonic rat brain in utero. For each experimental set, three representative cases are illustrated (indicated on the z-axis). The different neuron phenotypes are represented as the percentage on the total number of transplanted nerve cells observed in the relevant brain. Granule cells generated by embryonic progenitors in the lower brainstem are not included in these counts that were performed on sections encompassing mesencephalon and forebrain, where no granule neurons were found. gc, Granule cells; mli, molecular layer interneurons; pc, Purkinje cells; mn, multipolar neurons.
Fate of embryonic neocortical progenitors transplanted to the embryonic CNS in utero
Both embryonic and postnatal cerebellar progenitors showed a preferential incorporation into defined regions of the recipient forebrain (Fig. 1), suggestive of site-specific interactions between donor cells and local cues. Alternatively, however, it is possible that the distribution of transplanted cells was influenced by mechanical constraints linked to the injection procedure. To address this issue, we performed control experiments in which E12 neocortical progenitors were transplanted to the embryonic CNS in utero.
As shown in Figure 5A-F, neocortical progenitors showed a wide distribution throughout the host forebrain, including many regions in which cerebellar donor cells did not engraft, such as neocortex (Fig. 5A-C), hippocampus (Fig. 5D), and olfactory bulb (Fig. 5F). In addition, most of the grafted neocortical cells displayed distinct structural features according to their integration site (Fig. 5B-F), suggesting that their morphological maturation was influenced by local cues. Thus, the region-specific distribution of cerebellar progenitors within the host forebrain cannot be attributed to particular conditions related to the transplantation procedure.
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Figure 5. A-F, Fate of embryonic neocortical precursors grafted to the embryonic CNS in utero. Micrograph A shows EGFP-positive embryonic neocortical cells (some are pointed by arrowheads) engrafted in the host neocortex. Some of such neurons (B, C) show the morphology of stellate cells and can be immunolabeled by anti-parvalbumin antibodies. Many of these grafted cells show site-specific morphological features, such as presumptive pyramidal neurons in hippocampal CA1 (D), medium-sized spiny neurons in the striatum (E), and olfactory bulb interneurons (F). PV, Parvalbumin, EGFP, enhanced green fluorescent protein. Scale bars: B-E, 20 µm; A, F, 50 µm.
Fate of postnatal and embryonic cerebellar progenitors homotopically engrafted in the embryonic cerebellum
Despite the high frequency of heterotopically incorporated elements, EGFP-positive cells were found in the cerebellum only in five brains receiving postnatal progenitors and in four grafted with embryonic donor cells. In postnatal cerebellar transplants, aggregates of proliferating EGL progenitors were present in the region of the deep nuclei (Fig. 6A). Mature granule neurons were correctly positioned in the internal granular layer of several cortical lobules (Fig. 6B). These granule cells displayed characteristic clawed dendrites (Fig. 6E) and were immunolabeled by antibodies against the
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Figure 6. A-M, Fate of postnatal (A-I) and embryonic (J-M) cerebellar cells homotopically engrafted in the embryonic cerebellum in utero. Micrograph A, taken from a rat transplanted with postnatal donor cells, shows a survey of the host cerebellum, stained with anti-parvalbumin antibodies (dn, deep cerebellar nuclei; cc, cerebellar cortex): note the large aggregates of EGFP-positive cells in the region of the deep nuclei. Numerous EGFP-positive cells are also located in the granular layer (B), where they develop into mature granule cells. Such neurons display typical ascending axons (arrowheads in C, D) and parallel fibers (arrows) running along the longitudinal axis of the folium. In addition, they bear characteristic short clawed dendrites (E) and express the
Embryonic cerebellar cells that settled in the host cerebellum comprised rare Purkinje cells or deep nuclei neurons, usually <10 cells per animal, except for one cerebellum that contained several dozens of Purkinje cells. Purkinje cells, scattered over different host lobules, were correctly positioned in their cortical layer and bore characteristic monoplanar dendritic trees and thin axons traversing the granular layer (Fig. 6J,K). Nuclear neurons were located within the central gray matter, displayed the typical multipolar morphology (Fig. 6L), and were surrounded by numerous parvalbumin- or calbindin-immunolabeled Purkinje cell synaptic boutons (Fig. 6M). Finally, two of these cerebella also contained mature granule cells (data not shown), localized in the recipient granular layer or ectopically positioned in the region of the deep nuclei. These neurons displayed the same morphological features and anti-
Fate of postnatal cerebellar progenitors transplanted to organotypic explants of the embryonic CNS
In line with a previous report (Jankovski et al., 1996
The cerebellar region of the brainstem-cerebellar explants (Chédotal et al., 1997
Numerous EGFP-positive cells engrafted in the brainstem-cerebellar explants and generated neurons and glia. Neuronal types comprised granule cells and molecular layer interneurons. Granule cells showed a similar behavior to that observed in vivo, with large clusters of densely packed precursors, numerous migrating cells radiating into the host tissue (Fig. 7A), and mature elements with typical morphology (Fig. 7B). Molecular layer interneurons were readily identified by their morphology and parvalbumin immunolabeling (Fig. 7C). In addition, their perikaryal size [97.5 ± 19 µm2; mean ± SD; minimum (min), 62 µm2; maximum (max), 156 µm2; n = 59] was equivalent to that of their host counterparts (101.7 ± 19.8 µm2; min, 64 µm2; max, 160 µm2; n = 47). In four different explants maintained in vitro for 15-25 d we counted 6426 EGFP-positive cells (4776 neurons and 1650 glia). Neuronal phenotypes comprised 4307 granule cells (90%) and 469 molecular layer interneurons (10%). As shown in Figure 8A, the distribution of neuronal phenotypes was essentially similar in all the cultures, with no clear differences between cerebellar and extracerebellar regions.
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Figure 7. A-H, Fate of postnatal (A-E) and embryonic (F-L) cerebellar cells transplanted to cerebellar (A-C, F-J) or neocortical explants (D, E, K, L). In the cerebellar explants, postnatal cerebellar progenitors form dense aggregates (A) with numerous migrating elements (some are pointed by arrowheads). Mature granule cells, bearing typical morphological features, are shown in B, whereas parvalbumin-immunopositive molecular layer interneurons are displayed in C. D and E illustrate the neuronal phenotypes generated by postnatal cerebellar cells grafted to neocortical explants, granule cells (arrowheads in D) and molecular layer interneurons (arrowheads in E). The latter are also labeled by anti-parvalbumin antibodies (E). The micrographs F and G show EGFP-positive Purkinje cells derived from embryonic precursors intermingled with their host counterparts (asterisks in G) in the cerebellar cortex of the explant. Examples of EGFP-positive multipolar neurons in cerebellar explants are illustrated in H-J. Some of these neurons (arrowhead in H and I) are localized in the deep nuclei of the explant, and can be stained by anti-neurofilament antibodies (arrow in I points to an immunolabeled host neuron). In neocortical explants embryonic cerebellar progenitors also generate parvalbumin-immunopositive Purkinje cells (arrows in K) with variably shaped dendritic trees and granule cells (K, L, some are pointed by arrowheads in K). PV, Parvalbumin; NF, neurofilament; EGFP, enhanced green fluorescent protein. Scale bars: B, C, 20 µm; A, F, G, K, 25 µm; L, 30 µm; D, E, H-J, 50 µm.
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Figure 8. A-D, Quantitative analysis of the phenotypic repertoire generated by postnatal (A, B) and embryonic (C, D) cerebellar cells transplanted to cerebellar (A, C) or neocortical (B, D) explants. Each histogram shows the distribution of neuron phenotypes generated by grafted cells in four representative explants from each experimental set (indicated on the z-axis of each histogram). The different cell types are represented as the percentage on the total number of transplanted neurons observed in the relevant explant. gc, Granule cells; mli, molecular layer interneurons; pc, Purkinje cells; mn, multipolar neurons; cb, brainstem-cerebellar explant; ncx, neocortical explant.
Similar results were obtained when the postnatal cerebellar progenitors were grafted to neocortical explants, although a lower amount of cells survived in this condition. Among 1167 EGFP-positive cells from four different cultures there were 972 neurons and 195 glial cells (Fig. 7D,E), including 912 granule and (93.8%) and 60 parvalbumin-immunopositive molecular layer interneurons (6.2%) (Fig. 8B). Hence, the results of these in vitro experiments confirmed those obtained in vivo by showing that postnatal cerebellar progenitors are strictly committed to generate postnatal cerebellar phenotypes.
Fate of embryonic cerebellar progenitors transplanted to organotypic explants of the embryonic CNS
Embryonic cerebellar progenitors also engrafted in the brainstem-cerebellar explants and produced different mature phenotypes. Numerous Purkinje cells were dispersed in the extracerebellar regions of the explant or well integrated in the cerebellar cortex, and they developed mature features in parallel with their host counterparts (Fig. 7F,G). Similar to the in vivo experiments, these explants also contained a population of medium-sized or large EGFP-positive multipolar neurons (Fig. 7H-J). These cells, frequently labeled by anti-neurofilament antibodies, were present all over the explant, including the deep nuclei (Fig. 7H,I), where they were mixed to host neurons and innervated by Purkinje axons (data not shown). Morphometric evaluation of these neurons revealed a mean cell body size of 322.6 µm2 (±141.7; mean ± SD; min, 110 µm2; max, 836 µm2; n = 88). This value was clearly higher than that of molecular layer interneurons (see above; Student's t test; p < 0.0001), but also significantly lower than that of host neurofilament-immunopositive deep nuclei nerve cells (mean perikaryal size, 385 ± 117.7 µm2; min, 191 µm2; max, 836 µm2; n = 82; Student's t test; p = 0.002). Comparison of the frequency distributions of cell body sizes indicated that the EGFP-positive cells include the large neurofilament-positive neurons plus a subset of medium sized nerve cells that are not stained by SMI-32 antibodies. These data corroborate the conclusion drawn from in vivo experiments that this category of transplanted cells encompasses deep nuclei neurons and a minority of other cell types that could not be definitely identified.
In addition to these phenotypes, EGFP-positive cells also generated parvalbumin-immunopositive molecular layer interneurons and granule cells (data not shown). These types, however, represented a minority of the transplanted cells. Indeed, among 6980 cells from four explants we counted 4515 neurons (64.7%) and 2465 glial cells (35.3%). Neuronal phenotypes included 1997 Purkinje cells (44.2%), 1395 multipolar neurons (31%), 1095 granule cells (24.2%), and 28 molecular layer interneurons (0.6%). As shown in Figure 8C, granule cells were not uniformly distributed in the four examined explants, being rare in several cases. Thus, although E12 cerebellar cells grafted to homotopic-homochronic explants are able to produce all cerebellar phenotypes, they preferentially adopt earlier generated identities.
In a parallel set of experiments we tested whether E12 cerebellar progenitors can integrate in explants of neocortex, a region where they rarely penetrate in vivo. Transplanted cells were viable also in these explants and acquired different neuronal and glial phenotypes. The vast majority of EGFP-positive nerve cells were Purkinje cells, with variably shaped dendritic trees (Fig. 7K) and granule cells displaying typical morphological features (Fig. 7K,L). In contrast, other phenotypes, such as multipolar neurons or molecular layer interneurons, were extremely rare. Cell counts (Fig. 8D) yielded 5996 neurons and 2461 glial cells, which included 5261 granule cells (87.7%), 683 Purkinje cells (11.3%), 35 molecular layer interneurons (0.6%), and 17 multipolar neurons (0.3%). On the whole, the results of in vitro experiments further confirm the conclusion that embryonic cerebellar cells are committed to cerebellar fates, but, contrary to their postnatal counterparts, they have the potential to generate all major cerebellar types. Nevertheless, the ratio of mature phenotypes that will be actually generated is influenced by the environment in which the transplanted cells incorporate.
DISCUSSION
To assess the differentiative potential of cerebellar precursors at different developmental stages, we examined the fate of embryonic and postnatal progenitors after heterotopic-heterochronic transplantation to the embryonic CNS. Our results show that: (1) cerebellar cells of both ages engraft and produce mature neurons and glia that survive long after transplantation in wide areas of the host brain, but they show a characteristic region-specific distribution; (2) most ectopically located cells adopt cerebellar identities; (3) embryonic progenitors are able to generate all cerebellar neuron phenotypes, whereas postnatal ones are restricted to granule cells and molecular layer interneurons. Altogether, these observations indicate that cerebellar progenitors are regionally specified to local fates already at E12, and their potential is gradually restricted toward late-generated identities as development advances.
Distribution of transplanted cerebellar cells into the host CNS
Both embryonic and postnatal cerebellar cells transplanted in utero integrate in the host brainstem, diencephalon, and in some telencephalic structures, but they are seldom found in the cerebral cortex. Such a distribution pattern is consistent with another report concerning E14 donor cells (Olsson et al., 1998
Different types of neural progenitors transplanted into the developing brain incorporate in ectopic positions, but each donor population preferentially engrafts into specific recipient regions (Brüstle et al., 1995
EGFP-positive cells incorporate in the cerebellum only in some cases. It is likely that very few proliferating donor cells engraft into the E16 cerebellar anlage, because of its small dimensions and distant position from the transplantation site in the forebrain ventricles. Indeed, cerebellar localization of transplanted cells has been rarely reported (Lim et al., 1997
Mature phenotypes of transplanted cerebellar cells
Another goal of our work was to determine whether heterotopically transplanted cerebellar cells adopted site-specific phenotypes. Respecification of grafted precursors toward local identities has been shown for several progenitor cell populations (Brüstle et al., 1995
The intraventricular injection of single cell suspensions and the wide dispersion of transplanted cells through the host parenchyma indicate that, in our experiments, proliferating cerebellar progenitors were effectively exposed to the host environment. Nevertheless, most transplanted cells generated cerebellar neuron phenotypes, which could be readily identified by morphological criteria and cell-specific markers. Only a minority of the EGFP-positive multipolar neurons derived from E12 progenitors escaped a reliable classification. These cells lacked clear distinctive characteristics, but they were consistently observed in all recipient regions, including homotopic cerebellar explants, and they did not display definite site-specific features. Furthermore, although some of the markers used in this study also stained defined populations of extracerebellar neurons, we never observed any grafted cerebellar cells displaying unusual host-specific expression of such markers (e.g., calbindin- or parvalbumin-immunopositive granule cells). Thus, although we could not identify every single transplanted cell, and we cannot exclude that a lean minority of them actually changed their fate or expressed host-specific markers, our results indicate that respecification of cerebellar progenitors toward heterotypic phenotypes was a rare event, limited to embryonic progenitors.
Previous observations limited to 4 d after transplantation also indicate that mid-hindbrain progenitors become regionally specified between E10.5 and E13.5 (Olsson et al., 1997
The differentiative potential of embryonic and postnatal cerebellar cells
Despite their specification toward cerebellar fates, embryonic and postnatal progenitors show different developmental potentialities. Embryonic precursors transplanted to the early postnatal cerebellum produce a variety of mature phenotypes, whereas EGL progenitors are restricted to a granule cell fate (Gao and Hatten, 1994
In the different transplants, postnatal precursors always produced granule cells and molecular layer interneurons. In contrast, the phenotypic repertoire generated by the embryonic donors was not constant in all conditions. Both in heterotopic positions in vivo and in the cerebellar explants they preferentially adopted earlier-generated identities, whereas granule cells and molecular layer interneurons were less frequent. Nevertheless, the latter phenotypes were abundant in other conditions, most notably granule cells in the cerebellum in vivo or in neocortical explants, showing that embryonic cell suspensions did contain progenitors able to produce these types. As shown for rhombic lip cells (Alder et al., 1996
It is unclear whether such cues select distinct subsets of precursors already oriented toward specific identities or they provide instructive information to multipotent progenitors. Some studies indicate that individual cerebellar lineages get separated during early embryonic development (Alder et al., 1996
FOOTNOTES Received April 1, 2002; revised May 8, 2002; accepted June 10, 2002.
This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Ministero della Sanità Progetto Alzheimer (300RFA00/01-05) (F.R., L.M.), and Consiglio Nazionale delle Ricerche (Progetto Strategico basi biologiche delle malattie degenerative del sistema nervoso centrale). We are indebted to Dr. Masaru Okabe for the gift of EGFP mice and to Dr. Nathaniel Heintz for providing us with the RU49 mRNA probe. We also thank Mrs. Luisella Milano, Pina Dellisanti, and Graziella Milano for precious technical assistance.
Correspondence should be addressed to Ferdinando Rossi, Rita Levi Montalcini Centre for Brain Repair, Department of Neuroscience, University of Turin, Corso Raffaello 30, I-10125 Turin, Italy. E-mail: ferdinando.rossi{at}unito.it
